Histology A Text and Atlas With Correlated Cell and Molecular Biology 9th 9781975181512, 1975181514

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NINTH EDITION

HISTOLOGY A TEXT AND ATLAS With Correlated Cell and Molecular Biology

Wojciech Pawlina

Walking in freezing winter cold weather (−22°C) on his driveway in Rochester, Minnesota contemplating on snow-white histologic structures: white blood cells, white adipocytes, white pulp of the spleen, white matter of the brain and spinal cord, white muscle fibers, and perhaps corpus albicans, and tunica albuginea. (Photograph by Kevin J. Ness.)

Acquisitions Editor: Crystal Taylor Development Editor: Andrea Vosburgh/Deborah Bordeaux Freelance Editor: Kathleen H. Scogna Production Project Manager: Kirstin Johnson Marketing Manager: Danielle Klahr Manager, Graphic Arts & Design: Steve Druding Art Director: Jennifer Clements Manufacturing Coordinator: Margie Orzech-Zarenko Prepress Vendor: S4Carlisle Publishing Services Top cover image: Courtesy of Drs. Daniel Berger and Jeff W. Lichtman, Harvard University, Cambridge, MA Ninth Edition Copyright © 2024 Wolters Kluwer. Copyright © 2020, 2016 Wolters Kluwer Health. Copyright © 2011, 2006, 2003 Lippincott Williams & Wilkins. Copyright © 1995, 1989 Williams & Wilkins. Copyright © 1985 Harper & Row, Publisher, J. B. Lippincott Company. All rights reserved. This book is protected by copyright. No part of this book may be reproduced or transmitted in any form or by any means, including as photocopies or scanned-in or other electronic copies, or utilized by any information storage and retrieval system without written permission from the copyright owner, except for brief quotations embodied in critical articles and reviews. Materials appearing in this book prepared by individuals as part of their official duties as U.S. government employees are not covered by the above-mentioned copyright. To request permission, please contact Wolters Kluwer at Two Commerce Square, 2001 Market Street, Philadelphia, PA 19103, via email at [email protected], or via our website at shop.lww.com (products and services). 9 8 7 6 5 4 3 2 1 Printed in Mexico

Library of Congress Cataloging-in-Publication Data ISBN-13: 978-1-975181-51-2 ISBN-10: 1-975181-51-4 Library of Congress Control Number: 2023907331

This work is provided “as is,” and the publisher disclaims any and all warranties, express or implied, including any warranties as to accuracy, comprehensiveness, or currency of the content of this work. This work is no substitute for individual patient assessment based upon healthcare professionals’ examination of each patient and consideration of, among other things, age, weight, gender, current or prior medical conditions, medication history, laboratory data, and other factors unique to the patient. The publisher does not provide medical advice or guidance, and this work is merely a reference tool. Healthcare professionals, and not the publisher, are solely responsible for the use of this work including all medical judgments and for any resulting diagnosis and treatments. Given continuous, rapid advances in medical science and health information, independent professional verification of medical diagnoses, indications, appropriate pharmaceutical selections and dosages, and treatment options should be made and healthcare professionals should consult a variety of sources. When prescribing medication, healthcare professionals are advised to consult the product information sheet (the manufacturer’s package insert) accompanying each drug to verify, among other things, conditions of use, warnings, and side effects and identify any changes in dosage schedule or contraindications, particularly if the medication to be administered is new, infrequently used, or has a narrow therapeutic range. To the maximum extent permitted under applicable law, no responsibility is assumed by the publisher for any injury and/or damage to persons or property, as a matter of products liability, negligence law or otherwise, or from any reference to or use by any person of this work. shop.lww.com

This edition is dedicated to Teresa Pawlina, my wife, colleague, and best friend, whose love, patience, and endurance created a safe haven for working on this textbook and to my children Conrad Pawlina and Stephanie Pawlina Fixell, whose stimulation and excitement are always contagious and to my grandchildren Alexander Conrad Fixell and Zofia Marie Pawlina, whose capability to learn new life skills is simply breathtaking.

PREFACE Histology: A Text and Atlas With Correlated Cell and Molecular Biology continues its tradition This ninth edition of

of introducing Health Professions students to the foremost world of histology with cell and molecular biology combined. In addition, to better understand the nature of cells and tissues, the presented material is immersed in basic anatomy, embryology, and physiology and is accompanied by relevant clinical commentaries. As in previous editions, this book is a combination “text-atlas” in that the standard textbook descriptions of histologic concepts are supplemented by an array of schematics, tissue and cell images, and clinical photographs. The separate atlas sections conclude each chapter to provide large-format, labeled atlas plates with detailed legends that highlight and summarize the elements of microscopic anatomy. is, therefore, “two books in one.” This edition of is intended to serve as a reliable resource and clinical viewpoint for those who seek to understand histology from medical, dental, graduate, undergraduate, and other health professions perspective. Inclusion of current and up-to-date information provides a solid framework on which to build further scientific exploration and clinical application. As a student resource, it should not be approached with the goal of memorizing detailed facts but rather as a guide for learning by extracting from all explanations key concepts that will serve future academic pursuits. The following improvements have been made to this edition:

Histology: A Text and Atlas Histology: A Text and Atlas With Correlated Cell and Molecular Biology

All figures in this book have been carefully reviewed, revised, and updated. Several new figures have been added to show the latest interpretation of important concepts based on

recent discoveries in molecular and cellular research. All drawings maintain a uniform style throughout the chapters with a palette of eye-pleasing colors. Several new conceptual drawings have been aligned with photomicrographs or electron micrographs, a feature carried over from previous editions that has received wide acclaim from reviewers, students, and faculty members. In addition, all atlas plates have been renumbered to be consistent with chapter numbers. . Text material from the eighth edition has been carefully revised and updated to include the latest advancements in cellular and molecular biology, stem cell biology, cellular markers, and cell signaling. The ninth edition focuses on key concepts to help students comprehend these rapidly increasing fields. To accommodate reviewers’ suggestions, the ninth edition integrates new information in cell biology with clinical correlates, which readers will see as new clinical information items highlighted in blue text and in clinical correlations and functional considerations folders. For example, the last few years of the COVID-19 pandemic has sparked interest about the changes in normal tissue when infected by the severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2) virus. Several chapters contain descriptions of these changes with underlying explanations of cellular and molecular mechanisms and clinical features presented by patients. Additional changes include the following:

Cellular and molecular biology content has been updated

A new discussion on the mononuclear phagocyte system and the cell biology of resident tissue macrophages has been added. The latest research findings in immune cell activation have been incorporated. Updated cellular biology topics include beige adipose tissue, the epithelial–mesenchymal transition, conjunctiva-associated lymphatic tissue, biogenesis and function of peroxisomes, and exosomes as the newest discovered form of cell-to-cell communication.

New, more detailed information about the histology of the female and male external genitalia has been included. The skin chapter has been supplemented and updated with many new images and discussion on skin color and skin aging. With the constant improvement in microscopic methods, a new basic discussion on three-dimensional (3D) microscopy methods was incorporated in the methods chapter.

“Histology 101” sections have been revised and updated. These sections contain clear and concise summaries

for a quick review of the material listed in a “sticky notes” format posted on the notebook pages at the end of each chapter. The bullet-point format is designed specifically for students needing a quick review and is especially useful for quiz and examination preparation. These reader-friendly sections allow fast information retrieval with concepts and facts grouped on separate sticky notes. The colored sticky notes have been designed with ample free space to allow readers to write their own notes to complement the bulleted points. . Similar to the previous editions of this book, the aim is to provide ready access to important concepts and essential information. Changes introduced in previous editions, such as bolded key terms, clinical information in blue text, pages with color-coded edges, and a fresh design for clinical correlation folders, were all enthusiastically approved by the new generation of textbook users and have been maintained in this edition. Essential terms within each specific section are introduced within the text in eye-catching, oversized, bold, red font. Text containing clinical information and the latest research findings is presented in blue, with terminology pertaining to diseases, conditions, symptoms, or causative mechanisms highlighted in oversized bold blue font. Each clinical folder contains updated clinical text with even more illustrations and drawings and is easily found within

Reader-friendly innovations have been implemented

each chapter. As in previous editions, all changes have been made with students in mind. The author–editor team strived for clarity and concision to aid student comprehension of the subject matter, familiarity with the latest information, and application of newfound knowledge.

Wojciech Pawlina

ACKNOWLEDGMENTS Dr. Michael

I remain grateful to the creator of this book, , my mentor, colleague, and dear friend, for the ability to carry on his vision for teaching histology. Many changes in histology education have occurred in the last two decades. In today’s medical curricula, histology courses continue to lose their identity as they become integrated into larger didactic blocks. In the digital world of virtual histology, often driven by self-directed instructions, there is a need for a comprehensive textbook from which students can pick small chunks of knowledge for their specific learning assignments across many disciplines. The classical descriptive histology of the past is no longer sufficient for understanding the structure and function of cells and tissues. Modern histology is increasingly becoming the study of cell and molecular biology, a trend we had foreseen in the early 2000s. From the fifth edition of this textbook (2006), we increased emphases on topics of cell and molecular biology that are aligned with histology; as acknowledged by adding the subtitle “ ” to the fifth and subsequent editions of this textbook. Dr. Ross vision to provide the best quality modern histology text with superior imaging integrated with the most recent advances in cell and molecular biology and supporting clinical facts remained unchanged. Changes to the ninth edition arise largely from comments and suggestions of students from around the world who have taken the time and effort to send me messages about what they like about the book and, more importantly, how the book might be improved to help them better learn histology. I have also received thoughtful comments from my first-year histology students who often direct me to explore new discoveries and achievements in the many fields related to histology. I am

H. Ross

With Correlated Cell and Molecular Biology

grateful to them for their keen sense of sharpening this work. Also, many of my colleagues who teach histology, cell biology, immunology, and physiology courses have helped improve this new edition. Many have suggested a stronger emphasis on clinical relevance and new discoveries, especially in cell and molecular biology, which I strive to include as new research emerges. Others have provided new photomicrographs, electron micrographs, access to their virtual slide collections, proposals for new tables, and have suggested which existing diagrams and figures need to be redrawn. Specifically, I owe my thanks to the following reviewers, who have spent time to provide me with constructive feedback that had an impact on this current edition.

Jan Aten, PhD

Amsterdam University Medical Center Amsterdam, The Netherlands

Stefanie Attardi, PhD

Oakland University William Beaumont School of Medicine Rochester, Michigan

Barış Baykal, MD

Gülhane Faculty of Medicine University of Health Sciences Ankara, Türkiye

Paul B. Bell, Jr., PhD University of Oklahoma Norman, Oklahoma

Jalaluddin Bin Mohamed, MBBS, PhD

National Defence University of Malaysia Kuala Lumpur, Malaysia

David E. Birk, PhD

University of South Florida, College of Medicine Tampa, Florida

Christy Bridges, PhD

Mercer University School of Medicine Macon, Georgia

Craig A. Canby, PhD

Des Moines University Des Moines, Iowa

Stephen W. Carmichael, PhD

Mayo Clinic College of Medicine and Science Rochester, Minnesota

Yasmin Carter, PhD

University of Massachusetts Chan Medical School Worcester, Massachusetts

Pike See Cheah, PhD

Universiti Putra Malaysia Serdang, Selangor, Malaysia

Kevin N. Christensen, MD Winona Health Winona, Minnesota

Sookja K. Chung, PhD

Macau University of Science and Technology Taipa, Macau

John Clancy, Jr., PhD

Loyola University Medical Center Maywood, Illinois

Rita Colella, PhD

University of Louisville School of Medicine Louisville, Kentucky

Iris M. Cook, PhD

State University of New York Westchester Community College Valhalla, New York

Robert D. Cottrell, MHS, PA (ASCP)CM

Quinnipiac University School of Health Sciences Hamden, Connecticut

Dongmei Cui, MD, PhD

University of Mississippi Medical Center Jackson, Mississippi

Eduard I. Dedkov, MD, PhD

Cooper Medical School of Rowan University Camden, New Jersey

Andrea Deyrup, MD, PhD

University of South Carolina School of Medicine Greenville Greenville, South Carolina

Lori B. Dribin, PhD

Nova Southeastern University Fort Lauderdale, Florida

Jennifer Eastwood, PhD

Burrell College of Osteopathic Medicine Las Cruces, New Mexico

Rodrigo Enrique Elizondo-Omaña, MD, PhD

Autonomous University of Nuevo León, School of Medicine Monterrey, Nuevo León, Mexico

Tamira Elul, PhD

Touro University College of Osteopathic Medicine Vallejo, California

Francis A. Fakoya, MBChB, MSc, PhD

St. George’s University School of Medicine

True Blue, Grenada, West Indies

Bruce E. Felgenhauer, PhD

University of Louisiana at Lafayette Lafayette, Louisiana

G. Ian Gallicano, PhD

Georgetown University School of Medicine Washington, District of Columbia

Joaquin J. Garcia, MD

Mayo Clinic College of Medicine and Science Rochester, Minnesota

Mathangi Gilkes, MBBS, MSc

St. George’s University School of Medicine True Blue, Grenada, West Indies

Ferdinand Gomez, MS

Herbert Wertheim College of Medicine Florida International University Miami, Florida

Amos Gona, PhD

University of Medicine & Dentistry of New Jersey New Brunswick, New Jersey

Ervin M. Gore, PhD

Middle Tennessee State University Murfreesboro, Tennessee

Joseph P. Grande, MD, PhD

Mayo Clinic College of Medicine and Science Rochester, Minnesota

Joseph A. Grasso, PhD

University of Connecticut Health Center Farmington, Connecticut

Shannon Haley, MD, PhD

Thomas Jefferson University Philadelphia, Pennsylvania

Brian H. Hallas, PhD

New York Institute of Technology Old Westbury, New York

Arthur R. Hand, DDS

University of Connecticut School of Dental Medicine Farmington, Connecticut

Robert J. Hillwig, MD

Kentucky College of Osteopathic Medicine University of Pikeville Pikeville, Kentucky

Charlene Hoegler, PhD

Pace University—Pleasantville Campus Pleasantville, New York

Christopher Horst Lillig, PhD University of Greifswald Greifswald, Germany

Michael Hortsch, PhD

University of Michigan Medical School Ann Arbor, Michigan

Jim Hutson, PhD

Texas Tech University Lubbock, Texas

John-Olov Jansson, MD, PhD University of Gothenburg Gothenburg, Sweden

Malsawmzuali (JC) Joute Chawngvawr, MS

Cardiovascular Research, Mayo Clinic Rochester, Minnesota

Cynthia J. M. Kane, PhD

University of Arkansas for Medical Sciences Little Rock, Arkansas

Punnose K. Kattil, MBBS, MD

Mayo Clinic College of Medicine and Science Rochester, Minnesota

Nazanin Yeganeh Kazemi, MD, PhD

Mayo Clinic College of Medicine and Science Rochester, Minnesota

Michael J. Kern, PhD

Medical University of South Carolina Charleston, South Carolina

G. M. Kibria, MD

National Defence University of Malaysia Kuala Lumpur, Malaysia

Thomas S. King, PhD

University of Texas Health Science Center at San Antonio San Antonio, Texas

Bruce M. Koeppen, MD, PhD

Quinnipiac University Frank H. Netter MD School of Medicine North Haven, Connecticut

Andrew Koob, PhD

University of Wisconsin—River Falls River Falls, Wisconsin

Craig Kuehn, PhD

Western University of Health Sciences

Pomona, California

Ton La, MD, JD, LLM

Baylor College of Medicine Houston, Texas

Nirusha Lachman, PhD

Mayo Clinic College of Medicine and Science Rochester, Minnesota

Gavin R. Lawson, PhD

Western University of Health Sciences Pomona, California

Susan LeDoux, PhD

University of South Alabama Mobile, Alabama

Karen Leong, MD

Drexel University College of Medicine Philadelphia, Pennsylvania

Kenneth M. Lerea, PhD

New York Medical College Valhalla, New York

Frank Liuzzi, PhD

Lake Erie College of Osteopathic Medicine Bradenton, Florida

Donald J. Lowrie, Jr., PhD

University of Cincinnati College of Medicine Cincinnati, Ohio

Kuo-Shyan Lu, PhD

National Taiwan University College of Medicine Taipei, Taiwan

Andrew T. Mariassy, PhD

Nova Southeastern University College of Medical Sciences Fort Lauderdale, Florida

Geoffrey W. McAuliffe, PhD

Rutgers Robert Wood Johnson Medical School Piscataway, New Jersey

Kevin J. McCarthy, PhD

Louisiana State University Health Sciences Center Shreveport Shreveport, Louisiana

David L. McWhorter, PhD

Georgia Campus—Philadelphia College of Osteopathic Medicine Suwanee, Georgia

Fabiola Medeiros, MD

Cedars-Sinai Medical Center Los Angeles, California

William D. Meek, PhD

College of Osteopathic Medicine Oklahoma State University Tulsa, Oklahoma

Björn Meister, MD, PhD Karolinska Institutet Stockholm, Sweden

Amir A. Mhawi, DVM, PhD

Saba University School of Medicine Saba, Dutch Caribbean

Siobhan Moyes, PhD

University of Plymouth Plymouth, United Kingdom

Frank E. Nelson, PhD

Temple University Philadelphia, Pennsylvania

Christine E. Niekrash, DMD

Frank H. Netter MD School of Medicine Quinnipiac University North Haven, Connecticut

Diego F. Nino, PhD

Louisiana State University Health Sciences Center Delgado Community College New Orleans, Louisiana

Sasha N. Noe, DO, PhD Saint Leo University Saint Leo, Florida

Mohammad (Reza) Nourbakhsh, PhD University of North Georgia Dahlonega, Georgia

Ivón T. C. Novak, PhD

National University of Córdoba Córdoba, Argentina

Joanne Orth, PhD

Temple University School of Medicine Downingtown, Pennsylvania

Fauziah Othman, DVM, PhD

Universiti Putra Malaysia Serdang, Selangor, Malaysia

Claus Oxvig, PhD

Aarhus University Aarhus C, Denmark

Scott Paterson, PhD

University of Bristol Bristol, United Kingdom

Malin Petersson, MD

Karolinska Institutet Stockholm, Sweden

Thomas E. Phillips, PhD University of Missouri Columbia, Missouri

Stephen R. Planck, PhD

Oregon Health & Science University Portland, Oregon

Harry H. Plymale, PhD

San Diego State University San Diego, California

Rebecca L. Pratt, PhD

Oakland University William Beaumont School of Medicine Rochester, Michigan

Margaret Pratten, PhD

Medical School, University of Nottingham Nottingham, United Kingdom

Rongsun Pu, PhD

Kean University East Brunswick, New Jersey

Edwin S. Purcell, PhD

University of Medicine and Health Sciences Basseterre, St. Kitts & Nevis

Romano Regazzi, PhD

Faculty of Biology and Medicine University of Lausanne

Lausanne, Switzerland

Herman Reid, DVM, MD

Saba University School of Medicine Saba, Dutch Caribbean

Mary Rheuben, PhD

Michigan State University East Lansing, Michigan

Michael S. Risley, PhD

Albert Einstein College of Medicine—Jack and Pearl Resnick Campus Bronx, New York

Melvin G. Rosenfeld, PhD

New York University School of Medicine New York, New York

Jeffrey L. Salisbury, PhD

Mayo Clinic College of Medicine and Science Rochester, Minnesota

David K. Saunders, PhD

University of Northern Iowa Cedar Falls, Iowa

Roger C. Searle, PhD

School of Medical Sciences, Newcastle University Newcastle, United Kingdom

Lorenzo R. Sewanan, MD, PhD Yale School of Medicine New Haven, Connecticut

Allen A. Smith, PhD

Barry University Miami Shores, Florida

Carles Solsona, PhD

Faculty of Medicine, University of Barcelona Barcelona, Spain

Jamil Talukder, DVM, PhD

University of Wisconsin—Stout Menomonie, Wisconsin

Sehime G. Temel, MD, PhD Uludağ University Bursa, Türkiye

Barry Timms, PhD

Sanford School of Medicine, University of South Dakota Vermillion, South Dakota

James J. Tomasek, PhD

University of Oklahoma Health Science Center Oklahoma City, Oklahoma

John Matthew Velkey, PhD University of Michigan Ann Arbor, Michigan

Suvi Kristiina Viranta-Kovanen, PhD University of Helsinki Helsinki, Finland

Robert Waltzer, PhD

Belhaven University Jackson, Mississippi

Scott A. Weed, PhD

West Virginia University School of Medicine Morgantown, West Virginia

Taylor M. Weiskittel, MD, PhD

Mayo Clinic College of Medicine and Science

Rochester, Minnesota

Brandon A. Wilbanks, PhD

Mayo Clinic College of Medicine and Science Rochester, Minnesota

Anne-Marie Williams, PhD

School of Medical Sciences, University of Tasmania Hobart, Australia

Joan W. Witkin, PhD

College of Physicians and Surgeons, Columbia University New York, New York

Robert W. Zajdel, PhD

State University of New York Upstate Medical University Syracuse, New York As in every new edition, several colleagues have made especially notable contributions to this textbook. I am extremely grateful to Dr. from the National University of Córdoba in Argentina for providing a detailed review of the immune system with many helpful suggestions for improvements; to Dr. for editing and adding helpful comments to the chapter on lymphatic system; to Dr. from the University of Michigan Medical School for providing guidance in obtaining permission to use their outstanding virtual microscopy slide collection; to Dr. from Winona Health for providing original histologic images of skin specimens and for interesting discussions on dermatopathology; to Dr. from the Mayo Clinic College of Medicine and Science who provided me with many ideas and feedback for improvements; to Dr. ış , a Turkish translation editor from the University of Health Sciences, Gülhane Faculty of Medicine in Ankara, Türkiye, for providing a list of improvements he compiled while translating the eighth edition; and to the many other clinicians, researchers, and

Ivón T. C. Novak

Nazanin Yeganeh Kazemi Michael Hortsch

Kevin N. Christensen

Lachman

Bar Baykal

Nirusha

educators who gave me permission to use their original, unique digital images; electron micrographs; photomicrographs; and 3D reconstruction images in this edition. They are all acknowledged in the appropriate figure legends. When Wolters Kluwer initiated this ninth edition in early 2020, we had no idea that this revision would take longer than expected. The unforeseen factor, the COVID-19 pandemic, impacted our schedule as new demands on developing emergency online virtual resources took precedence over the scheduled activities of many histology educators. With pandemic restrictions, the limited access to our medical school laboratories, specimens and specimen processing facilities, and imaging equipment delayed many scheduled material developments for this edition. Despite these challenges, there have been many bright spots in the development of this edition. , senior acquisitions editor, is acknowledged for her longlasting (more than 20 years) support and encouragement throughout the development and improvement of this book. , freelance editor, as in all previous editions since the fourth edition (2003), edited the manuscript and provided comments and suggestions with honest feedback and constructive advice. For any author, a relationship built on mutual respect with a trusted editor is essential for a successful textbook. I am fortunate to experience such a relationship while working with Kathleen. I was once again privileged that from the Dragonfly Media Group (Baltimore, Maryland), one of the most talented medical illustrators working today, whom I have referred to in previous edition as the “Michelangelo of Histology’s Sistine Chapel,” agreed to work on this ninth edition. Rob added several new illustrations and improved many old illustrations in this edition. His commitment and willingness to work as an artist–author team provided an unprecedented creative dynamic that has made all the

Crystal Taylor

Kathleen H. Scogna

Rob Duckwall

difference. Rob has made each and every drawing an unparalleled work of fine art. I also wish to extend my appreciation to , the art director, for relabeling and replacing images in the text and atlas sections of this book. Finally, special thanks go to , development editor, who joined the team for this ninth edition, for managing all revised chapters and figures.

Clements

Jennifer

Deborah Bordeaux

CONTENTS Preface Acknowledgments 1

Methods

OVERVIEW OF METHODS USED IN HISTOLOGY TISSUE PREPARATION HISTOCHEMISTRY AND CYTOCHEMISTRY MICROSCOPY Folder 1.1 Clinical Correlation: Frozen Sections Folder 1.2 Clinical Correlation: Frozen Sections Folder 1.3 Functional Considerations: Feulgen Microspectrophotometry Folder 1.4 Functional Considerations: Proper Use of the Light Microscope HISTOLOGY 2

Cell Cytoplasm

OVERVIEW OF THE CELL AND CYTOPLASM MEMBRANOUS ORGANELLES NONMEMBRANOUS ORGANELLES INCLUSIONS CYTOPLASMIC MATRIX Folder 2.1 Clinical Correlation: Lysosomal Storage Diseases

Folder 2.2 Clinical Correlation: Abnormalities in Microtubules and Filaments Folder 2.3 Clinical Correlation: Abnormal Duplication of Centrioles and Cancer HISTOLOGY 3

The Cell Nucleus

OVERVIEW OF THE NUCLEUS NUCLEAR COMPONENTS CELL RENEWAL CELL CYCLE CELL DEATH Folder 3.1 Clinical Correlation: Cytogenetic Testing Folder 3.2 Clinical Correlation: Regulation of Cell Cycle and Cancer Treatment HISTOLOGY 4

Tissues: Concept and Classification

OVERVIEW OF TISSUES EPITHELIUM CONNECTIVE TISSUE MUSCLE TISSUE NERVE TISSUE HISTOGENESIS OF TISSUES IDENTIFYING TISSUES Folder 4.1 Clinical Correlation: Ovarian Teratomas HISTOLOGY

5

Epithelial Tissue

OVERVIEW OF EPITHELIAL STRUCTURE AND FUNCTION CLASSIFICATION OF EPITHELIUM CELL POLARITY THE APICAL DOMAIN AND ITS MODIFICATIONS THE LATERAL DOMAIN AND ITS SPECIALIZATIONS IN CELL-TO-CELL ADHESION THE BASAL DOMAIN AND ITS SPECIALIZATIONS IN CELL-TOEXTRACELLULAR MATRIX ADHESION GLANDS EPITHELIAL–MESENCHYMAL TRANSITION EPITHELIAL CELL RENEWAL Folder 5.1 Clinical Correlation: Epithelial Metaplasia Folder 5.2 Clinical Correlation: Primary Ciliary Dyskinesia (Immotile Cilia Syndrome) Folder 5.3 Clinical Correlation: Junctional Complexes as a Target of Pathogenic Agents Folder 5.4 Functional Considerations: Basement Membrane and Basal Lamina Terminology Folder 5.5 Functional Considerations: Mucous and Serous Membranes HISTOLOGY Atlas Plates PLATE 5.1 Simple Squamous and Simple Cuboidal Epithelia PLATE 5.2 Simple and Stratified Epithelia PLATE 5.3 Stratified Epithelia and Epithelioid Tissues 6

Connective Tissue

OVERVIEW OF CONNECTIVE TISSUE

EMBRYONIC CONNECTIVE TISSUE CONNECTIVE TISSUE PROPER CONNECTIVE TISSUE FIBERS EXTRACELLULAR MATRIX CONNECTIVE TISSUE CELLS Folder 6.1 Clinical Correlation: Collagenopathies Folder 6.2 Clinical Correlation: Sun Exposure and Molecular Changes in Photoaged Skin Folder 6.3 Clinical Correlation: Role of Myofibroblasts in Wound Repair Folder 6.4 Functional Considerations: The Mononuclear Phagocyte System Folder 6.5 Clinical Correlation: The Role of Mast Cells and Basophils in Allergic Reactions HISTOLOGY Atlas Plates PLATE 6.1 Loose and Dense Irregular Connective Tissue PLATE 6.2 Dense Regular Connective Tissue, Tendons, and Ligaments PLATE 6.3 Elastic Fibers and Elastic Lamellae 7

Cartilage

OVERVIEW OF CARTILAGE HYALINE CARTILAGE ELASTIC CARTILAGE FIBROCARTILAGE CHONDROGENESIS AND CARTILAGE GROWTH REPAIR OF HYALINE CARTILAGE Folder 7.1 Clinical Correlation: Osteoarthritis

Folder 7.2 Clinical Correlation: Malignant Tumors of the Cartilage: Chondrosarcomas HISTOLOGY Atlas Plates PLATE 7.1 Hyaline Cartilage PLATE 7.2 Hyaline Cartilage and the Developing Skeleton PLATE 7.3 Elastic Cartilage PLATE 7.4 Fibrocartilage 8

Bone

OVERVIEW OF BONE GENERAL STRUCTURE OF BONES TYPES OF BONE TISSUE CELLS OF BONE TISSUE BONE FORMATION BIOLOGIC MINERALIZATION AND MATRIX VESICLES BONE AS A TARGET OF ENDOCRINE HORMONES AND AS AN ENDOCRINE ORGAN BIOLOGY OF BONE REPAIR Folder 8.1 Clinical Correlation: Joint Diseases Folder 8.2 Clinical Correlation: Osteoporosis Folder 8.3 Clinical Correlation: Nutritional Factors in Bone Formation Folder 8.4 Functional Considerations: Hormonal Regulation of Bone Growth HISTOLOGY Atlas Plates PLATE 8.1 Bone, Ground Section PLATE 8.2 Bone and Bone Tissue

PLATE 8.3 PLATE 8.4 PLATE 8.5 9

Endochondral Bone Formation I Endochondral Bone Formation II Intramembranous Bone Formation

Adipose Tissue

OVERVIEW OF ADIPOSE TISSUE WHITE ADIPOSE TISSUE BROWN ADIPOSE TISSUE BEIGE ADIPOSE TISSUE TRANSDIFFERENTIATION OF ADIPOSE TISSUE Folder 9.1 Clinical Correlation: Obesity Folder 9.2 Clinical Correlation: Adipose Tissue Tumors Folder 9.3 Clinical Correlation: PET Scanning and Brown Adipose Tissue Interference HISTOLOGY Atlas Plates PLATE 9.1 Adipose Tissue 10

Blood

OVERVIEW OF BLOOD PLASMA ERYTHROCYTES LEUKOCYTES THROMBOCYTES COMPLETE BLOOD COUNT FORMATION OF BLOOD CELLS (HEMOPOIESIS) BONE MARROW

Folder 10.1 Clinical Correlation: ABO and Rh Blood Group Systems Folder 10.2 Clinical Correlation: Hemoglobin in Patients With Diabetes Folder 10.3 Clinical Correlation: Hemoglobin Disorders Folder 10.4 Clinical Correlation: Inherited Disorders of Neutrophils; Chronic Granulomatous Disease Folder 10.5 Clinical Correlation: Hemoglobin Breakdown and Jaundice Folder 10.6 Clinical Correlation: Cellularity of the Bone Marrow HISTOLOGY Atlas Plates PLATE 10.1 Erythrocytes and Granulocytes PLATE 10.2 Agranulocytes and Red Marrow PLATE 10.3 Erythropoiesis PLATE 10.4 Granulopoiesis 11

Muscle Tissue

OVERVIEW AND CLASSIFICATION OF MUSCLE SKELETAL MUSCLE CARDIAC MUSCLE SMOOTH MUSCLE Folder 11.1 Functional Considerations: Muscle Metabolism and Ischemia Folder 11.2 Clinical Correlation: Muscular Dystrophies— Dystrophin and Dystrophin-Associated Proteins Folder 11.3 Clinical Correlation: Myasthenia Gravis Folder 11.4 Functional Considerations: Comparison of the Three Muscle Types

HISTOLOGY Atlas Plates PLATE PLATE PLATE PLATE PLATE PLATE

12

11.1 11.2 11.3 11.4 11.5 11.6

Skeletal Muscle I Skeletal Muscle II and Electron Microscopy Myotendinous Junction Cardiac Muscle Cardiac Muscle, Purkinje Fibers Smooth Muscle

Nerve Tissue

OVERVIEW OF THE NERVOUS SYSTEM COMPOSITION OF NERVE TISSUE THE NEURON SUPPORTING CELLS OF THE NERVOUS SYSTEM: THE NEUROGLIA ORIGIN OF NERVE TISSUE CELLS ORGANIZATION OF THE PERIPHERAL NERVOUS SYSTEM ORGANIZATION OF THE AUTONOMIC NERVOUS SYSTEM ORGANIZATION OF THE CENTRAL NERVOUS SYSTEM RESPONSE OF NEURONS TO INJURY Folder 12.1 Clinical Correlation: Parkinson Disease Folder 12.2 Clinical Correlation: Demyelinating Diseases / 406 Folder 12.3 Clinical Correlation: Reactive Gliosis: Scar Formation in the Central Nervous System Folder 12.4 Clinical Correlation: Cognitive Impairments After COVID-19 Infections HISTOLOGY Atlas Plates PLATE 12.1 Sympathetic and Dorsal Root Ganglia

PLATE PLATE PLATE PLATE 13

12.2 Peripheral Nerve 12.3 Cerebrum 12.4 Cerebellum 12.5 Spinal Cord

Cardiovascular System

OVERVIEW OF THE CARDIOVASCULAR SYSTEM HEART GENERAL FEATURES OF ARTERIES AND VEINS ARTERIES CAPILLARIES ARTERIOVENOUS SHUNTS VEINS ATYPICAL BLOOD VESSELS LYMPHATIC VESSELS Folder 13.1 Clinical Correlation: Atherosclerosis Folder 13.2 Clinical Correlation: Hypertension Folder 13.3 Clinical Correlation: Coronary Heart Disease HISTOLOGY Atlas Plates PLATE 13.1 Heart PLATE 13.2 Aorta PLATE 13.3 Muscular Arteries and Medium Veins PLATE 13.4 Arterioles, Venules, and Lymphatic Vessels 14

Immune System and Lymphatic Tissues and Organs

OVERVIEW OF THE IMMUNE AND LYMPHATIC SYSTEMS CELLS OF THE IMMUNE SYSTEM LYMPHATIC TISSUES AND ORGANS DIFFUSE LYMPHATIC TISSUE AND LYMPHATIC NODULES LYMPH NODES THYMUS SPLEEN Folder 14.1 Functional Considerations: Origin of the Names T Lymphocyte and B Lymphocyte Folder 14.2 Clinical Correlation: Hypersensitivity Reactions Folder 14.3 Clinical Correlation: Human Immunodeficiency Virus (HIV) and Acquired Immunodeficiency Syndrome (AIDS) Folder 14.4 Clinical Correlation: Reactive (Inflammatory) Lymphadenitis HISTOLOGY Atlas Plates PLATE 14.1 Palatine Tonsil PLATE 14.2 Lymph Node I PLATE 14.3 Lymph Node II PLATE 14.4 Spleen I PLATE 14.5 Spleen II PLATE 14.6 Thymus 15

Integumentary System

OVERVIEW OF THE INTEGUMENTARY SYSTEM SKIN EPIDERMIS CELLS OF THE EPIDERMIS DERMIS

CELLS OF THE DERMIS HYPODERMIS SENSORY NERVE RECEPTORS OF THE SKIN EPIDERMAL SKIN APPENDAGES SKIN AGING Folder 15.1 Clinical Correlation: Cancers of Epidermal Origin Folder 15.2 Clinical Correlation: Mohs Micrographic Surgery Folder 15.3 Functional Considerations: Skin Color Folder 15.4 Clinical Correlation: Clinical Significance of Skin Color Variation Folder 15.5 Functional Considerations: Hair Growth and Hair Characteristics Folder 15.6 Clinical Correlation: Sweating and Disease Folder 15.7 Clinical Correlation: Skin Repair HISTOLOGY Atlas Plates PLATE 15.1 Skin I PLATE 15.2 Skin II PLATE 15.3 Apocrine and Eccrine Sweat Glands PLATE 15.4 Sweat and Sebaceous Glands PLATE 15.5 Integument and Sensory Organs PLATE 15.6 Hair Follicle and Nail 16

Digestive System I: Oral Cavity and Associated Structures

OVERVIEW OF THE DIGESTIVE SYSTEM ORAL CAVITY TONGUE

TEETH AND SUPPORTING TISSUES SALIVARY GLANDS Folder 16.1 Clinical Correlation: The Genetic Basis of Taste Folder 16.2 Clinical Correlation: Classification of Permanent (Secondary) and Deciduous (Primary) Dentition Folder 16.3 Clinical Correlation: Dental Caries Folder 16.4 Clinical Correlation: Salivary Gland Tumors HISTOLOGY Atlas Plates PLATE 16.1 Lip and Mucocutaneous Junction PLATE 16.2 Tongue I PLATE 16.3 Tongue II—Foliate Papillae and Taste Buds PLATE 16.4 Submandibular Gland PLATE 16.5 Parotid Gland PLATE 16.6 Sublingual Gland 17

Digestive System II: Esophagus and Gastrointestinal Tract

OVERVIEW OF THE ESOPHAGUS AND GASTROINTESTINAL TRACT ESOPHAGUS STOMACH SMALL INTESTINE LARGE INTESTINE Folder 17.1 Clinical Correlation: Pernicious Anemia and Peptic Ulcer Disease Folder 17.2 Clinical Correlation: Zollinger–Ellison Syndrome Folder 17.3 Functional Considerations: The Gastrointestinal Endocrine System

Folder 17.4 Functional Considerations: Digestive and Absorptive Functions of Enterocytes Folder 17.5 Functional Considerations: Immune Functions of the Alimentary Canal Folder 17.6 Clinical Correlation: The Pattern of Lymph Vessel Distribution and Diseases of the Large Intestine Folder 17.7 Clinical Correlation: Colorectal Cancer HISTOLOGY Atlas Plates PLATE 17.1 Esophagus PLATE 17.2 Esophagus and Stomach, Cardiac Region PLATE 17.3 Stomach I PLATE 17.4 Stomach II PLATE 17.5 Gastroduodenal Junction PLATE 17.6 Duodenum PLATE 17.7 Jejunum PLATE 17.8 Ileum PLATE 17.9 Colon PLATE 17.10 Appendix PLATE 17.11 Anal Canal 18

Digestive System III: Liver, Gallbladder, and Pancreas

LIVER GALLBLADDER PANCREAS Folder 18.1 Clinical Correlation: Lipoproteins Folder 18.2 Clinical Correlation: Congestive Heart Failure, Acetaminophen Overdose, and Liver Necrosis

Folder 18.3 Clinical Correlation: Insulin Production and Alzheimer Disease Folder 18.4 Functional Considerations: Insulin Synthesis, an Example of Post-translational Processing HISTOLOGY Atlas Plates PLATE 18.1 Liver I PLATE 18.2 Liver II PLATE 18.3 Gallbladder PLATE 18.4 Pancreas 19

Respiratory System

OVERVIEW OF THE RESPIRATORY SYSTEM NASAL CAVITIES PHARYNX LARYNX TRACHEA BRONCHI BRONCHIOLES ALVEOLI BLOOD SUPPLY LYMPHATIC VESSELS NERVES Folder 19.1 Clinical Correlation: Common Conditions Affecting the Nasal Mucosa Folder 19.2 Clinical Correlation: Squamous Metaplasia in the Respiratory Tract Folder 19.3 Clinical Correlation: Asthma Folder 19.4 Clinical Correlation: Cystic Fibrosis

Folder 19.5 Clinical Correlation: Chronic Obstructive Pulmonary Disease and Pneumonia HISTOLOGY Atlas Plates PLATE 19.1 Olfactory Mucosa PLATE 19.2 Larynx PLATE 19.3 Trachea PLATE 19.4 Bronchioles and End Respiratory Passages PLATE 19.5 Terminal Bronchiole, Respiratory Bronchiole, and Alveolus 20

Urinary System

OVERVIEW OF THE URINARY SYSTEM GENERAL STRUCTURE OF THE KIDNEY KIDNEY TUBULE FUNCTION INTERSTITIAL CELLS HISTOPHYSIOLOGY OF THE KIDNEY BLOOD SUPPLY LYMPHATIC VESSELS NERVE SUPPLY URETER, URINARY BLADDER, AND URETHRA Folder 20.1 Functional Considerations: Kidney and Vitamin D Folder 20.2 Clinical Correlation: Antiglomerular Basement

Membrane Antibody–Induced Glomerulonephritis; Goodpasture Syndrome Clinical Correlation: Renin–Angiotensin– Aldosterone System and Hypertension Clinical Correlation: Examination of the Urine— Urinalysis

Folder 20.3 Folder 20.4

Folder 20.5 Functional Considerations: Structure and Function of Aquaporin Water Channels Folder 20.6 Functional Considerations: Antidiuretic Hormone Regulation of Collecting Duct Function HISTOLOGY Atlas Plates PLATE 20.1 Kidney I PLATE 20.2 Kidney II PLATE 20.3 Kidney III PLATE 20.4 Kidney IV PLATE 20.5 Ureter PLATE 20.6 Urinary Bladder 21

Endocrine Organs

OVERVIEW OF THE ENDOCRINE SYSTEM PITUITARY GLAND (HYPOPHYSIS) HYPOTHALAMUS PINEAL GLAND THYROID GLAND PARATHYROID GLANDS ADRENAL GLANDS Folder 21.1 Functional Considerations: Regulation of Pituitary Gland Secretion Folder 21.2 Clinical Correlation: Principles of Endocrine Diseases Folder 21.3 Clinical Correlation: Pathologies Associated With Antidiuretic Hormone Secretion Folder 21.4 Clinical Correlation: Abnormal Thyroid Function Folder 21.5 Clinical Correlation: Chromaffin Cells and Pheochromocytoma

Folder 21.6 Functional Considerations: Biosynthesis of Adrenal Hormones HISTOLOGY Atlas Plates PLATE 21.1 Pituitary I PLATE 21.2 Pituitary II PLATE 21.3 Pineal Gland PLATE 21.4 Parathyroid and Thyroid Glands PLATE 21.5 Adrenal Gland I PLATE 21.6 Adrenal Gland II 22

Male Reproductive System

OVERVIEW OF THE MALE REPRODUCTIVE SYSTEM TESTIS SPERMATOGENESIS SEMINIFEROUS TUBULES INTRATESTICULAR DUCTS EXCURRENT DUCT SYSTEM ACCESSORY SEX GLANDS PROSTATE GLAND SEMEN PENIS AND SCROTUM Folder 22.1 Functional Considerations: Hormonal Regulation of Spermatogenesis Folder 22.2 Clinical Correlation: Factors Affecting Spermatogenesis Folder 22.3 Clinical Correlation: Sperm-Specific Antigens and the Immune Response

Folder 22.4 Clinical Correlation: Benign Prostatic Hypertrophy and Cancer of the Prostate Folder 22.5 Clinical Correlation: Mechanism of Erection and Erectile Dysfunction HISTOLOGY Atlas Plates PLATE 22.1 Testis I PLATE 22.2 Testis II PLATE 22.3 Efferent Ductules and Epididymis PLATE 22.4 Spermatic Cord and Ductus Deferens PLATE 22.5 Prostate Gland PLATE 22.6 Seminal Vesicle 23

Female Reproductive System

OVERVIEW OF THE FEMALE REPRODUCTIVE SYSTEM OVARY UTERINE TUBES UTERUS PLACENTA VAGINA EXTERNAL GENITALIA MAMMARY GLANDS Folder 23.1 Clinical Correlation: Polycystic Ovary Disease Folder 23.2 Clinical Correlation: In Vitro Fertilization Folder 23.3 Functional Considerations: Summary of Hormonal Regulation of the Ovarian Cycle Folder 23.4 Clinical Correlation: The Placenta Folder 23.5 Clinical Correlation: Cervical Cytology: The Pap Test

Folder 23.6 Clinical Correlation: Cervical Cancer and Human Papillomavirus Infection Folder 23.7 Functional Considerations: Lactation and Infertility HISTOLOGY Atlas Plates PLATE 23.1 Ovary I PLATE 23.2 Ovary II PLATE 23.3 Corpus Luteum PLATE 23.4 Uterine Tube PLATE 23.5 Uterus I PLATE 23.6 Uterus II PLATE 23.7 Cervix PLATE 23.8 Placenta I PLATE 23.9 Placenta II PLATE 23.10 Vagina PLATE 23.11 Mammary Gland Inactive Stage PLATE 23.12 Mammary Gland, Late Proliferative and Lactating Stages 24

Eye

OVERVIEW OF THE EYE GENERAL STRUCTURE OF THE EYE MICROSCOPIC STRUCTURE OF THE EYE ACCESSORY STRUCTURES OF THE EYE Folder 24.1 Clinical Correlation: Glaucoma Folder 24.2 Clinical Correlation: Retinal Detachment Folder 24.3 Clinical Correlation: Age-related Macular Degeneration

Folder 24.4 Clinical Correlation: Clinical Imaging of the Retina Folder 24.5 Clinical Correlation: Color Blindness Folder 24.6 Clinical Correlation: Conjunctivitis HISTOLOGY Atlas Plates PLATE 24.1 Eye I PLATE 24.2 Eye II: Retina PLATE 24.3 Eye III: Anterior Segment PLATE 24.4 Eye IV: Sclera, Cornea, and Lens 25

Ear

OVERVIEW OF THE EAR EXTERNAL EAR MIDDLE EAR INTERNAL EAR Folder 25.1 Clinical Correlation: Otosclerosis Folder 25.2 Clinical Correlation: Hearing Loss— Vestibular Dysfunction Folder 25.3 Clinical Correlation: Vertigo HISTOLOGY Atlas Plates PLATE 25.1 Ear PLATE 25.2 Cochlear Canal and Organ of Corti Index

1

METHODS

OVERVIEW OF METHODS USED IN HISTOLOGY TISSUE PREPARATION

Hematoxylin and Eosin Staining With Formalin Fixation Other Fixatives Other Staining Procedures

HISTOCHEMISTRY AND CYTOCHEMISTRY

Chemical Composition of Histologic Samples Chemical Basis of Staining

Enzyme Digestion Enzyme Histochemistry Immunocytochemistry Hybridization Techniques Autoradiography Expansion Microscopy

MICROSCOPY

Light Microscopy Examination of a Histologic Slide Preparation in the Light Microscope Other Optical Systems Super-Resolution Microscopy Electron Microscopy Atomic Force Microscopy Virtual Microscopy Clinical Correlation: Frozen Sections Functional Considerations: Feulgen Microspectrophotometry Clinical Correlation: Monoclonal Antibodies in Medicine Functional Considerations: Proper Use of the Light Microscope

Folder 1.1 Folder 1.2 Folder 1.3 Folder 1.4 HISTOLOGY

OVERVIEW OF METHODS USED IN HISTOLOGY

The objective of a histology course is to lead the student to understand the microanatomy of cells, tissues, and organs and to correlate structure with function.

Histology [Gr., histos, tissue; Gr., logia, science], also called microscopic anatomy, is the scientific study of microscopic structures of tissues and organs of the body. Modern histology is not only a descriptive science but also includes many aspects of molecular and cell biology, which help describe cell organization and function. The methods used by histologists are extremely diverse. Much of the histology course content can be framed in terms of light microscopy. Today, students in histology laboratories use either or, with increasing frequency, , where digitized microscopic specimens are viewed on a computer screen or mobile devices. In the past, more detailed interpretation of microscopic structures was done with the —both the and the . Now, the and a variety of can provide images that are comparable or higher (e.g., AFM) in resolution to those obtained with the TEM. Both EM and AFM, because of their greater resolution and useful magnification, are often the last step in data acquisition from many auxiliary techniques of cell and molecular biology. However, usage of super-resolution techniques is increasing in cell and molecular biology research, mainly because high-resolution images can be captured directly from living cells using fluorescence microscopy. These include the following:

microscopes microscopes

light virtual

electron microscope (EM) transmission electron microscope (TEM) scanning electron microscope (SEM) atomic force microscope (AFM) superresolution microscopic techniques

auxiliary techniques

Variety of staining methods to enhance contrast of microscopic images Histochemistry and cytochemistry techniques Immunocytochemistry and hybridization techniques Autoradiography Organ and tissue culture Cell and organelle separation by differential centrifugation Specialized tissue preparation (e.g., in expansion microscopy) Specialized microscopic techniques (e.g., super-resolution microscopy)

Specialized optical systems (e.g., polarizing microscope, confocal scanning microscope, or light sheet fluorescence microscope) Image analysis software that allow for three-dimensional reconstruction of cells and organelles The student may feel removed from such techniques and experimental procedures because direct experience with them is usually not available in current curricula. Nevertheless, it is important to know about these specialized procedures and the types of data they yield.

This chapter provides a survey of methods and offers an explanation of how the data provided by these methods can help the student acquire a better understanding of cells, tissues, and organ function.

One problem that students of histology face is understanding the nature of the two-dimensional (2D) image of a histologic slide or an electron micrograph and how the image relates to the three-dimensional (3D) structure from which it came. To bridge this conceptual gap, we first briefly describe how glass slides and electron microscopic specimens are produced and examined. In addition, we discuss several new methods of light and electron microscopy that have been developed to reconstruct 3D cellular objects from imaged structures.

TISSUE PREPARATION Hematoxylin and Eosin Staining With Formalin Fixation

The routinely prepared hematoxylin and eosin–stained section is the specimen most commonly studied. The slide set given to each student to study with the light microscope consists mostly of formalin-fixed, paraffinembedded, hematoxylin and eosin (H&E)-stained specimens. Nearly all of the light micrographs in the Atlas section of this book are of slides from actual student sets. Also, most photomicrographs used to illustrate tissues and organs in

histology lectures and conferences are taken from such slides. Other staining techniques are sometimes used to demonstrate specific cell and tissue components; several of these methods are discussed later.

The first step in preparation of a tissue or organ sample is fixation to preserve structure. Fixation, usually by a chemical or mixture of chemicals, permanently preserves the tissue structure for subsequent treatments. Specimens should be immersed in fixative immediately after they are removed from the body. Fixation is used to

terminate cell metabolism; prevent enzymatic degradation of cells and tissues by autolysis (self-digestion); kill pathogenic microorganisms such as bacteria, fungi, and viruses; and harden the tissue as a result of either cross-linking or denaturing protein molecules.

Formalin, a 37% aqueous solution of formaldehyde, at various

dilutions and in combination with other chemicals and buffers, is the most commonly used fixative. Formaldehyde preserves the general structure of the cell and extracellular components by reacting with the amino groups of proteins (most often crosslinked lysine residues). Because formaldehyde does not significantly alter their 3D structure, proteins maintain their ability to react with specific antibodies. This property is important in immunocytochemical staining methods (see pages 810). The standard commercial solution of formaldehyde buffered with phosphates (pH 7) acts relatively slowly but penetrates the tissue well. However, because it does not react with lipids, it is a poor fixative of cell membranes.

In the second step, the specimen is prepared for embedding in paraffin to permit sectioning. Preparing a specimen for examination requires its infiltration with an embedding medium that allows it to be thinly sliced,

typically in the range of 5–15 μm (1 μm = 1/1,000 mm; Table 1.1). The specimen is after fixation and in a series of alcohol solutions of ascending concentration as high as 100% alcohol to remove water. In the next step, , organic solvents such as xylol or toluol, which are miscible in both alcohol and , are used to remove the alcohol before infiltration of the specimen with melted paraffin.

washed

dehydrated clearing

paraffin

TABLE 1.1 Commonly Used Linear Equivalents 1 picometer

=

0.01 angstrom (Å)

1 angstrom

=

0.1 nanometer (nm)

10 angstroms

=

1.0 nanometer

1 nanometer

=

1,000 picometers (pm)

1,000 nanometers

=

1.0 micrometer (μm)

1,000 micrometers

=

1.0 millimeter (mm)

When the melted paraffin is cool and hardened, it is trimmed into an appropriately sized block. The block is then attached to a specially designed slicing machine—a —and cut with a steel knife. The resulting sections are then mounted on glass slides using a as an adhesive. Mounting medium is a solution that hardens into a permanent mount that keeps the specimen attached to the glass and prevents deterioration of the specimen over time (i.e., darken, fade, leach, crystalize, etc.). The most commonly used nonaqueous permanent mounting media include toluene-based synthetic resins (Permount), Canada balsam (a turpentine made from the resin of the balsam fir tree), and many others. Aqueous-based media are often used in immunocytochemistry and include glycerol-based products and gelatin.

mounting medium

microtome

In the third step, the specimen is stained to permit examination.

Because paraffin sections are colorless, the specimen is not yet suitable for light microscopic examination. To color or stain the tissue sections, the paraffin must be dissolved out, again with xylol or toluol, and the slide must then be

rehydrated through a series of solutions of descending alcohol concentration. The tissue on the slides is then stained with in water. Because the counterstain, , is more soluble in alcohol than in water, the specimen is again dehydrated through a series of alcohol solutions of ascending concentration and stained with eosin in alcohol. Figure 1.1 shows the results of staining with hematoxylin alone, eosin alone, and hematoxylin with counterstain eosin. After staining, the specimen is then passed through xylol or toluol to a nonaqueous mounting medium and covered with a coverslip to obtain a permanent preparation.

hematoxylin

eosin

FIGURE 1.1. Hematoxylin and eosin (H&E) staining .

This series of specimens from the pancreas are serial (adjacent) sections that demonstrate the effect of H&E used alone and H&E used in combination. This photomicrograph reveals the staining with hematoxylin only. Although there is a general overall staining of the specimen, those components and structures that have a high affinity for the dye are most heavily stained—for example, the nuclear DNA and areas of the cell containing cytoplasmic RNA. In this photomicrograph, eosin, the counterstain, likewise has an overall staining effect when used alone. Note, however, that the nuclei are less conspicuous than in the specimen stained with hematoxylin alone. After the specimen is stained with hematoxylin and then prepared for staining with eosin in alcohol solution, the hematoxylin that is not tightly bound is lost, and the eosin then stains those components to which it has a high affinity. This photomicrograph reveals the combined staining effect of H&E. ×480.

a.

b.

c.

Other Fixatives

Formalin does not preserve all cell and tissue components. Although H&E-stained sections of formalin-fixed specimens are convenient to use because they adequately display general structural features, they cannot elucidate the specific chemical composition of cell components. Also, many components are lost in the preparation of the specimen. To retain these components and structures, other fixation methods must be used. These methods are generally based on a clear understanding of the chemistry involved. For instance, the use of alcohols and organic solvents in routine preparations removes neutral lipids. To retain neutral lipids, such as those in adipose cells, frozen sections of formalin-fixed tissue and dyes that dissolve in fats must be used; to retain membrane structures, special fixatives containing heavy metals that bind to the phospholipids, such as permanganate and osmium, are used (Folder 1.1). The routine use of as a fixative for electron microscopy is the primary reason for the excellent preservation of membranes in electron micrographs.

osmium tetroxide

FOLDER 1.1

CLINICAL CORRELATION: FROZEN SECTIONS

Sometimes, a pathologist may be asked to immediately evaluate tissue obtained during surgery, especially when instant pathologic diagnosis may determine how the surgery will proceed. There are several indications to perform such an evaluation, routinely known as a . Most commonly, a surgeon in the operating room requests a frozen section when no preoperative diagnosis was available or when unexpected intraoperative findings must be identified. In addition, the surgeon may want to know whether all of a pathologic mass within the healthy tissue limit has been removed and whether the margin of the surgical resection is free of diseased tissue. Frozen sections are also done in combination with other procedures such as endoscopy or thin-needle biopsy to confirm whether the obtained biopsy material will be usable in further pathologic examinations. Three main steps are involved in frozen-section preparation:

frozen section

Freezing the tissue sample. Small tissue samples are frozen either by using compressed carbon dioxide or by immersion in a

cold fluid (isopentane) at a temperature of −50°C. Freezing can

be achieved in a special high-efficiency refrigerator. Freezing makes the tissue solid and allows sectioning with a microtome. . Sectioning is usually performed inside a cryostat, a refrigerated compartment containing a microtome. Because the tissue is frozen solid, it can be cut into extremely thin (5–10 μm) sections. The sections are then mounted on glass slides. . Staining is done to differentiate cell nuclei from the rest of the tissue. The most common stains used for frozen sections are hematoxylin and eosin (H&E), methylene blue (Fig. F1.1.1), and periodic acid–Schiff (PAS) stains.

Sectioning the frozen tissue Staining the cut sections

FIGURE F1.1.1. Evaluation of a specimen obtained during surgery by the frozen-section technique. a. This

photomicrograph shows a specimen obtained from the large intestine that was prepared by the frozen-section technique and stained with methylene blue. ×160. Part of the specimen was fixed in formalin and processed as a routine hematoxylin and eosin (H&E) preparation. Examination of the frozen section revealed it to be normal. This diagnosis was later confirmed by examining the routinely prepared H&E specimen. ×180. (Courtesy of Dr. Daniel W. Visscher.)

b.

The entire process of preparation and evaluation of frozen sections may take as little as 10 minutes to complete. The total time to obtain results largely depends on the transport time of the tissue from the operating room to the pathology laboratory, the pathologic technique used, and the experience of the pathologist. The findings are then directly communicated to the surgeon waiting in the operating room.

Other Staining Procedures

Hematoxylin and eosin are used in histology primarily to display structural features. Despite the advantages of H&E staining, the procedure does not adequately reveal certain structural components of histologic sections such as elastic material, reticular fibers, basement membranes, and lipids. When it is desirable to display these components, other staining procedures, most of them selective, can be used. These procedures include the use of orcein and resorcin-fuchsin for elastic material and silver impregnation for reticular fibers and basement membrane material. The chemical bases of many staining methods are not completely understood. Understanding the basic concepts of a staining procedure is often more important than knowing precisely all the steps that are involved in that process.

HISTOCHEMISTRY AND CYTOCHEMISTRY

Specific chemical procedures can provide information about the function of cells and the extracellular components of tissues. Histochemical and cytochemical procedures may be based on specific binding of a dye, use of a fluorescent dye–labeled antibody with a particular cell component, or the inherent enzymatic activity of a cell component. In addition, many large molecules present in cells can be localized by the process of autoradiography, in which radioactively tagged precursors of the molecule are incorporated by cells and tissues before

fixation. Many of these procedures can be used with both light microscopic and electron microscopic preparations. Before discussing the chemistry of routine staining and histochemical and cytochemical methods, it is useful to examine briefly the nature of a routinely fixed and embedded section of a specimen.

Chemical Composition of Histologic Samples

The chemical composition of a tissue ready for routine staining differs from living tissue. The components that remain after fixation consist mostly of large molecules that do not readily dissolve, especially after treatment with the fixative. These large molecules, particularly those that react with other large molecules to form macromolecular complexes, are usually preserved in a tissue section. Examples of such large macromolecular complexes include the following:

Nucleoproteins formed from nucleic acids bound to protein Intracellular cytoskeletal proteins complexed with associated proteins Extracellular proteins in large insoluble aggregates, bound to similar molecules by cross-linking molecules, as in collagen fiber formation

of

neighboring

Membrane phospholipid–protein (or carbohydrate) complexes These molecules constitute the structure of cells and tissues—that is, they make up the formed elements of the tissue. They are the basis for the organization that is seen in tissue with the microscope. In many cases, a structural element is also a functional unit. For example, in the case of proteins that make up the contractile filaments of muscle cells, the filaments are the visible structural components and the actual participants in the contractile process. The RNA of the cytoplasm is visualized as part of a structural component (e.g., ergastoplasm of

secretory cells, Nissl bodies of nerve cells) and is also the actual participant in the synthesis of protein.

Many tissue components are lost during the routine preparation of H&E-stained sections.

Although nucleic acids, proteins, and phospholipids are mostly retained in tissue sections, many tissue components are also lost. Small proteins and small nucleic acids, such as transfer RNA, are generally lost during the preparation of the tissue. As previously described, neutral lipids are usually dissolved by the organic solvents used in tissue preparation. Other large molecules also may be lost, for example, by being hydrolyzed because of the unfavorable pH of the fixative solutions. Examples of large molecules lost during routine fixation in aqueous fixatives are

glycogen (an intracellular storage carbohydrate common in liver and muscle cells) and proteoglycans and glycosaminoglycans (extracellular complex carbohydrates found in connective tissue).

These molecules can be preserved, however, by using a nonaqueous fixative for glycogen or by adding specific binding agents to the fixative solution that preserve extracellular carbohydrate-containing molecules.

Soluble components, ions, and small molecules are also lost during the preparation of paraffin sections.

Intermediary metabolites, glucose, sodium, chloride, and similar substances are lost during preparation of routine H&E paraffin sections. Many of these substances can be studied in special preparations, sometimes with considerable loss of structural integrity. These small soluble ions and molecules do not make up the formed elements of a tissue; they participate in synthetic processes or cellular reactions. When they can be preserved and demonstrated by specific methods, they provide invaluable information about cell metabolism, active transport, and other vital cellular processes. Water, a highly versatile molecule, participates in these reactions and processes and

contributes to the stabilization of macromolecular structure through hydrogen bonding.

Chemical Basis of Staining

Acidic and Basic Dyes Hematoxylin and eosin are the most commonly used dyes in histology. An acidic dye, such as eosin, carries a net negative charge on its colored portion and is described by the general formula [Na+ dye− ]. A carries a on its colored portion and is described by the general formula [dye+ Cl− ]. does not meet the definition of a strict basic dye but has properties that closely resemble those of a basic dye. The color of a dye is not related to whether it is basic or acidic, as can be noted by the examples of basic and acidic dyes listed in Table 1.2.

basic dye Hematoxylin

net positive charge

TABLE 1.2 Some Basic and Acidic Dyes Dye Basic Dyes

Color

Methyl green

Green

Methylene blue

Blue

Pyronin G

Red

Toluidine blue

Blue

Acid fuchsin

Red

Aniline blue

Blue

Eosin

Red

Orange G

Orange

Acidic Dyes

Basic dyes react with anionic components of cells and tissue (components that carry a net negative charge).

Anionic components

include the phosphate groups of nucleic acids, the sulfate groups of glycosaminoglycans, and the carboxyl groups of proteins. The ability of such anionic groups to react with a basic dye is called . Tissue components that stain with hematoxylin also exhibit basophilia. The reaction of the anionic groups varies with pH:

basophilia [Gr., base-

loving]

high pH

At a (about 10), all three groups are ionized and available for reaction by electrostatic linkages with the basic dye. At a (5–7), sulfate and phosphate groups are ionized and available for reaction with the basic dye by electrostatic linkages. At a (below 4), only sulfate groups remain ionized and react with basic dyes.

slightly acidic to neutral pH

low pH

Therefore, staining with basic dyes at a specific pH can be used to focus on specific anionic groups; because the specific anionic groups are found predominantly on certain macromolecules, the staining serves as an indicator of these macromolecules. As mentioned, is not, strictly speaking, a basic dye. It is used with a (i.e., an intermediate link between the tissue component and the dye). The mordant causes the stain to resemble a basic dye. The linkage in the is not a simple electrostatic linkage; when sections are placed in water, hematoxylin does not dissociate from the tissue. Hematoxylin is best suited to staining sequences in which it is followed by aqueous solutions of acidic dyes. True basic dyes, as distinguished from hematoxylin, are not generally used in sequences in which the basic dye is followed by an acidic dye. True basic dye would dissociate from the tissue during the aqueous washes between the basic and acidic dye solutions.

hematoxylin mordant

mordant–hematoxylin complex

tissue–

Acidic dyes react with cationic groups in cells and tissues, particularly with the ionized amino groups of proteins.

cationic groups with an acidic dye is acidophilia [Gr., acid-loving]. Reactions of cell and The reaction of

called tissue components with acidic dyes are neither as specific nor as precise as reactions with basic dyes. Although electrostatic linkage is the major factor in the primary binding of an acidic dye to the tissue, it is not the only one; because of this, acidic dyes are sometimes used in combinations to color different tissue constituents selectively. For example, three acidic dyes are used in the : aniline blue, acid fuchsin, and orange G. These dyes selectively stain collagen, ordinary cytoplasm, and red blood cells, respectively. Acid fuchsin also stains nuclei. In other multiple acidic dye techniques, hematoxylin is used to stain nuclei first, and then acidic dyes are used to stain cytoplasm and extracellular fibers selectively. The selective staining of tissue components by acidic dyes is attributable to relative factors such as the size and degree of aggregation of the dye molecules and the permeability and “compactness” of the tissue. Basic dyes can also be used in combination or sequentially (e.g., methyl green and pyronin to study protein synthesis and secretion), but these combinations are not as widely used as acidic dye combinations.

Mallory staining technique

A limited number of substances within cells and the extracellular matrix display basophilia. Substances that display basophilia include the following:

Heterochromatin and nucleoli of the nucleus (chiefly because of ionized phosphate groups in nucleic acids of both) Cytoplasmic components such as the ergastoplasm (also because of ionized phosphate groups in ribosomal RNA) Extracellular materials such as the complex carbohydrates of the matrix of cartilage (because of ionized sulfate groups)

Staining with acidic dyes is less specific, but more substances within cells and the extracellular matrix exhibit acidophilia.

Substances that exhibit acidophilia include the following:

cytoplasmic filaments intracellular membranous components extracellular fibers

Most , especially those of muscle cells Most and much of the otherwise unspecialized cytoplasm Most (primarily because of ionized amino groups)

Metachromasia Certain basic dyes react with tissue components that shift their normal color from blue to red or purple; this absorbance change is called metachromasia. The underlying mechanism for metachromasia is the presence of polyanions within the tissue. When these tissues are stained with a concentrated basic dye solution, such as toluidine blue, the dye molecules are close enough to form dimeric and polymeric aggregates. The absorption properties of these aggregations differ from those of the individual nonaggregated dye molecules. Cell and tissue structures that have high concentrations of ionized sulfate and phosphate groups—such as the ground substance of cartilage, heparin-containing granules of mast cells, and rough endoplasmic reticulum of plasma cells—exhibit metachromasia. Therefore, toluidine blue will appear purple to red when it stains these components.

Aldehyde Groups and the Schiff Reagent The ability of bleached basic fuchsin (Schiff reagent) to react with aldehyde groups results in a distinctive red color and is the basis of the periodic acid–Schiff and Feulgen reactions. The periodic acid–Schiff (PAS) reaction stains carbohydrates and carbohydrate-rich macromolecules. It is used to demonstrate glycogen in cells, mucus in various cells and tissues, the basement membrane that underlies epithelia, and reticular fibers in connective tissue. The Schiff reagent is also used in

Feulgen stain,

which relies on a mild hydrochloric hydrolysis to stain DNA. The PAS reaction is based on the following facts:

acid

Hexose rings of carbohydrates contain adjacent carbons, each of which bears a hydroxyl (−OH) group. Hexosamines of glycosaminoglycans contain adjacent carbons, one of which bears an −OH group, whereas the other bears an amino (−NH2) group. Periodic acid cleaves the bond between these adjacent carbon atoms and forms aldehyde groups. These aldehyde groups react with the Schiff reagent to give a distinctive magenta color. The PAS staining of basement membrane (Fig. 1.2) and reticular fibers is based on the content or association of proteoglycans (complex carbohydrates associated with a protein core). PAS staining is an alternative to silver impregnation methods, which are also based on reaction with the sugar molecules in the proteoglycans.

FIGURE 1.2. Photomicrograph of kidney tissue stained by the periodic acid–Schiff (PAS) method. This histochemical method demonstrates and localizes carbohydrates and carbohydrate-rich macromolecules. The basement membranes are PAS-positive as evidenced by the magenta staining of these sites. The kidney tubules ( ) are sharply delineated by the stained basement membrane surrounding the tubules. The glomerular capillaries ( ) and the epithelium of Bowman capsule ( ) also show PAS-positive basement membranes. The specimen was counterstained with hematoxylin to visualize cell nuclei. ×320.

BC

C

T

Feulgen reaction

The is based on the cleavage of purines from the deoxyribose of DNA by mild acid hydrolysis; the sugar ring then opens with the formation of aldehyde groups. Again, the newly formed aldehyde groups react with the Schiff reagent to impart its distinctive magenta color. The reaction of the Schiff reagent with DNA is , meaning that the product of this reaction is measurable and proportional to the amount of DNA. This property makes it ideal for use in spectrophotometric methods to quantify the amount of DNA in the nucleus of a cell. RNA does not stain with the Schiff reagent because it lacks deoxyribose.

stoichiometric

Enzyme Digestion

Enzyme digestion of a section adjacent to one stained for a specific component—such as glycogen, DNA, or RNA—can be used to confirm the identity of the stained material. Intracellular material that stains with the PAS reaction may be identified as glycogen by pretreatment of sections with diastase or amylase. Elimination of the staining after these treatments positively identifies the stained material as glycogen. Similarly, pretreatment of tissue sections with deoxyribonuclease (DNAse) will abolish the Feulgen staining in those sections, and treatment of sections of protein secretory epithelia with ribonuclease (RNAse) will abolish the staining of the ergastoplasm with basic dyes.

FOLDER 1.2

FUNCTIONAL CONSIDERATIONS: FEULGEN MICROSPECTROPHOTOMETRY

Feulgen microspectrophotometry is a technique developed to study DNA increases in developing cells and to analyze ploidy —that

is, the number of times the normal DNA content of a cell is multiplied (a normal, nondividing cell is said to be ; a sperm or egg cell is ). Two techniques, for tissue sections and for isolated cells, are used to quantify the amount of nuclear DNA. Static cytometry of Feulgen-stained sections of tumors uses microspectrophotometry coupled with a digitizing imaging system to measure the absorption of light emitted by cells and cell clusters at 560-nm wavelength. In contrast, flow cytometry uses instrumentation able to scan only single cells flowing past a sensor in a liquid medium. This technique provides rapid, quantitative analysis of a single cell based on the measurement of fluorescent light emission. Currently, Feulgen microspectrophotometry is used to study changes in the DNA content in dividing cells undergoing differentiation. It is also used clinically to analyze abnormal chromosomal number (i.e., ploidy patterns) in malignant cells. Some malignant cells that have a largely diploid pattern are said to be well differentiated; tumors with these types of cells have a better prognosis than tumors with (nonintegral multiples of the haploid amount of DNA) and tetraploid cells. Feulgen microspectrophotometry has been particularly useful in studies of specific adenocarcinomas (epithelial cancers), breast cancer, kidney cancer, colon and other gastrointestinal cancers, endometrial (uterine epithelium) cancer, and ovarian cancer. It is one of the most valuable tools for pathologists in evaluating the metastatic potential of these tumors and in making prognostic and treatment decisions.

haploid

flow cytometry

diploid

static cytometry

aneuploid

Enzyme Histochemistry

Histochemical methods are also used to identify and localize enzymes in cells and tissues. To localize enzymes in tissue sections, special care must be taken in fixation to preserve the enzyme activity. Usually, mild aldehyde fixation is the preferred method. In these procedures, the reaction product of the enzyme activity, rather than the enzyme itself, is visualized. In general, a , either a dye or a heavy metal, is used to trap or bind the reaction product of the enzyme by precipitation at the site of reaction. In a typical reaction to display a hydrolytic

reagent

capture

enzyme, the tissue section is placed in a solution containing a substrate (AB) and a trapping agent (T) that precipitates one of the products as follows:

where AT is the trapped end product and B is the hydrolyzed substrate. By using such methods, the lysosome, first identified in differential centrifugation studies of cells, was equated with a vacuolar component seen in electron micrographs. In lightly fixed tissues, the acid hydrolases and esterases contained in lysosomes react with an appropriate substrate. The reaction mixture also contains lead ions to precipitate (e.g., lead phosphate derived from the action of acid phosphatase). The precipitated reaction product can then be observed with both light and electron microscopy. Similar histochemical procedures have been developed to demonstrate alkaline phosphatase, adenosine triphosphatases (ATPases) of many varieties + + (including the Na /K ATPase that is the enzymatic basis of the sodium pump in cells and tissues), various esterases, and many respiratory enzymes (Fig. 1.3a).

FIGURE 1.3. Electron and light microscopic histochemical procedures. a. This electron micrograph shows localization of membrane ATPase in epithelial cells of rabbit gallbladder. Dark areas

visible on the electron micrograph show the location of the enzyme ATPase. This enzyme is detected in the plasma membrane at the lateral

domains of epithelial cells, which correspond to the location of sodium pumps. These epithelial cells are involved in active transport of molecules across the plasma membrane. ×26,000. This photomicrograph shows macrophages stained with a histochemical method using peroxidase-labeled antibodies and 3,3′-diaminobenzidine (DAB) reagent. A paraffin-embedded section of mouse kidney with renal vascular hypertension disease was stained for the presence of F4/80+ specific marker protein expressed only on the surface of macrophages. Initially, sections were exposed to primary rat anti-mouse F4/80+ antibodies followed by incubation with secondary goat anti-rat immunoglobulin G (IgG) antibodies labeled with horseradish peroxidase. The specimen was washed and treated with a buffer containing DAB. A brown precipitate (product of DAB oxidation by horseradish peroxidase) is localized in the areas where macrophages are present. The specimen was counterstained with hematoxylin to visualize cell nuclei. ×400. (Courtesy of Dr. Joseph P. Grande.)

b.

One of the most common histochemical methods (often used in conjunction with immunocytochemistry) employs horseradish peroxidase for enzyme-mediated antigen detection. A widely used substrate for horseradish peroxidase is 3,3′-diaminobenzidine (DAB), a colorless organic compound that produces a brown insoluble product at the site of enzymatic reaction (Fig. 1.3b). The product of this enzymatic reaction can be easily localized in cells, yielding high-resolution images in both light and electron microscopy.

Immunocytochemistry

The specificity of a reaction between an antigen and an antibody is the underlying basis of immunocytochemistry. Antibodies, also known as immunoglobulins, are glycoproteins that are produced by specific cells of the immune system in response to a foreign protein or antigen. In the laboratory, antibodies can be purified from blood and conjugated (attached) to a fluorescent dye. In general, fluorescent dyes (fluorochromes) are chemicals that absorb light of different

wavelengths (e.g., ultraviolet light) and then emit visible light of a specific wavelength (e.g., green, yellow, red). , the most commonly used dye, absorbs ultraviolet light and emits green light. Antibodies conjugated with

Fluorescein

fluorescein can be applied to sections of lightly fixed or frozen tissues on glass slides to localize an antigen in cells and tissues. The reaction of antibody with antigen can then be examined and photographed with a fluorescence microscope or confocal microscope that produces a 3D reconstruction of the examined tissue (Fig. 1.4).

FIGURE 1.4. Confocal microscopy image of a rat cardiac muscle cell. This image was obtained with the confocal microscope using the

indirect immunofluorescence method. Two primary antibodies were used. The first primary antibody recognizes a specific lactate transporter (MCT1) and is detected with a secondary antibody conjugated with rhodamine ( ). The second primary antibody is directed against the transmembrane protein CD147, which is tightly associated with MCT1. This antibody was detected by a secondary antibody labeled with fluorescein ( ). The color is visible at the point at which the two labeled secondary antibodies exactly colocalize within the

red

green

yellow

cardiac muscle cell. This three-dimensional image shows that both proteins are distributed on the surface of the muscle cell, whereas the lactate transporter alone is visible deep to the plasma membrane. (Courtesy of Drs. Andrew P. Halestrap and Catherine Heddle.)

Two types of antibodies are used in immunocytochemistry: polyclonal antibodies that are produced by immunized animals and monoclonal antibodies that are produced by immortalized (continuously replicating) antibody-producing cell lines. In a typical procedure, a specific protein, such as actin, is isolated from a muscle cell of one species, such as a rat, and injected into the circulation of another species, such as a rabbit. In the immunized rabbit, the rat’s actin molecules are recognized by the rabbit immune system as a foreign antigen. This recognition triggers a cascade of immunologic reactions involving multiple groups (clones) of immune cells called . The cloning of B lymphocytes eventually leads to the production of anti-actin antibodies. Collectively, these represent mixtures of different antibodies produced by many clones of B lymphocytes that each recognize different regions of the actin molecule. The antibodies are then removed from the blood, purified, and conjugated with a fluorescent dye. They can now be used to locate actin molecules in rat tissues or cells. If actin is present in a cell or tissue, such as a fibroblast in connective tissue, then the fluorescein-labeled antibody binds to it, and the reaction is visualized by fluorescence microscopy. (Folder 1.3) are those produced by an consisting of a single group (clone) of identical B lymphocytes. The single clone that becomes a cell line is obtained from an individual with , a tumor derived from a single antibodyproducing plasma cell. Individuals with multiple myeloma produce a large population of identical, homogeneous antibodies with an identical specificity against an antigen. To produce monoclonal antibodies against a specific antigen, a mouse or rat is immunized with that antigen. The activated B lymphocytes are then isolated from the lymphatic tissue (spleen or lymph nodes) of the animal and fused with the myeloma cell line. This

lymphocytes polyclonal antibodies

Monoclonal antibodies antibody-producing cell line multiple myeloma

B

hybridoma,

fusion produces a antibody-secreting cell line. against rat actin molecules, from the lymphatic organs of with myeloma cells.

an immortalized individual To obtain monoclonal antibodies for example, the B lymphocytes immunized rabbits must be fused

FOLDER 1.3

CLINICAL CORRELATION: MONOCLONAL ANTIBODIES IN MEDICINE Monoclonal antibodies are now widely used in immunocytochemical techniques and also have many clinical applications. Monoclonal antibodies conjugated with radioactive compounds are used to detect and diagnose tumor metastasis in pathology, differentiate subtypes of tumors and stages of their differentiation, and in infectious disease diagnosis to identify microorganisms in blood and tissue fluids. Monoclonal antibodies conjugated with immunotoxins, chemotherapy agents, or radioisotopes are now being used to deliver therapeutic agents to specific tumor cells in the body.

Both direct and indirect immunocytochemical methods are used to locate a target antigen in cells and tissues. The oldest immunocytochemistry technique used for identifying the distribution of an antigen within cells and tissues is known as . This technique uses a fluorochrome-labeled (either polyclonal or monoclonal) that reacts with the antigen within the sample (Fig. 1.5a). As a one-step procedure, this method involves only a single labeled antibody. Visualization of structures is not ideal because of the low intensity of the signal emission. Direct immunofluorescence methods are now being replaced by the indirect method because of suboptimal sensitivity.

direct immunofluorescence primary antibody

FIGURE 1.5. Direct and indirect immunofluorescence. a.

In direct immunofluorescence, a fluorochrome-labeled primary antibody reacts with a specific antigen within the tissue sample. Labeled structures are then observed in the fluorescence microscope in which an excitation wavelength (usually ultraviolet light) triggers the emission of another wavelength. The length of this wavelength depends on the nature of the fluorochrome used for antibody labeling. The indirect method involves two processes. First, specific primary antibodies react with the antigen of interest. Second, the secondary antibodies, which are fluorochrome labeled, react with the primary antibodies. The visualization of labeled structures within the tissue is the same in both methods and requires the fluorescence microscope.

b.

Indirect immunofluorescence

provides much greater sensitivity than direct methods and is often referred to as the “sandwich” or “double-layer technique.” Instead of conjugating a fluorochrome with a specific (primary) antibody directed against the antigen of interest (e.g., a rat actin molecule), the fluorochrome is conjugated with a directed against a rat primary antibody (i.e., goat anti-rat antibody; Fig. 1.5b). When the fluorescein is conjugated directly with the specific primary antibody, the method is direct; when fluorescein is conjugated with a secondary antibody, the method is indirect. The indirect method considerably enhances the fluorescence signal emission from the

antibody

secondary

tissue. An additional advantage of the indirect labeling method is that a single secondary antibody can be used to localize the tissue-specific binding of several different primary antibodies (Fig. 1.6). For microscopic studies, the secondary antibody can be conjugated with different fluorescent dyes so that multiple labels can be shown in the same tissue section (see Fig. 1.4). Drawbacks of indirect immunofluorescence are that it is expensive, labor-intensive, and not easily adapted to automated procedures.

FIGURE 1.6. Microtubules and nuclear-specific histone proteins visualized by immunocytochemical methods using expansion microscopy . Distribution of microtubules (elements of the cell

cytoskeleton labeled in green) and nuclear-specific phospho-histone H3 (Ser10) proteins (labeled in magenta) obtained from the HeLa human cervical cancer cell line can be studied in vitro using the fluorescence microscope. By use of indirect immunofluorescence techniques, microtubules were labeled with primary rabbit anti–αtubulin polyclonal antibodies and visualized by secondary goat antirabbit antibodies conjugated with green fluorescent dye (Alexa Fluor 488). The histone proteins were labeled with monoclonal mouse anti– phospho-histone H3 (Ser10) primary antibodies and visualized by

secondary goat anti-mouse antibodies conjugated with fluorescent dye (CF633). DNA in the nucleus has been counterstained with a nonspecific blue stain (DAPI stain). The microtubule network is well visualized because of the high resolution afforded by the expansion microscopy procedure (expansion factor: 4.2). (Photomicrograph courtesy of Drs. Yongxin Zhao and Edward S. Boyden, Massachusetts Institute of Technology, Cambridge, MA.)

It is also possible to conjugate polyclonal or monoclonal antibodies with other substances, such as enzymes (e.g., horseradish peroxidase), that convert colorless substrates (e.g., DAB) into an insoluble product of a specific color that precipitates at the site of the enzymatic reaction. The staining that results from this can be observed in the light microscope (see Fig. 1.3b) with either direct or indirect immunocytochemical methods. In another variation, colloidal gold or ferritin (an iron-containing molecule) can be attached to the antibody molecule. These electron-dense markers can be visualized directly with the EM.

immunoperoxidase method

Hybridization Techniques

Hybridization is a method of localizing messenger RNA (mRNA) or DNA by hybridizing the sequence of interest to a complementary strand of a nucleotide probe. In general, the term hybridization describes the ability of single-stranded RNA or DNA molecules to interact (hybridize) with complementary sequences. In the laboratory, hybridization requires the isolation of DNA or RNA, which is then mixed with a complementary nucleotide sequence (called a ). Hybrids are detected most often using a radioactive label attached to one component of the hybrid. Binding of the probe and sequence can take place in a solution or on a nitrocellulose membrane. In , the binding of the nucleotide probe to the DNA or RNA sequence of interest is performed within cells or tissues, such as cultured cells or whole embryos. This technique allows the localization of specific nucleotide sequences as small as 10–20 copies of mRNA or DNA per cell.

probe

hybridization

nucleotide

in situ

Several nucleotide probes are used in in situ hybridization. can be as small as 20–40 base pairs. Single- or double-stranded DNA probes are much longer and can contain as many as 1,000 base pairs. For specific localization of mRNA, complementary RNA probes are used. These probes are labeled with radioactive isotopes (e.g., 32 P, 35 S, 3 H), a specifically modified nucleotide (digoxigenin), or biotin (a commonly used covalent multipurpose label). Radioactive probes can be detected and visualized by autoradiography. Digoxigenin and biotin are detected by immunocytochemical and cytochemical methods, respectively. The strength of the bonds between the probe and the complementary sequence depends on the type of nucleic acid in the two strands. The strongest bond is formed between a DNA probe and a complementary DNA strand and the weakest between an RNA probe and a complementary RNA strand. If a tissue specimen is expected to contain a minute amount of mRNA or a viral transcript, amplification for DNA or for RNA can be used. The amplified transcripts obtained during these procedures are usually detected using labeled complementary nucleotide probes using standard in situ hybridization techniques. Recently, fluorescent dyes have been combined with nucleotide probes, making it possible to visualize multiple probes at the same time (Fig. 1.7). This technique, called , is extensively used in the clinical setting for genetic testing. For example, a probe hybridized to metaphase chromosomes can be used to identify the chromosomal position of a gene. is used to simultaneously examine chromosomes, gene expression, and the distribution of gene products such as pathologic or abnormal proteins. Many specific are now commercially available and are used clinically in for cervical cancer or for the detection of human immunodeficiency virus (HIV)-infected cells. FISH is also used in to visualize chromosomes in fetal cells obtained from amniocentesis or chorionic villus sampling to detect chromosomal abnormalities. In addition, FISH is used to examine the chromosomes in the lymphocytes of astronauts to

Oligonucleotide probes

polymerase chain reaction (PCR) reverse transcription-PCR (RT-PCR)

fluorescence in situ hybridization (FISH)

FISH

diagnostic testing

fluorescent probes screening procedures prenatal

estimate their absorbed radiation dose after their return from space. The frequency of chromosome translocations in lymphocytes is proportional to the absorbed radiation dose.

FIGURE 1.7. Example of the fluorescence in situ hybridization (FISH) technique used in prenatal diagnostic testing. Interphase nuclei of cells obtained from amniotic fluid specimens were hybridized with two specific DNA probes. The orange probe (LSI 21) is locusspecific for chromosome 21, and the green probe (LSI 13) is locusspecific for chromosome 13. The right nucleus is from a normal amniotic fluid specimen and exhibits two green and two orange signals, which indicates two copies of chromosomes 13 and 21, respectively. The nucleus on the left has three orange signals, which indicate trisomy 21 (Down syndrome). DNA has been counterstained with a nonspecific blue stain (DAPI stain) to make the nucleus visible. ×1,250. (Courtesy of Dr. Robert B. Jenkins.)

Autoradiography

Autoradiography makes use of a photographic emulsion placed over a tissue section to localize radioactive material within tissues. Many small molecular precursors of larger molecules, such as the amino acids that make up proteins and the nucleotides that make up nucleic acids, may be tagged by incorporating a

radioactive atom or atoms into their molecular structure. The radioactivity is then traced to localize the larger molecules in cells and tissues. Labeled precursor molecules can be injected into animals or introduced into cell or organ 3 cultures. For instance, of 3 or can be introduced to living cells to study synthesis of DNA and subsequent cell division, synthesis of mRNA, and localization of protein synthesis in the cell. Other radioactive precursors can trace protein secretion by cells and localization of synthetic products within cells and in the extracellular matrix. Sections of specimens that have incorporated radioactive material are mounted on slides. In the dark, the slide is usually dipped in a melted photographic emulsion, thus producing a thin photographic film on the surface of the slide. After appropriate exposure in a light-tight box, usually for days to weeks, the exposed emulsion on the slide is developed by standard photographic techniques and permanently mounted with a coverslip. The slides may be stained either before or after exposure and development. The silver grains in the emulsion over the radioactively labeled molecules are exposed and developed by this procedure and appear as dark grains overlying the site of the radioactive emission when examined with the light microscope (Fig. 1.8a).

thymidine)

radioactive precursors RNA ( H-uridine)

DNA ( H-

FIGURE 1.8. Examples of autoradiography used in light and electron microscopy. a. Photomicrograph of a lymph node section from

an animal injected with tritiated [ 3H]thymidine. Some of the cells exhibit aggregates of metallic silver grains, which appear as small black particles ( ). These cells synthesized DNA in preparation for cell division and have incorporated the [ 3H]thymidine into newly formed DNA. Over time, the low-energy radioactive particles emitted from the [ 3H]thymidine strike silver halide crystals in a photographic emulsion covering the specimen (exposure) and create a latent image (much like light striking photographic film in a camera). During photographic development of the slide with its covering emulsion, the latent image—actually the activated silver halide in the emulsion—is reduced to the metallic silver, which then appears as black grains in the microscope. ×1,200. (Original slide specimen courtesy of Dr. Ernst Kallenbach.) Electron microscopic autoradiograph of the apical region of an intestinal absorptive cell. In this specimen, 125I bound to nerve growth factor (NGF) was injected into the animal, and the tissue was removed 1 hour later. The specimen was prepared in a manner similar to that for light microscopy. The relatively small size of the silver grains aids precise localization of the 125I–NGF complexes. Note that the silver grains are concentrated over apical invaginations ( ) and early endosomal tubular profiles ( ). ×32,000. (Electron micrograph courtesy of Dr. Marian R. Neutra.)

arrows

b.

inv

tub

These grains may be used simply to indicate the location of a substance, or they may be counted to provide semiquantitative

information about the amount of a given substance in a specific location. For instance, after injection of an animal with tritiated thymidine, cells that have incorporated this nucleotide into their DNA before they divide will have approximately twice as many silver grains overlying their nuclei as will cells that have divided after incorporating the labeled nucleotide. Autoradiography can also be performed using thin plastic sections for examination with the EM. Essentially, the same procedures are used, but as with all TEM preparation techniques, the processes are much more delicate and difficult; however, they also yield much greater resolution and more precise localization (Fig. 1.8b).

Expansion Microscopy

Expansion microscopy is a method for improving the resolution of light microscopy by implementing a specific preparation that physically expands the specimen. Expansion microscopy (ExM) refers to a process in which specimens are infiltrated with swellable polymers (hydrogels— very absorbent materials commonly found in baby diapers), which form hydrophilic polymer networks that are able to absorb large amounts of water and increase their volumes. The structure and physical integrity of these networks are due to the presence of strong and stable cross-links that allow the hydrogel to withstand expansion forces generated by the addition of water, thus stabilizing the gel. As a result of the of the specimen, the molecules within the cells, plasma membrane, and extracellular matrix are separated from each other equally in all directions. Preparation of the specimen for ExM involves the following steps (Fig. 1.9):

expansion

isotropic

FIGURE 1.9. Steps in tissue processing for expansion microscopy. This diagram shows the consecutive steps in preparation of a specimen for expansion microscopy. After conventional fixation (in formaldehyde), the specimen is treated with anchoring reagents that bind to proteins or other molecules of interest and molecular

probes conjugated with fluorescent labels. The addition of sodium acrylate monomers triggers development of a dense three-dimensional hydrogel polymer matrix. After mechanical homogenization that allows the cells to break open, the specimen, now embedded in a hydrogel matrix, is ready for physical expansion by the addition of water. Proteins of interest remain connected to the expanded polymer network, which pulls them apart. The integrity of the expanded gel is maintained by strong and stable cross-links that resist expansion forces generated by the addition of the water. After the expansion (approximately 4.5 times), the specimen is ready for examination with the fluorescence microscope.

Fixation. The fixation process for ExM is the same as for light microscopy immunostaining protocols. Anchoring. The cellular structure of interest is labeled with

a molecular probe (e.g., antibodies conjugated with fluorescein dye, fluorescent proteins, and others) and incubated with an anchoring reagent expressing specific binding sites for molecular probes and with gel monomers. In addition, some molecules (e.g., proteins or RNA) can also be anchored directly to the gel. . The specimen is infiltrated with gel monomers (i.e., sodium acrylate) that polymerize within the cells and tissues. The resulting polymers (sodium polyacrylate) form a dense 3D matrix that is firmly anchored to the cell molecules via binding sites on the anchoring reagent. . The polymer-embedded specimen undergoes mechanical homogenization. This process, which breaks open the cells, involves denaturation and/or digestion of structural molecules by specific proteases. . A solvent is added to the specimen (water in the case of sodium polyacrylate polymers), causing expansion of specimen in all three dimensions (more than 100-fold in volume).

Gelation

Mechanical homogenization Expansion

Following this preparation, the expanded specimen is ready for examination using standard fluorescence microscopy.

Expansion microscopy, which utilizes inexpensive chemicals and commonly used optical microscopes, provides superresolution imaging by increasing the size of the observed specimen.

The main advantage of ExM lies in its ability to separate tightly spaced molecules that were not initially detectable as separate structures because of the light microscope’s innate resolution and diffraction limitations. In expanded specimens, these molecules are pulled far enough apart to be easily resolved without changing the resolution limits of the optical instrument. Linear expansion to 4.5 times is possible, which correlates to an increase in resolution in the range of 60–70 nm. Interestingly, after an initial expansion, the specimen can be subject to repeated expansions with a second swellable polymer network. This process, called , may be used to expand biologic specimens up to 20 times and obtain images of cells and tissues with a resolution of approximately 25 nm when viewed with conventional fluorescence microscopy (Fig. 1.10).

iterative expansion

microscopy (iExM)

FIGURE 1.10. Comparison of photomicrographs of mammary gland tissue from light microscopy, fluorescence microscopy, and expansion microscopy . All images were obtained from serial sections

of the same tissue and processed according to specific microscopy techniques. All images were obtained with the same 40× objective lens. This photomicrograph of a routine hematoxylin and eosin (H&E)-stained section shows the mammary duct ( ) and surrounding connective tissue ( ). There is noticeable increase of cell layers within the ductal epithelium ( ) that is indicative of usual ductal hyperplasia (UDH). Nuclei are stained blue with hematoxylin, and the pink elongated line beneath the epithelium represents connective tissue fibers stained with eosin. Note that the small and more

a.

CT

Ep

D

CT

intensely stained nuclei within the belong to infiltrating lymphocytes. ×460. This immunofluorescent image was obtained from the section stained for vimentin intermediate filaments. The vimentin proteins stained in magenta were labeled with polyclonal chicken antivimentin primary antibodies and visualized by secondary goat antichicken antibodies conjugated with fluorescent dye (Alexa Fluor 488). DNA has been counterstained with a nonspecific blue stain (DAPI stain) to make the nuclei visible. At this magnification, many nuclei are difficult to discern because of borderline resolution. ×460. Adjacent section of the same tissue shown in image b was processed for expansion microscopy. The specimen was embedded in the polyacrylate polymer and expanded 4.25 times. The image shows part of the section included within the rectangle in image that was photographed at the same magnification ×460. Because of expansion of the tissue, resolution of this image is markedly improved compared with the routine immunofluorescent image taken at the same magnification. (Photomicrographs courtesy of Drs. Yongxin Zhao and Edward S. Boyden, Massachusetts Institute of Technology, Cambridge, MA.)

b.

c.

b

Recently, ExM protocols have been applied to routine H&E slide preparations of pathologic specimens to convert glass slides into ExM-compatible preparations. This method, known as , allows for the optical microscopic examination and diagnosis of diseases that previously required electron microscopy.

expansion pathology (ExPath)

MICROSCOPY Light Microscopy A microscope, whether simple (one lens) or compound (multiple lenses), is an instrument that magnifies an image and allows visualization of greater detail than is possible with the unaided eye. The simplest microscope is a magnifying glass or a pair of reading glasses. The —that is, the distance by which two objects must be separated to be seen as two objects —is determined by the spacing of the photoreceptor cells in the retina. The role of a microscope is to magnify an image to a level at which the retina can resolve the information that would otherwise be below its limit of

resolving power of the human eye (0.2 mm)

resolution. Table 1.3 compares the resolution of the eye with that of various instruments.

TABLE 1.3 Eye Versus Instrument Resolution Distance Between Resolvable Points Human eye

0.2 mm

Bright-field microscope

0.2 μm

Super-resolution optical microscope

10–100 nm

Scanning electron microscope

2.5 nm

Transmission electron microscope Theoretical

0.05 nm

Tissue section

1.0 nm

Atomic force microscopy

50.0 pm

Resolving power is the ability of a microscope lens or optical system to produce separate images of closely positioned objects. Resolution depends not only on the optical system but also on the wavelength of the light source and other factors such as specimen thickness, quality of fixation, and staining intensity. With light of wavelength 540 nm (see Table 1.1), a green-filtered light to which the eye is extremely sensitive, and appropriate objective and condenser lenses, the greatest attainable is approximately (see Folder 1.4, page 15, for method of calculation). This theoretical resolution depends on all conditions being optimal.

resolving power of a bright-field microscope 0.2 μm The ocular or eyepiece lens magnifies the image produced by the objective lens, but it cannot increase resolution. FOLDER 1.4

FUNCTIONAL CONSIDERATIONS: PROPER USE OF THE LIGHT MICROSCOPE

This brief introduction to the proper use of the light microscope is directed to those students who will use the microscope for the routine examination of tissues. If the following comments appear elementary, it is only because most users of the microscope fail to use it to its fullest advantage. Despite the availability of today’s fine equipment, relatively little formal instruction is given on the correct use of the light microscope. Expensive and highly corrected optics perform optimally only when the illumination and observation beam paths are centered and properly adjusted. The use of proper settings and proper alignment of the optic pathway will contribute substantially to the recognition of minute details in the specimen and to the faithful display of color for the visual image and for photomicrography. is one key to good microscopy and is incorporated in the design of practically all modern laboratory and research microscopes. Figure F1.4.1 shows a typical light path and the controls for alignment on a modern laboratory microscope; it should be referred to in following the instructions given here to provide appropriate illumination in your microscope.

Köhler illumination

FIGURE F1.4.1. Diagram of a typical light microscope. drawing shows a cross-sectional view operating components, and light path.

of

the

microscope,

This its

alignment steps

The necessary to achieve good Köhler illumination are few and simple: Focus the specimen. Close the field diaphragm. Focus the condenser by moving it up or down until the outline of its field diaphragm appears in sharp focus. Center the field diaphragm with the centering controls on the (condenser) substage. Then, open the field diaphragm until the light beam covers the full field observed. Remove the eyepiece (or use a centering telescope or a phase telescope accessory if available) and observe the exit pupil of the objective. You will see an illuminated circular field that has a radius directly proportional to the numeric aperture of the objective. As you close the condenser diaphragm, its outline will appear in this circular field. For most stained materials, set the condenser diaphragm to cover approximately two-thirds of the objective aperture. This setting results in the best compromise between resolution and contrast (contrast simply being the intensity difference between dark and light areas in the specimen). Using only these five simple steps, the image obtained will be as good as the optics allow. Now, let us find out why. First, why do we adjust the field diaphragm to cover only the field observed? Illuminating a larger field than the optics can “see” only leads to internal reflections or stray light, resulting in more “noise” or a decrease in image contrast. Second, why do we emphasize the setting of the condenser diaphragm—that is, the illuminating aperture? This diaphragm greatly influences the resolution and the contrast with which specimen detail can be observed. For most practical applications, the is determined by the following equation:

resolution

where

d λ

    = point-to-point distance of resolved detail (in nm),      = wavelength of light used (green = 540 nm), NA = numeric aperture or sine of half angle picked up by the objective or condenser of a central specimen point

multiplied by the refractive index of the medium between objective or condenser and specimen. How do wavelength and numeric aperture directly influence resolution? Specimen structures diffract light. The diffraction angle is directly proportional to the wavelength and inversely proportional to the spacing of the structures. According to physicist Ernst Abbé, a given structural spacing can be resolved only when the observing optical system (objective) can see some of the diffracted light produced by the spacing. The larger the objective’s aperture, the more diffracted the light that participates in the image formation, resulting in resolution of smaller detail and sharper images. Our simple formula, however, shows that the condenser aperture is just as important as the objective aperture. This point is only logical when you consider the diffraction angle for an oblique beam or one of higher aperture. This angle remains essentially constant but is presented to the objective in such a fashion that it can be picked up easily. How does the aperture setting affect the contrast? Theoretically, the best contrast transfer from object to image would be obtained by the interaction (interference) between nondiffracted and all the diffracted wave fronts. For the transfer of contrast between full transmission and complete absorption in a specimen, the intensity relationship between diffracted and nondiffracted light would have to be 1:1 to achieve full destructive interference (black) or full constructive interference (bright). When the condenser aperture matches the objective aperture, the nondiffracted light enters the objective with full intensity, but only part of the diffracted light can enter, resulting in decreased contrast. In other words, closing the aperture of the condenser to two-thirds of the objective aperture brings the intensity relationship between diffracted and nondiffracted light close to 1:1 and thereby optimizes the contrast. Closing the condenser aperture (or lowering the condenser) beyond this equilibrium will produce interference phenomena or image artifacts such as diffraction rings or artificial lines around specimen structures. Most microscope techniques used for the enhancement of contrast—such as dark field, oblique illumination, phase contrast, or modulation contrast —are based on the same principle (i.e., they suppress or reduce the intensity of the nondiffracted light to improve an inherently low contrast of the specimen). By observing the steps outlined earlier and maintaining clean lenses, the quality and fidelity of visual images will vary only with the performance capability of the optical system.

light microscopes

Various are available for general and specialized use in modern biologic research. Their differences

are based largely on such factors as the wavelength of specimen illumination, physical alteration of the light coming into or leaving the specimen, and specific analytic processes that can be applied to the final image. Modern light microscopes are expensive and immensely complex, combining a growing number of optical, photomechanical, and electronic components. However, (i.e., UC2, µCube, etc.) are being designed that may be more widely available. Many of the light-, electron-, and nonoptical instruments used in education and research and their applications are described briefly in the following sections.

low-cost, 3D-printed, open-source light microscopes

The microscope used by most students and researchers is the bright-field microscope. The bright-field microscope is the direct descendant of the microscopes that became widely available in the 1800s and inaugurated the first major era of histologic research. The bright-field (light) microscope (Fig. F1.4.1 in Folder 1.4) essentially consists of

light source condenser lens stage objective lens ocular lens

a for illumination of the specimen (e.g., a substage lamp), a to focus the beam of light at the level of the specimen, a on which the slide or other specimen is placed, an to gather the light that has passed through the specimen, and an (or a pair of ocular lenses in the more commonly used binocular microscopes) through which the image formed by the objective lens may be examined directly. A specimen to be examined with the bright-field microscope must be sufficiently thin for light to pass through it. Although some light is absorbed while passing through the specimen, the optical system of the bright-field microscope does not produce a useful level of contrast in unstained specimens. For this reason, various staining methods as discussed earlier are used.

Examination of a Histologic Slide Preparation in the Light Microscope

Organs are three-dimensional, whereas histologic sections are only two-dimensional. As discussed in the earlier “Tissue Preparation” section, every tissue sample prepared for light microscopic examination must be sliced into thin sections. Thus, 2D sections are obtained from an original 3D sample of tissue. One of the most challenging aspects for students using the microscope to study histology is the ability to mentally reconstruct the “missing” third dimension. For example, slices in different planes through an orange are shown in Figure 1.11. Note that each cut surface (indicated by the dotted line) of the whole orange reveals different sizes and surface patterns, depending on the orientation of the cut. Thus, it is important when observing a given section cut through the orange to be able to mentally reconstruct the organization of the structure and its component parts. An example of a histologic structure—in this case, a kidney renal corpuscle—is shown as it would appear in different sectional planes (see Fig. 1.11). Note the marked differences in size, orientation, and organization of surrounding tissue in each section of the renal corpuscle. By examining a number of such 2D sections, it is possible to perceive the 3D configuration of the examined structure.

FIGURE 1.11. Example of sections from an orange and a kidney renal corpuscle. The solid color lines drawn on the intact orange indicate the plane of section that correlates with each cut surface. Similarly, different sections through a kidney renal corpuscle, (marked with ), which is also a spherical structure, show differences in appearance. The size and internal structural appearance are reflected in the plane of section.

dotted lines

Artifacts in histologic slides can be generated in all stages of tissue preparation. The preparation of a histologic slide requires a series of steps beginning with the collection of the specimen and ending with the placement of the coverslip. During each step, an (a defect caused by an error in the preparation process) may be introduced. In general, artifacts that appear on the finished glass slide are linked to methodology, equipment, or reagents used during preparation. The inferior purity of chemicals and reagents used in the process (fixatives, reagents, and stains), imperfections in the execution of the methodology (too short or too long intervals of fixation, dehydration, embedding, or staining, or careless mounting and placement of the coverslip), or improper equipment (e.g., a microtome with a defective blade) can produce artifacts in the final preparation. It is important for students to recognize that not every slide in their slide collection is perfect and to be familiar with the most common artifacts found on their slides.

artifact

Other Optical Systems Besides bright-field microscopy, which is commonly used for routine examination of histologic slides, other optical systems (described later) are used in clinical and research laboratories. Some of them are used to enhance the contrast without staining (such as phase contrast microscopes), whereas others are designed to visualize structures using specific techniques such as immunofluorescence (light sheet fluorescence and confocal microscopes). The serial images obtained by these microscopes can be captured in layers and three-dimensionally reconstructed using a variety of 3D image analysis software.

The phase contrast microscope enables examination of unstained cells and tissues and is especially useful for living cells. The phase contrast microscope takes advantage of small

differences in the refractive index of different parts of a

cell or tissue sample. Light passing through areas of relatively high refractive index (denser areas) is deflected and becomes out of phase with the rest of the beam of light that has passed through the specimen. The phase contrast microscope adds other induced, out-of-phase wavelengths through a series of optical rings in the condenser and objective lenses, essentially abolishing the amplitude of the initially deflected portion of the beam and producing contrast in the image. Dark portions of the image correspond to dense portions of the specimen; light portions of the image correspond to less dense portions of the specimen. The phase contrast microscope is used to examine living cells and tissues (such as cells in tissue culture) and extensively to examine unstained semithin (approximately 0.5 μm) sections of plastic-embedded tissue. Two modifications of the phase contrast microscope are the , which also allows quantification of tissue mass, and the (using Nomarski optics), which is especially useful for assessing surface properties of cells and other biologic objects.

interference microscope

differential interference microscope

In dark-field microscopy, no direct light from the light source is gathered by the objective lens. In dark-field microscopy, only light that has been scattered

or diffracted by structures in the specimen reaches the objective. The dark-field microscope is equipped with a special condenser that illuminates the specimen with strong, oblique light. Thus, the field of view appears as a dark background on which small particles in the specimen that reflect some light into the objective appear bright. The effect is similar to that of dust particles seen in the light beam emanating from a slide projector in a darkened room. The light reflected off the dust particles reaches the retina of the eye, thus making the particles visible. The resolution of the dark-field microscope cannot be better than that of the bright-field microscope, using, as it does, the same wavelength source. Smaller individual particles can be

detected in dark-field images, however, because of the enhanced contrast that is created. The dark-field microscope is useful in examining autoradiographs, in which the developed silver grains appear white in a dark background. Clinically, is useful in examining urine for crystals, such as those of uric acid and oxalate, and in demonstrating specific bacteria such as , particularly , the microorganism that causes , a sexually transmitted disease.

dark-field microscopy

spirochetes

Treponema pallidum

syphilis The fluorescence microscope makes use of the ability of certain molecules to fluoresce under ultraviolet light.

Fluorescent molecules are those that emit light of wavelengths in the visible range when exposed to an ultraviolet (UV) source. The is used to display naturally occurring fluorescent (autofluorescent) molecules such as vitamin A and some neurotransmitters. Because autofluorescent molecules are not numerous, the microscope’s most common application is the display of introduced fluorescence, as in the detection of antigens or antibodies in immunocytochemical staining procedures (see Fig. 1.6). Specific can also be injected into an animal or directly into cells and used as tracers. Such methods have been useful in studying intercellular (gap) junctions, tracing the pathway of nerve fibers in neurobiology, and detecting fluorescent growth markers of mineralized tissues. Various filters are inserted between the UV light source and the specimen to produce monochromatic or nearmonochromatic (single-wavelength or narrow-band-wavelength) light. A second set of filters inserted between the specimen and the objective allows only the narrow band of wavelength of the fluorescence to reach the eye or to reach an image sensor in the digital recording device.

fluorescence microscope

fluorescent molecules (fluorophores)

Light sheet fluorescence microscopy utilizes a thin plane of light to optically section a transparent specimen labeled with fluorescent molecules.

Light sheet fluorescence microscopy (LSFM) utilizes a light sheet that is formed by a flat laser beam. This thin sheet of light is formed in the focal plane and optically sections

transparent specimen labeled with fluorescent dyes. The fluorescent light emitted from the specimen is then collected perpendicularly to the light path by the objective of the microscope and recorded by an imaging sensor (e.g., a chargecoupled device or CCD). The specimen is illuminated only in a at a time, avoiding excitation from out-offocus areas of the specimen. The light sheet itself can be static or dynamically formed by a moving (scanning) laser beam approximating a light sheet over a short period of time. By moving the sample through the light sheet, images can be recorded in layers and three-dimensionally reconstructed (Fig. 1.12).

single focal plane

FIGURE 1.12. Light sheet fluorescence microscopy (LSFM) image of cells expressing galanin neuropeptide in the spinal cord of an adult male rat. a. This photomicrograph shows a three-dimensional (3D) rendering of an LSFM image of the rat spinal cord at the L3 and L4 vertebral levels. Within the rectangle , note the immunofluorescent

labeling of galanin neuropeptide expressed in the spinothalamic cells. This neuropeptide was detected by the indirect immunofluorescence method using polyclonal rabbit anti-galanin primary antibodies and then visualized by using goat anti-rabbit secondary antibodies

conjugated with fluorescein dye (Alexa Fluor 647). After immunostaining, the specimen was cleared in dibenzyl ether (DBE) and the transparent tissue was imaged in the horizontal plane using bidirectional LSFM. Stacks of TIFF images were collected at 4 μm optical intervals and linked together using specialized imaging software. The reconstructed image was then artificially colored in . The 3D rendering allows the image to be rotated and examined from all possible directions. ×10. This image represents a higher magnification of galanin-positive spinothalamic cells with the spinal cord background shown in the subtracted. Note that galaninpositive cells are in close proximity to the central canal. ×22. High-magnification view of galanin-expressing spinothalamic cells showing their interconnecting pattern. ×110. (Courtesy of Drs. Aleisha M. Moore, Michael N. Lehman, and Lique M. Coolen.)

green

b. rectangle

c.

Fluorescence microscopy is currently one of the more powerful and versatile techniques available for studies of biologic specimens. Most modern research laboratories utilize fluorescence microscopy as a primary tool in biologic research. Fluorescent molecules (fluorophores) have been engineered to absorb light at a specific wavelength and to emit light at a longer wavelength. These molecules appear very bright and are readily distinguishable in tissue sections from other background signals. In addition, with the development of genetically encoded , it has become possible to visualize and create images of protein expression, localization, and activity in living cells. The combination of fluorescence and confocal microscopic techniques with fast data processing hardware allows investigators to render the images in three dimensions.

fluorescent proteins (FPs)

The ultraviolet microscope uses quartz lenses with an ultraviolet light source. The image in the ultraviolet (UV) microscope depends on the

absorption of UV light by molecules in the specimen. The UV source has a wavelength of approximately 200 nm. Thus, the UV microscope may achieve a resolution of 0.1 μm. The general principle behind UV microscopy resembles that of a spectrophotometer; the results are usually recorded photographically. The specimen cannot be inspected directly

through an ocular lens because the UV light is not visible and is injurious to the eye. UV microscopy is useful in detecting nucleic acids, specifically the purine and pyrimidine bases of the nucleotides. It is also useful for detecting proteins that contain certain amino acids. Using specific illuminating wavelengths, UV spectrophotometric measurements are commonly made through the UV microscope to determine quantitatively the amount of DNA and RNA in individual cells. As described in Folder 1.2 on page 7, is used clinically to evaluate the degree of ploidy (multiples of normal DNA quantity) in sections of tumors.

Feulgen microspectrophotometry

The confocal scanning microscope combines components of a light optical microscope with a scanning system to dissect a specimen optically. The confocal scanning microscope allows visualization of a

biologic specimen in three dimensions. The two lenses in the confocal microscope (objective and phototube lens) are perfectly aligned to focus light from the focal point of one lens to the focal point of the other lens. The major difference between a conventional and a confocal microscope is the addition of a (pinhole) that is jugate with the point of the lens; therefore, it is . This precisely positioned pinhole allows only “in-focus” light to pass into a photomultiplier (detector) device, whereas the “out-of-focus” light is blocked from entering the detector (Fig. 1.13). This system has the capability to obtain exceptional resolution (0.2–0.5 μm) and clarity from a thin section of a biologic sample simply by rejecting out-of-focus light.

focal

detector aperture

con confocal

FIGURE 1.13. Diagram of the in-focus and out-of-focus emitted light in the confocal microscope. a. This diagram shows the path

of the laser beam and emitted light when the imaging structure is directly at the focus of the lens. The screen with a pinhole at the other side of the optical system of the confocal microscope allows the light from the structure in focus to pass through the pinhole. The light is then translated into an image by computer software. Because the focal point of the objective lens of the microscope forms a sharp image at the level at which the pinhole is located, these two points are referred to as . This diagram shows the path of the laser beam and the emitted light, which is out of focus in relation to the pinhole. Thus, the light from the specimen that gets blocked by the pinhole is never detected.

confocal points b.

The light source in a confocal microscope comes from an illuminating laser light system that is strongly convergent and therefore produces a high-intensity excitation light in the form of a shallow scanning spot. A mirror system is used to move the laser beam across the specimen, illuminating a single spot at a time (Fig. 1.14). Many single spots in the same focal plane are scanned, and a computer software program reconstructs the image from the data recorded during scanning. In this aspect, confocal microscopy resembles the imaging process in a computerized axial tomography (CAT) scan.

FIGURE 1.14. Structure of the confocal microscope and diagram of the beam path . The light source for the confocal microscope comes from a laser. The laser beam ( red line ) travels to the tissue sample via a dichroic beam splitter and then to two movable scanning mirrors; these mirrors scan the laser beam across the sample in both x and y directions. Finally, the laser beam enters the fluorescence microscope and travels through its optical system to illuminate an examined tissue sample. The emitted light by the illuminated tissue sample ( ) travels back through the optical system of the microscope, through both scanning mirrors, passes through the beam splitter, and is focused onto the pinhole. The light that passes through the pinhole is received and registered by the detector attached to a computer that builds the image one pixel at a time.

blue line

Furthermore, by using only the narrow depth of the in-focus image, it is possible to create multiple images at varying depths within the specimen. Thus, one can literally dissect

layer by layer through the thickness of the specimen. It is also possible to use the computer to make 3D reconstructions of a series of these images. Because each individual image located at a specific depth within the specimen is extremely sharp, the resulting assembled 3D image is equally sharp. Moreover, once the computer has assembled each sectioned image, the reconstructed 3D image can be animated for viewing on the computer from any orientation desired (see Fig. 1.4).

The polarizing microscope exploits the fact that highly ordered molecules or arrays of molecules can rotate the angle of the plane of polarized light. The polarizing microscope is a simple modification of the light microscope in which a polarizing filter (the polarizer) is located between the light source and the specimen, and a second polarizer (the analyzer) is located between the objective lens and the viewer. Both the polarizer and the analyzer can be rotated; the difference between their angles of rotation is used to determine the degree by which a structure affects the beam of polarized light. The ability of a crystal or paracrystalline array to rotate the plane of polarized light is called (double refraction). Striated muscle and the crystalloid inclusions in testicular interstitial cells (Leydig cells), among other common structures, exhibit birefringence.

birefringence

Super-Resolution Microscopy Conventional optical microscopes have an inherent limitation in resolving power because of the wavelength of light. The resolution, which is defined by the minimum point-to-point distance between two distinguishable details, is restricted by the diffraction limit of the light. Diffraction causes the light signal from the specimen to spread as it travels to the eye of the observer or other light detector devices. As discussed earlier, the resolution of an optical microscope with an optimal alignment of the objective and condenser lenses is

limited to 0.2 μm; therefore, it is unable to resolve many detailed cellular structures.

Newer super-resolution microscopy techniques are able to overcome the resolution limit of conventional light microscopy. For decades, researchers have been searching for techniques that could exceed the resolution limit of the optical microscope. Recent conceptual advances and technical innovation resulted in increases in optical resolution from 0.2 μm to ~10 nm. Any microscopy technique that increases the resolution of a conventional light microscope dictated by the diffraction barrier by at least a factor of 2 is called . Several super-resolution microscopy techniques have been developed to study living cells under fluorescence light microscopy. In general, there are three methods that are utilized in super-resolution microscopy:

microscopy

super-resolution

Single-molecule localization methods,

which include photoactivated localization microscopy (PALM), fluorescence photoactivated localization microscopy (FPALM), and stochastic optical reconstruction microscopy (STORM). These methods involve the use of photoactivatable and photoswitchable fluorescent molecules that can transition between the state of dark and bright emission when exposed to specific wavelengths of light. Computer analysis of combined data obtained from thousands of single-molecule intensity profiles and microscope diffraction profile are converted into an image with a resolution between 10 and 20 nm. , which are based on extracting fine structural details from the interference of a structure with predetermined illumination patterns. Because this method utilizes spatial frequencies, which are also limited by diffraction, SIM microscopy can only improve the resolution by a factor of 2 (resolution ~100 nm). , which include stimulated emission depletion (STED) microscopy and isotropic stimulated emission

Structured illumination microscopy (SIM) methods

Point-scanning methods

depletion (isoSTED) microscopy. These methods are based on laser scanning confocal microscopy with the addition of a depletion laser, which stimulates excited molecules to revert back to the ground state. Using STED microscopy, image resolution of 30–80 nm can be achieved. Super-resolution microscopy methods offer new opportunities for revealing details of cellular structures in living cells at a higher resolution that was not previously achievable with conventional fluorescence microscopy.

Electron Microscopy In general, two kinds of EMs can provide morphologic and analytic data about cells and tissues: the and the . The primary improvement in the EM versus the light microscope is that the wavelength of the EM beam is approximately 1/2,000th that of the light microscope beam, thereby increasing resolution by a factor of 103 .

electron microscope (TEM) microscope (SEM)

transmission scanning electron

The TEM uses the interaction of a beam of electrons with a specimen to produce an image. The optics of the TEM are, in principle, similar to those of the light microscope (Fig. 1.15), except that the TEM uses a beam of electrons rather than a beam of light. The principle of the microscope is as follows:

FIGURE 1.15. Diagram comparing the optical paths in different types of microscopes. For better comparison among all three types of microscopes, the light microscope ( left ) is shown as if it were turned upside down. Note that in both the transmission electron microscope ( TEM ) and the scanning electron microscope ( SEM ), specimens need to be inserted into a high-vacuum (10 −4 to 10 −7 Pa) environment.

cathode, electron emitter, electron electrons

An electron source ( ), such as a heated tungsten filament, emits electrons. The negatively charged are attracted toward an , which is the positively charged electron collector. An electrical difference between the cathode cover and the anode imparts an accelerating voltage of between 20,000 and 200,000 volts to the electrons, creating the . The beam then passes through a series of that serve the same function as the glass lenses of a light microscope.

gun anode

lenses

electron beam electromagnetic

condenser lens shapes and changes the diameter of the electron beam that reaches the specimen plane. The beam that The

has passed through the specimen is then focused and magnified by an and then further magnified by one or more . The final image is viewed on a phosphorcoated or captured on a . Portions of the specimen through which electrons have passed appear bright; dark portions of the specimen have absorbed or scattered electrons because of their inherent density or because of heavy metals added during specimen preparation. Often, an electron detector with a light-sensitive sensor such as a is placed above or below the viewing screen to observe the image in real time on a monitor. This allows images or videos to be archived in digital format for storage on computers.

objective lens projector lenses fluorescent screen

photographic plate

charge-coupled device (CCD)

Specimen preparation for transmission electron microscopy is similar to that for light microscopy except that it requires finer methods.

The principles used in the preparation of sections for viewing with the TEM are essentially the same as those used in light microscopy, with the added constraint that at every step one must work with specimens three to four orders of magnitude smaller or thinner than those used for light microscopy. The TEM, which has an electron beam wavelength of approximately 0.1 nm, has a theoretical resolution of 0.05 nm. Because of the exceptional resolution of the TEM, the quality of fixation—that is, the degree of preservation of subcellular structure—must be the best achievable.

Routine preparation of specimens for transmission electron microscopy begins with glutaraldehyde fixation followed by a buffer rinse and fixation with osmium tetroxide. Glutaraldehyde, a dialdehyde, preserves protein constituents by cross-linking them; osmium tetroxide reacts with lipids, particularly phospholipids. The osmium also imparts electron density to cell and tissue structures because it is a heavy metal, thus enhancing subsequent image formation in the TEM. Ideally, tissues should be perfused with buffered glutaraldehyde before excision. More commonly, tissue pieces no

more than 1 mm3 are fixed microscope specimens, which The dehydration process is microscopy, and the tissue resin, usually an polymerized.

for the TEM (compared with light may be measured in centimeters). identical to that used in light is infiltrated with a monomeric , that is subsequently

epoxy resin The plastic-embedded tissue is sectioned on specially designed microtomes using diamond knives. Because of the limited penetrating power of electrons, sections for routine TEM range from 50 nm to no more than 150 nm. Also, for the reason that abrasives used to sharpen steel knives leave unacceptable scratches on sections viewed in the TEM, with a nearly perfect cutting edge are used. Sections cut by the diamond knife are much too thin to handle; they are floated away from the knife edge on the surface of a fluid-filled trough and picked up from the surface onto plastic-coated copper mesh grids. The grids have 50–400 holes per inch or special slots for viewing serial sections. The beam passes through the specimen and then through the holes in the copper grid, and the image is then focused on the viewing screen, CCD, or photographic film.

diamond knives

Routine staining of transmission electron microscopy sections is necessary to increase the inherent contrast of cell structures so that details are readily visible and photographable. In general, TEM sections are stained by adding materials of great density, such as ions of heavy metals, to the specimen. may be bound to the tissues during fixation or dehydration or by soaking the sections in solutions of ions after sectioning. , routinely used in the fixative, binds to the phospholipid components of membranes, imparting additional density to the membranes. is often added to the alcohol solutions used in dehydration to increase the density of components of cell junctions and other sites. Sequential soaking in solutions of and is routinely used to stain

Heavy metal ions

Osmium tetroxide

Uranyl nitrate

uranyl acetate

lead citrate

sections before viewing with the TEM to provide highresolution, high-contrast electron micrographs. Sometimes, special staining is required to visualize results of histocytochemical or immunocytochemical reactions with the TEM. Phosphatase and esterase procedures are used for this purpose (see Fig. 1.3). Substitution of a for the fluorescent dye that has been conjugated with an antibody allows the adaptation of immunocytochemical methods to TEM. Similarly, routine have been refined for use with TEM (see Fig. 1.8b). These methods have been particularly useful in elucidating the cellular sources and intracellular pathways of certain secretory products, the location on the cell surface of specific receptors, and the intracellular location of ingested drugs and substrates.

heavy metal–

containing compound

EM

autoradiography techniques

Freeze fracture is a special method of sample preparation for transmission electron microscopy; it is especially important in the study of membranes. Freeze fracture is a special method of sample preparation that physically breaks apart (fractures) a frozen specimen to reveal its internal structures. The tissue to be examined may be fixed or unfixed; if it has been fixed, then the fixative is washed out of the tissue before proceeding. A cryoprotectant such as glycerol is allowed to infiltrate the tissue, and the tissue is then rapidly frozen to about −160°C. Ice crystal formation is prevented by the use of cryoprotectants, rapid freezing, and extremely small tissue samples. The frozen tissue is then placed in a vacuum in the freeze fracture apparatus and struck with a knife edge or razor blade.

The fracture plane passes preferentially through the hydrophobic portion of the plasma membrane, exposing the interior of the plasma membrane. The resulting fracture of the surfaces. The surface of the extracellular space is called the protoplasm (cytoplasm) is

plasma membrane produces two new membrane that is backed by the the ; the face backed by called the . The specimen

E-face

P-face

is then coated, typically with evaporated platinum and carbon, to create a replica of the fracture surface. The tissue is dissolved, and the surface replica, not the tissue itself, is picked up on grids to be examined with the TEM. Such a replica displays planar views of the internal organization of membranes with details at the macromolecular level (see Fig. 2.5, page 34). One of the most common uses of the freeze fracture technique is to examine zonula occludens junctions, where integral membrane proteins bind cells together (see Fig. 5.15c, page 141).

In scanning electron microscopy, the electron beam does not pass through the specimen but is scanned across its surface.

In many ways, the images obtained from the SEM more closely resemble those seen on a television screen than on the TEM monitor. They are 3D in appearance and portray the surface structure of an examined sample. For the examination of most tissues, the sample is fixed, dehydrated by critical point drying, coated with an evaporated gold–carbon film, mounted on an aluminum stub, and placed in the vacuum chamber of the SEM. For mineralized tissues, it is possible to remove all the soft tissues with bleach and then examine the structural features of the mineral. Scanning is accomplished by the same type of raster that scans the electron beam across the face of a television tube. Electrons reflected from the surface ( ) and electrons forced out of the surface ( ) are collected by one or more detectors and reprocessed to form a high-resolution 3D image of a sample surface (see Fig. 1.15). In earlier models of microscopes, images were captured on a high-resolution cathode ray tube (CRT) or photographic plate; modern instruments, however, capture digital images using sensitive detectors and CCD for display on a high-resolution computer monitor. Other detectors can be used to measure X-rays emitted from the surface, cathodoluminescence of molecules in the tissue below the surface, and very-low-energy Auger electrons emitted at the surface.

backscattered electrons secondary electrons

The scanning-transmission electron microscope (STEM) combines features of the TEM and SEM to allow electron-probe X-ray microanalysis. The SEM configuration can be used to produce a transmission image by inserting a grid holder at the specimen level, collecting the transmitted electrons with a detector, and reconstructing the image on a CRT. This latter configuration of a SEM or facilitates the use of the instrument for . Detectors can be fitted to the microscope to collect the Xrays emitted as the beam bombards the section; with appropriate analyzers, a map can be constructed that shows the distribution in the sections of elements with an atomic number above 12 and a concentration sufficient to produce enough X-rays to analyze. Semiquantitative data can also be derived for elements in sufficient concentration. Thus, both the TEM and the SEM can be converted into sophisticated analytical tools in addition to being used as “optical” instruments.

scanning-transmission electron microscope (STEM) electron-probe X-ray microanalysis

Three-dimensional electron microscopy is used for reconstructing entire cells, their connections, and their organelles.

Single 2D images obtained from TEM impose several limitations in interpreting different cellular structures, such as their size, shape, number, and relationship to neighboring structures. However, single 2D images (in serial sections) can be used to recreate 3D models of a structure of interest. Reconstructed 3D models of imaged structures allow researchers to obtain new information on volume, surface area, spatial distribution, and contact area between different structures. In the last decade, the has rapidly developed to include several powerful imaging modalities. In general, 3D reconstruction is possible with both TEM and SEM that allows multiple views of specimens to be collected and analyzed using 3D reconstruction software. Several methods, such as serial section TEM, focused ion beam SEM, serial block-face SEM, image segmentation, and 3D

microscopy (3DEM)

three-dimensional electron

reconstructions of cells and tissues, are currently utilized for obtaining 3D images at the ultrastructural level. 3D reconstruction is especially helpful in , a field that specializes in studying the brain’s structural and functional connections between nerve cells. With the recent imaging of the entire network of synaptic connections within the brain of (fruit fly), mapping of all neural connections in the human brain using 3DEM techniques may be possible in the distant future. The following approaches to 3D visualization at the EM level are frequently used:

connectomics

Drosophila melanogaster

Serial section TEM (SSTEM).

This technique involves sectioning epoxy resin–embedded tissues using an ultramicrotome and collecting the arrays of consecutive aligned sections throughout the specimen. Each section from the collected ribbons is placed on a grid and separately imaged using TEM. The advantage of using SSTEM is that all individual sections can be preserved, archived, and reimaged. Using image analysis software, EM images can be aligned, segmented, and modeled to generate 3D images of selected structures. . This technique takes advantage of dual beam instruments with two imaging columns. The first column is a standard electron column found in SEMs, and the second ion column generates positively charged ions (frequently, liquid metal gallium is used as a source of ions). Both columns have scan coils, which allow the beams of electrons and ions to move across the surface of the imaging sample. As the high-momentum ion beam is moved over the specimen, it mills away (removes) a very thin layer of the specimen. The imaging beam of electrons follows, allowing the SEM detector to capture backscattered and emitted secondary electrons to visualize the block surface between rounds of milling. A series of images are generated that are assembled into 3D images. . The basic principle of this technique is the presence of an ultramicrotome within the vacuum chamber of a SEM that removes thin slices of the embedded specimen. The top of the block face is imaged before

Focused ion beam SEM (FIBSEM)

Serial block-face SEM (SBFSEM)

the surface is shaved away by a diamond knife at a specified depth, revealing a new block face, which is again imaged. This cyclical repetition captures an automatically aligned stack of serial images of tissues sequentially removed from the block. Collected images are then reconstructed using 3D image analysis software. Specific organelles of interest can be segmented out and recreated in 3D (Fig. 1.16).

FIGURE 1.16. Serial block-face scanning electron microscopy (SBFSEM) image of the cell membrane and nuclear envelope. This

image of human embryonic kidney cells growing in culture (HEK-293 cell line) was obtained using SBFSEM. Cells were routinely prepared for SEM, and an epoxy resin block containing the sample was placed in the vacuum chamber containing an ultramicrotome. The top of the block face was imaged by an electron beam before the block face was cut off by the microtome at a thickness of ~20 nm, revealing a new block face that was again imaged. This process was repeated until ~100 images of sequential sections were collected. The outlines the cell of interest for three-dimensional (3D) reconstruction. The black inclusions in the cytoplasm represent gold nanoparticles taken up by cells. The block-imaged face and subsequent serial sections that were scanned and removed are shown. The collected 3D data set was manually segmented by tracing the nuclear envelope shown in panel ( ) and the cell membrane shown in panel ( ). Panel shows a superimposed reconstructed 3D image of the cell membrane and the nucleus within the cell. ×8,000 (panel d). (Courtesy of Dr. Louis J. Maher and Brandon A. Wilbanks, Mayo Clinic, Rochester, MN.)

a.

dashed

line

b blue structure c green structure

d

Image segmentation allows for visualization of structures of interest by assigning a label to every pixel associated with the structure In generating 3D models, image segmentation allows for visualizing selected structures of interest. In this process, individual pixels are assigned to objects, giving them a “label.” Segmentation can be performed manually by a researcher or automatically using computer algorithms. For example, identifying and assigning all pixels related to the nuclear membrane to a “nuclear membrane” label allows the user to tract the pixel distribution throughout serial sections and reconstruct them into a 3D object (Fig. 1.16).

Manual segmentation

is a labor-intensive and time-consuming process. It is often done by tracing selected structures with a computer mouse, pen, or stylus on a drawing tablet or touch-sensitive screen. The user then “paints” labels onto the 3D stack of images. uses image based on user-defined or preexisting criteria. Most commonly, a algorithm is the most

Automatic segmentation segmentation algorithms basic thresholding segmentation

successful in achieving good 3D imaging. During this procedure, the researcher selects a specific threshold value of pixel intensity, and all pixels with an intensity equal to or above the selected intensity value are automatically assigned to an object label. This approach is especially suited to images with highly contrasted and selectively stained structures of interest (i.e., individual neurons with biocytin stain or where osmium tetroxide staining is low in the lumen of T tubules compared with the surrounding cytoplasm of skeletal muscle cells). Automatic segmentation is the fastest method of generating 3D models. However, it often does not work in reconstructions of tissue samples because of the heterogeneous distribution of pixel intensities in the sample. Currently, fully automated segmentation protocols are still in development. More powerful algorithms are being developed that, in the future, will fully automate 3DEM image analysis.

Atomic Force Microscopy

The AFM has emerged as one of the most powerful tools for studying surface topography at molecular and atomic resolution. One newer microscope that has proved most useful for biologic studies is the atomic force microscope (AFM). It is a nonoptical microscope that works in the same way as a fingertip, which touches and feels the skin of our face when we cannot see it. The sensation from the fingertip is processed by our brain, which is able to deduce surface topography of the face while touching it. In the AFM, an ultrasharp, pointed probe, approaching the size of a single atom at the tip, scans the specimen following parallel lines along the -axis, repeating the scan at small intervals along the -axis. The sharp tip is mounted at the end of a highly flexible so that the tip deflects the cantilever as it encounters the “atomic force” on the surface of the specimen (Fig. 1.17). The upper surface of the cantilever is reflective, and a laser beam is directed off the

x y cantilever

cantilever to a diode. This arrangement acts as an “optical lever” because extremely small deflections of the cantilever are greatly magnified on the diode. The AFM can work with the tip of the cantilever touching the sample ( ), or the tip can tap across the surface ( ) much like the cane used by individuals with visual impairment (see Fig. 1.17, insets).

contact mode tapping mode

FIGURE 1.17. Diagram of the atomic force microscope (AFM).

An extremely sharp tip on a cantilever is moved over the surface of a biologic specimen. The feedback mechanism provided by the piezoelectric scanners enables the tip to be maintained at a constant force above the sample surface. The tip extends down from the end of a laser-reflective cantilever. A laser beam is focused onto the cantilever. As the tip scans the surface of the sample, moving up and down with the contour of the surface, the laser beam is deflected off the cantilever into a photodiode. The photodiode measures the changes in laser beam intensities and then converts this information into electrical current. Feedback from the photodiode is processed by a computer as a surface image and also regulates the piezoelectric scanner. In contact mode ( ), the electrostatic or surface tension forces drag the scanning tip over the surface of the sample. In tapping mode ( ), the tip of the cantilever oscillates. The latter mode allows visualization of soft and fragile samples while achieving a high resolution.

left inset right inset

z

As the tip moves up and down in the -axis as it traverses the specimen, the movements are recorded on the diode as movements of the reflected laser beam. A piezoelectric device under the specimen is activated in a sensitive feedback loop with the diode to move the specimen up and down so that the laser beam is centered on the diode. As the tip dips down into a depression, the piezoelectric device moves the specimen up to compensate, and when the tip moves up over an elevation, the device compensates by lowering the specimen. The current to the piezoelectric device is interpreted as the -axis, which along with the - and -axes renders the topography of the specimen at a molecular, and sometimes an atomic, resolution (Fig. 1.18).

x

y

z

FIGURE 1.18. Atomic force microscopic image of a single DNA molecule. This image was obtained in the contact mode in which the

sharp scanning tip “bumps” up and down as it is moved back and forth over the surface of the sample. The sample lies on an ultrasmooth mica surface. An individual molecule of DNA easily produces enough of a bump to be detected. Thickenings along the DNA molecule are produced by proteins bound to the molecule, and these thickenings produce an even larger movement of the scanning tip. The scan field measures 540 by 540 nm. The length of the DNA molecule ranges from 0 to 40 nm. ×185,000. (Courtesy of Dr. Gabriela Bagordo, JPK Instruments AG, Berlin, Germany.)

A major advantage of the AFM for examining biologic specimens is that unlike high-resolution optical instruments

(i.e., TEM or SEM), the specimen does not have to be in a vacuum; it can even be submerged in water. Thus, it is feasible to image living cells and their surrounding environments.

Virtual Microscopy

Virtual microscopy is a digital procedure that is an alternative to the examination of glass slides using a light microscope. Virtual microscopy integrates conventional light microscopy with digital technologies. Using optical image acquisition systems with automatic focus, glass slides are scanned to create 2D digital files that typically are stored on dedicated virtual microscopy servers (Fig. 1.18). The process of scanning involves acquiring images from a glass slide. Different systems acquire images either as tiles or as linear strips that are stitched together to create a virtual slide. The is a digital representation of a glass slide, which can be viewed remotely without a light microscope. Glass slides are commonly digitized in a single focal plane (e.g., 40× objective lens), but they can be captured in multiple focal planes. Many commercially available software packages called provide Internet access to viewers for exploring digital slides on any network device in a manner similar to light microscopy. Virtual microscopes offer new possibilities for specimen viewing and handling that are not available on a standard light microscope. These include the following:

virtual slide

microscopes

virtual

Remote viewing of any digitized slide on any network device (e.g., tablet computers, smartphones) containing a virtual microscopy viewer Seamless progressive zooming in and out (usually ranging from 0.06 to 40×) Switching with ease between very low- and high-power magnifications without altering the field of view or plane of focus

An orientation (navigation) thumbnail image of the whole slide that shows the location of the main screen image on the slide in real time (This orientation image remains present on the screen even while zooming.) A magnified glass thumbnail image that displays a digitally enlarged view of the region correlated to the position of the pointer on the screen Additional features such as drag, rotate, and measuring tools; arrays of color adjustment; and a focus feature to choose between different planes in images captured at multifocal planes From an educational perspective, students using virtual microscopes are able to compare side-by-side images of different tissues and/or the same tissues stained by different stains. An important feature not available on light microscopes is the ability of students or instructors to make personalized annotations on each virtual slide, including freehand drawings as well as typed text. These annotations can be easily saved as overlay files with the virtual microscopy slides. In addition, virtual microscopy facilitates collaborative and team-based learning approaches between multiple students sharing a virtual microscope in a laboratory environment (see Fig. 1.19).

FIGURE 1.19. Virtual microscopy.

Glass slides are scanned using a high-resolution automated slide scanner to create digital files that are stored typically in dedicated virtual microscopy servers. The virtual slide is a digital representation of a glass slide and can be displayed by using a specialized software viewer referred to as a virtual microscope. Virtual slides are distributed over a computer network or the Internet for remote viewing. Note that the virtual slides may be viewed individually or in groups on any mobile device, such as tablet computers or smartphones with virtual microscopy applications.

Virtual microscopy is also utilized in pathology education, research, and remote pathology practice ( ). It can be performed in a virtual environment by sharing virtual slides online among pathology specialists. Telepathology is currently used for many different applications, including histopathology diagnoses that are rendered from a distance. Use of digitalized slides (virtual microscopy) is preferred; however, in some developing countries, an analog imaging is still used in telepathology.

telepathology

METHODS

OVERVIEW OF METHODS USED IN HISTOLOGY Histology (microscopic anatomy)

is the scientific study of microscopic structures of tissues and organs of the body. (for viewing glass slides) and (for viewing digitized microscopic specimens on a computer screen or mobile device) are the most commonly taught methods for examining cells, tissues, and organs in histology courses.

Light microscopy microscopy

TISSUE PREPARATION

virtual

hematoxylin and eosin (H&E)

Routinely prepared -stained sections of -fixed tissue are the specimens most commonly examined for histologic studies with the . The first step in preparation of a tissue sample is , which preserves structure and prevents enzymatic degradation. In the second step, the , cleared, and then or epoxy resins to permit sectioning. In the third step, the on a glass slide and to permit light microscope examination. Specific preparations are required for in which specimens are infiltrated with hydrogels that cause physical expansion of specimens. Steps in specimen preparation for the are similar to that for light microscopy except that they require different fixatives (glutaraldehyde and osmium tetroxide), embedded media (plastic and epoxy resins), and staining dyes (heavy metals).

formalin

light

microscope fixation

specimen is dehydrated embedded in paraffin specimen is mounted stained

microscopy (ExM)

electron microscope (TEM)

expansion

transmission

STAINING PROCEDURES Eosin is charge .

acidic dye

net negative

an (pink) and carries a It reacts with positively charged cationic groups in cells and tissues, particularly amino groups of proteins (eosinophilic structures). acts as a (blue) and carries a . It reacts with negatively charged ionized phosphate groups in nucleic acids (basophilic structures). The stains carbohydrates and carbohydrate-rich molecules a distinctive magenta color. It is used to demonstrate glycogen in cells, mucus in cells and tissues, the basement membrane, and reticular fibers in connective tissue. is based on the specificity of the reaction between an antigen and an antibody that is conjugated either to a fluorescent dye (for light microscopy) or gold particles (for electron microscopy). Both and are used to locate a target antigen in cells and tissues. and procedures are based on of a dye with a particular cell component exhibiting . is a method of localizing mRNA or DNA by hybridizing the sequence of interest to a complementary strand of a nucleotide probe.

Hematoxylin positive charge

basic dye

net

periodic acid–Schiff (PAS) reaction

Immunocytochemistry

direct indirect immunocytochemical methods Histochemical cytochemical specific binding inherent enzymatic activity Hybridization

Fluorescence in situ hybridization (FISH) procedure

utilizes fluorescent dyes combined with nucleotide probes to visualize multiple probes at the same time. This technique is used extensively in genetic testing. makes use of a photographic emulsion placed over a tissue section to localize radioactive material within tissues.

Autoradiography

MICROSCOPY

Correct interpretation of microscopic images is important because organs are 3D, whereas histologic sections are only 2D. is the ability of a microscope lens or optical system to produce separate images of closely positioned objects. The resolving power of a (most commonly used by students and researchers) is about 0.2 μm. In addition to bright-field microscopy, other optical systems include , , , , , and . (TEMs; theoretical resolving power of 0.05 nm) use the interaction of a beam of electrons with a specimen to produce an image. (SEMs; resolving power of 2.5 nm) use electrons reflected or forced out of the specimen surface that are collected by detectors and reprocessed to form an image of a sample surface. (3DEM) is used for the reconstruction of entire structures including cells, their connections, and their organelles. The most frequently used 3DEM methods are , and . (AFMs; resolving power of 50 pm) are nonoptical microscopes that utilize an ultrasharp, pointed probe ( ) that is dragged across the surface of a specimen. The up-and-down movements of the cantilever are recorded and transformed into a graphic image.

Resolving power

field microscope

bright-

phase contrast microscopy dark-field microscopy fluorescence microscopy confocal scanning microscopy ultraviolet microscopy super-resolution microscopy Transmission electron microscopes Scanning electron microscopes

Three-dimensional electron microscopy

serial section TEM (SSTEM), focused ion beam SEM (FIBSEM) serial block-face SEM (SBFSEM) Atomic force microscopes cantilever

2

CELL CYTOPLASM

OVERVIEW OF THE CELL AND CYTOPLASM MEMBRANOUS ORGANELLES

Plasma Membrane Signaling Processes Membrane Transport and Vesicular Transport Endosomes Exosomes Lysosomes Proteasome-Mediated Degradation Rough Endoplasmic Reticulum Smooth Endoplasmic Reticulum Golgi Apparatus Mitochondria Peroxisomes

NONMEMBRANOUS ORGANELLES

Microtubules Actin Filaments Intermediate Filaments Centrioles and Microtubule-Organizing Centers Basal Bodies

INCLUSIONS CYTOPLASMIC MATRIX Folder 2.1 Clinical Correlation: Lysosomal Storage Diseases Folder 2.2 Clinical Correlation: Abnormalities in Microtubules and Filaments Folder 2.3 Clinical Correlation: Abnormal Duplication of Centrioles and Cancer HISTOLOGY

OVERVIEW OF THE CELL AND CYTOPLASM

Cells are the basic structural and functional units of all multicellular organisms. The processes we normally associate with the daily activities of organisms—protection, ingestion, digestion, absorption of metabolites, elimination of wastes, movement, reproduction, and even death—are all reflections of similar processes occurring within each of the billions of cells that constitute the human body. To a large extent, cells of different types use similar mechanisms to synthesize protein, transform energy, and move essential substances into the cell. They use the same kinds of molecules to engage in contraction, and they duplicate their genetic material in the same manner.

Specific functions are identified with specific structural components and domains within the cell. Some cells develop one or more of these functions to such a degree of specialization that they are identified by the function and the cell structures associated with them. For example, although all cells contain contractile filamentous proteins, some cells, such as , contain large amounts of these proteins in specific arrays. This allows them to carry out their specialized function of contraction at both the cellular and tissue level. The specialized activity or function of a cell may be reflected not only by the presence of a larger amount of the specific structural component performing the activity but also by the shape of the cell, its organization with respect to other similar cells, and its products (Fig. 2.1).

muscle cells

FIGURE 2.1. Histologic features of different cell types.

These three photomicrographs show different types of cells in three different organs of the body. The distinguishing features include size, shape, orientation, and cytoplasmic contents that can be related to each cell’s specialized activity or function. Epithelial cells in the kidney. Note several shapes of epithelial cells: columnar cells with well-defined borders in the collecting duct ( ), squamous cells in the thin segment ( ) of the nephron, and even more flattened cells lining blood vessels, the vasa recta ( ) in the kidney. ×380. Dorsal root ganglion cells. Note the large size of these nerve cell bodies and the large, pale (euchromatic) nuclei ( ) with distinct nucleoli. Each ganglion cell is surrounded by flattened satellite cells ( ). The size of the ganglion cell and the presence of a euchromatic nucleus, prominent nucleolus, and Nissl bodies (rough endoplasmic reticulum visible as darker granules within the cytoplasm) reflect the extensive synthetic activity required to maintain the exceedingly

TS

a.

b.

CD

S

N

VR

c.

long processes (axons) of these cells. ×380. Smooth muscle cells of the small intestine. Note that these cells are typically elongated, fusiform-shaped, and organized in a parallel array. The nuclei are also elongated to conform to the general shape of the cell. ×380.

Cells can be divided into two major compartments: the cytoplasm and the nucleus. In general, the cytoplasm is the part of the cell located outside the nucleus. The cytoplasm contains organelles (“little organs”), cytoskeleton (made of polymerized proteins that form microtubules, intermediate filaments, and actin filaments), and inclusions suspended in an aqueous gel called the cytoplasmic matrix. The matrix consists of a variety of solutes, including inorganic ions (Na+ , K+ , Ca2+ ) and organic molecules such as intermediate metabolites, carbohydrates, lipids, proteins, and RNAs. The cell controls the concentration of solutes within the matrix, which influences the rate of metabolic activity within the cytoplasmic compartment. The nucleus is the largest organelle within the cell and contains the genome along with the enzymes necessary for DNA replication and RNA transcription. The cytoplasm and nucleus not only play distinct functional roles but also work in concert to maintain the cell’s viability. The structure and function of the nucleus is discussed in Chapter 3, The Cell Nucleus, page 87.

Organelles are described as membranous (membrane-limited) or nonmembranous.

Organelles include the membrane systems of the cell and the membrane-limited compartments that perform the metabolic, synthetic, energy-requiring, and energy-generating functions of the cell as well as nonmembranous structural components. All cells have the same basic set of intracellular organelles, which can be classified into two groups: (1) with plasma membranes that separate the internal environment of the organelle from the cytoplasm and (2) without plasma membranes. The membranes of membranous organelles form vesicular, tubular, and other structural patterns within the cytoplasm that may be convoluted (as in smooth endoplasmic reticulum) or plicated (as in the inner mitochondrial membrane). These membrane configurations greatly increase the surface area on which essential physiologic and biochemical reactions take place. The spaces enclosed by the organelles’ membranes constitute the in which substrates, products, and other substances are segregated or concentrated. In addition, each type of organelle contains a set of unique proteins; in membranous organelles, these proteins are either incorporated into their membranes or sequestered within their lumens. For example, the enzymes of lysosomes are separated by a specific enzyme-resistant membrane from the cytoplasmic matrix because their hydrolytic activity would be detrimental to the cell. In nonmembranous organelles, the unique proteins usually self-assemble into polymers that form the structural elements of the . Besides organelles, the cytoplasm contains , structures that are not usually surrounded by a plasma membrane. They consist of such diverse materials as crystals, pigment granules, lipids, glycogen, and other stored waste products (for details, see pages 81-82). Membranous organelles include the following:

organelles

nonmembranous organelles

membranous

intracellular

microcompartments

inclusions

cytoskeleton

Plasma (cell) membrane, a lipid bilayer that forms the cell boundary as well as the boundaries of many organelles within the cell Rough endoplasmic reticulum (rER), a region of endoplasmic reticulum associated with ribosomes and the site of protein synthesis and modification of newly synthesized proteins Smooth endoplasmic reticulum (sER), a region of endoplasmic reticulum involved in

detoxifying xenobiotics (foreign drugs or chemicals) and synthesis of lipids and steroids but not associated with ribosomes , a membranous organelle composed of multiple flattened cisternae responsible for modifying, sorting, and packaging proteins and lipids for intracellular or extracellular transport , membrane-bounded compartments interposed within endocytic pathways that have the major function of sorting proteins delivered to them via endocytic vesicles and redirecting them to different cellular compartments for their final destination

Golgi apparatus Endosomes

Exosomes,

small (average 100 nm in diameter), endosome-derived, membrane-bound vesicles that originate within the lumen of . They are released via exocytosis into the extracellular space. Exosomes carry nucleic acids, proteins, lipids, and metabolites secreted by cells to the extracellular space. They act as mediators for near- and long-distance communication between cells. , small organelles containing digestive enzymes that are formed from endosomes by targeted delivery of unique lysosomal membrane proteins and lysosomal enzymes —including , , and —that are involved in both endocytosis and exocytosis and vary in shape and the material that they transport , organelles that provide most of the energy to the cell by producing adenosine triphosphate (ATP) in the process of oxidative phosphorylation , small organelles involved in an oxidative type of metabolism. They are involved in the degradation of fatty acids and production and degradation of reactive oxygen intermediates.

multivesicular bodies

Lysosomes Transport vesicles vesicles Mitochondria Peroxisomes

pinocytic vesicles endocytic vesicles

coated

Nonmembranous organelles include the following:

Microtubules, which together with actin and intermediate filaments form elements of the cytoskeleton and continuously elongate (by adding tubulin dimers) and shorten (by removing tubulin dimers), a property referred to as dynamic instability Filaments, which are also part of the cytoskeleton and can be classified into two groups —actin filaments, which are flexible chains of actin molecules, and intermediate filaments, which are rope-like fibers formed from a variety of proteins—provide tensile strength to withstand tension and confer resistance to shearing forces. Centrioles, or short, paired cylindrical structures found in the center of the microtubule-organizing center (MTOC) or centrosome and whose derivatives give rise to basal bodies of cilia Ribosomes, structures essential for protein synthesis and composed of ribosomal RNA (rRNA) and ribosomal proteins (including proteins attached to membranes of the rER and proteins free in the cytoplasm) , which are protein complexes that enzymatically degrade damaged and unnecessary proteins into small polypeptides and amino acids

Proteasomes

An outline of the key features of cellular organelles and inclusions is provided in Table 2.1. The normal function and related pathologies of the organelles are summarized in Table 2.2.

of Organelles and Cytoplasmic Inclusions: A Key to Light Microscopic TABLE 2.1 Review and Electron Microscopic Identification Organelle or Inclusion Nucleus

Size (μm) Light Microscopic Features

Electron Microscopic Features

3–10

Largest organelle within the cell with distinct boundary Often visible nucleoli and chromatin pattern regions

Nucleolus

Surrounded by two membranes (nuclear envelope) containing nuclear pore complexes and perinuclear cisternal space Regions with condensed and diffused chromatin pattern (heterochromatin and euchromatin)

1–2

Roughly circular, basophilic region within the nucleus Visible in living cells throughout interphase with interference microscopy

Dense, nonmembranous structure containing fibrillar and granular material

Organelle or Inclusion Plasma membrane

Size (μm) Light Microscopic Features

Electron Microscopic Features

0.008–0.01

Not visible

External membrane and membranes surrounding membranous organelles of cell; two inner and outer electron-dense layers separated by intermediate electron-lucent layer

Area ~5–10

Often observed as basophilic region of cytoplasm referred to as

Flattened sheets, sacs, and tubes of membranes with attached ribosomes

Throughout cytoplasm

Golgi apparatus

Not visible Cytoplasm in region of sER may exhibit distinct eosinophilia

Flattened sheets, sacs, and tubes of membranes attached ribosomes

Area ~5–10

Stack of flattened membrane sheets, often adjacent to one side of nucleus

Secretory vesicles

Sometimes observed as “negative staining” region Appears as network in heavy metal –stained preparations Visible in living cells with interference microscopy

0.05–1.0

Observed only when vesicles are very large (e.g., zymogen granules in pancreas)

Mitochondria

Many relatively small, membranebounded vesicles of uniform diameter, often polarized on one side of cell

0.2–7

Sometimes observed in favorable situations (e.g., liver or nerve cells) as miniscule, dark dots; visible in living cells stained with vital dyes (e.g., Janus Green)

Endosomes

Two-membrane system: outer membrane and inner membrane arranged in numerous folds (cristae) In steroid-producing cells, inner membrane arranged in tubular cristae

0.02–0.5

Not visible

Exosomes

Tubulovesicular structures with subdivided lumen containing electron-lucent material or other smaller vesicles

0.03–0.1

Not visible

Intraluminal vesicles inside the multivesicular bodies (MVBs). They are released into extracellular space by fusion of MVBs with the cell membrane.

0.2–0.5

Visible only after special enzyme histochemical staining

Membrane-bounded vesicles, often electron dense

0.1–0.5

Visible only after special enzyme histochemical staining

Membrane-bounded vesicles, often with electron-dense crystalloid inclusions

0.006–0.025

Only observed when organized into large structures (e.g., muscle fibrils)

Long, linear staining pattern with width and features characteristic of each filament type

0.025

Not visible

Minute dark dots, often associated with the rER

0.015

Not visible

Difficult to distinguish from other matrix proteins

0.010–0.040

Observed as a “purple haze” region of cytoplasm metachromasia with toluidine blue–stained specimen

Nonmembranous, extremely dense grape-like inclusions

0.2–5, up to 80

Readily visible when extremely large (e.g., in adipocytes) Appear as large empty holes in section (lipid itself is usually removed by embedding solvents)

Nonmembranous inclusions Generally appear as a void in the section

rER sER

Lysosomes Peroxisomes Cytoskeletal elements Ribosomes Proteasomes Glycogen Lipid droplets

ergastoplasm

without

rER, rough endoplasmic reticulum; sER, smooth endoplasmic reticulum.

TABLE 2.2 Organelles and Cytoplasmic Inclusions: Functions and Pathologies Organelle or Inclusion Nucleus Nucleolus Plasma membrane rER

sER Golgi apparatus Secretory vesicles Mitochondria Endosomes Exosomes Lysosomes Peroxisomes Cytoskeletal elements Ribosomes

Functions

Pathologies

Storage and use of genome

Inherited genetic diseases; environmentally induced mutations

Synthesis of rRNA and partial assembly of ribosomal subunits Involved in regulation of cell cycle

Werner syndrome (premature aging disease) Malfunctions of cell cycle leading to cancerogenesis

Ion and nutrient transport Recognition of environmental signal Cell-to-cell and cell-toextracellular matrix adhesions

Cystic fibrosis Intestinal malabsorption syndromes Lactose intolerance

Binds ribosomes engaged in translating mRNA for proteins destined for secretion or for membrane insertion Also involved in chemical modifications of proteins and membrane lipid synthesis

Pseudoachondroplasia Calcium pyrophosphate dihydrate crystal deposition disease (CPPD disease; pseudogout)

Involved in lipid and steroid metabolism

Hepatic endoplasmic reticular storage disease

Chemical modification of proteins Sorting and packaging of molecules for secretion or transport to other organelles

I-cell disease Polycystic kidney disease

Transport and storage of secreted proteins to plasma membrane

Lewy bodies of Parkinson disease Proinsulin diabetes

Aerobic energy supply (oxidative phosphorylation, ATP) Initiation of apoptosis

Mitochondrial myopathies such as MERRF syndrome, MELAS syndrome, Kearns–Sayre syndrome, and Leber hereditary optic atrophy

Transport of endocytosed material Biogenesis of lysosomes

M-6-P receptor deficiency (I-cell disease)

Transport vehicles for intercellular communication and material exchange between cells

Depending on their transported cargo, they can promote neoplasia, tumor growth, metastasis formation, and resistance to therapy.

Digestion of macromolecules

Lysosomal storage diseases (see Folder 2.1, Clinical Correlation: Lysosomal Storage Diseases)

Oxidative digestion (e.g., fatty acids), lipid biosynthesis, metabolism of reactive oxygen intermediates

Zellweger spectrum diseases (peroxisomal biogenesis disorders): Zellweger syndrome, Refsum disease, and X-linked adrenoleukodystrophy

Various functions, including cell motility, cell adhesions, and intracellular and extracellular transport Maintenance of cellular skeleton

Immotile cilia syndrome, Alzheimer disease, epidermolysis bullosa

Synthesis of protein by translating protein-coding sequence from mRNA

Ribosomal dysfunction in Alzheimer disease; Diamond–Blackfan anemia Many antibiotics act selectively on bacterial ribosomes: for example, tetracyclines, aminoglycosides (gentamicin, streptomycin).

Organelle or Inclusion Proteasomes Glycogen Lipid droplets

Functions

Pathologies

Degradation of unnecessary and damaged proteins that are labeled for destruction with ubiquitin

Diseases characterized by cytoplasmic accumulation of misfolded proteins: Parkinson disease, Alzheimer disease, Angelman syndrome, inclusion body myopathies

Short-term storage of glucose in the form of branched polymer Found in liver, skeletal muscle, and adipose tissue

Several known glycogen-storage diseases, including major groups of hepatic–hypoglycemic and muscle-energy pathophysiologies

Storage of esterified forms of fatty acids as high-energy storage molecules

Lipid storage diseases such as Gaucher and Niemann–Pick disease, liver cirrhosis

ATP, adenosine triphosphate; MELAS, mitochondrial encephalopathy, lactic acidosis, and stroke-like episodes; MERRF, myoclonic epilepsy with ragged red fiber; mRNA, messenger RNA; rER, rough endoplasmic reticulum; ROIs, reactive oxygen intermediates; rRNA, ribosomal RNA; sER, smooth endoplasmic reticulum.

MEMBRANOUS ORGANELLES Plasma Membrane

The plasma membrane is a lipid-bilayered structure visible with transmission electron microscopy. The plasma membrane (cell membrane, plasmalemma) is a dynamic structure that actively participates in many physiologic and biochemical activities essential to cell function and survival. When the plasma membrane is properly fixed, sectioned, stained, and viewed in cross section with the transmission electron microscope (TEM), it appears as two electrondense layers separated by an intermediate, electron-lucent (nonstaining) layer (Fig. 2.2). The total thickness of the plasma membrane is about 8–10 nm.

FIGURE 2.2. Electron micrograph of microvilli on the apical surface of an absorptive cell.

This electron micrograph shows the apical portion of absorptive cells with microvilli. Note that at this magnification, the plasma membrane displays its characteristic appearance, showing two electrondense lines separated by an electron-lucent intermediate layer. The glycoproteins of the glycocalyx can be seen extending from the tips of the microvilli into the lumen. The relationship between the outer plasma membrane leaflet and the glycocalyx is particularly well demonstrated. Glycoproteins of the glycocalyx include terminal digestive enzymes such as dipeptidases and disaccharidases. ×100,000. (Courtesy of Dr. Ray C. Henrikson.)

The plasma membrane is composed of an amphipathic lipid layer containing embedded integral membrane proteins with peripheral membrane proteins attached to its surfaces. The current interpretation of the molecular organization of the plasma membrane is referred to as the modified fluid–mosaic model (Fig. 2.3). The membrane consists primarily of phospholipid, cholesterol, and protein molecules. The lipid molecules form a lipid bilayer

with an amphipathic character (it is both hydrophobic and hydrophilic). The fatty acid chains of the lipid molecules face each other, making the inner portion of the membrane (i.e., having no affinity for water). The surfaces of the membrane are formed by the polar head groups of the lipid molecules, thereby making the surfaces (i.e., they have an affinity for water). Lipids are distributed asymmetrically between the

hydrophobic

hydrophilic

lipid bilayer,

inner and outer leaflets of the among different biologic membranes.

and their composition varies considerably

FIGURE 2.3. Diagram of a plasma membrane showing the modified fluid–mosaic model.

The plasma membrane is a lipid bilayer consisting primarily of phospholipid molecules, cholesterol, and protein molecules. The hydrophobic fatty acid chains of phospholipids face each other to form the inner portion of the membrane, whereas the hydrophilic polar heads of the phospholipids form the extracellular and intracellular surfaces of the membrane. Cholesterol molecules are incorporated within the gaps between phospholipids equally on both sides of the membrane. Note the elevated area of the lipid raft that is characterized by a high concentration of glycosphingolipids and cholesterol. It contains large numbers of integral and peripheral membrane proteins. The raft protrudes above the level of asymmetrically distributed phospholipids in the membrane bilayer ( ). Carbohydrate chains attach to both integral and peripheral membrane proteins to form glycoproteins as well as to polar phospholipid heads to form glycolipids.

indicated by the different

colors of the phospholipid heads

In most plasma membranes, protein molecules constitute approximately half of the total membrane mass. Most of the proteins are embedded within the lipid bilayer or pass through the lipid bilayer completely. These proteins are called . The other types of protein— —are not embedded within the lipid bilayer. They are associated with the plasma membrane by strong ionic interactions, mainly with integral proteins on both the extracellular and intracellular surfaces of the membrane (see Fig. 2.3). In addition, on the extracellular surface of the plasma membrane, carbohydrates may be attached to proteins, thereby forming , or to lipids of the bilayer, thereby forming . These surface molecules constitute a layer at the surface of the cell, referred to as the or (see Fig. 2.2). They help establish extracellular microenvironments at the membrane surface that have specific functions in metabolism, cell recognition, and cell association and serve as receptor sites for hormones.

peripheral membrane proteins glycolipids

cell coat

integral membrane proteins

glycoproteins glycocalyx

Microdomains of the plasma membrane, known as lipid rafts, control the movement and distribution of proteins within the lipid bilayer.

The fluidity of the plasma membrane is not revealed in static electron micrographs. Experiments reveal that the membrane behaves as though it were a two-dimensional lipid fluid. For many years, it was thought that integral membrane proteins moved freely within the plane of the membrane; this movement was compared to the movement of icebergs floating in the ocean (see Fig. 2.3). However, the distribution and movement of proteins within the lipid bilayer is not as random as once thought. The plasma membrane appears to be patchy with localized regions that are distinct in structure and function and vary in thickness and composition. These localized regions contain high concentrations of and and are called . Because of the high concentration of cholesterol and the presence of longer, highly saturated fatty acid chains, the lipid raft area is thicker and exhibits less fluidity than the surrounding plasma membrane (Fig. 2.4).

glycosphingolipids

lipid rafts

cholesterol

Cholesterol is the dynamic “glue” that holds the raft together; its removal from the raft results in dispersal of raft-associated lipids and proteins.

FIGURE 2.4. Image of lipid rafts obtained with tapping-mode atomic force microscopy (AFM).

This image shows a 5-nm-thick lipid bilayer spread on a mica support. The bilayer is composed of dioleoyl phosphatidylcholine (dioleoyl-PC), sphingomyelin, and cholesterol. Sphingomyelin and cholesterol together form lipid rafts represented on the image by the ; the are the nonraft background of the bilayer. Because the sphingomyelin molecules are longer than the dioleoyl-PC molecules, the rafts protrude from the nonraft background by about 0.8 nm, and the AFM is sensitive enough to detect this protrusion. The black regions represent the mica support. The image also shows molecules of the toxin VacA ( ), which preferentially bind to protein receptors on the raft domains. The area depicted in this image is 800 nm 2. (Courtesy of Drs. Nicholas A. Geisse, Timothy L. Cover, Robert M. Henderson, and J. Michael Edwardson.)

pink areas

areas

Helicobacter pylori

blue-purple

white particles

In general, there are two types of lipid rafts:

Planar lipid rafts

flotillins

contain a family of 47-kDa proteins known as as well as specific lipids and cholesterol. Flotillins are regarded as the molecular markers of lipid rafts and are considered to be scaffolding proteins. They also participate in the recruitment of specific membrane proteins into the rafts and work as active partners in various signaling pathways. , or (“little caves”), represent small (50–100 nm in diameter), flask-shaped invaginations of the plasma membrane containing 18- to 24-kDa integral membrane proteins called . Oligomerization of caveolins produces the caveolin scaffold essential to creating invaginations within the plasma membrane. These proteins also bind to cholesterol and a variety of proteins involved in signal transduction. Caveolae are prominent in (see page 370), where they contain various Ca2+ channels, Na+ /Ca2+ exchangers, and G protein–coupled receptors involved in ligandmediated regulation of intracellular Ca2+ levels.

Caveolar rafts

caveolae caveolins

smooth muscle cells

Lipid rafts contain a variety of integral and peripheral membrane proteins involved in cell signaling. They can be viewed as “ ” floating in the ocean of lipids. Each individual raft is equipped with all of the necessary elements (receptors, coupling factors, effector enzymes, and substrates) to receive and convey specific signals. Signal transduction in lipid rafts occurs more rapidly and efficiently because of the close proximity of interacting proteins. In addition, different signaling rafts allow for the separation of specific signaling molecules from each other. In bacterial and viral infections, the initial contact of the microorganism with the cell occurs at the lipid raft. For example, some bacteria (e.g., , ) hijack the rafts with their signaling mechanism and use them to support their own entry into the cell. Many bacteria use rafts to avoid phagocytosis and subsequent destruction in lysosomes. In other cases, invading bacteria use raft-associated receptors to generate vacuoles made of raft components. These vacuoles are then used to transport bacteria into the cell without the risk of being detected by phagocytic compartments.

signaling platforms

Shigella flexneri Salmonella

typhimurium

Integral membrane proteins can be visualized with the freeze fracture tissue preparation technique. The existence of proteins within the substance of the plasma membrane (i.e., integral proteins) was confirmed by the preparation technique known as freeze fracture. When tissue is prepared for electron microscopy (EM) by the freeze fracture process (Fig. 2.5a), membranes typically split or cleave along the hydrophobic plane (i.e., between the two lipid layers) to expose two interior faces of the membrane, an E-face and a P-face (Fig. 2.5b). For details on tissue preparation using freeze fracture technique, see Chapter 1, Methods, page 22.

FIGURE 2.5. Freeze fracture examination of the plasma membrane. a. View of the plasma membrane seen on edge, with arrow indicating the preferential plane of splitting of the lipid bilayer through the hydrophobic portion of the membrane. When the membrane splits, some proteins are carried with the outer leaflet, although most are retained within the inner leaflet. b. View of the plasma membrane

with the leaflets separating along the cleavage plane. The surfaces of the cleaved membrane are coated, forming replicas; the replicas are separated from the tissue and examined with the transmission electron microscope (TEM). Proteins appear as bumps. The replica of the inner leaflet is called the ; it is backed by cytoplasm ( rotoplasm). A view of the outer leaflet is called the ; it is backed by the xtracellular space. Electron micrograph of a freeze fracture replica shows the E-face of the membrane of one epithelial cell and the P-face of the membrane of the adjoining cell. The cleavage plane has jumped from the membrane of one cell to the membrane of the other cell, as indicated by the clear space (intercellular space) across the middle of the figure.

E-face

P-face

e

p

c.

Note the paucity of particles in the E-face compared with the P-face, from which the majority of the integral membrane proteins project. (Courtesy of Dr. Giuseppina d’Elia Raviola.)

E-face p

P-face

e

The is backed by the xtracellular space, whereas the is backed by the cytoplasm ( rotoplasm). The numerous particles seen on the E- and P-faces with the TEM represent the integral proteins of the membrane. Usually, the P-face displays more particles, thus more protein, than the E-face (Fig. 2.5c).

Integral membrane proteins have important functions in cell metabolism, regulation, integration, and cell signaling.

Six broad categories of membrane proteins have been defined in terms of their function: pumps, channels, receptors, linkers, enzymes, and structural proteins (Fig. 2.6). These categories are not mutually exclusive (e.g., a structural membrane protein may simultaneously serve as a receptor, an enzyme, a pump, or any combination of these functions):

FIGURE 2.6. Different functions of integral membrane proteins.

The six major categories of integral membrane proteins are shown in this diagram: pumps, channels, receptors, linkers, enzymes, and structural proteins. These categories are not mutually exclusive. A structural membrane protein involved in cell-to-cell junctions might simultaneously serve as a receptor, enzyme, linker, or a combination of these functions.

Pumps transport certain ions, such as Na , actively across membranes. Pumps also transport +

metabolic precursors of macromolecules, such as amino acids and sugars, across membranes, either by themselves or linked to the Na+ pump. allow the passage of small ions, molecules, and water across the plasma membrane in either direction (i.e., passive diffusion). Gap junctions formed by aligned channels in the membranes of adjacent cells permit passage of ions and small molecules involved in signaling pathways from the cytoplasm of one cell to the cytoplasm of adjacent cells. allow recognition and localized binding of ligands (molecules that bind to the extracellular surface of the plasma membrane) in processes such as hormonal stimulation, coated vesicle endocytosis, and antibody reactions. Receptors that bind to signaling molecules transmit the signal through a sequence of molecular switches (i.e., second messengers) to the cell’s internal signaling pathways, thereby initiating a physiologic response. anchor the intracellular cytoskeleton to the extracellular matrix. Examples of linker proteins include the family of integrins that link cytoplasmic actin filaments to an extracellular matrix protein (fibronectin). have a variety of roles. ATPases have specific roles in ion pumping: ATP synthase is the major protein of the inner mitochondrial membrane, and digestive enzymes such as disaccharidases and dipeptidases are integral membrane proteins. are visualized by the freeze fracture method, especially where they form junctions with neighboring cells. Often, certain proteins and lipids are concentrated

Channels

Receptor proteins Linker proteins Enzymes

Structural proteins

in localized regions of the plasma membrane to carry out specific functions. Examples of such regions can be recognized in polarized cells such as epithelial cells.

Integral membrane proteins move within the lipid bilayer of the membrane. Particles bound to the membrane can move on the surface of a cell; even integral membrane proteins, such as enzymes, may move from one cell surface to another (e.g., from apical to lateral) when barriers to flow, such as cell junctions, are disrupted. The fluidity of the membrane is a function of the types of phospholipids in the membrane and variations in their local concentrations. As previously mentioned, lipid rafts containing integral membrane proteins may move to a different region of the plasma membrane. The movement of an integral protein anchored on a lipid raft makes signaling more precise and prevents nonspecific interactions. The lateral migration of proteins is often limited by physical connections between membrane proteins and intracellular or extracellular structures. Such connections may exist among proteins associated with cytoskeletal elements and portions of the membrane proteins that extend into the adjacent cytoplasm, the cytoplasmic domains of membrane proteins, and peripheral proteins associated with the extracellular matrix and the integral membrane proteins that extend from the cell surface (i.e., the extracellular domain). Through these connections, proteins can be localized or restricted to specialized regions of the plasma membrane or can act as transmembrane linkers between intracellular and extracellular filaments (see the next section).

The plasma membrane undergoes continuous remodeling.

The cell membrane and internal plasma membranes of organelles are continually remodeling. Video microscopy reveals that the plasma membrane undergoes bulging, invagination, budding, tubulation, ruffling, fusion, and fission. These processes are essential for virtually all the major functions of the cell, including synthesis and degradation of molecules, endocytosis, exocytosis, signaling, immunologic defense mechanisms, cell division, and migration. In general, there are two types of membrane remodeling:

excludes cytoplasm endocytic vesicles

Remodeling that from the lumen of forming vesicles or tubules, such as during the formation of (Fig. 2.7). This process involves proteins residing on the inner (cytoplasmic) surface of the membrane (i.e., clathrin, caveolins, COP-I and COP-II, and others) that help change the shape of the plasma membrane into vesicle-like invaginations (see page 41). The family of membrane scission proteins is essential to mediate the liberation of the vesicles from the plasma membrane. Similar invaginations can be found in skeletal muscle cells, where are connected to the external cell environment. In smooth muscle cells, and endocytic vesicles help regulate Ca2+ homeostasis.

dynamin

T tubules caveolae

FIGURE 2.7. Remodeling process of the plasma membrane. a.

Examples of plasma membrane remodeling that excludes cytoplasm from the lumen of forming vesicles (i.e., endocytic vesicles or caveolae) are shown here. This process involves proteins such as clathrin, caveolins, COP-I, and COP-II that help in the formation of coated vesicles. The dynamin family of membrane scission proteins is essential to liberate the vesicles from the plasma membrane. Tubular invaginations can also be found in skeletal muscle cells, where the lumen of the T-tubule is connected to the external cell environment. Examples of plasma membrane remodeling that includes cytoplasm within the newly formed structures are shown here. This process occurs in budding vesicles from cell membranes or intracellular structures. The liberation of vesicles or tubular structures filled with cytoplasm is controlled by endosomal sorting complex required for transport (ESCRT) complexes of proteins. Note that the separation of membranes mediated by ESCRT occurs from the inside surface of the membrane contiguous with the cytoplasm, whereas vesicles and tubules that exclude cytoplasm are liberated from the plasma membrane by the dynamin family of membrane scission proteins.

b.

includes cytoplasm viral particles

Remodeling that within the newly formed structures. This process occurs when cytoplasmic-embedded bud from the cell membrane (Fig. 2.8). As the cell membrane envelops the viral particles and surrounds them with cytoplasm, the connection of this forming vesicle to the cell membrane narrows. The membrane constricts until the budding vesicles are connected to the cell by only a thin, cytoplasm-filled stalk (membrane neck) that must be severed to liberate the vesicle. Formation of vesicles containing viral particles can also occur inside the cell from intracellular membranes such as rER, Golgi, or membrane-bounded replication complexes for the virus (Fig. 2.8). Remodeling of the membrane and the liberation of vesicles or scission of tubules filled with cytoplasm are controlled by complexes of proteins. ESCRT complexes also participate in the formation of , , , , , and . In addition, cell division requires remodeling of the plasma membrane during cytoplasmic separation ( ) into two daughter cells. The separation of membranes mediated by ESCRT occurs from the surface of the membrane that is contiguous with the inside of the cytoplasm-filled membrane neck (see Fig. 2.7). This mechanism is opposite to that of the dynamin family of membrane scission proteins, which cleaves the membrane neck by constricting it from the outside (see Fig. 2.7).

endosomal sorting complex required for transport (ESCRT) multivesicular bodies (MVBs) exosomes microvesicles apoptotic bodies resealing of the post-mitotic nuclear envelope closure of autophagosomes cytokinetic abscission

FIGURE 2.8. Budding vesicles containing viral particles.

This electron micrograph shows a kidney epithelial cell in culture derived from the African green monkey cell line (Vero E6) infected by a coronavirus (severe acute respiratory syndrome [SARS]-CoV). Vero E6 cells are susceptible to virus infection because they have a deficiency in the interferon gene cluster and cyclin-dependent kinase inhibitor genes; therefore, they do not secrete α or β interferon. Three to five days after inoculation with the SARS-CoV virus, Vero E6 cells were harvested, inactivated by γ-irradiation, and processed for standard EM examination. Arrowheads indicate budding vesicles into the cisternae of rough endoplasmic reticulum or Golgi apparatus that contain viral particles (varions). These cisternae represent cellular compartments in which vesicles containing viral particles are stored within the cytoplasm before the entire compartment migrates to the cell surface to release its contents into the extracellular space. The area labeled represents viral intracellular inclusions of the viral nucleocapsid. The large cisternae labeled represent double-membrane vesicles that serve as replication complexes for this virus. ×55,000. (Courtesy of Dr. Cynthia Goldsmith, Centers for Disease Control and Prevention, Atlanta, Georgia).

VNC

Cell injury

RC

often manifests as morphologic changes in the cell’s plasma membrane that result in the formation of . These are dynamic cell protrusions of the plasma membrane that are commonly observed in acute cell injury, in dividing and dying cells, and during cell movement. Blebbing is caused by the detachment of the plasma membrane from underlying actin filaments of the cell cytoskeleton. that act on actin filaments such as phalloidin and cytochalasin B cause extensive membrane blebbing. In addition, in such as human immunodeficiency virus (HIV), Ebola, and human T-lymphotropic virus, the ESCRT complex is “hijacked” during virus budding at the surface of infected host cells, where they catalyze the scission of the membrane stalk that connects the budding virus to the host cell.

plasma membrane blebs

viral infections

Signaling Processes

Cytoskeletal toxins

Cell signaling involves integral membrane proteins such as cell surface receptors, cell surface channels, and molecules released from exosomes. Cell signaling is the process by which extracellular stimuli are received, processed, and

conveyed by the cell to regulate its own physiologic responses. A single cell may receive many different signals at the same time, and it needs to integrate all information into a unified action plan. Signaling processes often are involved in the regulation of gene expression, exocytosis, endocytosis, differentiation, cell growth and death, cytoskeletal reorganization, movement, contraction, and/or cell relaxation. Individual cells also send out signaling molecules to other cells both near (e.g., neurotransmitters in nerve synapses) and far away (e.g., hormones acting on distant cells). are mechanisms by which cells respond to the external environment. They are hierarchical cascades of molecular events that mediate tissue and cell specificity, allow for amplification and modulation of the signal, and are involved in biochemical and physiologic regulation. They are initiated by external (also referred to as or ) that can be soluble, act locally (autocrine or paracrine control as discussed in Chapter 21, Endocrine Organs, pages 816818), or be transmitted to cellular targets via blood vasculature (endocrine signaling). They can also be insoluble, tethered to cell membranes, or localized in the extracellular matrix. Signaling molecules in sensory systems often are of exogenous origin (i.e., odorants, mechanical signals, vibration, light). The majority of signaling pathways are initiated by the binding of primary messengers to specific receptors, which exist in an inactive state in the absence of ligands. Signals from receptors are conveyed to target molecules inside the cell by the . Receptors are typically classified into three groups, which are discussed in earlier sections and later chapters: (page 34), , and (see Chapter 21, Endocrine Organs, pages 817-818). The latter group includes members of the G-protein–linked receptor family (see Chapter 12, Nerve Tissue, page 400), the enzyme-linked (catalytic) receptor family (see Chapter 21, Endocrine Organs, page 816), and the integrin family of cell-to-extracellular matrix receptors (see Chapter 5, Epithelial Tissue, page 146). represents an intercellular communication pathway in which functional proteins, metabolites, and nucleic acids are delivered to recipient cells using exosomes as transfer vehicles. Exosomes represent very small, membrane-bound secreted into extracellular space by virtually every prokaryotic and eukaryotic cell. The detailed structure and function of exosomes are discussed on pages 47-48. Exosomes are present in the blood and all body fluids and transfer messages and cargo molecules from the parent cells to the target destination via autocrine, paracrine, and endocrine mechanisms. Exosomes with their cargo molecules interact with a target cell, fusing either directly with the plasma membrane of recipient cells (mediated by SNAREs and Rab interactions) or using receptor–ligand-mediated interaction pathways, that is, phagocytosis, macropinocytosis, or micropinocytosis (pages 39-40). Exosomes then release their contents directly into the cytoplasm of the target cell, initiating downstream signaling cascades. After delivering their cargo, exosomes undergo degradation in the cytoplasm of the recipient cell in a typical endosomal pathway.

Signal transduction pathways

primary messengers

surface receptors

signaling molecules

ligands

second messenger system intracellular receptors cell

channel proteins

Exosomal signaling

cargo vesicles

Activation of cell surface receptors leads to posttranslational modifications, which contribute to the amplification of the signal. There are several posttranslational modifications of intracellular proteins that contribute to the amplification of a signal that the cell receives. These include the following:

Phosphorylation (addition of phosphate groups—PO ) Glycosylation (addition of a diverse selection of sugar moieties) Acetylation (attaching acetyl functional groups—COCH ) Methylation (adding methyl groups—CH ) Nitrosylation (reaction of nitric oxide [NO] with protein-free cysteine residues) 4

3−

3

3

Ubiquitination (attaching ubiquitin protein) SUMOylation (addition of small ubiquitin-related modifier [SUMO] protein) Common to the activation of cell surface receptors is the triggering of kinase-linked cascades of intracellular reactions. and are families of enzymes that mediate the phosphorylation and dephosphorylation of cellular proteins, respectively. Phosphorylation of seryl, threonyl, or tyrosyl residues can alter activity, levels, or subcellular location of proteins. Multiple protein kinases exist in cells and are classified as follows:

Protein kinases

protein phosphatases

Second messenger-dependent protein kinases,

such as cyclic adenosine monophosphate (cAMP)-dependent protein kinase (PKA, see Fig. 13.12), cyclic granulocyte/monocyte progenitor (cGMP)-dependent protein kinase (PKG, see Fig. 13.12), and calcium/calmodulindependent kinases, including myosin light chain kinase (MLCK, see Fig. 11.28) , such as enzymes of the mitogen-activated protein kinase (MAPK, see Fig. 3.21), cyclin-dependent kinase (Cdk, see Fig. 3.11), and protein tyrosine kinase (see Fig. 21.4)

Second messenger-independent protein kinases

Consequently, the intracellular spatial–temporal patterns of specific phosphorylation events are tightly linked to many of the cellular responses highlighted in subsequent chapters.

Membrane Transport and Vesicular Transport

Substances that enter or leave the cell must traverse the plasma membrane. Some substances (small, fat-soluble, uncharged molecules and gases) cross the plasma membrane by simple or passive diffusion down their concentration gradient without expenditure of metabolic energy and without help of transport proteins (Fig. 2.9). All other molecules require membrane transport proteins to provide them with individual passage across the plasma membrane.

FIGURE 2.9. Movement of molecules across the plasma membrane. Fat-soluble and other small, uncharged molecules (in green ) cross the plasma membrane by simple diffusion down their concentration gradient. Other molecules require membrane transport proteins to provide them with individual passage across the plasma membrane. Small water-soluble molecules (in blue ) require highly selective carrier

proteins to transfer them across the plasma membrane. After binding with a molecule, the carrier protein undergoes a series of conformational changes and releases the molecule on the other side of the membrane. If the process requires energy, it is called (e.g., transport of H +

active transport

ions against their concentration gradient). If the process does not require energy, it is called (e.g., glucose transport). Ions and other small charged molecules (in ) are transported across the plasma membrane by ion-selective channel proteins. In neurons, for instance, ion transport is regulated by membrane potentials (voltage-gated ion channels); in skeletal muscle cells, neuromuscular junctions possess ligand-gated ion channels.

passive transport

purple

There are generally two classes of transport proteins:

Carrier proteins

transfer small, water-soluble molecules. They are highly selective, often transporting only one type of molecule. After binding to a molecule designated for transport, the carrier protein undergoes a series of conformational changes and releases the molecule on the other side of the membrane (see Fig. 2.9). Some carrier proteins, such as the Na+ /K+ pump or H+ pump, require energy for of molecules against their concentration or electrochemical gradient. Other carrier proteins, such as glucose carriers, do not require energy and participate in . also transfer small, water-soluble molecules. In general, channels are made of transmembrane proteins with several membrane-spanning domains that create hydrophilic channels through the plasma membrane. Usually, channel proteins contain a that partially penetrates the membrane bilayer and serves as an ion-selectivity filter. The pore domain is responsible for exquisite ion selectivity, which is achieved by regulation of its three-dimensional structure (see Fig. 2.9). Channels are ion-selective and are regulated on the basis of the cell’s needs. Channel protein transport can be regulated by membrane potentials (e.g., in neurons), neurotransmitters (e.g., such as acetylcholine receptors in muscle cells), or mechanical stress (e.g., in the internal ear).

Channel proteins

active transport passive transport

pore

domain

voltage-gated ion channels ligand-gated ion channels mechanically gated ion channels

Vesicular transport maintains the integrity of the plasma membrane and also provides for the transfer of molecules between different cellular compartments. Some substances enter and leave cells by vesicular transport, a process that involves configurational changes in the plasma membrane at localized sites and subsequent formation of vesicles from the membrane or fusion of vesicles with the membrane (Fig. 2.10).

FIGURE 2.10. Endocytosis and exocytosis.

These processes are the two major forms of vesicular transport. Endocytosis brings molecules and other substances into the cell. In exocytosis, synthesized molecules and other substances leave the cell. Endocytosis is associated with the formation and budding of vesicles from the plasma membrane; exocytosis is associated with the fusion of vesicles originating from intracellular organelles with the plasma membrane, and it is a primary secretory modality.

The major mechanism by which large molecules enter, leave, and move within the cell is called . Vesicles formed by budding from the plasma membrane of one compartment fuse with the plasma membrane of another compartment. Within the cell, this process ensures intercompartmental transfer of the vesicle contents. Vesicular transport involving the cell membrane may also be described in more specific terms:

vesicle budding

Endocytosis is the general term for processes of vesicular transport in which substances enter the cell. Endocytosis controls the composition of the plasma membrane and the cellular response to changes in the external environment. It also plays key roles in nutrient uptake, cell signaling, and cell shape changes. is the general term for processes of vesicular transport in which substances (cargo molecules) leave the cell. Exocytosis is also the process by which all cells deliver the intracellular plasma membrane (that forms cytoplasmic vesicles) to the cell surface. Both processes can be visualized with the EM.

Exocytosis

Exocytosis and endocytosis are coupled together: When exocytosis is abolished, no endocytosis takes place. Fusion of a vesicle with the plasma membrane releases cargo proteins into the extracellular space. Following exocytosis, the vesicular membrane and its associated proteins are retrieved from the plasma membrane through endocytosis, which recycles vesicles and prevents secretory cells from swelling or shrinking. Recent experimental studies revealed that tetanus or botulinum neurotoxins that also in nerve terminals. These studies indicate that exocytosis and endocytosis are coupled together and that the proteins mediating exocytosis and vesicular membrane fusion (i.e., ; see pages 42-44) have a role in .

block exocytosis

Endocytosis

initiating endocytosis

block endocytosis SNARE proteins

Endocytosis is the cellular process that facilitates the uptake of membrane proteins, fluids, nutrients, lipids, and signaling molecules from the extracellular environment into the cell by endocytic vesicles. Following endocytosis, the contents of endocytic vesicles and their membrane components are either recycled to the cell surface or are transported to late endosomes for future degradation.

Uptake of fluid and macromolecules during endocytosis depends in general on three different mechanisms. Some endocytic mechanisms require special proteins during vesicle formation. The best-known protein that interacts with the plasma membrane in vesicle formation is clathrin. Although the presence of clathrin is certainly important for endocytic vesicle formation, many vesicles are formed in a clathrin-independent manner utilizing different proteins (i.e., caveolins or flotillins). Therefore, endocytosis can be classified as either or . In general, three major mechanisms of endocytosis are recognized in the cell. They include , , and . Pinocytosis can occur via two different pathways, micropinocytosis and macropinocytosis, which are discussed separately:

clathrin dependent clathrin independent pinocytosis [Gr., cell drinking] phagocytosis [Gr., cell eating] receptor-mediated endocytosis Micropinocytosis

is the nonspecific ingestion of fluid and small protein molecules via small vesicles, usually smaller than 150 nm in diameter. Micropinocytosis is performed by virtually every cell in the organism, and it is (i.e., it involves a continuous dynamic formation of small vesicles at the cell surface) (Fig. 2.11a). The vesicle formation in micropinocytosis is usually associated with the presence of and proteins that are found in lipid rafts. Caveolin-1 and caveolin-2 are found in all nonmuscle cells, except neurons and white blood cells, whereas caveolin-3 is muscle cell specific. Flotillin-1 and flotillin-2 are found in vesicles distinct from caveolae. Also, mechanoenzymes such as , a GTPase family of membrane scission proteins, are involved in pinocytic vesicle scission (the process of pinching off from the plasma membrane). Pinocytic vesicles are visible with the TEM, and they have a smooth surface. They are especially numerous in the endothelium of blood vessels (Fig. 2.11b) and in smooth muscle cells. Because caveolin-1 forms complexes (of 14–16 monomers) that effect changes in membrane curvature leading to vesicle formation, micropinocytosis does not require clathrin. Micropinocytosis also does not require remodeling of the actin cytoskeleton and therefore may be referred to as and .

constitutive

flotillin

caveolin

dynamin

independent endocytosis

clathrin-independent

actin-

FIGURE 2.11. Pinocytosis. a.

Micropinocytosis involves the dynamic formation of small vesicles at the cell surface. First, substances to be pinocytosed (e.g., small soluble proteins, colloidal tracers) make contact with the extracellular surface of the plasma membrane. Next, the surface becomes invaginated, and dynamin (a GTPase) pinches off the vesicle from the membrane to become a pinocytic vesicle within the cell. Pinocytosis of certain substances may be associated with caveolin. This electron micrograph shows numerous smooth-surfaced pinocytic vesicles within the cytoplasm of endothelial cells of a blood vessel. Also membrane ruffles are visible on this image. They are essential in the formation of large macropinosomes. ×55,000. Macropinocytosis involves the rearrangement of the plasma membrane and underlying actin cytoskeleton to form surface membrane ruffles that entrap large volumes of extracellular fluid. The large vesicles (macropinosomes) enter the cell cytoplasm, undergo maturation, and either fuse with early lysosomes or return to the plasma membrane for recycling.

b.

c.

Macropinocytosis

represents a nonspecific uptake mechanism for extracellular fluids, solutes, nutrients, and antigens. In this actin-dependent process, the actin cytoskeleton is rearranged at the plasma membrane, leading to formation of . The membrane ruffles become elongated and then fold back onto the plasma membrane to entrap extracellular fluid. They give rise to large (>0.2 μm in diameter) endocytic vacuoles called (see Fig. 2.11c). The large fluid-carrying capacity of macropinosomes is utilized by cells of the immune system (e.g., macrophages and dendritic cells) in order to sample as much of their extracellular environment as possible. The amount of solutes and membranes internalized during macropinocytosis exceeds that of any other endocytic pathways. Macropinocytosis is a regulated process and occurs in response to various growth factors, such as macrophage colony-stimulating factor-1 (CSF-1), epidermal growth factor (EGF), or platelet-derived growth factor (PDGF). The macropinosomes undergo a defined sequence of maturation steps in which their contents are either degraded in the late endosome or lysosome or recycled back to the plasma membrane. Because of the initial increase in actin cytoskeleton remodeling in distinct regions of the cell surface leading to formation of plasma membrane ruffles, macropinocytosis is referred to as but . is the ingestion of large particles such as cell debris, bacteria, and other foreign materials. In this nonselective process, the plasma membrane sends out pseudopodia to engulf phagocytosed particles into large vesicles (larger than approximately 250 nm in diameter) called . Phagocytosis is performed mainly by a specialized group of cells belonging to the mononuclear phagocyte system (MPS). Phagocytosis is generally a receptor-mediated process in which receptors on the cell surface recognize non–antigenbinding domains (Fc fragments) of antibodies coating the surface of an invading microorganism or cell (Fig. 2.12a). Phagocytosis is also triggered by recognition of that are commonly expressed on pathogen surfaces by toll-like receptors (page 309). PAMP recognition leads to activation of nuclear factor kappa B (NF-κB) transcription factor, which regulates genes that control cell responses in phagocytosis. Nonbiologic materials, such as inhaled carbon particles, inorganic dusts, and asbestos fibers, as well as biologic debris from inflammation, wound healing, and dead cells are sequestered by cells of the mononuclear phagocyte system (MPS) without involvement of Fc receptors (Fig. 2.12b). This process does not require clathrin

surface membrane ruffles

macropinosomes

Phagocytosis

clathrin-independent

actin-dependent endocytosis

phagosomes

pathogen-associated molecular patterns (PAMPs)

for phagosome formation. Because of the initial extension of pseudopods by the plasma membrane that contributes to the formation of phagosome, the actin cytoskeleton must be rearranged in a process that requires depolymerization and repolymerization of the actin filaments. Thus, phagocytosis is referred to as but .

clathrin-independent

endocytosis

actin-dependent

FIGURE 2.12. Phagocytosis. a.

This drawing shows the steps in the phagocytosis of a large particle, such as a bacterium, that has been killed as a result of an immune response. The bacterium is surrounded by antibodies attached to the bacterial surface antigens. F c receptors on the surface of the plasma membrane of phagocytic cells recognize the F c portion of the antibodies. This interaction triggers rearrangement of the actin cytoskeleton. Depolymerizations and repolymerizations of actin filaments produce temporary projections of the plasma membrane called . They surround the phagocytosed particle, forming a phagosome. By targeted delivery of lysosomal enzymes, a phagosome matures into a lysosome that digests its phagocytosed contents. Nonbiologic materials such as inhaled carbon particles, inorganic dusts, and asbestos fibers, as well as cellular debris resulting from inflammation, are internalized without involvement of antibodies and F c receptors. These particles are bound to multiple receptors on the plasma membrane.

pseudopodia

b.

Receptor-mediated endocytosis

allows entry of specific molecules into the cell. In this mechanism, receptors for specific molecules, called , accumulate in welldefined regions of the cell membrane. These regions, which are represented by the lipid rafts in the plasma membrane, eventually become (Fig. 2.13a). The name is derived from these regions’ appearance in the EM as an accumulation of electrondense material that represents aggregation of molecules on the cytoplasmic surface of the plasma membrane. Cargo receptors recognize and bind to specific molecules that come in contact with the plasma membrane. Clathrin molecules then assemble into a basket-like cage that helps change the shape of the plasma membrane into a vesicle-like invagination (Fig. 2.13b). Clathrin interacts with the cargo receptor via , which are instrumental in selecting appropriate cargo molecules for transport into the cells. Thus, selected cargo proteins and their receptors are pulled from the extracellular space into the lumen of a forming vesicle. The large (100-kDa) mechanoenzyme GTPase from the family of membrane scission proteins, called , mediates the liberation of forming clathrin-coated vesicles from the plasma membrane during receptor-mediated endocytosis. The type of vesicle formed as a result of receptor-mediated endocytosis is referred to as a , and the process itself is known as . Clathrin-coated vesicles are also involved in

pit

cargo receptors coated pits clathrin

clathrin

adaptor proteins (adaptin, AP180) dynamin

clathrin-dependent endocytosis

coated

coated vesicle

the movement of the cargo material from the plasma membrane to early endosomes and from the Golgi apparatus to the early and late endosomes.

FIGURE 2.13. Receptor-mediated endocytosis. a.

This diagram shows the steps in receptormediated endocytosis, a transport mechanism that allows selected molecules to enter the cell. Cargo receptors recognize and bind specific molecules that come in contact with the plasma membrane. Cargo receptor–molecule complexes are recognized by adaptin, a protein that helps select and gather appropriate complexes in specific areas of the plasma membrane for transport into cells. Clathrin molecules then bind to the adaptin–cargo receptor–molecule complex to assemble into a shallow basket-like cage and form a coated pit. Clathrin interactions then assist the plasma membrane to change shape to form a deep depression, a fully formed coated pit that becomes pinched off from the plasma membrane by the protein complex dynamin as a coated vesicle (i.e., budding from the membrane). Selected cargo proteins and their receptors are thus pulled from the extracellular space into the lumen of a forming coated vesicle. After budding and internalization of the vesicle, the coat proteins are removed and recycled for further use. The uncoated vesicle travels to its destination to fuse with a cytoplasmic organelle. Electron micrograph of the cytoplasmic surface of the plasma membrane of A431 cells prepared by the quick-freeze, deep-etch technique. This image shows coated pits and clathrin-coated vesicles in different stages of their formation. Note that the coated pits and clathrin-coated vesicles are formed in areas devoid of actin filaments. The small uniform pinocytic vesicles do not have a clathrin coat and are located in close proximity to actin filaments. ×200,000. (Courtesy of Dr. John E. Heuser, Washington University School of Medicine.)

b.

Exocytosis The movement of secretory vesicles to the plasma membrane is essential for normal cell function. The fusion of secretory vesicles with the plasma membrane is a complex process and involves various types of proteins and lipids. Understanding the underlying molecular mechanisms of exocytosis and membrane fusion provides a solid foundation for pharmacologic treatment of many diseases.

Exocytosis is the process by which a vesicle moves from the cytoplasm to the plasma membrane, where it discharges its contents to the extracellular space.

A variety of molecules produced by the cell for export are initially delivered from the site of their formation to the Golgi apparatus. The next step involves sorting and packaging the secretory product into transport vesicles that are destined to fuse with the plasma membrane in a process known as . Intracellular traffic of these vesicles is achieved by the presence of specific proteins on their surface (coatomers such as COP-I and COP-II) that mediate their movements (see page 56). The molecules that travel this route are often chemically modified (e.g., glycosylated, sulfated) as they pass through different cellular compartments. The membrane that is added to the plasma membrane by exocytosis is recovered into the cytoplasmic compartment by an endocytic process. There are two general pathways of exocytosis:

exocytosis

constitutive pathway

In the , substances designated for export are continuously delivered in transport vesicles to the plasma membrane. Proteins that leave the cell by this process are secreted immediately after their synthesis and exit from the Golgi apparatus, as seen in the secretion of immunoglobulins by plasma cells and of procollagen by fibroblasts. This pathway is present to some degree in all cells. The TEM reveals that these cells lack secretory granules. In the , specialized cells, such as endocrine and exocrine cells and neurons, concentrate secretory proteins and transiently store them in secretory vesicles within the cytoplasm (Fig. 2.14). In this case, a regulatory event (hormonal or neural stimulus) must be activated for secretion to occur, as in the release of secretory vesicles by chief cells of the gastric mucosa and by acinar cells of the pancreas. The signaling stimulus causes a transient influx of Ca2+ into the cytoplasm, which in turn stimulates secretory vesicles to fuse with the plasma membrane and discharge their contents (Fig. 2.15). In the past, secretory vesicles containing inactive precursor (zymogen) were called zymogen granules.

regulated secretory pathway

FIGURE 2.14. Photomicrograph of secretory cells of the pancreas.

Note that secretory vesicles containing protein ready for secretion fill the apical portion of the cells. This process requires an external signaling mechanism for the cell to discharge the accumulated granules. ×860.

FIGURE 2.15. Diagram showing two pathways for exocytosis. Newly synthesized proteins are synthesized in the rough endoplasmic reticulum ( rER ). After their initial posttranslational

modification, they are delivered in COP-II-coated vesicles to the Golgi apparatus. After additional modification in the Golgi apparatus, sorting, and packaging, the final secretory product is transported to the plasma membrane in vesicles that form from the Golgi network ( ). Note that retrograde transport is present between Golgi cisternae and is mediated by the COP-I-coated vesicle. Two distinct pathways are recognized. indicate the constitutive pathway in which proteins leave the cell immediately after their synthesis. In cells using this pathway, almost no secretory product accumulates, and thus, few secretory vesicles are present in the cytoplasm. indicate the regulated secretory pathway in which protein secretion is regulated by hormonal or neural stimuli. In cells using this pathway, such as the pancreatic acinar cells in Figure 2.14, secretory proteins are concentrated and transiently stored in secretory vesicles within the cytoplasm. After appropriate stimulation, the secretory vesicles fuse with the plasma membrane and discharge their contents.

Blue arrows

arrows

trans-

TGN

Red

In addition to excretory pathways, proteins can be transported between the Golgi apparatus and other organelles along endosomal pathways. These pathways are used for delivery of organelle-specific proteins, such as lysosomal structural proteins, into the appropriate organelles.

The precise targeting of vesicles to the appropriate cellular compartment is initially controlled by docking proteins, and specificity is ensured by interactions between soluble NSF attachment receptor (SNARE) proteins. As discussed previously, newly formed vesicles that bud off from the donor membrane (such as cell membrane or Golgi cisternae) can fuse with a number of possible target membranes within the cell. Shortly after budding and shedding its clathrin coat, a vesicle must be targeted to the appropriate cellular compartment. A can be likened to a taxi driver in a large city who successfully delivers a passenger to the proper street address. In the cell, the street address is recognized by bound to the membrane of the traveling vesicle. Rab-GTPase interacts with located on the target membrane. This initial interaction provides recognition of the vesicle and recruits the necessary number of tethering proteins to dock the incoming vesicle. The between Rab-GTPase and its receptor immobilizes the vesicle near the target membrane (Fig. 2.16).

targeting mechanism Rab-GTPase tethering proteins

docking complex

FIGURE 2.16. Steps in formation, targeting, docking, and fusion of transport vesicles with the target membrane. (1) Lipid raft with cargo receptors ready to interact with cargo protein. Note the presence of the specific targeting protein v-SNARE. (2) Initial step in vesicle formation: The binding of the adaptin complex and clathrin forms a coated pit. (3) Formation (budding) of the fully assembled coated vesicle. (4) Transport of the coated vesicle to its destination. (5) Disassembly of clathrin coat. Note the expression of Rab-GTPase activity. (6) Tethering of the vesicle to the target membrane by the interaction between Rab-GTPase and tethering proteins. (7) Beginning of the docking process (recruitment of tethering proteins). (8) Formation of the docking complex between Rab-GTPase and its protein in the target membrane: v-SNAREs on the immobilized vesicle interact with t-SNAREs on the target membrane to form the trans -SNARE complex. (9) Fusion of the vesicle to the target membrane, trans-SNARE becomes cis-SNARE complex. (10) Discharge of the cargo protein into the early endosomal compartment and disassembly of the cis complex by the interaction of the NSF/α-SNAP protein complex. (11) Recycling of v-SNAREs in the transport vesicles for use in another round of vesicle targeting and fusion.

The SNARE family of small transmembrane proteins (name derived from “Soluble NSF Attachment REceptor”) is expressed on both the vesicles and target membranes to mediate accurate vesicle trafficking and subsequent membrane fusion. SNAREs are originally grouped according to their location within the vesicle or target membrane. A vesicle-specific SNARE called v-SNARE interacts with the target plasma membrane that contains a target-specific SNARE called t-SNARE. When a vesicle reaches its destination membrane, both groups of SNARE proteins located on separate membranes must recognize each other and assemble into a tight α-helical configuration called the trans-SNARE complex. Successful assembly of the transSNARE complex guarantees the specificity of interaction between a particular vesicle and its target membrane. It also pulls the vesicle and plasma membrane together, initiating a membrane fusion.

After the membrane fuses, proteins of the trans-SNARE complexes are located in this single fused membrane and are now referred as . These complexes are dismantled with the help of the 𝛂 and recycled for use in another round of vesicle fusion. The SNARE proteins and their interactions have been extensively studied in neuromuscular junctions and other nerve terminals. In nerve terminals, three specific SNARE proteins control trafficking and fusion of synaptic vesicles (containing neurotransmitter) with the presynaptic plasma membrane:

cis-SNARE complexes NSF/ -SNAP protein complex

Synaptobrevin is an 18-kDa integral membrane protein found in the synaptic vesicle (vSNARE). Syntaxin is a 33-kDa integral membrane protein found in the presynaptic plasma membrane (t-SNARE). SNAP-25 is a 23-kDa peripheral membrane protein covalently attached to the intracellular surface of the presynaptic plasma membrane via modified lipid (palmitic acid) in the process called palmitoylation. SNAP-25 is essential for neurotransmitter release from synaptic terminals and it is considered a t-SNARE protein.

trans

Interactions of these three SNARE proteins are required for the formation of -SNARE complexes and neurotransmitter release. Their intracellular domains have an ability to form coiled-coil structures. All three SNARE proteins contribute their own coiled-coil regions for the formation of the -SNARE complex, creating a parallel four-helical bundle. Synaptobrevin and syntaxin each contribute a single helical region, and SNAP-25 contributes two helical regions to form the complex. Malfunction of any one of these three proteins leads to neurotransmitter release defects in nerve endings. For instance, , produced by the anaerobic bacterium , blocks neuromuscular transmission. This toxin binds to the neuronal cell membrane and is subsequently endocytosed. Next, the toxin penetrates the membrane of the endocytic vesicle to enter the cytoplasm of the nerve terminal at the neuromuscular junction. There are seven distinct serotypes (A to G) of botulinum toxins, with each toxin cleaving the SNARE proteins at different sites. This prevents the release of the neurotransmitter acetylcholine from the neuromuscular terminal and depolarization of muscle cell. Serotypes B, D, F, and G cleave ; serotypes A, C, and E cleave ; and serotype C cleaves . In humans, the A, B, and E serotypes are responsible for , a life-threatening disease characterized by progressive muscle weakness. Symptoms include descending paralysis that begins with the muscles controlling eye movement, facial expression, and swallowing and spreads subsequently to the upper limbs, thorax (respiratory muscles), and lower limbs. Paralysis of respiratory muscles (e.g., diaphragm) hampers breathing and eventually results in respiratory failure. The botulin toxin serotypes A and B are used therapeutically to treat patients with nerve and muscle disorders. Injection of a small amount of botulin toxin into specific muscles is used in ophthalmology for treating (excessive blinking) or (not aligned eyes). In strabismus, the toxin is used to paralyze the muscle on one side of the eye that is pulling the eye into the abnormal position. In such as , uncontrollable repetitive skeletal muscle contractions as well as are also treated by injections of botulin toxin. In addition, injection of extremely small quantities of botulin toxin ( or ) into muscles of facial expression is used for the cosmetic treatment of facial wrinkles. Another anaerobic bacterium, , produces , which causes . Tetanospasmin cleaves (v-SNARE protein) and prevents the release of inhibitory neurotransmitters (mainly glycine and γ-aminobutyric acid [GABA]) from synaptic vesicles at the inhibitory motor nerve endings in the central nervous system. The physiologic function of inhibitory neurotransmitters is to decrease and modulate excitatory activity of motor neurons. With the loss of this inhibition, motor neurons

trans

botulinum neurotoxin

Clostridium botulinum

botulism

syntaxin

synaptobrevin

blepharospasm

dystonia gastrointestinal smooth muscle and sphincter spasms onabotulinumtoxinA botox Clostridium tetani tetanus synaptobrevin

SNAP-25

strabismus movement disorders tetanospasmin toxin

excessively stimulate muscle contractions, producing stiffness and rigidity (particularly in the jaw and neck muscles), painful muscle contractions, and muscle spasms. It is important to mention here that SNARE proteins are also involved in initiating endocytosis. For example, synaptobrevin binds to clathrin adaptor protein (AP180); SNAP-25 binds to intersectin, a protein that coordinates the traffic of endocytic vesicles; and syntaxin binds to dynamin.

Endosomes

Endosomes represent a network of membrane-enclosed cytoplasmic compartments that are essential in sorting endocytosed material and membrane proteins for either transport to the cell surface or degradation in lysosomes. TEM of the cell cytoplasm reveals the presence of membrane-enclosed compartments associated with all endocytic pathways (Fig. 2.17). These compartments, called early sorting endosomes or early endosomes, are restricted to a portion of the cytoplasm near the cell membrane. The sorting function of early endosomes enables the return of many vesicles budding from their surface to the plasma membrane. Early endosomes are formed de novo from the invagination of the plasma membrane, forming vesicles containing cell surface proteins and soluble content from the extracellular space. These newly formed endocytic vesicles directly merge with each other or with preexisting early endosomes. In addition, vesicles originating from the trans-Golgi network (TGN) and endoplasmic reticulum can also contribute to the formation and content of the early endosomes.

FIGURE 2.17. Electron micrograph of an early endosome. This deep-etch electron the structure of an early endosome in Dictyostelium . Early endosomes are located

micrograph shows near the plasma membrane and, as in many other sorting compartments, have a typical tubulovesicular structure. The tubular portions contain the majority of integral membrane proteins destined for membrane recycling, whereas the luminal portions collect secretory cargo proteins. The lumen of the endosome is subdivided into multiple compartments, or , by the invagination of its membrane and undergoes frequent changes in shape. ×15,000. (Courtesy of Dr. John E. Heuser, Washington University School of Medicine.)

cisternae

The contents of vesicles incorporated into early endosomal compartments remain in this compartment. As the early endosome matures, it becomes more acidic and sinks into deeper regions of the cytoplasm to become or . The ESCRT

late-sorting endosomes

late endosomes

cytoplasmic protein complex controls membrane remodeling (pages 35-36), which is the basis of the sorting and maturation of early endosomes into late endosomes. The sorting function continues in late endosomes. The endosomal membrane of some late endosomes undergoes inward invagination to generate intraluminal vesicles. These structures, called intracellular or multivesicular endosomes, are also sorted. Some mature into (where their contents will be degraded), and others fuse with the plasma membrane to release intraluminal vesicles by exocytosis. These extracellular vesicles originating from MVBs are called .

multivesicular bodies (MVBs) lysosomes exosomes Endosomes can be viewed either as stable cytoplasmic organelles or as temporary structures formed by endocytosis. Recent experimental observations of endocytic pathways conducted in vitro and in vivo suggest two different models that explain the origin and formation of the endosomal compartments in the cell:

stable compartment model

The describes early and late endosomes as stable cellular organelles that maintain their connection to the external environment of the cell and Golgi apparatus by vesicular transport. Coated vesicles formed at the plasma membrane fuse only with early endosomes because they express specific surface receptors; these receptors remain part of the early endosomal membrane. The suggests that early endosomes are formed de novo from endocytic vesicles originating from the plasma membrane. Therefore, the composition of the early endosomal membrane changes progressively as some components are recycled between the cell surface and the Golgi apparatus. This maturation process leads to the formation of late endosomes and, subsequently, lysosomes. As this compartment matures, specific receptors present on early sorting endosomes (e.g., for coated vesicles) are removed by recycling, degradation, or inactivation.

maturation model

Both models complement rather than contradict each other in describing, identifying, and studying the pathways of internalized molecules.

The major function of early endosomes is to sort and recycle proteins internalized by endocytic pathways. As their name implies, early endosomes sort proteins that have been internalized by endocytic processes. The sorting process is primarily regulated by ESCRT (pages 35-36). ESCRT represents an assembly of four separate protein subcomplexes (ESCRT 0 through III) that mediate the scission of membrane necks from the inside. The shape and geometry of the tubules and vesicles emerging from the early endosome create an environment where localized changes in pH constitute the basis of the endosome sorting mechanism. This mechanism includes the dissociation of ligands from their receptor proteins. In addition, the narrow diameter of the tubules and vesicles may also aid in sorting large molecules, which can be mechanically prevented from entering specific sorting compartments. After sorting, most proteins are rapidly recycled or destined for digestion, and the excess membrane is returned to the plasma membrane.

The fate of the internalized ligand–receptor complex depends on the sorting and recycling ability of the early endosome. The following four pathways for processing internalized ligand–receptor complexes are present in the cell:

The receptor is recycled and the ligand is degraded. Surface receptors allow the cell to

bring in substances selectively through the process of endocytosis. This pathway occurs most often in the cell; it is important because it allows surface receptors to be recycled. Most ligand–receptor complexes dissociate in the acidic pH of the early endosome. The receptor, most likely an integral membrane protein (see page 35), is recycled to the surface via vesicles that bud off the ends of narrow-diameter tubules of

the early endosome. Ligands are usually sequestered in the spherical vacuolar part of the endosome that will later form MVBs, which will transport the ligand to late endosomes for further degradation in the lysosome (Fig. 2.18a). This pathway is utilized by the , , and a variety of and their receptors.

lowdensity lipoprotein (LDL)–receptor complex insulin–glucose transporter (GLUT) receptor complex peptide hormones

FIGURE 2.18. Fate of receptor and ligand in receptor-mediated endocytosis. This diagram shows four major pathways along which the fate of internalized ligand–receptor complexes is determined. a. The internalized ligand–receptor complex dissociates, the receptor is recycled to

the cell surface, and the ligand is directed to late endosomes and eventually degraded within lysosomes. This processing pathway is used by the LDL–receptor complex, insulin–GLUT receptor complex, and a variety of peptide hormone–receptor complexes. LDL, low-density lipoprotein; GLUT, glucose transporter. Both internalized receptor and ligand are recycled. Ligand–receptor complex dissociation does not occur, and the entire complex is recycled to the surface. An example is the iron–transferrin–transferrin receptor complex that uses this processing pathway. Once iron ( 3+) is released from transferrin in the endosome, the transferrin–transferrin receptor complex returns 3+ ions are reduced to to the cell surface, where transferrin is released. In early endosomes, the 2 the ferrous state ( +) by ferrireductase and are released into the cell cytoplasm. The internalized ligand–receptor complex dissociates in the early endosome. The free ligand and the receptor are directed to the late endosomal compartment for further degradation. This pathway is used by many growth factors (e.g., the EGF–receptor complex). EGF, epidermal growth factor. The internalized ligand–receptor complex is transported through the cell. Dissociation does not occur, and the entire complex undergoes transcytosis and release at a different site of the cell surface. This pathway is used during secretion of immunoglobulins (secretory IgA) into saliva. The antibody IgA–receptor complex is internalized at the basal surface of the secretory cells in the salivary gland and released at the apical surface. IgA, immunoglobulin A.

b. Fe

Fe

Fe

c.

d.

Both receptor and ligand are recycled. Ligand–receptor complex dissociation does not always accompany receptor recycling. For example, the low pH of the endosome dissociates iron from the iron-carrier protein transferrin, but transferrin remains associated with

its receptor. However, transferrin is released once the transferrin–receptor complex returns to the cell surface. At neutral extracellular pH, transferrin must again bind iron to be recognized by and bound to its receptor. A similar pathway is recognized for , which are recycled to the cell surface with a foreign antigen protein attached to them (Fig. 2.18b). . This pathway has been identified for and its receptor. Like many other proteins, EGF binds to its receptor on the cell surface. The complex is internalized and carried to early endosomes. Here, EGF dissociates from its receptor, and both are sorted, packaged in separate vesicles, and transferred to the late endosome, often detected in the MVBs. From there, both ligand and receptor are destined to be degraded within lysosomes (Fig. 2.18c). . This pathway is used for secretion of into the saliva and human milk. During this process, commonly referred to as , substances can be altered as they are transported across the epithelial cell (Fig. 2.18d). Transport of maternal immunoglobulin G (IgG) across the placental barrier into the fetus also follows a similar pathway.

histocompatibility complex (MHC) I and II molecules Both receptor and ligand are degraded growth factor (EGF)

Both receptor and ligand are transported through the cell immunoglobulins (secretory IgA) transcytosis

major

epidermal

Early and late endosomes differ in their cellular localization, morphology, and state of acidification and function. Early and late endosomes are localized in different areas of the cell. Early endosomes can be found in the more peripheral cytoplasm, whereas late endosomes are often positioned near the Golgi apparatus and the nucleus. An early endosome has a tubulovesicular structure: The lumen is subdivided into cisternae separated by invaginations of its membrane. Within early endosomes, proteins destined to be transported to late endosomes are sorted and separated from proteins destined for recycling and packaged into vesicles. Early endosomes exhibit only a slightly more acidic environment (pH 6.2–6.5) than the cell cytoplasm. In contrast, have a more complex structure and often exhibit onion-like internal membranes. Invagination of late endosomal membranes results in the formation of containing . The pH of late endosomes is more acidic, averaging 5.5. TEM studies reveal specific vesicles that transport substances between early and late endosomes. MVBs either mature into lysosomes to be degraded or are relocated to fuse with the plasma membrane to release exosomes and other contents by exocytosis (Fig. 2.19).

MVBs

late endosomes intraluminal vesicles

FIGURE 2.19. Schematic diagram of endosomal compartments of the cell. This diagram fate of protein ( red circles ) endocytosed from the cell surface and destined for

shows the lysosomal destruction. Proteins are first found in endocytic (coated) vesicles that deliver them to early endosomes, which are located in the peripheral part of the cytoplasm. Because of the sorting

capability of early endosomes, receptors are usually recycled to the plasma membrane, and endocytosed proteins are transported via vesicles to late endosomes positioned near the Golgi apparatus and the nucleus. The proteins transported to late endosomes will eventually be degraded in lysosomes. Note that the late endosomal compartment makes vesicles in which the endosomal membrane undergoes inward invaginations to generate intraluminal vesicles. These intracellular multivesicular bodies ( ) typically mature into lysosomes where their contents will be degraded or fused with the cell membrane to release intraluminal vesicles (now called exosomes) and other contents by exocytosis. The acidification scale ( ) illustrates changes in pH from early endosomes to lysosomes. The acidification is accomplished by the active transport of protons into endosomal compartments.

MVBs

left

Late endosomes destined to become lysosomes receive newly synthesized lysosomal enzymes that are targeted via the mannose-6-phosphate (M-6-P) receptor. Some endosomes also communicate with the vesicular transport system of the rER. This pathway provides constant delivery of newly synthesized lysosomal enzymes, or hydrolases. A hydrolase is synthesized in the rER as an enzymatically inactive precursor called a prohydrolase. This heavily glycosylated protein then folds in a specific way so that a signal patch is formed and exposed on its surface. This recognition signal is created when specific amino acids are brought into close proximity by the three-dimensional folding of the protein. The signal patch on a protein destined for a lysosome is then modified by several enzymes that attach to the prohydrolase surface. M-6-P acts as a target for proteins possessing an . M-6-P receptors are present in early and late endosomes, lysosomes, and the Golgi apparatus, which is involved in sorting and retrieving secreted prohydrolases destined for transport to endosomes (Fig. 2.20). The acidic environment of late endosomes causes the release of prohydrolases from the M-6-P receptors, which are recycled back (retrograde transport) to the TGN. Prohydrolases are next activated by cleavage and by removing phosphate groups from the mannose residues.

mannose-6-phosphate (M-6-P) M-6-P receptor

FIGURE 2.20. Pathways for delivery of newly synthesized lysosomal enzymes. Lysosomal enzymes (such as lysosomal hydrolases) are synthesized and glycosylated within the rough endoplasmic reticulum ( rER ). The enzymes then fold in a specific way so that a signal patch is formed to which mannose-6phosphate ( M-6-P ) is added. This additional modification allows the enzyme to be targeted to specific proteins that possess M-6-P receptor activity. M-6-P receptors are present in the trans -Golgi network ( TGN ) of the Golgi apparatus, where the lysosomal enzymes are sorted and packaged into vesicles later transported to the early or late endosomes. The M-6-P receptors are recycled back to the Golgi apparatus.

The contents of late endosomes are degraded in lysosomes; however, intraluminal vesicles found in multivesicular bodies are often released as exosomes into the extracellular matrix. In general, substances transported to late endosomes are eventually degraded in lysosomes in a default process that does not require any additional signals. Because late endosomes mature into lysosomes, they are also called . Late endosomes may fuse with each other or with mature lysosomes. Video microscopy allows researchers to observe the complex behavior of these organelles. A population of late endosomes may undergo further transformation. Invagination of late endosomal membranes results in the formation of (they reside within

prelysosomes

intraluminal vesicles

the lumen of the late endosomes). These vesicles contain cytosolic components and certain proteins derived from the invaginated late endosomal membrane. The formation of intraluminal vesicles is aided by ESCRT cytoplasmic protein complex (pages 35-36).

Exosomes

Exosomes originate from multivesicular bodies and represent a novel mode of intercellular communication. Exosomes represent very small (40–100 nm in diameter) endosome-derived membrane-bound vesicles secreted by cells into the extracellular space. These nano-sized vesicles are released from the cell when MVBs fuse with the plasma membrane (Fig. 2.21). High concentrations of exosomes are present in all body fluids, including blood, lymph, cerebrospinal fluid, vitreous body, interstitial fluids, saliva, breast milk, amniotic fluid, semen, and urine.

FIGURE 2.21. Origin, structure, and contents of exosomes .

Exosomes are small, membrane-bound vesicles derived from the lumen of multivesicular bodies (MVBs). They are released via exocytosis into the extracellular space. Exosomes carry a cargo of nucleic acids (fragments of DNA and several types of RNAs), proteins, amino acids, and metabolites as well as several classes of transmembrane proteins that are released by cells into the extracellular space. Exosomes act as mediators for near- and longdistance communication between cells. Note that the tetraspanin family of transmembrane proteins (CD9, CD63, CD81, and CD82) and membrane-associated proteins (such as ALIX, TSG101, flotillin-1, and clathrin) are used as general markers of exosomes. Exosomes may alter signal transduction, antigen presentation, immune responses, and other cellular processes in target cells.

The presence of exosomes in the extracellular space was discovered in the late 1980s when they were initially characterized as cellular waste products. Recent studies confirm that exosomes are functional vesicles that act as transport vehicles for near- and long-distance intercellular communication and material exchange between cells. Exosome membranes contain various transmembrane proteins originating from the plasma membrane of the cell of origin (i.e., adhesion molecules, receptors, and signaling molecules). Exosomes also carry a cargo of nucleic acids (fragments of DNA and several types of RNAs), proteins, lipids, cytokines, transcription factor receptors, growth factors, and other bioactive substances that can be delivered to target cells (see Fig. 2.21). Because the tetraspanin family of transmembrane proteins (such as CD9, CD63, CD81, and CD82) and membrane-associated proteins (such as ALIX, TSG101, flotillin-1, and clathrin) are most common, they are often used as general markers of exosomes. However, many more cell markers specific to the cell type from which the exosome originated have been identified. Exosomes released from cancer cells can promote neoplasia, tumor growth, metastasis formation, and resistance to therapy. For instance, exosomes originating from breast and prostate cancer cells induce neoplastic transformation of other cells through the transfer of their miRNA cargo. The delivery of the exosome contents is achieved by the selective binding of exosomes to cell surface receptors (triggering specific intracellular signaling), by endocytosis, or by the direct transfer of intra-exosomal contents, such as messenger RNA (mRNA), into the recipient cells by fusion of the exosome with the cell membrane. Exosomes, therefore, represent a novel mode of intercellular communication in signal transduction, antigen presentation, immune responses, and many other cellular processes. Clinical research into exosomes is rapidly evolving. Exosomes offer a novel approach for effective vaccine development. Recently, that drive the expression of the immunogenic COVID-19 viral nucleocapsid and spike proteins have been tested. This vaccine shows long-lasting cellular and humoral responses, demonstrating that exosome-based mRNA formulations represent a new approach to protecting against COVID-19 and other infectious diseases. In addition, exosomes are being tested for delivery of gene-based agents, such as DNA and RNA, to patients with genetic disorders and for targeted delivery of therapeutic agents encapsulated into exosomes.

exosome-based mRNA vaccines

Lysosomes

Lysosomes are digestive organelles that were recognized only after histochemical procedures were used to demonstrate lysosomal enzymes. Lysosomes are organelles rich in hydrolytic enzymes such as proteases, nucleases, glycosidases, lipases, and phospholipases. A lysosome represents a major digestive compartment in the cell that degrades macromolecules derived from endocytic pathways as

well as from the cell itself in a process known as autophagy (removal of cytoplasmic components, particularly membrane-bounded organelles, by digesting them within lysosomes). For more information about autophagy, see pages 50-53. The , formulated almost a half century ago, postulated that lysosomes arise as complete and functional organelles budding from the Golgi apparatus. These newly formed lysosomes were termed in contrast to , which had already fused with incoming endosomes. However, the primary and secondary lysosome hypothesis has proved to have little validity as new research data allow a better understanding of the details of protein secretory pathways and the fate of endocytic vesicles. It is now widely accepted that in a complex series of pathways that converge at the late endosomes, transforming them into lysosomes. These pathways are responsible for . As stated earlier, lysosomal enzymes are synthesized in the rER and sorted in the Golgi apparatus based on their binding ability to M-6-P receptors (see pages 46-47).

original hypothesis for lysosomal biogenesis

secondary lysosomes

primary lysosomes

lysosomes are formed targeted delivery of newly synthesized lysosomal enzymes and structural lysosomal membrane proteins into late endosomes

Lysosomes have a unique membrane that is resistant to the hydrolytic digestion occurring in their lumen.

Lysosomes contain a collection of hydrolytic enzymes and are surrounded by a unique membrane that resists hydrolysis by their own enzymes (Fig. 2.22). The has an unusual phospholipid structure that contains cholesterol and a unique lipid called . Most of the structural lysosomal membrane proteins are classified into , and . LAMPs, LGPs, and LIMPs represent more than 50% of the total membrane proteins in lysosomes and are highly glycosylated on the luminal surface. Sugar molecules cover almost the entire luminal surface of these proteins, thus protecting them from digestion by hydrolytic enzymes. Lysobisphosphatidic acids within the lysosomal membrane may play an important role in restricting the activity of hydrolytic enzymes directed against the membrane. The same family of membrane proteins is also detected + in late endosomes. In addition, lysosomes and late endosomes contain that transport H+ ions into the lysosomal lumen, maintaining a low pH (~4.7). The lysosomal membrane also contains that transport the final products of digestion (amino acids, sugars, nucleotides) to the cytoplasm, where they are used in the synthetic processes of the cell or are exocytosed.

lysosomal membrane

lysobisphosphatidic acid lysosome-associated membrane proteins (LAMPs), lysosomal membrane glycoproteins (LGPs) lysosomal integral membrane proteins (LIMPs)

transport proteins

proton (H ) pumps

FIGURE 2.22. Schematic diagram of a lysosome.

This diagram shows a few selected lysosomal enzymes residing within a lysosome and their respective substrates. The major lysosomal membrane-specific proteins, as well as a few other proteins associated with membrane transport, are also shown. LAMP, lysosome-associated membrane protein; LGP, lysosomal membrane glycoprotein; LIMP, lysosomal integral membrane protein.

chloroquine lysosomotropic agent

Certain drugs can affect lysosomal function. For example, , an agent used in the treatment and prevention of malaria, is a that accumulates in the lysosomes. It raises the pH of the lysosomal content, thereby inactivating many lysosomal enzymes. The action of chloroquine on lysosomes accounts for its antimalarial activity; the

concentrates in the acidic food vacuole of the malaria parasite (Plasmodium falciparum) and interferes with its digestive processes, eventually killing the parasite. Lysosomal membrane proteins are synthesized in the rER and have a specific lysosomal targeting signal. drug

As mentioned previously, the intracellular trafficking leading to the delivery of many soluble lysosomal enzymes to late endosomes and lysosomes involves the M-6-P signal and its receptor. All membrane proteins destined for lysosomes (and late endosomes) are synthesized in the rER and transported to and sorted in the Golgi apparatus. However, because they do not contain M-6-P signals, they must be targeted to lysosomes by a different mechanism. The targeting signal for integral membrane proteins is represented by a short cytoplasmic Cterminus domain, which is recognized by adaptin protein complexes and packaged into clathrin-coated vesicles. These proteins reach their destination by one of two pathways:

constitutive secretory pathway

In the , LIMPs exit the Golgi apparatus in coated vesicles and are delivered to the cell surface. From there, they are endocytosed and, via the early and late endosomal compartments, finally reach lysosomes (Fig. 2.23).

FIGURE 2.23. Lysosome biogenesis.

This diagram shows regulated and constitutive pathways for delivery of lysosomal-specific membrane proteins into early and late endosomes. The lysosomal membrane possesses highly glycosylated specific membrane proteins that protect the membrane from digestion by lysosomal enzymes. These lysosome-specific proteins are synthesized in the rough endoplasmic reticulum, transported to the Golgi apparatus, and reach their destination by two pathways. indicate the constitutive secretory pathway in which certain lysosomal

Blue arrows

membrane proteins exit the Golgi apparatus and are delivered to the cell surface. From there, they are endocytosed and, via the early and late endosomal compartments, finally reach lysosomes. indicate the endosomal Golgi-derived coated vesicle secretory pathway. Here, other lysosomal proteins, after sorting and packaging, exit the Golgi apparatus in clathrin-coated vesicles to fuse with early and late endosomes.

Green

arrows

Golgi-derived coated vesicle secretory pathway

In the , LIMPs, after sorting and packaging, exit the Golgi apparatus in clathrin-coated vesicles (see Fig. 2.23). These transport vesicles travel and fuse with late endosomes as a result of interaction between endosome-specific components of v-SNARE and t-SNARE docking proteins (see pages 42-43).

Three different pathways deliver material for intracellular digestion in lysosomes. Depending on the nature of the digested material, different pathways deliver material for digestion within the lysosomes (Fig. 2.24). In the digestion process, most of the digested material comes from endocytic processes; however, the cell also uses lysosomes to digest its own obsolete parts, nonfunctional organelles, and unnecessary molecules. Three pathways for digestion exist:

FIGURE 2.24. Pathways of delivery of materials for digestion in lysosomes. Most of the small extracellular particles are internalized by both receptor-mediated endocytosis and pinocytosis. These two endocytic pathways are labeled with red arrows . Large extracellular particles such as bacteria and cellular debris are delivered for cellular digestion via the phagocytic pathway ( blue arrows ). The cell also uses lysosomes to digest its own organelles and other intracellular proteins via the autophagic pathway ( green arrows ). Intracellular particles are isolated from the cytoplasmic matrix by the isolation membrane of the smooth endoplasmic reticulum ( sER ), transported to lysosomes, and subsequently degraded.

Extracellular large particles such as bacteria, cell debris, and other foreign materials are engulfed in the process of phagocytosis. A phagosome, formed as the material is

internalized within the cytoplasm, subsequently receives hydrolytic enzymes to become a late endosome, which matures into a lysosome. such as extracellular proteins, plasma membrane proteins, and ligand–receptor complexes are internalized by and . These particles follow the endocytic pathway through early and late endosomal compartments and are finally degraded in lysosomes. such as entire organelles, cytoplasmic proteins, and other cellular components are isolated from the cytoplasmic matrix by endoplasmic reticulum membranes, transported to lysosomes, and degraded. This process is called .

Extracellular small particles endocytosis Intracellular particles

pinocytosis

receptor-mediated autophagy

In addition, some cells (e.g., osteoclasts involved in bone resorption and neutrophils involved in acute inflammation) may release lysosomal enzymes directly into the extracellular space to digest components of the extracellular matrix.

Lysosomes in some cells are recognizable in the light microscope because of their number, size, or contents. The numerous azurophilic granules of neutrophils (white blood cells) represent lysosomes and are recognized in aggregate by their specific staining. Lysosomes that contain phagocytized bacteria and fragments of damaged cells are often recognized in macrophages. Hydrolytic breakdown of the contents of lysosomes often produces a debris-filled vacuole called a residual body that may remain for the entire life of the cell. For example, in neurons, residual bodies are called age pigment or lipofuscin granules. Residual bodies are a normal feature of cell aging. The absence of certain lysosomal enzymes can cause the pathologic accumulation of undigested substrate in residual bodies. This can lead to several disorders collectively termed (see Folder 2.1).

FOLDER 2.1

lysosomal storage diseases

CLINICAL CORRELATION: LYSOSOMAL STORAGE DISEASES Many genetic disorders have been identified in individuals who have mutations in a gene that encodes lysosomal proteins. These diseases are termed lysosomal storage diseases (LSDs) and are characterized by dysfunctional lysosomes. The defective protein in most cases is a hydrolytic enzyme or its cofactor; less commonly, lysosomal membrane proteins or proteins that are involved in sorting, targeting, and transporting lysosomal proteins are defective. The result is an accumulation in cells of the specific products that lysosomal enzymes normally use as substrates in their reactions. These undigested, accumulated products disrupt the normal function of the cell and lead to its death. Currently, 49 disorders are known LSDs with a collective incidence of about 1 in 7,000 live births. The life expectancy across the entire group of people with these disorders is 15 years. The first LSD was described in 1881 by British ophthalmologist Warren Tay, who reported symptoms of retinal abnormalities in a 12-month-old infant with severe neuromuscular symptoms. In 1896, U.S. neurologist Bernard Sachs described a patient with similar eye symptoms found earlier by Tay. This disease is now known as . It is caused by the absence of one enzyme, a lysosomal galactosidase (β-hexosaminidase) that catalyzes a step in lysosomal breakdown of gangliosides in neurons. The resulting accumulation of the GM 2 ganglioside that is found within concentric lamellated structures in residual bodies of neurons interferes with normal cell function. Children born with LSDs usually appear normal at birth; however, they soon show clinical signs of the disease. They often experience slower growth, show changes in facial features, and develop bone and joint deformities that lead to significant restrictions of limb movement. They may lose already attained skills such as speech and learning ability. Behavioral problems as well as severe

Tay–Sachs disease

intellectual disability may occur. They are prone to frequent lung infections and heart disease. Some children have enlarged internal organs such as the liver and spleen (hepatosplenomegaly). The most common LSDs in children are Gaucher disease, Hurler syndrome (mucopolysaccharidosis [MPS] I), Hunter syndrome (MPS II), and Pompe disease. Not long ago, LSDs were seen as neurodegenerative disorders without any potential treatment. In the last two decades, there has been limited success in treating the symptoms of LSDs. Considerable effort has been devoted to genetic research and finding methods to replace the missing enzymes that cause various forms of LSD. , which requires the cellular delivery of a manufactured recombinant enzyme, is available for some LSDs such as cystinosis and Gaucher disease. Enzymes have also been supplied by transplantation of bone marrow containing normal genes from an unaffected person. Success of the enzyme replacement therapy is often limited by insufficient biodistribution of recombinant enzymes and high costs. Recently, emerging strategies for the treatment of LSDs include in which chaperone molecules are delivered to affected cells. In some cases, synthetic chaperones can assist in the folding of mutated enzymes to improve their stability and advance their lysosomal delivery. In the future, the combination of different therapies such as enzyme replacement, pharmacologic chaperone, and with the development of newborn screening tests will enable early detection and improve clinical outcome of patients with LSDs.

Enzyme replacement therapy

pharmacologic chaperone therapy

gene transfer therapies

Summary of Common Lysosomal Storage Diseases Disease Protein Deficiency Disorders of Sphingolipid Degradation

Accumulating Product (or Defective Process)

Gaucher disease

Glucocerebrosidase

Glucosylceramide

Tay–Sachs disease

β-Hexosaminidase, α-subunit

GM2 ganglioside

Sandhoff disease

β-Hexosaminidase, β-subunit

GM2 ganglioside, oligosaccharides

Krabbe disease

Galactosylceramidase

Gal-ceramide, galsphingosine

Niemann–Pick disease A, B

Sphingomyelinase

Sphingomyelin

Aspartylglycosaminuria

Aspartylglycosaminidase

Disorders of Glycoprotein Degradation

N-linked

oligosaccharides

α-Mannosidosis

α-Mannosidase

α-Mannosides

Hurler syndrome (MPS I)

α-L-iduronidase

Dermatan sulfate, heparan sulfate

Hunter syndrome (MPS II)

L-iduronate sulfatase

Dermatan sulfate, heparan sulfate

GalNAc 4-sulfatase/arylsulfatase B

Dermatan sulfate

Disorders of Glycosaminoglycan Degradation

Maroteaux–Lamy syndrome (MPS VI)

Other Disorders of Single Enzyme Deficiency Pompe disease (glycogenosis II)

α-1,4-Glucosidase

Glycogen

Wolman disease (familial xanthomatosis)

Acid lipase

Cholesterol esters, triglycerides

Canavan disease

Aspartoacylase

N-acetylaspartic

(aspartoacylase deficiency)

acid

Disorders of Lysosomal Biogenesis Inclusion-cell (Icell) disease, mucolipidosis II

GlcNAc-1-phosphotransferase (GlcNAcPTase); leads to defective sorting of most soluble hydrolytic lysosomal enzymes

Disorders of the Lysosomal Membrane

Lysosomal hydrolases are not present in lysosomes

Danon disease

LAMP2

Presence of autophagic vacuoles

Cystinosis

Cystinosin (cystine transporter)

Cystine

LAMP2, lysosome-associated membrane protein 2; MPS I, II, VI, mucopolysaccharidosis type I, II, VI.

Autophagy Autophagy represents the major cellular pathway in which a number of cytoplasmic proteins, organelles, and other cellular 2.25). This important process catabolic cell functions and organelles. Digested components and development.

structures are degraded in the lysosomal compartment (Fig. maintains a well-controlled balance between anabolic and permits the cell to eliminate unwanted or unnecessary of organelles are recycled and reused for normal cell growth

FIGURE 2.25. Three autophagic pathways for degradation of cytoplasmic constituents. In macroautophagy, a portion of the cytoplasm or an entire organelle is surrounded by an intracellular membrane of the endoplasmic reticulum to form a double-membraned autophagosome vacuole. After fusion with a lysosome, the inner membrane and the contents of the vacuole are degraded. In microautophagy , cytoplasmic proteins are internalized into lysosomes by invagination of the lysosomal membrane. Chaperone-mediated autophagy to lysosomes is the most selective process for degradation of specific cytoplasmic proteins. It requires assistance of proteins called chaperones . The chaperone protein (hsc73) binds to the protein and helps transport it into the lysosomal lumen, where it is finally degraded.

Cytoplasmic proteins and organelles are substrates for lysosomal degradation in the process of autophagy. Autophagy plays an essential role during starvation, cellular differentiation, cell death, and cell aging. Applying genetic screening tests originally developed for yeasts, researchers have discovered a number (approximately 33) of in the mammalian cell genome and have been able to trace the activation or inhibition of these genes under specific conditions. The presence of adequate nutrients and growth factors stimulates enzymatic activity of a serine/threonine kinase known as . High mTOR activity exerts an inhibitory effect on autophagy.

genes)

target of rapamycin (mTOR)

autophagy-related genes (Atg

mammalian

The opposite occurs in nutrient starvation, hypoxia, and high temperatures, where lack of mTOR activity causes activation of Atg genes. This results in the formation of an that initiates the process of autophagy. Generally, autophagy can be divided into three well-characterized pathways:

protein kinase autophagy–regulatory complex Macroautophagy,

Atg1

or simply autophagy, is a nonspecific process in which a portion of the cytoplasm or an entire organelle is first surrounded by a double or multilamellar intracellular membrane of endoplasmic reticulum, called the , to form a vacuole called an . This process is aided by proteins encoded by several Atg genes. First, the complex containing attaches to a portion of the endoplasmic reticulum and localizes in the isolation membrane. Subsequently, is recruited and bound to the membrane. Together, these proteins change the shape of the isolation membrane, which bends to enclose and seal an organelle destined for digestion within the lumen of the autophagosome. Once the autophagosome is completed, the Atg12– Atg5–Atg16L complex and Atg8 dissociate from this structure. After targeted delivery of lysosomal enzymes, the autophagosome matures into a lysosome. The isolation membrane disintegrates within the hydrolytic compartment of a lysosome. Macroautophagy occurs in the liver during the first stages of starvation (Fig. 2.26).

autophagosome

isolation membrane Atg12–Atg5–Atg16L proteins

Atg8

FIGURE 2.26. Electron micrograph of nutrient-starved mouse embryonic fibroblasts. This electron micrograph shows several autophagosomes ( AP ). Note that autophagosomes are double-membraned structures containing undigested intracellular organelles, such as mitochondria or fragments of the endoplasmic reticulum ( arrowheads ). After the autophagosome fuses with a lysosome, it forms an

AL

autolysosome ( ) that degrades the enclosed materials including the inner autophagosomal membrane. , mitochondria, , rough endoplasmic reticulum. ×26,300. (Courtesy of Drs. Chieko Kishi-Itakura and Noboru Mizushima.)

M

rER

Microautophagy

is also a nonspecific process in which cytoplasmic proteins are degraded in a slow, continuous process under normal physiologic conditions. In microautophagy, small cytoplasmic soluble proteins are internalized into the lysosomes by invagination of the lysosomal membrane. is the only selective process of protein degradation and requires assistance from specific cytosolic chaperones such as a called . This process is activated during nutrient deprivation and requires the presence of targeting signals on the degraded proteins and a specific receptor on the lysosomal membrane. Chaperone-mediated direct transport resembles the process of protein importation to various other cellular organelles: hsc73 binds to the protein and assists in its transport through the lysosomal membrane into the lumen, where it is finally degraded. Chaperone-mediated autophagy is responsible for the degradation of approximately 30% of cytoplasmic proteins in organs such as the liver and kidney.

Chaperone-mediated autophagy protein hsc73

heat-shock chaperone

Proteasome-Mediated Degradation In addition to the lysosomal pathway of protein degradation, cells are able to destroy proteins without involvement of lysosomes. Such a process occurs within large cytoplasmic or nuclear protein complexes called . They represent ATP-dependent protease complexes that destroy proteins that have been specifically tagged for this pathway. is used by cells to destroy abnormal proteins that are misfolded, are denatured, or contain abnormal amino acids. This pathway also degrades normal short-lived regulatory proteins that need to be rapidly inactivated and degraded, such as mitotic cyclins that regulate cell cycle progression, transcriptional factors, tumor suppressors, and tumor promoters.

Proteasome-mediated degradation

proteasomes

Proteins destined for proteasome-mediated degradation must be recognized and specifically tagged by the polyubiquitin chain. Degradation of a protein in the proteasome-mediated pathway involves two successive steps:

Polyubiquitination,

in which proteins targeted for destruction are repeatedly tagged by covalent attachments of a small (8.5-kDa) protein called . The tagging reaction is catalyzed by three ubiquitin ligases called . In a cascade of enzymatic reactions, the targeted protein is first marked by a single ubiquitin molecule. This creates a signal for consecutive attachment of several other ubiquitin molecules, resulting in a linear chain of ubiquitin conjugates. A protein target for destruction within the proteasome must be labeled with at least four ubiquitin molecules in the form of a that serves as a degradation signal for proteasome complex. . Each proteasome consists of a hollow cylinder, shaped like a barrel, containing a that facilitates the multicatalytic protease activity in which polyubiquitinated proteins are degraded into small polypeptides and amino acids. On both ends of the CP cylinder are two . The RP that forms the lid of the barrel recognizes polyubiquitin tags, unfolds the protein, and regulates its entry into the destruction chamber. The RP on the opposite side (on the base) of the barrel releases short peptides and amino acids after degradation of the protein is completed. Free ubiquitin molecules are released by and recycled (Fig. 2.27).

E3

ubiquitin ubiquitin-activating enzymes E1, E2, and

polyubiquitin chain Degradation of the tagged protein by the 26S proteasome complex 20S core particle (CP) 19S regulatory particles (RPs)

deubiquitinating (DUB) enzymes

FIGURE 2.27. Proteasome-mediated degradation.

This degradation pathway involves tagging proteins destined for destruction by a polyubiquitin chain and its subsequent degradation in a proteasome complex with the release of free reusable ubiquitin molecules. Ubiquitin in the presence of adenosine triphosphate ( ) is activated by a complex of three ubiquitin-activating enzymes (E1, E2, and E3) to form a single polyubiquitin chain that serves as the degradation signal for the 26S proteasome complex. The regulatory particle (19S RP) that forms the lid of the main protein destruction chamber (20S core particle [CP]) recognizes polyubiquitin tags, unfolds the protein, and inserts and regulates its entry into the destruction chamber. The regulatory particle on the opposite side of the chamber releases short peptides and amino acids after degradation of the protein is completed. Free ubiquitin molecules are released by deubiquitinating enzymes ( ) and recycled. ADP, adenosine diphosphate.

ATP

DUBs

Two groups of pathologic conditions are associated with malfunction of proteasomemediated degradation. The first group results from a loss of proteasome function because of

mutations in the genes encoding ubiquitin-activating enzymes. This leads to a decrease in protein degradation and their subsequent accumulation in the cell cytoplasm (e.g., in and ). The second group is caused by overexpression of proteins involved in the proteasome-mediated degradation pathway that causes accelerated degradation of cellular proteins (e.g., infections with human papillomavirus). Use of a

Angelman syndrome

Alzheimer disease

specific proteasome inhibitor has been successful in treating multiple myeloma, and researchers hope to develop additional inhibitors for the treatment of other diseases.

Rough Endoplasmic Reticulum

The protein synthetic system of the cell consists of the rough endoplasmic reticulum and ribosomes. The cytoplasm of a variety of cells engaged chiefly in protein synthesis stains intensely with basic dyes. The basophilic staining is caused by the presence of RNA. The portion of the cytoplasm that stains with the basic dye is called . The ergastoplasm in secretory cells (e.g., pancreatic acinar cells) is the light microscopic image of the organelle called the . With the TEM, the rER appears as a series of interconnected, membrane-limited, flattened sacs called , with particles studding the exterior surface of the membrane (Fig. 2.28). These particles, called , are attached to the membrane of the rER by ribosomal docking proteins. Ribosomes measure 15–20 nm in diameter and consist of a small and large subunit. Each subunit contains of different lengths as well as numerous types of proteins. In many instances, the rER is continuous with the outer membrane of the nuclear envelope (see the next section). Groups of ribosomes form short spiral arrays called or (Fig. 2.29) in which many ribosomes are attached to a thread of .

ergastoplasm

rough endoplasmic reticulum (rER) cisternae ribosomes ribosomal RNA (rRNA) polyribosomes polysomes messenger RNA (mRNA)

FIGURE 2.28. Electron micrograph of the rough endoplasmic reticulum (rER). This image of the rER in a chief cell of the stomach shows the membranous cisternae ( C ) closely packed in parallel arrays. Polyribosomes are present on the cytoplasmic surface of the membrane surrounding the cisternae. The appearance of a ribosome-studded membrane is the origin of the term rough endoplasmic reticulum. A few ribosomes are free in the cytoplasm. M, mitochondrion. ×50,000.

FIGURE 2.29. Electron micrograph of the rough endoplasmic reticulum (rER) and polyribosome complexes. This image shows a small section of the rER adjacent to the nucleus sectioned in two planes. The reticulum has turned within the section. Thus, in the upper right and left , the membranes of the reticulum have been cut at a right angle to their surface. In the center , the reticulum has twisted and is shown as in an aerial view (from above the membrane). The large spiral cytoplasmic assemblies ( arrows ) are chains of ribosomes that form polyribosomes that are actively engaged in translation of the mRNA molecule. ×38,000.

Protein synthesis involves transcription and translation.

transcription pre-mRNA

The production of proteins by the cell begins within the nucleus with , in which the genetic code for a protein is transcribed from DNA to . After posttranscriptional modifications of the pre-mRNA molecule—which includes RNA cleavage, excision of introns, rejoining of exons, and capping by the addition of poly(A) tracks at the 3′ end and a methylguanosine cap [M(7) GPPP] at the 5′ end—the resulting molecule leaves the nucleus and migrates into the cytoplasm (Fig. 2.30). Transcription is followed by , in which the coded message contained in the mRNA is read by ribosomal complexes to form a polypeptide. A typical single cytoplasmic mRNA molecule binds to many ribosomes spaced as close as 80 nucleotides apart, thus forming a or . A polysome attached to the cytoplasmic surface of the rER can

translation

complex

polysome

mRNA

polyribosome

translate a single mRNA molecule and simultaneously produce many copies of a particular protein. In contrast, reside within the cytoplasm. They are not associated with any intracellular membranes and are structurally and functionally identical to polysomes of the rER.

free ribosomes

FIGURE 2.30. Summary of events during protein synthesis.

Protein synthesis begins within the nucleus with transcription, during which the genetic code for a protein is transcribed from DNA to messenger RNA (mRNA) precursors. After posttranscriptional modifications of the pre-mRNA molecule— which include RNA cleavage, excision of introns, rejoining of exons, and capping by addition of poly(A) tracks at the 3′ end and methylguanosine cap at the 5′ end—the resulting mRNA molecule leaves the nucleus and enters the cytoplasm. In the cytoplasm, the mRNA sequence is read by the ribosomal complex in the process of translation to form a polypeptide chain. The first group of 15–60 amino acids on the amino-terminus of a newly synthesized polypeptide forms a signal sequence (signal peptide) that directs the protein to its destination (i.e., lumen of rough endoplasmic reticulum [ ]). The signal peptide interacts with a signal recognition particle ( ), which arrests further growth of the polypeptide chain until its relocation toward the rER membrane. Binding of the SRP to a docking protein on the cytoplasmic surface of the rER aligns the ribosome with the translocator protein. Binding of the ribosome to the translocator causes dissociation of the SRP–docking protein complex away from the ribosome, and protein synthesis is resumed. The translocator protein guides the polypeptide chain into the lumen of the rER cisterna. The signal sequence is cleaved from the polypeptide by signal peptidase and is subsequently digested by signal peptide peptidases. On completion of protein synthesis, the ribosome detaches from the translocator protein.

rER

SRP

prokaryotic (bacterial) and eukaryotic

The differences between the structure of were exploited by researchers, who discovered chemical compounds (antibiotics) that bind to bacterial ribosomes. Antibiotics are able to kill bacteria without harming the cells of the infected individual. Several types of antibiotics, such as aminoglycosides (streptomycin), macrolides (erythromycin), lincosamides (clindamycin), tetracyclines, and chloramphenicol, inhibit protein synthesis by binding to different portions of bacterial ribosomes.

ribosomes

Signal peptides direct the posttranslational transport of a protein.

Most proteins that are synthesized for export or that will become a part of specific organelles (such as the plasma membrane, mitochondrial matrix, endoplasmic reticulum, or nucleus) require sorting signals that direct proteins to their correct destinations. These are often found in the sequence of the first group of 15 –60 amino acids on the amino-terminus of a newly synthesized protein. Signal sequences can be compared to airline tags on luggage. Just as the tags ensure that baggage moves correctly from one aircraft to another at airports, so signal peptides ensure that the newly synthetized protein is properly identified as it passes through the organelles of the cell. During this transit, a series of synthetic events and posttranslational modifications occur before the polypeptides ultimately arrive at their proper destination. For example, almost all proteins that are transported to the endoplasmic reticulum have a signal sequence consisting of 5–10 hydrophobic amino acids on their amino-termini. The signal sequence of the nascent peptide interacts with a , which arrests further growth of the polypeptide chain. The complex containing the SRP– polyribosome complex that arrests polypeptide synthesis is then relocated toward the rER membrane. Binding of the SRP to a on the cytoplasmic surface of rER aligns the ribosome with the , an integral membrane protein of the rER. Binding of the ribosome to the protein translocator causes dissociation of the SRP–docking protein complex from the ribosome and rER membrane, releasing the translational block and allowing the ribosome to resume protein synthesis (see Fig. 2.30). The translocator protein inserts the polypeptide chain into its aqueous pore, allowing the newly formed protein to be discharged into the lumen of the rER cisterna. For simple secretory proteins, the polypeptide continues to be inserted by the translocator into the lumen as it is synthesized. The signal sequence is cleaved from the polypeptide by signal peptidase residing on the cisternal face of the rER membrane, even before the synthesis of the entire chain is completed. For integral membrane proteins, sequences along the polypeptide may instruct the forming protein to pass back and forth through the membrane, creating the functional domains that the protein will exhibit once it is inserted into the membrane. On completion of protein synthesis, the ribosome detaches from the translocator protein and is again free in the cytoplasm.

signal sequences (signal peptides)

signal recognition particle (SRP)

translocator

docking protein

The posttranslational modification and sequestration of proteins within the rER is the first step in the export of proteins destined to leave the cell.

As polypeptide chains are synthesized by the membrane-bound polysomes, the protein is injected into the lumen of the rER cisterna, where it is further modified posttranslationally by enzymes. These modifications include core glycosylation, disulfide bond and internal hydrogen bond formation, folding of the newly synthesized protein with the help of molecular chaperones, and partial subunit assembly. Proteins are then concentrated within the lumen of neighboring cisternae of rER, or they are carried to another part of the cell in the continuous channels of the rER. Some antibiotics, such as collectively act on rER to inhibit -linked glycosylation in glycoprotein synthesis. By preventing glycoprotein synthesis, these compounds impede the formation of the surface coat (glycocalyx) of virus-infected cells and the protective viral capsid of replicating viruses. The name tunicamycin ( coat) reflects this antibiotic’s mechanism of action. Although tunicamycin is a powerful antibiotic against gram-positive bacteria and has strong antiviral activity, it has no therapeutic use because of its high toxicity. Except

and D),

N

Lat., tunica,

tunicamycin (A, B, C,

for the few proteins that remain permanent residents of the rER membranes and those proteins secreted by the constitutive pathway, the newly synthesized proteins are normally delivered to the Golgi apparatus within minutes. A few diseases are characterized by an inability of the rER to export posttranslationally modified proteins to the Golgi apparatus. For example, in , a single amino acid substitution renders the rER unable to export α1-antitrypsin (A1AT). This leads to decreased activity of A1AT in the blood and lungs and abnormal deposition of defective A1AT within the rER of liver hepatocytes, resulting in (chronic obstructive pulmonary disease) and impaired liver function. In cells in which the constitutive pathway is dominant—namely, plasma cells and activated fibroblasts—newly synthesized proteins may accumulate in the rER cisternae, causing their engorgement and distention. The rER also serves as a in the process of protein production. If the newly synthesized protein is not properly posttranslationally modified or is misfolded, it is then exported from the rER back to the cytoplasm via the mechanism of retrotranslocation. Here, defective proteins are deglycosylated, polyubiquitinated, and degraded within proteasomes (see page 53).

α1-antitrypsin deficiency emphysema

quality checkpoint

The rER is most highly developed in active secretory cells.

The rER is particularly well developed in those cells that synthesize proteins destined to leave the cell (secretory cells) as well as in cells with large amounts of plasma membrane, such as neurons. Secretory cells include glandular cells, activated fibroblasts, plasma cells, odontoblasts, ameloblasts, and osteoblasts. The rER is not limited, however, to secretory cells and neurons. Virtually every cell of the body contains profiles of rER. However, they may be few in number, a reflection of the amount of protein the cell secretes, and dispersed so that in the light microscope, they are not evident as areas of basophilia. The rER is most highly developed in active secretory cells because secretory proteins are synthesized exclusively by the ribosomes of the rER. In all cells, however, the ribosomes of the rER also synthesize proteins that are to become permanent components of lysosomes, Golgi apparatus, rER, or nuclear envelope (these structures are discussed in the next sections) or integral components of the plasma membrane.

Coatomers mediate bidirectional traffic between the rER and Golgi apparatus.

Two classes of coated vesicles are involved in the transport of protein from and to the rER. A protein coat similar to clathrin surrounds vesicles transporting proteins between the rER and the Golgi apparatus (page 41). However, unlike clathrins, which mediate bidirectional transport from and to the plasma membrane, one class of proteins is involved only in from the rER to the -Golgi network (CGN), the Golgi cisternae closest to the rER. Another class of proteins mediates from the CGN back to the rER (Fig. 2.31). These two classes of proteins are called or .

anterograde transport

cis

retrograde transport coatomers

COPs

FIGURE 2.31. Anterograde and retrograde transport between the rough endoplasmic reticulum (rER) and cis-Golgi network (CGN). Two classes of coated vesicles are involved in protein

transport to and from the rER. These vesicles are surrounded by COP-I and COP-II protein coat complexes, respectively. COP-II is involved in anterograde transport from the rER to the CGN, and COPI is involved in retrograde transport from the CGN back to the rER. After a vesicle is formed, the coat components dissociate from the vesicle and are recycled to their site of origin. The COP-I protein coat is also involved in retrograde transport between cisternae within the Golgi apparatus (see Fig. 2.15).

COP-I

mediates transport vesicles originating in the CGN back to the rER (Fig. 2.32a). This mediates a salvage operation that returns rER proteins mistakenly transferred to the CGN during normal anterograde transport. In addition, COP-I is also responsible for maintaining retrograde transport between the Golgi cisternae.

retrograde transport

FIGURE 2.32. Electron micrograph of COP-I- and COP-II-coated vesicles. a. This image shows COP-I-coated vesicles that initiate retrograde transport from the cis -Golgi network (CGN) to the rough endoplasmic reticulum (rER). In this image, taken from cells prepared by the quick-freeze, deep-etch technique, note the structure of the CGN and emerging vesicles. ×27,000. b. Image of COP-

II-coated vesicles that are responsible for anterograde transport. Note that the surface coat of

these vesicles is different from that of clathrin-coated vesicles. ×50,000. (Courtesy of Dr. John E. Heuser, Washington University School of Medicine.)

COP-II is responsible for anterograde transport, forming rER transport vesicles destined for the CGN (Fig. 2.32b). COP-II assists in the physical deformation of rER membranes into sharply curved buds and the further separation of vesicles from the rER membrane. Most proteins produced in the rER use COP-II-coated vesicles to reach the CGN.

Shortly after formation of COP-I- or COP-II-coated vesicles, the coats dissociate from the newly formed vesicles, allowing the vesicle to fuse with its target. The coat components then are recycled to their site of origin.

“Free” ribosomes synthesize proteins that will remain in the cell as cytoplasmic structural or functional elements. Proteins targeted to the nucleus, mitochondria, or peroxisomes are synthesized on free ribosomes and released into the cytosol. Some have a short targeting signal (e.g., for peroxisomes, the targeting signal consists of three amino acid motif, Ser-Lys-Leu) and may use specialized proteins (soluble chaperones) to guide them to specific organelles (pages 64-66). However, most proteins synthesized on free ribosomes lack a signal sequence and thus remain in the cytosol. Cytoplasmic basophilia is associated with cells that produce large amounts of protein that will remain in the cell. Such cells and their products include developing red blood cells (hemoglobin), developing muscle cells (the contractile proteins actin and myosin), nerve cells (neurofilaments), and keratinocytes of the skin (keratin). In addition, most enzymes of the mitochondrion are synthesized by free polysomes and transported into that organelle. in these cells was formerly called ergastoplasm and is caused by the presence of large amounts of RNA. In this case, the ribosomes and polysomes are free in the cytoplasm (i.e., they are not attached to membranes of the endoplasmic reticulum). The large basophilic bodies of nerve cells, which are called , consist of both rER and large numbers of free ribosomes (Fig. 2.33). All ribosomes contain RNA; it is the phosphate groups of the RNA of the ribosomes, not the membranous component of the endoplasmic reticulum, that account for basophilic staining of the cytoplasm.

Basophilia

Nissl bodies

FIGURE 2.33. Electron micrograph of a nerve cell body showing the rough endoplasmic reticulum (rER). This image shows rER profiles as well as numerous free ribosomes located between

the membranes of the rER. Collectively, the free ribosomes and membrane-attached ribosomes are responsible for the characteristic cytoplasmic basophilia (Nissl bodies) observed in the light microscope in the perinuclear cytoplasm of neurons. ×45,000.

Smooth Endoplasmic Reticulum

The sER consists of short anastomosing tubules that are not associated with ribosomes. Cells with large amounts of smooth endoplasmic reticulum may exhibit distinct cytoplasmic

eosinophilia (acidophilia) when viewed in the light microscope. The sER is structurally similar to the rER but lacks the ribosome docking proteins. It tends to be tubular rather than sheet-like, and it may be separate from the rER or an extension of it. The sER is abundant in cells that function in (i.e., cells that synthesize fatty acids and phospholipids), and it proliferates in hepatocytes when animals are challenged with lipophilic drugs. The sER is well developed in cells that synthesize and , such as in adrenocortical cells and testicular Leydig (interstitial) cells (Fig. 2.34). In skeletal and cardiac muscle, the sER is also called the .

lipid metabolism

steroids

secrete sarcoplasmic reticulum

It sequesters Ca2+ , which is essential for the contractile process and is closely apposed to the plasma membrane invaginations that conduct the contractile impulses to the interior of the cell. The sER is also involved in de novo biogenesis of peroxisomes (pages 64-65).

FIGURE 2.34. Electron micrograph of the smooth endoplasmic reticulum (sER).

This image shows numerous profiles of sER in an interstitial (Leydig) cell of the testis, a cell that produces steroid hormones. The sER seen here is a complex system of anastomosing tubules. The small, dense objects are glycogen particles. ×60,000.

The sER is the principal organelle involved in the detoxification of the xenobiotics. The sER is particularly well developed in the liver and contains a variety of detoxifying enzymes related to cytochrome P450. These enzymes are anchored directly into sER plasma

membranes (especially in the liver). The cytochrome P450 enzymatic compound represents a group of heme-containing enzymes that participate in the metabolism of many xenobiotics (foreign drugs or chemicals), steroids, and carcinogens. Cytochrome P450 enzymes modify and detoxify hydrophobic compounds such as drugs, pesticides, and carcinogens by chemically converting them into water-soluble conjugated products that can be eliminated from the body. In essence, cytochrome P450 enzymes in the sER of the liver control the speed at which drugs are metabolized and the duration for which the drugs are present in the body. Cytochrome P450 compound is also important in the synthesis of steroid hormones (i.e., estrogen and testosterone), fatty acids, and sterols (such as cholesterol and bile acids). Characterizing an individual’s enzymes may provide information about optimal therapeutic drug dosage levels, thus preventing complications of overdose or, conversely, ineffective treatment. For example, the that encodes a member of the cytochrome P450 enzyme family metabolizes the anticoagulant and other drugs such as phenytoin, tolbutamide, and ibuprofen. The ability to metabolize drugs is genetically determined, and a person can be classified as an ultrarapid, rapid, extensive, intermediate, or poor metabolizer. By identifying the specific gene variant an individual possesses and thus the type of metabolizer the person is, the warfarin dosage can be tailored to prevent internal bleeding, a common and potentially dangerous complication of this drug. Such personalization of therapeutics has been called . The degree to which the liver is involved in detoxification at any given time may be estimated by the amount of sER present in liver cells. The sER is also involved in

cytochrome P450

CYP2C9 gene

warfarin (blood thinner)

personalized medicine

lipid and steroid metabolism,

glycogen metabolism, and membrane formation and recycling. Because of these widely disparate functions, numerous other enzymes—including hydrolases, methylases, glucose-6-phosphatase, ATPases, and lipid oxidases—are associated with the sER, depending on its functional role.

Golgi Apparatus

The Golgi apparatus is well developed in secretory cells and does not stain with hematoxylin or eosin. The Golgi apparatus was described more than 100 years ago by the histologist Camillo Golgi. In studies of osmium-impregnated nerve cells, he discovered an organelle that formed networks around the nucleus. It was also described as well developed in secretory cells. Changes in the shape and location of the Golgi apparatus relative to its secretory state were described even before it was viewed with the EM and before its functional relationship to the rER was established. It is active both in cells that secrete protein by exocytosis and in cells that synthesize large amounts of membrane and membrane-associated proteins such as nerve cells. In the light microscope, secretory cells that have a large Golgi apparatus (e.g., plasma cells, osteoblasts, and cells of the epididymis) typically exhibit a clear area partially surrounded by ergastoplasm (Fig. 2.35). In the EM, the Golgi apparatus appears as a series of stacked, flattened, membrane-limited sacs or cisternae and tubular extensions embedded in a network of microtubules near the microtubule-organizing center or MTOC (see pages 75-77). Small vesicles involved in vesicular transport are seen in association with the cisternae.

FIGURE 2.35. Photomicrograph of plasma cells.

This photomicrograph of a plastic-embedded specimen showing the lamina propria of the small intestine is stained with toluidine blue. The plasma cells, where appropriately oriented, exhibit a clear area in the cytoplasm near the nucleus. These negatively stained regions ( ) represent extensive accumulation of membranous cisternae that belong to the Golgi apparatus. The surrounding cytoplasm is deeply metachromatically stained because of the presence of ribosomes associated with the extensive rough endoplasmic reticulum (rER). ×1,200.

arrows

The Golgi apparatus is polarized both morphologically and functionally. The flattened cisternae located closest to the rER represent the forming face, or the ; the cisternae located away from the rER represent the maturing face, or the (Figs. 2.36 and 2.37). The cisternae located between the TGN and CGN are commonly referred as .

(CGN) Golgi network (TGN)

the medial-Golgi network

cis-Golgi network trans-

FIGURE 2.36. Electron micrograph of the Golgi apparatus. This electron micrograph shows the extensive Golgi apparatus in an islet cell of the pancreas. The flattened membrane sacs of the Golgi apparatus are arranged in layers. The cis- Golgi network ( CGN ) is represented by the flattened vesicles on the outer convex surface, whereas the flattened vesicles of the inner convex region constitute the trans-Golgi network (TGN). Budding off the TGN are several vesicles (1) . These vesicles are released (2) and eventually become secretory vesicles (3) . ×55,000.

FIGURE 2.37. Electron micrograph of Golgi cisternae. a. This transmission electron micrograph shows a quick-frozen isolated Golgi apparatus replica from a cultured Chinese hamster ovary (CHO) cell line. The trans -Golgi cisternae are in the process of coated vesicle formation. b. Incubation of the trans-Golgi cisternae with the coatomer-depleted cytosol shows a decrease in vesicle formation activity. Note the lack of vesicles and the fenestrated shape of the trans -Golgi cisternae. ×85,000. (Courtesy of Dr. John E. Heuser, Washington University School of Medicine.)

The Golgi apparatus functions in the posttranslational modification, sorting, and packaging of proteins. Small COP-II-coated transport vesicles carry newly synthesized proteins (both secretory and membrane) from the rER to the CGN. From there, they travel within transport vesicles from one cisterna to the next. The vesicles bud from one cisterna and fuse with the adjacent cisternae (Fig. 2.38).

FIGURE 2.38. The Golgi apparatus and vesicular trafficking.

The Golgi apparatus contains several stacks of flattened cisternae with dilated edges. The Golgi cisternae form separate functional compartments. The closest compartment to the rough endoplasmic reticulum ( ) represents the Golgi network (CGN), to which COP-II-coated transport vesicles originating from the rER fuse and deliver newly synthesized proteins. Retrograde transport from the CGN to the rER, as well as retrograde transport between Golgi cisternae, is mediated by COP-I-coated vesicles. Once proteins have been modified within the CGN, the transport vesicles bud off dilated ends of this compartment, and proteins are transferred into -Golgi cisternae. The process continues; in the same fashion, proteins are translocated into the Golgi cisternae and further into the Golgi network ( ), where they are sorted into different transport vesicles that deliver them to their final destinations.

TGN

rER

medial trans-

cis

trans-

As proteins and lipids travel through the Golgi stacks, they undergo a series of that involve remodeling of -linked oligosaccharides previously added in the rER. In general, oligosaccharides present in glycoproteins and glycolipids are trimmed and translocated. Proteins and lipids undergo glycosylation by several carbohydrate-processing enzymes that add, remove, and modify sugar moieties of oligosaccharide chains. M-6-P is added to those proteins destined to travel to late endosomes and lysosomes (see pages 44-46). In addition, glycoproteins are phosphorylated and sulfated. The proteolytic cleavage of certain proteins is also initiated within the cisternae.

posttranslational modifications

N

Four major pathways of protein secretion from the Golgi apparatus disperse proteins to various cell destinations.

As noted, proteins exit the Golgi apparatus from the TGN. This network and the associated tubulovesicular array serve as the sorting station for shuttling vesicles that deliver proteins to the following locations (Fig. 2.39):

FIGURE 2.39. Summary of events in protein trafficking from the trans-Golgi network (TGN). The tubulovesicular array of the TGN serves as the sorting station for transporting vesicles that deliver proteins to the following destinations: (1) apical plasma membrane (i.e., epithelial cells); (2) apical region of the cell cytoplasm where proteins are stored in secretory vesicles (i.e., secretory cells); (3) early or late endosomal compartment; (4) selected proteins containing lysosomal signals, which are targeted to lysosomes; (5) lateral plasma membrane (i.e., epithelial cells); (6) basal plasma membrane (i.e., epithelial cells); (7) proteins destined for apical, basal, and lateral surfaces of plasma membrane, which are delivered to the basal plasma membrane (i.e., in hepatocytes); (8) all proteins endocytosed and sorted in early endosomes; (9) apical plasma membrane from early endosomes; (10) lateral plasma membrane; and (11) basal plasma membrane. Note the two targeting mechanisms of proteins to different surfaces of plasma membrane. In epithelial cells, proteins are directly targeted from the TGN into the appropriate cell surface as shown in steps 1 , 5 , and 6 . In hepatocytes, all proteins are secreted first to the basal cell surface, after which they are distributed to the appropriate cell surface via the endosomal compartment as shown in steps 7 –11 .

Apical plasma membrane.

Many extracellular and membrane proteins are delivered to this site. This constitutive pathway most likely uses non–clathrin-coated vesicles. In most cells, secretory proteins destined for the apical plasma membrane have specific sorting signals that guide their sorting process in the TGN. Proteins are then delivered to the apical cell surface. . Proteins targeted to the basolateral domain also have a specific sorting signal attached to them by the TGN. This constitutive pathway uses vesicles coated with unique proteins that bind to epithelium-specific adaptor proteins. The transported membrane proteins are continuously incorporated into the basolateral cell surface. This type of targeting is present in most polarized epithelial cells. In liver hepatocytes, however, the process of protein sorting into the basolateral and apical domains is quite different. All integral plasma membrane proteins that are destined for

Basolateral plasma membrane

both apical and basolateral domains are first transported from the TGN to the basolateral plasma membrane. From there, both proteins are endocytosed and sorted into early endosomal compartments. Basolateral proteins are recycled back into the basolateral membrane, whereas apical proteins are transported across the cytoplasm to the apical cell membrane via transcytosis. . Most proteins destined for organelles bear specific signal sequences. They are sorted in the TGN and delivered to specific organelles. However, TGN sorting mechanisms are never completely accurate. For instance, about 10% of LIMPs instead travel directly into early or late endosomes, traveling an extended route via the apical plasma membrane (see Fig. 2.23), and from there move back into the endosomal pathways. Enzymes destined for lysosomes using M-6-P markers (see pages 46-47) are delivered into early or late endosomes as they develop into mature lysosomes. . Proteins that were aggregated or crystallized in the TGN as a result of changes in pH and Ca2+ concentration are stored in large . These vesicles undergo a maturation process in which secretory proteins are retained within the vesicle. All other nonsecretory proteins are recycled into the endosomal compartment or TGN in clathrin-coated vesicles (see Fig. 2.38). Mature secretory vesicles eventually fuse with the plasma membrane to release the secretory product by exocytosis. This type of secretion is characteristic of highly specialized secretory cells found in exocrine glands.

Endosomes or lysosomes

Apical cytoplasm

secretory vesicles

Sorting and packaging of proteins into transport vesicles occurs in the trans-Golgi network. Proteins that arrive in the TGN are distributed to different intracellular locations within transport vesicles. The intracellular destination of each protein depends on the sorting signals that are incorporated within the polypeptide chain of the protein. The actual sorting and packaging of proteins in TGN is primarily based on sorting signals and physical properties.

Sorting signals

are represented by the linear array of amino acid or associated carbohydrate molecules. This type of signal is recognized by the sorting machinery, which directs the protein into the appropriately coated transport vesicle. are important for packaging functionally associated protein complexes. These groups of proteins are first partitioned into separate lipid rafts that are later incorporated into transport vesicles destined for a targeted organelle.

Physical properties

Mitochondria

Mitochondria are abundant in cells that generate and expend large amounts of energy. Mitochondria were also known to early cytologists who observed them in cells vitally stained with Janus Green B. It is now evident that mitochondria increase in number by division throughout interphase, and their divisions are not synchronized with the cell cycle. Video microscopy confirms that mitochondria can both change their location and undergo transient changes in shape. They may therefore be compared to mobile power generators as they migrate from one area of the cell to another to supply needed energy. Because mitochondria generate ATP, they are more numerous in cells that use large amounts of energy, such as striated muscle cells and cells engaged in fluid and electrolyte transport. Mitochondria also localize at sites in the cell where energy is needed, as in the middle piece of sperm cells, the intermyofibrillar spaces in striated muscle cells, and adjacent to the basolateral plasma membrane infoldings in the cells of the proximal convoluted tubule of the kidney.

Mitochondria evolved from aerobic bacteria that were engulfed by eukaryotic cells. Mitochondria are believed to have evolved from an aerobic prokaryote (Eubacterium)

that lived symbiotically within primitive eukaryotic cells. This hypothesis received support with

the demonstration that mitochondria possess their own genome, increase their numbers by division, and synthesize some of their structural (constituent) proteins. is a small, closed circular molecule that encodes 13 enzymes involved in the oxidative phosphorylation pathway, 2 mitochondrial rRNAs that are essential components of its own translational apparatus, and 22 transfer RNAs (tRNAs) used in the translation of the mitochondrial mRNA. The mitochondrial in nature and is . Mitochondria possess a , including the synthesis of their own ribosomes. The remainder of the mitochondrial proteins is encoded by nuclear DNA; new polypeptides are synthesized by free ribosomes in the cytoplasm and then imported into mitochondria with the help of two protein complexes. These include and . Translocation of proteins through mitochondrial membranes requires energy and assistance from several specialized chaperone proteins.

Mitochondrial DNA

(mtDNA)

from the mother

DNA is haploid complete system for protein synthesis

inherited exclusively

translocase of the outer mitochondrial membrane (TOM) complexes translocase of the inner mitochondrial membrane (TIM) complexes Mitochondria are present in all cells except red blood cells and terminal keratinocytes.

The number, shape, and internal structure of mitochondria are often characteristic for specific cell types. When present in large numbers, mitochondria contribute to the acidophilia of the cytoplasm because of the large amount of membrane they contain. Mitochondria may be stained specifically by histochemical procedures that demonstrate some of their constituent enzymes, such as those involved in ATP synthesis and electron transport.

Mitochondria possess two membranes that delineate distinct compartments.

Mitochondria display a variety of shapes, including spheres, rods, elongated filaments, and even coiled structures. All mitochondria, unlike other organelles described earlier, possess two membranes (Fig. 2.40). The surrounds a space called the . The is in close contact with the cytoplasm. The space between the two membranes is called the . The following structural components of mitochondria possess specific characteristics related to their functions.

matrix

inner mitochondrial membrane outer mitochondrial membrane intermembrane space

FIGURE 2.40. Structure of the mitochondrion. a.

This electron micrograph shows a mitochondrion in a pancreatic acinar cell. Note that the inner mitochondrial membrane forms the cristae ( ) through a series of infoldings, as is evident in the region of the . The outer mitochondrial membrane is a smooth continuous envelope that is separate and distinct from the inner membrane. ×200,000. Schematic diagram showing the components of a mitochondrion. Note the location of the elementary particles ( ), the shape of which reflects the three-dimensional structure of adenosine triphosphate ( ) synthase.

inset ATP

arrow

C

b.

Outer mitochondrial membrane. This 6- to 7-nm-thick smooth membrane contains many voltage-dependent anion channels (also called mitochondrial porins). These large

channels (approximately 3 nm in diameter) are permeable to uncharged molecules as large as 5 kDa. Thus, small molecules, ions, and metabolites can enter the intermembrane space but cannot penetrate the inner membrane. The environment of the intermembrane space is therefore similar to that of cytoplasm with respect to ions and small molecules. The outer membrane possesses receptors for proteins and polypeptides that translocate into the intermembrane space. It also contains several enzymes, including phospholipase A2, monoamine oxidase, and acetyl coenzyme A (CoA) synthase. . The TEM reveals that this membrane is thinner than the outer mitochondrial membrane. It is arranged into numerous (folds) that significantly increase the inner membrane surface area (see Fig. 2.40). These folds project into the matrix that constitutes the inner compartment of the organelle. In some cells involved in steroid metabolism, the inner membrane may form tubular or vesicular projections into the matrix. The inner membrane is rich in the phospholipid , which makes the membrane impermeable to ions. The membrane forming the cristae contains proteins that have three major functions: performing the of the respiratory electron transport chain, , and of metabolites into and out of the matrix. The enzymes of the are attached to the inner membrane and project their heads into the matrix (see Fig. 2.40). With the TEM, these enzymes appear as tennis racquet–shaped structures called . Their heads measure about 10 nm in diameter and contain enzymes that carry out oxidative phosphorylation, which generates ATP. . This space is located between the inner and outer membranes and contains specific enzymes that use the ATP generated in the inner membrane. These enzymes include creatine kinase, adenylate kinase, and . The latter is an important factor in initiating apoptosis (see pages 104-107). . The mitochondrial matrix is surrounded by the inner mitochondrial membrane and contains the soluble enzymes of the and the enzymes involved in . The major products of the matrix are CO2 and reduced NADH, which is the source of electrons for the electron transport chain. Mitochondria contain dense that store Ca2+ and other divalent and trivalent cations. These granules increase in number and size when the concentration of divalent (and trivalent) cations increases in the cytoplasm. Mitochondria can accumulate cations against a concentration gradient. Thus, in addition to ATP production, mitochondria also regulate the concentration of certain ions of the cytoplasmic matrix, a role they share with the sER. The matrix also contains mitochondrial DNA, ribosomes, and tRNAs.

Inner mitochondrial membrane

cristae

cardiolipin oxidation reactions synthesizing ATP regulating transport respiratory chain elementary

particles Intermembrane space Matrix

cytochrome c

fatty acid β-oxidation matrix granules

citric acid cycle (Krebs cycle)

Mitochondria contain the enzyme system that generates ATP by means of the citric acid cycle and oxidative phosphorylation. Mitochondria generate ATP in a variety of metabolic pathways, including oxidative phosphorylation, the citric acid cycle, and β-oxidation of fatty acids. The energy generated from these reactions, which take place in the mitochondrial matrix, is represented by hydrogen ions (H+ ) derived from reduced NADH. These ions drive a series of located within the inner mitochondrial membrane that transfer H+ from the matrix to the intermembrane space (Fig. 2.41). These pumps constitute the of respiratory enzymes (see Fig. 2.40). The transfer of H+ across the inner mitochondrial membrane establishes an . This gradient creates a that causes the movement of H+ to occur down its electrochemical gradient through a large, membrane-bound enzyme called . ATP synthase provides a pathway across the inner mitochondrial membrane in which H+ ions are used to drive the energetically unfavorable reactions leading to synthesis of ATP. This movement of protons back to the mitochondrial matrix is referred to as . The newly produced ATP is transported from the matrix to the intermembrane space by the voltage gradient–driven located in the inner mitochondrial membrane. From

proton motive force

proton pumps electron transport chain electrochemical proton gradient large ATP synthase

ATP/ADP exchange protein

chemiosmotic coupling

voltage-dependent anion channels (VDACs)

here, ATP leaves the mitochondria via in the outer membrane to enter the cytoplasm. At the same time, ADP produced in the cytoplasm rapidly enters the mitochondria for recharging.

FIGURE 2.41. Schematic diagram illustrating how mitochondria generate energy. The diagram indicates the adenosine triphosphate ( ATP ) synthase complex and the electron transport chain of proteins located in the inner mitochondrial membrane. The electron transport chain generates a proton gradient between the matrix and intermembrane space that is used to produce ATP. Numbers represent sequential proteins involved in the electron transport chain and ATP production: 1 , NADH dehydrogenase complex; 2 , ubiquinone; 3 , cytochrome b–c 1 complex; 4 , cytochrome c ; 5 , cytochrome oxidase complex; and 6 , ATP synthase complex. ADP , adenosine diphosphate.

mitochondrial defects

Several are related to defects in enzymes that produce ATP. Metabolically active tissues that use large amounts of ATP, such as muscle cells and neurons, are the most commonly affected. For example, is characterized by muscle weakness, ataxia, seizures, and cardiac and respiratory failure. Microscopic examination of muscle tissue from affected patients shows aggregates of abnormal mitochondria that provide a ragged appearance of red muscle fibers. MERRF is caused by mutation of the mitochondrial DNA gene encoding tRNA for lysine. This defect produces two abnormal complexes in the electron transport chain of respiratory enzymes affecting ATP production.

fibers (MERRF)

myoclonic epilepsy with ragged red

Mitochondria decide whether the cell lives or dies.

Experimental studies indicate that mitochondria sense cellular stress and are capable of deciding whether the cell lives or dies by initiating (programmed cell death). The major cell death event generated by the mitochondria is the release of cytochrome from the mitochondrial intermembranous space into the cell cytoplasm. Changes in the at the outer mitochondrial membrane are responsible for this release. This event, regulated by the proapoptotic (see pages 106-107), initiates the cascade of proteolytic enzymatic reactions that lead to apoptosis. The Bcl-2 family of proteins thus controls cell death primarily by regulating permeability of the outer mitochondrial membrane, which leads to the irreversible release of cytochrome , subsequent caspase activation, and apoptosis. However, in certain conditions (e.g., translational modifications), the Bcl-2 proteins may act as antiapoptotic agents.

apoptosis

dependent anion channels (VDACs)

Bcl-2 protein family

c voltage-

c

Mitochondria undergo morphologic changes related to their functional state. TEM studies show mitochondria in two distinct configurations. In the orthodox configuration, the cristae are prominent, and the matrix compartment occupies a large part of the total mitochondrial volume. This energized mitochondrion configuration is observed in

c

healthy cells. In this configuration, most of the cytochrome is sequestered within the cristae and is resistant to release by agents that disrupt the mitochondrial outer membrane. Matrix remodeling to the results in depolarization of mitochondrial membranes. This configuration is characterized by unfolded cristae that are not easily recognized in the TEM. The matrix is reduced in volume and appears more concentrated, whereas the intermembrane space increases to as much as 50% of the total volume of the organelle. These changes expose cytochrome to the intermembrane space, facilitating its release from the mitochondria during apoptosis.

condensed configuration c

Peroxisomes

Peroxisomes are single-membrane-bound organelles containing a variety of oxidative enzymes. Peroxisomes (historically called microbodies) are dynamic, multifunctional membrane-limited

spherical organelles with an oxidative type of metabolism. When observed in TEM, they resemble small (0.1–0.5 μm in diameter) vesicles distributed throughout the cell cytoplasm. Because peroxisomes are involved in cellular lipid metabolism, they interact with the ER, mitochondria, lysosomes, and lipid droplet inclusions. Recent studies indicate that peroxisomes have inter-organelle membrane contact sites that allow the exchange of metabolites, lipids, and proteins with the ER, mitochondria, and lysosomes. Although abundant in liver and kidney cells, peroxisomes are found in almost all cells in the body. They vary in size and number and typically range from 102 to 103 peroxisomes per cell. The number of peroxisomes in a cell increases in response to diet, drugs, and hormonal stimulation. In most animals, but not humans, peroxisomes also contain urate oxidase (uricase), which often appears as a characteristic . Because peroxisomes contain , particularly catalase and other peroxidases, researchers originally thought that the primary function of these organelles was the metabolism of , particularly . Almost all oxidative enzymes produce ROIs during oxidation reactions that are toxic 22 to cells. Some cells, such as phagocytic cells, use ROIs to immobilize and kill ingested live bacteria (see pages 310-311). The catalase in peroxisomes carefully regulates the , thus protecting the cell. For example, hydrogen peroxide is broken down into water (H2O) and oxygen (O2). Oxidative enzymes are particularly important in liver cells (hepatocytes), where they perform a variety of detoxification processes. For instance, peroxisomes in hepatocytes are responsible for detoxifying ingested alcohol by converting it to acetaldehyde. However, recent evidence suggests that peroxisomes play a primary function in lipid metabolism, contributing to the breakdown and detoxification of fatty acids. Although medium and long chain fatty acids are mainly oxidized in mitochondria, peroxisomes almost exclusively metabolize very long chain fatty acids ( and branched chain fatty acids ( . In addition, peroxisomes in mammalian cells are involved in lipid biosynthesis of cholesterol, dolichol, and fatty acid–linked phospholipids (e.g., ether phospholipids found in the myelin sheath). In the liver, peroxisomes are also involved in the synthesis of bile acids, which are derived from cholesterol. As mentioned earlier, β-oxidation also occurs in mitochondria. Peroxisomal β-oxidation in some cells may equal that occurring in mitochondria and thus aids in maintaining metabolic fatty acid cell homeostasis. A summary of the functions of peroxisomes is shown in Figure 2.42.

(H O )

crystalloid core inclusion oxidative enzymes reactive oxygen intermediates (ROIs) hydrogen peroxide

removal of ROIs

acids)

β-oxidation of very long chain fatty α-oxidation of branched chain fatty acids)

FIGURE 2.42. Schematic diagram of a peroxisome.

This diagram shows peroxisomal enzymes residing within the peroxisome matrix and their respective metabolic pathways. Selected peroxisomal membrane proteins are also shown. The crystalloid core of oxidases may not be present in all species. ABCD1, ATP-binding cassette (ABC) transporters in subfamily D; ADHAPR, alkyl dihydroxyacetone phosphate reductase; ADHAPS, alkyl dihydroxyacetone phosphate synthase; DHAPAT; dihydroxyacetone phosphate acyltransferase; PMP-70, peroxisomal membrane protein-70; ROI, reactive oxygen intermediate.

In addition to their metabolic roles, peroxisomes have recently been implicated in cellular signaling in various metabolic pathways. Different levels of peroxisomal reactive oxygen species in peroxisomes may influence intracellular processes promoting autophagy, improving cell survival, and modulating cellular immunity.

Peroxisomal biogenesis is complex; they may arise from preexisting peroxisomes, or they can be synthesized de novo. The biogenesis of peroxisomes is still incompletely understood. In general, two models of biogenesis are generally accepted:

classical model

In the , peroxisomes arise from preexisting peroxisomes through growth and division. This observation is supported by TEM studies of pharmacologically induced proliferation of peroxisomes in the liver. In the , peroxisomes are generated de novo by acquiring membrane proteins and lipids from the sER and their matrix proteins from the cytoplasm. De novo formation of peroxisomes has been confirmed in human cells using immunofluorescent techniques with labeled peroxin molecules.

alternative model

De novo formation is initiated by the formation of pre-peroxisomal vesicles (PPVs) that bud off from the surface of sER (Fig. 2.43). At least two classes of PPVs have been identified containing different populations of , a family of 32 known peroxisomal proteins required for the assembly and function of peroxisomes. Protein segregation within the two PPV classes prevents premature assembly of peroxisomes. These early precursors of peroxisomes undergo fusion and are further targeted for delivery of specific peroxisomal matrix proteins and additional peroxisomal membrane proteins synthesized on free ribosomes in the cytoplasm. All proteins destined for peroxisomes must have a simple three amino acid

peroxins

Ser-Lys-Leu

motif, , known as the carboxy-terminus (PTS1).

peroxisomal targeting signal (PTS)

attached to its

FIGURE 2.43. De novo formation of peroxisomes.

Peroxisomal membrane proteins are synthesized on free ribosomes in the cytoplasm. Initially, they are incorporated into the smooth endoplasmic reticulum (sER). The region of sER rich in peroxisomal membrane proteins buds off the sER to form preperoxisomal vesicles. Two classes of vesicles (V 1 and V 2 ) containing different classes of peroxisomal membrane proteins are depicted in the initial stage of peroxisome formation. Segregation of proteins into separate vesicles prevents their interaction and the premature assembly of peroxisomes. Fusion of the two classes of vesicles results in a precursor of the peroxisome. This precursor is further targeted for delivery of specific peroxisomal matrix and membrane proteins. Delivery of these proteins to the peroxisome is aided by soluble cytoplasmic receptor (chaperone) proteins. Chaperones bind to respective peroxisomal proteins in the cytoplasm, transport them to the place of docking (Pex3 and Pex16), and insert them either into the peroxisomal membrane (Pex19) or act as import receptors (Pex5 or Pex7) to guide matrix proteins into the lumen. After the insertion of proteins, chaperones are recycled back into the cytoplasm. Note that proteins destined for the peroxisomes have a peroxisomal targeting signal.

Peroxisomal membrane proteins Pex19

are inserted into the peroxisomal membrane with the aid of peroxin 19 ( ) protein. This soluble chaperone binds to peroxisomal membrane proteins in the cytoplasm and transports them to the place of docking (Pex3 and Pex16) and insertion into the peroxisomal membrane. After insertion, Pex19 is recycled back to the cytoplasm. also utilize peroxins ( and ) as import receptors. These receptors bind peroxisomal matrix proteins in the cytoplasm and transport them to docking receptors (Pex3 and Pex16) at the peroxisomal membrane, where the proteins are delivered to the peroxisomal lumen. The receptors recycle back to the cytoplasm. In general, peroxisomal disorders are caused by impaired peroxisomal activity or lack of peroxisomes because of defective peroxisomal biogenesis. Various human metabolic disorders are caused by mutations in the encoding the (peroxins) required for peroxisome biogenesis and function. These mutations result in the inability to import peroxisomal matrix and membrane proteins into the organelle. are associated with impaired peroxisomal lipid metabolism (e.g., resulting in the accumulation of very long chain fatty acids, branched chain fatty acids) and defective synthesis of ether lipids and bile acids. include and The most common inherited Zellweger spectrum disorders are , , and . Zellweger syndrome is the most severe of these three disorders. It causes craniofacial malformations, hepatomegaly, neurologic and developmental abnormalities, retinal degeneration, and deafness. Most infants affected by Zellweger syndrome do not survive past 1 year of age. The clinical features of neonatal adrenoleukodystrophy and infantile Refsum

Peroxisomal matrix proteins

Pex5

PEX genes

Pex7

peroxisomal proteins Peroxisomal disorders

Peroxisomal biogenesis disorders Zellweger spectrum disorders (ZSDs) rhizomelic chondrodysplasia punctata type 1 (RCDP1). cerebrohepatorenal Zellweger syndrome neonatal adrenoleukodystrophy (NALD) infantile Refsum disease

disease are similar to those of Zellweger syndrome; however, these disorders progress more slowly. Mutations in Pex3, Pex16, and Pex19 genes cause the most severe phenotypes, which result in the complete absence of peroxisomes. Therapies for peroxisomal disorders have been unsatisfactory to date.

NONMEMBRANOUS ORGANELLES Microtubules Microtubules

are nonbranching and rigid hollow tubes of polymerized protein that can rapidly assemble and equally rapidly disassemble. In general, microtubules are found in the cytoplasm, where they originate from the . They grow from the MTOC located near the nucleus and extend toward the cell periphery. Microtubules are also present in cilia and flagella, where they form the axoneme and its anchoring basal body; in centrioles and the mitotic spindle; and in elongating processes of the cell, such as those in growing axons. Microtubules are involved in numerous essential cellular functions:

microtubule-organizing center (MTOC)

Intracellular vesicular transport

(i.e., movement of secretory vesicles, endosomes, and lysosomes). Microtubules create a system of connections within the cell, frequently compared with railroad tracks originating from a central station, along which vesicular movement occurs.

Movement of cilia and flagella Attachment of chromosomes to the mitotic spindle and their movement meiosis Maintenance of cell shape, particularly its asymmetry Regulatory effect on cell elongation and movement (migration)

during mitosis and

Although microtubules may exert a regulatory effect on cell elongation and movement, they are not essential for these functions, which are mediated by actin polymerization (see pages 69-70). Microtubules play an indirect role by regulating actin polymerization, organizing transport of vesicles to the leading edge of migrating cells, and facilitating disassembly of focal adhesions (see pages 160-161). In addition, microtubules may restrain cell locomotion by slowing the retraction of the trailing edge (tail) of the migrating cell, thus influencing the direction of cell migration.

Microtubules are elongated polymeric structures composed of equal parts of α-tubulin and β-tubulin. Microtubules measure 20–25 nm in diameter. The wall of the microtubule is approximately 5 nm thick and consists of 13 circularly arrayed globular dimeric tubulin molecules. The tubulin dimer has a molecular weight of 110 kDa and is formed from an α-tubulin and a βtubulin molecule, each with a molecular weight of 55 kDa (Fig. 2.44). The dimers polymerize in an end-to-end fashion, head to tail, with the α molecule of one dimer bound to the β molecule of the next dimer in a repeating pattern. Longitudinal contacts between dimers link them into a linear structure called a . Axial periodicity seen along the 5-nmdiameter dimers corresponds to the length of the protein molecules. A small, 1-μm segment of microtubule contains approximately 16,000 tubulin dimers.

protofilament

FIGURE 2.44. Polymerization of microtubules.

left

On the , the diagram depicts the process of polymerization of tubulin dimers during microtubule assembly. Each tubulin dimer consists of an αtubulin and β-tubulin subunit. The plus (+) end of the microtubule is the growing end to which tubulin dimers bound to guanosine triphosphate ( ) molecules are incorporated into a curved sheet, which in turn closes into a tube. Incorporated tubulin dimers hydrolyze GTP, which releases the phosphate groups to form polymers with guanosine diphosphate ( )-tubulin molecules. The minus (−) end of the microtubule contains a ring of γ-tubulin, which is necessary for microtubule nucleation. This end is usually embedded within the microtubule-organizing center (MTOC) and possesses numerous capping proteins. On the is a diagram showing that each microtubule contains 13 tubulin dimers within its cross section.

GTP

GDP

right

Microtubules grow from γ-tubulin rings within the MTOC that serve as nucleation sites for each microtubule. Microtubule formation can be traced to hundreds of γ-tubulin rings that form an integral part of the MTOC and function as templates for the correct assembly of microtubules. Their nucleation pattern initiated in the MTOC can be studied in vitro (Fig. 2.45). The α- and β-tubulin dimers are added to a γ-tubulin ring in an end-to-end fashion. The most simplistic model used in the past described microtubule assembly as a process of adding tubulin dimers one by one onto the growing end of a fully formed microtubule. However, a

number of experimental studies using cryoelectron microscopy have shown that the initial assembly occurs from a curved sheet made of tubulin dimers, which in turn closes into a tube at the growing end of the microtubule (see Fig. 2.44).

FIGURE 2.45. Nucleation activity of microtubules observed in vitro using immunocytochemical methods. The behavior of microtubules obtained from human breast cancer cells can be studied in vitro

by measuring their nucleation activity. Microtubules were labeled with a mixture of anti-α-tubulin and anti-β-tubulin monoclonal antibodies (primary antibodies) and visualized by secondary antibodies conjugated with fluorescein dye (fluorescein isothiocyanate–goat antimouse immunoglobulin G). Polymerization of tubulin dimers is responsible for the formation of more than 120 microtubules visible on this image. They originate from the microtubule-organizing center (MTOC) and extend outward approximately 20–25 μm in a uniform radial array. ×1,400. (Photomicrograph courtesy of Drs. Wilma L. Lingle and Vivian A. Negron.)

guanosine triphosphate (GTP)

Polymerization of tubulin dimers requires the presence of 2+ . Each tubulin molecule binds GTP before it is incorporated into the forming and microtubule. The tubulin dimers containing GTP have a conformation that favors stronger lateral interactions between dimers resulting in polymerization. At some point, GTP is hydrolyzed to guanosine diphosphate (GDP). As a result of this polymerization pattern, microtubules are polar structures because all of the dimers in each protofilament have the same orientation. Each microtubule possesses a that corresponds to α-tubulin; in the cell, it is usually embedded in the MTOC and often stabilized by actin-capping proteins (see Fig. 2.44). The of microtubules corresponds to β-tubulin and extends the cell periphery. Tubulin dimers dissociate from microtubules in the steady state, which creates a pool of free tubulin dimers in the cytoplasm. This pool is in equilibrium with the polymerized tubulin in the microtubules; therefore, polymerization and depolymerization are in equilibrium. The equilibrium can be shifted in the direction of depolymerization by exposing the cell or isolated microtubules to low temperatures or high pressure. Repeated exposure to alternating low and high temperatures is the basis of the purification technique for tubulin and microtubules. The speed of polymerization or depolymerization can also be modified by interaction with specific . These proteins, such as MAP-1, MAP-2, MAP-3, and MAP-4; MAP-τ; and TOG-ρ, regulate microtubule assembly and anchor the microtubules to specific organelles. MAPs are also responsible for the existence of stable populations of nondepolymerizing microtubules in the cell, such as those found in cilia and flagella.

Mg

nongrowing (–) end end

growing (+)

microtubule-associated proteins (MAPs)

The length of microtubules constantly changes as dimers are added or removed in a process of dynamic instability. Microtubules observed in cultured cells with real-time video microscopy appear to be constantly growing toward the cell periphery by addition (polymerization) of tubulin dimers and then suddenly shrinking in the direction of the MTOC by removal (depolymerization) of tubulin dimers (Fig. 2.46). This constant remodeling process, known as , is linked to a pattern of GTP hydrolysis during the microtubule assembly and disassembly process. The tubulin dimers bound to GTP at the growing (+) end of the microtubule protect it from disassembly. In contrast, tubulin dimers bound to GDP are prone to depolymerization that leads to rapid microtubule disassembly and shrinking. During disassembly, the tubulin dimers bound to GDP lose lateral interaction with each other and protofilaments of the tubulin dimers curl away from the end of the microtubule, producing “split ends” (see Fig. 2.46). The process of switching from a growing to a shrinking microtubule is often called a .

dynamic instability

microtubule catastrophe

FIGURE 2.46. Depolymerization of microtubules.

Microtubules are dynamic structures involved in a constant remodeling process known as dynamic instability. They elongate by addition (polymerization) of tubulin dimers bound to guanosine triphosphate ( ) and then quickly shrink by removal (depolymerization) of tubulin dimers that hydrolyzed GTP. The tubulin dimers bound to guanosine diphosphate ( ) are prone to depolymerization by losing lateral interactions between each other. This allows for protofilaments to curl away from the end of the microtubule. Note the arrangement of tubulin dimers in a single protofilament highlighted in .

GDP

GTP

pink

The MTOC can be compared to a feeding chameleon, which fires its long, projectile tongue to make contact with potential food. The chameleon then retracts its tongue back into its mouth and repeats this process until it is successful in obtaining food. The same strategy of “firing” dynamic microtubules from the MTOC toward the cell periphery and subsequently retracting them enables microtubules to search the cytoplasm. When the fired microtubule

encounters stabilization factors (such as MAPs), it is captured and changes its dynamic behavior. This allows the cell to establish an organized system of microtubules linking peripheral structures and organelles with the MTOC. As mentioned earlier, the association of a microtubule with MAPs (e.g., within the axoneme of a cilium or flagellum) effectively blocks this dynamic instability and stabilizes the microtubules. In certain cells, such as neurons, some microtubules that nucleated at the MTOC can be released by the action of a called . Short, detached polymers of microtubules are then transported along existing microtubules by molecular motor proteins such as kinesins. The structure and function of microtubules in mitosis and in cilia and flagella are discussed later in this chapter and in Chapter 5, Epithelial Tissue, pages 128-134.

selective stabilization process

microtubule-severing protein

katanin

Microtubules can be visualized with a variety of imaging methods.

EM of both in vitro isolated microtubules and in vivo microtubules within the cell cytoplasm is an essential tool for examining their structure and function. Microtubules can be readily visualized with the TEM as shown in Figure 2.47. High-resolution images of microtubules have been obtained with cryoelectron microscopy aided by tomographic reconstruction of their unique molecular structure (Fig. 2.48). In addition, high-resolution images of microtubules can also be obtained using atomic force microscopy. In the past, microtubules were observed in the light microscope by using special stains, polarization, or phase contrast optics. Because of the limited resolution of the light microscope, microtubules may now be easily distinguished from other components of the cell cytoskeleton with immunocytochemical methods using tubulin antibodies conjugated with fluorescent dyes (Fig. 2.49).

FIGURE 2.47. Electron micrographs of microtubules. a. Micrograph showing microtubules (arrows) of the mitotic spindle in a dividing cell. On the right , the microtubules are attached to chromosomes. ×30,000. b. Micrograph of microtubules ( arrows ) in the axon of a nerve cell. In both cells, the microtubules are seen in longitudinal profile. ×30,000.

FIGURE 2.48. Three-dimensional reconstruction of an intact microtubule.

This image was obtained using cryoelectron microscopy. Tomographic (sectional) images of a frozen hydrated microtubule were collected and digitally reconstructed at a resolution of 8 Å. The helical structure of the α-tubulin molecules is recognizable at this magnification. ×3,250,000. (Courtesy of Dr. Kenneth Downing.)

FIGURE 2.49. Staining of microtubules with fluorescent dye.

This confocal immunofluorescent image shows the organization of the microtubules within an epithelial cell in tissue culture. In this example, the specimen was immunostained with three primary antibodies against tubulin ( ), centrin ( ), and kinetochores ( ) and then incubated in a mixture of three different fluorescently tagged secondary antibodies that recognized the primary antibodies. Nuclei were stained ( ) with a fluorescent molecule that intercalates into the DNA double helix. Note that the microtubules are focused at the microtubule-organizing center (MTOC) or centrosome ( ), located adjacent to the nucleus. The cell is in the S phase of the cell cycle, as indicated by the presence of both large unduplicated kinetochores and smaller pairs of duplicated kinetochores. ×3,000. (Courtesy of Drs. Wilma L. Lingle and Vivian A. Negron.)

red

light blue

red

green dark blue

Movement of intracellular organelles is generated by molecular motor proteins associated with microtubules. In cellular activities that involve movement of organelles and other cytoplasmic structures —such as transport vesicles, mitochondria, and lysosomes—microtubules serve as guides to the appropriate destinations. attach to these organelles or structures and ratchet along the microtubule track (Fig. 2.50). The energy required for the ratcheting movement is derived from ATP hydrolysis. Two families of molecular motor proteins have been identified that allow for unidirectional movement:

Molecular motor proteins

FIGURE 2.50. Molecular motor proteins associated with microtubules.

Microtubules serve as guides for molecular motor proteins. These adenosine triphosphate (ATP)-driven microtubule-associated motor proteins are attached to moving structures (such as organelles) that ratchet them along a tubular track. Two types of molecular motors have been identified: dyneins that move along microtubules toward their minus (−) end (i.e., toward the center of the cell) and kinesins that move toward their plus (+) end (i.e., toward the cell periphery).

Dyneins constitute one family of molecular motors. They move along the microtubules toward the minus (–) end of the tubule. Therefore, cytoplasmic dyneins are capable of transporting organelles from the cell periphery toward the MTOC. One member of the dynein family, axonemal dynein, is present in cilia and flagella. It is responsible for the sliding of one microtubule against an adjacent microtubule within the axoneme, thus effecting their movement. , members of the other family, move along the microtubules ; therefore, they are capable of moving organelles from the cell center toward the cell periphery.

Kinesins end

toward the plus (+)

Both dyneins and kinesins are involved in mitosis and meiosis. In these activities, dyneins move the chromosomes along the microtubules of the mitotic spindle. Kinesins are simultaneously involved in movement of polar microtubules. These microtubules extend from one spindle pole past the metaphase plate and overlap with microtubules extending from the opposite spindle pole. Kinesins located between these microtubules generate a sliding movement that reduces the overlap, thereby pushing the two spindle poles apart toward each daughter cell (Fig. 2.51).

FIGURE 2.51. Distribution of kinesin-like motor protein within the mitotic spindle. This confocal immunofluorescent image shows a mammary gland epithelial cell in anaphase of mitosis. Each mitotic spindle pole contains two centrioles ( green ). A mitosis-specific kinesin-like molecule called Eg5 ( red ) is associated with the subset of the mitotic spindle microtubules that connect the kinetochores ( white ) to the spindle poles. The motor action of Eg5 is required to separate the sister chromatids ( blue ) into the daughter cells. This cell was first immunostained with three primary antibodies against Eg5 ( red ), centrin ( green ), and kinetochores ( white ) and then incubated in three different fluorescently tagged secondary antibodies that recognize the primary antibodies. Chromosomes were stained with a fluorescent molecule that intercalates into the DNA double helix. ×3,500. (Courtesy of Drs. Wilma L. Lingle and Vivian A. Negron.)

Actin Filaments

Actin filaments are present in virtually all cell types. Actin molecules (42 kDa) are abundant and may constitute as much as 20% of the total protein

of some nonmuscle cells (Fig. 2.52). Similar to the tubulin in microtubules, actin molecules also assemble spontaneously by polymerization into a linear helical array to form filaments 6–8 nm in diameter. They are thinner, shorter, and more flexible than microtubules. Free actin molecules in the cytoplasm are referred to as , in contrast to the polymerized actin of the filament, which is called . An actin filament or microfilament is a polarized structure; its fast-growing end is referred to as the , and its slow-growing end is referred to as the .

end

plus (barbed) end

G-actin (globular actin) F-actin (filamentous actin) minus (pointed)

FIGURE 2.52. Distribution of actin filaments in pulmonary artery endothelial cells in culture. Cells were fixed and stained with NDB-phallacidin stain conjugated with fluorescein dye.

Phallacidin binds and stabilizes actin filaments, preventing their depolymerization. Note the accumulation of actin filaments at the periphery of the cell just beneath the plasma membrane. These cells were also stained with two additional dyes: a mitochondria-selective dye (MitoTracker Red) that allows the visualization of mitochondria ( ) in the middle of the cell and DAPI stain that reacts with nuclear DNA and exhibits fluorescence over the nucleus. ×3,000. (Courtesy of Molecular Probes, Inc., Eugene, OR.)

blue

red

Actin is preferentially added to the plus end of the actin filament, and it dissociates from the minus end. The dynamic process of actin polymerization that occurs mainly at the plus end of the actin 2+ , and filament requires the presence of + , . After each G-actin molecule is incorporated into the filament, ATP is hydrolyzed to ADP. However, the phosphate group release from the ATP hydrolysis is not immediate, and the transient form of actin bound to ADP and the free phosphate group persist in filaments (Fig. 2.53).

K Mg

ATP

FIGURE 2.53. Polymerization of actin filaments. Actin filaments are polarized structures. Their fast-growing end is referred to as the plus (+) or barbed end ; the slow-growing end is referred to as the minus (−) or pointed end . The dynamic process of actin polymerization requires energy in the form of an adenosine triphosphate ( ATP ) molecule that is hydrolyzed to adenosine diphosphate ( ADP ) after a G-actin molecule is incorporated into the filament. The phosphate groups are not immediately released; therefore, a transient form of actin bound to ADP–P i is detectable in the filament.

Under physiologic conditions, G-actin molecules are preferentially incorporated into the plus end and preferentially dissociated from the minus end of the actin filament. Thus, each new G-actin molecule that is added to the plus end travels the length of the actin filament as additional actin molecules are added behind it and eventually leaves the actin filament from the minus end. This phenomenon is called (see Fig. 5.5). When the rate at which free G-actin is added at the plus end is greater than the rate of subunit loss at the minus end, the filament appears to grow. Conversely, when the rate at

treadmilling

which free G-actin is added is lower than the rate of subunit loss, the actin filament appears to shrink. When the rate at which G-actin is added is equal to the rate of its dissociation at the minus end, the length of filament is unchanged. This state is referred to as the . The control and regulation of the polymerization process depends on the local concentration of G-actin and the interaction of , which can prevent or enhance polymerization. Several natural toxins that have been found and isolated from mushrooms, fungi, and sponges bind to actin filaments and affect their polymerization and disassembly. , a seven-amino-acid polypeptide found in the death cap mushroom ( ), inhibits actin filament disassembly by stabilizing adjacent actin molecules within the filament. Because of its strong binding to F-actin, phalloidin molecules linked to fluorescent tags are used as staining reagents for microscopic visualization of actin filaments. The produced by a variety of fungi bind to the plus end of actin filaments to prevent actin filament assembly and disassembly at that end. , a toxin produced by the Red Sea sponge ( ), binds to G-actin monomers to inhibit their polymerization into actin filaments. , a small compound present in the marine sponge ( ) found in the Fuji and Palau islands, stabilizes actin monomers, thereby enhancing polymerization and assembly of actin filaments.

steady state of treadmilling

actin-binding proteins (ABPs)

Phalloidin phalloides

Amanita

cytochalasins

Latrunculin A

Latrunculia magnifica Jasplakinolide Jaspis johnstoni

Actin-binding proteins are responsible for assembly, disassembly, and organization of actin filaments. In addition to controlling the rate of polymerization of actin filaments, ABPs are responsible for the filaments’ organization. For example, a number of proteins can modify or act on actin filaments to give them various specific characteristics:

Actin-bundling proteins

cross-link actin filaments into parallel arrays, creating actin filament bundles. An example of this modification occurs inside the microvillus, where actin filaments are cross-linked by the actin-bundling proteins and . This cross-linkage provides support and imparts rigidity to the microvilli. cut long actin filaments into shorter fragments. An example of such a protein is , a 90-kDa ABP that normally initiates actin polymerization but at high Ca2+ concentrations causes severing and capping of the actin filaments, converting an actin gel into a fluid state. , an 18-kDa protein involved in rapid remodeling of the actin cytoskeleton, severs actin filaments to create free plus and minus ends that are available for polymerization or depolymerization of free actin molecules. block further addition of actin molecules by binding to the free end of an actin filament. An example is , which can be isolated from skeletal and cardiac muscle cells. Tropomodulin binds to the free end of actin myofilaments, regulating the length of the filaments in a sarcomere. proteins are responsible for cross-linking actin filaments with each other. An example of such proteins can be found in the cytoskeleton of erythrocytes. Several proteins—such as , , , and —are involved in cross-linking actin filaments. belong to the myosin family, which hydrolyzes ATP to provide the energy for movement along the actin filament from the minus end to the plus end. Some cells, such as muscle cells, are characterized by the size, amount, and nature of the filaments and actin motor proteins they contain. There are two types of filaments ( ) present in muscle cells: 6- to 8-nm actin filaments (called ; Fig. 2.54) and 15-nm filaments (called ) of myosin II, which is the predominant protein in muscle cells. is a double-headed molecule with an elongated rod-like tail. The specific structural and functional relationships among actin, myosin, and other ABPs in muscle contraction are discussed in Chapter 11, Muscle Tissue, pages 349-352.

fascin

Actin filament–severing proteins gelsolin Actin-capping proteins Actin cross-linking

Actin motor proteins myofilaments filaments

fimbrin

Cofilin

tropomodulin

spectrin adducin protein 4.1

protein 4.9

thick filaments Myosin II

thin

FIGURE 2.54. Thin filament organization and structure in cardiac cells. a. Immunofluorescence micrograph of a chick cardiac myocyte stained for actin ( green ) to show the thin filaments and for tropomodulin ( red ) to show the location of the slow-growing (−) ends of the thin filaments. Tropomodulin appears as regular striations because of the uniform lengths and alignment of the thin filaments in sarcomeres. ×320. b. Diagram of a thin filament. The polarity of the thin

filament is indicated by the fast-growing (+) end and the slow-growing (−) end. Only a portion of the entire thin filament is shown for clarity. Tropomodulin is bound to actin and tropomyosin at the slow-growing (−) end. The troponin complex binds to each tropomyosin molecule every seven actin monomers along the length of the thin filament. (Courtesy of Drs. Velia F. Fowler and Ryan Littlefield.)

myosin I

In addition to myosin II, nonmuscle cells contain , a protein with a single globular domain and short tail that attaches to other molecules or organelles. Extensive studies have revealed the presence of a variety of other nonmuscle myosin isoforms that are responsible for motor functions in many specialized cells, such as melanocytes, kidney and intestinal absorptive cells, nerve growth cones, and inner ear hair cells.

Actin filaments participate in a variety of cell functions. Actin filaments are often grouped in bundles close to the plasma

membrane. Functions of

these membrane-associated actin filaments include the following.

Anchorage and movement of membrane proteins.

Actin filaments are distributed in threedimensional networks throughout the cell and are used as anchors within specialized cell junctions such as focal adhesions. on absorptive epithelial cells. Actin filaments may also help maintain the shape of the apical cell surface (e.g., the apical of actin filaments serves as a set of tension cables beneath the cell surface). . Locomotion is achieved by the force exerted by actin filaments undergoing polymerization at their growing ends. This mechanism is used in many migrating cells, particularly in transformed cells of invasive tumors. As a result of actin polymerization at their , cells extend processes from their surface by pushing the plasma membrane ahead of the growing actin filaments. Although the leading edge is extending, the actin filaments in the (tail) of the cell undergo depolymerization, causing its retraction. The leading edge extensions of a crawling cell are called ; they contain elongating organized bundles of actin filaments with their plus ends directed toward the plasma membrane. . These processes can be observed in many other cells that exhibit small protrusions called , located around their surface. As in lamellipodia, these protrusions contain loose aggregations of 10–20 actin filaments organized in the same direction, again with their plus ends directed toward the plasma membrane. Actin filaments are also essential in cytoplasmic streaming (i.e., the streamlike movement of cytoplasm that can be observed in cultured cells).

Formation of the structural core of microvilli terminal web Locomotion of cells leading edge

lamellipodia Extension of cell processes

listeriosis

trailing edge

filopodia

Listeria monocytogenes

In , an infection caused by , the actin polymerization machinery of the cell is hijacked by the invading pathogen and utilized for its intracellular movement and dissemination throughout the tissue. Following internalization into the host phagosome (see Fig. 2.24), lyses the membrane of the phagosome and escapes into the cytoplasm. Within the cytoplasm, one end of the bacterium triggers polymerization of the host cell’s actin filaments, which propels it through the

L. monocytogenes

cell like a space rocket, leaving a characteristic tail of polymerized actin behind. Actin polymerization allows bacteria to pass into a neighboring cell by forming protrusions in the host plasma membrane.

Intermediate Filaments

Intermediate filaments play a supporting or general structural role. These rope-like filaments are called intermediate because their diameter of 8–10 nm is between those of actin filaments and microtubules. Nearly all intermediate filaments consist of subunits with a molecular weight of about 50 kDa. Some evidence suggests that many of the stable structural proteins in intermediate filaments evolved from highly conserved enzymes, with only minor genetic modification.

Intermediate filaments are formed from nonpolar and highly variable intermediate filament subunits.

Unlike those of microfilaments and microtubules, the protein subunits of intermediate filaments show considerable diversity and tissue specificity. In addition, they do not possess enzymatic activity and form nonpolar filaments. Intermediate filaments also do not typically disappear and re-form in the continuous manner characteristic of most microtubules and actin filaments. For these reasons, intermediate filaments are believed to play a primarily structural role within the cell and to compose the cytoplasmic link of a tissuewide continuum of cytoplasmic, nuclear, and extracellular filaments (Fig. 2.55).

FIGURE 2.55. Electron micrograph of the apical part of an epithelial cell demonstrating intermediate filaments. This electron micrograph, obtained using the quick-freeze, deep-etch technique, shows the terminal web ( TW ) of an epithelial cell and underlying intermediate filaments ( IF ). The long, straight actin filament cores or rootlets ( R ) extending from the microvilli are cross-

linked by a dense network of actin filaments containing numerous actin-binding proteins. The network of intermediate filaments can be seen beneath the TW anchoring the actin filaments of the microvilli. ×47,000. (Reprinted with permission from Hirokawa N, Keller TC 3rd, Chasan R, et al. Mechanism of brush border contractility studied by the quick-freeze, deep-etch method. 1983;96:1325– 1336.)

J Cell Biol.

Intermediate filament proteins are characterized by a conserved central rod-shaped domain with highly variable globular domains at either end (Fig. 2.56). Although the

various classes of intermediate filaments slightly differ in the amino acid sequence of the rod-shaped domain and show some variation in molecular weight, they all share a homologous region that is important in filament self-assembly. Intermediate filaments are assembled from a pair of that twist around each other to form . Next, two coiled-coil dimers twist around each other in antiparallel fashion (parallel but pointing in opposite directions) to generate a of two coiled-coil dimers, thus forming the nonpolarized unit of the intermediate filaments (see Fig. 2.56). Each tetramer, acting as an individual unit, is aligned along the axis of the filament. The ends of the tetramers are bound together to form the free ends of the filament. This assembly process provides a stable, staggered, helical array in which filaments are packed

helical monomers

staggered tetramer

coiled-coil dimers

together and additionally stabilized by lateral binding interactions between adjacent tetramers.

FIGURE 2.56. Polymerization and structure of intermediate filaments.

Intermediate filaments are self-assembled from a pair of monomers that twist around each other in parallel fashion to form a stable dimer. Two coiled-coil dimers then twist around each other in antiparallel fashion to generate a staggered tetramer of two coiled-coil dimers. This tetramer forms the nonpolarized unit of the intermediate filaments. Each tetramer, acting as an individual unit, aligns along the axis of the filament and binds to the free end of the elongating structure. This staggered helical array is additionally stabilized by lateral binding interactions between adjacent tetramers.

Intermediate filaments are a heterogeneous group of cytoskeletal elements found in various cell types. Intermediate filaments are organized into six major classes on the basis of gene structure, protein composition, and cellular distribution (Table 2.3).

TABLE 2.3 Classes of Intermediate Filaments, Their Location, and Associated Diseases

Type of Protein Class 1 and 2: Acid cytokeratins Basic cytokeratins

Molecular Where Found Weight (kDa) Keratins 40–64

All epithelial cells

Epidermolysis bullosa simplex

52–68

All epithelial cells

Keratoderma disorders caused by keratin mutations Meesmann corneal dystrophy

Class 3: Vimentin and Vimentin-Like Vimentin 55 Cells of mesenchymal origin (including endothelial cells, myofibroblasts, some smooth muscle cells) and some cells of neuroectodermal origin

Desmin 53 Glial 50–52 fibrillary acidic protein (GFAP) Peripherin 54 Class 4: Neurofilaments Neurofilament 68 L (NF-L) Neurofilament M (NF-M) Neurofilament H (NF-H) Nestin 𝛂 -Internexin Synemin 𝛂 / 𝛃a

Syncoilin Paranemin Class 5: Lamins Lamin A/C b Lamin B

Examples of Associated Diseases

Desmin-related myopathy (DRM) Dilated cardiomyopathy Alexander disease Amyotrophic lateral sclerosis (ALS)

Muscle cells; coassembles with nestin, synemin, and paranemin

 

Neuroglial cells (mainly astrocytes; to lesser degree, ependymal cells), Schwann cells, enteric glial cells, satellite cells of sensory ganglia, and pituicytes

 

Peripheral neurons

 

Neurons Coassembles with NF-M or NF-H

Charcot–Marie– Tooth disease Parkinson disease

110

Neurons Coassembles with NF-L

 

130

Neurons Coassembles with NF-L

 

240

Neural stem cells, some cells of neuroectodermal origin, muscle cells Coassembles with desmin

 

68

Neurons

 

182

Muscle cells Coassembles with desmin

 

64

Muscle cells

 

178

Muscle cells Coassembles with desmin

 

62–72

Nucleus of all nucleated cells

Emery–Dreifuss muscular dystrophy

65–68

Nucleus of all nucleated cells

Limb girdle muscular dystrophy

Eye lens fiber cells

Juvenile-onset

Class 6: Beaded Filaments Phakinin 49

(CP49) c Filensin (CP115)

115

Coassembles with filensin

cataracts Congenital cataracts

Eye lens fiber cells Coassembles with phakinin

 

aSynemin α and synemin β represent two alternative transcripts of bLamin C is a splice product of lamin A. cThe molecular weight of filensin/phakinin heterodimer is 131 kDa.

the DMN gene.

Classes 1 and 2. These are the most diverse groups of intermediate filaments and are called keratins (cytokeratins). These classes contain more than 50 different isoforms and account for most of the intermediate filaments (about 54 genes of the total 70 human intermediate filament genes are linked to keratin molecules). Keratins only assemble as heteropolymers; an (class 1) and a (class 2) molecule form a heterodimer. Each keratin pair is characteristic of a particular type of epithelium; however, some epithelial cells may express more than one pair. Keratin filaments are found in different cells of epithelial origin and are divided into three expression groups: , , and , also called . Hard keratins are found in skin appendages, such as hair and nails. Keratin filaments span the cytoplasm of epithelial cells and, via desmosomes, connect with keratin filaments in neighboring cells. Keratin subunits do not coassemble with other classes of intermediate filaments; therefore, they form a distinct cell-specific and tissue-specific recognition system. . This group contains four proteins: , the most widely distributed intermediate filament protein in the body, and vimentin-like proteins such as , , and . They represent a diverse family of cytoplasmic filaments found in many cell types. In contrast to keratins, class 3 proteins (with the exception of desmin) preferentially form homopolymeric filaments containing only one type of intermediate protein. Vimentin is the most abundant intermediate filament found in all mesoderm-derived cells, including fibroblasts (Fig. 2.57); desmin is characteristic of muscle cells; GFAP is found in glial cells (highly specific for astrocytes), and peripherin is found in many peripheral nerve cells.

acid cytokeratin

basic cytokeratin

keratins of simple epithelia keratins of stratified epithelia structural keratins hard keratins Class 3 glial fibrillary acidic protein (GFAP)

vimentin peripherin

desmin

FIGURE 2.57. Distribution of intermediate filaments in human fetal lung fibroblasts. Distribution of vimentin ( red ) and actin filaments ( green ) is shown in cultured fibroblasts from

human fetal lung. Vimentin is an intermediate filament protein expressed in all cells of mesenchymal origin. In cultured fibroblasts, vimentin filaments are visible centrally within the cell cytoplasm, whereas the actin filaments are aggregated primary near the cell surface. This immunofluorescent image was obtained using indirect immunofluorescence techniques in which vimentin filaments were treated with mouse antivimentin primary antibodies followed by goat antimouse secondary antibodies conjugated with Texas red fluorescent dye. Actin filaments were counterstained with phalloidin conjugated with a green fluorescent dye. Nuclei were stained blue with Hoechst fluorescent stain. ×3,500. (Reprinted with permission from Michael W. Davidson, Florida State University.)

Class 4.

neurofilaments

Historically, this group has been called ; they contain intermediate filament proteins that are expressed mostly in the axons of nerve cells. The three types of neurofilament proteins are of different molecular weights: (a lowweight protein), (a medium-weight protein), and (a high-weight protein). They coassemble to form a heterodimer that contains one NF-L molecule along with one of the other two proteins. All three proteins form neurofilaments that extend from the cell body into the ends of axons and dendrites, providing structural support. However, genes for class 4 proteins also encode several other intermediate filament proteins. These include and 𝛂 in nerve cells as well as , , and in

NF-M

nestin

-internexin

NF-H

synemin syncoilin

NF-L

paranemin

muscle cells. heteropolymers.

Members

of

this

group

preferentially

coassemble

in

tissues

as

Class 5. Lamins, specifically nuclear lamins, form a network-like structure that is associated with the nuclear envelope. Lamins are represented by two types of proteins, lamin A and lamin B. In contrast to other types of intermediate filaments found in the cytoplasm, lamins are located within the nucleoplasm of almost all differentiated cells in the body. A description of their structure and function can be found on page 94. . This is a lens-specific group of intermediate filament, or “ ,” containing two proteins, and . The periodic bead-like surface appearance of these filaments is attributed to the globular structure of the carboxy-terminus of the filensin molecule, which projects out from the assembled filament core.

Class 6

phakinin

beaded filaments

filensin

Intermediate filament–associated proteins are essential for the integrity of cell-tocell and cell-to-extracellular matrix junctions. A variety of intermediate filament–associated proteins function within the cytoskeleton as integral parts of the molecular architecture of cells. Some proteins, such as those of the plectin family, possess binding sites for actin filaments, microtubules, and intermediate filaments and are thus important in the proper assembly of the cytoskeleton. Lamins, the intermediate filaments in the nucleus, are associated with numerous proteins in the inner nuclear membrane, including emerin, lamin B receptor (LBR), nurim, and several lamina-associated polypeptides. Some of these proteins have multiple binding sites to

intermediate filaments, actin, chromatin, and signaling proteins; thus, they function in chromatin organization, gene expression, nuclear architecture, and cell signaling and provide an essential link between the nucleoskeleton and cytoskeleton of the cell. Another important family of intermediate filament–associated proteins consists of , , and . These proteins form the attachment plaques for intermediate filaments, an essential part of and . The interaction of intermediate filaments with cell-to-cell and cell-to-extracellular matrix junctions provides mechanical strength and resistance to extracellular forces. Table 2.4 summarizes the characteristics of the three types of cytoskeletal filaments.

desmoplakin-like proteins

plakoglobins

desmosomes

desmoplakins hemidesmosomes

TABLE 2.4 Summary Characteristics of Three Types of Cytoskeletal Elements Shape

Actin Filaments (Microfilaments)

Intermediate Filaments

Double-stranded linear helical array Rope-like fibers

Diameter (nm) Basic protein subunit Enzymatic activity Polarity Assembly process Source of

6–8

8–10

Monomer of G-actin (MW 42 kDa)

Various intermediate filam

ATP hydrolytic activity

None

Yes; minus (−) or pointed end is slow-growing end. Plus (+) or barbed end is faster growing end.

Nonpolar structures

Monomers of G-actin are added to growing filament. Polymerization requires presence of K +, Mg 2+, and ATP, which is hydrolyzed to ADP after each G-actin molecule is incorporated into the filament.

Two pairs of monomers form two coiled-coil dimers t a staggered tetramer, wh filament and binds to th structure.

ATP

N/A

energy required for assembly Characteristics Associated proteins Location in cell Major functions

Thin, flexible filaments

Strong, stable structures

Variety of ABPs with different functions: fascin = bundling; gelsolin = filament severing; CP protein = capping; spectrin = cross-linking; myosin I and II = motor functions

Intermediate filament–ass microtubules, actin, and desmoplakins and plakogl to desmosomes and hemide

Core of microvilli Terminal web Concentrated beneath plasma membrane Contractile elements of muscles Contractile ring in dividing cells

Extend across cytoplasm co hemidesmosomes In nucleus just beneath in

Provide essential components (sarcomeres for muscle cells)

Provide mechanical strengt

ABP, actin-binding protein; ADP, adenosine diphosphate; ATP, adenosine triphosphate; GDP, guanosine diphosphate; GTP, guanosine triphosphate; MAP, microtubule-associated protein; MTOC, microtubuleorganizing center; MW, molecular weight; N/A, not applicable.

Centrioles and Microtubule-Organizing Centers

Centrioles represent the focal point around which the MTOC assembles. Centrioles, visible in the light microscope, are paired, short, rod-like cytoplasmic cylinders built from nine microtubule triplets. In resting cells, centrioles have an orthogonal orientation: One centriole in the pair is arrayed at a right angle to the other. Centrioles are usually found close to the nucleus, often partially surrounded by the Golgi apparatus, and associated with a zone of amorphous, dense pericentriolar material. The region of the cell containing the centrioles and pericentriolar material is called the microtubule-organizing center or centrosome (Fig. 2.58). The MTOC is the region where most microtubules are formed and from which they are then directed to specific destinations within the cell. The MTOC controls the number, polarity, direction, orientation, and organization of microtubules formed during the interphase of the cell cycle. During mitosis, duplicated MTOCs serve as mitotic spindle poles. Development of the MTOC itself depends solely on the presence of centrioles. When centrioles are missing, the MTOCs disappear, and formation of microtubules is severely impaired.

FIGURE 2.58. Structure of the microtubule-organizing center (MTOC) .

This diagram shows the location of the MTOC in relation to the nucleus and the Golgi apparatus. In some species, the MTOC is tethered to the nuclear envelope by a contractile protein, the nucleus–basal body connector ( ). The MTOC contains the centrioles and an amorphous protein matrix with an abundance of γ-tubulin rings. Each γ-tubulin ring serves as the nucleation site for the growth of a single microtubule. Note that the minus (−) end of the microtubule remains attached to the MTOC, and the plus (+) end represents the growing end directed toward the plasma membrane.

NBBC

The pericentriolar matrix of MTOC contains numerous ring-shaped structures that initiate microtubule formation. The MTOC contains centrioles and an amorphous pericentriolar matrix of more than 200 proteins, including γ-tubulin that is organized in ring-shaped structures. Each γ-tubulin ring serves as the starting point (nucleation site) for the growth of one microtubule that is assembled from tubulin dimers; α- and β-tubulin dimers are added with specific orientation to the γ-tubulin ring. The minus end of the microtubule remains attached to the MTOC, and the plus end represents the growing end directed toward the plasma membrane (see Fig. 2.58).

Centrioles provide basal bodies for cilia and flagella and align the mitotic spindle during cell division.

Although centrioles were discovered more than a century ago, their precise functions, replication, and assembly are still under intense investigation. The known functions of centrioles can be organized into two categories:

Basal body formation. One of the important functions of the centriole is to provide basal

bodies, which are necessary for the assembly of cilia and flagella (Fig. 2.59). Basal bodies are formed either de novo without contact with the preexisting centrioles (the ) or by duplication of existing centrioles (the ). About 95% of the centrioles are generated through the acentriolar pathway. Both pathways give rise to multiple immediate precursors of centrioles, known as , which mature as they migrate to the appropriate site near the apical cell membrane, where they become (Fig. 2.60). The basal body acts as the organizing center for a cilium. Microtubules grow upward from the basal body, pushing the cell membrane outward, and elongate to form the mature cilium. The process of centriole duplication is described on pages 76-79.

acentriolar pathway

centriolar pathway procentrioles

basal bodies

FIGURE 2.59. Basal bodies and cilia.

This electron micrograph shows the basal bodies and cilia in cross-sectional profile as seen in an oblique section through the apical part of a ciliated cell in the respiratory tract. Note the 9 + 2 microtubule arrangement of the cilia in which nine microtubules at the periphery of the cilia surround two central microtubules. The basal bodies lack the central tubule pair. On several cross sections, the basal foot is visible as it projects laterally from the basal body ( ). ×28,000. (Courtesy of Patrice C. Abell-Aleff.)

asterisks

FIGURE 2.60. Two pathways of basal body formation.

In the centriolar pathway, a pair of existing centriole serves as an organizing center for the duplication of new centrioles. Utilizing this pathway, ciliated cells have the ability to assemble large number of centrioles in the vicinity of an old mature centriole. In the acentriolar pathway, which plays a major role in the formation of basal bodies in ciliated cells, new centrioles are formed de novo from fibrous granules located in close proximity to nonmicrotubular structures called deuterosomes. Both pathways give rise to procentrioles, which mature as they migrate to the appropriate site near the apical cell membrane, where they become basal bodies. Fibrous granules contribute to the formation of the striated rootlet. (Based on Hagiwara H, Ohwada N, Takata K. Cell biology of normal and abnormal ciliogenesis in the ciliated epithelium. . 2004;234:101–139.)

Int Rev Cytol

Mitotic spindle formation.

During mitosis, the position of centrioles determines the location of mitotic spindle poles. Centrioles are also necessary for the formation of a fully functional MTOC, which nucleates mitotic spindle–associated microtubules. For instance, are formed around each individual centriole in a star-like fashion. They are crucial in establishing the axis of the developing mitotic spindle. In some animal cells, the mitotic spindle itself (mainly kinetochore microtubules) is formed by MTOC-independent mechanisms and consists of microtubules that originate from the chromosomes. Data from experimental studies indicate that in the absence of centrioles, astral microtubules fail to develop, causing errors in mitotic spindle orientation (Fig. 2.61). Thus, the primary role of centrioles in mitosis is to position the mitotic spindle properly by recruiting the MTOC from which astral microtubules can grow and establish the axis for the developing spindle.

astral microtubules

FIGURE 2.61. Mitotic spindle during normal cell division and in cells lacking centrioles. a. This schematic drawing shows the orientation of the mitotic spindle in a normal cell undergoing mitosis. Note the positions of the centrioles and the distribution of the spindle microtubules. MTOC, microtubule-organizing center. b. In a cell that lacks centrioles, mitosis occurs and a mitotic spindle containing only kinetochore microtubules mitotic spindle lack astral microtubules, which position mitosis. Such a misoriented spindle is referred to as Marshall WF, Rosenbaum JL. How centrioles work: lessons 2000;12:119–125.)

is formed. However, both poles of the the spindle in the proper plane during an . (Based on from green yeast. .

anastral bipolar spindle Curr Opin Cell Biol

The dominant feature of centrioles is the cylindrical array of triplet microtubules with associated proteins. The TEM reveals that each rod-shaped centriole is about 0.2 μm long and consists of nine triplets of microtubules that are oriented parallel to the long axis of the organelle and run in slightly twisted bundles (Fig. 2.62). The three microtubules of the triplet are fused, with adjacent microtubules sharing a common wall. The innermost or A microtubule is a complete ring of 13 protofilaments containing α- and β-tubulin dimers; the middle and outer B and C microtubules, respectively, appear C-shaped because they share tubulin dimers with each other and with the A microtubule. The microtubules of the triplets are not equal in length. The C microtubule of the triplet is usually shorter than A and B.

FIGURE 2.62. Electron micrograph showing parent and daughter centrioles in a fibroblast. Note that the transverse-sectioned centriole in each of the pairs reveals the triplet configuration of microtubules. The lower right centriole represents a mid-longitudinal section, whereas the upper left

centriole has also been longitudinally sectioned but along the plane of its wall. ×90,000. (Courtesy of Drs. Manley McGill, D. P. Highfield, T. M. Monahan, and Bill R. Brinkley.)

The microtubule triplets of the centriole surround an internal lumen. The distal part of the lumen (away from the nucleus) contains a 20-kDa Ca2+ -binding protein, (Fig. 2.63). The proximal part of the lumen (close to the nucleus) is lined by , which provides the template for the arrangement of the triplet microtubules. In addition, a family of newly discovered , , , and molecules as well as protein complexes have been localized with the centrioles. Other proteins, such as , form a ring of molecules that appears to link the distal end of the centriole to the plasma membrane. Filamentous connections between the centriole pair have been identified in human lymphocytes. In other organisms, two protein bridges, the and , connect each centriole in a pair (see Fig. 2.63).

δ- ε- ζz-

fibers

centrin γ-tubulin pericentrin protein p210

η-tubulin

proximal

distal connecting

FIGURE 2.63. Schematic structure of centrioles.

In nondividing cells, centrioles are arranged in pairs in which one centriole is aligned at a right angle to the other. One centriole is also more mature (generated at least two cell cycles earlier) than the other centriole, which was generated in the previous cell cycle. The mature centriole is characterized by the presence of satellites and appendages. Centrioles are located in close proximity to the nucleus. The basic components of each centriole are microtubule triplets that form the cylindrical structure surrounding an internal lumen. The proximal part of the lumen is lined by γ-tubulin, which provides the template for nucleation and arrangement of the microtubule triplets. The distal part of each lumen contains the protein centrin. In some species, two protein bridges, the proximal and distal connecting fibers, connect each centriole in a pair. In some species, but not in humans, the proximal end of each centriole is attached to the nuclear envelope by a contractile protein known as the nucleus–basal body connector ( ).

NBBC

In dividing cells, these connections participate in segregating the centrioles to each daughter cell. In some organisms, the proximal end of each centriole is attached to the nuclear envelope by contractile proteins called . Their function is to link the centriole to the mitotic spindle poles during mitosis. In human cells, the centrosome–nucleus connection appears to be maintained by filamentous structures of the cytoskeleton. A distinctive feature of mammalian centrioles is the difference between individual centrioles in the pair. One centriole (termed the ) contains stalk-like satellite processes and sheet-like appendages whose function is not known (see Fig. 2.63). The other centriole (termed the ) does not possess satellites or appendages.

nucleus–basal body connectors (NBBCs)

centriole

mature

immature centriole Centrosome duplication is synchronized with cell cycle events and linked to the process of ciliogenesis.

Centrosome dynamics, such as duplication or formation of basal bodies for ciliogenesis, are synchronized with cell cycle progression. Cilia are assembled during the G1 phase; they are most abundant in G0 and are disassembled before the cell enters the M phase. These events are depicted in Figure 2.64, which shows the relationships between centrosome duplication, primary cilium formation, and progression through the cell cycle.

FIGURE 2.64. Association of the centrosome duplication and primary cilium formation with the cell cycle. After a cell emerges from mitosis, it possesses a single centrosome (microtubule-

organizing center [MTOC]) surrounded by amorphous pericentriolar material. The primary cilium formation first occurs during the G 1 phase in which the centrosome migrates toward the cell membrane and initiates the process of ciliogenesis. Necessary structural and transport proteins are acquired and activated to build the primary cilium axoneme (9 + 0) directly on top of the mature centriole. During the end of the G 1 phase, as well as in G 0 , the primary cilium functions as an external receiver antenna sensing and interpreting signals from the extracellular environment. Duplication of centrioles begins near the transition between the G 1 and S phases of the cell cycle, and the two centrioles are visible in S phase. During the late G 2 phase, centrioles reach their full maturity, whereas the primary cilium is disassembled. This allows centrioles to migrate away from the cell membrane and participate in mitotic spindle formation. Once cell division is complete, the centrioles can proceed to ciliary reassembly in the G 1 phase. (Based on Santos N, Reiter JF. Building it up and taking it down: the regulation of vertebrate ciliogenesis. . 2008;237:1972–1981.)

Dev Dyn

Because each daughter cell receives only one pair of centrioles after cell division, the daughter cells must duplicate existing centrioles prior to cell division. In most somatic cells, duplication of centrioles begins near the transition between the G1 and S phases of the cell cycle. This event is closely associated with the activation of the during the S phase of the cell cycle (see Fig. 3.11). This complex directly phosphorylates the nucleus-chaperoning protein , which is responsible for initiating the duplication of centrioles. In most cells, duplication begins with the splitting of a centriole pair, followed by the appearance of a small mass of fibrillar and granular material at the proximal lateral end of each original centriole. Because the existing pair of centrioles serves as a core for new organelle formation, this process of centriole duplication is referred as the (see Fig. 2.60). In this pathway, the coalesce into dense spherical structures called , and they give rise to the (or bud), which gradually enlarges to form a right angle appendage to the parent (see Fig. 2.60). Microtubules begin to develop in the mass of fibrous granules as it grows (usually during the S to late G2 phases of the cell

complex

pathway deuterosomes

nucleophosmin/B23

fibrous granules procentriole

cyclin E–Cdk2

centriolar

cycle), appearing first as a ring of nine single tubules, then as doublets, and finally as triplets. As procentrioles mature during the S and G2 phases of the cell cycle, each parent –daughter pair migrates around the nucleus. Before the onset of mitosis, centrioles with surrounding amorphous pericentriolar material position themselves on opposite sides of the nucleus and produce astral microtubules. In doing so, they define the poles between which the bipolar mitotic spindle develops. The important difference between duplication of centrioles during mitosis and during ciliogenesis is the fact that during mitosis, only one daughter centriole buds from the lateral side of parent organelle, whereas during ciliogenesis, as many as 10 centrioles may develop around the parent centriole.

Basal Bodies

Development of cilia on the cell surface requires the presence of basal bodies, structures derived from centrioles. Each cilium requires a basal body. The generation of centrioles, which occurs during the process of ciliogenesis, is responsible for the production of basal bodies. The newly formed centrioles migrate to the apical surface of the cell and serve as organizing centers for the assembly of the microtubules of the cilium. The core structure (axoneme) of a motile cilium is composed of a complex set of microtubules consisting of two central microtubules surrounded by nine microtubule doublets (9 + 2 configuration). The organizing role of the basal body differs from that of the MTOC. The axonemal microtubule doublets are continuous with the A and B microtubules of the basal body from which they develop by addition of αand β-tubulin dimers at the growing plus end. A detailed description of the structure of cilia, basal bodies, and the process of ciliogenesis can be found in Chapter 5, Epithelial Tissue, pages 137-139.

FOLDER 2.2

CLINICAL CORRELATION: ABNORMALITIES IN MICROTUBULES AND FILAMENTS

Abnormalities related to the organization and structure of microtubules, actin, and intermediate filaments underlie many pathologic disorders. These abnormalities lead to defects in the cytoskeleton and can produce a variety of defects related to intracellular vesicular transport, intracellular accumulations of pathologic proteins, and impairment of cell mobility.

Microtubules

Defects in the organization of microtubules and microtubule-associated proteins can immobilize the cilia of respiratory epithelium, interfering with the ability of the respiratory system to clear accumulated secretions. This condition, known as (see Folder 5.2, page 139), also causes dysfunction of microtubules, which affects sperm motility and leads to male sterility. It may also cause infertility in women because of impaired ciliary transport of the ovum through the oviduct. Microtubules are essential for vesicular transport (endocytosis and exocytosis) as well as cell motility. Certain drugs, such as , bind to tubulin molecules and prevent their polymerization. Used in the treatment of acute attacks of gout, this prevents neutrophil migration and decreases their ability to respond to urate crystal deposits in the tissues. and represent another family of drugs that bind to microtubules and inhibit the formation of the mitotic spindle essential for cell division. These drugs are used as antimitotic and antiproliferative agents in cancer therapy. Another drug, , is used in chemotherapy for breast cancer. It stabilizes microtubules, preventing them from depolymerizing (an action opposite to that of colchicine), and thus arrests cancer cells in various stages of cell division.

Kartagener syndrome

colchicine

Vinblastine

vincristine (Oncovin)

paclitaxel (Taxol)

Actin Filaments

Actin filaments play essential roles in various stages of leukocyte migration as well as the phagocytic functions of various cells. Some chemical substances isolated from fungi, such as and , prevent actin polymerization by binding to the plus end of the actin filament, inhibiting lymphocyte migration, phagocytosis, and cell division (cytokinesis). Several toxins of poisonous mushrooms, such as , also bind to actin filaments, stabilizing them and preventing their depolymerization. Conjugated with fluorescein

cytochalasin B

cytochalasin D

phalloidin

dyes, derivatives of the phallotoxin family (i.e., NDB-phallacidin) are frequently used in the laboratory to stain actin filaments (see Figs. 2.47 and 2.51). Prolonged exposure of the cell to these substances can disrupt the dynamic equilibrium between F-actin and G-actin, causing cell death.

Intermediate Filaments

As noted, the molecular structure of intermediate filaments is tissue specific and consists of many different types of proteins. Several diseases are caused by defects in the proper assembly of intermediate filaments. These defects have also been induced experimentally by mutations in intermediate filament genes in laboratory animals. Changes in neurofilaments within brain tissue are characteristic of , which produces containing neurofilaments and other microtubule-associated proteins. Another disorder of the central nervous system, , is associated with mutations of the GFAP gene. The pathologic feature of this disease is the presence of , which are formed by accumulations of intermediate filament protein GFAP and other associated proteins within the cytoplasm of astrocytes. Altered GFAP prevents the assembly not only of intermediate filaments but also of other proteins that contribute to the structural integrity and function of astrocytes. In addition, bundles of Rosenthal fibers interfere with the successful completion of astrocyte mitosis and cell divisions. Infants with Alexander disease develop leukoencephalopathy (infection of the brain) with macrocephaly (abnormally large head), seizures, and psychomotor impairment, leading to death usually within the first decade of life. A prominent feature of is the presence of eosinophilic intracytoplasmic inclusions composed predominantly of keratin intermediate filaments. These inclusions, called , are visible in light microscopy within the hepatocyte cytoplasm (Fig. F2.2.1).

Alzheimer disease

fibers

neurofibrillary tangles Alexander disease

Rosenthal

alcoholic liver cirrhosis Mallory bodies

FIGURE F2.2.1. Photomicrograph of Mallory bodies.

Accumulation of keratin intermediate filaments forming intracellular inclusions is frequently associated with specific cell injuries. In alcoholic liver cirrhosis, hepatocytes exhibit such inclusions ( ), which are known as

arrows

Mallory bodies. Lymphocytes and macrophages responsible for an intense inflammatory reaction surround cells containing Mallory bodies. ×900.

INCLUSIONS

Inclusions contain products of metabolic activity of the cell and consist largely of pigment granules, lipid droplets, and glycogen. Inclusions are cytoplasmic or nuclear structures with characteristic staining properties

that are formed from the metabolic products of cell. They are considered nonmoving and nonliving components of the cell. Some of them, such as pigment granules, are surrounded by a plasma membrane; others (e.g., lipid droplets or glycogen) instead reside within the cytoplasmic or nuclear matrix.

Lipofuscin

is a brownish-gold pigment visible in routine hematoxylin and eosin (H&E) preparations. It is easily seen in nondividing cells such as neurons and skeletal and cardiac muscle cells. Lipofuscin accumulates over time in most eukaryotic cells as a result of cellular senescence (aging); thus, it is often called the . Lipofuscin is a conglomerate of oxidized lipids, phospholipids, metals, and organic molecules that accumulate within the cells as a result of oxidative degradation of mitochondria and lysosomal digestion. Phagocytic cells such as macrophages may also contain lipofuscin, which accumulates from the digestion of bacteria, foreign particles, dead cells, and their own organelles. Experimental studies indicate that lipofuscin accumulation may be an accurate indicator of cellular stress. is an found within the cytoplasm of many cells. It is most likely formed by the indigestible residues of hemoglobin, and its presence is related to phagocytosis of red blood cells. Hemosiderin is most easily demonstrated in the spleen, where aged erythrocytes are phagocytosed, but it can also be found in alveolar macrophages in the lung tissue, especially following pulmonary infection accompanied by a small hemorrhage into the alveoli. It is visible in light microscopy as a deep brown granule, more or less indistinguishable from lipofuscin. Hemosiderin granules can be differentially stained using histochemical methods for iron detection. is a highly branched polymer used as a storage material for glucose. It is not stained in routine H&E preparations. However, it may be seen in the light microscope with special fixation and staining procedures (such as toluidine blue or the periodic acid– Schiff [PAS] method). Liver and striated muscle cells, which usually contain large amounts of glycogen, may display unstained regions where glycogen is located. Glycogen appears in the EM as granules 25–30 nm in diameter or as clusters of granules that often occupy significant portions of the cytoplasm (Fig. 2.65).

“wear-and-tear”

pigment

Hemosiderin

Glycogen

iron-storage complex

FIGURE 2.65. Electron micrographs of a liver cell with glycogen inclusions. a. Lowmagnification electron micrograph showing a portion of a hepatocyte with part of the nucleus ( N , upper left). Glycogen (G) appears as irregular electron-dense masses. Profiles of rough endoplasmic reticulum ( rER ) and mitochondria ( M ) are also evident. ×10,000. b. This higher magnification electron micrograph reveals glycogen ( G ) as aggregates of small particles. Even the smallest aggregates ( arrows ) appear to be composed of several smaller glycogen particles. The density of the glycogen is considerably greater than that of the ribosomes ( lower left ). M, mitochondria. ×52,000.

Lipid inclusions (fat droplets) are usually nutritive inclusions that provide energy for

cellular metabolism. The lipid droplets may appear in a cell for a brief time (e.g., in intestinal absorptive cells) or may reside for a long period (e.g., in adipocytes). In adipocytes, lipid inclusions often constitute most of the cytoplasmic volume, compressing the other formed organelles into a thin rim at the margin of the cell. Lipid droplets are usually extracted by the organic solvents used to prepare tissues for both light microscopy and EM. What is seen as a fat droplet in light microscopy is actually a hole in the cytoplasm that represents the site from which the lipid was extracted. In individuals with genetic defects of enzymes involved in lipid metabolism, lipid droplets may accumulate in abnormal locations or in abnormal amounts. Such diseases are classified as . contained in certain cells are recognized in the light microscope. In humans, such inclusions are found in the Sertoli (sustentacular) and Leydig (interstitial) cells of the testis. With the TEM, crystalline inclusions have been found in many cell types and in virtually all parts of the cell, including the nucleus and most cytoplasmic organelles. Although some of these inclusions contain viral proteins, storage material, or cellular metabolites, the significance of others is not clear.

lipid storage diseases Crystalline inclusions

FOLDER 2.3

CLINICAL CORRELATION: ABNORMAL DUPLICATION OF CENTRIOLES AND CANCER

One of the critical components of normal cell division is the precise redistribution of chromosomes and other cell organelles during mitosis. Following replication of chromosomal DNA in the S phase of the cell cycle, centrioles undergo a single round of duplication that is closely coordinated with cell cycle progression. During mitosis, centrioles are responsible for forming the bipolar mitotic spindle, which is essential for equal segregation of chromosomes between daughter cells. Alterations of mechanisms regulating centriole duplication may lead to multiplication and abnormalities of centrioles and surrounding centrosomes (microtubule-organizing center [MTOC]). Cells with multiple centrosomes that overcome tumor-suppress or protein (p53)mediated cell cycle arrest and spindle assembly checkpoint inhibition can enter cell divisions with distortions of the mitotic spindle (i.e., the presence of multipolar or misoriented spindles) (Fig. F2.3.1), leading to abnormal sorting of chromosomes during cell division. Multipolar cell divisions lead to aneuploidy, resulting in cell death.

FIGURE F2.3.1. Multipolar mitotic spindle in a tumor cell. a. Electron micrograph of an invasive breast tumor cell showing abnormal symmetrical tripolar mitotic spindle in the metaphase of cell division. ×16,000. b. This drawing composed of color tracings of microtubules ( red ), mitotic spindle poles ( green ), and metaphase chromosomes ( blue ) (obtained from six nonadjacent

serial sections of dividing tumor cell) shows more clearly the organization of this abnormal mitotic spindle. Detailed analysis and three-dimensional reconstruction of the spindle revealed that each spindle pole had at least two centrioles and that one spindle pole was composed of two distinct but adjacent foci of microtubules. (Reprinted with permission from Lingle WL, Salisbury JL. Altered centrosome structure is associated with abnormal mitoses in human breast tumors. . 1999;155:1941–1951.)

Am J

Path

However, some cancer cells can cluster their extra centrosomes into two poles and then undergo cell division resulting in viable daughter cells. Centrosome clustering, which depends on interaction of astral microtubules with the cell membrane, can lead to aneuploidy and defective asymmetric cell division. Centrosome clustering is most likely a unique requirement for the survival of certain tumor cells in which increased numbers of centrioles are frequently observed. Also, additional centrosomes may lose pericentriolar matrix proteins essential for microtubule nucleation (i.e., γ-tubulin). This process silences MTOC activity in extra centrosomes, blocking their participation in spindle formation. The resulting changes in chromosomal number may increase the activity of oncogenes or decrease protection from tumor suppressor genes. These changes are known to promote malignant cell transformation.

CYTOPLASMIC MATRIX

The cytoplasmic matrix is a concentrated aqueous gel consisting of molecules of different sizes and shapes. The cytoplasmic matrix (ground substance or cytosol) shows little specific structure by light microscopy or conventional TEM and has traditionally been described as a concentrated aqueous solution containing molecules of different size and shape (e.g., electrolytes, metabolites, RNA, and synthesized proteins). In most cells, it is the largest single compartment. The cytoplasmic matrix is the site of physiologic processes that are fundamental to the cell’s existence (protein synthesis and degradation, breakdown of nutrients). Studies with high-voltage EM (HVEM) of 0.25- to 0.5-μm sections reveal a complex three-dimensional structural network of thin and . This network provides a structural substratum on which cytoplasmic reactions occur, such as those involving free ribosomes, and along which regulated and directed cytoplasmic transport and movement of organelles occur.

microtrabecular strands

linkers

CELL CYTOPLASM

cross-

OVERVIEW OF THE CELL AND CYTOPLASM

Cells are the basic structural and functional units of all multicellular organisms. Cells have two major compartments: the cytoplasm (which contains organelles and inclusions surrounded by cytoplasmic matrix ) and the nucleus (which contains the

genome). Organelles are metabolically active complexes or compartments that are classified as and .

membranous

nonmembranous organelles

PLASMA MEMBRANE

plasma membrane

The is an amphipathic lipid-bilayered structure visible with the transmission electron microscope (TEM). It is composed of phospholipids, cholesterol, embedded integral membrane proteins, and associated peripheral membrane proteins. have important functions in cell metabolism, regulation, and integration. They include pumps, channels, receptor proteins, linker proteins, enzymes, and structural proteins. represent microdomains in the plasma membrane that contain high concentrations of cholesterol and glycosphingolipids. They are movable signaling platforms that carry integral and peripheral membrane proteins. The and of organelles are . Cytoplasm can be included or excluded in the remodeling process that is mediated by complexes of proteins (i.e., family of or proteins). The plasma membrane invaginates, which allows for . Vesicle budding permits molecules to enter the cell ( ), leave the cell ( ), or travel within the cell cytoplasm in transport vesicles.

Integral membrane proteins Lipid rafts

cell membrane remodeling

internal plasma membranes continuously ESCRT dynamin vesicle budding endocytosis exocytosis

MEMBRANE TRANSPORT AND VESICULAR TRANSPORT Small molecules (fat-soluble, uncharged) and gases cross the plasma membrane by without energy expenditure. All other molecules require (carrier proteins or channels) for passage across the plasma membrane. requires energy because molecules are transported across the plasma membrane against their concentration or electrochemical gradient. requires carrier proteins but does not consume energy. is the cellular uptake of fluids and macromolecules. It is dependent on three different mechanisms: (both micro- and macropinocytotic uptake of fluids and solutes in small or large vesicles, respectively); (uptake of large particles); and (uptake of specific molecules that bind to receptors). and represent a network of membrane-enclosed cytoplasmic compartments that are essential in sorting endocytosed material and membrane proteins for either transport to the cell surface or degradation in lysosomes. during receptor-mediated endocytosis involves interaction with the protein , which assembles in basket-like cages visible in the EM as or . is the process of cellular secretion in which transport vesicles, when fused with plasma membrane, discharge their contents into the extracellular space. are necessary for membrane fusion in exocytosis and for endocytosis initiation. are very small endosome-derived membrane-bound vesicles secreted by cells into extracellular space. They act as transport vehicles for near- and long-distance

simple (passive) diffusion membrane transport proteins Active transport Endocytosis Early

pinocytosis receptor-mediated endocytosis late endosomes

Vesicle formation clathrin pits coated vesicles Exocytosis SNARE proteins Exosomes

Passive transport

phagocytosis

coated

intercellular communication and material exchange between cells. is an ongoing process in which the contents of transport vesicles are continuously delivered and discharged at the plasma membrane. In , the contents of vesicles are stored within the cell and released pending hormonal or neural stimulation.

Constitutive exocytosis regulated secretory exocytosis

DEGRADATION OF PROTEINS Lysosomes

are digestive organelles containing hydrolytic enzymes that degrade substances derived from endocytosis and from the cell itself (autophagy). They have a unique membrane made of specific structural proteins resistant to hydrolytic digestion. by receiving newly synthesized lysosomal proteins (enzymes and structural proteins) that are targeted via the lysosomal targeting signals. The endosomal membrane of some to generate intraluminal vesicles. These intracellular are either degraded in lysosomes or fused with the plasma membrane to release intraluminal vesicles into extracellular space as . are nonmembranous organelles that also function in degradation of proteins. They represent cytoplasmic protein complexes that destroy damaged (misfolded) or unnecessary proteins that have been labeled for destruction with without the involvement of lysosomes.

Lysosomes develop from endosomes P) Proteasomes

mannose-6-phosphate (M-6late endosomes undergoes inward invagination multivesicular bodies (MVBs) exosomes

ubiquitin

ENDOPLASMIC RETICULUM

rough endoplasmic reticulum (rER) ribosomes

The represents a region of endoplasmic reticulum associated with . It is the site of protein synthesis and posttranslational modification of newly synthesized proteins. The rER is most highly developed in active secretory cells and is visible in light microscopy as a basophilic region ( ). The consists of anastomosing tubules that are not associated with ribosomes. It contains (liver) and enzymes for glycogen and . sER also serves as a Ca2+ reservoir in skeletal muscle cells.

ergastoplasm smooth endoplasmic reticulum (sER) cytochrome P450 detoxifying enzymes lipid metabolism

GOLGI APPARATUS AND OTHER MEMBRANOUS ORGANELLES Golgi apparatus

The represents a series of stacked, flattened cisternae and functions in the posttranslational modification, sorting, and packaging of proteins directed to four major cellular destinations: and , and (for storage and/or secretion). are elongated, mobile organelles containing the of respiratory enzymes to generate adenosine triphosphate (ATP). They are abundant in cells that generate and expend large amounts of energy, and they regulate (programmed cell death). are small organelles involved in an oxidative type of metabolism. They degrade , synthesize ether phospholipids, and produce and degrade such as H2O2.

lysosomes Mitochondria

apical cytoplasm

Peroxisomes fatty acids oxygen intermediates

apical and basolateral plasma membrane, endosomes electron transport chain apoptosis reactive

MICROTUBULES

Microtubules are elongated, rigid hollow tubes (20–25 nm in diameter) α-tubulin and β-tubulin . They originate from γ-tubulin rings

composed of within the microtubule-organizing center (MTOC), and their length changes dynamically as tubulin dimers are added or removed in a constant remodeling process known as . Microtubules form tracts for intracellular and ; they are also responsible for the movement of and and for the maintenance of cell shape. along microtubules is generated by molecular motor proteins ( and ). are paired, short, rod-like cytoplasmic cylinders built from . They represent the focal point around which the MTOC assembles, and they provide for cilia and flagella and align the mitotic spindle during cell division.

dynamic vesicular transport mitotic spindles cilia flagella

instability

Movement of intracellular organelles dyneins kinesins Centrioles microtubule triplets basal bodies

nine

ACTIN FILAMENTS Actin filaments

(microfilaments) are thinner (6–8 nm in diameter), shorter, and more flexible than microtubules. They are composed of polymerized molecules that form . Actin filaments are also responsible for , movement of membrane proteins, formation of the structural core of microvilli, and cell motility through the creation of cell extensions ( and ). (myosin family), which hydrolyze ATP to provide energy for movement along the actin filament, are responsible for muscle contraction.

G-actin (globular actin) F-actin (filamentous actin) cell-to-extracellular matrix attachment (focal adhesions) lamellipodia filopodia Actin motor proteins

INTERMEDIATE FILAMENTS

Intermediate filaments

are rope-like filaments (8–10 nm in diameter) that add stability to the cell and interact with cell junctions (desmosomes and hemidesmosomes). Intermediate filaments are formed from nonpolar and highly variable intermediate filament subunits that include (found in epithelial cells), (mesodermally derived cells), (muscle cells), (nerve cells), (nucleus), and (eye lens).

lamins

INCLUSIONS

keratins vimentin desmin neurofilament proteins beaded filament proteins

Inclusions contain products of metabolic activity of the cell and consist largely of pigment granules (lipofuscin is the most common “wear-and-tear” pigment ), lipid droplets , and glycogen .

3

THE CELL NUCLEUS

OVERVIEW OF THE NUCLEUS NUCLEAR COMPONENTS Chromatin Nucleolus Nuclear Envelope Nucleoplasm

CELL RENEWAL CELL CYCLE

Phases and Checkpoints Within the Cell Cycle Regulation of the Cell Cycle

Mitosis Meiosis

CELL DEATH

Nonprogrammed Cell Death: Necrosis Programmed Apoptotic Cell Death: Apoptosis and Anoikis Programmed Nonapoptotic Cell Death Clinical Correlation: Cytogenetic Testing Clinical Correlation: Regulation of Cell Cycle and Cancer Treatment

Folder 3.1 Folder 3.2 HISTOLOGY

OVERVIEW OF THE NUCLEUS

The nucleus is a membrane-limited compartment that contains the genome (genetic information) in eukaryotic cells. The nucleus contains genetic information, together with the machinery for DNA replication and RNA transcription and processing. The nucleus of a nondividing cell, also called an , consists of the following components:

interphase cell Chromatin is nuclear material organized as euchromatin heterochromatin. It contains DNA associated with roughly

or an equal mass of various nuclear proteins (e.g., histones) that are necessary for DNA to function. The (pl., ) is a small area within the nucleus that contains DNA in the form of transcriptionally active ribosomal RNA (rRNA) genes, RNA, and proteins. The nucleolus is the site of rRNA synthesis and contains regulatory cell cycle proteins. The is a double membrane system that surrounds the nucleus of the cell. It consists of an inner and an outer membrane separated by a perinuclear cisternal space and

nucleolus

nuclear envelope

nucleoli

nuclear pores

perforated by . The outer membrane of the nuclear envelope is continuous with that of the rough-surfaced endoplasmic reticulum (rER) and is often studded with ribosomes (Fig. 3.1).

FIGURE 3.1. Nucleus and its relationship to the rough-surfaced endoplasmic reticulum (rER). a. The nuclear wall consists of a

double membrane envelope that surrounds the nucleus. The outer membrane is continuous with the membranes of the rER; thus, the perinuclear space communicates with the rER lumen. The inner membrane is adjacent to nuclear intermediate filaments that form the nuclear lamina. This electron micrograph, prepared by the quick-freeze, deep-etch technique, shows the nucleus, the large spherical object, surrounded by the nuclear envelope. Note that the outer membrane possesses ribosomes and is continuous with the rER. ×12,000. (Courtesy of Dr. John E. Heuser, Washington University School of Medicine.)

b.

nucleoplasm is nuclear content other than the chromatin and

The nucleolus.

A simple microscopic evaluation of the nucleus provides a great deal of information about a cell’s well-being. Evaluation of nuclear size, shape, and structure plays an important role in cancer. For instance, have visible nuclear alterations. These include the following findings:

dying cells

Karyolysis,

or the disappearance of nuclei due to complete dissolution of DNA by increased activity of DNAse

Pyknosis, or condensation of chromatin leading to shrinkage of the nuclei (they appear as dense basophilic masses) Karyorrhexis, or fragmentations of nuclei (these changes are usually preceded by pyknosis)

NUCLEAR COMPONENTS Chromatin

Chromatin, a complex of DNA and proteins, is responsible for the characteristic basophilia of the nucleus. Each eukaryotic cell contains about 6 billion bits of information encoded within a DNA molecule, which measures about 1.8 m in length. The length of the DNA molecule is 100,000 times longer than that of the nuclear diameter. Therefore, DNA must be highly folded and tightly packed in the cell nucleus. This is accomplished by the formation of a unique nucleoprotein complex called . The chromatin complex consists of DNA and structural proteins. Further folding of chromatin, such as that which occurs during mitosis, produces structures called . Each human cell contains 46 chromosomes. Chromatin proteins include five basic proteins called along with other . A unique feature of chromatin packaging is that it permits the transcriptional machinery to access those regions of the chromosomes required for gene expression.

chromatin

histones

chromosomes nonhistone proteins

Sequencing the human genome was successfully completed in 2003. The human genome encompasses the entire length of human DNA that contains the genetic information packaged in all 46 chromosomes. Sequencing of the human genome took approximately 13 years and was successfully completed in 2003 by the Human Genome Project. The human genome contains a 2.85 billion base pair consensus sequence of nucleotides, which are arranged in approximately 23,000 protein-coding genes. For years, it was thought that genes were usually present in two copies in a genome. However, recent discoveries have revealed that large segments of DNA can vary in numbers of copies. For instance, genes that were thought to always occur in two copies per genome have sometimes one, three,

copy number variations (CNVs)

or more copies. Such are widespread in the human genome and most likely lead to genetic imbalances. Previously defined as a segment of DNA involved in producing a polypeptide chain, a is now defined as a union of genomic sequences encoding a coherent set of potentially overlapping functional products.

gene

In general, two forms of chromatin are found in the nucleus: a condensed form called heterochromatin and a dispersed form called euchromatin.

In most cells, chromatin does not have a homogeneous appearance; rather, clumps of densely staining chromatin are embedded in a more lightly staining background. The densely staining material is highly condensed chromatin called , and the lightly staining material (where most transcribed genes are located) is a dispersed form called . It is the phosphate groups of the chromatin DNA that are responsible for the characteristic basophilia of chromatin (page 6). There are two recognizable types of heterochromatin: constitutive and facultative. contains the same regions of genetically inactive, highly repetitive sequences of DNA that are condensed and consistently packaged in the same regions of the chromosome when compared with other cells. Large amounts of constitutive heterochromatin are found in chromosomes near the centromeres and telomeres. is also condensed and is not involved in the transcription process. In contrast to constitutive heterochromatin, facultative heterochromatin is not repetitive, and its location within the nucleus and chromosomes varies when compared with other cells. Facultative heterochromatin may undergo active transcription in certain cells (see the description of Barr bodies on page 91) owing to specific conditions, such as certain cell cycle stages, nuclear localization changes (i.e., migration from the center to the periphery), or the active transcription of only one allele of a gene (monoallelic gene expression). Heterochromatin is found in the following three locations (Fig. 3.2):

heterochromatin euchromatin

Constitutive heterochromatin

Facultative heterochromatin

FIGURE 3.2. Electron micrographs of nuclei from two different cell types. The large electron micrograph shows the nucleus of a nerve

cell. Two nucleoli are included in the plane of section. The nucleus of this active cell, exclusive of the nucleoli, comprises almost entirely extended chromatin or euchromatin. ×10,000. The smaller nucleus belongs to a circulating lymphocyte (the entire cell is shown in the micrograph). It is a relatively inactive cell. Note the paucity of cytoplasm and cytoplasmic organelles. The chromatin in the nucleus is largely condensed (heterochromatin). The lighter areas represent euchromatin. ×13,000.

inset.

Marginal chromatin is found at the periphery of the nucleus; the structure that light microscopists formerly referred to as the nuclear membrane actually consists largely of marginal chromatin. Karyosomes are discrete bodies of chromatin irregular in size and shape that are found throughout the nucleus. Nucleolar-associated chromatin is chromatin found in association with the nucleolus.

Heterochromatin stains with hematoxylin and basic dyes; it is also readily displayed with the Feulgen stain (a specific histochemical reaction for the deoxyribose of DNA, page 6) and fluorescent vital dyes such as Hoechst dyes and propidium iodide. It is the heterochromatin that accounts for the conspicuous staining of the nucleus in hematoxylin and eosin (H&E) preparations. Euchromatin is not evident in the light microscope. It is present within the nucleoplasm in the “clear” areas between and around the heterochromatin. In routine electron micrographs, there is no sharp delineation between euchromatin and heterochromatin; both have a granular, filamentous appearance, but the euchromatin is less tightly packed. Euchromatin indicates active chromatin—that is, chromatin that is stretched out so that the genetic information in the DNA can be read and transcribed. It is prominent in metabolically active cells, such as neurons and liver cells. Heterochromatin predominates in metabolically inactive cells, such as small circulating lymphocytes and sperm cells, or in cells that produce one major product, such as plasma cells.

The smallest units of chromatin structure are macromolecular complexes of DNA and histones called nucleosomes. Nucleosomes are found in both euchromatin and heterochromatin and in chromosomes. These 10-nm-diameter particles represent the first level of chromatin folding and are formed by the coiling of the DNA molecule around a protein core. This step shortens the DNA molecule by approximately sevenfold relative to the unfolded DNA molecule. The core of the nucleosome consists of eight (called an ). Two loops of DNA (~146 nucleotide pairs) are wrapped around the core octamer. The DNA

histone molecules

octamer

extends between each particle as a 2-nm filament that joins adjacent nucleosomes. When chromatin is extracted from the nucleus, the nucleosomal substructure of chromatin is visible in transmission electron microscopy (TEM) and is often described as “ ” (Fig. 3.3a).

beads on a string

FIGURE 3.3.

a.

Packaging of chromatin into the chromosomal structure. Sequential steps in the packaging of nuclear chromatin are shown in this diagram, beginning with the DNA double helix and ending with the highly

b.

condensed form found in chromosomes. Structure of human metaphase chromosome 2 as visible in atomic force microscopic image. ×20,000. (Courtesy of Dr. Tatsuo Ushiki.)

In the next step of chromatin folding, a long strand of nucleosomes is coiled to produce a . Six nucleosomes form one turn in the coil of the chromatin fibril, which is approximately 40-fold shorter than unfolded DNA. Long stretches of 30-nm chromatin fibrils are further organized into (containing 15,000–100,000 base pairs), which are anchored into a or composed of nonhistone proteins. In heterochromatin, the chromatin fibers are tightly packed and folded on each other; in euchromatin, the chromatin fibrils are more loosely arranged.

30-nm chromatin fibril

loop domains

chromosome scaffold

nuclear matrix

In dividing cells, chromatin is condensed and organized into discrete bodies called chromosomes. During mitotic division, chromatin fibers formed from chromatin loop domains attached to a flexible protein scaffold undergo condensation to form chromosomes [Gr., colored bodies]. Each chromosome is formed by two chromatids that are joined together at a point called the centromere (Fig. 3.3b). The double nature of the chromosome is produced in the preceding synthetic (S) phase of the cell cycle (see pages 97-99), during which DNA is replicated in preparation for the next mitotic division. The area located at each end of the chromosome is called the . Telomeres shorten with each cell division. Recent studies indicate that telomere length is an important indicator of the life span of the cell. To survive indefinitely (become “immortalized”), cells must activate a mechanism that maintains telomere length. For example, in cells that have been transformed into malignant cells, an enzyme called is present that adds repeated nucleotide sequences to the telomere ends. Expression of this enzyme has been shown to extend the life span of cells, thus promoting cell growth. Telomerase is being studied as a potential target for use as an anticancer treatment. With the exception of the mature gametes, the egg and sperm, human cells contain organized as (each chromosome in the pair has the same shape and size). Twenty-two pairs have identical chromosomes (i.e., each

telomere

telomerase

pairs

46 chromosomes

23 homologous

chromosome of the pair contains the same portion of the genome) and are called . The 23rd pair of chromosomes are the , designated and . Females contain two X chromosomes (46,XX); males contain one X and one Y chromosome (46,XY). The chromosomal number, 46, is found in most of the somatic cells of the body and is called the number. To simplify the description of chromosomal number and DNA changes during mitosis and meiosis, we use the lowercase letter for chromosome number and lowercase letter for DNA content. Diploid chromosomes have the amount of DNA immediately after cell division. They have twice that amount after the S phase (see pages 99-101). As a result of , eggs and sperm have only 23 chromosomes, the haploid number, as well as the haploid amount of DNA. The somatic chromosome number and the diploid amount of DNA are restored at by the fusion of the sperm nucleus with the egg nucleus (see pages 102-103).

autosomes sex chromosomes

X

Y

diploid (2n)

(2d)

meiosis (1n)

(d) (4d)

(n)

(1d)

(2n) (2d) fertilization In a karyotype, chromosome pairs are sorted according to their size, shape, and emitted fluorescent color.

A preparation of chromosomes derived from mechanically ruptured, dividing cells that are then fixed, plated on a microscope slide, and stained is called a . In the past, chromosomes were routinely stained with Giemsa stain; however, with the recent development of in situ hybridization techniques, the fluorescent in situ hybridization (FISH) procedure is now more often used to visualize a chromosomal spread. These spreads are observed with fluorescence microscopes, and computercontrolled cameras are then used to capture images of the chromosome pairs. Image-processing software is used to sort the chromosome pairs according to their morphology to form a (see Fig. F3.1.1a). A variety of molecular probes that are now commercially available are used in to diagnose disorders caused by chromosomal abnormalities, such as nondisjunctions, transpositions (see Fig. F3.1.1a), deletions (see Fig. F3.1.1b), and duplications of specific gene sites. Karyotypes are also used for prenatal determination of sex in fetuses and for prenatal diagnosis of certain genetic diseases (see Fig. 1.7).

metaphase spread

karyotype

cytogenetic testing

The Barr body represents a region of facultative heterochromatin that can be used to identify the sex of a fetus. Some chromosomes are repressed in the interphase nucleus and exist only in the tightly packed heterochromatic form. One X chromosome of the female is an example of such a chromosome and

can be used to identify the sex of a fetus. This chromosome was discovered in 1949 by Barr and Bartram in nerve cells of female cats, where it appears as a well-stained round body, now called the , adjacent to the nucleolus. In females, the Barr body represents a region of that is condensed and not involved in the transcription process. During embryonic development, one randomly chosen X chromosome in the female zygote undergoes chromosome-wide chromatin condensation, and this state is maintained throughout the lifetime of the organism. Although the Barr body was originally found in sectioned tissue, it was subsequently shown that any relatively large number of cells prepared as a smear (e.g., scrapings of the oral mucous membrane from the inside of the cheeks or neutrophils from a blood smear) can be used to search for the Barr body. In cells of the oral mucous membrane, the Barr body is located adjacent to the nuclear envelope. In neutrophils, the Barr body forms a drumstick-shaped appendage on one of the nuclear lobes (Fig. 3.4). In both sections and smears, many cells must be examined to find those whose orientation is suitable for the display of the Barr body.

Barr body

facultative heterochromatin

FIGURE 3.4.

Photomicrograph of a neutrophil from a female patient’s blood smear. The second X chromosome of the female patient is repressed in the interphase nucleus and can be demonstrated in the neutrophil as a drumstick-appearing appendage ( ) on a nuclear lobe. ×250.

Nucleolus

arrow

The nucleolus is the site of ribosomal RNA (rRNA) synthesis and initial ribosomal assembly. The nucleolus is a nonmembranous region of the nucleus that surrounds transcriptionally active rRNA genes. It is the primary site of ribosomal production and assembly. The nucleolus varies in size but is particularly well developed in cells active in

protein synthesis. Some cells contain more than one nucleolus (Fig. 3.5). The nucleolus has three morphologically distinct regions:

FIGURE 3.5. Electron micrograph of the nucleolus. This nucleolus from a nerve cell shows fibrillar centers ( FC ) surrounded by the fibrillar ( F ) and granular ( G ) materials. Such a network of both materials is referred to as the nucleolonema . The ribosomal RNA (rRNA), DNA-containing genes for the rRNA, and specific proteins are localized in the interstices of the nucleolonema. ×15,000.

Fibrillar centers

contain DNA loops of five different chromosomes (13, 14, 15, 21, and 22) that contain rRNA genes, RNA polymerase I, and transcription factors. contains ribosomal genes that are actively undergoing transcription and large amounts of rRNA. represents the site of initial ribosomal assembly and contains densely packed preribosomal particles.

Fibrillar material (pars fibrosa)

Granular material (pars granulosa)

The network formed by the granular and the fibrillar materials is called the . rRNA is present in both granular and fibrillar material and is organized, respectively, as both granules and extremely fine filaments packed tightly together. Genes for the ribosomal subunits are localized in the interstices of this network and are transcribed by RNA polymerase I. After further processing and modification of rRNA by small nucleolar RNAs (snoRNAs), the subunits of rRNA are assembled using ribosomal proteins imported from the cytoplasm. The partially assembled ribosomal subunits (preribosomes) are exported from the nucleus via nuclear pores for full assembly into mature ribosomes in the cytoplasm.

nucleolonema

The nucleolus is involved in the regulation of the cell cycle. Nucleostemin is a p53-binding protein found within the nucleolus that regulates the cell cycle and influences cell differentiation. As cellular differentiation progresses, the level of this protein decreases. The presence of nucleostemin in suggests that it could play a role in their (Folder 3.2). In addition, DNA, RNA, and retroviruses and their viral proteins interact with the nucleolus and cause redistribution of fibrillar and granular materials during the course of viral infection. These viruses may use components of the nucleolus as part of their own replication process. Evidence suggests that viruses may target the nucleolus and its components to favor viral transcription and translation and perhaps alter the cell cycle to promote viral replication.

malignant cells uncontrolled proliferation

FOLDER 3.1

CLINICAL CORRELATION: CYTOGENETIC TESTING Cytogenetic testing is an important component in the diagnosis and evaluation of genetic disorders and refers to the analysis of chromosomes. Chromosomal abnormalities occur in ~0.5% of all live births and are detected in ~50% of first-trimester miscarriages (spontaneous abortions) and 95% of various tumor cells. Chromosome analysis can be performed on peripheral blood, bone marrow, tissues (such as skin or chorionic villi obtained from biopsies), and cells obtained from amniotic fluid during amniocentesis. Studies of chromosomes begin with the extraction of whole chromosomes from the nuclei of dividing cells. These chromosomes are then placed on glass slides, hybridized with special fluorescence

probes (fluorescent in situ hybridization [FISH] technique), and examined under a microscope. A single fluorescent DNA probe produces a bright microscopic signal when the probe is hybridized to a specific part of a particular chromosome. To obtain an image of all of the chromosomes, a mixture of different probes is used to produce different colors in each chromosome. Karyotypes labeled by this method allow cytogeneticists to perform a comprehensive analysis of changes in the number of chromosomes and chromosomal abnormalities, such as additions or deletions. The paired chromosomes are numbered in the karyotype, and the male sex is indicated by the presence of chromosomes X and Y (Fig. F3.1.1a). The inset in Figure F3.1.1a shows the XX chromosome pair as it appears in the female.

white box

FIGURE F3.1.1. Karyotypes obtained with the fluorescent in situ hybridization technique. a. Karyotype of a normal male. The white box inset shows the XX chromosome pair of a normal female. The red box inset reveals an abnormality in chromosomes 14 and 8. (Courtesy of the Applied Imaging International Ltd., Newcastle upon Tyne, United Kingdom.) b. A metaphase spread from a patient with Prader-Willi/Angelman syndrome. The yellow box inset shows the enlarged pair of chromosome 15. (Courtesy of Dr. Robert B. Jenkins.) Sometimes, part of a chromosome will break off and attach to another chromosome. When this happens, it is referred to as a . Note that the inset in Figure F3.1.1a shows a translocation between chromosomes 8 and 14 (t8;14). It is clearly visible in this color image that a part of the original chromosome 8 ( ) is now attached to chromosome 14, and a small portion of chromosome 14 ( ) is now part of chromosome 8.

translocation

aqua blue region

red box

red region

Such chromosomal translocations are present in lymphomas (cancers of blood cells), such as acute myeloid leukemia (AML), non-Hodgkin lymphoma (NHL), and Burkitt lymphoma. In Figure F3.1.1b, a metaphase spread obtained from cultured lymphocytes of a patient with suspected has been hybridized with several DNA probes reacting with chromosome 15 (an enlarged chromosomal pair from chromosome 15 is shown in the inset). The probe (D15Z1) indicates the centromere of chromosome 15. The adjacent probe (D15S10) reacts with the PWS/AS region of chromosome 15. Deletion of this region is associated with PWS/AS. Note that one homolog of chromosome 15 has lost that region (no orange signal is visible). The third probe (PML) recognizes the distal long arm of chromosome 15 and is visible in both chromosomes. Severe intellectual disability, muscular hypotonia, short stature, hypogonadism, and insulin-resistant diabetes are characteristics of PWS/AS. When the deletion is inherited from the mother, patients develop AS; when inherited from the father, patients develop PWS. This preparation is counterstained with DAPI that reacts with double-stranded DNA and exhibits fluorescence.

Prader-Willi/Angelman

syndrome (PWS/AS)

yellow box

orange

green

red

blue

FOLDER 3.2

CLINICAL CORRELATION: REGULATION OF CELL CYCLE AND CANCER TREATMENT Understanding the details of cell cycle regulation has had an impact on cancer research and has contributed to the development of new treatments. For instance, inactivation of tumor suppressor genes has been shown to play a role in the growth and division of cancer cells. The proteins encoded by these genes are used by the cell throughout several DNA-damage checkpoints. For instance, mutations in the and are associated with an increased risk for bilateral breast cancer and ovarian cancer. Both protein products of these tumor suppressor genes—namely, BRCA-1 and BRCA-2 proteins—are directly involved in multiple cellular processes in response to DNA damage, including checkpoint activation, gene transcription, and repair of DNA double-strand breaks. Together with , which is involved in the homologous recombination and repair of DNA, they maintain the stability of the human genome. The defective BRCA proteins are unable to interact with RAD-51. By screening patients for mutations in these genes, enhanced screening and prophylactic mastectomy and/or oophorectomy can be offered to those who test positive for these mutations. It is also now known why in some individuals, make their tumors resistant to radiotherapy. DNA damage caused by therapeutic radiation procedures is detected by DNA-damage checkpoints, which cause cancer cells to be arrested in the cell

breast cancer susceptibility gene 1 (BRCA-1) cancer susceptibility gene 2 (BRCA-2)

breast

RAD-51 protein

p53 mutations

cycle. However, these cells will not die because of the absence of functional p53, which triggers apoptosis.

The nucleolus stains intensely with hematoxylin and basic dyes and metachromatically with thionine dyes. The relation of basophilia and metachromasia of the nucleolus to the phosphate groups of the nucleolar RNA is confirmed by predigestion of specimens with ribonuclease (RNAse), which abolishes the staining. As mentioned earlier, DNA is present in the nucleolus; however, its concentration is below the detection capability of the Feulgen reaction. Thus, when examined in the light microscope, nucleoli appear Feulgen-negative with Feulgenpositive nucleolus-associated chromatin that often rims the nucleolus.

Nuclear Envelope

The nuclear envelope, formed by two membranes with a perinuclear cisternal space between them, separates the nucleoplasm from the cytoplasm. The nuclear envelope provides a selectively permeable membranous

barrier between the nuclear compartment and the cytoplasm, and it encloses the chromatin. The nuclear envelope is assembled from two (outer and inner) nuclear membranes with a between them. The perinuclear clear cisternal space is continuous with the cisternal space of the rER (see Fig. 3.1). The two membranes of the envelope are perforated at intervals by that mediate the active transport of proteins, ribonucleoproteins, and RNAs between the nucleus and the cytoplasm. The membranes of the nuclear envelope differ in structure and functions:

cisternal space

perinuclear

nuclear pores

outer nuclear membrane

The closely resembles the membrane of the ER and in fact is continuous with the rER membrane (Fig. 3.6). Polyribosomes are often attached to ribosomal docking proteins present on the cytoplasmic side of the outer nuclear membrane.

FIGURE 3.6. Structure of the nuclear lamina. a.

This schematic drawing shows the structure of the nuclear lamina adjacent to the inner nuclear membrane. The cut window in the nuclear lamina shows the DNA within the nucleus. Note that the nuclear envelope is pierced by nuclear pore complexes, which allow for selective bidirectional transport of molecules between the nucleus and the cytoplasm. b. Electron micrograph of a portion of the nuclear lamina from a oocyte. It is formed by intermediate filaments (lamins) that are arranged in a square lattice. ×43,000. (Adapted from Aebi U, Cohn J, Buhle L, et al. The nuclear lamina is a meshwork of intermediate-type filaments. . 1986;323:560–564.)

Xenopus

Nature

inner nuclear membrane nuclear (fibrous) lamina

The is supported by a rigid network of intermediate protein filaments attached to its inner surface called the (see Fig. 3.6). In addition, the inner nuclear membrane contains specific lamin receptors and several lamina-associated proteins that bind to chromosomes and secure the attachment of the nuclear lamina.

The nuclear lamina is formed by intermediate filaments and lies adjacent to the inner nuclear membrane.

nuclear lamina

The , a thin, electron-dense intermediate filament network-like layer, resides underneath the nuclear membrane. In addition to its supporting or “nucleoskeletal” function, nuclear lamina is essential in many nuclear activities, such as DNA replication, transcription, and gene regulation. If the membranous component of the nuclear envelope is disrupted by exposure to detergent, the nuclear lamina remains, and the nucleus retains its shape. The major components of the lamina, as determined by biochemical isolation, are , a specialized type of nuclear intermediate filament (see pages 73-75), and . Nuclear lamina is essentially composed of lamin A and lamin C proteins that form intermediate filaments. These filaments are cross-linked into an orthogonal lattice (see Fig. 3.6), which is attached mainly via lamin B protein to the inner nuclear membrane through its interactions with lamin receptors. The family of lamin receptors includes (34 kDa) that binds both lamin A and B; (29 kDa) that binds lamin A; and a 58-kDa that, as its name suggests, binds lamin B. Unlike other cytoplasmic intermediate filaments, lamins disassemble during mitosis and reassemble when mitosis ends. The nuclear lamina appears to serve as scaffolding for chromatin, chromatin-associated proteins, nuclear pores, and the membranes of the nuclear envelope. In addition, it is involved in nuclear organization, cell cycle regulation, differentiation, and gene expression. Impairment in nuclear lamina architecture or function is associated with certain genetic diseases (laminopathies) and apoptosis. Mutations in cause tissue-specific diseases that affect striated muscle, adipose tissue, peripheral nerve or skeletal development, and premature aging. Two hereditary forms of are associated with mutations in either lamins or lamin receptors. The X-linked recessive form of EDMD is caused by mutations of , whereas the autosomal dominant form of EDMD is caused by mutations in lamin A/C. In general, EDMD is characterized by early-onset contractures of major tendons, slowly progressive muscle weakness, muscle wasting in the upper and lower limbs, and cardiomyopathy (weakening of the heart muscle).

associated proteins

nuclear lamins

nurim lamin B receptor (LBR)

lamin-

emerin

lamin A/C

Emery–Dreifuss muscular dystrophy (EDMD)

emerin

The nuclear envelope has an array of openings called nuclear pores. At numerous sites, the paired membranes of the nuclear envelope are punctuated by 70- to 80-nm “openings” through the envelope. These are formed from the merging of the inner and outer membranes of the nuclear envelope. With an ordinary TEM, a diaphragm-like structure appears to cross the pore opening (Fig. 3.7). Often, a small, dense body is observed in the center of the opening (Fig. 3.8). Because such profiles are thought to represent either ribosomes or other protein complexes (transporters) captured during their passage through the pore at the time of fixation, the term is commonly used to describe this feature.

nuclear pores

central plug/transporter

FIGURE 3.7. Electron micrograph of the nuclear envelope. Note the visible nuclear pore complexes ( arrows ) and the two membranes that

constitute the nuclear envelope. At the periphery of each pore, the outer and inner membranes of the nuclear envelope appear continuous. ×30,000.

FIGURE 3.8. Cryoelectron tomography of the nuclear pore complex (NPC). These surface renderings of electron tomograms obtained from the frozenhydrated Dictyostelium nuclei show detailed structure of the NPC. ×320,000. a. Cytoplasmic face of the NPC shows eight protein fibrils

arranged around the central channel. They protrude from the cytoplasmic ring subunits and point toward the center of the structure. Note a presence of the central plug or transporter within the central pore, which represents either ribosomes or other protein transporters captured during their passage through the NPC. Nuclear face of the NPC shows the nucleoplasmic ring subunits connected by nuclear filaments with the basket indicated in color. (Adapted from Beck M, Förster F, Ecke M, et al. Nuclear pore complex structure and dynamics revealed by cryoelectron tomography. . 2004;306:1387–1390.)

brown

b.

Science

With special techniques—such as negative staining and highvoltage TEM, or, recently, cryoelectron tomography—the nuclear pore exhibits additional structural detail (see Fig. 3.8). Eight multidomain protein subunits arranged in an octagonal at the periphery of each pore form a cylinder-like structure known as the . The NPC, 6 which has an estimated total mass of 125 × 10 Da, is composed of approximately 50 different NPC proteins collectively referred to as . This central framework is inserted between the and the (Fig. 3.9). From the cytoplasmic ring, eight short protrude into the cytoplasm and point toward the center of the structure. The nucleoplasmic ring complex anchors a

framework

nuclear pore complex (NPC)

nucleoporins (Nup proteins) cytoplasmic ring

central

nuclear ring protein fibrils nuclear

basket (or nuclear “cage” that resembles a fish trap) assembled from eight thin 50-nm-long filaments joined distally by an adjustable terminal ring 30–50 nm in diameter (see Fig. 3.9). The cylinder-shaped central framework encircles the central pore of the NPC, which acts as a close-fitting diaphragm or gated channel. In addition, each NPC contains one or more water-filled channels for the transport of small molecules.

FIGURE 3.9. Sagittal section of the nuclear pore complex.

Cryoelectron tomographic view of a sagittal section of the nuclear pore complex—shown in Figure 3.8—is compared with a schematic drawing of the complex. Note that the central plug/transporter has been removed from the central pore. ×320,000. Each pore contains eight protein subunits arranged in an octagonal central framework at the periphery of the pore. These subunits form a nuclear pore complex that is inserted between two cytoplasmic and nucleoplasmic rings. Eight short protein fibrils protrude from the cytoplasmic rings into the cytoplasm. The nuclear ring anchors a basket assembled from eight thin filaments joined distally into the terminal ring. The diameter of the ring can be adjusted to meet nuclear pore transport requirements. The cylindrical central framework encircles the central pore, which acts as a close-fitting diaphragm. (Adapted from Beck M, Förster F, Ecke M, et al. Nuclear pore complex structure and dynamics revealed by cryoelectron tomography. . 2004;306:1387– 1390.)

Science

The nuclear pore complex nucleocytoplasmic transport.

mediates

bidirectional

Various experiments have shown that the NPC regulates the passage of proteins between the nucleus and the cytoplasm. The significance of the NPC can be readily appreciated, as the nucleus does not carry out protein synthesis. Ribosomal proteins are partially assembled into ribosomal subunits in the nucleolus and are transported through nuclear pores to the cytoplasm. Conversely, nuclear proteins, such as histones and lamins, are produced in the cytoplasm and are transported through nuclear pores into the nucleus. Transport through the NPC largely depends on the size of the molecules:

Large molecules

(such as large proteins and macromolecular complexes) depend on the presence of an attached signal sequence called the for passage through the pores. Proteins labeled with NLS destined for the nucleus then bind to a soluble cytosolic receptor called a that directs them from the cytoplasm to an appropriate NPC. They are then actively transported through the pore by a . Export of proteins and RNA from the nucleus is similar to the import mechanism into the nucleus. Proteins that possess a bind in the nucleus to (a protein that moves molecules from the nucleus into the cytoplasm) and to a GTP molecule. Protein –exportin–GTP complexes pass through the NPC into the cytoplasm where GTP is hydrolyzed and the NES protein is released. The NPC transports proteins and all forms of RNA as well as ribosomal subunits in their fully folded configurations. (200 in aggrecan. Note also that versican has identical GAG molecules (chondroitin sulfate) attached to

the core molecule, whereas aggrecan has a mixture of chondroitin sulfate and keratan sulfate attached to the core protein. Syndecan-1 is a transmembrane proteoglycan that attaches the cell membrane to the extracellular matrix.

Proteoglycans are found in the ground substance of all connective tissues and also as membrane-bound molecules on the surface of many cell types. Transmembrane proteoglycans, such as , link cells to ECM molecules (see Fig. 6.18). Syndecan-1 carries 3–5 heparan sulfate and chondroitin sulfate chains that allow for interaction with a large variety of ligands including fibroblast growth factors (FGFs), vascular endothelial growth factor (VEGF), TGF-β, and ECM molecules, such as fibronectin. As an example of this function, syndecan-1 is expressed on the surface of B lymphocytes at two different phases of their development. Syndecan-1 molecules are first expressed during early development when lymphocytes are attached to the matrix protein of the bone marrow as they undergo differentiation. The loss of expression of this proteoglycan coincides with the release of the B lymphocyte into the circulation. The second time B lymphocytes express syndecan-1 is during its differentiation into a plasma cell within the connective tissue. Syndecan-1 anchors the plasma cell to the ECM proteins of the connective tissue. (1,000–2,500 kDa) is another important extracellular proteoglycan. Its molecules are noncovalently bound to the long molecule of hyaluronan (like bristles to the backbone in a bottle brush); this binding is facilitated by linking proteins. To each aggrecan core protein, multiple chains of chondroitin sulfate and keratan sulfate are covalently attached through the trisaccharide linker. The most common proteoglycans are summarized in Table 6.4. Aggrecan is a cartilage-specific proteoglycan and a critical component in cartilage structure and the function of joints.

syndecan-1

Aggrecan

Proteoglycans

TABLE 6.4

EGF, epithelial growth factor.

Multiadhesive glycoproteins play an important role in stabilizing the ECM and linking it to cell surfaces. Multiadhesive glycoproteins represent a small but important group of proteins residing in the ECM. They are multidomain and multifunctional molecules that play an important role in stabilizing the ECM and linking it to the cell surface. They possess binding sites for a variety of ECM proteins, such as collagens, proteoglycans, and GAGs; they also interact with cell surface receptors, such as integrin and laminin receptors (Fig. 6.19). Multiadhesive glycoproteins regulate and modulate functions of the ECM related to cell movement and cell migration as well as stimulate cell proliferation and differentiation. Among the best characterized multiadhesive glycoproteins are the following:

FIGURE 6.19. Common multiadhesive glycoproteins.

These proteins reside in the extracellular matrix and are important in stabilizing the matrix and linking it to the cell surface. They are multifunctional molecules of different shapes and possess multiple binding sites for a variety of extracellular matrix proteins, such as collagens, proteoglycans, and GAGs. Note that multiadhesive proteins interact with basal membrane receptors, such as integrin and laminin receptors.

Fibronectin

(250–280 kDa) is the most abundant glycoprotein in connective tissue. Fibronectins are dimer molecules formed from two similar peptides linked by disulfide bonds at a carboxy-terminus to form 50-nm-long arms (see Fig. 6.19). Each molecule contains several binding domains that interact with different ECM molecules (e.g., heparan sulfate; collagen types I, II, and III; fibrin; hyaluronan; and fibronectin) and integrin, a cell surface receptor. Binding to a cell surface receptor activates fibronectin, which then assembles into fibrils. Fibronectin plays an important role in cell attachment to the ECM. At least 20 different fibronectin molecules have been identified to date. (140–400 kDa) is present in basal and external laminae. It possesses binding sites for collagen type IV molecules, heparan sulfate, heparin, entactin, laminin, and the laminin receptor on the cell surface. The process of basal lamina assembly and the role of the laminin in this process are described in Chapter 5, Epithelial Tissue (see pages 155-158). (280 kDa per monomer) appears during embryogenesis, but its synthesis is switched off in mature tissues. It reappears during wound healing and is also found within musculotendinous junctions and malignant tumors. Tenascin is a disulfide-linked dimer molecule that consists of six chains joined at their amino-terminus (see Fig. 6.19). It has binding sites for fibrinogen, heparin, and EGF-like growth factors; thus, it participates in cell attachment to the ECM. (44 kDa) is present in the ECM of bone. It binds to osteoclasts and attaches them to the underlying bone surface. Osteopontin plays an important role in sequestering calcium and promoting calcification of the ECM.

Laminin

Tenascin

Osteopontin

Important multiadhesive glycoproteins found in the ECM of connective tissue are summarized in Table 6.5.

TABLE 6.5 Multiadhesive Glycoproteins

CAM, cell adhesion molecule; ECM, extracellular matrix; EGF, epithelial growth factor.

CONNECTIVE TISSUE CELLS

Connective tissue cells can be resident or wandering. The cells that make up the resident cell population

are relatively stable; they typically exhibit little movement and can be regarded as permanent residents of the tissue. These resident cells include

fibroblasts and a closely related cell type, the myofibroblast; macrophages; adipocytes; mast cells; and adult stem cells. The wandering cell population or transient cell population

consists primarily of cells that have migrated into the tissue from the blood in response to specific stimuli. These include

lymphocytes, plasma cells,

neutrophils, eosinophils, basophils, and monocytes.

Fibroblasts and Myofibroblasts

The fibroblast is the principal cell of connective tissue. Fibroblasts are responsible for the synthesis of collagen,

elastic, and reticular fibers and the complex carbohydrates of the ground substance. Research suggests that a single fibroblast is capable of producing all of the ECM components. Fibroblasts reside in close proximity to collagen fibers. In routine H&E preparations, however, often, only the nucleus is visible. It appears as an elongated or disc-like structure, sometimes with a nucleolus evident. The thin, pale-staining, flattened processes that form the bulk of the cytoplasm are usually not visible, largely because they blend with the collagen fibers. In some specially prepared specimens, it is possible to distinguish the cytoplasm of the cell from the fibrous components (Fig. 6.20a). When ECM material is produced during active growth or in wound repair (in ), the cytoplasm of the fibroblast is more extensive and may display basophilia as a result of increased amounts of rER associated with protein synthesis (Fig. 6.20b). When examined with the TEM, the fibroblast cytoplasm exhibits profiles of rER and a prominent Golgi apparatus (Fig. 6.21).

fibroblasts

activated

FIGURE 6.20. Fibroblasts in connective tissue. a.

Photomicrograph of a connective tissue specimen in a routine hematoxylin and eosin (H&E)-stained, paraffin-embedded preparation shows nuclei of fibroblasts ( ). ×600. During wound repair, the activated fibroblasts ( ) exhibit more basophilic cytoplasm, which is readily observed with the light microscope. ×500.

F F

b.

FIGURE 6.21. Electron micrograph of fibroblasts. The processes of several fibroblasts are shown. The nucleus of one fibroblast is in the upper right of the micrograph. The cytoplasm contains conspicuous profiles of rough endoplasmic reticulum ( rER ). The cisternae of the reticulum are distended, indicating active synthesis. The membranes of the Golgi apparatus ( G ) are seen in proximity to the rER . Surrounding the cells are collagen fibrils ( CF ), almost all of which have been cut in cross section and thus appear as small dots at this magnification. ×11,000.

The myofibroblast displays properties of both fibroblasts and smooth muscle cells. The myofibroblast is an elongated, spindly connective tissue

cell not readily identifiable in routine H&E preparations. It is characterized by the presence of bundles of actin filaments with associated actin motor proteins such as nonmuscle myosin (page 76). Expression of (α-SMA; actin isoform found in vascular smooth muscles) in myofibroblasts is regulated by TGF-β1. The actin bundles transverse the cell cytoplasm, originating and terminating on the opposite sides of the plasma membrane. The site where actin fibers attach to the plasma membrane also serves as a cell-to-ECM anchoring junction and is called a ; it resembles the focal adhesion found in epithelial cells (see pages 160-162). The arrangement of actin bundles and their attachment sites form a , in which forces generated by the contraction of intracellular actin bundles are transmitted to the ECM. With the TEM, the myofibroblast displays characteristics typical of a fibroblast along with characteristics of smooth muscle cells. In addition to rER and Golgi profiles, the myofibroblast contains bundles of longitudinally disposed actin filaments and dense bodies similar to those observed in smooth muscle cells (Fig. 6.22). As in the smooth muscle cell, the nucleus often shows an undulating surface profile, a phenomenon associated with cell contraction. The myofibroblast differs from the smooth muscle cell in that it lacks a surrounding basal lamina (smooth muscle cells are surrounded by a basal or external lamina). Also, it usually exists as an isolated cell, although its processes may contact the processes of other

α-smooth muscle actin

fibronexus

mechanotransduction system

myofibroblasts. Such points of contact exhibit gap junctions, indicating intercellular communication.

FIGURE 6.22. Electron micrograph of a myofibroblast. The cell exhibits some features of a fibroblast, such as areas with a moderate amount of rough endoplasmic reticulum ( rER ). Compare with Figure 6.21. Other areas, however, contain aggregates of thin filaments and cytoplasmic densities ( arrows ), features that are characteristic of smooth muscle cells. The arrowheads indicate longitudinal profiles of collagen fibrils. ×11,000.

Macrophages

Macrophages are phagocytic cells derived from monocytes that contain an abundant number of lysosomes. Connective tissue macrophages, also known as tissue histiocytes, are derived from blood cells called monocytes. Monocytes migrate from the bloodstream into the connective tissue, where they differentiate into macrophages. In the light microscope and with conventional stains, tissue macrophages are difficult to identify unless they display obvious evidence of —for example, visible ingested material within their cytoplasm. Another feature that assists in identifying macrophages is an indented or kidneyshaped nucleus (Fig. 6.23a). Lysosomes are abundant in the cytoplasm and can be revealed by staining for acid phosphatase activity (both in the light microscope and with the TEM); a positive reaction is a further aid in the identification of the macrophage. With the TEM, the surface of the macrophage exhibits numerous folds and finger-like projections (Fig. 6.23b). The surface folds engulf the substances to be phagocytosed. The lysosomes of the macrophage, along with the surface cytoplasmic projections, are the structures most indicative of the specialized phagocytic capability of the cell. The macrophage may also contain endocytotic vesicles, phagolysosomes, and other evidence of phagocytosis (e.g., residual bodies).

phagocytic activity

FIGURE 6.23. Photomicrograph and electron micrograph of a macrophage. a. This photomicrograph shows several macrophages (M) in

the connective tissue from an area of wound healing. They can be distinguished from other cells by a presence of an indented or kidneyshaped nucleus (similar to that of monocytes in the blood vessels). Note several mature neutrophils ( ) with segmented nuclei located in the connective tissue that surround blood vessel ( ) filled with red and white blood cells in the of the image. ×480. The most distinctive electron micrograph features of the macrophage are its population of endocytotic vesicles, early and late endosomes, lysosomes, and phagolysosomes. The surface of the cell reveals a number of finger-like projections, some of which may be sections of surface folds. ×10,000.

N center

BV

b.

The rER, smooth ER (sER), and Golgi apparatus support the synthesis of proteins involved in the cell’s phagocytic and digestive functions, as well as its secretory functions. Secretory products leave the cell by both the constitutive and regulated exocytotic pathways. Regulated secretion can be activated by phagocytosis, immune complexes, complement, and signals from lymphocytes (including the release of , biologically active molecules that influence the activity of other cells). The secretory products released by the macrophage include a wide variety of substances related to the immune response, anaphylaxis, and inflammation. The release of neutral proteases and GAGases (enzymes that break down GAGs) facilitates the migration of macrophages through the connective tissue.

lymphokines

Macrophages are antigen-presenting cells and play an important role in immune response reactions.

Although the main function of the macrophage is phagocytosis, either as a defense activity (e.g., phagocytosis of bacteria) or as a cleanup operation (e.g., phagocytosis of cell debris), it also plays an important role in immune response reactions. Macrophages have specific proteins on their surface known as molecules that + allow them to interact with . When macrophages engulf a foreign cell, antigens—short polypeptides (7–10 amino acids long) from the foreign cell—are displayed on the surface of the MHC II molecules. If a CD4+ T lymphocyte

major histocompatibility complex II (MHC II) helper CD4 T lymphocytes

recognizes the displayed antigen, it becomes activated, triggering an immune response (see Chapter 14, Immune System and Lymphatic Tissues and Organs, pages 492-493). Because macrophages “present” antigens to helper CD4+ T lymphocytes, they are known as .

antigen-presenting cells (APCs) Macrophages arrive after neutrophils at the site of tissue injury and undergo differentiation. At the site of tissue injury, the first cells to reach the injured area are neutrophils. They are the first cells to recognize foreign organisms or infectious agents and initiate destruction by either reactive oxygen intermediates or oxygenindependent killing mechanisms (see pages 310-311). During this destruction process, large amounts of secretory products and cellular debris are generated at the site of injury. In addition, microorganisms that survived the action of neutrophils may also be present. After 24 hours, monocytes from blood vessels enter the site of injury and differentiate into macrophages, where they remain until inflammation resolves. Initially, the macrophage’s objective is to kill microorganisms that have survived the attack of neutrophils. Simultaneously, macrophages are activated by the interaction with several molecules produced by neutrophils and invading microorganisms. During this process, macrophages undergo a series of functional, morphologic, and biochemical modifications triggered by various gene activations.

Classically activated macrophages (M1 macrophages) promote inflammation, the destruction of ECM, and apoptosis.

Activation by interferon γ (IFN-γ), tumor necrosis factor α (TNF-α), or by bacterial lipopolysaccharide (LPS) creates the or . These macrophages have the capacity, through the production of nitric oxide (NO) and other intermediates, to destroy microorganisms at the site of inflammation. They also secrete interleukin (IL)-12, which stimulates helper CD4+ T lymphocytes. In turn, helper T cells secrete IL-2, which stimulates cytotoxic CD8+ T lymphocytes to arrive at the site of inflammation. In summary,

classically activated macrophage

M1 macrophage

chronic inflammation

tissue injury

M1 macrophages elicit and . When macrophages encounter large foreign bodies, they may fuse to form a large cell with as many as 100 nuclei that engulfs the foreign body. These multinucleated cells are called .

foreign-body giant cells (Langhans cells) Alternatively activated macrophages (M2 macrophages) assist in the resolution of inflammation and promote rebuilding of ECM, cell proliferation, and angiogenesis.

When the inflammatory stimulus is removed from the site of tissue injury, the body switches into a repair mode that includes the removal of cell debris, synthesis of components of new ECM, and revascularization of the injured tissue. During this period, macrophages are activated by cytokines IL-4, IL-5, IL-10, or IL-13. These types of cells are called . M2 macrophages are anti-inflammatory in that they assist in . They secrete IL-4 to promote differentiation of B lymphocytes into plasma cells and VEGF to stimulate angiogenesis. M2 macrophages also secrete ECM components (e.g., fibronectin and other multiadhesive glycoproteins). They promote owing to their anti-inflammatory, proliferative, and angiogenic activities. M2 macrophages are also efficient at combating parasitic infections (i.e., schistosomiasis). In addition to their beneficial activities, M2 macrophages are involved in pathogenesis of and .

activated macrophages or M2 macrophages inflammation

alternatively resolution of

wound repair

asthma

allergy

Mast Cells

Mast cells develop in the bone marrow and differentiate in connective tissue. Mast cells are large, ovoid, connective tissue cells (20–30 μm in diameter) with a spherical nucleus and cytoplasm filled with large, intensely basophilic granules. They are not easily identified in human tissue sections unless special fixatives are used to preserve the granules. After glutaraldehyde fixation, mast cell granules can be displayed with basic dyes,

such as toluidine blue. It stains the granules intensely and metachromatically because they contain heparin, a highly sulfated proteoglycan (Fig. 6.24a). The cytoplasm displays small amounts of rER, mitochondria, and a Golgi apparatus. The cell surface contains numerous microvilli and folds.

FIGURE 6.24. Mast cell. a.

Photomicrograph of a mast cell stained with toluidine blue. The granules stain intensely and, because of their numbers, tend to appear as a solid mass in some areas. The nucleus of the cell is represented by the pale-staining area. ×1,250.

b. This electron micrograph shows the cytoplasm of a mast cell that is virtually filled with granules. Note a small lymphocyte present in the upper left of the figure. ×6,000.

mast cell

The is related, but not identical, to the basophil, a white blood cell that contains similar granules (Table 6.6). They both arise from a pluripotential in the bone marrow. (MCPs) initially circulate in the peripheral blood as agranular cells of monocytic appearance. After migrating into the connective tissue, these immature cells differentiate and produce their characteristic granules (Fig. 6.24b). In contrast, (BaPs) differentiate and remain within the circulatory system. The surface of mature mast cells expresses a large number of ɛ c to which immunoglobulin E (IgE) antibodies are attached. Binding of a specific antigen to exposed IgE antibody molecules on the mast cell surface leads to an aggregation of Fc receptors. This triggers mast cell activation, which results in granule exocytosis (degranulation) and the release of granule content into the ECM. Mast cells can also be activated by an IgE-independent mechanism during complement protein activation.

hemopoietic Mast cell progenitors

stem cell (HSC)

basophil progenitors high-affinity F receptors (Fc RI)

of Features Characteristic of Mast TABLE 6.6 Comparison Cells and Basophils Characteristic Features

Mast Cells

Basophils

Origin

Hemopoietic stem cell

Hemopoietic stem cell

Site of differentiation

Connective tissue

Bone marrow

Cell divisions

Yes (occasionally)

No

Cells in circulation

No

Yes

Life span

Weeks to months

Days

Size

20–30 μm

7–10 μm

Shape of nucleus

Round

Segmented (usually

bilobar) Granules

Many, large, metachromatic

Few, small, basophilic

High-affinity surface receptors for IgE antibodies (FcεRI)

Present

Present

Marker of cellular activity

Tryptase

Not yet established

IgE, immunoglobulin E; FcεRI, Fc receptors.

Two types of human mast cells have been identified based on morphologic and biochemical properties. Most mast cells in the connective tissue of the skin, intestinal submucosa, mammary glands, and axillary lymph nodes contain cytoplasmic granules with a lattice-like internal structure. These cells contain granule-associated tryptase and chymase and are referred to as . In contrast, TC mast cells in the lungs and intestinal mucosa have granules with a scroll-like internal structure. These cells produce only tryptase and are termed . T Nearly equivalent concentrations of each type are found in nasal mucosa.

MC mast cells or connective tissue mast cells

MC mast cells or mucosal mast cells

Mast cells are especially numerous in the connective tissues of skin and mucous membranes but are not present in the brain and spinal cord. Connective tissue mast cells (MCTC mast cells) are distributed chiefly in the connective tissue of skin in the vicinity of small blood vessels, hair follicles, sebaceous glands, and sweat glands. Mast cells are also present in the capsules of organs and the connective tissue that surrounds the blood vessels of internal organs. A notable exception is the central nervous system. Although the (sheets of connective tissue that surround the brain and spinal cord) contain mast cells, the connective tissue around the small blood vessels within the brain and spinal cord is devoid of mast cells. The absence of mast cells protects the brain and spinal cord from the potentially disruptive effects of the edema caused by allergic reactions. Mast cells are also numerous in the thymus

meninges

and, to a lesser degree, in other lymphatic organs, but they are not present in the spleen.

FOLDER 6.3

CLINICAL CORRELATION: ROLE OF MYOFIBROBLASTS IN WOUND REPAIR An important role of myofibroblasts occurs during the process of wound healing. A clean surgical skin incision begins the healing process when a blood clot containing fibrin and blood cells fills the narrow space between the edges of the incision. The inflammatory process , which begins as early as 24 hours after the initial injury, contains the damage to a small area, aids in the removal of injured and dead tissues, and initiates the deposition of new ECM proteins. During the initial phases of inflammation, neutrophils and monocytes infiltrate the injury (maximum infiltration by neutrophils occurs in the first 1–2 days after injury). Monocytes transform into macrophages (they usually replace neutrophils by day 3 after injury; page 312). At the same time, in response to local growth factors, fibroblasts and vascular endothelial cells begin to proliferate and migrate into the delicate fibrin matrix of the blood clot, forming the , a specialized type of tissue characteristic of the repair process. Usually by day 5 after injury, the fully developed granulation tissue bridges the incision gap. It is composed mainly of large numbers of small vessels, fibroblasts, and myofibroblasts and variable numbers of other inflammatory cells. Migrating fibroblasts exert tractional forces on the ECM, reorganizing it along lines of stress. Under the influence of growth factors such as transforming growth factor β1 (TGF-β1) and mechanical forces, fibroblasts undergo differentiation into . This process can be visualized by monitoring the synthesis of α smooth muscle actin (α-SMA). This type of actin is not present in the cytoplasm of fibroblasts (Fig. F6.3.1). The myofibroblasts generate and maintain a steady contractile force (similar to that of smooth muscle cells) that causes shortening of the connective tissue fibers and wound closure. At the same time, myofibroblasts synthesize and lay down collagen fibers and other ECM components that are responsible for tissue remodeling.

tissue

granulation

myofibroblasts

FIGURE F6.3.1. Fibroblasts and myofibroblasts in culture.

This immunofluorescence image shows wild-type 3T3 fibroblasts cultured on a collagen lattice. Under the stimulation of certain growth factors such as TGF-β1, some fibroblasts differentiate to myofibroblasts, expressing α-SMA, the marker of myofibroblast differentiation. Cells were stained with fluorescein-labeled phalloidin to visualize F-actin filaments ( ), and α-SMA were labeled with primary antibodies against α-SMA and visualized with secondary goat anti-mouse antibodies conjugated with FITC ( ). Co-localization of α-SMA with F-actin is indicated by the color. Note that some cells have completed their differentiation, and others are in the early stages. ×1,000. , α smooth muscle actin; , tumor growth factor β1. (Courtesy of Dr. Boris Hinz.)

green

TGF-β1

α-SMA

red yellow

During the second week of wound healing, the number of cells in tissue undergoing repair decreases; most of the myofibroblasts undergo apoptosis and disappear, resulting in a that has very few cellular elements. In some pathologic conditions, myofibroblasts persist and continue the process of remodeling. This continued remodeling causes formation, resulting in excessive connective tissue contracture. Extensive numbers of myofibroblasts are found in most contractive diseases of connective tissue (fibromatoses). For example, is characterized by the thickening of palmar aponeurosis, which leads to progressive flexion contracture of the fourth and fifth digits of the hand (Fig. F6.3.2). If scar tissue grows beyond the boundaries of the original wound and does not regress, it is called a . Its formation is more common among African Americans than other ethnic groups.

connective

tissue scar hypertrophic scar palmar fibromatosis (Dupuytren disease) keloid

FIGURE F6.3.2. Hand of a patient with Dupuytren disease .

Dupuytren disease is an example of a contractive disease of connective tissue of the palm. The most commonly affected areas— near the crease of the hand close to the base of the ring and small fingers—form contracted fibrous cords, which are infiltrated by an extensive number of myofibroblasts. Most patients report problems when they try to place the affected hand on a flat surface. In

severe cases, the fingers are permanently flexed and interfere with everyday activities, such as washing hands or placing the hand into a pocket. (Courtesy of Dr. Richard A. Berger.)

FOLDER 6.4

FUNCTIONAL CONSIDERATIONS: THE MONONUCLEAR PHAGOCYTE SYSTEM The originally proposed classification of macrophages, monocytes, and their precursors into the mononuclear phagocyte system (MPS) was based on their common morphology, function, origin, and cell kinetics. Recent studies challenging the classic MSP concept that all macrophages are derived from monocytes propose that resident macrophages are instead maintained entirely by selfrenewal. MPS cells represent a network of phagocytic cells primarily involved in phagocytosis and antigen processing and antigen presentation. MSP cells are composed of monocytes, macrophages, and dendritic cells and are classified depending on their physical and functional characteristics. are circulating cells and can easily travel from the blood into the tissue compartment where they differentiate into macrophages. Directed by signaling molecules, migrate within target tissues toward a destination point, where they react with foreign substances, pathogens, and microorganisms to initiate an immune response. They also secrete chemokines and cytokines and express various receptors, such as pattern recognition receptors (PRRs), receptors for complement, and receptors for F c fragments of immunoglobulins

Monocytes

macrophages

Dendritic cells

(Ig). are antigen-presenting cells with phagocytic functions in the adaptive immune response As mentioned earlier, the original classification was built on the presumption that all macrophages are derived from monocytes that originate from bone marrow hematopoietic stem cells. It is now accepted that cells of the MPS arise from two distinct populations of progenitor cells in separate waves during hematopoiesis. The early population derives from the originating from the (extraembryonic primitive hematopoietic wave). These embryonic macrophages are first seen at 3–4 weeks of gestation and develop independently from blood monocytes. They migrate to developing tissues and organs and become . The second population originates from (definitive hematopoietic wave) and represents

cells

erythro-myeloid progenitor yolk sac

resident macrophages bone marrow hematopoietic stem cells monocyte-derived

macrophages . Monocytes first appear in the circulation much later in development, at about the 17th week of gestation. Therefore, it is important to understand that MPS cells represent a mixed type of cell population with different embryologic origins. Both populations of macrophages are antigen-presenting cells; however, they demonstrate different phenotypes and divergent functionality. Yolk sac–derived resident macrophages are self-sustaining and represent a resident self-renewing phagocytic population in the tissues, whereas monocyte-derived macrophages are replenished from an influx of circulating monocytes. Resident tissue macrophages also adapt to each tissue environment to perform specific functions. For example, represent small, stellate cells located primarily along the capillaries of the central nervous system that function as phagocytic cells. They arise from erythro-myeloid progenitor cells in the yolk sac and migrate and differentiate in the central nervous system during the embryonic and perinatal stages of development. Similarly, macrophages of the liver ( ), lungs ( ), and skin ( ) are also yolk sac derived; their acquisition of tissue-specific macrophage phenotypes correlates with the development and maturation of organs where they reside. Microglial cells in the brain and Langerhans cells in the skin cannot be replenished by bone marrow–derived monocytes. Similarly, derived from the fusion of granulocyte/macrophage progenitor cells (GMP) are also included in the MPS. Also, fibroblasts of the subepithelial sheath of the lamina propria of the intestine and uterine endometrium have been shown to differentiate into cells with morphologic, enzymatic, and functional characteristics of resident connective tissue macrophages. The various cells of the MPS are listed in the following table.

microglia

cells

alveolar macrophages

Kupffer cells Langerhans cells

osteoclasts

Cells of the Mononuclear Phagocyte System Cells

Location

Resident macrophages

Connective tissue, spleen, lymph nodes, bone marrow, adipose tissue, and thymus

Monocytes and their precursors in bone marrow: monoblasts and promonocytes

Blood and bone marrow

Dendritic cells

Lymph nodes, spleen

Stellate sinusoidal macrophages (Kupffer cell)

Liver

Alveolar macrophages

Lungs

Mesangial cells

Kidney

Placental macrophages (Hofbauer cell)

Placenta

Pleural and peritoneal macrophages

Serous cavities

Microglia

Central nervous system

Langerhans cells

Epidermis of skin, oral mucosa, foreskin, female genital epithelium

Intestinal macrophages

Gastrointestinal tract: submucosa and lamina propria

Peritubular and interstitial macrophages

Testis

Osteoclasts (originate from hemopoietic progenitor cells)

Bone

Fibroblast-derived macrophages (originate from mesenchymal cells)

Lamina propria of intestine, endometrium of uterus

Multinucleated giant cells (e.g., foreign-body giant cells, Langhans giant cells; originate from fusion of several macrophages)

Pathologic granulomas: suture granuloma, tuberculosis

Most mast cell secretory products (mediators of inflammation) are stored in granules and released at the time of mast cell

activation. Mast cells contain intensely basophilic granules that store chemical substances known as . Mediators produced by mast cells are divided into two categories: that are stored in secretory granules and released upon cell activation and (mostly lipids and cytokines) that are often absent in the resting cells, although they are produced and secreted by activated mast cells. found inside mast cell granules are the following:

preformed mediators synthesized mediators

mediators of inflammation

newly

Preformed mediators

Histamine is a biogenic amine that increases the permeability of small blood vessels, causing edema in the surrounding tissue and a skin reaction demonstrated by an itching sensation. In addition, it increases mucus production in the bronchial tree and prompts contraction of smooth muscle in the pulmonary airways. Histamine’s effects can be blocked by . These competitive inhibitors have a similar chemical structure and bind to histamine receptors without initiating histamine’s effects. is a sulfated GAG that is an anticoagulant. Its expression is limited essentially to the granules of mast cells and basophils. When heparin unites with antithrombin III and platelet factor IV, it can block numerous coagulation factors. On the basis of its anticoagulant properties, heparin is useful for the treatment of . It also interacts with FGF and its receptor to induce signal transduction in the cells. (tryptase and chymase). is selectively concentrated in the secretory granules of human mast cells (but not basophils). It is released by mast cells together with histamine and serves as a marker of mast cell activation. plays an important role in generating angiotensin II in response to vascular tissue injury. Mast cell chymase also activates MMPs and induces apoptosis of vascular smooth muscle cells, particularly in the area of atherosclerotic lesions.

antihistaminic agents Heparin

thrombosis

Serine proteases

Chymase

Tryptase

Eosinophil chemotactic factor (ECF) chemotactic factor (NCF), which attract

neutrophil

and eosinophils and neutrophils, respectively, to the site of inflammation. The secretions of eosinophils counteract the effects of histamine and leukotrienes.

Newly synthesized mediators include the following: Leukotriene C (LTC4 ) is released from the mast cell and then cleaved in the ECM, yielding two active leukotrienes—D (LTD4 ) and E (LTE4 ). They represent a family of modified

lipids conjugated to glutathione (LTC4) or cysteine (LTD4 and LTE4). Leukotrienes are released from mast cells during anaphylaxis (see Folder 6.5 for a description of anaphylaxis) and promote inflammation, including eosinophil migration and the increase in vascular permeability. Similar to histamine, leukotrienes trigger prolonged constriction of smooth muscle in the pulmonary airways, causing . The bronchoconstrictive effects of leukotrienes develop more slowly and last much longer than the effects of histamine. Bronchospasm caused by leukotrienes can be prevented by (blockers), but not by antihistaminic agents. The leukotriene receptor antagonists are among the most prescribed drugs for the management of ; they are used for both the treatment and prevention of acute asthma attacks.

bronchospasm

leukotriene receptor antagonists asthma

FOLDER 6.5

CLINICAL CORRELATION: THE ROLE OF MAST CELLS AND BASOPHILS IN ALLERGIC REACTIONS

Exposure to a specific antigen (allergen) that reacts with IgE antibodies bound to the surface of mast cells or basophils via their high-affinity receptors (FcεRI) initiates mast cell activation. This type of IgE-dependent activation triggers a cascade of events, resulting in an . These reactions can occur as immediate hypersensitivity reactions (usually within seconds to minutes after exposure to an allergen), late-phase reactions, or chronic allergic inflammations.

allergic reaction

immediate hypersensitivity reaction

The involves IgEmediated release of histamine and other mediators from mast cells and also from basophils. The clinical symptoms caused by these mediators vary, depending on which organ system is affected. The release of mediators in the superficial layers of the skin can manifest as erythema (redness), swelling and itching, or pain sensations. Respiratory symptoms include sneezing, rhinorrhea (runny nose), increased production of mucus, coughing, bronchospasm (constriction of bronchi), and pulmonary edema. Individuals with these symptoms often complain of tightness in the chest, shortness of breath, and wheezing. The gastrointestinal tract can also be affected with symptoms of nausea, vomiting, diarrhea, and abdominal cramping. In highly sensitive individuals, the antigen injected by an insect can trigger a massive discharge of mast cells and basophil granules that affect more than one system. This condition is known as . Dilation and increased permeability of systemic blood vessels can cause . This often-explosive, life-threatening reaction is characterized by significant hypotension (decreased blood pressure), decreased circulating blood volume (leaky vessels), and smooth muscle cell constriction in the bronchial tree. The individual has difficulty breathing and may exhibit a rash as well as nausea and vomiting. Symptoms of anaphylactic shock usually develop within 1–3 minutes, and immediate treatment with vasoconstrictors such as epinephrine is required. Clinical assessment of the activation of basophils in systemic anaphylactic reactions is not possible because an assay for a specific cellular marker released by basophils (and not by other cells such as mast cells) has not yet been developed. Following resolution of the signs or symptoms of an immediate hypersensitivity reaction, an affected individual may develop a 6–24 hours later. The symptoms of these reactions may include redness, persistent swelling of the skin, nasal discharge, sneezing, and coughing, usually accompanied by an elevated white blood cell count. These symptoms usually last a few hours and then disappear within 1–2 days of the initial allergen exposure. In the respiratory system, the late-phase reaction is believed to be responsible for the development of persistent asthma. If the exposure to an allergen is persistent (for instance, by a dog-owning patient who is allergic to dogs), it can result in . Tissues in such individuals accumulate a variety of immune cells such as eosinophils and T lymphocytes that cause more tissue damage and prolong inflammation. This can lead to permanent structural and functional changes in the affected tissue.

shock

anaphylaxis

late-phase allergic reaction

chronic allergic inflammation

anaphylactic

Tumor necrosis factor α (TNF-α)

is a major cytokine produced by mast cells. It increases expression of adhesion molecules in endothelial cells and has antitumor effects. Several

interleukins (IL-4, IL-3, IL-5, IL-6, IL-8, and IL16), growth factors (GM-CSF), and prostaglandin D2 (PGD2 )

are also released during mast cell activation. These mediators are not stored in granules but are synthesized by the cell and released immediately into the ECM. Mediators released during mast cell activation as a result of interactions with allergens are responsible for a variety of symptoms and signs that are characteristic of .

reactions

allergic

Basophils

Basophils that develop and differentiate in the bone marrow share many features with mast cells. Basophils are granulocytes that circulate in the bloodstream

and comprise less than 1% of peripheral white blood cells (leukocytes). Developmentally, they represent a separate lineage from mast cells, despite sharing a common precursor cell in the bone marrow. Basophils develop and mature in the and are released into the circulation as mature cells. They also have many other common features with mast cells, such as , an ability to secrete similar mediators, and an abundance of c on their cell membranes. They participate in allergic reactions (see Folder 6.5) and, together with mast cells, release histamine, heparin, heparan sulfate, ECF, NCF, and other mediators of inflammation. In contrast to mast cells, basophils do not produce PGD2 and IL-5. Basophils and their features are discussed in more detail in Chapter 10, Blood (pages 313-314).

bone marrow

basophilic secretory granules high-affinity F receptors for IgE antibodies

Adipocytes

The adipocyte is a connective tissue cell specialized to store neutral fat and produce a variety of hormones. Adipocytes differentiate from mesenchymal stem cells and

gradually accumulate fat in their cytoplasm. They are located throughout loose connective tissue as individual cells and groups of cells. When they accumulate in large numbers, they are called . Adipocytes are also involved in the synthesis of a variety of hormones, inflammatory mediators, and growth factors. This specialized connective tissue is discussed in Chapter 9, Adipose Tissue, page 280.

adipose tissue

Adult Stem Cells and Pericytes

Niches of adult stem cells are located in various tissues and organs. Many tissues in mature individuals contain reservoirs of stem cells called adult stem cells. Compared with embryonic stem cells, adult stem cells cannot differentiate into multiple lineages. They are usually capable of differentiating only into lineage-specific cells. Adult stem cells are found in many tissues and organs, residing in specific sites referred to as . Cells residing within niches in various tissues and organs (excluding the bone marrow) are called . They have been identified in the gastrointestinal tract —for instance, in the stomach (isthmus of the gastric gland), small and large intestines (base of the intestinal gland), and many other areas. Bone marrow represents a unique reservoir of stem cells. In addition to containing (see Chapter 10, Blood, see pages 321-322), bone marrow also contains at least two other populations of stem cells: a heterogeneous population of that appear to have broad developmental capabilities and that can generate chondrocytes, osteoblasts, adipocytes, muscle cells, and endothelial cells. MAPCs are adult counterparts of embryonic stem cells. Niches of adult stem cells, called , are found in the loose connective tissue of adults.

niches cells (HSCs)

tissue stem

hemopoietic stem cells

multipotent adult progenitor cells (MAPCs) bone marrow stromal cells (BMSCs) cells

mesenchymal stem

These cells give rise to differentiated cells that function in the repair and formation of new tissue, such as in wound healing and the development of new blood vessels (neovascularization).

The vascular pericytes that partially wrap around capillaries and venules are mesenchymal stem cells. Pericytes, also called adventitial cells or perivascular cells, are typically wrapped, at least partially, around capillaries and venules (Fig. 6.25). Their nuclei are shaped similarly to that of endothelial cells (i.e., flattened but curved to conform to the tubular shape of the vessel). Several observations support the interpretation that vascular pericytes are indeed mesenchymal stem cells. Experimental studies show that in response to external stimuli, pericytes express a cohort of proteins similar to those of stem cells in the bone marrow. Pericytes are surrounded by basal lamina material that is continuous with the basal lamina of the capillary endothelium; thus, they are not truly located in the connective tissue compartment. The role of pericytes as has been confirmed experimentally in studies that demonstrate the ability of cultured pericytes from retinal capillaries to differentiate into a variety of cells, including osteoblasts, adipocytes, chondrocytes, and fibroblasts.

cells

mesenchymal stem

FIGURE 6.25. Electron micrograph of a small blood vessel . The nucleus at the upper left belongs to the endothelial cell that forms

right

the wall of the vessel. At the is another cell, a pericyte, which is in intimate relation to the endothelium. Note that the basal lamina ( ) covering the endothelial cell divides ( ) to surround the pericyte. ×11,000.

BL

arrow

TEM studies demonstrate that pericytes surrounding the smallest venules have cytoplasmic characteristics almost identical to those of the endothelial cells of the same vessel. Pericytes associated with larger venules have characteristics of smooth muscle cells of the tunica media of small veins. In fortuitous sections cut parallel to the long axis of venules, the distal portion and proximal portion of the same pericyte exhibit characteristics of endothelial cells and smooth muscle cells, respectively. These studies suggest that during the development of new vessels, cells with characteristics of may differentiate into smooth muscle found in the vessel wall.

pericytes The fibroblasts and blood vessels within healing wounds develop from mesenchymal stem cells associated with the tunica adventitia of venules.

Autoradiographic studies of wound healing using parabiotic (crossed-circulation) pairs of animals have established that mesenchymal stem cells located in the tunica adventitia of venules and small veins are the primary source of new cells in healing wounds. In addition, fibroblasts, pericytes, and endothelial cells in portions of the connective tissue adjacent to the wound divide and give rise to additional cells that form new connective tissue and blood vessels.

Lymphocytes, Plasma Cells, and Other Cells of the Immune System

Lymphocytes are principally involved in immune responses. Connective tissue lymphocytes are the smallest of the wandering cells in the connective tissue (see Fig. 6.24b). They have a thin rim of cytoplasm surrounding a deeply staining, heterochromatic nucleus. Often, the cytoplasm of connective

tissue lymphocytes may not be visible. Normally, small numbers of lymphocytes are found in the connective tissue throughout the body. The number increases dramatically, however, at the sites of tissue caused by pathogenic agents. Lymphocytes are most numerous in the lamina propria of the respiratory and gastrointestinal tracts, where they are involved in immunosurveillance against pathogens and foreign substances that enter the body by crossing the epithelial lining of these systems.

inflammation

Lymphocytes are a heterogeneous population of at least three major functional cell types: T cells, B cells, and natural killer (NK) cells. At the molecular level, lymphocytes are characterized by the expression of specific molecules on the plasma membrane known as a cluster of differentiation (CD) proteins. CD proteins recognize specific ligands on target cells. proteins are present only on specific types they are considered specific marker proteins. these specific markers, lymphocytes can be three functional cell types.

Because some CD of lymphocytes, On the basis of classified into

T lymphocytes are characterized by the presence of the CD2, CD3, CD5, and CD7 marker proteins and T-cell receptors (TCRs). These cells have a long life span and are effectors in cell-mediated immunity. B lymphocytes are characterized by the presence of CD9, CD19, and CD20 proteins and attached IgM and IgD. These cells recognize antigens, have a variable life span, and are effectors in . are non–T, non–B lymphocytes that express CD16a, CD56, and CD94 proteins not found in other lymphocytes. These cells neither produce Igs nor express TCR on their surface. Thus, NK lymphocytes are not antigen specific. Similar in action to T lymphocytes, however, they destroy virus-infected cells and some tumor cells by a cytotoxic mechanism.

antibody-mediated (humoral) immunity NK lymphocytes

In response to the presence of antigens, lymphocytes become activated and may divide several times, producing clones of themselves. In addition, clones of B lymphocytes mature into plasma cells. A description of lymphocytes and their functions during immune response reactions is presented in Chapter 14, Immune System and Lymphatic Tissues and Organs (see pages 487491).

Plasma cells are antibody-producing cells derived from B lymphocytes. Plasma cells are a prominent constituent of loose connective tissue at sites where antigens tend to enter the body (e.g., the gastrointestinal and respiratory tracts). They are also a normal component of salivary glands, lymph nodes, and hematopoietic tissue. Once derived from its precursor, a plasma cell has only limited migratory ability and a somewhat short life span of 10–30 days. The plasma cell is a relatively large, ovoid cell (20 μm) with a considerable amount of cytoplasm. The cytoplasm displays strong basophilia because of an extensive rER (Fig. 6.26a). The Golgi apparatus is usually prominent because of its relatively large size and lack of staining. It appears in light microscope preparations as a clear area in contrast to the basophilic cytoplasm.

B-lymphocyte

FIGURE 6.26. Plasma cell. a.

This photomicrograph shows the typical features of a plasma cell as seen in a routine hematoxylin and eosin (H&E) preparation. Note clumps of peripheral heterochromatin alternating with clear areas of euchromatin in the nucleus. Also note the negative Golgi ( ) and basophilic cytoplasm. ×5,000. Electron micrograph shows that an extensive rough endoplasmic reticulum ( ) occupies most of the cytoplasm of the plasma cell. The Golgi apparatus ( ) is also relatively large, a further reflection of the cell’s secretory activity. ×15,000.

rER

b.

arrows

G

nucleus

The is spherical and typically offset or eccentrically positioned. It is small—not much larger than the nucleus of the lymphocyte. It exhibits large clumps of peripheral heterochromatin alternating with clear areas of euchromatin. This arrangement has traditionally been described as resembling a or , with the heterochromatin resembling the spokes of the wheel or the numbers on a clock (Fig. 6.26b). The heterochromatic nucleus of the plasma cell is somewhat surprising, given the cell’s

cartwheel

analog clock face

function in synthesizing large amounts of protein. However, because the cells produce large amounts of only one type of protein—a —only a small segment of the genome is exposed for transcription.

specific antibody Eosinophils, monocytes, and neutrophils are also observed in connective tissue.

Certain cells rapidly migrate from the blood to enter the connective tissue, particularly neutrophils and monocytes in response to tissue injury. Their presence generally indicates an acute inflammatory reaction. In these reactions, neutrophils migrate into the connective tissue in substantial numbers, followed by large numbers of monocytes. As noted, the monocytes then differentiate into macrophages. A description of these cells and their roles is found in Chapter 10, Blood (see pages 305-314). The , which functions in and , is also described. Eosinophils may be observed in normal connective tissue, particularly the lamina propria of the intestine, as a result of chronic immunologic responses that occur in these tissues.

reactions

eosinophil parasitic infections

allergic

CONNECTIVE TISSUE

OVERVIEW OF CONNECTIVE TISSUE Connective tissue

forms a continuous compartment throughout the body that connects and supports other tissue. It is bounded by the basal laminae of various epithelia and by the external laminae of muscle cells and nerve-supporting cells. Connective tissue comprises a diverse group of within a tissue-specific . ECM contains protein fibers and .

extracellular matrix (ECM) ground substance

cells

Classification of connective tissue is primarily based on the composition and organization of its extracellular components and on its functions: , and .

tissue proper

embryonic, connective specialized connective tissue

EMBRYONIC CONNECTIVE TISSUES Mesenchyme

is derived from embryonic mesoderm and gives rise to various connective tissues of the body. It contains a loose network of spindle-shaped cells that are suspended in a viscous ground substance containing fine collagen and reticular fibers. is present in the umbilical cord. It contains widely separated spindle-shaped cells embedded in a gelatin-like, hyaluronan-rich ECM; its ground substance is called .

Mucous connective tissue

Wharton jelly

CONNECTIVE TISSUE PROPER

Connective tissue proper is divided into loose and dense connective tissue. Dense connective tissue is further subdivided into dense irregular and dense regular connective tissue. Loose connective tissue is characterized by large numbers of cells of various types embedded in an abundant gel-like ground substance with loosely arranged fibers. It typically surrounds glands, various tubular organs, and blood vessels and is found beneath the epithelia that cover internal and external body surfaces. contains few cells (primary fibroblasts), randomly distributed bundles of collagen fibers, and relatively little ground substance. It provides significant strength and allows organs to resist excessive stretching and distension.

Dense irregular connective tissue

Dense regular connective tissue

is characterized by densely packed, parallel arrays of collagen fibers with cells (tendinocytes) aligned between the fiber bundles. It is the main functional component of tendons, ligaments, and aponeuroses.

CONNECTIVE TISSUE FIBERS

connective tissue

There are three principal types of : collagen, reticular, and elastic fibers. are the most abundant structural components of connective tissue. They are flexible, have a high-tensile strength, and are formed from that exhibit a characteristic 68-nm banding pattern. involves events that occur both within the fibroblasts (production of procollagen molecules) and outside the fibroblasts in the ECM (polymerization of collagen molecules into fibrils, which are assembled into larger collagen fibers). are composed of and provide a supporting framework for cells in various tissues and organs (abundant in lymphatic tissues). In the lymphatic and hemopoietic tissues, reticular fibers are produced by specialized . In most other tissues, reticular fibers are produced by fibroblasts. are produced by fibroblasts, chondrocytes, endothelial cells, and smooth muscle cells. They allow tissues to respond to stretch and distension. Elastic fibers are composed of cross-linked molecules associated with a network of , which are made of fibrillin and fibrillinassociated proteins (EMILINs and MAGPs).

fibers Collagen fibers

fibrils Collagen fiber formation Reticular fibers

collagen

type III collagen reticular cells

Elastic

fibers

microfibrils

elastin fibrillin

EXTRACELLULAR MATRIX ECM

The provides mechanical and structural support for connective tissue, influences extracellular communication, and provides pathways for cell migration. In addition to protein fibers, the ECM contains ground substance that is rich in , hydrated , and . are the most abundant heteropolysaccharide components of ground substance. These molecules are composed of long-chain unbranched polysaccharides containing many sulfate and carboxyl groups. They covalently bind to core proteins to form that are responsible for the physical properties of ground substance. The largest and longest GAG molecule is . By means of special , proteoglycans indirectly bind to hyaluronan, forming giant macromolecules called . The binding of water and other molecules (e.g., growth factors) to proteoglycan aggregates regulates the movement and migration of macromolecules, microorganisms, and metastatic cancer cells in the ECM. (e.g., fibronectin, laminin, and tenascin) are multifunctional molecules that possess binding sites for a variety of ECM proteins (e.g., collagens, proteoglycans, and GAGs). They also interact with cell surface receptors, such as integrin and laminin receptors.

glycosaminoglycans glycoproteins GAGs

(GAGs)

proteoglycans multiadhesive

proteoglycans

link proteins proteoglycan aggregates

hyaluronan

Multiadhesive glycoproteins

CONNECTIVE TISSUE CELLS Connective tissue cells are classified as part of the (relatively stable, nonmigratory) or the (or )

resident

cell

population wandering

transient cell

population (primarily cells that have migrated from blood vessels). Resident cells include fibroblasts (and myofibroblasts), macrophages, adipocytes, mast cells, and adult stem cells. Wandering (transient) cells include lymphocytes,

plasma cells, neutrophils, eosinophils, basophils, and monocytes (described in Chapter 10, Blood, see pages 305-314). are the principal cells of connective tissue. They are responsible for the synthesis of collagen and other components of the ECM. Fibroblasts that express actin filaments and associated actin motor proteins such as nonmuscle myosin are called . are phagocytic cells derived from that contain an abundant number of lysosomes and play an important role in immune response reactions. are specialized connective tissue cells that store neutral fat and produce a variety of hormones (see Chapter 9, Adipose Tissue, page 280). develop in the bone marrow and differentiate in connective tissue. They contain basophilic granules that store mediators of inflammation. Upon activation, mast cells synthesize leukotrienes, interleukins, and other inflammation-promoting cytokines. reside in specific locations (called ) in various tissues and organs. They are difficult to distinguish from other cells of connective tissue.

Fibroblasts

myofibroblasts Macrophages

monocytes

Adipocytes

Mast cells

Adult stem cells niches

PLATE 6.1 LOOSE AND DENSE IRREGULAR CONNECTIVE TISSUE Loose and dense irregular connective tissue represents two of the several types of connective tissue. The others are namely cartilage, bone, blood, adipose tissue, and reticular tissue.

Loose connective tissue is characterized by a relatively high proportion of cells within a matrix of thin and sparse collagen fibers. In contrast, dense irregular connective tissue

contains few cells, almost all of which are fibroblasts that are responsible for the formation and maintenance of the abundant collagen fibers that form the matrix of this tissue. The cells that are typically associated with loose connective tissue are , the collagen-forming cells, and those cells that function in the immune system and those of the body’s general defense system. Thus, in loose connective tissue, there are, to varying degrees, lymphocytes, macrophages, eosinophils, plasma cells, and mast cells.

fibroblasts

Loose and dense irregular connective tissue , mammary gland, human, hematoxylin and eosin (H&E) ×175; insets ×350.

This micrograph shows at low magnification both loose connective tissue ( ) and dense irregular connective tissue ( ) for comparative purposes. The surrounds the glandular epithelium ( ). The consists mainly of thick bundles of collagen fibers with few cells present, whereas the loose connective tissue has a relative paucity of fibers and a considerable number of cells. The is a higher magnification of the dense connective tissue. Note that only a few cell nuclei are present relative to the larger expanse of collagen fibers. The , revealing the glandular epithelium and surrounding loose connective tissue, shows very few fibers but large numbers of cells. Typically, the cellular component of loose connective tissue contains a relatively small proportion of fibroblasts but large numbers of lymphocytes, plasma cells, and other connective tissue cell types.

DICT

LCT

connective tissue dense irregular connective tissue upper inset

GE

loose

lower inset

Loose connective tissue Mallory trichrome ×250.

connective tissue LCT

, colon, monkey,

loose

This micrograph reveals an extremely cellular ( ), also called lamina propria, which is located between the intestinal glands of the colon. The simple, columnar, mucus-secreting epithelial cells seen here represent the glandular tissue. The Mallory stain colors cell nuclei and collagen . Note how the cells are

red

blue

surrounded by a framework of blue-stained collagen fibers. Also shown in this micrograph is a band of smooth muscle, the muscularis mucosa ( ) of the colon and below that, seen in part, is ( ) that forms the submucosa of the colon. Typically, the collagen fibers ( ) that lie just below the epithelial cells ( ) at the luminal surface are more concentrated and thus appear prominently in the micrograph.

MM

connective tissue DICT Ep

dense irregular

CF

Loose connective tissue

, colon, monkey,

Mallory trichrome ×700.

boxed

Shown at higher magnification is the area in the adjacent figure. The base of the epithelial cells ( ) is seen on each side of the micrograph. The ( ) appear as thin threads that form a stroma surrounding the cells. The mixture of cells that are present here consists of ( ), ( ), fibroblasts, smooth muscle cells, macrophages ( ), and occasional mast cells.

fibers CF lymphocytes ML plasma cells P

CF, collagen fibers DICT, dense irregular connective tissue Ep, epithelial cells GE, glandular epithelium L, lymphocyte LCT, loose connective tissue M, macrophage MM, muscularis mucosa P, plasma cells

Ep

collagen

PLATE 6.2 DENSE REGULAR CONNECTIVE TISSUE, TENDONS, AND LIGAMENTS Dense regular connective tissue

is distinctive in that its fibers are very densely packed and are organized in parallel array into fascicles. The collagen fibrils that make up the fibers are also arranged in an ordered parallel array. , which attach muscle to bone, and , which attach bone to bone, are examples of this type of tissue. Ligaments are similar to tendons in most respects, but their fibers and the organization of the fascicles tend to be less ordered. In tendons as well as ligaments, the fascicles are separated from one another by dense irregular connective tissue, the , through which travel vessels and nerves. Also, a fascicle may be partially divided by connective tissue septa that extend from the endotendineum and contain the smallest vessels and nerves. Some of the fascicles may be grouped into larger functional units by a thicker, surrounding connective tissue, the . Finally, the fascicles and groups of fascicles are surrounded by dense irregular connective tissue, the . The fibroblasts, also called in tendons, are elongated cells that possess exceedingly thin, sheet-like cytoplasmic processes that reside between and embrace adjacent fibers. The margins of the cytoplasmic processes contact those of neighboring tendon cells, thus forming a syncytium-like cytoplasmic network. The most regular dense connective tissue is that of the stroma of the cornea of the eye (see Chapter 24, Eye, see pages 983987). In this tissue, the collagen fibrils are arranged in parallel in lamellae that are separated by large, flattened fibroblasts. Adjacent lamellae are arranged at approximately right angles to one another, thus forming an . The extreme regularity of fibril size and fibril spacing in each lamella, in conjunction with the orthogonal array of the lamellae, is believed to be the basis of corneal transparency.

Tendons

ligaments

endotendineum peritendineum epitendineum

tendinocytes

orthogonal array

Dense regular connective tissue

, tendon, longitudinal section, human, hematoxylin and eosin (H&E) ×100.

epitendineum Ept

tendon fascicles

This specimen includes the surrounding dense irregular connective tissue of the tendon, the ( ). The

TF

( ) that make up the tendon are surrounded by a less dense connective tissue than that associated with the epitendineum. In longitudinal sections such as this, the connective tissue that surrounds the individual fascicles, the ( ), seems to disappear at certain points, with the result that one fascicle appears to merge with a neighboring fascicle. This is due to an obliqueness in the plane of section rather than an actual merging of fascicles. The collagen that makes up the bulk of the tendon fascicle has a homogeneous appearance as a result of the orderly packing of the individual collagen fibrils. The nuclei of the tendinocytes appear as elongate profiles arranged in linear rows. The cytoplasm of these cells blends in with the collagen, leaving only the nuclei as the representative feature of the cell.

endotendineum Ent

Dense regular connective tissue

, tendon,

longitudinal section, human, H&E ×400.

tendinocyte nuclei TC

This higher magnification micrograph shows the ordered single-file array of the ( ) along with the intervening collagen. The latter has a homogeneous appearance. The cytoplasm of the cells is indistinguishable from the collagen, as is typical in H&E paraffin specimens. The variation in nuclear appearance is due to the plane of section and the position of the nuclei within the thickness of the section. A small blood vessel ( ) coursing within the endotendineum is also present in the specimen.

BV

Dense regular connective tissue

, tendon,

cross section, human, H&E ×400.

This specimen is well preserved, and the densely packed collagenous fibers appear as a homogeneous field, even though the fibers are viewed on their cut ends. The nuclei appear irregularly scattered, as opposed to their more uniform pattern in the longitudinal plane. This is explained by examining the in the figure, which is meant to represent an arbitrary cross-sectional cut of the tendon. Note the irregular spacing of the nuclei that are in the plane of the cut. Lastly, several small blood vessels ( ) are present within the ( ) within a fascicle.

dashed line

endotendineum Ent

BV, blood vessel Ent, endotendineum

lower left

BV

Ept, epitendineum TC, tendinocyte nuclei TF, tendon fascicle dashed line, arbitrary cross-sectional cut of tendon

PLATE 6.3 ELASTIC FIBERS AND ELASTIC LAMELLAE Elastic fibers

are present in loose and dense connective tissue throughout the body but in lesser amounts than collagenous fibers. Elastic fibers are not conspicuous in routine hematoxylin and eosin (H&E) sections but are visualized readily with special staining methods. (The following selectively color elastic material: Weigert elastic tissue stain, ; Gomori aldehyde fuchsin stain, ; Verhoeff hematoxylin elastic tissue stain, ; and modified Taenzer-Unna orcein stain, .) By using a combination of the special elastic stains and counterstains, such as H&E, not only the elastic fibers but also the other tissue components may be revealed, thus allowing to study the relationships between the elastic material and other connective tissue components. occurs in both fibrous and lamellar forms. In loose and dense connective tissue and elastic cartilage (see Plate 7.3, page 234), the elastic material is in fibrous form. Similarly, the elastic ligaments that connect the cervical vertebrae and that are particularly prominent in grazing animals have a mixture of elastic and collagenous fibers in a tightly packed array. In the major, largest diameter arteries (e.g., aorta, pulmonary, common carotid, and other primary branches of the aorta), the consists of fenestrated layers of elastic tissue alternating with layers containing smooth muscle cells and collagenous tissue. This allows stretching and elastic rebound to assist in the propulsion of the blood. All arteries and most large arterioles have an that supports the delicate endothelium and its immediately subjacent connective tissue. It should be noted that both the collagen and elastic components of the tunica media are produced by the smooth muscle cells of this layer.

brown

black

purple-violet

blue-black

red-

Elastic material

tunica media

internal elastic membrane

Elastic fibers

, dermis, monkey, Weigert ×160.

This shows the connective tissue of the skin, referred to as the , stained to show the nature and distribution of the ( ), which appear . The ( ) have been stained by eosin, and the two fiber types are easily differentiated. The connective tissue at the of the figure, close to the epithelium (the papillary layer of the dermis), contains thin elastic fibers (see ) as well as less coarse

dermis

purple

elastic fibers E collagen fibers C upper left

top

lower portion

collagen fibers. The shows considerably heavier elastic and collagen fibers. Also note that many of the elastic fibers appear as short rectangular profiles. These profiles simply represent fibers traveling through the thickness of the section at an oblique angle to the path of the knife. Careful examination will also reveal a few fibers that appear as dot-like profiles. They represent crosssectioned elastic fibers. Overall, the elastic fibers of the dermis have a three-dimensional interlacing configuration, thus showing a variety of forms.

Elastic fibers

, mesentery, rat, Weigert ×160.

This is a whole mount specimen of mesentery prepared to show the connective tissue elements and differentially stained to reveal elastic fibers. The ( ) appear as thin, long, crisscrossing, and branching threads without discernible beginnings or endings and with a somewhat irregular course. Again, the ( ) are contrasted by their eosin staining and appear as long, straight profiles that are considerably thicker than the elastic fibers.

elastic fibers E collagen fibers C

Elastic lamellae

, elastic artery, monkey,

Weigert ×80.

Elastic material also occurs in sheets or lamellae rather than string-like fibers. This figure shows the wall of an elastic artery (pulmonary artery) that was stained to show the elastic material. Each of the is a that is organized in the form of a fenestrated sheet or membrane. The plane of section is such that the elastic membranes are seen on the edge. This specimen was not subsequently stained with H&E. The empty-appearing spaces between elastic layers contain collagen fibers and smooth muscle cells, but they remain essentially unstained. In the muscular layer of blood vessel, both elastin and collagen are secreted by the smooth muscle cells. Tissues of the body containing large amounts of elastic material are limited in distribution to the walls of elastic arteries and some ligaments that are associated with the spinal column.

lines

BV, blood vessel C, collagen fibers D, duct of sweat gland

lamella of elastic material

wavy

E,

elastic fibers

7

CARTILAGE

OVERVIEW OF CARTILAGE HYALINE CARTILAGE ELASTIC CARTILAGE FIBROCARTILAGE CHONDROGENESIS AND CARTILAGE GROWTH REPAIR OF HYALINE CARTILAGE Folder 7.1 Clinical Correlation: Osteoarthritis Folder 7.2 Clinical Correlation: Malignant Tumors of the Cartilage: Chondrosarcomas HISTOLOGY

OVERVIEW OF CARTILAGE

Cartilage is a form of connective tissue composed of cells called chondrocytes and a highly specialized extracellular matrix. Cartilage is an avascular tissue that consists of chondrocytes and an extensive extracellular matrix. More than 95% of cartilage volume consists of extracellular matrix, which is a functional element of this tissue. The chondrocytes are sparse but essential participants in the production and maintenance of the matrix (Fig. 7.1).

FIGURE 7.1. General structure of hyaline cartilage.

This photomicrograph of a routine hematoxylin and eosin (H&E) preparation of hyaline cartilage shows its general features. Note the extensive extracellular matrix that separates a sparse population of chondrocytes. ×450.

The extracellular matrix in cartilage is not only solid and firm but also somewhat pliable, which accounts for its resilience. Because there is no vascular network within cartilage, the composition of the extracellular matrix is crucial to the survival of the chondrocytes. The large ratio of to in the cartilage matrix permits diffusion of substances between blood vessels in the surrounding connective tissue and the chondrocytes dispersed within the matrix, thus maintaining the viability of the tissue. Close interactions are seen between two classes of structural molecules that possess contrasting biophysical characteristics: the meshwork of tension-resisting collagen fibrils and the large amounts of heavily hydrated proteoglycan aggregates. The latter are extremely weak in shear and allow the cartilage to bear weight,

glycosaminoglycans (GAGs)

type II collagen fibers

especially at points of movement such as synovial joints. Because it maintains this property even while growing, cartilage is a key tissue in the development of the fetal skeleton and in most growing bones. Three types of cartilage that differ in appearance and mechanical properties are distinguished on the basis of the characteristics of their matrix:

Hyaline cartilage is characterized by matrix-containing type II collagen fibers, GAGs, proteoglycans, and multiadhesive glycoproteins. Elastic cartilage is characterized by elastic fibers and elastic lamellae in addition to the matrix material of hyaline cartilage. Fibrocartilage is characterized by abundant type I collagen fibers as well as the matrix material of hyaline cartilage. Table 7.1 lists the locations, functions, and features of each type of cartilage.

TABLE 7.1 Summary of Cartilage Features Features

Hyaline Cartilage

Elastic Ca

Fetal skeletal tissue, epiphyseal plates, articular surface of synovial joints, costal cartilages of rib cage, cartilages of nasal cavity, larynx (thyroid, cricoid, and arytenoids), rings of trachea, and plates in bronchi

Pinna of ex (Eustachi cornicula

 

Location Function Presence of perichondrium Undergoes calcification Main cell types present Characteristic features of extracellular matrix Growth Repair

Resists compression Provides cushioning, smooth, and low-friction surface for joints Provides structural support in respiratory system (larynx, trachea, and bronchi) Forms foundation for development of fetal skeleton and further endochondral bone formation and bone growth

Provides fl

Yes (except articular cartilage and epiphyseal plates)

Yes

Yes (e.g., during endochondral bone formation, during aging process)

No

Chondroblasts and chondrocytes

Chondroblas

Type II collagen fibrils and aggrecan monomers (the most important proteoglycan)

Type II col

Interstitially and appositionally, very limited in adults Very limited capability, commonly forms a scar, resulting in fibrocartilage forma

HYALINE CARTILAGE

Hyaline cartilage is distinguished by a homogeneous, amorphous matrix. The matrix of hyaline cartilage appears glassy in the living state, hence the name hyaline [Gr. hyalos, glassy]. Throughout the cartilage matrix are spaces called lacunae. Located within these lacunae are the chondrocytes. Hyaline cartilage is not a simple, inert, homogeneous substance but a complex living tissue. It provides a low-friction surface, participates in lubricating synovial joints, and distributes applied forces to the underlying bone. Although its capacity for repair is limited, under normal circumstances, it shows no evidence of abrasive wear over a lifetime. An exception is articular cartilage, which, in many individuals, breaks down with age (Folder 7.1). The macromolecules of hyaline cartilage matrix consist of collagen (predominantly type II fibrils and other cartilagespecific collagen molecules), proteoglycan aggregates containing GAGs, and multiadhesive glycoproteins (noncollagenous proteins). Figure 7.2 illustrates the relative distribution of the various components that constitute cartilage matrix.

FIGURE 7.2. Molecular composition of hyaline cartilage.

Cartilage’s net weight is 60%–80% intercellular water, which is bound by proteoglycan aggregates. About 15% of the total weight is attributed to collagen molecules, of which type II collagen is the most abundant. Chondrocytes occupy only 3%–5% of the total cartilage mass.

FOLDER 7.1

CLINICAL CORRELATION: OSTEOARTHRITIS

Osteoarthritis , a degenerative joint disease, is one of the most common types of joint diseases.

The pathogenesis of osteoarthritis is unknown, but it is related to aging and injury of articular cartilage. Most individuals show some evidence of this disease by age 65. The disease is characterized by with various degrees of and . Osteoarthritis commonly affects weight-bearing joints: hips, knees, lower lumbar vertebra, and joints of the hand and foot. There is a decrease in proteoglycan content, which reduces the intercellular water content in the cartilage matrix. Chondrocytes also

chronic joint pain of the articular cartilage

joint deformity

destruction

play an important role in the pathogenesis of osteoarthritis. Interleukin-1 (IL-1) and tumor necrosis factor α (TNF-α) stimulate the production of metalloproteinases and inhibit synthesis of type II collagen and proteoglycans by chondrocytes. In the early stages of the disease, the superficial layer of the articular cartilage is disrupted. Eventually, destruction of the cartilage extends to the bone, where the exposed subchondral bone becomes a new articular surface. These changes result in a progressive reduction of mobility and increased pain with joint movement. There is no cure for osteoarthritis, and treatment focuses on relieving pain and stiffness to allow a greater range of joint movement. Osteoarthritis may stabilize with age, but more often, it slowly progresses with eventual long-term disability.

Hyaline cartilage matrix is produced by chondrocytes and contains three major classes of molecules. Three classes of molecules exist in hyaline cartilage matrix:

Collagen molecules.

Collagen is the major matrix protein. Four types of collagen participate in the formation of a three-dimensional meshwork of relatively thin (20-nm diameter) and short matrix fibrils. constitutes the bulk of the fibrils (see Fig. 7.2), facilitates fibril interaction with the matrix proteoglycan molecules, regulates the fibril size, and organizes the collagen fibrils into a three-dimensional hexagonal lattice that is crucial to its successful mechanical function. In addition, is also found in the matrix, mainly at the periphery of the chondrocytes where it helps to attach these cells to the matrix framework. Because types II, VI, IX, X, and XI are found in significant amounts only in the cartilage matrix, they are referred to as . (Review the types of collagen in Table 6.2.) . The ground substance of hyaline cartilage contains three kinds of GAGs: , , and . As in loose connective tissue matrix, the chondroitin and keratan sulfate of the cartilage matrix are joined to a to form a . The most important proteoglycan monomer in hyaline cartilage is , also known as cartilage-specific proteoglycan core protein, which in humans is encoded by the ACAN gene. It has a molecular weight of 250 kDa. Each molecule contains approximately 100 chondroitin sulfate chains and as many as 60 keratan sulfate molecules. Because of the presence of the sulfate groups, aggrecan molecules have a large negative charge with an affinity for water molecules. Each linear hyaluronan molecule is associated with a large number of aggrecan molecules (>300), which are bound to the hyaluronan by link proteins at the N-terminus of the molecule to form large . These highly charged proteoglycan aggregates are bound to the collagen matrix fibrils by electrostatic interactions and multiadhesive glycoproteins (Fig. 7.3). The entrapment of these aggregates within the intricate matrix of collagen fibrils is responsible for the unique biomechanical properties of hyaline cartilage. Cartilage matrix also contains other proteoglycans (e.g., decorin, biglycan, and fibromodulin). These proteoglycans do not form aggregates but bind to other molecules and help stabilize the matrix.

Type II collagen type IX collagen type XI collagen type VI collagen

molecules Proteoglycans hyaluronan chondroitin sulfate keratan sulfate protein proteoglycan monomer aggrecan

proteoglycan aggregates

type X collagen

cartilage-specific collagen

core

FIGURE 7.3. Molecular structure of hyaline cartilage matrix.

This schematic diagram shows the relationship of proteoglycan aggregates to type II collagen fibrils and chondrocytes in the matrix of hyaline cartilage. A hyaluronan molecule forming a linear aggregate with many proteoglycan monomers is interwoven with a network of collagen fibrils. The proteoglycan monomer (such as aggrecan) consists of ~180 glycosaminoglycans joined to a core protein. The end of the core protein contains a hyaluronan-binding region that is joined to the hyaluronan by a link protein. Isogenous groups of chondrocytes are dispersed in extracellular matrix.

Multiadhesive glycoproteins, also referred to as noncollagenous and nonproteoglycanlinked glycoproteins, influence interactions between the chondrocytes and the matrix molecules. Examples of such proteins are anchorin CII (cartilage annexin V), a small 34kDa molecule that functions as a collagen receptor on chondrocytes, and tenascin and fibronectin (see Table 6.5, page 197), which also help anchor chondrocytes to the matrix. Multiadhesive glycoproteins have clinical value as markers of cartilage turnover and degeneration.

Hyaline cartilage matrix is highly hydrated to provide resilience and diffusion of small metabolites. Like other connective tissue matrices, cartilage matrix is highly hydrated. Between 60% and 80% of the net weight of hyaline cartilage is intercellular water (see Fig. 7.2). Much of this water is bound tightly to the , which create a high osmotic swelling pressure. These large hydrodynamic domains in the matrix are accountable for imparting resilience to the cartilage. The network of collagen type II fibers is not only responsible for the shape and tensile strength of hyaline cartilage but also provides a framework to resist the swelling pressure from aggrecan molecules. Some of the water is bound loosely enough to allow diffusion of small metabolites to and from the chondrocytes. In articular cartilage, both transient and regional changes occur in water content during joint movement and when the joint is subjected to pressure. The high degree of hydration and the movement of water in the matrix allow the cartilage matrix to respond to varying pressure loads and contribute to the cartilage’s weight-bearing capacity. Throughout life, cartilage undergoes continuous as the cells replace matrix molecules lost through degradation. Normal matrix turnover depends on the ability of the chondrocytes to detect changes in matrix composition. The chondrocytes respond by synthesizing appropriate types of new molecules. In addition, the matrix acts as a signal transducer for the embedded chondrocytes. Thus, pressure loads applied to the cartilage, as in synovial joints, create mechanical, electrical, and chemical signals that help direct the synthetic

aggrecan–hyaluronan aggregates

internal remodeling

activity of the chondrocytes. As the body ages, however, the composition of the matrix changes, and the chondrocytes lose their ability to respond to these stimuli.

Chondrocytes are specialized cells that produce and maintain the extracellular matrix. In hyaline cartilage, chondrocytes are distributed either singularly or in clusters called isogenous groups (Fig. 7.4). When the chondrocytes are present in isogenous groups, they represent cells that have recently divided. As the newly divided chondrocytes produce the matrix material that surrounds them, they are dispersed. They also secrete metalloproteinases (MMPs), enzymes that degrade cartilage matrix, allowing the cells to expand and reposition themselves within the growing isogenous group.

FIGURE 7.4. Photomicrograph of a typical hyaline cartilage specimen stained with hematoxylin and eosin . The upper portion of the micrograph shows dense connective tissue (DCT) overlying the perichondrium ( P ), from which new cartilage cells are derived. A slightly basophilic layer of growing cartilage ( GC ) underlying the perichondrium contains chondroblasts and immature chondrocytes that

display little more than the nucleus residing in an empty-appearing lacuna. This layer represents deposition of new cartilage (appositional growth) on the surface of the existing hyaline cartilage. Mature chondrocytes with clearly visible nuclei ( ) reside in the lacunae and are well preserved in this specimen. They produce the cartilage matrix that shows the dark-staining capsule or territorial

N

TM

IM

matrix ( ) immediately surrounding the lacunae. The interterritorial matrix ( ) is more removed from the immediate vicinity of the chondrocytes and is less intensely stained. Growth from within the cartilage (interstitial growth) is reflected by the chondrocyte pairs and clusters that are responsible for the formation of isogenous groups ( ). ×480.

rectangles

The appearance of chondrocyte cytoplasm varies according to chondrocyte activity. Chondrocytes that are active in matrix production display areas of cytoplasmic basophilia, which are indicative of protein synthesis, and clear areas, which indicate their large Golgi apparatus (Fig. 7.5). Chondrocytes secrete not only the collagen present in the matrix but also all of the GAGs and proteoglycans. In older, less active cells, the Golgi apparatus is smaller; clear areas of cytoplasm, when evident, usually indicate sites of extracted lipid droplets and glycogen stores. In such specimens, chondrocytes also display considerable distortion resulting from shrinkage after the glycogen and lipid are lost during the preparation of the tissue. In the transmission electron microscope (TEM), the active chondrocyte displays numerous profiles of rough-surfaced endoplasmic reticulum (rER), a large Golgi apparatus, secretory granules, vesicles, intermediate filaments, microtubules, and actin microfilaments (Fig. 7.6).

FIGURE 7.5. Photomicrograph of young, growing cartilage. This specimen was preserved in glutaraldehyde, embedded in plastic, and stained with hematoxylin and eosin (H&E). The chondrocytes, especially those in the upper part of the photomicrograph, are well preserved. The cytoplasm is deeply stained, exhibiting a distinct and relatively homogeneous basophilia. The clear areas ( arrows ) represent sites of the Golgi apparatus. ×520.

FIGURE 7.6. Electron micrograph of a young, active chondrocyte and surrounding matrix. The nucleus ( N ) of the chondrocyte is eccentrically located, like those in Figure 7.5, and the cytoplasm displays numerous and somewhat dilated profiles of rough-surfaced endoplasmic reticulum ( rER ), Golgi apparatus ( G ), and mitochondria ( M ). The large amount of rER and the extensive Golgi apparatus

indicate that the cell is actively engaged in the production of cartilage matrix. The numerous dark particles in the matrix contain proteoglycans. The particularly large particles adjacent to the cell are located in the region of the matrix that is identified as the capsule or territorial matrix. ×15,000. (Courtesy of Dr. H. Clarke Anderson.)

Components of the hyaline cartilage matrix are not uniformly distributed. Because the proteoglycans of hyaline cartilage contain a high concentration of bound sulfate groups, ground substance stains with basic dyes and hematoxylin (Plate 7.1, page 230). Thus, the basophilia and metachromasia seen in stained sections of cartilage provide information about the distribution and relative concentration of sulfated proteoglycans. However, under

closer examination, the matrix does not stain uniformly. Rather, three different regions are described based on the staining property of the matrix (Fig. 7.7).

FIGURE 7.7. Diagram of cartilage matrices.

Note the areas of capsular, interterritorial matrices. The characteristics of each are described in the text. endoplasmic reticulum.

territorial, and rough-surfaced

rER,

capsular (pericellular) matrix

The is a ring of more densely staining matrix located immediately around the chondrocyte (see Fig. 7.4). It contains the highest concentration of sulfated proteoglycans, hyaluronan, biglycans, and several multiadhesive glycoproteins (e.g., fibronectin, decorin, and laminin). The capsular matrix contains almost exclusively type VI collagen that forms a tightly woven enclosure around each chondrocyte. Type VI collagen binds to integrin receptors on the cell surface and anchors the chondrocytes to the matrix. A higher concentration of type IX collagen is also present in the capsular matrix. The is a region that is more removed from the immediate vicinity of the chondrocytes. It surrounds the isogenous group and contains a randomly arranged network of type II collagen fibrils with smaller quantities of type IX collagen. It also has a lower concentration of sulfated proteoglycans and stains less intensely than the capsular matrix. The is a region that surrounds the territorial matrix and occupies the space between groups of chondrocytes.

territorial matrix

interterritorial matrix

In addition to these regional differences in the concentration of sulfated proteoglycans and distribution of collagen fibrils, there is a decrease in proteoglycan content that occurs as cartilage ages, which is also reflected by staining differences.

Hyaline cartilage provides a model for the developing skeleton of the fetus. In early fetal development, hyaline cartilage is the precursor of bones that develop by the process of endochondral ossification (Fig. 7.8). Initially, most long bones are represented by cartilage models that resemble the shape of the mature bone (Plate 7.2, page 232). During the developmental process, in which most of the cartilage is replaced by bone, residual

cartilage at the proximal and distal ends of the bone serves as growth sites called . This cartilage remains functional as long as the bone grows in length (Fig. 7.9). In a fully grown individual, a remnant of cartilage from the developing skeleton is found on the articular surface of joints (articular cartilage) and in the rib cage (costal cartilages). Hyaline cartilage also exists in the adult as the skeletal unit in the trachea, bronchi, larynx, and nose.

epiphyseal growth plates (epiphyseal discs)

FIGURE 7.8. Photomicrograph of several cartilages that form the initial skeleton of the foot. The hyaline cartilage of developing tarsal bones will be replaced by bone as endochondral

ossification proceeds. In this early stage of development, synovial joints are being formed between developing tarsal bones. Note that nonarticulating surfaces of the hyaline cartilage models of tarsal bones are covered by perichondrium, which also contributes to the development of joint capsules. Also, a developing tendon ( ) is evident in the indentation of the cartilage seen on the side of the micrograph. ×85.

T

left

FIGURE 7.9. Photomicrograph of the proximal end of a growing long bone.

A disc of hyaline cartilage—the epiphyseal plate—separates the more proximally located epiphysis from the funnelshaped diaphysis located distal to the plate. The articular cartilage on the surface of the epiphysis contributes to the synovial joint and is also composed of hyaline cartilage. The cartilage of the epiphyseal plate disappears when lengthwise growth of the bone is completed, but the articular cartilage remains throughout life. The spaces within the bone are occupied by marrow. ×85.

A firmly attached connective tissue, the perichondrium, surrounds hyaline cartilage. The perichondrium is a dense irregular connective tissue composed of cells that

are indistinguishable from fibroblasts. In many respects, the perichondrium resembles the capsule that surrounds glands and many organs. It also serves as the source of new cartilage cells. When actively growing, the perichondrium appears divided into an inner cellular layer, which gives rise to new cartilage cells, and an outer fibrous layer. This division is not always evident, especially in perichondrium that is not actively producing new cartilage or in very slow-growing cartilage. The changes that occur during the differentiation of new chondrocytes in growing cartilage are illustrated in Figure 7.4.

Hyaline cartilage of articular joint surfaces does not possess a perichondrium.

Hyaline cartilage that covers the articular surfaces of movable joints is termed articular cartilage. In general, the structure of articular cartilage is similar to that of hyaline cartilage. However, the free, or articular, surface has no perichondrium. Also, on the opposite surface, the cartilage contacts the bone, and there is no perichondrium. Articular cartilage is a remnant of the original hyaline cartilage template of the developing bone, and it persists throughout adult life. In adults, the articular cartilage is 2- to 5-mm thick and is divided into four zones (Figs. 7.10 and 7.11):

FIGURE 7.10. Diagram and photomicrograph of articular cartilage. a.

This diagram shows the organization of the collagen network and chondrocytes in the various zones of articular cartilage. Photomicrograph of normal articular cartilage from an adult. The superficial zone ( ) exhibits elongated and flattened chondrocytes. The intermediate zone ( ) contains round chondrocytes. The deep zone ( ) contains chondrocytes arranged in short columns. The calcified zone ( ), which borders the subchondral bone, exhibits small chondrocytes surrounded by the calcified matrix. Also, this zone is lighter staining than the matrix of the more superficial zones. The separates calcified zone from subchondral bone. ×160.

DZ

IZ

tidemark

CZ

SZ

b.

FIGURE 7.11.

Photomicrograph of articular cartilage obtained from a tibial surface of a 12-week-old rat knee joint. This specimen is stained with safranin-O, Fast Green, and hematoxylin that are commonly used in histologic articular cartilage examination. The superficial (tangential) zone is stained light green due to a high condensation of type II collagen fibrils that are arranged in fascicles parallel to the free surface. Both intermediate (transitional) and deep (radial) zones are stained intensely red by safranin-O due to a high concentration of cartilage-specific proteoglycans (mainly sulfated glycosaminoglycans). The calcified zone is stained light green and contains collagen fibrils embedded in calcified matrix with a few small chondrocytes. Note that the calcified zone is separated from the deep zone by the tidemark (chondro-osseus junction) that is traced by the and by the cement line from subchondral bone, which is indicated by the . Subchondral bone that exhibits a typical osteonal pattern is stained deep blue. ×240. (Reprinted with permission from Schultz M, Molligan J, Schon L, et al. Pathology of the calcified zone of articular cartilage in post-traumatic osteoarthritis in rat knees. . 2015;10[3]:e0120949.)

line

yellow line

superficial (tangential) zone

white

PLoS One

The is a pressure-resistant region closest to the articular surface. It contains numerous elongated and flattened chondrocytes surrounded by a condensation of type II collagen fibrils that are arranged in fascicles parallel to the free surface. The lies below the superficial zone and contains round chondrocytes randomly distributed within the matrix. Collagen fibrils are less organized and are arranged in a somewhat oblique orientation to the surface.

intermediate (transitional) zone

deep (radial) zone

The is characterized by small, round chondrocytes that are arranged in short columns perpendicular to the free surface of the cartilage. The collagen fibrils are positioned between columns parallel to the long axis of the bone (see Fig 7.11). The is characterized by a calcified matrix with the presence of small chondrocytes. This zone is separated from the deep (radial) zone by a smooth, undulating, heavily calcified line called the . Above this line, proliferation of chondrocytes within the cartilage lacunae provides new cells for interstitial growth. In articular cartilage renewal, chondrocytes migrate from this region toward the joint surface. The calcified zone rests on the (a layer of bone just below the articular cartilage), and its junction is clearly defined by the (see Fig 7.11). In response to , active calcification is triggered in the subchondral bone, resulting in the formation of a thicker subchondral bone plate.

calcified zone

cement line

tidemark (chondro-osseus junction)

subchondral bone joint injury

The renewal process of mature articular cartilage is very slow. This slow growth is a reflection of the highly stable type II collagen network and the long half-life of its proteoglycan molecules. Also, in healthy articular cartilage, metalloproteinase (MMP-1 and MMP-13) activity is low.

ELASTIC CARTILAGE

Elastic cartilage is distinguished by the presence of elastin in the cartilage matrix. In addition to containing the normal components of hyaline cartilage matrix, elastic cartilage matrix also contains a dense network of branching and anastomosing elastic fibers

and interconnecting sheets of elastic material (Fig. 7.12 and Plate 7.3, page 234). These fibers and lamellae are best demonstrated in paraffin sections with special stains, such as resorcin-fuchsin and orcein. The elastic material gives the cartilage elastic properties in addition to the resilience and pliability that are characteristic of hyaline cartilage.

FIGURE 7.12. Photomicrograph of elastic cartilage from the epiglottis.

This specimen was stained with orcein and reveals elastic fibers, stained brown, within the cartilage matrix. The elastic fibers are of various sizes and constitute a significant part of the cartilage. Chondrocyte nuclei are evident in many of the lacunae. The perichondrium is visible at the of the photomicrograph. ×180.

top

Elastic cartilage is found in the external ear, walls of the external acoustic meatus, auditory (Eustachian) tube, and epiglottis of the larynx. The cartilage in all of these locations is surrounded by a perichondrium similar to that found around most hyaline cartilage. Unlike hyaline cartilage, which calcifies with aging, the matrix of elastic cartilage does not calcify during the aging process.

FIBROCARTILAGE

Fibrocartilage consists of chondrocytes and their matrix material in combination with dense connective tissue. Fibrocartilage is a combination of dense regular connective tissue and hyaline cartilage. The chondrocytes are dispersed among the collagen fibers singularly, in rows, and in isogenous groups (Fig. 7.13 and Plate 7.4, page 236). These chondrocytes appear similar to the chondrocytes of hyaline cartilage, but they have considerably less cartilage matrix material. There is also no surrounding perichondrium as in hyaline and elastic cartilage. In a section containing fibrocartilage, a population of cells with rounded nuclei and a small amount of surrounding amorphous matrix material can typically be seen. These nuclei belong to chondrocytes. Within the fibrous areas are nuclei that are flattened or elongated. These are fibroblast nuclei.

FIGURE 7.13. Photomicrograph of fibrocartilage from an intervertebral disc. The collagen fibers are stained green in this Gomori trichrome preparation. The tissue has a fibrous appearance and contains a relatively small number of fibroblasts with elongated nuclei ( arrows ) as well as more numerous chondrocytes with dark round nuclei. The chondrocytes exhibit close spatial groupings and are arranged either in rows among the collagen fibers or in isogenous groups. ×60. Inset. Higher magnification of an isogenous group. Chondrocytes are contained within lacunae. Typically, there is little cartilage matrix surrounding the chondrocytes. ×700.

Fibrocartilage is typically present in intervertebral discs, the pubic symphysis, articular discs of the sternoclavicular and temporomandibular joints, menisci of the knee joint, triangular fibrocartilage complex of the wrist, and certain places where tendons attach to bones. The presence of fibrocartilage in these sites indicates that resistance to both compression and shearing forces is required of the tissue. The cartilage serves much like a shock absorber. The degree to which such forces occur is reflected in the amount of cartilage matrix material present.

Extracellular matrix of fibrocartilage is characterized by the presence of both type I and type II collagen fibrils.

The cells in fibrocartilage synthesize a wide variety of extracellular matrix molecules not only during its development stage but also during its mature, fully differentiated state. This allows the fibrocartilage to respond to changes in the external environment (such as mechanical forces, nutritional changes, and changing levels of hormones and growth factors). The contains significant quantities of both

extracellular matrix of fibrocartilage

type I

collagen (characteristic of connective tissue matrix) and type II collagen

(characteristic of hyaline cartilage). The relative proportions of these collagens can vary. For example, menisci of the knee joint contain only a small quantity of type II collagen, whereas the intervertebral disc contains equal amounts of type I and type II collagen fibers. The ratio of type I to type II collagen in fibrocartilage changes with age. In older individuals, there is more type II collagen because of the metabolic activity of chondrocytes, which continuously produce and discharge type II collagen fibrils into the surrounding matrix. In addition, the extracellular matrix of fibrocartilage contains larger amounts of (a proteoglycan monomer secreted by fibroblasts) than aggrecan (produced by chondrocytes). Versican can also bind to hyaluronan to form highly hydrated proteoglycan aggregates (see Table 6.4, page 196). is associated with proteolytic degradation of proteoglycan aggregates present within the extracellular matrix of the fibrocartilage.

versican

Intervertebral disc degeneration

CHONDROGENESIS AND CARTILAGE GROWTH

Most cartilage arises from mesenchyme during chondrogenesis. Chondrogenesis, the process of cartilage development, begins

with the aggregation of chondroprogenitor mesenchymal cells to form a mass of rounded, closely apposed cells. In the head, most of the cartilage arises from aggregates of ectomesenchyme derived from neural crest cells. The site of hyaline cartilage formation is recognized initially by an aggregate of mesenchymal or ectomesenchymal cells known as a . Expression of triggers differentiation of these cells into , which then secrete cartilage matrix (expression of SOX-9 coincides with secretion of type II collagen). The chondroblasts progressively move apart as they deposit matrix. When they are completely surrounded by matrix material, the cells are called . The mesenchymal tissue immediately surrounding the chondrogenic nodule gives rise to the perichondrium. Chondrogenesis is regulated by many molecules, including extracellular ligands, nuclear receptors, transcription factors, adhesion molecules, and matrix proteins. Specifically, aggrecan, a cartilage matrix proteoglycan, plays an important role during chondrogenesis. Experimental studies reveal that aggrecan is required for chondrocyte differentiation from chondroprogenitor cells. Mutations of the in humans cause early growth cessation of epiphyseal cartilages in long bones (despite normally timed puberty). This condition is linked to , a skeletal disorder characterized by severe short stature (dwarfism) with advanced bone maturation and early onset of osteoarthritis (see Folder 7.1). Furthermore, the growth and development of the cartilage skeleton are influenced by biomechanical forces. These forces not only regulate the shape, regeneration, and aging of cartilage but also modify cell-to-extracellular matrix interactions within the cartilage.

transcription factor SOX-9

chondrogenic nodule

chondroblasts

chondrocytes

aggrecan gene (ACAN) spondyloepimetaphyseal dysplasia

Cartilage is capable of two kinds of growth: appositional and interstitial.

With the onset of matrix secretion, cartilage growth continues via a combination of two processes:

Appositional growth, cartilage Interstitial growth,

the process that forms new cartilage at the surface of existing the process that forms new cartilage within an existing cartilage

mass

New cartilage cells produced during appositional growth are derived from the inner portion of the surrounding perichondrium. The cells resemble fibroblasts in form and function, producing the collagen component of the perichondrium (type I collagen). When cartilage growth is initiated, however, the cells undergo a differentiation process guided by expression of the transcription factor SOX-9. The cytoplasmic processes disappear, the

nucleus becomes rounded, and the cytoplasm increases in amount and prominence. These changes result in the cell becoming a chondroblast. Chondroblasts function in cartilage matrix production, including secretion of type II collagen. The new matrix increases the cartilage mass, whereas new fibroblasts are produced simultaneously to maintain the cell population of the perichondrium. New cartilage cells produced during interstitial growth arise from the division of chondrocytes within their lacunae (see Fig. 7.4). This is possible only because the chondrocytes retain the ability to divide and the surrounding matrix is distensible, thus permitting further secretory activity. Initially, the daughter cells of the dividing chondrocytes occupy the same lacuna. As a new matrix is secreted, a partition is formed between the daughter cells; at this point, each cell occupies its own lacuna. With continued secretion of matrix, the cells move even farther apart from each other. The overall growth of cartilage thus results from the interstitial secretion of new matrix material by chondrocytes and by the appositional secretion of matrix material by newly differentiated chondroblasts (Folder 7.2).

FOLDER 7.2

CLINICAL CORRELATION: MALIGNANT TUMORS OF THE CARTILAGE: CHONDROSARCOMAS

Chondrosarcomas

are generally slow-growing malignant tumors characterized by secretion of cartilage matrix. Approximately 3.6% of primary bone tumors diagnosed in the United States each year are chondrosarcomas. These tumors are the second most common matrix-producing tumors of bone after osteosarcomas (malignant bone-forming tumors). They occur more commonly in men than women and usually affect individuals aged 45 years and older. Chondrosarcomas originate predominantly in the axial skeleton (and most commonly involve vertebrae, pelvic bones, ribs, scapulae, and sternum) and in the metaphyses of the proximal ends of long bones (most often, the femur and humerus). The most common symptom reported by patients is a deep pain, often present for months and typically dull in character. Because cartilaginous tissue is compressed inside the bone, in most cases, the initial growth of a tumor cannot be palpated. Radiographs, computed tomography (CT), and magnetic resonance imaging (MRI) scans are essential for the initial diagnosis and later for the evaluation of the extent of deep intramedullary tumors. Chondrosarcomas are classified by grades that strongly correlate with prognosis. Microscopically, grade 1 represents the least aggressive and grade 3 represents the most aggressive tumor. Most chondrosarcomas (90%) are pathologically classified as conventional (grades 1 and 2); they rarely metastasize and are composed of hyaline cartilage that infiltrates the bone marrow cavity and surrounds existing bony trabeculae (Fig. F7.2.1). Multiple chondroblasts that are often binucleated with pleomorphic and hyperchromatic nuclear patterns are frequently seen in a single lacuna. The cartilaginous matrix may also undergo mineralization and subsequent endochondral ossification. Metastatic spread to lungs and lymph nodes is more frequently associated with grade 3 lesions.

FIGURE F7.2.1. Photomicrograph of a chondrosarcoma (grade 1) from the epiphysis of the long bone stained with hematoxylin and eosin (H&E). This photomicrograph shows a tissue mass

of chondrosarcoma infiltrating intertrabecular spaces of the bone marrow. Note the presence of malignant chondrocytes in various stages of maturity. Small area of active bone marrow is visible in the corner of the image. ×240. (Courtesy of Dr. Fabiola Medeiros.)

upper left

Immunohistochemical localization of collagen types can be used to determine the stage of tissue differentiation, which may be a prognostic indicator, and confirm the diagnosis. The presence of and the proteoglycan in biopsies indicates mature tumors associated with good prognosis. Conversely, the presence of indicates changes in the extracellular matrix toward dedifferentiated (fibrous) types of tumor with poorer prognosis. In addition, , which is essential for differentiation of mesenchymal cells into chondroblasts during normal fetal development, is expressed in chondrosarcomas. Treatment of chondrosarcoma is primarily surgical: The tumor is widely excised. Chemotherapy and radiation play limited roles in treatment. Patients with adequately resected low-grade tumors have an excellent survival rate.

collagen types II and X

transcription factor SOX-9

REPAIR OF HYALINE CARTILAGE

Cartilage has a limited ability for repair.

aggrecan

collagen type I

Cartilage can tolerate considerably intense and repetitive stress. However, when damaged, cartilage manifests a striking inability to heal, even in the most minor injuries. This lack of response to injury is attributable to the avascularity of cartilage, the immobility of the chondrocytes, and the limited ability of mature chondrocytes to proliferate. Some repair can occur but only if the defect involves the perichondrium. In these injuries, repair results from the activity of the located in the perichondrium. Even then, however, few cartilage cells, if any, are produced. Repair mostly involves the production of dense connective tissue. At the molecular level, cartilage repair is a tentative balance between deposition of in the form of scar tissue and repair by expression of . However, in adults, new blood vessels commonly develop at the site of the healing wound that stimulates the growth of bone rather than actual cartilage repair. The limited ability of cartilage to repair itself can cause significant problems in cardiothoracic surgery, such as coronary artery bypass graft surgery, when costal cartilage must be cut to enter the chest cavity. A variety of treatments may improve the healing of articular cartilage, including perichondrial grafts, autologous cell transplantation, insertion of artificial matrices, and application of growth factors.

pluripotential progenitor cells

type I collagen collagens

cartilage-specific

When hyaline cartilage calcifies, it is replaced by bone. Hyaline cartilage is prone to calcification, a process in which calcium phosphate crystals become embedded in the cartilage matrix. The matrix of hyaline cartilage undergoes calcification as a regular occurrence in three well-defined situations:

The portion of articular cartilage that is in contact with bone tissue in growing and adult bones, but not the surface portion, is calcified. Calcification always occurs in cartilage that is about to be replaced by bone (endochondral ossification) during an individual’s growth period. Hyaline cartilage in the adult calcifies with time as part of the aging process. In most situations, given sufficient time, cartilage that calcifies is replaced by bone. For example, in older individuals, portions of the cartilage rings in the trachea are often replaced by bone tissue (Fig. 7.14). Chondrocytes normally derive all of their nutrients and dispose of wastes by diffusion of materials through the matrix. When the matrix becomes heavily calcified, diffusion is impeded and the chondrocytes swell and die. The ultimate consequence of this event is the removal of the calcified matrix and its replacement by bone.

FIGURE 7.14. Photomicrograph of a tracheal ring from an elderly individual, stained with hematoxylin and eosin (H&E). The darker, somewhat basophilic areas on the left side of the micrograph represent normal cartilage matrix ( C ). The lighter and more eosinophilic areas represent bone tissue ( B ) that has replaced the original cartilage matrix. A large marrow cavity has formed within the cartilage structure and is visible in the center of the micrograph. ×75. Many investigators believe the process of cartilage removal involves a specific cell type designated as a . This cell is described as resembling an osteoclast in both morphology and lytic function. Early studies of chondroclast structure and function were carried out on the developing mandible, in which the resorption of Meckel cartilage is not followed by bone replacement (endochondral ossification). have also been observed on the deep surface of resorbed articular cartilage in some joint diseases. For instance, these multinucleated cells have been identified on both calcified and noncalcified articular cartilage erosions in . Recent immunocytochemical studies on chondroclasts obtained from pathologic joint specimens revealed that chondroclasts express the . It is likely that , which are capable to resorb cartilage and are found wherever cartilage is being removed.

chondroclast

Chondroclasts

rheumatoid arthritis osteoclast-type phenotype chondroclasts are mature osteoclasts

CARTILAGE

OVERVIEW OF CARTILAGE

Cartilage is a solid, firm, and somewhat pliable form of connective tissue composed of chondrocytes and a highly specialized extracellular matrix (comprises 95% of cartilage volume). Chondrocytes reside within lacunae surrounded by extracellular matrix. Cartilage is an avascular structure ; therefore, the composition of the extracellular

matrix is essential for diffusion of substances between chondrocytes and blood vessels in the surrounding connective tissue. There are three major types of cartilage: , , and .

hyaline cartilage elastic cartilage

fibrocartilage

HYALINE CARTILAGE

extracellular matrix

The homogeneous, amorphous of hyaline cartilage is produced by chondrocytes and appears glassy. contains three classes of molecules: (mainly type II and other cartilage-specific collagens, e.g., types VI, IX, X, XI); , which contain glycosaminoglycans (GAGs); and . The ground substance of hyaline cartilage contains three types of GAGs: , , and . The last two bind to a core protein to form a . Aggrecan is the most abundant proteoglycan monomer in hyaline cartilage. molecules interact with a large number of aggrecan molecules to form large . Their negative charges bind and hold large amounts of water molecules. are distributed either singularly or in clusters called . Extracellular matrix surrounding individual chondrocytes ( ) or the isogenous group ( ) varies in collagen content and staining properties. The surrounds the territorial matrix and occupies the space between isogenous groups. A firmly attached connective tissue, the , surrounds hyaline cartilage. It is not present on the free, or articular, surfaces of articular cartilage in synovial joints. Hyaline cartilage is a key tissue in the development of the fetal skeleton ( ) and in most growing bones ( ).

Hyaline cartilage matrix proteoglycan aggregates glycoproteins chondroitin sulfate keratan sulfate proteoglycan monomer Hyaluronan proteoglycan aggregates Chondrocytes groups territorial matrix interterritorial matrix

collagen molecules multiadhesive hyaluronan

isogenous

capsular matrix

perichondrium

endochondral ossification

ELASTIC CARTILAGE

epiphyseal growth plates

Elastic cartilage contains normal components of hyaline cartilage matrix with the addition of a dense network of elastic fibers and interconnecting sheets of elastic material . The presence of elastin in the extracellular matrix gives elastic cartilage great flexibility to endure repeated bending.

external ear, middle ear does not calcify

Elastic cartilage is found in the (auditory tube), and . It is always surrounded by the perichondrium. The elastic cartilage matrix during the aging process.

larynx

FIBROCARTILAGE Fibrocartilage

is a combination of dense regular connective tissue and hyaline cartilage. Fibrocartilage is typically present in intervertebral discs, the pubic symphysis, insertion of tendons, and structures within certain joints (e.g., menisci of the knee joint). The of fibrocartilage contains varying amounts of both and . In addition, ground substance contains larger amounts of than aggrecan molecules.

extracellular matrix type II collagen fibrils versican

type I

CHONDROGENESIS AND CARTILAGE GROWTH Most

cartilage

chondrogenesis

arises

from mesenchyme during . Expression of triggers differentiation of mesenchymal cells into cartilage-producing cells called . Cartilage is capable of two kinds of growth: (forms new cartilage at the surface of an existing cartilage) and (forms new cartilage by mitotic division of chondrocytes within an existing cartilage mass).

transcription factor SOX-9

chondroblasts appositional growth interstitial growth

REPAIR OF HYALINE CARTILAGE

limited ability for repair . Repair calcification and is replaced by

Owing to its avascular nature, cartilage has mostly involves the production of dense connective tissue. In the aging process, hyaline cartilage is prone to bone.

PLATE 7.1 HYALINE CARTILAGE

Hyaline cartilage is an avascular form of connective tissue composed of cells called chondrocytes and a highly specialized homogeneous-appearing extracellular matrix. The hyaline matrix contains type II collagen molecules, proteoglycan aggregates , and multiadhesive glycoproteins . In addition to type II collagen that constitutes the bulk of the fibrils, the hyaline matrix contains sufficient amounts of type VI, IX, X, and XI collagens called cartilage-specific collagens . All collagen molecules interact with each other in a three-

dimensional felt-like arrangement. The matrix is highly hydrated—>60% of its net weight consists of water, most of which is bound to proteoglycan aggregates (aggrecan monomers bound to a long hyaluronan molecule). Hyaline cartilage is found in the adult as the structural framework for the larynx, trachea, and bronchi; it is found on the articular ends of the ribs and on the surfaces of synovial joints. In addition, hyaline cartilage constitutes much of the fetal skeleton and plays an important role in the growth of most bones. At most sites in the body, except for synovial joint surfaces, hyaline cartilage is surrounded by dense irregular connective tissue called the . Hyaline cartilage displays both , the addition of new cartilage at its surface by chondroblasts, and , the division and differentiation of chondrocytes within its extracellular matrix. The newly divided cells produce a new cartilage matrix, thus expanding the volume of the cartilage from inside. Therefore, the overall growth of

perichondrium

appositional growth interstitial growth

cartilage results from the interstitial secretion of new matrix by chondrocytes and by the appositional secretion of matrix by newly differentiated chondroblasts.

Hyaline cartilage

, trachea, human, hematoxylin and eosin (H&E) ×450.

This micrograph reveals hyaline cartilage from the trachea as seen in a routinely prepared specimen. The cartilage appears as an avascular expanse of matrix material and a population of cells called ( ). The chondrocytes produce the matrix; the space each chondrocyte occupies is called a ( ). Surrounding the cartilage and in immediate apposition to it is a cover of connective tissue, the ( ). The perichondrium serves as a source of new chondrocytes during of the cartilage. Often, the perichondrium reveals two distinctive layers: an outer, more fibrous layer and an inner, more cellular layer. The inner, more cellular layer, containing chondroblasts and chondroprogenitor cells, provides for external growth. contains collagenous fibrils masked by ground substance in which they are embedded; thus, the fibrils are not evident. The matrix also contains, among other components, sulfated glycosaminoglycans that exhibit basophilia with hematoxylin or other basic dyes. Also, the matrix material immediately surrounding a lacuna tends to stain more intensely with basic dyes. This region is referred to as a ( ). Not uncommonly, the matrix may appear to stain more intensely in localized areas ( ) that look much like the capsule matrix. This results from the inclusion of a capsule within the thickness of the section, but not the lacuna it surrounds. Frequently, two or more chondrocytes are located extremely close to one another, separated by only a thin partition of matrix. These are isogenous cell clusters that arise from a single predecessor cell. The proliferation of new chondrocytes by this means with the consequent addition of matrix results in of the cartilage.

chondrocytes Ch

P

lacuna L perichondrium appositional growth

Cartilage matrix

capsule Cap asterisks

interstitial growth

Hyaline cartilage

, trachea, human, H&E ×160.

The hyaline cartilage in this micrograph is from a specimen obtained shortly after death and kept cool during fixation. The procedure reduces the loss of its negatively charged sulfate groups; thus, the matrix is stained more heavily with hematoxylin. Also, note the very distinct and deeply stained capsules ( ) surrounding the chondrocytes. The capsule represents the site where the sulfated glycosaminoglycans are most concentrated. In contrast to the basophilia of the cartilage matrix, the ( ) is stained with eosin. The lightly stained region between the perichondrium and the deeply stained matrix is the matrix that has not yet matured. It has fewer sulfate groups.

arrows

perichondrium P

Hyaline cartilage

, trachea, human, H&E ×850.

rectangle

This higher magnification micrograph reveals the area within the in the figure. The chondrocytes ( ) in the of the micrograph represent an isogenous group and are producing matrix material for interstitial growth. A prominent capsule is not yet evident. The lightly stained basophilic area reveals immature chondrocytes ( ) within the perichondrium ( ). Closest to the cartilage matrix, within the ( ), are several chondrocytes that exhibit just barely detectable cytoplasm and elongated nuclei ( ). These cells are formative chondrocytes that are just beginning to, or will shortly, produce matrix material. In contrast, the nuclei near the of the micrograph are fibroblast nuclei ( ); they belong to the outer layer of the perichondrium. Note how attenuated their nuclei are compared with the formative chondroblast nuclei of the inner perichondrial layer.

lower left

Ch

upper part

perichondrium FChP

P

arrows

bottom edge

Fib

Cap, capsule Ch, chondrocytes FCh, formative chondrocytes Fib, fibroblasts L, lacuna P, perichondrium arrows, immature chondrocytes asterisk, capsule of a lacuna (lacuna and its chondrocyte not included within the thickness of the section)

PLATE 7.2 HYALINE CARTILAGE AND THE DEVELOPING SKELETON Hyaline cartilage

is present as a precursor to bones that develop in the fetus by the process of endochondral ossification. This cartilage is replaced by bone tissue, except where one bone contacts another, as in a movable joint. In these locations, cartilage persists and covers the end of each bone as articular cartilage, providing a smooth, well-lubricated surface against which the end of one bone moves on the other in the joint. In addition, cartilage, being capable of interstitial growth, persists in weight-supporting bones and other long bones as a growth plate as long as growth in length occurs. The role of hyaline cartilage in bone growth is considered briefly here and in more detail in Plates 8.3 and 8.4.

Developing skeleton

, fetal foot, rat, hematoxylin and eosin (H&E) ×85.

cartilagesL C

This section shows the ( ) that will ultimately become the bones of the foot. In several places, developing ligaments ( ) can be seen where they join the cartilages. The nuclei of the fibroblasts within the ligaments are just barely perceptible. They are aligned in rows and are

separated from other rows of fibroblasts by collagenous material. The hue and intensity of the color of the cartilage matrix, except at the periphery, are due to the combined uptake of the H&E. The collagen of the matrix stains with eosin; however, the presence of sulfated glycosaminoglycans results in staining by hematoxylin. The matrix of cartilage that is about to be replaced by bone, such as that shown here, becomes impregnated with calcium salts, and the calcium is also receptive to staining with hematoxylin. The many enlarged lacunae (seen as light spaces within the matrix where the chondrocytes have fallen out of the lacunae) are due to hypertrophy of the chondrocytes, an event associated with calcification of the matrix. Thus, where these large lacunae are present, that is, in the center region of the cartilage, the matrix is heavily stained. This figure also shows that the cartilage is surrounded by perichondrium, except where it faces a ( ). Here, the bare cartilage forms a surface. Note that the joint cavity is a space between the cartilages whose boundaries are completed by ( ). The connective tissue at the surface of the cavity constitutes the synovial membrane in the adult and contributes to the formation of a lubricating fluid (synovial fluid) that is present in the joint cavity. All surfaces that will enclose the adult joint cavity are derived originally from the mesenchyme. Synovial fluid is a viscous substance containing, among other things, hyaluronan and glycosaminoglycans; it can be considered an exudate of interstitial fluid. The synovial fluid could be considered an extension of the extracellular matrix, as the joint cavity is not lined by an epithelium.

joint cavity JC

connective tissue CT

Developing skeleton

, fetal finger, human, thionine–picric acid ×30.

This photomicrograph shows a developing long bone of the finger and its articulation with the distal and proximal bones. Before the stage shown here, each bone consisted entirely of a hyaline cartilaginous structure similar to the cartilages seen in the previous figure but shaped like the long bones into which they would develop. Here, only the ends, or epiphyses, of the bone remain as cartilage, the ( ). The shaft, or diaphysis, has become a cylinder of bone tissue ( ) surrounding the ( ). The dark region at the ends of the marrow cavity is calcified cartilage ( ) that is being replaced by bone. The bone at the ends of the marrow cavity constitutes the . With this staining method, the calcified cartilage appears . The newly formed metaphyseal bone is admixed with the degenerating calcified cartilage and is difficult to define at this low magnification; it has the same color as the diaphyseal bone. With the continued proliferation of cartilage, the bone grows in length. Later, the cartilage becomes calcified; bone is then produced and occupies the site of the resorbed cartilage. With the cessation of cartilage proliferation and its replacement by bone, growth of the bone stops, and only the cartilage at the articular surface remains. The details of this process are explained in Plates 8.3 and 8.4 that depict endochondral bone formation.

epiphyseal cartilage B

C

marrow cavity MC arrowhead metaphysis

B, bone C, cartilage CT, connective tissue JC, joint cavity L, ligament MC, marrow cavity arrowhead, calcified cartilage

yellow-brown

dark brown

PLATE 7.3 ELASTIC CARTILAGE Elastic cartilage

has a matrix containing elastic fibers and elastic lamellae in addition to type II collagen and other components found in the extracellular matrix of hyaline cartilage. It is found in the auricle of the external ear, auditory tube, epiglottis, and other parts of the larynx (i.e., cuneiform cartilages, vocal processes of arytenoid cartilages). The elastic material imparts properties of elasticity, as distinguished from resiliency, which are not shared by hyaline cartilage. Elastic cartilage is surrounded by perichondrium, and it, too, increases in size by both appositional and interstitial growth. Unlike hyaline cartilage, however, elastic cartilage does not normally undergo the calcification process.

Elastic cartilage ×80.

, epiglottis, human, hematoxylin and eosin (H&E) and orcein stains

elastic cartilage EC

This section of the epiglottis contains ( ) as the centrally located, purplestained tissue. The essential components of the cartilage, namely, the matrix containing elastic

perichondrium

glands (

MG

purple PC AT

fibers, which stains , and the light, unstained lacunae surrounded by matrix, are evident in this low-magnification micrograph. The perimeter of the cartilage is covered by ( ); its fibrous character is just barely visible in this figure. The epiglottis contains many small perforations (epiglottic foramina); note the presence of adipose tissue ( ) within these openings. Adipose tissue in this micrograph is visible within the boundaries of the elastic cartilage. Above and below the elastic cartilage is connective tissue, and each surface of the epiglottis is formed by stratified squamous nonkeratinized epithelium ( ). Mucous ) can be seen in the connective tissue at the of this figure.

SE

bottom

Elastic cartilage ×400.

, epiglottis, human, H&E and orcein stains ×250; inset

elastic cartilage

This shows an area of the at higher magnification. The elastic fibers appear as , elongated profiles within the matrix. They are most evident at the edges of the cartilage, but they are obscured in some deeper parts of the matrix, where they blend with the elastic material that forms a honeycomb about the lacunae. ( ) are also apparent in the adipose tissue ( ), between the adipocytes. Some of the lacunae in the cartilage are arranged in pairs separated by a thin plate of matrix. The plate of matrix appears as a bar between the adjacent lacunae. This is a reflection of interstitial growth by the cartilage, in that the adjacent cartilage cells are derived from the same parent cell. They have moved away from each other and secreted a plate of cartilage matrix between them to form two lacunae. Most ( ) shown in this figure occupy only part of the lacuna. This is, in part, due to shrinkage, but it is also due to the fact that older chondrocytes contain large lipid droplets that are lost during tissue preparation. The shrinkage of chondrocytes within the lacunae or their loss during preparation causes the lacunae to stand out as light, unstained areas against the darkly stained matrix. The shows the elastic cartilage at still higher magnification. Here, the elastic fibers ( ) are again evident as elongated profiles, chiefly at the edges of the cartilage. Most chondrocytes in this part of the specimen show little shrinkage. Many of the cells display a typically rounded nucleus, and the cytoplasm is evident. Note, again, that some lacunae contain two chondrocytes, indicating interstitial growth.

purple

Elastic fibers E

AT

chondrocytes Ch

inset

AT, adipose tissue Ch, chondrocytes E, elastic fiber EC, elastic cartilage MG, mucous gland PC, perichondrium SE, stratified squamous nonkeratinized epithelium

E

PLATE 7.4 FIBROCARTILAGE Fibrocartilage

is a combination of dense irregular connective tissue and hyaline cartilage. It has a matrix with large bundles of type I collagen in addition to type II collagen. The amount of cartilage varies, but in most locations, the cartilage cells and their matrix occupy a lesser portion of the tissue mass. Fibrocartilage is found at the intervertebral discs, pubic symphysis, knee joint, mandibular joint, sternoclavicular joint, and shoulder joint. It may also be present along the grooves or insertions for tendons and ligaments. Its presence is associated with sites where resilience is required in dense connective tissue to help absorb sudden physical impact, that is, where resistance to both compressive and shearing forces is required in the tissue. Histologically, fibrocartilage appears as small fields of cartilage blending almost imperceptibly with regions of dense fibrous connective tissue. It is usually identified by the presence of aggregates of rounded chondrocytes (isogenous groups) among bundles of collagen fibers and by the basophilic staining of the capsular matrix material and territorial matrix secreted by these cells. No perichondrium is present.

Fibrocartilage

, intervertebral disc, human, Mallory trichrome ×160.

fibrocartilage

This is a low-magnification view of . The Mallory method stains collagen . The tissue has a fibrous appearance, and at this low magnification, the nuclei of the ( ) appear as small, elongated, or spindle-shaped bodies. There are relatively few fibroblasts present, as is characteristic of dense connective tissue. The ( ) are more numerous and exhibit close spatial groupings, that is, . Some of the chondrocytes appear as elongated clusters of cells, whereas others appear in single-file rows. The matrix material immediately surrounding the chondrocytes has a homogeneous appearance and is thereby distinguishable from the fibrous connective tissue.

light blue

fibroblasts F chondrocytes Ch isogenous groups

Fibrocartilage

, intervertebral disc, human, Mallory trichrome ×700.

rectangle

This figure shows the area circumscribed by the in the micrograph above at higher magnification. The chondrocytes are contained within lacunae ( ), and their cytoplasm stains deeply. The surrounding material is scant and blends into the dense connective tissue. Cartilage matrix material can be detected best by observing the larger group of chondrocytes at the of this figure and then observing this same area in the previous figure. Note the light homogeneous area around the cell nest in the lower power view. This is the region of cartilage matrix. At the greater magnification of this figure, it is possible to see that some of the collagen fibers are incorporated in the matrix, where they appear as wispy bundles.

cartilage matrix left

Ch, chondrocytes F, fibroblast arrows, lacunae

arrows

8 OVERVIEW OF BONE GENERAL STRUCTURE OF BONES Bone as an Organ Outer Surface of Bones Bone Cavities

TYPES OF BONE TISSUE Mature Bone Immature Bone

CELLS OF BONE TISSUE

Osteoprogenitor Cells Osteoblasts Osteocytes Bone-Lining Cells Osteoclasts

BONE FORMATION

Intramembranous Ossification Endochondral Ossification

BONE

Growth of Endochondral Bone Development of the Osteonal (Haversian) System

BIOLOGIC MINERALIZATION AND MATRIX VESICLES BONE AS A TARGET OF ENDOCRINE HORMONES AND AS AN ENDOCRINE ORGAN BIOLOGY OF BONE REPAIR Folder 8.1 Clinical Correlation: Joint Diseases Folder 8.2 Clinical Correlation: Osteoporosis Folder 8.3 Clinical Correlation: Nutritional Factors in Bone Formation Folder 8.4 Functional Considerations: Hormonal Regulation of Bone Growth HISTOLOGY

OVERVIEW OF BONE

Bone is a connective tissue characterized by a mineralized extracellular matrix. Bone is a specialized form of connective tissue that, like other connective

tissues, consists of cells and extracellular matrix. The feature that distinguishes bone from other connective tissues is the mineralization of its matrix, which produces an extremely hard tissue capable of providing and . The mineral is calcium phosphate in the form of [Ca10(PO4)6(OH)2]. By virtue of its mineral content, bone also serves as a . Both calcium and phosphate can be mobilized from the bone matrix and taken up by the blood as needed to maintain appropriate levels throughout the body. Thus, in addition to support and protection, bone plays an important secondary role in the homeostatic regulation of blood calcium levels.

support hydroxyapatite crystals phosphate

protection

storage site for calcium and

Bone matrix contains mainly type I collagen along with other matrix (noncollagenous) proteins. The major structural component of bone matrix is type I collagen and, to a lesser extent, type V collagen. Trace amounts of other types such as type III, XI, and XIII collagens have also been found in the matrix. All collagen molecules constitute about 90% of the total weight of the bone matrix proteins. The matrix also contains other matrix (noncollagenous) proteins that constitute the of bone. As a minor component of bone, constituting only 10% of the total weight of bone matrix proteins, they are essential to bone development, growth, remodeling, and repair. Both the collagen and the ground substance become mineralized to form bone tissue. The four main groups of noncollagenous proteins found in the bone matrix are the following:

ground substance

Proteoglycan macromolecules contain a core protein with various numbers of covalently attached side chains of glycosaminoglycans (hyaluronan, chondroitin sulfate, and keratan sulfate). Some proteoglycans, such as keratan sulfate, contain osteoadherin, a bonespecific protein that strongly binds to hydroxyapatite crystals. Proteoglycans contribute to the compressive strength of bone. They are also responsible for binding growth factors, and in certain conditions, they may inhibit mineralization. Proteoglycans are described in detail in Chapter 6, Connective Tissue (Table 6.3, page 193). are responsible for attachment of bone cells and collagen fibers to the mineralized ground substance. Some important are ,

Multiadhesive glycoproteins

glycoproteins

osteonectin

podoplanin (E11) dentin matrix protein (DMP) bone sialoproteins osteopontin BSP-1 BSP-2 Bone-specific, vitamin K–dependent proteins osteocalcin protein S matrix Gla-protein (MGP) Growth factors cytokines bone morphogenic proteins (BMPs) sclerostin interleukins (IL-1, IL-6) BMP-7 osteogenic protein-1 (OP-1)

which serves as a glue between the collagen and hydroxyapatite crystals; , which is produced exclusively by osteocytes in response to mechanical stress; , which is critical for bone matrix mineralization; , such as (also known as ), which mediates attachment of cells to bone matrix; and , which mediates cell attachment and initiates calcium phosphate formation during the mineralization process. serve a variety of functions and include , which captures calcium from the circulation and attracts and stimulates osteoclasts in bone remodeling; , which assists in the removal of cells undergoing apoptosis; and , which participates in the development of vascular calcifications. and are small regulatory proteins and include insulin-like growth factors (IGFs), tumor necrosis factor α (TNF-α), transforming growth factor β (TGF-β), platelet-derived growth factors (PDGFs), , (a BMP antagonist), and . BMPs are unique because they induce the differentiation of mesenchymal cells into osteoblasts, the bone-producing cells. Recombinant human , also known as , is now used clinically to induce bone growth after bone surgery involving large bone defects, spinal fusions, or implantation of graft materials.

Bone matrix contains lacunae connected by a network of canaliculi. Within the bone matrix are spaces called lacunae (sing., lacuna), each of which contains a bone cell or osteocyte. The osteocyte extends numerous processes into small tunnels called canaliculi. Canaliculi course through the mineralized matrix, connecting adjacent lacunae and allowing contact between the cell processes of neighboring osteocytes (Plate 8.1, page 270). In this manner, a continuous network of canaliculi and lacunae-containing cells and their processes is formed throughout the entire mass of mineralized tissue. Electron micrographs show that osteocyte processes communicate by gap junctions. Bone tissue depends on the osteocytes to maintain viability. In addition to osteocytes, four other cell types are associated with bone:

Osteoprogenitor cells are derived from mesenchymal stem cells; they give rise to osteoblasts. Osteoblasts secrete the extracellular matrix of bone; once the cell is surrounded by its secreted matrix, it is referred to as an osteocyte. Bone-lining cells remain on the bone surface when there is no active growth. They are derived from those osteoblasts that remain after bone deposition ceases. Osteoclasts resorb bone and are present on bone surfaces where bone is being removed or remodeled (reorganized) or where bone has been damaged.

Osteoprogenitor cells and osteoblasts are developmental precursors of the osteocyte. Osteoclasts are phagocytotic cells derived from fusion of hemopoietic progenitor cells in bone marrow that give rise to the neutrophilic granulocyte and monocyte lineages. Each of these cells is described in more detail later.

GENERAL STRUCTURE OF BONES Bone as an Organ

Bones are the principal organs of the musculoskeletal system; bone tissue is the structural component of bones. Typically, a bone consists of bone tissue and other specialized connective tissues, including cartilage, hemopoietic tissue, fat tissue, and associated blood vessels and nerves. Bones, muscles, and joints form the musculoskeletal system. Its primary functions include supporting the body, allowing motion, and protecting vital organs. The

musculoskeletal system is made up of bones (the adult human skeletal comprises ~206 bones), skeletal muscles, tendons, cartilages, ligaments, joints, and other connective tissue components that support and connect these elements.

Bones are classified according to shape; the location of spongy and compact bone varies with bone shape.

Spongy and compact bone tissues are located in specific parts of bones. The distribution of these tissues within bones contributes to their shape and is therefore an important factor in how bones are classified. On the basis of shape, bones can be classified into four groups:

Long bones

are longer in one dimension than other bones and consist of a shaft and two ends (e.g., the tibia and the metacarpals). A schematic diagram of a long bone sectioned longitudinally through the shaft is shown in Figure 8.1.

FIGURE 8.1. Structure of a typical long bone.

The diaphysis (shaft) of a long bone in the adult contains yellow bone marrow in a large marrow cavity surrounded by a thick-walled tube of compact bone. A small amount of spongy bone may line the inner surface of the compact bone. The proximal and distal ends, or epiphyses, of the long bone consist chiefly of spongy bone with a thin outer shell

of compact bone. The expanded or flared part of the diaphysis nearest the epiphysis is referred to as the metaphysis. Except for the articular surfaces that are covered by hyaline (articular) cartilage, indicated in , the outer surface of the bone is covered by a fibrous layer of connective tissue called the periosteum.

blue

Short bones are nearly equal in length and diameter (e.g., the carpal bones of the hand). Flat bones are thin and plate-like (e.g., the bones of the calvaria [skullcap], scapula, and the sternum). A flat bone consists of two layers of relatively thick compact bone with an intervening layer of spongy bone. have a shape that does not fit into any one of the three groups just described; the shape may be complex (e.g., a vertebra), or the bone may contain air spaces or sinuses (e.g., the ethmoid bone).

Irregular bones

Long bones have a shaft, called the diaphysis, and two expanded ends, each called an epiphysis (Fig. 8.2). The articular surface of the epiphysis is covered with hyaline cartilage. The flared portion of the bone between the diaphysis and the epiphysis is called the metaphysis. It extends from the diaphysis to the epiphyseal line. A large cavity filled with bone marrow, called the marrow or medullary cavity, forms the inner portion of the bone. In the shaft, almost the entire thickness of the bone tissue is compact; at most, only a small amount of spongy bone faces the marrow cavity. At the ends of the bone, the reverse is true. Here, the spongy bone is extensive, and the compact bone consists of little more than a thin outer shell (see Fig. 8.2).

FIGURE 8.2. Epiphysis of an adult long bone.

This photograph shows a longitudinally sectioned proximal epiphysis of the femur after the bone was processed by alkaline hydrolysis. The interior of the bone exhibits a spongy configuration and represents spongy (cancellous) bone. It consists of numerous interconnecting bony trabeculae separated by a labyrinth of interconnecting marrow spaces. The three-dimensional orientation of bony trabeculae is not random but is correlated with the magnitude and directionality of hip joint loads (forces acting on the hip joint and transmitted on the head of the femur). The outer portion of the bone has a solid structure and represents compact (dense) bone. It is particularly well visualized in the diaphysis, which encloses the bone marrow cavity. from the rectangular area shows enlargement of the boundary between spongy and compact bone.

Inset

Short bones possess a shell of compact bone and have spongy bone and a marrow space on the inside. Short bones usually form movable joints with their neighbors; like long bones, their articular surfaces are covered with hyaline cartilage. Elsewhere, , a fibrous connective tissue capsule, covers the outer surface of the bone.

periosteum To allow movement and stability during locomotion, different bones are connected by different types of joints. If the bone forms a freely movable joint (also called a synovial joint), the contact areas of the two bones are referred to as articular surfaces. The articular surfaces are covered by hyaline cartilage, also called articular cartilage, because of its location and function. The space between articulating bones is called the joint cavity; it contains a lubricating fluid (synovial fluid). Articular cartilage is not covered with perichondrium.

The details of articular cartilage are discussed in Chapter 7, Cartilage (pages 221-223) and Folder 8.1 (page 241).

FOLDER 8.1

CLINICAL CORRELATION: JOINT DISEASES Inflammation of the joints or arthritis can be caused by many factors and can produce varying degrees of pain and disability. Arthritis is caused by a pathologic response of articular cartilage to injury. Simple trauma to a joint by a single incident or by repeated insult can damage the articular cartilage, causing it to calcify. Eventually, the cartilage is replaced by bone. This process can lead to (i.e., bony fusion in the joint and subsequent loss of motion). The foot and knee joints of runners and football players and hand and finger joints of stringed-instrument players are especially vulnerable to this condition. Immune responses or infectious processes that localize in joints, as in or , can also damage the articular cartilages, producing both severe joint pain and gradual ankylosis. Surgical replacement of the damaged joint with a prosthetic joint can often relieve the pain and restore joint motion in seriously debilitated individuals. Another common cause of damage to articular cartilages is the deposition of crystals of uric acid in the joints, particularly those of the toes and fingers. This condition is known as or, more simply, . Gout has become more common due to the widespread use of thiazide diuretics in the treatment of hypertension. In genetically predisposed individuals, gout is the most common side effect of these drugs. Gout causes severe pain due to the presence of sharp, needle-like uric acid crystals in the joint. Damage to the cartilage also causes the formation of calcareous deposits that deform the joint and limit its motion.

ankylosis

rheumatoid arthritis

tuberculosis

arthritis

articular capsule

gouty

gout

The that surrounds the joint cavity is composed of an outer fibrous layer of dense irregular connective tissue and inner layer of that is attached to the edges of . Although the synovial membrane lines the joint cavity, it is not considered an epithelium. The synovial membrane consists of two types of mesenchymally derived synoviocytes: type A, macrophage-like cells, and type B, fibroblast-like cells. In most synovial joints, the articular capsule is reinforced from the exterior by ligaments. Some synovial joints have fibrocartilaginous structures superimposed between the articulating surfaces (i.e., menisci in the knee joint or the intraarticular disc in the temporomandibular joint). In addition to movable joints, bones can be connected by or . These joints have no joint cavity and are held together by either fibrocartilage (i.e., intervertebral discs or pubic symphysis permitting slight movements), irregular connective tissue (i.e., sutures of the skull), or cartilage (between certain bones of the rib cage). The ability of the bone to perform its skeletal function is attributable to the bone tissue, regular and irregular connective tissue (i.e., ligaments, joint capsule), and the articular (hyaline) cartilage in the synovial joints. The surgical replacement of joints due to , the most frequent idiopathic disease of joints characterized by degeneration of articular cartilage, is called an . During this procedure, the damaged articular surface with underlying bone tissue is removed and replaced with an artificial joint (called a ) made of metal, plastic, or ceramic materials designed to mimic the normal anatomic structures of the joint. Due to excellent biocompatibility, a titanium metal prosthesis (often coated with hydroxyapatite crystals, growth factors, or both) is commonly used. They develop a strong structural and functional connection (osseointegration) between living bone and the surface of the implant.

articular cartilages

slightly movable

joints

arthroplasty

synovial membrane

immovable

osteoarthritis

prosthesis

Outer Surface of Bones

Bones are covered by periosteum, a sheath of dense fibrous connective tissue containing osteoprogenitor cells. Bones are covered by a periosteum except in areas where they articulate with another bone.

In the latter case, the articulating surface is covered by cartilage. The periosteum that

covers an actively growing bone consists of an outer fibrous layer that resembles other dense irregular connective tissues and an inner, more cellular layer that is in direct contact with bone. The cellular layer contains , including osteoprogenitor cells. If active bone formation is not in progress on the bone surface, the fibrous layer is the main component of the periosteum, and the inner layer is not well defined. The relatively few cells that are present, the , are, however, capable of undergoing division and becoming osteoblasts under the appropriate stimulus. , a 90-kDa extracellular matrix protein secreted by periosteal cells, is a key regulator of periosteal responses to mechanical stress, injury, and bone repair. In general, the collagen fibers of the periosteum are arranged parallel to the surface of the bone in the form of a capsule. The character of the periosteum is different where ligaments and tendons attach to the bone. Collagen fibers from these structures extend obliquely or at right angles to the long axis of the bone, where they are continuous with the collagen fibers of the extracellular matrix. These fibers are called or . They extend into the outer circumferential and interstitial lamellae but usually do not enter the osteons.

skeletal stem cells periosteal cells

Periostin

perforating

Sharpey fibers

Bone Cavities

Bone cavities are lined by endosteum, a layer of connective tissue cells that contains osteoprogenitor cells. The lining tissue of both the compact bone facing the marrow cavity and the trabeculae of spongy bone within the cavity is referred to as endosteum. The endosteum is often only one cell layer thick and consists of osteoprogenitor cells that can differentiate into bone matrix–secreting cells (osteoblasts) and bone-lining cells. Osteoprogenitor cells and bonelining cells are difficult to distinguish at the microscopic level. They are both flattened in shape with elongated nuclei and indistinguishable cytoplasmic features. Because of their location within the bone cavities, they are frequently called .

endosteal cells The marrow cavity and the spaces in spongy bone contain bone marrow. Red bone marrow consists of blood cells in different stages of development and a network of

reticular cells and fibers that serve as a supporting framework for the developing blood cells and vessels. As an individual grows, the amount of red marrow does not increase proportionately with bone growth. In later stages of growth and in adults, when the rate of blood cell formation has diminished, the tissue in the marrow cavity consists mostly of fat cells; it is then called . In response to appropriate stimuli, such as extreme blood loss, yellow marrow can revert to red marrow. In the adult, red marrow is normally restricted to the spaces of spongy bone in a few locations, such as the sternum and the iliac crest. Diagnostic bone marrow samples and marrow for transplantation are obtained from these sites.

yellow marrow

TYPES OF BONE TISSUE

Bone tissue is classified as either compact (dense) or spongy (cancellous). If a bone is cut, two distinct structural arrangements of bone tissue can be recognized (see Fig. 8.2 and Plate 8.2, page 272). A compact, dense layer forms the outside of the bone ( ); a sponge-like meshwork consisting of (thin, anastomosing spicules of bone tissue) forms the interior of the bone ( ). The spaces within the meshwork are continuous and, in a living bone, are occupied by marrow and blood vessels.

compact bone

Mature Bone

trabeculae spongy bone

Mature bone is composed of structural units called osteons (Haversian systems).

osteons Haversian systems lamella

Mature bone is largely composed of cylindrical units called or (Fig. 8.3). The osteons consist of (sing., ) of bone matrix surrounding a central canal, the , which contains the vascular and nerve supply of the osteon. Canaliculi containing the processes of osteocytes are generally arranged in a radial pattern with respect to the canal (Plate 8.1, page 270). The system of canaliculi that opens to the osteonal canal also serves for the passage of substances between the osteocytes and blood vessels. Between the osteons are remnants of previous concentric lamellae called (see Fig. 8.3). Because of this organization, mature bone is also called .

concentric lamellae osteonal (Haversian) canal interstitial lamellae lamellar bone

FIGURE 8.3. Diagram of a section of compact bone removed from a long bone.

The concentric lamellae and the Haversian canal that they surround constitute an osteon (Haversian system). One of the Haversian systems in this diagram is drawn as an elongated cylindrical structure rising above the plane of the bone section. It consists of several concentric lamellae that have been partially removed to show the perpendicular orientation of collagen fibers in adjacent layers. Interstitial lamellae result from bone remodeling and formation of new Haversian systems. The inner and outer surfaces of the compact bone in this diagram show additional lamellae—the outer and inner circumferential lamellae—arranged in broad layers. Both the inner circumferential lamellae and the spongy bone on the internal surface of the compact bone are covered by a thin layer of endosteum, which faces the bone marrow spaces. The outer surface of the bone is covered by periosteum that contains a thicker layer of

connective tissue. Branches of nutritional arteries and small veins accompanied by nerves are shown within the Haversian and Volkmann canals. These vessels and nerves also supply the periosteum and endosteum.

The long axis of an osteon is usually parallel to the long axis of the bone. The collagen fibers in the concentric lamellae in an osteon are laid down parallel to one another in any given lamella but in different directions in adjacent lamellae. This arrangement gives the cut surface of lamellar bone the appearance of plywood and imparts great strength to the osteon. Lamellar bone is also found at sites other than the osteon. follow the entire inner and outer circumferences of the shaft of a long bone, appearing much like the growth rings of a tree (see Fig. 8.3). are channels in lamellar bone through which blood vessels and nerves travel from the periosteal and endosteal surfaces to reach the osteonal (Haversian) canal; they also connect osteonal canals to one another (Fig. 8.4). They usually run at approximately right angles to the long axis of the osteons and of the bone (see Fig. 8.3). Volkmann canals are not surrounded by concentric lamellae, a key feature in their histologic identification.

Circumferential lamellae Perforating (Volkmann) canals

FIGURE 8.4. Three-dimensional reconstruction of Haversian and Volkmann canals from a compact bone. a. This photograph shows enlargement of the interphase between compact and spongy bone from a diaphysis of the femur. b. Using high-resolution quantitative computed tomography (CT), a three-

dimensional reconstruction of the Haversian and Volkmann canals was obtained from a small area of the compact bone indicated on the adjacent photograph. Note that all Haversian canals run parallel to each other in the same direction and are interconnected by perpendicularly oriented Volkmann canals. ×180. (Courtesy of Dr. Mark Knackstedt, Australian National University.)

Mature spongy bone is structurally similar to mature compact bone. Mature spongy bone is similar in structure to mature compact bone except that the tissue is arranged as trabeculae or spicules; numerous interconnecting marrow spaces of various sizes are present among the bone tissue. The matrix of the bone is lamellated. Arteries that enter the marrow cavity through the nutrient foramina supply blood to the shaft of long bones. Nutrient foramina are openings in the bone through which blood vessels pass to reach the marrow. The greatest numbers of nutrient foramina are found in the diaphysis and epiphysis (Fig. 8.5). Metaphyseal arteries supplement the blood supply to the bone. Veins that drain the blood from bone exit through the nutrient foramina or through the bone tissue of the shaft and continue out through the periosteum.

FIGURE 8.5. Diagram showing the blood supply of an adult long bone.

The nutrient artery and the epiphyseal arteries enter the bone through nutrient foramina. These openings in the bone arise developmentally as the pathways of the principal vessels of periosteal buds. Metaphyseal arteries arise from periosteal vessels that become incorporated into the metaphysis as the bone grows in diameter.

nutrient arteries

The that supply the diaphysis and epiphysis arise developmentally as the principal vessels of the periosteal buds. The metaphyseal arteries, in contrast, arise developmentally from periosteal vessels that become incorporated into the metaphysis during the growth process (i.e., through the widening of the bone).

The blood supply to bone tissue is essentially centrifugal.

The blood that nourishes bone tissue moves from the marrow cavity into and through the bone tissue and out via periosteal veins; thus, its flow is in a centrifugal direction. With respect to nourishment of the bone tissue itself, Volkmann canals provide the major route of entry for vessels to pass through the compact bone. The smaller blood vessels enter the Haversian canals, which contain a single arteriole and a venule or a single capillary. A lesser blood supply to the outermost portions of the compact bone is provided by the branches of periosteal arteries (see Fig. 8.5). Bone tissue lacks lymphatic vessels; lymphatic drainage occurs only from the periosteum.

Immature Bone

Bone tissue initially formed in the skeleton of a developing fetus is called It differs from mature bone in several respects (Fig. 8.6):

immature bone.

FIGURE 8.6. Diagram of immature and mature compact and spongy bone. a. Immature (woven) bone does not display an organized lamellar appearance because of the interlacing arrangement of the collagen fibers. The cells tend to be randomly arranged. b. The cells in mature compact bone are organized in a circular manner that reflects the lamellar structure of the Haversian system. Resorption canals in mature bone are lined by osteoclasts (in cutting cones) and have their long axes oriented in the same direction as the Haversian canals. Mature spongy bone represents a meshwork of trabeculae (thin, anastomosing spicules of bone tissue). The spaces within the meshwork are continuous and, in a living bone, are occupied by bone marrow.

c.

Immature bone does not display an organized lamellated appearance. On the basis of its collagen fiber arrangement, such bone is designated . Nonlamellar bone is also referred to as or because of the interlacing arrangement of the collagen fibers. Immature bone contains relatively more cells per unit area than does mature bone. The cells in immature bone tend to be randomly arranged, whereas cells in mature bone are usually arranged with their long axes in the same direction as the lamellae. The matrix of immature bone has more ground substance than does the matrix of mature bone. The matrix in immature bone stains more intensely with hematoxylin, whereas the matrix of mature bone stains more intensely with eosin.

bundle bone

woven bone

nonlamellar

Although not evident in typical histologic sections (Fig. 8.7), immature bone is not heavily mineralized when it is initially formed, whereas mature bone undergoes prolonged secondary mineralization. The secondary mineralization of mature bone is evident in microradiographs of ground sections that show younger Haversian systems to be less mineralized than older Haversian systems (see Fig. 8.25).

FIGURE 8.7. Photomicrographs of decalcified immature and mature bone. a. Decalcified immature bone, stained with H&E, showing the relationship of cells to the extracellular matrix. The immature bone has more cells, and the matrix is not layered in osteonal arrays. ×130. b. This cross section of decalcified mature compact bone stained with H&E shows several osteons ( O ) with concentric lamellae.

The Haversian canals contain blood vessels, nerve, and connective tissue. Osteocytes undergo considerable shrinkage during routine slide preparation, revealing empty lacunae with a small nucleus attached to their walls. Mature bone has fewer osteocytes per unit area than immature bone. Note the presence of interstitial lamellae between neighboring osteons. ×160.

Immature bone

forms more rapidly than mature bone. Although mature bone is clearly the major bone type in the adult and immature bone is the major bone type in the fetus, areas of immature bone are present in adults, especially where bone is being remodeled. Areas of immature bone are common in the alveolar sockets of the adult oral cavity and where tendons insert into bones. It is this immature bone in the alveolar sockets that makes orthodontic corrections possible in adults.

CELLS OF BONE TISSUE As noted previously, five designated cell types are associated with bone tissue: osteoprogenitor cells, osteoblasts, osteocytes, bone-lining cells, and osteoclasts. With the exception of the osteoclast, each of these cells may be regarded as a differentiated form of the same basic cell type (Fig. 8.8). Each undergoes transformation from a less mature form to a more mature form in relation to functional activity (growth of bone). In contrast, the osteoclast originates from a different cell line and is responsible for bone resorption, an activity associated with bone remodeling.

FIGURE 8.8. Schematic drawing of cells associated with bone.

All cells except osteoclasts originate from the mesenchymal stem cells, which differentiate into osteoprogenitor cells, osteoblasts, and finally osteocytes and bone-lining cells. Bone-lining cells on external bone surfaces are part of the periosteum, hence the term . Bone-lining cells on internal bone surfaces are frequently called endosteal cells. Note that osteoprogenitor cells and bone-lining cells have a similar microscopic appearance and are often difficult to distinguish from each other. Osteoclasts originate from hemopoietic progenitor cells, which differentiate into bone-resorbing cells. The specific details of osteoclast differentiation are illustrated in Figure 8.15.

periosteal cells

Osteoprogenitor Cells

The osteoprogenitor cell is derived from mesenchymal stem cells. Osteogenesis, the process of new bone formation, is essential to normal bone function. It requires a population of renewable osteoprogenitor cells (osteoblast precursor cells) that are responsive to molecular stimuli that transform them into bone-forming cells. Osteoprogenitor cells are derived from mesenchymal stem cells in the bone marrow that have the potential to differentiate into many different cell types, including fibroblasts, osteoblasts, adipocytes, chondrocytes, and muscle cells. The key factor that triggers differentiation of osteoprogenitor cells is a transcription factor called , also called . This protein prompts the expression of genes that are characteristic of the phenotype of the osteoblast. IGF-1 and IGF-2 stimulate osteoprogenitor cell proliferation and differentiation

factor alpha-1 (CBFA1)

core binding runt-related transcription factor 2 (RUNX2)

into osteoblasts. As noted on page 239, BMPs also play a role in the differentiation into osteoblasts. Several clinical studies have demonstrated that assists in the healing of bone fractures by increasing bone tissue regeneration. This effect is related to increased cellular differentiation of osteoprogenitor cells after stimulation with an electromagnetic field. This approach is now being used as an effective tissue-engineering strategy to treat bone defects, accelerate fracture repair, and help vertebrae fuse after spinal fusion surgery.

stimulation (PEMF)

pulsed electromagnetic field

The osteoprogenitor cell is a resting cell that can differentiate into an osteoblast and secrete bone matrix. Osteoprogenitor cells are found on the external and internal surfaces of bones and may also reside in the microvasculature supplying bone. Morphologically, they resemble the periosteal cells that form the innermost layer of the periosteum and the endosteal cells that line the marrow cavities, the osteonal (Haversian) canals, and the perforating (Volkmann) canals. In growing bones, osteoprogenitor cells appear as flattened or squamous cells with lightly staining, elongated, or ovoid nuclei and inconspicuous acidophilic or slightly basophilic cytoplasm. Electron micrographs reveal profiles of rough-surfaced endoplasmic reticulum (rER) and free ribosomes as well as a small Golgi apparatus and other organelles.

Osteoblasts

The osteoblast is the differentiated bone-forming cell that secretes bone matrix. Like its close relatives, the fibroblast and the chondroblast, the osteoblast is a versatile secretory cell that retains the ability to divide. It secretes both type I collagen (which constitutes 90% of the protein in bone) and bone matrix proteins, which constitute the initial unmineralized bone, or osteoid. Bone matrix proteins produced by the osteoblast include calcium-binding proteins, such as osteocalcin and osteonectin; multiadhesive glycoproteins, such as bone sialoproteins (BSP-1 [osteopontin] and BSP-2); thrombospondins; various proteoglycans and their aggregates; and tissue nonspecific alkaline phosphatase (TNAP). Circulating levels of TNAP and osteocalcin are used clinically

as markers of osteoblast activity. The osteoblast is also responsible for the calcification of bone matrix. The calcification process appears to be initiated by the osteoblast through the secretion into the matrix of small, 50- to 250-nm, membrane-limited . The vesicles are actively secreted only during the period in which the cell produces the bone matrix. The structure and function of these vesicles are discussed later in this chapter (pages 259263). Osteoblasts are recognized in the light microscope by their cuboidal or polygonal shape and their aggregation into a single layer of cells lying in apposition to the forming bone (Fig. 8.9). The newly deposited matrix is not immediately calcified. It stains lightly or not at all compared with the mature mineralized matrix, which stains heavily with eosin. Because of this unique staining property of the newly formed matrix, osteoblasts appear to be separated from the bone by a light band. This band represents the , the nonmineralized matrix.

matrix vesicles

osteoid

FIGURE 8.9. Photomicrograph of a growing bone spicule stained with Mallory-Azan. Osteocytes are embedded within the bone matrix of the spicule, which is stained dark blue . These cells are

metabolically active, laying down the unmineralized bone matrix (osteoid). A number of osteoblasts are aligned on the right side of the spicule. Between these cells and the calcified bone spicule is a thin, light blue–stained layer of osteoid. This is the uncalcified matrix material produced by the osteoblasts. One of the cells ( ) has virtually surrounded itself by its osteoid product; thus, it can now be called an osteocyte. On the left side of the spicule, the nongrowing part, are inactive osteoblasts. The cells exhibit flattened nuclei and attenuated cytoplasm. ×550.

arrow

active osteoblast

The cytoplasm of an is markedly basophilic, and the Golgi apparatus, because of its size, is sometimes observed as a clear area adjacent to the nucleus. Small, periodic acid–Schiff (PAS)-positive granules are observed in the cytoplasm, and a strong alkaline phosphatase reaction associated with the cell membrane can be detected by appropriate histochemical staining. In contrast to the secreting osteoblasts found in active matrix deposition, are flat or attenuated cells that cover the bone surface. These cells resemble osteoprogenitor cells. Osteoblasts respond to mechanical stimuli to mediate the changes in bone growth and bone remodeling. As osteoid deposition occurs, the osteoblast is eventually surrounded by osteoid matrix and thereby becomes an osteocyte.

osteoblasts

inactive

Not every osteoblast is designated to become an osteocyte. Only 10% to 20% of osteoblasts differentiate into osteocytes. Most osteoblasts undergo apoptosis. Others transform into inactive cells and become either periosteal or endosteal bone-lining cells (see Fig. 8.8).

Osteoblast processes communicate with other osteoblasts and with osteocytes by gap junctions.

At the electron microscope level, osteoblasts exhibit thin cytoplasmic processes that penetrate the adjacent osteoid produced by the cell and are joined to similar processes of adjacent osteocytes by gap junctions. This early establishment of junctions between an osteoblast and adjacent osteocytes (as well as between adjacent osteoblasts) allows neighboring cells within the bone tissue to communicate. The osteoblast cytoplasm is characterized by abundant rER and free ribosomes (Fig. 8.10). These features are consistent with its basophilia observed in the light microscope as well as with its role in the production of collagen and proteoglycans for the extracellular matrix. The Golgi apparatus and surrounding regions of the cytoplasm contain numerous vesicles with a flocculent content that is presumed to consist of matrix precursors. These vesicles are the PAS-staining granules seen in light microscopy. The matrix vesicles, also produced by the osteoblast, appear to arise by an ectosomal pathway, originating as spherelike outgrowths that pinch off from the apical plasma membrane or microvilli to become free in the matrix. Other cell organelles include numerous rod-shaped mitochondria and occasional dense bodies and lysosomes.

FIGURE 8.10. Electron micrograph showing active bone formation. This electron micrograph is similar to the growing surface of the bone spicule in the preceding light micrograph (see Fig. 8.9). The marrow cavity ( M ) with its developing blood cells is seen in the lower right corner. Osteoprogenitor cells ( Opc ) are evident between the marrow and the osteoblasts ( Ob ). They exhibit elongated or ovoid nuclei. The osteoblasts are aligned along the growing portion of the bone, which is covered by a layer of osteoid ( Os ). In this same region, one of the cells ( upper right corner) embedded within the osteoid exhibits a small process ( arrow ). This cell, because of its location within the osteoid, can now be called an osteocyte ( Oc ). The remainder of the micrograph ( upper left ) is composed of calcified bone matrix ( CB ). Within the matrix are canaliculi ( C ) containing osteocyte processes. The boundary between two adjacent lamellae ( L ) of previously formed bone is evident as an irregular dark line. ×9,000.

Osteocytes

The osteocyte is the mature bone cell enclosed by bone matrix that was previously secreted as an osteoblast. When completely surrounded by osteoid or bone matrix, the osteoblast is referred to as an osteocyte (see Fig. 8.9). The process of transformation from osteoblast to osteocyte takes

approximately 3 days. During this time, the osteoblast produces a large amount of extracellular matrix (nearly 3 times its own cellular volume), reduces its cell volume by roughly 70% in comparison to the volume of the original osteoblast, decreases size and number of organelles, and develops long cell processes that radiate from its cell body. Each osteocyte develops on average about 50 cell processes. Following bone matrix mineralization, each osteocyte occupies a space, or , that conforms to the shape of the cell. Osteocytes’ cytoplasmic processes are enclosed by within the matrix (Fig. 8.11). They communicate with processes of neighboring osteocytes and bone-lining cells by means of formed by a family of bone-expressed connexins. Osteocytes also communicate indirectly with distant osteoblasts, endothelial cells of bone marrow vasculature, pericytes of blood vessels, and other cells through the expression of various signaling molecules, such as nitric oxide or glutamate transporters. In addition to typical cell-to-cell communication (gap junctions are discussed in Chapter 5, Epithelial Tissue, pages 150-151), osteocyte processes contain (the unopposed half of gap junction channels) that provide communication between cells and extracellular matrix.

lacuna

gap junctions

canaliculi

hemichannels

FIGURE 8.11. Osteocyte lacunae with extensive network of canaliculi . This scanning electron micrograph of an acid-etched, resin-embedded sample of bone from a 4-month-old mouse shows a network of canaliculi interconnecting three osteocyte lacunae ( OL ) and endosteal cells. In this method, resin

fills the osteocyte lacunae, canaliculi, osteoid, and bone marrow spaces but does not penetrate mineralized bone matrix. Phosphoric acid is usually used to remove the mineral, leaving behind a resin cast. The of the image is occupied by bone marrow cells ( ), which are separated from bone tissue by the endosteum ( ). ×2,000. (Courtesy of Dr. Lynda Bonewald.)

upper part

EOS

BM

In hematoxylin and eosin (H&E)-stained sections, the canaliculi and their processes are not discernible. However, in ground sections, the canaliculi are readily evident (Plate 8.1, page 270). Osteocytes are typically smaller than their precursor cells because of their reduced perinuclear cytoplasm. Often, in routinely prepared microscopic specimens, the cell is highly distorted by shrinkage and other artifacts that result from decalcification of the matrix prior to sectioning the bone. In such instances, the nucleus may be the only prominent feature. In well-preserved specimens, osteocytes exhibit less cytoplasmic basophilia than osteoblasts, but little additional cytoplasmic detail can be seen (Plate 8.2, page 272).

Osteocytes are metabolically active and multifunctional cells that respond to mechanical forces applied to the bone. In the past, osteocytes were considered passive cells responsible only for maintaining the bone matrix. Recent discoveries show that osteocytes are metabolically active and multifunctional cells. They are involved in the process of in which they respond to mechanical forces applied to the bone. Decreased mechanical stimuli (e.g., immobilization, muscle weakness, weightlessness in space) cause bone loss, whereas increased mechanical stimuli promote bone formation. Due to the slight flexibility of bone, mechanical forces applied to the bone (e.g., to the femur or tibia during walking) cause interstitial fluid to flow out of the canaliculi and lacunae on the compressed side of the bone. Movement of interstitial fluid through the canalicular system generates a at the moment when the stress is applied. The streaming potential opens voltage-gated calcium channels in the membranes of the osteocytes over which the tissue fluid flows. Resulting increases in intracellular calcium, adenosine triphosphate (ATP), nitric oxide concentration, and prostaglandin E2 (PGE2) synthesis alter expression of and genes responsible for bone formation. The shear stress of the fluid flow also induces the opening of that allow release of accumulated intracellular molecules into the extracellular space of the canaliculi. In addition, expression of the IGF-1 gene results in the increased production of IGF-1, which promotes conversion of osteoprogenitor cells into osteoblasts. Thus, the regions of a bone under the most stress will have the largest deposition of new bone. The osteocyte also has primary cilium that recent studies suggest may play a possible role in detecting the flow of interstitial fluid within the lacuna and may be involved in mechanosensation and molecular signaling (see Chapter 5, Epithelial Tissue, pages 135-136 for detailed information about the primary cilium’s structure and function). An osteocyte responds to reduced mechanical stress by secreting . The empty space surrounding osteocytes is the result of enzymatic degradation of bone matrix by MMP. Increased mechanical stress activates molecular mechanisms similar to those found in the matrix-producing osteoblasts. Thus, the osteocytes are responsible for reversible remodeling of their pericanalicular and perilacunar bone matrix. This process is called .

mechanotransduction

transient electrical potential (streaming potential)

hemichannels

metalloproteinases (MMPs)

c-fos

cox-2

matrix

osteocytic remodeling Osteocytes appear in different functional states during the osteocytic remodeling of their perilacunar and pericanalicular microenvironment. Electron microscopy has revealed osteocytes in various functional states related to the osteocytic remodeling process. Indeed, there is histologic and microradiographic evidence

(i.e., enlarged lacunae and reduced radiodensity) that the osteocyte can remodel the surrounding bone matrix. As mentioned earlier, osteocytes can modify their microenvironment (the volume of their lacunae or diameter of their canaliculi) in response to environmental stimuli. Because the surface area of lacunae and canaliculi inside the bone is several orders of magnitude greater than the surface area of the bone itself, removal of minute amounts of mineralized matrix by each osteocyte would have significant effects on circulating levels of calcium and phosphates. Three functional states, each with a characteristic morphology, have been identified based on the appearance of osteocytes in electron micrographs:

Quiescent osteocytes

exhibit a paucity of rER and a markedly diminished Golgi apparatus (Fig. 8.12a). An osmiophilic lamina representing mature calcified matrix is seen in close apposition to the cell membrane.

FIGURE 8.12. Electron micrographs of three different functional stages of an osteocyte. a. Relatively quiescent osteocyte that contains only a few profiles of rough endoplasmic reticulum ( rER ) and a few mitochondria ( M ). The cell virtually fills the lacuna that it occupies; the arrows indicate where cytoplasmic processes extend into canaliculi. Hydroxyapatite crystals have been lost from the matrix, which is ordinarily mineralized ( MM ), but some hydroxyapatite crystals fill the pericellular space. The hydroxyapatite crystals obscure the other substances within the pericellular space. The dark band marking the boundary of the lacuna is the osmiophilic lamina ( OL ). ×25,000. b. A formative osteocyte containing larger amounts of rER and a large Golgi apparatus ( G ). Of equal importance is the presence of a small amount of osteoid in the pericellular space within the lacuna. The osteoid shows profiles of collagen fibrils ( arrows ) not yet mineralized. The lacuna of a formative osteocyte is not bounded by an osmiophilic lamina. ×25,000. c. A resorptive osteocyte containing a substantial amount of rER, a large Golgi apparatus, mitochondria ( M ), and lysosomes ( L ). The pericellular space is devoid of collagen fibrils and may contain some flocculent material. The lacuna containing a resorptive osteocyte is bounded by a less conspicuous osmiophilic lamina ( OL ). ×25,000.

Formative osteocytes

show evidence of matrix deposition and exhibit certain characteristics similar to those of osteoblasts. Thus, the rER and Golgi apparatus are more abundant, and there is evidence of osteoid in the pericellular space within the lacuna (Fig. 8.12b). , like formative osteocytes, contain numerous profiles of endoplasmic reticulum and a well-developed Golgi apparatus. Moreover, lysosomes are conspicuous (Fig. 8.12c). The concept of degradation of bone by the resorptive osteocytes (previously called ) has fallen out of favor because it is not the dominant form of bone resorption. However, some osteocytic perilacunar and canalicular remodeling has been observed, and it may act as a backup system for calcium and phosphate ion homeostasis, especially during lactation, stress, and reproductive cycles. It is also possible that changes in the size of the lacunae and canaliculi or surrounding extracellular matrix might modulate mechanotransduction and osteoblasts’ responses to mechanical forces applied to bone.

Resorptive osteocytes

osteocytic osteolysis

Osteocytes are long-living cells and their deaths are attributed to apoptosis, degeneration/necrosis, senescence (old age), or bone remodeling activity of the osteoclasts. The natural in humans is estimated to be about . The percentage of dead osteocytes in bone increases with age, from 1% at birth to 75% in the eighth decade of life. It is hypothesized that when the age of an individual exceeds the upper limit of the life span of the osteocyte, these cells may die (senescence) and their lacunae and canaliculi may fill with mineralized tissue.

life span of osteocytes

Bone-Lining Cells

10–20 years

Bone-lining cells are derived from osteoblasts and cover bone that is not remodeling.

In sites where remodeling is not occurring, the bone surface is covered by a layer of flat cells with attenuated cytoplasm and a paucity of organelles beyond the perinuclear region (Fig. 8.13a). These cells are designated simply as . Bone-lining cells on external bone surfaces are called , and those lining internal bone surfaces are often called (see Fig. 8.8). Gap junctions are present where the bonelining cell processes contact one another (Fig. 8.13b).

endosteal cells

periosteal cells

bone-lining cells

FIGURE 8.13. Electron micrograph of bone-lining cells. a. The cytoplasm of a bone-lining cell located on the surface of a spicule of mature bone is very attenuated and contains small amounts of rough endoplasmic reticulum ( rER ) and free ribosomes. A gap junction is seen between the two adjacent

bone-lining cells. In addition, cytoplasmic processes are clearly seen where they pass through the matrix of unmineralized bone (osteoid). A fat cell of the marrow is also present. ×8,900. (Reprinted with permission from Miller SC, Bowman BM, Smith JM, et al. Characterization of endosteal bone-lining cells from fatty marrow bone sites in adult beagles. 1980;198:163–173.) Highmagnification photomicrograph of a similar bone spicule stained with H&E, included for orientation purposes. The bone-lining cells (endosteal cells) on the surface of the spicule are indicated by the . ×350. Electron micrograph of the cytoplasm of two bone-lining cells observed at higher magnification. The gap junction is clearly seen where the two cells are in apposition. The edge of a fat cell is seen at the of the electron micrograph; its lipid, thin rim of cytoplasm, plasma membrane, and external lamina are also evident. ×27,000.

Anat Rec.

arrows

b.

Inset.

top

Bone-lining cells represent a population of cells that are derived from osteoblasts. They are thought to function in the maintenance and nutritional support of the osteocytes embedded in the underlying bone matrix and regulate the movement of calcium and phosphate into and out of the bone. These suggested roles are based on the observation that the cell processes of bone-lining cells extend into the canalicular channels of the adjacent bone (see Fig. 8.13b) and communicate by means of gap junctions with osteocytic processes. In these respects, bone-lining cells are somewhat comparable to osteocytes.

Osteoclasts

The osteoclast is responsible for bone resorption.

Osteoclasts

are large, multinucleated cells found at sites where bone is being removed. They rest directly on the bone tissue where resorption is taking place (Fig. 8.14). As a result of osteoclast activity, a shallow bay called a can be observed in the bone directly under the osteoclast. The cell is conspicuous not only because of its large size but also because of its marked acidophilia. It also exhibits a strong histochemical reaction for acid phosphatase because of the numerous lysosomes that it contains. One of these enzymes, the 35-kDa iron-containing , is used clinically as a marker of osteoclast activity and differentiation.

phosphatase (TRAP)

resorption bay (Howship lacuna)

tartrate-resistant acid

FIGURE 8.14. Photomicrograph of an osteoclast on a bone spicule. This Mallory-stained specimen shows a spicule made of calcified cartilage (stained light blue ) and a covering of bone tissue (stained dark blue ). An osteoclast on the left side of the spicule has resorbed bone tissue and lies in a depression (Howship lacuna) in the spicule. The light band between the osteoclast and the bone spicule corresponds to the ruffled border of the osteoclast. The arrows on the nongrowing surface indicate cytoplasm of inactive bone-lining cells (osteoprogenitor cells). In contrast, bone is being deposited on the opposite side of the spicule, as evidenced by the presence of osteoblasts on this surface and newly formed osteocytes just below the surface of the spicule. ×550.

Osteoclasts are derived from the fusion of mononuclear hemopoietic progenitor cells under the influence of multiple cytokines. Contrary to what was once thought, osteoclasts are not related to osteoblasts. They are derived from the fusion of mononuclear hemopoietic cells, namely, granulocyte/macrophage progenitor cells (GMP, CFU-GM) that give rise to granulocyte and monocyte cell lineages

(see Fig. 10.19). Osteoclast formation occurs in close association with stromal cells in bone marrow. These cells secrete essential cytokines for differentiation of both osteoclasts and macrophages from GMP progenitor cells, including monocyte colony-stimulating factor (MCSF), tumor necrosis factor (TNF), and several interleukins. Initially, cells committed to become osteoclasts (osteoclast precursors) express two important transcription factors, and ; later, a receptor molecule called is expressed on their surface. The RANK receptor interacts with the produced and expressed on the stromal cell surface (Fig. 8.15). The is essential for osteoclast differentiation and maturation. Alternatively, during , activated T lymphocytes can produce both membrane-bound and soluble RANKL molecules. Therefore, inflammatory processes can stimulate osteoclastmediated bone resorption. This pathway can be blocked by , which serves as a “decoy” receptor for RANKL. Lack of available ligand affects the RANK–RANKL signaling pathway and acts as a potent inhibitor of osteoclast formation. OPG is produced mainly by osteoblasts and is regulated by many bone metabolic regulators, such as IL-1, TNF, TGF-β, and vitamin D. PGE2 is secreted by stressed osteocytes and stimulates the production of RANKL; however, active osteoblasts in the region of bone deposition produce OPG that inactivates RANKL. Thus, regions where osteoblasts are depositing new bone will have little or no osteoclastic activity in contrast to surrounding regions with higher osteoclastic activity. All substances that promote bone remodeling by osteoclast differentiation and bone resorption act through the OPG/RANKL system in the bone marrow. Both OPG and RANKL are detected in free form in the blood, and their concentrations can be measured for diagnostic purposes and to monitor therapy of many bone diseases.

fos NF-κB (RANK) (RANKL) molecule RANKL signaling mechanism inflammation

creceptor activator of nuclear factor-κB RANK ligand RANK– osteoprotegerin (OPG)

FIGURE 8.15. The origin of osteoclasts. Osteoclasts are derived from fusion of granulocyte/monocyte progenitor cells (GMP, CFU-GM), which originate from multipotential common myeloid progenitor cells ( CMP, CFU-GEMM ). GMP cells also give rise to the granulocyte and monocyte cell lineages such as neutrophil progenitor ( NoP, CFU-G ) and monocyte progenitor ( MoP, CFU-M ) cells. Osteoclast formation occurs in close association with stromal cells in bone marrow, which secrete monocyte colony-stimulating factor ( M-CSF ), tumor necrosis factor ( TNF ), and several interleukins ( ILs ). Osteoclast precursors express c-fos , NFκB, and receptor molecules called RANK ( receptor activator of nuclear factor-κB). The signal generated by the interaction of the RANK receptor with the RANK ligand ( RANKL ) molecule is essential for osteoclast differentiation and maturation. During inflammation, T lymphocytes produce both soluble and membrane-bound RANKL molecules, which increase bone resorption. These pathways can be blocked by osteoprotegerin ( OPG ). Note that activated T lymphocytes can stimulate formation of osteoclasts by producing both membrane-bound and soluble RANKL molecules.

Newly formed osteoclasts undergo an activation process to become bone-resorbing cells. The newly formed osteoclast must be activated to become a bone-resorbing cell. During this

process, it becomes highly polarized. When actively resorbing bone, osteoclasts exhibit three specialized regions:

ruffled border

The is the part of the cell in direct contact with bone. Its numerous deep plasma membrane infoldings form microvillous-type structures that increase the surface area available for exocytosis of hydrolytic enzymes and secretion of protons by ATPdependent proton pumps as well as endocytosis of degradation products and bone debris. The ruffled border stains less intensely than the remainder of the cell and often appears as a light band adjacent to the bone at the resorption site (see Fig. 8.14). At the electron microscopic level, hydroxyapatite crystals from the bone substance are observed between the processes of the ruffled border (Fig. 8.16). Internal to the ruffled border and in close proximity are numerous mitochondria and lysosomes. The nuclei are typically located in the part of the cell more removed from the bone surface. In this same region are profiles of rER, multiple stacks of Golgi apparatus, and many vesicles.

FIGURE 8.16. Electron micrograph of an osteoclast. This micrograph shows a segment of bone surface ( B ) and a portion of an osteoclast that is in apposition to the partially digested bone. The resorption front ( RF ) of the osteoclast possesses numerous infoldings of the plasma membrane. When viewed in the light microscope, these infoldings are evident as the ruffled border. When the plane of section is parallel to the infoldings ( asterisks ), a broad, nonspecialized expanse of cytoplasm is seen. The cytoplasm of the osteoclast contains numerous mitochondria ( M ), lysosomes, and Golgi apparatus, all of which are functionally linked with the resorption and degradation of the bone matrix. In the upper part of the figure, some collagen fibrils are evident; the arrows indicate where 68-nm cross-banding is visible. ×10,000.

clear zone

The (sealing zone) is a ring-like perimeter of cytoplasm adjacent to the ruffled border that demarcates the bone area being resorbed. Essentially, the clear zone is a compartment at the site of the ruffled border where resorption and degradation of the matrix occurs. It contains abundant actin filaments but essentially lacks other organelles. The actin filaments are arranged in a ring-like structure surrounded on both sides by actin-binding proteins such as vinculin and talin (Fig. 8.17). The plasma membrane at the site of the clear zone contains cell and that provide a tight seal between the plasma membrane and mineralized matrix of the bone. Several classes of (i.e., αvβ3 vitronectin receptor, α2β1 type I collagen receptor, or αvβ1 vitronectin/fibrinogen receptor) help maintain the seal.

molecules

extracellular matrix adhesion integrin extracellular receptors

FIGURE 8.17. Schematic drawing of an osteoclast.

This drawing shows the structure of the osteoclast and its three regions: the ruffled border, clear zone, and basolateral region. Note that the clear zone contains abundant actin filaments arranged in a ring-like structure surrounded on both sides by actin-binding proteins such as vinculin and talin. The plasma membrane at the site of the clear zone contains cell-to-extracellular matrix adhesion molecules (integrin receptors) that provide a tight seal between the plasma membrane and mineralized matrix of the bone. The pathways for proton and chloride transport are described in the text.

basolateral region

The functions in the exocytosis of digested material (see Fig. 8.17). Transport vesicles containing degraded bone material endocytosed at the ruffled border fuse here with the cell membrane to release their contents. TRAP has been found within these vesicles, suggesting its role in the fragmentation of endocytosed material.

Osteoclasts resorb bone tissue by releasing protons and lysosomal hydrolases into the constricted microenvironment of the extracellular space.

osteoclast lysosomal enzymes cathepsin K

Some, if not most, of the vesicles in the are lysosomes. Their contents are released into the extracellular space in the clefts between the cytoplasmic processes of the ruffled border, a clear example of functioning outside the cell. Once liberated, these hydrolytic enzymes, which include (a cysteine protease) and , degrade collagen and other proteins of the bone matrix. Before digestion can occur, however, the bone matrix must be decalcified through acidification of the bony surface, which initiates dissolution of the mineral matrix. The cytoplasm of the osteoclast contains , which produces carbonic acid (H2CO3) from carbon dioxide and water. Subsequently, the carbonic acid dissociates to bicarbonate (HCO3) and a proton (H+ ). With the help of , protons are transported through the ruffled border, generating a low pH (4–5) in the microenvironment of the resorption bay. This local acidic environment created in the extracellular space between the bone and the osteoclast is protected by the clear zone. coupled with facilitate the electroneutrality of the ruffled border membrane (see Fig. 8.17). Excess bicarbonate is removed by passive exchange with chloride ions via exchangers located at the basolateral membrane. The acidic environment initiates the degradation of the mineral component of bone (composed primarily of hydroxyapatites) to calcium ions, soluble inorganic phosphates, and water. When resorption of designated bone tissue is complete, osteoclasts undergo apoptosis. Recent studies indicate that many drugs used to inhibit bone resorption in osteoporosis (i.e., bisphosphonates and estrogens) promote (Folder 8.2).

matrix metalloproteinases

carbonic anhydrase II ATP-dependent proton pumps

Chloride channels

FOLDER 8.2

proton pumps chloride–carbonate protein

osteoclast apoptosis

CLINICAL CORRELATION: OSTEOPOROSIS Osteoporosis , which literally means porous bone, is the most common bone disease, affecting an estimated 75 million people in the United States, Europe, and Japan. It is characterized by progressive loss of normal bone density accompanied by the deterioration of its microarchitecture. Osteoporosis is caused by an imbalance between osteoclast-mediated bone resorption and osteoblastmediated bone deposition, resulting in decreased bone mass, enhanced bone fragility, and increased risk of fracture. In healthy individuals, osteoclast activity is primarily regulated by PTH and to a lesser degree by IL-1 and TNF. In addition, differentiation of osteoclast precursors is influenced by M-CSF and IL-6. Female hormones known as (especially estradiol) not only inhibit formation of these cytokines but also modulate the expression of RANKL, therefore limiting the activity of osteoclasts. In postmenopausal women in whom estrogen levels are reduced, secretion of these cytokines is increased, resulting in enhanced activity of osteoclasts leading to intensified bone resorption. Osteoporosis is estimated to be present in one-third of postmenopausal women and most of the elderly population. It results in more than 1.3 million fractures annually in the United States. There are three general types of osteoporosis.

estrogens

Type I primary osteoporosis occurs in postmenopausal women. Because this type appears at an earlier stage of life than type II, its long-term effect is usually more severe than osteoporosis that develops in the later years of life. Type II primary osteoporosis occurs in elderly individuals in their seventh or eighth decade of life and is the leading cause of serious morbidity and functional loss in this age group. Secondary osteoporosis develops as a result of drug therapy (i.e., corticosteroids) or disease processes that may affect bone remodeling, including malnutrition, prolonged immobilization, weightlessness (i.e., with space travel), and metabolic bone diseases (i.e., hyperparathyroidism, metastatic cancers).

Osteoporotic bone has a normal histologic structure; however, there is less tissue mass (Fig. F8.2.1), which results in weakened bones that are more prone to fractures following even minor trauma. Femoral head and neck fractures (commonly known as ), wrist fractures, and compressed vertebrae fractures are common injuries that frequently disable and confine an elderly person to a wheelchair. Individuals with fractures are at greater risk for death, not directly from the fracture, but from the complications immobilization, which included an increased risk of pneumonia, pulmonary thrombosis, and embolism.

hip fractures

FIGURE F8.2.1.

a.

Scanning electron micrograph of trabecular bone. This image shows a section from the trabecular bone obtained from a vertebral body of a healthy individual. This specimen was obtained from a vertebral body of an elderly woman showing extensive signs of osteoporosis. Compare the pattern of trabecular architecture in osteoporosis with normal vertebral bone. (Courtesy of Dr. Alan Boyd.)

b.

Treatment of individuals with osteoporosis includes an improved diet with vitamin D and calcium supplementation and moderate exercise to help slow further bone loss. In addition to diet and exercise, pharmacologic therapy directed toward slowing down bone resorption is often used. For postmenopausal women, several treatment options are available. (HRT) with estrogen and progesterone is approved for the prevention of osteoporotic fractures in postmenopausal women, but it is not considered a first-line therapy because of its association with an increased risk of cardiovascular disease, blood clots, and breast cancer. HRT is recommended for fracture prevention if a woman also has severe symptoms of menopause (such as hot flashes) or if she cannot tolerate other therapies and she does not have risk factors for or a history of cardiovascular disease or breast cancer. , such as raloxifene, bind to estrogen receptors and act as an estrogen agonist (mimicking estrogen action) in bone; in other tissues, it blocks the estrogen receptor action (acting as an estrogen antagonist). SERM therapy has the same beneficial effect as estrogen on bone tissue but does not cause the same adverse effects as estrogen in other tissues (such as increased risk of breast cancer). Other nonestrogen therapies include (i.e., alendronate or risedronate), which inhibit osteoclastic activity by inducing apoptosis of osteoclasts. Hormonal therapy in osteoporosis includes the use of (i.e., teriparatide), which has the same physiologic action on bone and kidneys as the hormone. In intermittent doses, it promotes bone formation by increasing osteoblastic activity and improving thickness of trabecular bone. Release of PTH is modified by physical exercise and depends on the intensity and duration of exercise. Short-duration, high-intensity exercise and long-duration, low-intensity exercise seem to have no impact on PTH secretion. Therapies targeting RANK, RANKL, and OPG molecules that control the development, commitment, differentiation, and function of cells in the osteoclast lineage are also available for patients with osteoporosis. Denosumab, a human , mimics the action of osteoprotegerin (OPG) which serves as a soluble decoy receptor for RANKL. Denosumab blocks RANK–RANKL signaling pathway, reduces the number of differentiating osteoclasts by inhibiting their activation and survival, thus preventing bone resorption.

Hormone replacement

therapy

Selective estrogen receptor modulators (SERMs) bisphosphonates

human parathyroid hormone recombinant

monoclonal antibody

Osteoclast function is regulated by many factors. Digested materials from the resorbed bone are transported in endocytic vesicles across the osteoclast. The content of the endocytic vesicles that originate at the ruffled border is released at the basolateral region (see Fig. 8.17), which is usually in contact with blood vessels. Therefore, numerous coated pits and coated vesicles are present at the ruffled border. Osteoclasts are observed at sites where bone remodeling is in progress. (The process of remodeling is described in more detail later in this chapter.) Thus, at sites where osteons are being altered or where bone is undergoing change during the growth process, osteoclasts are relatively numerous. secreted by the principal (chief) cells of the parathyroid glands is the most important regulator of calcium and phosphate levels in the extracellular fluid. Because osteoclasts do not have PTH receptors, PTH exerts only an indirect effect on osteoclasts. In contrast, osteocytes, osteoblasts, and T lymphocytes all have that activate adenyl cyclase, increasing intracellular levels of cyclic adenosine monophosphate (cAMP). Brief intermittent exposure to PTH increases bone mass through the cAMP/IGF-1 pathway in osteocytes and osteoblasts. However, prolonged, continuous exposure to

Parathyroid hormone (PTH)

PTH receptors

PTH increases the production of RANKL by T lymphocytes (see Fig. 8.15) and osteoblasts, leading to osteoclastic hyperactivity and eventually osteoporosis. Estrogen suppresses RANKL production by T lymphocytes. , secreted by the parafollicular cells of the thyroid gland, has the singular effect of reducing osteoclastic activity. Other molecules that play an important role in regulating osteoclast activity include cathepsin K, carbonic anhydrase II, and proteins encoding the proton pump (TCIRG1). Deficiency of these proteins causes , a rare congenital disease characterized by increased bone density and defective osteoclast function. In individuals with osteopetrosis, osteoclasts do not function properly, which causes bones to appear dense on X-ray; however, they are actually very fragile and break easily. Recent research indicates that both healthy and dying osteocytes communicate with osteoclasts to recruit them for bone remodeling. Osteocyte death through apoptosis occurring at sites of bone damage generates apoptotic bodies that express RANKL molecules. These molecules, acting through RANK–RANKL signaling pathways, increase osteoclastic activity (Table 8.1).

Calcitonin

osteopetrosis

TABLE 8.1 Summary Features of Osteoblasts, Osteocytes, and Osteoclasts Features

Osteoblast

Osteocyte

Bone surface; closing cone of resorption canals

Lacunae and canali

>5%

~95%

Deposits bone matrix; initiates mineralization by releasing matrix vesicles

Maintains bone mat regulates calciu

Cuboidal or polygonal, mononuclear cell; basophilic cytoplasm; negative Golgi

Small, oval, monon processes

Osteoprogenitor cell

Osteoblast

CBFA1 (RUNX2); IGF-1

Selection process

RANKL, PTH receptors

RANKL, PTH recepto

Weeks (~12 days)

Years (~10–20 yea

Osteocalcin; bone sialoprotein (BSP-2)

Dentin matrix prot protein); sclero (FGF-23)

 

Location Percentage of all cells in the bone Function Cell morphology Precursor cells Differentiation process/transcription factors Major hormonal/regulatory receptors Life span Biochemical markers

CBFA1, core binding factor alpha-1; GMP/CFU-GM, granulocyte/macrophage progenitor cells; IGF-1, insulin-like growth factor 1; NF-κB, nuclear factor-κB; PTH, parathyroid hormone; RANK, receptor activator of nuclear factor-κB; RANKL, RANK ligand molecule; RUNX2, runt-related transcription factor 2.

BONE FORMATION

The development of a bone is traditionally classified as endochondral or intramembranous. The distinction between endochondral and intramembranous formation rests on whether a cartilage model serves as the precursor of the bone (endochondral ossification) or whether the bone is formed by a simpler method, without the intervention of a cartilage precursor (intramembranous ossification). The bones of the extremities and those parts of the axial skeleton that bear weight (e.g., vertebrae) develop by endochondral ossification. The flat bones of the skull and face, the mandible, and the clavicle develop by intramembranous ossification. The existence of two distinct types of ossification does not imply that existing bone is either membranous bone or endochondral bone. These names refer to the mechanism by which a bone is initially formed. Because of the remodeling that occurs later, the initial bone tissue laid down by endochondral formation or by intramembranous formation is soon replaced. The replacement bone is established on the preexisting bone by appositional growth and is identical in both cases. Although the long bones are classified as being formed by endochondral formation, their continued growth involves the histogenesis of both endochondral and intramembranous bone, with the latter occurring through the activity of the periosteal (membrane) tissue.

only

Intramembranous Ossification

In intramembranous ossification, bone formation is initiated by condensation of mesenchymal cells that differentiate into osteoblasts. The first evidence of intramembranous ossification is seen around the eighth week of human development within embryonic connective tissue, the mesenchyme. Some of the spindle-shaped, pale-staining migrate and aggregate in specific areas (e.g., the region of flat bone development in the head), forming . This condensation of cells within the mesenchymal tissue initiates the process of intramembranous ossification (Fig. 8.18a). Mesenchymal cells in these ossification centers elongate and differentiate into . These cells express , which is essential for osteoblast differentiation and the expression of genes necessary for both intramembranous and endochondral ossification. The osteoprogenitor cell cytoplasm changes from eosinophilic to basophilic, and a clear Golgi area becomes evident. These cytologic changes result in the differentiated , which then secretes collagens (mainly type I collagen molecules), bone sialoproteins, osteocalcin, and other components of the bone matrix (osteoid). The osteoblasts accumulate at the periphery of the ossification center and continue to secrete osteoid at the center of the nodule. As the process continues, the osteoid undergoes mineralization, and the entrapped osteoblasts become (Fig. 8.18b). Within the bony matrix, osteocytes increasingly separate from one another as more matrix is produced, but they remain attached by thin cytoplasmic processes. With time, the matrix becomes mineralized, and the interconnecting cytoplasmic processes of osteocytes are contained within canaliculi.

mesenchymal cells

ossification centers

osteoprogenitor cells

CBFA1 transcription factor

osteoblast

osteocytes

FIGURE 8.18. Intramembranous ossification. a.

An ossification center appears in the mesenchymal connective tissue. It consists of aggregated mesenchymal-derived osteoprogenitor cells that further differentiate into bone-secreting cells, the osteoblasts. They begin to deposit unmineralized bone matrix, the osteoid. The osteoblasts accumulate at the periphery of the ossification center and continue to secrete osteoid toward the center of the nodule. As the process continues, osteoid undergoes mineralization and trapped osteoblasts become osteocytes. Osteocytes exhibit processes that communicate with each other and with osteoblasts. The newly formed tissue has a microscopic structure of immature (woven) bone with thick trabeculae lined by osteoblasts and endosteal cells. Further growth and remodeling of the bone results in replacement of woven bone by the inner and outer layers of compact bone with spongy bone between them. Spaces between trabeculae become occupied by bone marrow cells that arrive with blood vessels. Note that one space is lined by inactive endosteal cells and the other is lined by osteoblasts, osteoclasts, and endosteal cells, an indication of the active remodeling process.

b.

c.

d.

Initially, newly formed bone matrix appears in histologic sections as small, irregularly shaped spicules and trabeculae. Bone matrix appears in histologic sections as small, irregularly shaped spicules and trabeculae, which are characteristic of spongy bone. A number of the osteoprogenitor cells come into apposition with the initially formed spicules, become osteoblasts, and add more matrix (Fig. 8.19 and Plate 8.5, page 278). During this process, called , the spicules enlarge and become joined in a trabecular network with the general shape of the developing bone. Through continued mitotic activity, the osteoprogenitor cells maintain their numbers and thus provide a constant source of osteoblasts for growth of the bone spicules. The new osteoblasts, in turn, lay down bone matrix in successive layers, giving rise to (Fig. 8.18c). This immature bone, discussed on pages 242-243, is characterized internally by interconnecting spaces occupied by connective tissue and blood vessels. Further growth and remodeling result in replacement of woven bone by compact bone in the periphery and spongy bone in the center of the newly formed bone (Fig. 8.18d). Spaces between trabeculae become occupied by bone marrow cells that arrive with blood vessels. Bone tissue formed by the process just described is referred to as or .

appositional

growth

woven bone

intramembranous bone

membrane bone

FIGURE 8.19. Section of mandible developing by the process of intramembranous ossification.

This photomicrograph shows a section from a developing mandible stained with H&E. In this relatively early stage of development, the mandible consists of bone spicules of various sizes and shapes. The bone spicules interconnect with each other and form trabeculae, providing the general shape of the developing bone (no cartilage model is present). The numerous osteoblasts responsible for this growing region of spicules are seen at the surface of the newly deposited bone. The older, calcified portion of spicules contains osteocytes surrounded by bone matrix. In the of the figure, adjacent to the bone spicules, the connective tissue is very cellular and is developing into the early periosteum. ×250.

right portion

Endochondral Ossification

Endochondral ossification also begins with the proliferation and aggregation of mesenchymal cells at the site of the future bone. Under the influence of different growth factors and bone morphogenic proteins (BMPs) (see page 239), the mesenchymal cells initially express type II collagen and differentiate into chondroblasts that, in turn, produce cartilage matrix.

Initially, a hyaline cartilage model with the general shape of the bone is formed. Once established, the cartilage model (a miniature version of the future definitive

bone) grows by interstitial and appositional growth (Plate 8.3, page 274). The increase in the length of the cartilage model is attributed to interstitial growth. The increase in its width is largely the result of the addition of cartilage matrix produced by new chondrocytes that differentiate from the chondrogenic layer of the perichondrium surrounding the cartilage mass. Illustration of Figure 8.20 shows an early cartilage model.

1

FIGURE 8.20. Schematic diagram of developing long bone. Illustrations 1 to 10 depict longitudinal sections through the long bone. The process begins with the formation of a cartilage model ( 1 ); next, a periosteal (perichondrial) collar of bone forms around the diaphysis (shaft) of the cartilage model ( 2 ); next, the cartilaginous matrix in the shaft begins to calcify ( 3 ). Blood vessels and connective tissue cells then erode and invade the calcified cartilage ( 4 ), creating a primitive

marrow cavity in which remnant spicules of calcified cartilage remain at the two ends of the cavity. As a primary center of ossification develops, the endochondral bone is formed on spicules of calcified cartilage. The bone at the ends of the developing marrow cavity constitutes the metaphysis. Periosteal bone continues to form as a result of intramembranous ossification ( ); it can be recognized histologically because it is not accompanied by local cartilage erosion, or is the bone deposited on spicules of calcified cartilage. Blood vessels and perivascular cells invade the proximal epiphyseal cartilage ( ), and a secondary center of ossification is established in the proximal epiphysis ( ). A similar epiphyseal (secondary) ossification center forms at the distal end of the bone ( ), and epiphyseal cartilage is thus formed between each epiphysis and the diaphysis. With continued growth of the long bone, the distal epiphyseal cartilage disappears ( ), and finally, with cessation of growth, the proximal epiphyseal cartilage disappears ( ). The metaphysis then becomes continuous with the epiphysis. Epiphyseal lines remain where the epiphyseal plate last existed.

5

6

10

9

8

7

The first sign of ossification is the appearance of a cuff of bone around the cartilage model. At this stage, the perichondrial cells in the midregion of the cartilage model no longer give rise to chondrocytes. Instead, osteoblasts are produced. Thus, the connective tissue surrounding this portion of the cartilage is no longer functionally a perichondrium; rather, because of its altered role, it is now called the periosteum. Moreover, because the cells within this layer are differentiating into osteoblasts, an osteogenic layer can now be identified within the periosteum. Because of these changes, a layer of bone is formed around the cartilage model (Plate 8.3, page 274). This bone can be classified as either periosteal bone, because of its location, or intramembranous bone, because of its method of

bony collar 2 With the establishment of the periosteal bony collar, the chondrocytes in the midregion of the cartilage model become hypertrophic. As the chondrocytes enlarge, their surrounding cartilage matrix is resorbed, forming thin irregular cartilage plates between the hypertrophic cells. The hypertrophic cells begin to synthesize TNAP, RANKL, and vascular endothelial growth factor (VEGF); concomitantly, the surrounding cartilage matrix undergoes calcification (see illustration 3 of Fig. 8.20). The development. In the case of a long bone, a distinctive cuff of periosteal bone, the , is established around the cartilage model in the diaphyseal portion of the developing bone. The bony collar is shown in illustration of Figure 8.20.

calcification of the cartilage matrix should not be confused with mineralization that occurs in bone tissue.

The calcified cartilage matrix inhibits diffusion of nutrients, causing the degeneration, death, and possible transdifferentiation of the chondrocytes in the cartilage model.

With the degeneration, death, and possible transdifferentiation of the chondrocytes, much of the matrix breaks down, and neighboring lacunae become confluent, producing an increasingly large cavity. While these events are occurring, one or several blood vessels grow through the thin diaphyseal bony collar to vascularize the cavity (see illustration of Fig. 8.20).

4 Mesenchymal stem cells migrate into the cavity along the growing blood vessels. Mesenchymal stem cells residing in the developing periosteum migrate along the penetrating blood vessels and differentiate into osteoprogenitor cells in the bone marrow cavity. Hemopoietic stem cells (HSCs) also gain access to the cavity via the new vasculature, leaving the circulation to give rise to the marrow, which includes all blood cell progenitors. As the calcified cartilage breaks down and is partially removed, some remains as irregular spicules. When the osteoprogenitor cells come in apposition to the remaining calcified cartilage spicules, they become osteoblasts and begin to deposit bone matrix (osteoid) on the spicule framework. In addition, some osteoblasts may derive from the pool of reprogrammed hypertrophic chondrocytes that underwent chondrocyte-to-osteoblast transdifferentiation. Thus, bone formed in this manner may be described as endochondral bone. This initial site where bone begins to form in the diaphysis of a long bone is called the (see illustration of Fig. 8.20). The combination of bone, which is initially only a thin layer, and the underlying calcified cartilage is described as a . Histologically, mixed spicules can be recognized by their staining characteristics. Calcified cartilage tends to be basophilic, whereas bone is distinctly eosinophilic. With the Mallory stain, bone stains a deep blue, and calcified cartilage stains light blue (Fig. 8.21). Also, calcified cartilage no longer contains cells, whereas the newly produced bone may reveal osteocytes in the bone matrix. Such spicules persist for a short time before the calcified cartilage component is removed. The remaining bone component of the spicule may continue to grow by appositional growth, thus becoming larger and stronger, or it may undergo resorption as new spicules are formed.

primary ossification center mixed spicule

5

FIGURE 8.21. Photomicrograph of a mixed bone spicule formed during endochondral bone formation. In this Mallory-Azan–stained section, bone has been deposited on calcified cartilage spicules. In the center of the photomicrograph, the spicules have already grown to create an anastomosing trabecula. The initial trabecula still contains remnants of calcified cartilage, as shown by the light-blue staining of the calcified matrix compared with the dark-blue staining of the bone. In the upper part of the spicule, note a lone osteoclast ( arrow ) aligned near the surface of the spicule, where remodeling is about to be initiated. ×275.

Growth of Endochondral Bone

Endochondral bone growth begins in the second trimester of pregnancy and continues into early adulthood. The events described previously represent the early stage of endochondral bone formation that occurs in the fetus, beginning at about the 12th week of development. The continuing growth process that lasts into early adulthood is described in the following section.

Growth in length of long bones depends on the presence of epiphyseal cartilage. As the diaphyseal marrow cavity enlarges (see illustration 6 of Fig. 8.20), a

distinct zonation can be recognized in the cartilage at both ends of the cavity. This remaining

epiphyseal cartilage

cartilage, referred to as , exhibits distinct zones as illustrated in Figure 8.22 and Plate 8.4, page 276. During endochondral bone formation, the avascular cartilage is gradually replaced by vascularized bone tissue. This replacement is initiated by and is accompanied by expression of genes responsible for production of type X collagen and matrix metalloproteases (enzymes responsible for degradation of cartilage matrix). The , beginning with the zone most distal to the diaphyseal center of ossification and proceeding toward that center, are as follows:

vascular endothelial growth factor (VEGF)

zones in the epiphyseal cartilage

FIGURE 8.22. Longitudinal section through the diaphyseal side of the epiphyseal growth plate from a fetal metacarpal bone. Photomicrograph on the right shows active bone formation on the

diaphyseal side of the epiphyseal growth plate. The zonation is apparent in this H&E-stained specimen (×180) because chondrocytes undergo divisions, hypertrophy, and eventually apoptosis, leaving room for invading bone-forming cells. In the corresponding diagram on the , bone marrow cells have been removed, leaving osteoblasts, osteoclasts, and endosteal cells lining the internal surfaces of the bone. Note that calcified cartilage ( ) is present in the bone spicules.

blue

zone of reserve cartilage zone of proliferation

left

The exhibits no cellular proliferation or active matrix production. The is adjacent to the zone of reserve cartilage in the direction of the diaphysis. In this zone, the cartilage cells undergo divisions and organize into distinct columns. These cells are larger than those in the reserve zone and actively produce collagen (mainly types II and XI) and other cartilage matrix proteins. The contains 10- to 20-fold enlarged (hypertrophic) cartilage cells. The cytoplasm of these cells is clear, a reflection of the glycogen that they normally accumulate (which is lost during tissue preparation). Chondrocytes in this zone remain metabolically active; they continue to secrete type II collagen while increasing their secretion of type X collagen. Hypertrophic chondrocytes also secrete VEGF, which initiates

zone of hypertrophy

vascular invasion, and RANKL, which affects osteoclast development to maintain a balance between bone resorption and bone formation (see pages 250-251). The cartilage matrix is compressed to form linear bands between the columns of hypertrophied cartilage cells. In the , the hypertrophied cells begin to degenerate and the cartilage matrix becomes calcified. The calcified cartilage then serves as an initial scaffold for deposition of new bone. In the past, hypertrophic chondrocytes positioned in the more proximal part of this zone have been considered the terminal state of growth plate chondrocytes, having undergone degenerative maturation resulting in nuclear condensation and apoptosis. However, programmed cell death by apoptosis is not the only fate of hypertrophic chondrocytes. Recent research findings show that some hypertrophic chondrocytes may undergo chondrocyte-to-osteoblast transdifferentiation and directly become osteoblasts. The is the zone nearest the diaphysis. The calcified cartilage here is in direct contact with the connective tissue of the marrow cavity. In this zone, small blood vessels and accompanying osteoprogenitor cells invade the region previously occupied by the dying chondrocytes. They form a series of spearheads (see Fig. 8.22), leaving the calcified cartilage as longitudinal spicules. In cross section, the calcified cartilage resembles a honeycomb because of the absence of the cartilage cells. The invading blood vessels are the source of osteoprogenitor cells, which will differentiate into osteoblasts.

zone of calcified cartilage

zone of resorption

Bone deposition occurs on the cartilage spicules in the same manner as described for the formation of the initial ossification center. As bone is laid down on the calcified spicules, the cartilage is resorbed, ultimately leaving primary spongy bone. This spongy bone undergoes reorganization through osteoclastic activity and addition of new bone tissue, thus accommodating the continued growth and physical stresses placed on the bone. Shortly after birth, a develops in the proximal epiphysis. The cartilage cells undergo hypertrophy and degenerate. As in the diaphysis, calcification of the matrix occurs, and blood vessels and osteogenic cells from the perichondrium invade the region, creating a new marrow cavity (see illustration of Fig. 8.20). Later, a similar epiphyseal ossification center forms at the distal end of the bone (see illustration of Fig. 8.20). This center is also regarded as a secondary ossification center, although it develops later. With the development of the secondary ossification centers, the only cartilage that remains from the original model is the articular cartilage at the ends of the bone and a transverse disc of cartilage, known as the , which separates the epiphyseal and diaphyseal cavities (Plate 8.3, page 274).

secondary ossification center

7

8

epiphyseal growth plate Cartilage of the epiphyseal growth plate is responsible for maintaining the growth process.

For a bone to retain proper proportions and its unique shape, both external and internal remodeling must occur as the bone grows in length. The proliferative zone of the epiphyseal plate gives rise to the cartilage on which bone is later laid down. In reviewing the growth process, it is important to realize the following: The thickness of the epiphyseal plate remains relatively constant during growth. The amount of new cartilage produced (zone of proliferation) equals the amount resorbed (zone of resorption). The resorbed cartilage is, of course, replaced by spongy bone. Actual lengthening of the bone occurs when new cartilage matrix is produced at the epiphyseal plate. Production of new cartilage matrix pushes the epiphysis away from the diaphysis, elongating the bone. This incremental growth is followed by the same processes— namely, hypertrophy, calcification, resorption, and ossification—by which newly formed cartilage is replaced by bone tissue during fetal development.

Bone increases in width or diameter when appositional growth of new bone occurs between the cortical lamellae and the periosteum. The marrow cavity then enlarges by resorption of bone on the endosteal surface of the cortex of the bone. As bones elongate, remodeling is required. It consists of preferential resorption of bone in some areas and deposition of bone in other areas, as described previously and as outlined in Figure 8.23.

FIGURE 8.23. Diagram of external remodeling of a long bone. This diagram shows two periods during the growth of the bone. The younger bone profile (before remodeling) is shown on the left ; the older (after remodeling) on the right . Superimposed on the right side of the figure is the shape of the bone ( right half only) as it appeared at an earlier time. The bone is now longer, but it has retained its general shape. To grow in length and retain the general shape of the particular bone, bone resorption occurs on some surfaces, and bone deposition occurs on other surfaces, as indicated in the diagram. (Based on Ham AW, Some histophysiological problems peculiar to calcified tissues. 1952;24A:701–728.)

J Bone

Joint Surg Am.

When an individual achieves maximal growth, proliferation of new cartilage within the epiphyseal plate terminates. When the proliferation of new cartilage ceases, the cartilage that has already been produced in the epiphyseal plate continues to undergo the changes that lead to the deposition of new bone until, finally, there is no remaining cartilage. At this point, the epiphyseal and diaphyseal marrow cavities become confluent. The elimination of the epiphyseal plate is referred to as . In illustration of Figure 8.20, the lower epiphyseal

epiphyseal closure

9

10

cartilage is no longer present; in illustration , both epiphyseal cartilages are gone. Growth is now complete, and the only remaining cartilage is found on the articular surfaces of the bone. Vestigial evidence of the site of the epiphyseal plate is reflected by an consisting of bone tissue (see Fig. 8.2).

epiphyseal line

Development of the Osteonal (Haversian) System

Osteons typically develop in preexisting compact bone. Compact bone can take several different forms. Compact bone may be formed from fetal spongy bone by continued deposition of bone on the spongy bone spicules; it may be deposited directly as adult compact bone (e.g., the circumferential lamellae of an adult bone); or it might be older compact bone consisting of osteons and interstitial lamellae. The process in which new osteons are formed is referred to as .

internal remodeling During the development of new osteons, osteoclasts bore a tunnel, the resorption cavity, through compact bone. Formation of new osteons in compact bone initially involves the creation of a tunnel-like space, the resorption cavity, by osteoclast activity. This resorption cavity will have the dimensions of the new osteon. When osteoclasts have produced an appropriately sized cylindrical tunnel by resorption of compact bone, blood vessels and their surrounding connective tissue occupy the tunnel. As the tunnel is occupied, new bone deposition on its wall begins almost immediately. These two aspects of cellular activity—namely, osteoclast resorption and osteoblast synthesis—constitute a unit. A bone-remodeling unit consists of two distinct parts: an advancing (also called a ) and a (Fig. 8.24).

canal

bone-remodeling cutting cone

closing cone

resorption

FIGURE 8.24. Diagram of a bone-remodeling unit.

A bone-remodeling unit consists of an advancing cutting cone and a closing cone. The cutting cone formed by osteoclasts is responsible for boring the tunnel or resorption cavity through the compact bone. Its action is initiated within the compact bone at the of the diagram (in the area corresponding to section ). The cutting cone moves along osteons, in the direction indicated by the , to the area corresponding to section . Section shows the cross section through the cutting cone lined by osteoclasts ( ). The resorption cavity is the site where the future osteon is formed by the action of the closing cone, which consists of osteoblasts ( ). These cells begin to deposit the osteoid on the walls of the canal in

far left

arrow

purple cells

a

green cells

d

d

successive lamellae. Gradual formation of the new bone fills the resorption cavity. Note the deposition of the osteoid deep to the osteoblasts seen in sections and and, in sections and , the presence of the mineralized bone. As successive lamellae of bone are deposited, the canal ultimately attains the relatively narrow diameter of the mature Haversian canal lined by the endosteal cells ( ), like those shown in section . The growth-reversal line that appears at the outer limits of a newly formed osteon represents a border between the resorption activity of the cutting cone and the bony matrix not remodeled by this activity.

b

pink cells

c

a

b

a

The tip of the cutting cone consists of advancing osteoclasts closely followed by an advancing capillary loop and pericytes. It also contains numerous dividing cells that give rise to osteoblasts, additional pericytes, and endothelial cells. (Recall that osteoclasts are derived from mononuclear hemopoietic progenitor cells.) The osteoclasts drill a canal about 200 μm in diameter. This canal establishes the diameter of the future osteonal (Haversian) system. The cutting cone constitutes only a small fraction of the length of the bone-remodeling unit; thus, it is seen much less frequently than the closing cone. After the diameter of the future Haversian system is established, osteoblasts begin to fill the canal by depositing the organic matrix of bone (osteoid) on its walls in successive lamellae. This process is depicted in bone section in Figure 8.25 as the result of sequential injections of different fluorescent dyes into experimental animals to examine rate of bone remodeling. With time, the bone matrix in each of the lamellae becomes mineralized. As the successive lamellae of bone are deposited, , the canal ultimately attains the relatively narrow diameter of the adult osteonal canal (see Fig. 8.25).

from the periphery inward

FIGURE 8.25. Osteon formation during bone remodeling.

Bone remodeling was examined in an experimental animal (rat) by daily intraperitoneal injections of three different fluorescent dyes (calcein, xylenol orange, and alizarin complexone) every 9 days for 27 days. These stains have a high affinity for calcium ions and are incorporated into the bone matrix during the mineralization process. The bone tissue was fixed in paraformaldehyde solution and sectioned using a precision saw. To limit light scattering and absorption, the tissue was embedded in the optical clearing reagent 2,2 thiodiethanol (TDE). Each fluorescent marker has a different emission wavelength, as shown in panels a, b, and c. The overlay image of the entire newly formed osteon is shown in panel d. This sequence of fluorescent labeling shows the centripetal (directed toward the center) formation of the osteon from the periphery toward the osteonal canal. a. For the first 9 days, calcein was injected. This image shows green fluorescence (light emission between 497 and 557 nm) of the newly deposited bone matrix at the periphery of the resorption cavity (closing cone). b. Following calcein treatment, xylenol orange was injected for the second 9-day period. The orange color indicates fluorescence of the second wave of bone deposition that narrows the original resorption cavity. This image shows fluorescent light emission between 545 and 625 nm. c. Alizarin complexone was injected during the third 9-day period to label the newly calcified matrix. This image shows the layer of bone matrix deposition ( ; light emission between 610 and 690 nm) that outlines the boundary of the osteonal (Haversian) canal in the center of the newly formed osteon. d. This image shows an overlay of all three fluorescent zones extending from the earliest deposited outer (peripheral) lamellae of the osteon ( ) through the middle lamellae ( ) and to the most recently deposited inner lamellae ( ) near the osteonal canal. ×380. (Courtesy of Dr. Barıs¸ Baykal, University of Health Sciences, Gülhane Faculty of Medicine, Ankara, Türkiye).

′

red

orange label

green label red label

Compact adult bone contains Haversian systems of varying age and size. Microradiographic examination of a ground section of bone reveals that younger Haversian systems are less completely mineralized than older systems (Fig. 8.26). They undergo a progressive secondary mineralization that continues (up to a point) even after the osteon has been fully formed. Figure 8.26 also illustrates the dynamic internal remodeling of compact bone. In the adult, deposition balances resorption. In an older person, resorption

often exceeds deposition. If this imbalance becomes excessive, Folder 8.2).

osteoporosis

develops (see

FIGURE 8.26. Microradiograph of the cross section of a bone.

This 200-μm-thick cross section of bone from a healthy 19-year-old male shows various degrees of mineralization in different osteons. Mature compact bone is actively replacing immature bone, which is seen on the periosteal ( ) surface. The degree of mineralization is reflected by the shade of light and dark in the microradiograph. Thus, represent the highly mineralized tissue that deflects the Xrays and prevents them from striking the photographic film. Conversely, contain less mineral and, thus, are less effective in deflecting the X-rays. Note that the interstitial lamellae (the older bone) are very light, whereas some of the osteons are very dark (these are the most newly formed). The Haversian canals appear , as they represent only soft tissue. ×157. (Courtesy of Dr. Jenifer Jowsey.)

very light areas

upper

dark areas

black

BIOLOGIC MINERALIZATION AND MATRIX VESICLES

Biologic mineralization is a cell-regulated extracellular event under the control of osteoblasts.

osteoid mineralization

The organic matrix of bone, the , is produced by bone-forming cells: osteoblasts in bone and ameloblasts and odontoblasts in developing teeth. Once osteoid is laid down, osteoblasts initiate the process, which occurs in the of bone, cartilage, and in the dentin, cementum, and enamel of teeth. Except for enamel, the matrices of these structures contain collagen fibrils and ground substance. At the same time, mineralization is initiated within the collagen fibrils and in the ground substance surrounding them. In enamel, mineralization occurs within the extracellular matrix secreted by the enamel organ. synthesize the majority of extracellular matrix components and control the mineralization process by secreting such as osteocalcin, bone sialoproteins, and osteoadherin. Osteoblasts also modulate the local concentration of phosphate ions by regulating the activity of , which hydrolyzes phosphate groups from a variety of physiologic substrates. Despite the extracellular location of biologic mineralization, this process is controlled by osteoblasts and is regulated by membrane transporters, enzymes, and surrounding extracellular matrix proteins.

Osteoblasts

extracellular matrix

regulatory proteins tissue nonspecific alkaline phosphatase

(TNAP)

Local accumulation of Ca2+ and PO4 ions in the extracellular matrix is essential for initiation of mineralization.

In places where the mineralization of bone, cartilage, dentin, and cementum is initiated, the local concentration of Ca2+ and PO4 ions in the matrix must exceed the normal threshold level. Several events are responsible for this increase. The binding of extracellular Ca2+ by and other bone sialoproteins creates a high local concentration of this ion. In turn, the high Ca2+ concentration stimulates the osteoblasts to secrete , which increases the local concentration of PO4 ions. The high PO4 ions concentration stimulates further increases in Ca2+ concentration in areas where mineralization will be initiated.

osteocalcin

TNAP

Formation of hydroxyapatite crystals is initiated in the lumen of matrix vesicle and next spreads into extracellular matrix.

In areas with a high extracellular Ca2+ and PO4 ions concentration, osteoblasts begin the process by releasing small (50- to 200-nm) . These matrix vesicles represent that are released from the apical plasma membrane or microvilli of the osteoblast near the osteoblast–osteoid interface. The plasma membrane of matrix vesicles contains several membrane transporters and enzymes, and their lumen provides a nurturing microenvironment for calcium phosphate nucleation and subsequent crystal growth. Matrix vesicles contain , , carbonic anhydrases, and pyrophosphatases. TNAP and annexin A5 expressed on the surface of the matrix vesicle bind to type I collagen, which anchors the vesicle to the extracellular matrix. Annexin A5 is a channel protein for Ca2+ entry into the matrix vesicles. Influx of Ca2+ into the matrix vesicle is accompanied by + simultaneous transport of PO4 ions via the . The initial takes place within the matrix vesicles (Fig. 8.27). In this phase, the following events occur:

mineralization

ectosomes

matrix vesicles

annexins TNAPs

Na -phosphate cotransporter 3 (NPT3) matrix vesicle–mediated mineralization phase

FIGURE 8.27. Schematic representation of the mineralization processes and the role of matrix vesicles. Matrix vesicles are released from the osteoblast at the osteoblast–osteoid interface. The plasma membrane of matrix vesicles contains several proteins, including Ca channels (annexins), Na+– phosphate cotransporters ( NPT3 ), and tissue nonspecific alkaline phosphatases ( TNAP ). TNAP increases 2+

the extracellular concentration of PO 4 ions, which are transported into the vesicle via NPT3 cotransporters. Annexin A5 allows for influx of Ca 2+. In the matrix vesicle–mediated mineralization phase, both Ca 2+ and PO 4 ions accumulate in the vesicle lumen and initiate a stepwise process of hydroxyapatite crystals formation. Hydroxyapatite crystals emerge from the lumen into the extracellular matrix through holes punched in the vesicle membrane by phospholipases. Next, the collagen mineralization phase takes place outside the matrix vesicle, which results in the organization of hydroxyapatite crystals into mineralized nodules. High concentrations of Ca 2+, PO 4 ions, and Ca 2+-binding proteins in the extracellular matrix provide a favorable environment for hydroxyapatite crystals growth. They rapidly expand into spaces between collagen fibrils until they join neighboring crystals produced by other mineralized nodules. In this way, a wave of mineralization sweeps through the osteoid. , bone sialoprotein.

BSP

matrix vesicles amorphous calcium phosphate

The accumulate Ca2+ and PO4 ions that cause the local isoelectric point to increase, which results in formation of small (10 nm), noncrystalline spheroidal particles of [Ca9(HPO4)(PO4)5(OH)], also called calcium-deficient hydroxyapatite. Amorphous calcium phosphate undergoes further crystallization to [Ca8H2(PO4)6 · 5H2O]. In the presence of a high concentration of Ca2+ and PO4 ions, the octacalcium phosphate crystal grows within the matrix vesicle into insoluble, needle-like [Ca10(PO4)6(OH)2]. Hydroxyapatite crystals accumulate within the matrix vesicle. Phospholipases punch holes in the plasma membrane of the matrix vesicles through which elongating hydroxyapatite crystals exit and begin to emerge from the lumen into the surrounding extracellular matrix.

octacalcium phosphate

hydroxyapatite

crystals

collagen mineralization phase that takes place outside matrix vesicle, the mineralized nodules (Fig. 8.28). In this phase, the following

In the second hydroxyapatite crystals form events occur:

FIGURE 8.28. Electron micrograph of osteoid with mineralized nodules. This micrograph shows several mineralized nodules ( MN ) in different stages of formation surrounded by collagen fibrils in

the collagen mineralization phase of the osteoid. Mineralized nodules are formed by hydroxyapatite crystals that exited the lumen of matrix vesicles and continue to grow in size between collagen fibrils. Note that mineralized nodules have multiple connections with collagen fibrils ( ), which are remnants of the collagen binding sites of proteins (i.e., tissue nonspecific alkaline phosphatases [ ] and annexin A5) expressed on the surface of matrix vesicles. × 30,000. (Reprinted with permission from Amizuka N, Li M, Kobayashi M, et al. Vitamin K2, a gamma-carboxylating factor of gla-proteins, normalizes the bone crystal nucleation impaired by Mg-insufficiency. 2008;23:1353–1366.)

arrowheads

TNAP

Histol Histopathol.

High levels of Ca2+ , PO4 ions, and Ca2+ -binding proteins (including osteopontin [BSP-1], osteocalcin, and osteonectin) in the extracellular matrix provide a favorable environment for continuing . The hydroxyapatite crystals grow rapidly between collagen fibrils and proteoglycan ground substance molecules until they join neighboring crystals produced by other mineralized nodules. In this way, a sweeps through the osteoid. Complete rupture and occur later in this phase.

nucleation of hydroxyapatite crystals

wave of mineralization disintegration of matrix vesicles

BONE AS A TARGET OF ENDOCRINE HORMONES AND AS AN ENDOCRINE ORGAN

Bone serves as a reservoir for body calcium.

The maintenance of normal blood calcium levels is critical to health and life. Because bone serves as a reservoir for body calcium, its release and retrieval from the blood is closely monitored by endocrine hormones such as parathyroid hormone and calcitonin. Calcium may be delivered from the bone matrix to the blood if the circulating blood levels of calcium fall below a critical point (physiologic calcium concentration ranges from 8.9 to 10.1 mg/dL). Conversely, excess blood calcium may be removed from the blood and stored in bone. These processes are regulated by , secreted by the principal (chief) cells of the parathyroid glands, and , secreted by the parafollicular cells of the thyroid gland (Folder 8.4).

parathyroid hormone (PTH) calcitonin

PTH acts on the bone to increase low blood calcium levels to normal. PTH release results in the rapid mobilization of Ca from bone. Calcitonin acts on the bone to decrease elevated blood calcium levels to normal. PTH regulates the distribution of total body Ca . It stimulates both osteocytes and 2+

2+

osteoclasts (indirectly via RANK–RANKL signaling pathways because osteoclasts do not have PTH receptors) to resorb bone, thereby releasing calcium into the blood. As described previously (see pages 247-248), resorption of bone by osteocytes occurs during osteocytic

remodeling. PTH also reduces excretion of calcium by the kidney and stimulates absorption of calcium by the small intestine. PTH further acts to maintain homeostasis by stimulating the kidney to excrete the excess phosphate produced by bone resorption. inhibits bone resorption, specifically inhibiting the effects of PTH on osteoclasts. It is highly active in young individuals but decreases in activity as individuals age. The mechanisms by which PTH helps regulate serum calcium levels and bone resorption are more complex. PTH has an (it increases bone formation) as well as a (it causes bone resorption). Clinical trials in which PTH was administered in intermittent subcutaneous doses to postmenopausal women with osteoporosis have shown significant increases in bone formation and bone mineral density. Increases in the density of spongy (cancellous) bone due to PTH treatment were shown in the ilium, vertebral bodies, and the shafts of radial and femoral bones (see Folder 8.2). The possible mechanisms behind this counterintuitive anabolic action of PTH are most likely related to its dosing. Brief or intermittent treatment with PTH is anabolic; it stimulates bone deposition through cAMP/IGF1 pathways in osteocytes and osteoblasts. Conversely, prolonged and continuous treatment is catabolic; it increases production of RANKL molecules by osteoblasts and T lymphocytes, leading to activation of osteoclasts and bone resorption.

Calcitonin

catabolic action

anabolic action

Bone cells produce endocrine hormones that are involved in regulating phosphate and glucose metabolism.

Several newly discovered hormones produced by osteoblasts and osteocytes contribute to mineral and nutrient homeostasis. These hormones include the following:

Fibroblast growth factor 23 (FGF-23),

which is produced by osteocytes, regulates serum phosphate levels by altering the levels of active vitamin D and the activity of specific phosphate transporters in the kidney. FGF-23 is an important factor in aiding PTH in the disposal of excess phosphate released from hydroxyapatites during bone resorption. , which is produced by osteoblasts, is linked to a pathway regulating energy and glucose metabolism. It targets adipocytes and insulin-producing cells in the pancreas. In addition, osteocalcin has been shown to induce testosterone production in Leydig cells of the testes.

Osteocalcin

Both FGF-23 and osteocalcin function as classic endocrine hormones; that is, they are produced exclusively in bone tissue and act on distant target organs through a regulatory feedback mechanism. Understanding the endocrine role of bone tissue will improve diagnosis and management of osteoporosis, diabetes mellitus, and other metabolic disorders.

BIOLOGY OF BONE REPAIR

Bone can repair itself after injury either by a direct (primary) or indirect (secondary) bone healing process. Repair of bone fracture occurs by two processes: direct or indirect bone healing. Direct (primary) bone healing occurs when the fractured bone is surgically stabilized with

compression plates, thereby completely restricting movement between fractured fragments of bone. In this process, bone undergoes internal remodeling similar to that of mature bone. The cutting cones formed by the osteoclasts cross the fracture line and generate longitudinal resorption canals that are later filled by bone-producing osteoblasts residing in the closing cones (see pages 259-261 for details). This process results in the simultaneous generation of a bony union and the restoration of Haversian systems. involves responses from periosteum and surrounding soft tissues as well as endochondral and intramembranous bone formation. This type of bone repair occurs in fractures that are treated with nonrigid or semirigid bone fixation (i.e., treatment with casts, fracture braces, external fixation, intramedullary nailing, or

Indirect (secondary) bone healing

application of metal plates over the fracture gap). The major stages of indirect bone healing are shown in Figure 8.29.

FIGURE 8.29. Bone fracture and stages of bone healing process. a. View of healthy bone before fracture. b. The initial response to the injury produces a fracture hematoma that surrounds the ends

of the fractured bone. The ends of bone fragments undergo necrosis. An acute inflammatory reaction develops and is manifested by infiltration of neutrophils and macrophages, activation of fibroblasts, and proliferation of capillaries. The fracture hematoma is gradually replaced by granulation tissue. Fibrocartilage matrix is deposited. Newly formed fibrocartilage fills the gap at the fracture site producing a soft callus. This stabilizes and binds together the fractured ends of the bone. The osteoprogenitor cells from the periosteum differentiate into osteoblasts and begin to deposit new bone on the outer surface of the callus (intramembranous process) until new bone forms a bony sheath over the fibrocartilaginous soft callus. The cartilage in the soft callus calcifies and is gradually replaced by bone as in endochondral ossification. Newly deposited woven bone forms a bony hard callus. Bone remodeling of the hard callus transforms woven bone into the lamellar mature structure with a central bone marrow cavity. The hard callus is gradually replaced by compact bone through the action of osteoclasts and osteoblasts, which restores bone to its original shape.

c.

d.

e.

Bone fracture initiates an acute inflammatory response that is necessary for bone healing. The initial response to bone fracture is similar to the response to any injury that produces tissue destruction and hemorrhage. Initially, a fracture hematoma (a collection of blood that surrounds the fracture ends of the bones) is formed (Fig. 8.29b), and bone necrosis is seen at the ends of the fractured bone fragments. Injury to nearby soft tissues and degranulation of platelets from the blood clot secrete cytokines (e.g., TNF-α, IL-1, IL-6, IL-11, IL-18) and initiate an acute inflammatory response. This process is reflected by the infiltration of neutrophils followed by the migration of macrophages. Fibroblasts and capillaries subsequently proliferate and grow into the site of the injury. Also, specific mesenchymal stem cells arrive at the site of injury from the surrounding soft tissues and bone marrow. The fracture hematoma, which initially contained entrapped erythrocytes within a network of fibrin, is gradually replaced by , a type of newly formed loose connective tissue containing collagen type III and type II fibers. Both fibroblasts and periosteal cells participate in this phase of healing.

granulation tissue

FOLDER 8.3

CLINICAL CORRELATION: NUTRITIONAL FACTORS IN BONE FORMATION Both nutritional and hormonal factors affect the degree of bone mineralization. Calcium deficiency during growth causes rickets, a condition in which the bone matrix does not calcify normally. Rickets may be caused by insufficient amounts of dietary calcium or insufficient vitamin D (a steroid prohormone), which is needed for absorption of calcium by the intestines. An X-ray of a child with advanced rickets presents classic radiologic symptoms: bowed lower limbs (outward curve of long bones of the leg and thighs) and a deformed chest and skull (often having a distinctive “square” appearance). If rickets is not treated while a child is still growing, skeletal deformities and short stature may be permanent. In adults, the same nutritional or vitamin deficiency leads to . Although rickets and osteomalacia are no longer major health problems in populations where nutrition is adequate, they are among the most frequent childhood diseases in many developing countries. In addition to its influence on intestinal absorption of calcium, vitamin D is needed for normal calcification. Other vitamins known to affect bone are vitamins A and C. Vitamin A deficiency suppresses endochondral growth of bone; vitamin A excess leads to fragility and subsequent fractures of long bones. Vitamin C is essential for synthesis of collagen, and its deficiency leads to . The matrix produced in scurvy is unable to calcify. Another form of insufficient bone mineralization often seen in postmenopausal women is the condition known as (see Folder 8.2).

osteomalacia

scurvy

osteoporosis

Granulation tissue transforms into a fibrocartilaginous soft callus, which gives the fracture site a stable, semirigid structure. As the granulation tissue becomes denser, chondroblasts differentiate from the skeletal stem cells residing in periosteal lining. The newly produced cartilage matrix invades the periphery of granulation tissue. The dense connective tissue and newly formed cartilage grows and covers the bone at the fracture site, producing a (Fig. 8.29c). This callus will form even if the fractured parts are not in immediate apposition to each other, and it helps stabilize and bind together the fractured bone (Fig. 8.30).

soft callus

FIGURE 8.30.

a.

Photomicrograph of fractured long bone undergoing repair. This low-magnification photomicrograph of a 3-week-old bone fracture, stained with H&E, shows parts of the bone separated from each other by the fibrocartilage of the soft callus. At this stage, the cartilage undergoes endochondral ossification. In addition, the osteoblasts of the periosteum are involved in secretion of new bony matrix on the outer surface of the callus. On the of the microphotograph, the soft callus is covered by periosteum, which also serves as the attachment site for the skeletal muscle. ×35. Higher magnification of the callus from the area indicated by the in panel shows osteoblasts lining bone trabeculae. Most of the original fibrous and cartilaginous matrix at

b.

right

upper rectangle

a

this site has been replaced by bone. The early bone is deposited as an immature bone, which is later replaced by mature compact bone. ×300. Higher magnification of the callus from the area indicated by the in panel . A fragment of old bone pulled away from the fracture site by the periosteum is now adjacent to the cartilage. It will be removed by osteoclast activity. The cartilage will calcify and be replaced by new bone spicules as seen in panel . ×300.

lower rectangle

a

c.

b

A bony callus replaces fibrocartilage at the fracture site and allows for weight bearing. While the callus is forming, osteoprogenitor cells of the periosteum divide and differentiate into osteoblasts. The newly formed osteoblasts begin to deposit osteoid on the outer surface of the callus (intramembranous process) at a distance from the fracture. This new bone formation progresses toward the fracture site until new bone forms a bony sheath over the fibrocartilaginous callus. Osteogenic buds from the new bone invade the callus and begin to deposit bone within the callus, gradually replacing the original fibrous and cartilaginous callus with a (Fig. 8.29d). In addition, endosteal proliferation and differentiation occur in the marrow cavity, and bone grows from both ends of the fracture toward the center. When this bone unites, the bony union of the fractured bone, produced by the osteoblasts and derived from both the periosteum and endosteum, consists of spongy bone. As in normal endochondral bone formation, the spongy bone is gradually replaced by compact bone. The hard callus becomes more solid and mechanically rigid.

hard callus

The remodeling process restores the original shape of the bone.

Although the hard callus is a rigid structure providing mechanical stability to the fracture site, it does not fully restore the properties of normal bone. Bone remodeling of the hard callus must occur in order to transform the newly deposited woven bone into a lamellar mature bone. Subsequently, the bone marrow cavity must be restored. While compact bone is being formed, remnants of the hard callus are removed by the action of osteoclasts, and gradual restores the bone to its original shape (Fig. 8.29e).

bone remodeling

FOLDER 8.4

FUNCTIONAL CONSIDERATIONS: HORMONAL REGULATION OF BONE GROWTH Hormones other than PTH and calcitonin have major effects on bone growth. One such hormone is pituitary growth hormone (GH, somatotropin). This hormone stimulates growth in general and, especially, growth of epiphyseal cartilage and bone. It acts directly on osteoprogenitor cells, stimulating them to divide and differentiate. Chondrocytes in epiphyseal growth plates are regulated by insulin-like growth factor I (IGF-1), which is primarily produced by the liver in response to GH. In addition to IGF-1, insulin and thyroid hormones stimulate chondrocyte activity. Oversecretion in childhood, caused by a defect in the mechanism regulating GH secretion or a GHsecreting tumor in the pituitary gland, leads to , an abnormal increase in the length of bones. Absence or hyposecretion of GH in childhood leads to failure of growth of the long bones, resulting in . Absence or severe hyposecretion of thyroid hormone during development and infancy leads to failure of bone growth and dwarfism, a condition known as . When oversecretion of GH occurs in an adult, bones do not grow in length as a result of epiphyseal closure. Instead, abnormal thickening and selective overgrowth of hands, feet, mandible, nose, and intramembranous bones of the skull occurs. This condition, known as , is caused by increased activity of osteoblasts on bone surfaces.

pituitary dwarfism congenital hypothyroidism acromegaly In healthy individuals, bone healing

gigantism

usually takes 6–12 weeks, depending on the severity of the break and the specific bone that is fractured. The inflammatory process lasts approximately 1 week. It is typically accompanied by pain and swelling, and it leads to granulation tissue formation. The soft callus is formed approximately 2–3 weeks after fracture. The hard callus in which the fractured fragments are firmly united by new bone requires 3–4 months to develop. The process of bone remodeling may last from a few months to several years until the bone has completely returned to its original shape. Setting the bone (i.e., reapproximating the normal anatomic configuration) and holding the parts in place by internal fixation (by pins, screws, or plates) or by external fixation (by casts or by pins and screws) expedites the healing process.

BONE

OVERVIEW OF BONE

Bone is a specialized type of connective tissue characterized by a mineralized extracellular matrix that stores calcium and phosphate. Bone contributes to the skeleton , which supports the body, protects vital structures, provides the mechanical basis for body movement, and harbors bone marrow.

GENERAL STRUCTURE OF BONES

Bones are classified according to shape into long, short, flat, or irregular bones. Long bones are tubular in shape and consist of two ends (proximal and distal epiphyses ) and a long shaft (diaphysis ). Metaphysis is the junction between the diaphysis and the epiphysis. Bone is covered by periosteum , a connective tissue membrane that attaches to the outer surface by Sharpey fibers . Periosteum contains a layer of osteoprogenitor (periosteal) cells that can differentiate into osteoblasts. Bone cavities are lined by endosteum , a single layer of cells that contains osteoprogenitor (endosteal) cells, osteoblasts, and osteoclasts. Bones articulate with neighboring bones by synovial joints , a movable connection. The articular surfaces that form contact areas between two bones are covered by hyaline (articular) cartilage .

GENERAL STRUCTURE OF BONE TISSUE

Bone tissue formed during development is called immature (woven) bone . It from mature (lamellar) bone in its collagen fiber arrangement. Bone tissue is classified as either compact (dense) or spongy (cancellous).

differs

Compact bone lies outside and beneath the periosteum, whereas an internal, sponge-like meshwork of trabeculae forms spongy bone. is mostly composed of . These concentric lamellar structures are organized around an that contains the vascular and nerve supply of the osteon. are perpendicularly arranged and connect osteonal canals to one another. The between concentric lamellae contain that are interconnected with other osteocytes and the osteonal canal via .

Mature (lamellar) bone canals lacunae

osteons (Haversian systems) osteonal (Haversian) canal Perforating (Volkmann) osteocytes canaliculi

CELLS AND EXTRACELLULAR MATRIX Osteoprogenitor cells

derive from mesenchymal stem cells in the bone marrow. They differentiate under the influence of the core binding factor alpha-1 (CBFA1) transcription factor (RUNX2) into osteoblasts. differentiate from osteoprogenitor cells and secrete , an unmineralized bone matrix that undergoes mineralization triggered by matrix vesicles. are mature bone cells enclosed within of bone matrix. They communicate with other osteocytes by a network of long cell processes occupying

Osteoblasts Osteocytes

osteoid

lacunae

canaliculi , and they respond to mechanical forces applied to the bone. Osteoclasts differentiate from hemopoietic progenitor cells; they resorb bone matrix during bone formation and remodeling. They differentiate and mature under the control of the RANK–RANKL signaling mechanism . Bone matrix contains mainly type I collagen along with other noncollagenous proteins and regulatory proteins.

BONE FORMATION

endochondral intramembranous ossification

The development of bone is classified as (a cartilage model serves as the precursor of the bone) or (without involvement of a cartilage precursor). Flat bones of the skull, mandible, and clavicle develop by ; all other bones develop by endochondral ossification. In , the is formed. Next, osteoprogenitor cells surrounding this model differentiate into bone-forming cells that initially deposit bone on the cartilage surface (periosteal ) and later penetrate the diaphysis to form the . develop later within the epiphyses. Primary and secondary ossification centers are separated by the , seen in children and adolescents, that provides a source for new cartilage involved in bone growth. The has several zones (reserve cartilage, proliferation, hypertrophy, calcified cartilage, and resorption). Resorbed calcified cartilage is replaced by bone.

intramembranous

ossification endochondral ossification

hyaline cartilage model

bony collar primary ossification center epiphyseal growth

Secondary ossification centers plate epiphyseal growth plate

BONE GROWTH, REMODELING, AND REPAIR

the interstitial growth of cartilage appositional growth bone-remodeling units

Elongation of endochondral bone depends on on the epiphyseal growth plate. Bone increases in width (diameter) by of new bone that occurs between the compact bone and the periosteum. Bone is constantly being remodeled throughout life by composed of osteoclasts and osteoblasts. This process allows bone to change shape in response to mechanical load. Bone can repair itself after injury either by a or bone healing process. After injury, periosteal cells become activated to produce a , which is subsequently replaced by a .

direct (primary) indirect soft (fibrocartilage) hard (bony) callus

(secondary) callus

PHYSIOLOGIC ASPECTS OF BONE

reservoir for Ca

2+ in the body. Ca2+ may be removed from bone if the Bone serves as a circulating level of Ca2+ in the blood falls below the critical value. Likewise, excess Ca2+ may be removed from the blood and stored in bone. Maintenance of blood Ca2+ levels is regulated by , secreted by the parathyroid glands, and , secreted by the thyroid gland. stimulates both osteocytes and osteoclasts (indirectly via RANK–RANKL signaling pathways because osteoclasts do not have PTH receptors) to resorb bone, thereby increasing Ca2+ levels in the blood.

PTH

calcitonin

parathyroid hormone (PTH)

Calcitonin

inhibits bone resorption by inhibiting the effects of PTH on osteoclasts, thereby lowering blood Ca2+ levels.

PLATE 8.1 BONE, GROUND SECTION Bone

is a specialized connective tissue characterized by a mineralized extracellular matrix. Calcium phosphate, in the form of [Ca 10 (PO 4 ) 6 OH 2 ], is deposited along the collagen fibrils and in the proteoglycan ground substance. Bone serves as a storage site for calcium and phosphate, which can be released to the blood to maintain homeostatic levels. reside in lacunae in the bone matrix and extend fine cellular processes into canaliculi that connect the lacunae, thus forming a continuous network of cells within the mineralized tissue. Bones are organs of the skeletal system; bone tissue is the structural component of bones. Ground sections of bone are prepared from bone that has not been fixed but merely allowed to dry. Thin slices of the dried bone are then cut with a saw and further ground to a thinness that allows viewing in a light microscope. Slices may be treated with India ink to fill spaces that were formerly occupied by organic matter, for example, cells, blood vessels, and unmineralized matrix. A simpler method is to mount the ground specimen on a slide with a viscous medium that traps air in some of the spaces, as in the specimen in this plate. Here, some of the osteonal canals and a perforating canal are filled with the mounting medium, making them translucent instead of black. Specimens prepared in this manner are of value chiefly to display the architecture of the compact bone.

hydroxyapatite crystals

Osteocytes

Ground bone

, long bone, human, ×80.

This figure reveals a cross-sectioned area of a long bone at low magnification and includes the outer or peripheral aspect of the bone, identified by the presence of ( ). (The exterior or periosteal surface of the bone is not included in the micrograph.) To their are the ( ) or Haversian systems that appear as circular profiles. Between the osteons are ( ), the remnants of previously existing osteons. Osteons are essentially cylindrical structures. In the shaft of a long bone, the long axes of the osteons are oriented parallel to the long axis of the bone. Thus, a cross section through the shaft of a long bone would reveal the osteons in cross section, as in this figure. At the center of each osteon is an ( ) that contains blood vessels, connective tissue, and cells lining the surface of the bone material. Because the organic material is not retained in ground sections, the Haversian canals and other spaces will appear black, as they do here, if filled with India ink or air. Concentric layers of mineralized substance, the concentric lamellae, surround the Haversian canal and appear much the same as growth rings of a tree. The canal is also surrounded by concentric arrangements of lacunae. These appear as the small, dark, elongate structures. During the period of bone growth and during adult life, there is constant internal remodeling of bone. This involves the destruction of osteons and formation of new ones. The breakdown of an osteon is usually not complete; however, part of the osteon may remain intact. Moreover, portions of adjacent osteons may also be partially destroyed. The space created by the breakdown process is reoccupied by a new osteon. The remnants of the previously existing osteons become the interstitial lamellae. Blood vessels reach the Haversian canals from the marrow through other tunnels called ( ). In some instances, as here, Volkmann canals travel from one Haversian canal to another. Volkmann canals can be distinguished from Haversian canals in that they pass through lamellae, whereas Haversian canals are surrounded by concentric rings of lamellae.

circumferential lamellae CL

right

lamellae IL

osteons O

interstitial

osteonal (Haversian) canal HC

perforating

(Volkmann) canals VC

Ground bone-osteon

, long bone, human, ×300.

osteon

This figure shows a higher magnification micrograph of the labeled from the figure. It includes Haversian canal ( ) surrounded by concentric lamellae and some of the interstitial lamellae ( ) that are now seen at the of the micrograph (the micrograph has been reoriented). Note the ( ) and the fine thread-like profiles emanating from the lacunae. These thread-like profiles represent the canaliculi, spaces within the bone matrix that contain cytoplasmic processes of the

upper

IL

HC

lacunae L

bottom

osteocyte. The canaliculi of each lacuna communicate with canaliculi of neighboring lacunae to form a three-dimensional channel system throughout the bone.

Ground bone

, long bone, human, ×400.

circumferential lamellae

In a still higher magnification, the are found around the shaft of the long bone at the outer as well as the inner surface of the bone. The osteoblasts that contribute to the formation of circumferential lamellae at these sites come from the periosteum and endosteum, respectively, whereas the osteons are constructed from osteoblasts in the canal of the developing Haversian system. This figure reveals not only the lacunae ( ) and canaliculi but also the lamellae of the bone. The latter are just barely defined by the faint lines ( ) that extend across the micrograph. Collagenous fibers in neighboring lamellae are oriented in different directions. This change in orientation accounts for the faint line or interface between adjacent lamellae.

L

CL, circumferential lamellae HC, Haversian canal IL, interstitial lamellae L, lacuna O, osteon VC, Volkmann canal arrows, lamellar boundary

arrows

PLATE 8.2 BONE AND BONE TISSUE Bone

represents one of the specialized connective tissues. It is characterized by a mineralized extracellular matrix. It is the mineralization of the matrix that sets bone tissue apart from the other connective tissues and results in an extremely hard tissue that is capable of providing support and protection to the body. The mineral is calcium phosphate in the form of hydroxyapatite crystals. Bone also provides a storage site for calcium and phosphate. Both can be mobilized from the bone matrix and taken up by the blood as needed to maintain normal levels. Bone matrix contains type I collagen and, in small amounts, a number of other types of collagen, that is, types V, III, XI, and XIII. Other matrix proteins that constitute the ground substance of bone such as proteoglycan macromolecules, multiadhesive glycoproteins, growth factors, and cytokines are also present. Bone is typically studied in histologic preparations by removing the calcium content of the bone (decalcified bone), thus allowing it to be sectioned like other soft tissues. The orientation micrograph shows the proximal end of a decalcified humerus from an infant. The interior of the head of the bone, the ( ), consists of spongy (cancellous) bone made up of an anastomosing network of ( ) in the form of bone spicules. The outer portion consists of a dense layer of bone tissue known as ( ). Its thickness varies in different parts of the bone. The wider portion of the bone adjacent to the ( ) known as ( ) contains

ORIENTATION MICROGRAPH:

CB

epiphyseal growth plate EGP

epiphysis E trabeculae T

metaphysis M

compact bone spongy bone

diaphysis D bone marrow BM

SB

CB

( ). The shaft of this bone, the ( ), is also made up of compact bone ( ) and contains bone marrow cavity filled with ( ), which at this stage of life is in the form of active hemopoietic tissue. Cartilage is also a component of the bone, present as ( ) and as an epiphyseal growth plate ( ) in growing bones.

articular cartilage AC

Articular surface

EGP

, long bone, human, H&E ×178.

top right box

The articular surface of the epiphysis within the on the orientation micrograph containing articular cartilage and the underlying bone tissue is shown here at higher magnification. The lighter staining area is the ( ) of the glenohumeral (shoulder) joint. Note the presence of isogenous groups of ( ), a characteristic feature of growing cartilage. Below the cartilage is a darker staining area of ( ). It can be distinguished from the cartilage by the presence of ( ) and arrangement of the ( ). The osteocytes lie within the bone matrix but are typically recognized only by their nuclei. Because bone matrix is laid down in layers (lamellae), bone characteristically shows linear or circular patterns surrounding HCs. The irregular spaces seen within the bone tissue are ( ) that contain, in addition to blood vessels, the osteoclasts and osteoblasts. Presence of resorption canals indicates an active process of bone remodeling.

chondrocytes Ch compact bone CB Haversian canals HC

Compact bone

articular cartilage AC

osteocytes Oc resorption canals RC

, long bone, human, H&E ×135.

bottom right box

Bone from the diaphysis within the on the orientation micrograph is shown here at higher magnification. The outer surface of the bone is covered by dense connective tissue known as ( ). The remaining tissue in the micrograph is ( ).

periosteum P

compact bone CB Haversian canals

osteocytes Oc osteoclasts Ocl

HC

( ) are surrounded by the ( ) and are recognized by their nuclei within the bone matrix. Another feature worth noting in this growing bone is the presence of bone-resorbing cells known as ( ). They are large multinucleated cells found at sites in bone where remodeling is taking place (see Plate 8.4).

Spongy bone

, long bone, human, H&E ×135.

top left box

The area in the in the orientation micrograph containing spongy bone in the epiphysis is shown here at higher magnification. Although the bone tissue at this site forms a three-dimensional structure consisting of branching trabeculae, its structural organization and components are the same as that seen in compact bone. Note the nuclei of ( ). As bone matures, the bone tissue becomes reorganized and forms ( ), which consist of ( ) and surrounding layers (lamellae) of bone matrix. The two circular spaces are the ( ), in which bone tissue has been removed and will be replaced by new tissue in the form of osteons. The spaces surrounding the spongy bone contain bone marrow consisting mainly of adipocytes. Other cells that have the capacity to form bone or hemopoietic tissue are also present.

osteons O

osteocytes Oc Haversian canals HC resorption canals RC

AC, articular cartilage BM, bone marrow CB, compact bone Ch, chondrocytes D, diaphysis E, epiphysis EGP, epiphyseal growth plate HC, Haversian canal M, metaphysis O, osteons Oc, osteocytes Ocl, osteoclasts P, periosteum RC, resorption canal SB, spongy bone T, trabeculae

PLATE 8.3 ENDOCHONDRAL BONE FORMATION I Endochondral bone formation

involves a cartilage model that represents a cartilage precursor to the newly formed bone. The cartilage model appears as a miniature version of the future bone. Bone that arises through this process is formed by the simultaneous removal of the cartilage model and its replacement with bone tissue. The first sign of bone formation is the appearance of bone-forming cells around the shaft (diaphysis) of the cartilage model. The bone-forming cells, known as , are derived from osteoprogenitor cells in surrounding mesenchyme. They secrete collagens, bone sialoproteins, osteocalcin, and other components of the bone matrix. The initial deposition of these products is referred to as a and contains osteoid (unmineralized bone), which later becomes mineralized. With the initial establishment of this periosteal bony collar, the chondrocytes in the center of the cartilage model become hypertrophic (see ), which leads to their death (or transdifferentiation into osteoblasts), and the cartilage matrix in this region becomes calcified. At the same time, blood vessels grow through the thin bony collar and vascularize the center of the bone diaphysis, allowing infiltration of precursor cells of bone marrow. Osteoprogenitor cells enter the bone marrow cavity with blood vessels and differentiate into osteoblasts. In long bones, this process is repeated in the epiphyses of the cartilage model (see lower micrograph). The process of the actual deposition of bone is described and illustrated in the next plate.

osteoblasts

periosteal bony collar

upper figure

Developing bone

, fetal finger, monkey, H&E ×240.

epiphyses E

An early stage in the process of endochondral bone formation in the fetal digit is shown in this micrograph. Proximal and distal ( ) of this developing bone are made of cartilage. This bone of the fetal digit is connected by joints with other bones; note ( ) on both edges of this micrograph. The midregion of this long bone reveals that have undergone marked ( ). The cytoplasm of these chondrocytes appears very clear or washed out. Their nuclei, when included in the plane of section, appear as small, condensed basophilic bodies. Note how the cartilage matrix in this region is calcified and has been compressed into narrow linear bands of tissue surrounding the chondrocytes. The ( ) stains more intensely with hematoxylin in routine H&E preparation and appears darker. At this stage of development, bone tissue has been produced to form the early periosteal ( ) around the cartilage model. This bone tissue is produced by appositional growth from bone-forming cells that were derived from the mesenchyme in the tissue surrounding the cartilage. This process represents intramembranous bone formation, which will be described later.

joint cavities JC chondrocytes

hypertrophy HCh

calcified cartilage matrix CCM bony collar BC

Developing bone

, fetal finger, human, H&E ×60.

bone marrow cavity Cav

The bone shown in this micrograph represents a later stage of development. Most of the diaphysis of the bone contains the ( ) filled with marrow, part of which is highly cellular and represents accumulations of hemopoietic ( ). The nonstaining areas consist of adipose tissue, which occupies much of the remainder of the bone marrow cavity. The thin bony collar seen earlier has now developed into a relatively thicker mass of ( ). The part of the bone in which bone tissue is being deposited by ( ) formation is seen at both ends of the bone marrow cavity. Note that its eosinophilic character is similar to the diaphyseal bone. As these processes continue in the shaft of the bone, ( ) on both proximal and distal epiphyses are invaded by blood vessels and connective tissue from the periosteum (periosteal bud), and it undergoes the same changes that occurred earlier in the shaft (except that no periosteal bony collar is formed).

marrow cells BMC

bone

diaphyseal bone DB endochondral bone EB cartilages C

Developing bone

, proximal epiphysis of long bone, human, H&E ×60; inset

×200.

secondary ossification center SOC

This micrograph shows considerable developmental advancement beyond that of the bone in the above micrograph. A ( ) has been established in the proximal epiphysis of this long bone. At a slightly later time, a similar epiphyseal ossification center will form at the distal end of the bone. The process of endochondral bone formation occurs the same way as in the diaphysis. With time, these epiphyseal centers of ossification will increase in size to form much larger cavities ( ). The consequence of this activity is that an ( ) is formed. As visible on this micrograph, the epiphyseal growth plate, consisting of cartilage, separates the SOCs at the proximal end of the bone from the primary ossification center formed in the shaft of the bone. This cartilaginous plate is essential for the longitudinal growth of the bone and will persist until bone growth ceases. The shows the secondary ossification center at higher magnification. Within this area, new ( ) is already being produced. The new bone appears eosinophilic in contrast to the more basophilic appearance of the surrounding ( ). Note that its staining pattern of endochondral bone in the SOC is identical to the more abundant endochondral bone ( ) that replaces ( ) at the upper end of the diaphysis.

epiphyseal growth plate EGP

inset

endochondral bone EB

calcified cartilage CC

BC, bony collar BMC, bone marrow cells C, cartilage Cav, bone marrow cavity CC, calcified cartilage CCM, calcified cartilage matrix DB, diaphyseal bone

cartilage C

dashed line

EB

E, epiphysis EB, endochondral bone EGP, epiphyseal growth plate HCh, hypertrophic chondrocytes JC, joint cavity SOC, secondary ossification center dashed line, epiphyseal center of ossification

PLATE 8.4 ENDOCHONDRAL BONE FORMATION II

Endochondral bone formation is the principal process by which the long bones (e.g., the bones of the appendages and digits) increase in length to achieve their adult dimensions. As long as an epiphyseal growth plate exists between the primary (diaphyseal) and secondary (epiphyseal)

ossification centers, the bone will continue to grow in length. During bone growth, distinct zoning can be recognized in the epiphyseal growth plate at both ends of the early formed marrow cavity. In the part of the cartilage that is farthest away from the bone marrow cavity at both ends of the growing bone, individual chondrocytes, separated by cartilage matrix, have not yet began to participate in the bone-forming process. This region is called the zone of reserve cartilage. As these chondrocytes immerse in changes leading to their proliferation, hypertrophy,

and eventual death (or transdifferentiation to osteoblasts), their microscopic appearance and changes in extracellular matrix define different functional zones of endochondral bone formation.

Endochondral bone formation

, epiphysis of long bone, human, H&E ×80;

inset ×380.

This is a photomicrograph of an epiphysis at higher magnification than that seen in Plate 8.3. Different zones of the cartilage of the epiphyseal plate reflect the progressive changes that occur in active growth of endochondral bone. These zones are not sharply delineated, and the boundaries between them are somewhat arbitrary. They lead toward the bone marrow ( ) cavity, so that the first zone is furthest from the cavity. There are five zones:

BM

Zone of reserve cartilage (ZRC): The cartilage cells of this zone have not yet begun to participate in the growth of the bone; thus, they are reserve cells. These cells are small, usually only one to a lacuna, and not grouped. At some point, some of these cells will proliferate and undergo the changes outlined for the next zone. ( ): The cells of this zone undergo divisions and are increasing in number; they are slightly larger than the chondrocytes in the zone of reserve cartilage and are close to their neighbors; they begin to form rows. ( ): The cells of this zone are aligned in rows and are significantly larger than the cells in the preceding zone. ( ): In this zone, the cartilage matrix is impregnated with calcium salts. The calcified cartilage will serve as an initial scaffold for the deposition of the new bone. Chondrocytes positioned in the more proximal part of this zone undergo apoptosis. ( ): This zone is represented by eroded cartilage that is in direct contact with the connective tissue of the marrow cavity. Small blood vessels and accompanying osteoprogenitor cells invade the region previously occupied by the dying chondrocytes. They form a series of spearheads, leaving on both sides the ( ) as longitudinal spicules. Osteoprogenitor cells give rise to osteoblasts that begin lining the surfaces of exposed spicules. ( ) is then deposited on the surfaces of these calcified cartilage spicules by osteoblasts, thus forming as seen in the . Note the ( ), some of which are just beginning to produce bone in apposition to the calcified cartilage ( ). The lower right of the shows mixed spicules containing endochondral bone ( ) and calcified cartilage ( ). Several osteoblasts ( ), and an osteoclast ( ) are present on the surface of the spicules. An osteocyte ( ) already embedded in the bone matrix is also visible.

Zone of proliferation ZP Zone of hypertrophy ZH Zone of calcified cartilage ZCC Zone of resorption ZR Ob CC

Endochondral bone EB CC

inset

calcified cartilage CC

mixed spicules Oc

Ob

Endochondral bone formation

inset

Ocl

osteoblasts EB

, epiphysis of long bone, human, H&E ×150;

inset ×380.

calcified cartilage spicules on which bone has been deposited. In the lower portion of the figure, the spicules have already grown to create anastomosing bone trabeculae ( T ). These initial trabeculae still contain remnants of calcified cartilage, as shown by the bluish color of the cartilage matrix (compared with the red staining of the bone). Osteoblasts ( Ob ) are aligned on the surface of the spicules, where bone formation is active. Several osteocytes ( Oc ) already embedded in the bone matrix are also visible. The inset reveals several osteoclasts ( Ocl ) in higher magnification. They are in apposition to the spicules, which are mostly made of calcified cartilage. A small amount of bone is evident, based on the red-stained material. The light area ( arrow ) represents the ruffled border of the osteoclast. This is a higher magnification of the lower area from the above figure. It shows

BM, bone marrow CC, calcified cartilage EB, endochondral bone Ob, osteoblast Oc, osteocyte Ocl, osteoclast T, trabeculae ZCC, zone of calcified cartilage ZH, zone of hypertrophy

ZP, zone of proliferation ZR, zone of resorption ZRC, zone of reserve cartilage arrow, ruffled border of osteoclast

PLATE 8.5 INTRAMEMBRANOUS BONE FORMATION Intramembranous bone formation

is limited to those bones that are not required to perform an early supporting function, for example, the flat bones of the skull. This process requires the proliferation and differentiation of cells of the mesenchyme to become , the boneforming cells. They produce bone-specific extracellular matrix. This initial matrix, called , undergoes mineralization to form bone. As the osteoblasts continue to secrete their product, some are entrapped within their matrix and are then known as . They are responsible for maintenance of the newly formed bone tissue. The remaining osteoblasts continue the bone deposition process at the bone surface. They are capable of reproducing to maintain an adequate population for continued growth. This newly formed bone appears first as that enlarge and interconnect as growth proceeds, creating a three-dimensional trabecular structure similar in shape to the future mature bone. The interstices contain blood vessels and connective tissue (mesenchyme). As the bone continues to grow, remodeling occurs. This involves resorption of localized areas of bone tissue by in order to maintain appropriate shape in relation to size and to permit vascular nourishment during the growth process.

osteoid

osteoblasts

osteocytes

spicules

osteoclasts

Intramembranous bone formation

, fetal head, human, Mallory trichrome

×45.

bone spicules BS

A cross section of the developing mandible, as seen at this relatively early stage of development, consists of ( ) of various sizes and shapes. The bone spicules interconnect and, in three dimensions, have the general shape of the mandible. Other structures present that will assist in orientation include ( ), ( ), seen on the , and the ( ). The of the specimen shows the ( ) of the submandibular region of the neck. A large portion of the developing tongue is seen in the of the figure. The tongue consists largely of developing striated visceral muscle fibers arranged in a three-dimensional orthogonal array that is characteristic of this organ.

DT

Meckel cartilage MC left side epidermis Ep upper half

oral cavity OC

Intramembranous bone formation ×175.

developing teeth

bottom surface

, fetal head, human, Mallory trichrome

boxed area

upper

bone spicules BS

This higher magnification view of the in the micrograph shows the interconnections of the ( ) of the developing mandible. Within and around the spaces enclosed by the developing spicules is mesenchymal tissue. These mesenchymal cells contain stem cells that will form the vascular components of the bone as well as the osteoprogenitor cells that will give rise to new osteoblasts. The denser connective tissue ( ) will differentiate into the periosteum on one side of the developing mandible. Other structures shown in the field include numerous blood vessels ( ) and the enamel organ of a ( ).

developing

CT tooth DT

BV

Intramembranous bone formation

, fetal head, human, Mallory trichrome

×350.

lower left blue

This higher magnification micrograph of a portion of the field in the micrograph shows the distinction between newly deposited osteoid, which stains , and mineralized bone, which stains . are seen in two different levels of activity. Osteoblasts that are relatively inactive ( ) and are in apposition to wellformed osteoid exhibit elongate nuclear profiles and appear to be flattened on the surface of the osteoid. Those osteoblasts that are actively secreting new osteoid ( ) appear as tall, columnar-like cells adjacent to the osteoid. One of the spicules shows a cell completely surrounded by bone matrix; this is an osteoblast that has become trapped in its own secretions and is now an ( ). At this magnification, the embryonic tissue characteristics of the mesenchyme and the sparseness of the ( ) are well demonstrated. The highly cellular connective tissue ( ) on the of the micrograph is the developing perichondrium. Some of its cells have osteoprogenitor cell characteristics and will develop into osteoblasts to allow growth of the bone at its surface.

red

osteocyte Oc CT

AOb, active osteoblast BS, bone spicules BV, blood vessels CT, connective tissue DT, developing teeth Ep, epidermis IOb, inactive osteoblast MC, Meckel cartilage MeC, mesenchymal cells Oc, osteocyte OC, oral cavity

Osteoblasts IOb

mesenchymal cells MeC right margin

AOb

9

ADIPOSE TISSUE

OVERVIEW OF ADIPOSE TISSUE WHITE ADIPOSE TISSUE

Function of White Adipose Tissue Differentiation of Adipocytes Structure of Adipocytes and Adipose Tissue Regulation of Adipose Tissue

BROWN ADIPOSE TISSUE BEIGE ADIPOSE TISSUE TRANSDIFFERENTIATION OF ADIPOSE TISSUE Folder 9.1 Clinical Correlation: Obesity Folder 9.2 Clinical Correlation: Adipose Tissue Tumors Folder 9.3 Clinical Correlation: PET Scanning and Brown Adipose Tissue Interference HISTOLOGY

OVERVIEW OF ADIPOSE TISSUE

Adipose tissue is a specialized connective tissue that plays an important role in energy homeostasis. Individual fat cells, or adipocytes, and groups of adipocytes are found throughout loose connective tissue. Tissues in which adipocytes are the primary cell type are designated adipose tissue. Adipocytes play a key role in energy homeostasis.

For its survival, the body needs to ensure continuous delivery of energy despite highly variable supplies of nutrients from the external environment. To meet the body’s energy demands when nutrient supplies are low, adipose tissue efficiently stores excess energy. The body has a limited capacity to store carbohydrate and protein; therefore, energy reserves are stored within of adipocytes in the form of . Triglycerides represent a dynamic form of that is added when food intake is greater than energy expenditure and is tapped when energy expenditure is greater than food intake. The energy stored in adipocytes can be rapidly released for use at other sites in the body. Triglycerides are the most concentrated form of metabolic energy storage available to humans. Because triglycerides lack water, they have about twice the energy density of carbohydrates and proteins. The energy density of triglycerides is approximately 37.7 kJ/g (9 cal/g), whereas the density of carbohydrates and proteins is 16.8 kJ/g (4 cal/g). In the event of food deprivation, triglycerides are an essential source of water and energy. Some animals can rely solely on metabolic water obtained from fatty acid oxidation for the maintenance of their water balance. For instance, the hump of a camel consists largely of adipose tissue and is a source of water and energy for this desert animal. Adipocytes perform other important functions in addition to their role as fat storage containers. They also regulate energy metabolism by secreting autocrine, paracrine, and endocrine substances. Adipose tissue is considered a major . Considerable evidence links increased endocrine activity of adipocytes to the metabolic and cardiovascular complications associated with .

lipid droplets energy storage

triglycerides

endocrine organ

obesity Historically, there were only two major types of adipose tissue identified: white (unilocular) and brown (multilocular); however, recent classifications include a third type: beige (paucilocular) adipose tissue. The three types of adipose tissue, white adipose tissue, brown adipose tissue, and beige adipose tissue, are so named because of their color in the living state, which is attributable to their high mitochondrial density and high vascularization:

White adipose tissue is the predominant type in adult humans. Brown adipose tissue is present in large amounts in humans

during fetal life. It diminishes during the first decade after birth but continues to be present in varying amounts, mainly around internal organs. or “brite” (brown-in-white) adipose tissue consists of an accumulation of brown-like adipocytes within the subcutaneous white adipose tissue deposits in adult humans.

Beige adipose tissue

In general, white adipose tissue stores excess energy in lipids; in contrast, brown and beige adipose tissues dissipate energy through the production of heat. All adipose tissues produce biologically active substances to regulate energy homeostasis.

WHITE ADIPOSE TISSUE

White (unilocular) adipose tissue

represents at least 10% of the body weight of a normal

healthy individual.

Function of White Adipose Tissue

Functions of white adipose tissue include energy storage, insulation, cushioning of vital organs, and secretion of hormones. White adipose tissue forms a fatty layer of the subcutaneous (superficial) fascia called the panniculus adiposus [Lat. panniculus, a little garment; adipatus, fatty] in the

connective tissue beneath the skin. Because the thermal conductivity of adipose tissue is only about one-half that of skeletal muscle, the subcutaneous fascia provides significant thermal insulation against the cold by reducing the rate of heat loss. Concentrations of adipose tissue are found in the connective tissue under the skin of the abdomen, buttocks, axilla, and thigh. Sex differences in the thickness of this fatty layer in the skin of different parts of the body account, in part, for the differences in body contour between females and males. In both sexes, the is a preferential site for accumulation of adipose tissue; the nonlactating female is composed primarily of this tissue. In the lactating female, the mammary fat pad plays an important role in supporting breast function. It provides lipids and energy for milk production, but it is also a site for the synthesis of growth factors that modulate responses to steroid hormones and proteins that regulate mammary gland function. Internally, adipose tissue is preferentially located in the greater omentum, mesentery, and retroperitoneal space and is usually abundant around the kidneys. It is also found in the and between other tissues, where it fills in spaces. Adipose tissue functions as a cushion in the palms of the hands and the soles of the feet, beneath the visceral pericardium (around the outside of the heart), and in the orbits around the eyeballs. It retains this structural function even during reduced caloric intake; when adipose tissue elsewhere becomes depleted of lipid, this structural adipose tissue remains undiminished.

mammary fat pad mammary gland

bone marrow

White adipose tissue secretes a variety of adipokines, which include hormones, growth factors, and cytokines. White adipocytes actively synthesize and secrete adipokines, a group of biologically active

substances, which include hormones, growth factors, and cytokines (Fig. 9.1). For this reason, adipose tissue is regarded as an important player in energy homeostasis, adipogenesis, steroid metabolism, angiogenesis, and immune responses.

FIGURE 9.1. Major adipokines secreted by white adipose tissue.

This schematic drawing shows various types of adipokines secreted by white adipose tissue, including hormones (e.g., leptin), cytokines (e.g., insulin-like growth factor 1), and other molecules with specific biological functions (e.g., prostaglandins). Note secretion of exosomes, extracellular nanovesicles that facilitate intercellular communication. They contain small fragments of adipocyte RNA (microRNAs) responsible for the regulation of gene expression in distant organs, which, in turn, affects whole-body metabolism. , insulin-like growth factor 1; , interleukin-16; , 1 , prostaglandin I 1 ; transforming growth factor β; , tumor necrosis factor α; , tumor necrosis factor β.

IGF-1

TNF-α

IL-6

leptin [Gr. leptos, thin]

PGI TNF-β

TGF-β

A notable adipokine is , a 16-kDa peptide hormone discovered in 1994. Leptin is involved in the regulation of energy homeostasis and is primarily secreted by adipocytes. Small amounts of leptin are also produced in other organs (e.g., stomach, placenta, mammary glands, and ovaries). Leptin inhibits food intake and stimulates metabolic rate and loss of body weight. Thus, leptin fulfills the criteria for a that controls food intake when the body’s store of energy is sufficient. Leptin participates in an endocrine signaling pathway that communicates the energy state of adipose tissue to brain centers that regulate food uptake. It acts on the central nervous system by binding to specific receptors, mainly in the . Leptin also regulates the production of (testosterone, estrogens, and glucocorticoids). In addition, leptin communicates the fuel state of adipocytes from fat storage sites to other metabolically active tissues (i.e., from adipose tissue to muscle at a different site). In addition to leptin, adipose tissue secretes a variety of other adipokines, such as , , retinol-binding protein 4 (RBP-4), visfatin, apelin, plasminogen activator inhibitor 1 (PAI-1), tumor necrosis factors (TNFs), interleukin-6 (IL-6), monocyte chemotactic protein 1 (MCP-1), , and others. Some adipokines are also synthesized in other tissues. For example, AGE is synthesized in the liver; increased production of this peptide by adipose tissue contributes to hypertension (elevation of blood pressure), a frequent complication of obesity. Recently, were found in circulating intact exosomes (membrane-bound extracellular vesicles) secreted by white adipocytes. These miRNAs represent small (about 22 nucleotides) RNA molecules that contain intact genetic information specific to adipocytes. Exosomal miRNAs participate in various metabolic processes, such as glucose/lipid metabolism, insulin signaling, inflammation, and differentiation of white and brown adipocytes in various tissues. Adipocytes also help regulate the synthesis of sex hormones and glucocorticoids. Specific enzymes expressed in adipocytes convert the inactive forms of these hormones to their active forms. In this way, these enzymes influence the sex steroid profiles of obese individuals. Obesity associated with increased secretion of (tumor necrosis factor α [TNF-α], transforming growth factor β [TGF-β], insulin-like growth factor 1 [IGF-1]) and

circulating satiety

factor

hypothalamus

steroid hormones

adiponectin resistin

angiotensinogen (AGE)

microRNAs (miRNAs)

growth factors

cytokines

(IL-6 and prostaglandins) may be linked to metabolic abnormalities and development of diabetes. Table 9.1 presents a summary of the most important adipokines produced by white adipocytes and their functions.

of Molecules Synthesized and Secreted by White Adipose Tissue and TABLE 9.1 Summary Their Functions Molecule Acylationstimulating protein (ASP) Adiponectin Adipophilin Adipsin Angiotensinogen (AGE) and angiotensin II (AngII) Apelin Insulin-like growth factor 1 (IGF-1) Interleukin-6 (IL6) Leptin Plasminogen activator inhibitor 1 (PAI1) Prostaglandins I 2 and F 2 𝛂 (PGI 2 and PGF 2 𝛂) Resistin Retinol-binding protein 4 (RBP-4) Transforming growth factor 𝛃 (TGF- 𝛃) Tumor necrosis factor 𝛂 and 𝛃 (TNF- 𝛂, TNF- 𝛃) Visfatin

Major Function or Effect Influences the rate of triglyceride synthesis in adipose tissue

Stimulates fatty acid oxidation in liver and muscles Decreases plasma triglycerides and glucose concentrations and increases insulin sensitivity in cells Plays a role in the pathogenesis of familial combined hyperlipidemia Correlated with insulin resistance and hyperinsulinemia Serves as a specific marker for lipid accumulation in cells Regulates adipose tissue metabolism by facilitating fatty acid storage and stimulating triglyceride synthesis AGE: Precursor of vasoactive AngII, which regulates blood pressure and electrolyte levels in the serum and is also involved in the metabolism and differentiation of adipose tissue AngII: During development, inhibits differentiation of lipoblasts; in mature adipocytes, it regulates lipid storage Increases cardiac muscle contractility Decreases blood pressure Stimulates proliferation of a wide variety of cells and mediates many of the effects of growth hormone Interacts with cells of immune system and regulates glucose and lipid metabolism Decreases activity of adipose tissue in cancer and other wasting disorders Regulates appetite and body energy expenditure Signals to the brain about body fat stores Increases formation of new vessels (angiogenesis) Involved in blood pressure control by regulating vascular tone Potent inhibitor of bone formation Inhibits fibrinolysis (a process that degrades blood clots)

Helps regulate inflammation, blood clotting, ovulation, menstruation, and acid secretion

Increases insulin resistance Linked to obesity and type 2 diabetes Produced mainly by visceral adipose tissue Decreases insulin sensitivity and alters glucose homeostasis Regulates a wide variety of biological responses, including proliferation, differentiation, apoptosis, and development Interferes with insulin receptor signaling and is a possible cause of development of insulin resistance in obesity Produced by visceral adipose tissue; its level correlates with visceral adipose tissue mass Involved in regulation of body mass index Decreases blood glucose levels

Modified from Vázquez-Vela ME, Torres N, Tovar AR. White adipose tissue as endocrine organ and its role in obesity. . 2008;39:715–728.

Arch Med Res

Differentiation of Adipocytes

White adipocytes differentiate from mesenchymal stem cells under the control of PPARγ/RXR transcription factors. During embryonic development, white adipocytes derive from undifferentiated perivascular mesenchymal stem cells associated with the adventitia of small venules (Fig. 9.2). A transcription factor called peroxisome proliferator–activated receptor γ (PPARγ), in complex with the retinoid X receptor (RXR), plays a critical role in adipocyte differentiation and initiation of lipid metabolism. This complex induces the maturation of early lipoblasts (adipoblasts) or preadipocytes into mature fat cells of white adipose tissue. Most of the PPARγ target genes in adipose tissue influence lipogenic pathways and initiate the storage of triglycerides. Therefore, PPARγ/RXR is regarded as the “masterswitch” regulator in differentiation of white adipocytes.

FIGURE 9.2. Development of white and brown adipose tissue cells.

Brown and white adipose cells arise from distinctly different cellular lineages. White adipocytes derive from undifferentiated perivascular mesenchymal stem cells associated with the adventitia of small venules. By expressing PPARγ/RXR transcription factors, these cells will differentiate into early lipoblasts (preadipocytes)

committed to white adipocyte lineage development. Brown adipocytes also have a mesenchymal origin; however, they derive from common skeletal myogenic progenitor stem cells expressing myogenic factor 5 (Myf5) protein found in dermatomyotomes of developing embryos. By expressing PRDM16/PGC-1 transcription factors, these cells will differentiate into early lipoblasts committed to the brown adipocyte lineage development. Note that the developmental lineage of white adipocytes does not express myogenic factor and is Myf5 negative. Lipoblasts develop an external (basal) lamina and begin to accumulate numerous lipid droplets in their cytoplasm. In white adipose tissue, these droplets fuse to form a single large lipid droplet that eventually fills the mature cell, compressing the nucleus, cytoplasm, and cytoplasmic organelles into a thin rim around the droplet. In brown adipose tissue, the individual lipid droplets remain separate. Beige adipocytes are shown here arising from white adipocytes. Beige adipocytes also develop from progenitor cells as shown in Figure 9.9; however, transdifferentiation from white adipocytes is the most common developmental pathway.

White adipose tissue begins to form midway through fetal development. Lipoblasts initially develop from stromal–vascular cells along the small blood vessels in

the fetus and are free of lipids. These cells are committed to becoming adipocytes at this early stage by expressing PPARγ/RXR transcription factors. Collections of such cells are sometimes called . They are characterized by proliferating early lipoblasts and proliferating capillaries. Lipid accumulation in lipoblasts produces the typical morphology of adipocytes.

primitive fat organs

Early lipoblasts look like fibroblasts but develop small lipid inclusions and a thin external lamina. Transmission electron microscopy (TEM) studies reveal that early lipoblasts have an

elongated configuration, multiple cytoplasmic processes, and abundant endoplasmic reticulum and Golgi apparatus. As lipoblastic differentiation begins, vesicles increase in number, with a corresponding decrease in rough-surfaced endoplasmic reticulum (rER). Small appear at one pole of the cytoplasm. Pinocytotic vesicles and an also appear. The presence of an external lamina is a feature that further distinguishes adipocytes from proper connective tissue cells.

inclusions

lipid external lamina

Midstage lipoblasts become ovoid as lipid accumulation changes the cell dimensions.

With continued development, the early lipoblasts assume an oval configuration. The most characteristic feature at this stage is an extensive concentration of vesicles and around the nucleus and extending toward both poles of the cell. Glycogen particles appear at the periphery of the lipid droplets, and pinocytotic vesicles and basal lamina become more apparent. These cells are designated .

lipid droplets

small

midstage lipoblasts The mature adipocyte is characterized by a single large lipid inclusion surrounded by a thin rim of cytoplasm. In the late stage of differentiation, the cells increase in size and become more spherical. Small lipid droplets coalesce to form a single large lipid droplet that occupies the central portion of the cytoplasm. Smooth-surfaced endoplasmic reticulum (sER) is abundant, whereas rER is less prominent. These cells are designated late lipoblasts. Eventually, the lipid mass compresses the nucleus to an eccentric position, producing a signet-ring appearance in hematoxylin and eosin (H&E) preparations. Because these cells have a single lipid droplet, they are designated unilocular [Lat. unus, single; loculus, a little place] adipocytes or mature lipocytes.

Structure of Adipocytes and Adipose Tissue

Unilocular adipocytes are large cells, sometimes 100 μm or more in diameter. When isolated, white adipocytes are spherical, but they may appear polyhedral or oval when

crowded together in adipose tissue. Their large size is due to the accumulated lipid in the cell. The nucleus is flattened and displaced to one side of the lipid mass; the cytoplasm forms a thin rim around the lipid. In routine histologic sections, the lipid is lost through

extraction by organic solvents, such as xylene; consequently, adipose tissue appears as a delicate meshwork of polygonal profiles (Fig. 9.3). The thin strand of meshwork that separates adjacent adipocytes represents the cytoplasm of both cells and the extracellular matrix. The strand is usually so thin, however, that it is not possible to resolve its component parts in the light microscope.

FIGURE 9.3. White adipose tissue. a.

Photomicrograph of white adipose tissue showing its characteristic meshwork in a hematoxylin and eosin (H&E)-stained paraffin preparation. Each space represents a single large drop of lipid before its dissolution from the cell during tissue preparation. The surrounding eosin-stained material represents the cytoplasm of the adjoining cells and some intervening connective tissue. ×320. High-power photomicrograph of a glutaraldehydepreserved, plastic-embedded specimen of white adipose tissue. The cytoplasm of the individual adipose cells is recognizable in some areas, and part of the nucleus of one of the cells is included in the plane of section. A second nucleus ( ), which appears intimately related to one of the adipose cells, may actually belong to a fibroblast; it is difficult to tell with assurance. Because of the large size of adipose cells, the nucleus is infrequently observed in a given cell. A capillary and a small venule are also evident in the photomicrograph. ×950.

b.

arrow

Adipose tissue is richly supplied with blood vessels, and capillaries are found at the angles of the meshwork where adjacent adipocytes meet. Silver stains show that adipocytes are surrounded by reticular fibers (type III collagen), which are secreted by the adipocytes. Special stains also reveal the presence of unmyelinated nerve fibers and numerous mast cells. A summary of white adipose tissue features is listed in Table 9.2.

TABLE 9.2 Summary of Adipose Tissue Features Features

White Adipose Tissue

Brown Adipose Tissue

Beige Adipose

Location

Function Adipocyte morphology Precursor cells

Subcutaneous layer, mammary gland, greater omentum, mesenteries, retroperitoneal space, visceral pericardium, orbits (eye sockets), bone marrow cavity

Large amounts in newborns Remnants in adults at the retroperitoneal space, deep cervical and supraclavicular regions of the neck, interscapular, paravertebral regions of the back, mediastinum

Distributed thr body in vario subcutaneous tissue

Metabolic energy storage, insulation, cushioning, hormone production (adipokines), source of metabolic water

Heat production (thermogenesis UCP-1 dependent), hormone production (batokines)

Heat production (thermogenesi production (b

Unilocular, spherical, flattened nucleus, rim of cytoplasm Large diameter (15–150 μm)

Multilocular, spherical, round eccentric nucleus Smaller diameter (10–25 μm)

Paucilocular, s round nucleus Smaller diamete

Perivascular mesenchymal stem cells (Myf5 negative)

Common skeletal myogenic progenitor cells (Myf5 positive)

Perivascular me cells (Myf5 n

Specific Ob (leptin), HOXC8, and HOXC9 cellular markers Transcription PPARγ/RXR factors “master switch” in differentiation UCP-1 gene No expression Thermogenesis No existing mechanism Mitochondria Few, elongated, filamentous with

LHX8 and ZIC1

TMEM26 and TBX1

PRDM16/PGC-1

Low expression

Yes

Yes

UCP-1 dependent

Not completely dependent, ut cycling

Many, large, round, with well-developed cristae

Many, but less adipocytes, l with well-dev

Few sympathetic nerve fibers

High density of noradrenergic sympathetic nerve fibers

High density of sympathetic n

Few blood vessels

Highly vascularized tissue

Highly vascular

Decreased lipogenesis Increased lipoprotein lipase activity Transdifferentiation to brown adipose tissue

Increased lipogenesis Decreased lipoprotein lipase activity Increased heat production

Increased lipog Decreased lipop activity Increased heat

Throughout entire life from stromal perivascular cells

During fetal period from common skeletal myogenic progenitor cells Decreases in adult life

Throughout enti white-to-brow transdifferen novo from pre Induced in indi pheochromocyt

poorly developed cristae

Innervation Vascularization Response to environmental stress (i.e., cold exposure) Growth and differentiation

hibernoma, or exposure

The lipid mass in the adipocyte is not membrane bound. TEM reveals that the interface between the contained lipid and surrounding cytoplasm of the adipocyte is composed of a 5-nm-thick condensed layer of lipid reinforced by parallel measuring 5–10 nm in diameter. This layer separates the hydrophobic contents of the lipid droplet from the hydrophilic cytoplasmic matrix. The perinuclear cytoplasm of the adipocyte contains a small Golgi apparatus, free ribosomes, short profiles of rER, microfilaments, and intermediate filaments. Filamentous forms of mitochondria and multiple profiles of sER are also found in the thin rim of cytoplasm surrounding the lipid droplet (Fig. 9.4).

vimentin filaments

FIGURE 9.4. Electron micrograph showing portions of two adjacent adipose cells. The cytoplasm of the adipose cells reveals mitochondria ( M ) and glycogen (the latter appears as very dark particles). ×15,000. Upper inset. Attenuated cytoplasm ( Cy ) of two adjoining adipose cells. Each cell is separated by a narrow space containing external (basal) lamina and an extremely attenuated process of a fibroblast. ×65,000. Lower inset. The external (basal) lamina ( BL ) of the adipose cells appears as a discrete layer by which the cells are adequately separated from one another. F , fibroblast processes. ×30,000.

Regulation of Adipose Tissue It is almost impossible to separate regulation of adipose tissue from digestive processes and functions of the central nervous system. These interconnected hormonal and neural signals emanating from the adipose tissue, alimentary tract, and central nervous system form

brain–gut–adipose axis

the homeostasis (Fig. 9.5).

that

regulates

appetite,

hunger,

satiety,

and

energy

FIGURE 9.5. Regulation of energy homeostasis.

This schematic diagram shows the relationship of adipose tissue to the central nervous system and gastrointestinal system within the brain–gut– adipose axis that is responsible for regulating energy homeostasis.

The amount of an individual’s adipose tissue is determined by two physiologic systems: one associated with short-term weight regulation and the other with long-term weight regulation. The amount of adipose tissue in an individual is regulated by two physiologic systems. The first system, which is associated with short-term weight regulation, controls appetite and metabolism on a daily basis. Two small peptide hormones produced in the gastrointestinal tract—ghrelin, an appetite stimulant, and peptide YY (PYY), an appetite suppressant—have been linked to this system. The second system, which is associated with long-term weight regulation, controls appetite and metabolism on a continual basis (over months and years). Two major hormones influence this system, leptin and insulin, along with other hormones,

including thyroid hormone, glucocorticoids, and hormones of the pituitary gland (see Fig. 9.5).

Ghrelin and peptide YY control appetite as part of the short-term weight control system. Ghrelin is a small, 28-amino-acid polypeptide produced by gastric epithelial cells. In

addition to its appetite stimulatory role, it acts on the anterior lobe of the pituitary gland to release growth hormone. In humans, ghrelin functions through receptors located in the , increasing the sense of hunger. As such, it is considered a “mealinitiator” factor. A mutation of a gene located on chromosome 15 causes , in which an overproduction of ghrelin leads to morbid obesity. In individuals with this syndrome, compulsive eating and an obsession with food usually arise at an early age. The urge to eat in these individuals is physiologic, overwhelming, and very difficult to control. If not treated, these individuals often die before age 30 of complications attributable to obesity. The small, 36-amino-acid long gastrointestinal hormone is produced by the small intestine and plays an important role in promoting and maintaining weight loss by inducing a greater sense of fullness soon after a meal. It also acts through receptors in the that . It decreases food intake in individuals by inducing satiety or a sense of fullness and the desire to stop eating. In experimental clinical studies, the infusion of PYY into humans has been shown to reduce food intake by 33% over a period of 24 hours.

hypothalamus syndrome

Prader–Willi

peptide YY

hypothalamus

suppress appetite

Two hormones, leptin and insulin, are responsible for long-term regulation of body weight. The discovery of leptin and its Ob(Lep) gene, which, in human, resides on chromosome 7, has given some insight into the mechanism of energy homeostasis, provided a framework for studying the pathogenesis of obesity, the biological response to starvation, and helped with understanding of the neural mechanisms that control feeding. Leptin (a 16-kDa protein) is an adipose tissue hormone that plays a critical role in energy homeostasis, metabolism, and regulation of neuroendocrine functions. Leptin circulation in the body reflects on the adipose tissue mass and the amount of stored energy. It also guides the central nervous system to maintain the balance between the ingestion of food and the expenditure of energy. Leptin has an immediate effect on the brain for appetite regulation by binding with leptin receptors in the hypothalamus. This neuroendocrine system protects individuals from the risks associated with starvation or obesity. In addition, leptin stimulates fatty acid oxidation and reduces body fat accumulation in nonadipose tissues, resulting in improved insulin sensitivity. Leptin levels fall during starvation (i.e., individuals with ) and elicit adaptive responses, leading to reduced energy expenditure (e.g., cessation of menstruation, insulin resistance, and alterations of immune function). Most obese individuals have high endogenous levels of leptin. Mutations in the genes encoding leptin or its receptor cause in mice and humans, and leptin can effectively treat obesity in leptin-deficient patients. , the pancreatic hormone that , is also involved in the regulation of adipose tissue metabolism. It enhances the conversion of glucose into the triglycerides of the lipid droplet by the adipocyte. Like leptin, insulin by acting on brain centers in the hypothalamus. In contrast to leptin, insulin is required for the accumulation of adipose tissue. Antiobesity drug research is currently focusing on substances that can inhibit insulin and leptin signaling in the hypothalamus.

anorexia nervosa

morbid obesity regulates blood glucose levels

Insulin

weight

regulates

Deposition and mobilization of lipid are influenced by neural and hormonal factors. One of the major metabolic functions of adipose tissue involves the uptake of fatty acids from the blood and their conversion to triglycerides within the adipocyte. Triglycerides

are then stored within the cell’s lipid droplet. When adipose tissue is stimulated by neural or hormonal mechanisms, triglycerides are broken down into glycerol and fatty acids, a process called . The fatty acids pass through the adipocyte cell membrane to

mobilization

albumin

enter a capillary. Here, they are bound to the carrier protein and transported to other cells, which use fatty acids as metabolic fuel. is particularly important during periods of fasting and exposure to severe cold. During the early stages of experimental starvation in rodents, adipose cells in a denervated fat pad continue to deposit fat. Adipose cells in the intact contralateral fat pad mobilize fat. It is now known that (which is liberated by the endings of nerve cells of the sympathetic nervous system) initiates a series of metabolic steps that lead to the activation of . This enzyme splits triglycerides, which constitute more than 90% of the lipids stored in the adipocyte. This enzymatic activity is an early step in the mobilization of lipids. involves a complex system of hormones and enzymes that control fatty acid release from adipocytes. These include insulin, glucagon, growth hormone, thyroid hormones, and adrenal steroids. is an important hormone that promotes lipid synthesis by stimulating lipid synthesis enzymes (fatty acid synthase, acetyl-CoA carboxylase) and suppresses lipid degradation by inhibiting the action of hormone-sensitive lipase and thus blocking the release of fatty acids. , another pancreatic hormone, and from the pituitary gland both increase lipid utilization (lipolysis). increase lipogenesis (formation of lipids) followed by promoting lipolytic enzymes, which break down the stored lipids in adipocytes into free fatty acids. , such as cortisol, stimulate lipolysis in adipocytes to liberate free fatty acids and triglycerides for energy utilization. In addition, elevated levels of α ( ) have been implicated as a causative factor in the development of insulin resistance associated with obesity and diabetes.

Neural mobilization

lipase

Hormonal mobilization

norepinephrine

Insulin

growth hormone Thyroid hormones steroids factor TNF-α

Glucagon

Adrenal tumor necrosis

BROWN ADIPOSE TISSUE

Brown adipose tissue, abundant in newborns, is markedly reduced in adults. Brown adipose tissue, a key thermogenic tissue, is present in large amounts in the newborn,

which helps to offset the extensive heat loss that results from the newborn’s high surfaceto-mass ratio and to avoid lethal hypothermia (a major risk of death for preterm babies). In newborns, brown adipose tissue makes up about 5% of the total-body mass and is located on the back, along the upper half of the spine, and toward the shoulders. The amount of brown adipose tissue gradually decreases as the body grows, but it remains widely distributed throughout the first decade of life in the cervical, axillary, paravertebral, mediastinal, sternal, and abdominal regions of the body. It then disappears from most sites, except for regions around the kidney, adrenal glands, large vessels (e.g., aorta), and regions of the neck (deep cervical and supraclavicular), back (interscapular and paravertebral), and thorax (mediastinum). used to detect cancer cells based on their uptake of large amounts of radioactively labeled glucose (18 F-FDG) is able to detect patterns characteristic of brown adipose tissue within the region of the adult body described above (see Folder 9.3, page 293). These findings have been confirmed with tissue biopsies.

Positron emission tomography (PET)

Adipocytes of brown, multilocular adipose tissue contain numerous fat droplets. The cells of brown (multilocular) adipose tissue are smaller than those of white adipose tissue. The cytoplasm of each cell contains many small lipid droplets, hence the name multilocular. In contrast, white unilocular adipocytes contain only one large lipid droplet, and paucilocular [Lat. paucus, having few] adipocytes contain fewer lipid droplets. The nucleus of a mature brown adipocyte is typically located in an eccentric position within the cell, but it is not flattened like the nucleus of a white adipocyte. In routine H&E-stained sections, the cytoplasm of the brown adipocyte consists largely of empty vacuoles because the lipid that ordinarily occupies the vacuolated spaces is lost during preparation (Fig. 9.6). Brown adipocytes depleted of their lipid more closely resemble epithelial cells than connective tissue cells. The brown adipocyte contains numerous large spherical mitochondria

with numerous cristae, a small Golgi apparatus, and only small amounts of rER and sER. The mitochondria contain large amounts of cytochrome oxidase, which imparts the brown color to the cells.

FIGURE 9.6. Brown adipose tissue. a. Photomicrograph of brown adipose tissue from a newborn in a hematoxylin and eosin (H&E)-stained paraffin preparation. The cells contain fat droplets of varying size. ×150. b. This photomicrograph, obtained at a higher magnification, shows the brown adipose cells with round and often centrally located nuclei. Most of the cells are polygonal and closely packed, with numerous lipid droplets. In some cells, large lipid droplets displace nuclei toward the cell periphery. A network of collagen fibers and capillaries surrounds the brown adipose cells. ×320.

FOLDER 9.1

CLINICAL CORRELATION: OBESITY Obesity is epidemic in the United States. According to current estimates by the National Institutes of Health (NIH), about two-thirds of Americans are considered to be obese, and 300,000 die annually from obesity-related metabolic diseases (i.e., diabetes, hypertension, cardiovascular diseases, and cancer). An individual is considered obese when the percentage of body fat exceeds the average percentage for the individual’s age and sex. The prevalence of obesity has increased in the past decade from 12% to 18%. The increases are seen in both sexes and at all socioeconomic levels, with the greatest increase reported in the 18- to 29-year-old age group. The , expressed as weight (kg)/height (m) 2, is closely correlated with the total amount of body fat and is commonly used to classify overweight and obesity among adults. A BMI of 18.5–24.9 kg/m 2 is considered normal. A BMI of 25–29.9 kg/m 2 is considered overweight, and a BMI of ≥30 kg/m 2 is considered obese. Obesity is associated with an increased risk of mortality as well as with many diseases such as hypertension, cardiovascular diseases, diabetes, and cancer. It is a chronic condition related to both a person’s genetic makeup and their environment. encode the molecular components of the short- and long-term weight regulation systems, which include leptin, ghrelin, and other factors that regulate energy balance. In addition, several of these factors modulate glucose metabolism by adipose tissue and contribute to the development of insulin resistance, which is associated with . Intensive research directed toward adipocyte-derived proteins may in the future provide potential drugs for reducing obesity and overcoming insulin resistance. of adipose tissue from an obese individual shows hypertrophic adipocytes with a gigantic lipid droplet. Debris from damaged or dead adipocytes is often seen dispersed among hypertrophic adipocytes. Dead adipocytes are found ~30 times more often in obese than in nonobese individuals. Large macrophages are seen to infiltrate the obese adipose tissue; their roles are to remove damaged cells and cellular debris and to alter secretion of adipokines (Fig. F9.1.1). In addition, macrophages inhibit differentiation of adipocytes from their progenitor cells, leading to hypertrophy of the existing fat cells. Owing to the large size of the macrophages, as well as the length of time required for the removal of cellular debris, the obese adipose tissue shows signs of . The number of macrophages

body mass index (BMI)

Obesity genes

type 2 diabetes

Microscopic examination

chronic low-grade inflammation

positively correlates to the size of adipocytes and coincides with the emergence of insulin resistance.

FIGURE F9.1.1. Changes in adipocyte metabolism in obesity.

Adipocytes from obese individuals are hypertrophic and produce more leptin. Increased leptin secretion causes nonadipose tissue to become resistant to leptin. Hypertrophic adipocytes also secrete high amounts of fatty acids and adipokines that promote insulin resistance. This leads to pathologic accumulation of lipids in organs, such as the kidney (renal lipotoxicity), liver (nonalcoholic fatty liver disease), pancreas, and heart. (Modified from Vázquez-Vela ME, Torres N, Tovar AR. White adipose tissue as endocrine organ and its role in obesity. . 2008;39:715–728.)

Arch Med Res

Brown adipose tissue is subdivided into lobules by partitions of connective tissue, but the connective tissue stroma between individual cells within the lobules is sparse. The tissue has a rich supply of capillaries that enhance its color. Numerous unmyelinated, noradrenergic sympathetic nerve fibers are present among the fat cells. Brown adipose tissue features are listed in Table 9.2.

Brown adipocytes differentiate from mesenchymal skeletal myogenic progenitor (Myf5 positive) stem cells under the control of PRDM16/PGC-1 transcription factors. Brown adipocytes are also derived from mesenchymal stem cells but from a different cellular lineage than those differentiating into white adipocytes. Lineage tracing experiments show that brown adipose tissue and skeletal muscle derive from common skeletal myogenic progenitor stem cells found in dermatomyotomes of the developing embryo. These cells are characterized by expression of the myogenic lineage marker myogenic factor 5 (Myf5), which remains detectable in mature brown adipocytes and in all stages of their

differentiation. In contrast to white adipocytes, differentiation of brown adipocytes is influenced by a different pair of transcription factors. When the zinc-finger protein known as is activated, myogenic progenitor cells synthesize several members of the family of transcription factors, activating brown adipocyte differentiation and suppressing skeletal muscle development. Loss of PRDM16 from brown adipocyte precursors causes a loss of brown fat characteristics and promotes skeletal muscle differentiation. Therefore, is regarded as a “ ” regulator in differentiation of brown adipocytes. These factors, in turn, regulate the expression of genes that encode a specific mitochondrial protein called or (a 33-kDa inner mitochondrial membrane protein), which is essential for brown adipocyte metabolism (thermogenesis). Clinical observations confirm that under normal conditions, brown adipose tissue can expand in response to increased blood levels of . This becomes evident in patients with , an endocrine tumor of the adrenal medulla that secretes excessive amounts of epinephrine and norepinephrine. In these individuals, the UCP-1 gene is activated by norepinephrine stimulation, which also protects brown adipocytes by inhibiting apoptosis. In the past, it was thought that uncoupling proteins were expressed only in brown adipose tissue. Several similar uncoupling protein homologs are present in other tissues. UCP-2 is linked to hyperinsulinemia and obesity and may be involved in the regulation of body weight. UCP-3 is expressed in skeletal muscles and may account for the thermogenic effects of thyroid hormone. UCP-4 and UCP-5 are brain mitochondria–specific molecules.

PR domain containing 16 (PRDM16) PPARγ coactivator 1 (PGC-1)

master-switch uncoupling protein (UCP-1) pheochromocytoma

PRDM16/PGC-1

thermogenin

norepinephrine

FOLDER 9.2

CLINICAL CORRELATION: ADIPOSE TISSUE TUMORS

adipose tissue tumors

The study of the numerous varieties of benign and malignant provides further insight into, and confirmation of, the sequence of adipose tissue differentiation described earlier. As with epithelial tumors and tumors of fibroblast origin, the variety of adipose tissue tumors reflects the normal pattern of adipose tissue differentiation; that is, discrete tumor types can be described that consist primarily of cells resembling a given stage in normal adipose tissue differentiation. is the most common benign tumor of adipose tissue in adulthood. It is more common than all other soft-tissue tumors combined. Lipomas are subclassified by the morphology of the predominant cell in the tumor. For instance, consist of mature white adipocytes. have adipocytes surrounded by an excess of fibrous tissue, and contain adipocytes separated by an unusually large number of vascular channels. Most lipomas show structural chromosome aberrations that include balanced rearrangements, often involving chromosome 12. Lipomas are usually found in subcutaneous tissues in middle-aged and elderly individuals. They are characterized as well-defined, soft, and painless masses of mature adipocytes and are most often located in the subcutaneous fascia of the back, thorax, and proximal parts of the upper and lower limbs. Treatment of lipomas usually involves a simple surgical excision. Malignant tumors of adipose tissue, called , are rare. They are typically detected in older individuals and are mainly found in the deep adipose tissues of the lower limbs, abdomen, and shoulder area. Liposarcomas may contain both well-differentiated, mature adipocytes and early, undifferentiated cells (Fig. F9.2.1). Tumors containing more cells in earlier stages of differentiation are more aggressive and more frequently metastasize. Typically, liposarcomas are surgically removed, but if a tumor has already metastasized, chemotherapy and radiation therapy can be utilized as presurgical or postsurgical treatments.

Lipoma

Fibrolipomas angiolipomas

conventional lipomas

liposarcomas

FIGURE F9.2.1. Well-differentiated liposarcoma.

This photomicrograph was obtained from a tumor surgically removed from the retroperitoneal space of the abdomen. Well-differentiated liposarcoma is characterized by a predominance of mature adipocytes that vary in size and shape. They are interspersed between broad fibrous septa of connective tissue containing cells (most are fibroblasts) with atypical hyperchromatic nuclei. Relatively few scattered spindle cells with hyperchromatic and pleomorphic nuclei are found within the connective tissue. ×340. (Courtesy of Dr. Fabiola Medeiros.)

lipoma

hibernomas

Although the term relates primarily to white adipose tissue tumors, tumors of brown adipose tissue are also found. Not surprisingly, these are called . They are rare, benign, and slow-growing soft-tissue tumors of brown fat most commonly arising in the periscapular region, axillary fossa, neck, or mediastinum. Most hibernomas contain a mixture of white and brown adipose tissue; pure hibernomas are very rare.

Metabolism of lipid in brown adipose tissue generates heat in a process known as thermogenesis. Hibernating animals have large amounts of brown adipose tissue. The tissue serves as a ready source of lipid. When oxidized, it produces heat to warm the blood flowing through the brown fat on arousal from hibernation and in the maintenance of body temperature in the cold. This type of heat production is known as . Brown adipose tissue is also present in nonhibernating animals and humans and again serves as a source of heat. As in the mobilization of lipid in white adipose tissue, lipid is mobilized and heat is generated by brown adipocytes when they are stimulated by the sympathetic nervous system. This process, known as the response, is a target of current obesity research. Mechanisms to increase brown fat differentiation may potentially be an attractive treatment in both diet-induced and genetically acquired obesity.

nonshivering thermogenesis

human adaptive thermogenesis

Thermogenic activity of brown adipose tissue is facilitated by UCP-1 that is found in the inner mitochondrial membrane.

The mitochondria in eukaryotic cells produce and store energy as an electrochemical proton gradient across the inner mitochondrial membrane. As described earlier (see pages 61-64), this energy is used to synthesize adenosine triphosphate (ATP) when the protons return to the mitochondrial matrix through the ATP synthase enzyme located at the inner mitochondrial membrane. The unique large, round in the cytoplasm of brown adipose tissue cells contain , which uncouples the oxidation of fatty acids from the production of ATP. As a result, protons are allowed to travel from the intermembrane space back to the mitochondrial matrix along the gradient without passing through ATP synthase and thus without producing ATP. This can occur because an alternative pathway for the protons’ return is available through UCP-1 that facilitates proton transport across the inner mitochondrial membrane. The movement of protons from the inner mitochondrial compartment dissipates the mitochondrial proton gradient, thus uncoupling respiration from ATP synthesis. The energy produced by the mitochondria is then dissipated as heat in a process known as .

mitochondria uncoupling protein (UCP-1)

thermogenesis The metabolic activity of brown adipose tissue is regulated by the sympathetic nervous system and is related to ambient outdoor temperature. The metabolic activity of brown adipose tissue is largely regulated by norepinephrine released from sympathetic nerve terminals, which stimulates lipolysis and hydrolysis of triglycerides as well as increases mitochondrial expression and activity of UCP-1 molecules.

In experimental animals, UCP-1 activity has been shown to increase during cold stress. In humans, UCP-1 is responsible for the adaptive thermogenesis response, a regulated heat production that is triggered by changes in the external environment. In addition, cold stimulates glucose utilization in brown adipocytes by overexpression of glucose transporters (Glut-4). Clinical studies using in adults have shown a direct relationship between ambient temperature and the amount of brown fat accumulated in the body. An increase in the amount of brown adipose tissue has been reported on the neck and supraclavicular regions during the winter months, especially in lean individuals. This is supported by autopsy findings of larger amounts of brown fat in outdoor workers exposed to cold. Modern molecular imaging techniques now allow clinicians to precisely locate where brown fat is distributed in the body, which is essential for proper differential diagnosis of cancerous lesions (see Folder 9.3, page 293).

PET scans

FOLDER 9.3

CLINICAL CORRELATION: PET SCANNING AND BROWN ADIPOSE TISSUE INTERFERENCE Positron emission tomography, also called a “PET scan,” is a diagnostic tool that can locate malignant cells in the body. PET is based on the detection of high-energy γ rays created when positrons (subatomic particles of antimatter), produced during decay of radioactive

18-fluorine-2-fluoro-2-deoxy-d-glucose ( F-FDG)

materials, are encountered by electrons. The procedure requires the injection of a radioactive 18 tracer, most commonly . This radioactive glucose isotope is used in PET imaging because malignant cells metabolize glucose at a greater rate than normal cells. After injection of the isotope, a detector scans the entire body and records radiation emitted by the 18F-FDG tracer as it becomes incorporated within the body’s cells. A computer reassembles the signals into images, which are, in effect, biological maps of 18F-FDG distribution in the body. Recently, owing to greater diagnostic accuracy and improved biopsy methods, combined PET and computed tomography (CT) scanners are utilized more frequently. One drawback to PET imaging is that many normal tissues and benign lesions also show increased glucose metabolism and can thus be misinterpreted as malignant. For example, brown and beige adipose tissues, with its increased glucose uptake mediated by increased activity of glucose transporters, can be a potential source of of PET scans. Because both brown and beige adipose tissues are present in the neck, supraclavicular regions, and mediastinum (see pages 285-286), it is commonly observed on PET scans, especially in underweight patients and during winter months, when beige adipose tissue is more predominant. This 18F-FDG uptake most likely represents activated brown adipose tissue during increased sympathetic nerve activity related to cold stress. A typical PET image of brown/beige fat is usually bilateral and symmetrical; however, in the mediastinum, the image may be asymmetrical or focal and can mimic malignancy. False-positive results from brown/beige fat 18F-FDG uptake in these areas have been reported in young women undergoing scans for diagnosis and staging of breast cancer. Therefore, understanding that brown and beige fat can show increased radioactive tracer uptake is crucial for establishing an accurate diagnosis and avoiding false-positive results (Fig. F9.3.1).

false-positive interpretation

FIGURE F9.3.1. Coronal positron emission tomography/computed tomography (PET/CT) image of a healthy young woman. This upper part of the coronal section of a whole-body PET/CT scan shows extensive bilateral increased F-FDG uptake ( red color ) in the neck, supraclavicular, and upper axillary regions. Note that moderate increase in radioactive tracer uptake is also detectable in the myocardium ( yellow color ). Regions of extensive metabolic activity correlate with the 18

distribution pattern of low-density brown adipose tissue. PET/CT imaging allows for precise localization of increased 18F-FDG uptake areas and differentiation between brown adipose tissue tracer uptake and malignant tumor findings. (Courtesy of Dr. Jolanta Durski.)

Brown adipose tissue secretes active substances called batokines that contribute to the regulation of various body functions. Like white adipose tissue, brown adipose tissue actively synthesizes and secretes biologically active substances collectively called batokines (Fig. 9.7). They contribute to the regulation of various functions, such as thermogenic activity, immune activity,

vascularization, substrate utilization, and other functions related to whole-body energy expenditure and glucose homeostasis.

FIGURE 9.7. Major batokines secreted by brown/beige adipose tissue.

This schematic drawing shows various types of batokines secreted by brown and beige adipose tissue that contribute to the regulation of various functions, such as thermogenic activity, immune activity, vascularization, substrate utilization, and other functions. These include fibroblast growth factor-21 (FGF21), which stimulates transdifferentiation of adipose tissue; C-terminal fragment of the Slit 2 protein (Slit2C), which stimulates adipose thermogenesis; insulin-like growth factor–binding protein 2 (IGF-BP2), which influences bone growth; neuregulin-4 (NRG4), which promotes neuronal processes outgrowth; nerve growth factor (NGF) and S100b protein, which target sympathetic nervous endings promoting innervation; vascular endothelial growth factor A (VEGF-A), which induces vascularization of adipose tissue; bone morphogenetic protein 8B (BMP-8B), which acts locally on brown adipose tissue, enhancing thermogenesis and in the hypothalamus, activating the sympathetic nervous system; myostatin, which decreases performance of skeletal muscles; interleukin-6 (IL-6), insulin-like growth factor 1 (IGF-1), chemokine (C-X-C motif) ligand-14 (CXCL14), and growth/differentiation factor-15 (GDF15) that acts on immune system; prostaglandins (PGI2 and PGE2), which promote development of brown adipocytes; and exosomal microRNAs, which regulate gene expression in distant organs, thus affecting whole-body metabolism.

Several batokines act locally (via autocrine and paracrine mechanisms) to promote hypertrophy and hyperplasia of brown adipose tissue, including its vascularization and innervation, to enhance their own thermogenic activity. Examples of these molecules include nerve growth factor (NGF) and fibroblast growth factor 21 (FGF21) that increase sympathetic innervation and trigger cell division, and vascular endothelial growth factor A (VEGF-A) that promotes vascularization of brown adipose tissue. Some batokines act as endocrine factors to target peripheral tissues, such as white adipose tissue, lymphatic tissue, liver, pancreas, heart, and bone. These include FGF21, neuregulin-4 (NRG4), VEGF-A, myostatin, and bone morphogenetic protein 8B (BMP-8B). In addition, exosomal miRNA molecules secreted by brown adipocytes have systemic effects on body metabolism by acting on distant organs, such as the liver, skeletal muscles, heart, and others.

BEIGE ADIPOSE TISSUE

Beige adipose tissue exhibit cellular and molecular features that are intermediate between white and brown adipose tissue.

Recently, a new type of adipose tissue has been identified within the subcutaneous white adipose tissue throughout the body. This adipose tissue consists of pockets of brown-like adipocytes that were named or “ adipocytes. The morphology of these paucilocular cells is intermediate between that of white and brown adipocytes (they contain fewer lipid droplets than brown adipocytes). They have a thermogenic ability owing to the expression of the UCP-1 protein, although this activity is not completely UCP-1 dependent. When compared to brown adipocytes, beige adipocytes have much lower levels of UCP-1 protein expression in their mitochondria, but their UCP-1 is highly inducible in response to cold exposure or hormonal (norepinephrine) stimulation. It is postulated that increases in intracellular Ca2+ activate ATP synthesis for ATP-dependent metabolic thermogenesis to generate heat and increase energy expenditure. The comparison between all three types of adipose tissues and their UCP-1 distribution is shown in Figure 9.8.

beige adipocytes

brite” (brown-in-white)

FIGURE 9.8. Three types of adipose tissue with UCP-1 immunofluorescence. Photomicrographs of three different adipose tissues were obtained from C57BL/6 mice and routinely processed for hematoxylin and eosin (H&E) preparation. a. White adipose tissue from the subcutaneous fat pad. White adipocytes ( WA ) are tightly packed together and appear polyhedral in shape with large lipid droplets (represented by white spaces because lipids are washed out in routine slide preparation). Note a narrow rim of cytoplasm with flattened nuclei ( N ). A few blood vessels ( BV ) and smaller capillaries can be found in this section. b. Brown adipose tissue from the interscapular region. Brown adipocytes

are polygonal and closely packed, with numerous lipid droplets. Note that nuclei are round and typically pushed to the cell periphery by lipid droplets. Brown tissue has an extensive blood vessel ( ) network. Beige adipose tissue from the subcutaneous fat pad. Beige adipocytes ( ) form a pocket inclusion (see area indicated by ) within the white adipose tissue ( ). Corresponding insets provide pattern of UCP-1 distributions in each tissue. Note that white adipose tissue does not exhibit UCP-1 activity (no staining), brown tissue has an abundance of UCP-1, and beige adipose tissue exhibits staining in the area of accumulation of brown-like adipocytes (dispersed between white adipocytes [ ]). UCP-1 was visualized using an indirect immunofluorescence technique that utilizes primary goat polyclonal antibodies against the UCP-1 protein, followed by secondary anti-goat antibodies conjugated with Alexa Fluor 488 fluorescent stain. ×180. (Courtesy of Prof. Dr. Carlos A. Mandarim-de-Lacerda, Universidade do Estado do Rio de Janeiro, Brazil.)

BV

c.

BeA

dashed line

WA

WA

Beige adipocytes are genetically different from brown adipocytes. They develop from white adipocyte precursor stem cells; thus, they do not have the same skeletal myogenic pregenital cells origin as brown adipocytes (Fig. 9.9). Beige adipocytes are and express specific beige markers, such as t-box transcription factor-1 (tBX1) and transmembrane protein-26 (TMEM26) Research has shown that beige adipocytes are generated in two independent coexisting pathways:

.

Myf5 negative

FIGURE 9.9. Development of beige adipocytes.

Beige adipocytes are genetically different from brown adipocytes. There are two pathways by which they develop. The first and most common is the white-to-brown transdifferentiation pathway, which accounts for 80%–95% of beige adipocyte development. This process is induced after exposure to cold or norepinephrine stimulation. The second pathway includes de novo differentiation from perivascular mesenchymal stem cells (specific white adipose precursors) that reside within white adipose tissue. Precursor cells, especially those expressing and markers, have a greater propensity to proliferate and differentiate into beige adipocytes. Note that both white and beige adipocytes are myogenic factor 5 (Myf5) negative. The development of brown adipocytes progresses from common skeletal myogenic pregenital cells; therefore, they express Myf5 protein markers. Transdifferentiation of brown-to-beige adipocytes is questionable; there are only a few reports of Myf5 markers on beige adipocytes.

tBX1

TMEM26

White-to-brown transdifferentiation pathway (note that precursor cells for white adipose cells are Myf5 negative; see page 282). The vast majority (80%–95%) of newly formed beige adipocytes arise through this pathway. from the specific white adipose precursor cells that reside within white adipose tissue. White adipocyte precursor cells, especially those expressing tBX1 and TMEM26 markers, have a greater ability to proliferate and differentiate into beige adipocytes. PRDM16 seems to be a key transcriptional co-regulator in this process.

De novo differentiation

Beige adipocytes actively synthesize and secrete substances similar to brown adipocytes (see Fig. 9.7).

batokines,

biologically

active

TRANSDIFFERENTIATION OF ADIPOSE TISSUE

Adipocytes undergo white-to-brown and brown-to-white transformation in response to the thermogenic needs of an organism. Exposure to chronic cold temperatures increases the thermogenic demands of an organism. Studies have shown that in cold conditions, mature white adipocytes can transform into brown-like adipocytes to generate body heat. Conversely, brown-like adipocytes transform into white adipocytes when the energy balance is positive and the body requires an increase in triglyceride storage capacity. This phenomenon, known as , has been observed in experimental animals and humans. After 3–5 days of cold exposure, white adipose tissue in mice undergoes a “ ” to produce pockets of paucilocular, UCP1–positive within the white adipose tissue. This change in the phenotype of adipocytes occurs in the absence of cell division (no increase in DNA content) or apoptosis, suggesting that white adipocytes transform directly into brown-like (beige) adipocytes. These findings are also supported by observations of differential gene expression. In addition, mice with abundant natural or induced brown adipose tissue are resistant to obesity, whereas genetically modified mice without functional brown adipocytes are prone to and . The putative genome reprogramming mechanism that drives the browning phenomenon could be used for future therapeutic strategies aimed at controlling the amount of brown adipose tissue in the body. This discovery may lead to the control of obesity and type 2 diabetes.

beige adipocytes obesity

browning phenomenon

type 2 diabetes

transdifferentiation

White-to-brown transdifferentiation of adipose tissue is induced by cold exposure and physical activity. Cold exposure and physical activity induce conversion of white-to-brown adipocytes via several molecular pathways. Cold temperatures are sensed by the central nervous system, causing increased stimulation of the noradrenergic sympathetic nerve system. Increased amounts of brown/beige adipose tissue have been found in outdoor workers in northern countries and in individuals with pheochromocytoma (a noradrenaline-secreting tumor). Recent studies indicate that the density of the sympathetic nerves innervating white adipose tissue is one of the most important factors in white-to-brown transdifferentiation. Physical exercise stimulation is more complex and involves secretion of atrial and ventricular natriuretic peptides in the heart that act on the kidney, which, in turn, activate transcription factors essential for brown adipocyte differentiation. Other triggers of transdifferentiation include reprograming of adipose tissue genes by activating specific transcription factors (“master regulators”) and growth factors, such as FGF21. Many epidemiologic studies demonstrated an inverse correlation between the presence or amount of brown/beige adipose tissue and body weight as well as obesity-associated complications. In the future, signaling pathways and molecules involved in adipocyte transdifferentiation may open new avenues in pharmacologic treatment of obesity, diabetes, and other metabolic diseases.

ADIPOSE TISSUE

OVERVIEW OF ADIPOSE TISSUE Adipose tissue

is a specialized connective tissue that plays an important role in energy homeostasis (stores energy in lipid droplets in the form of triglycerides) and hormone production (adipokines and batokines). There are three types of adipose tissue: , , and adipose tissue.

white (unilocular) brown (multilocular)

beige (paucilocular)

WHITE ADIPOSE TISSUE

White adipose tissue represents at least 10% of body weight in a normal healthy adult. White adipose tissue with supporting collagen and reticular fibers forms the subcutaneous fascia , is concentrated in the mammary fat pads, and surrounds several internal organs. White adipocytes are very large cells (≥100 μm in diameter) with a single large

lipid droplet (unilocular), a thin rim of cytoplasm, and a flattened, peripherally displaced nucleus. A single large lipid droplet within the white adipocyte represents cytoplasmic inclusion and is not membrane bound. White adipose tissue secretes a variety of , which include hormones (e.g., leptin), growth factors, cytokines, and exosomal microRNAs (miRNAs). White adipocytes differentiate from perivascular mesenchymal stem (myogenic factor 5 [Myf5] negative) cells under the control of (“master switch” for white adipocyte differentiation). The amount of adipose tissue is regulated by two hormonal pathways: pathway ( and ) and pathway ( and ).

adipokines

regulation peptide YY leptin insulin

ghrelin

PPARγ/RXR transcription factors short-term weight long-term weight regulation

Triglycerides stored in adipocytes are released by lipases that are activated during neural mobilization (which involves norepinephrine released from sympathetic nerves) and/or hormonal mobilization (which involves glucagon and growth hormone ).

BROWN ADIPOSE TISSUE

Brown adipose tissue is abundant in newborns (5% of total-body mass) but is markedly reduced in adults. Brown adipocytes are smaller than white adipocytes and contain many lipid droplets (multilocular) and cytoplasm with a round nucleus and abundance of mitochondria. Brown adipose tissue secretes a variety of batokines , which include growth factors, cytokines, and exosomal miRNAs. Brown adipocytes differentiate from skeletal myogenic mesenchymal (Myf5 positive) stem cells under the control of (the “master switch” for brown adipocyte differentiation). Brown adipocytes express a specific mitochondrial protein called or , which is essential for brown adipocyte metabolism. Metabolism of lipids in brown adipose tissue generates heat ( ) by uncoupling the oxidation of fatty acids in the mitochondria from ATP production. The of brown adipose tissue is regulated by released from sympathetic nerves and is related to ambient outdoor temperature (cold weather increases the amount of brown adipose tissue).

(UCP-1)

thermogenin

metabolic activity

PRDM16/PGC-1 transcription factors uncoupling protein thermogenesis norepinephrine

BEIGE ADIPOSE TISSUE

Beige adipose tissue is widely distributed throughout the body in the form of brownlike adipocytes pockets within white adipose tissue Beige adipocytes have intermediate features between white and brown adipocytes, containing fewer lipid droplets (paucilocular) than brown adipocytes. Beige adipose tissue secretes a variety of batokines similar to that of brown adipose tissue. White adipocytes can undergo white-to-brown transdifferentiation in response to the

thermogenic needs of the body. Transdifferentiation is a major source of beige adipocytes in the body. Cold exposure, hormonal activity (norepinephrine), and physical activity induce white-to-brown transdifferentiation that increases the number of beige adipocytes in white adipose tissue. Beige adipocytes also express the ; however, thermogenic activity is not completely UCP1 dependent.

uncoupling protein (UCP-1)

PLATE 9.1 ADIPOSE TISSUE

Adipose tissue is widely distributed throughout the body and in varying amounts in different individuals. It is a specialized connective tissue consisting of triglyceride -storing cells called adipocytes . Adipose tissue is also considered an endocrine organ that secretes adipokines and batokines that include many factors with autocrine, paracrine, and endocrine

functions. Adipose tissue has a rich blood supply, which complements its metabolic and endocrine functions. Three types of adipose tissue are now recognized. The most common and abundant is referred to as . White adipocytes catabolize triglycerides, and when energy expenditure exceeds energy intake, are released into circulation. In addition, and fatty acids released from the adipocytes participate in glucose metabolism. White adipocytes are very large cells whose cytoplasm contains a single large vacuole in which the fat is stored in the form of triglycerides. When observed in a typical

white adipose tissue glycerol

fatty acids

hematoxylin and eosin (H&E) section, white adipose tissue appears as a mesh-like structure (see Orientation Micrograph). The second type is . It consists of smaller cells. Their cytoplasm is characterized by numerous vesicles that occupy much of the cells’ volume. It also is very richly vascularized. Brown adipose tissue is found in human newborns where it assists in maintaining body temperature. The third type is . Beige adipocytes represent pockets of brown-like adipocytes within white adipose tissue. They exhibit morphology intermediate between that of white and brown adipocytes and contain fewer vesicles filled with lipids than brown adipocytes. In general, white adipose tissue stores energy reserves in lipid droplets, whereas the metabolic function of brown and beige adipose tissue is lipid oxidation to produce heat. Shown here is white adipose tissue from the hypodermis of skin. It consists of numerous adipocytes closely packed in lobules. Dense irregular connective tissue ( ) surrounds the adipose tissue. The loss of the fat within the cell during routine H&E slide preparation gives the adipose tissue a mesh-like appearance. Note the small blood vessels ( ) observed at the periphery of the tissue. They provide a rich capillary network within the adipose tissue. Several sweat gland ducts ( ) are also present in the dense connective tissue.

brown adipose tissue

beige adipose tissue

ORIENTATION MICROGRAPH: DICT BV

White adipose tissue

SGD

, human, H&E ×363; inset ×700.

white adipose tissue DICT adipocytes A

This is a higher magnification micrograph of from the specimen shown in the orientation micrograph. It reveals portions of several lobules of adipose cells. Dense irregular connective tissue ( ) separates the lobules from surrounding structures. In well-preserved specimens, the ( ) have a spherical profile in which they exhibit a very thin rim of cytoplasm surrounding a single large fat-containing vacuole. Because the fat is lost during tissue preparation, one only sees the rim of cytoplasm and an almost clear space. Between the cells, there is an extremely thin, delicate connective tissue stroma holding the adipocytes together, and within this stroma are small blood vessels ( ), mostly capillaries and venules. Most of the nuclei that are observed within the adipose tissue belong to fibroblasts, adipocytes, or cells of small blood vessels. However, distinguishing between fibroblast nuclei and adipocyte nuclei is often difficult. The shows a

BV

inset

N

white adipocyte whose nucleus ( ) is relatively easy to identify. It appears to reside within the rim of cytoplasm ( ), giving the adipocyte the classic “signet-ring” appearance. A second nucleus ( ), partially out of the plane of section, appears to reside between the cytoplasmic rims of two adjacent cells. This is probably the nucleus of a fibroblast. Because of the relatively large size of the adipocyte, it is very infrequent that the nucleus of the cell is included in the plane of section of a given cell. Other cells that may be seen within the delicate connective tissue stroma are mast cells ( ).

Cy

N′

MC

Brown adipose tissue

, human, hematoxylin and eosin (H&E) ×450; inset

×1,100.

brown adipose tissue

The shown here consists of small fat cells that are very closely packed with minimal intercellular space. Because of this arrangement, it is hard to define individual cells at this magnification. At higher magnification (not shown), it is possible to identify some individual cells. One cell, whose boundaries could be identified at higher magnification, is circumscribed by a . Each cell contains many small, fat-containing vacuoles surrounded by cytoplasm. Included in this cell is its nucleus ( ). As noted, brown adipose tissue is highly vascularized, and in this specimen, one can see numerous blood vessels ( ) as evidenced by the red blood cells that they contain. It is even more difficult to distinguish fibroblasts within the lobule from nuclei of the fat cells. Even at higher magnification ( ), it is difficult to determine which nuclei belong to which cells. A capillary ( ) can be identified in the . Again, it is recognized by the presence of red blood cells. Where the lobules are slightly separated from one another ( ), small elongate nuclei of fibroblasts in the connective tissue septa can be recognized. These belong to fibroblasts in the connective tissue forming the septa.

dotted line

BV

inset

arrows

A, adipocytes BV, blood vessels C, capillary Cy, cytoplasm DICT, dense irregular connective tissue MC, mast cells N, nucleus of adipocyte N′, nucleus of fibroblast SGD, sweat gland ducts arrows, connective tissue septa

N

C

inset

10 OVERVIEW OF BLOOD PLASMA ERYTHROCYTES LEUKOCYTES

BLOOD

Neutrophils Eosinophils Basophils Lymphocytes Monocytes

THROMBOCYTES COMPLETE BLOOD COUNT FORMATION OF BLOOD CELLS (HEMOPOIESIS)

Monophyletic Theory of Hemopoiesis Development of Erythrocytes (Erythropoiesis) Kinetics of Erythropoiesis Development of Thrombocytes (Thrombopoiesis) Development of Granulocytes (Granulopoiesis) Kinetics of Granulopoiesis Development of Monocytes Development of Lymphocytes (Lymphopoiesis)

BONE MARROW Folder 10.1 Clinical Correlation: ABO and Rh Blood Group Systems Folder 10.2 Clinical Correlation: Hemoglobin in Patients With Diabetes Folder 10.3 Clinical Correlation: Hemoglobin Disorders Folder 10.4 Clinical Correlation: Inherited Disorders of Neutrophils; Chronic Granulomatous Disease Folder 10.5 Clinical Correlation: Hemoglobin Breakdown and Jaundice Folder 10.6 Clinical Correlation: Cellularity of the Bone Marrow

HISTOLOGY

OVERVIEW OF BLOOD

Blood is a fluid connective tissue that circulates through the cardiovascular system. Like other connective tissues, blood consists of cells and an extracellular component. Total blood volume in the average adult is approximately 6 L or 7%–8% of total body weight. The heart’s pumping action propels blood through the cardiovascular system to the body tissues. Blood’s many functions include the following:

Delivery of nutrients and oxygen directly or indirectly to cells Transport of wastes and carbon dioxide away from cells Delivery of hormones and other regulatory substances to and from cells and tissues Maintenance of homeostasis by acting as a buffer and participating in coagulation and thermoregulation Transport of humoral agents and cells of the immune system that protect the body from pathogenic agents, foreign proteins, and transformed cells (e.g., cancer cells)

Blood consists of cells and their derivatives and a proteinrich fluid called plasma. Blood cells and their derivatives include the following:

Erythrocytes, also called red blood cells Leukocytes, also known as white blood cells Thrombocytes, also termed platelets Plasma is the liquid extracellular material

that imparts fluid properties to blood. The relative volume of cells and plasma in whole blood is approximately 45% and 55%, respectively. The volume of packed erythrocytes in a sample of blood is called the or . The HCT is measured by centrifuging a blood sample to which anticoagulants have been added and then calculating the percentage of the centrifuge tube volume occupied by the

(PCV)

hematocrit (HCT)

packed cell volume

erythrocytes compared with that of the whole blood (Fig. 10.1). A is 39%–50% in men and 35%–45% in women; thus, 39%–50% and 35%–45% of the blood volume for men and women, respectively, consist of erythrocytes. Low hematocrit values often reflect reduced numbers of circulating erythrocytes (a condition called ) and may indicate significant blood loss caused by internal or external bleeding.

normal hematocrit

anemia

FIGURE 10.1. Blood composition. apparent

after

centrifuging

a

Blood composition is clearly small volume of blood in a

microhematocrit tube. The volume of packed erythrocytes occupies about 45% of whole blood (this fraction is called ). The thin layer between erythrocytes and plasma contains leukocytes and platelets; it is often referred to as a . The remaining volume (about 55%) consists of a , opaque fluid and represents protein-rich blood plasma.

hematocrit buffy coat pale yellow

Leukocytes and platelets constitute 1% of the blood volume.

In a blood sample that has been centrifuged, the cell fraction (the part of the sample that contains the cells) consists mainly of packed erythrocytes (~99%). The leukocytes and platelets are contained in a narrow, light-colored layer between the erythrocytes and plasma called the (see Fig. 10.1). As Table 10.1 indicates, there are nearly 1,000 times more erythrocytes (~5 × 1012 cells/L of blood) than leukocytes (~7 × 109 /L of blood).

buffy coat

TABLE 10.1 Formed Elements of the Blood Formed Elements Erythrocytes Leukocytes Agranulocytes

Cells/L in Adults Male Female

4.3–5.7 × 10 12

3.9–5.0 × 10 12

3.5–10.5 × 10 9

3.5–10.5 × 10 9

Lymphocytes

0.9–2.9 × 10 9

0.9–2.9 × 10 9

Monocytes

0.3–0.9 × 10 9

0.3–0.9 × 10 9

Granulocytes

 

 

Neutrophils

1.7–7.0 × 10 9

1.7–7.0 × 10 9

Eosinophils

0.05–0.5 × 10 9

0.05–0.5 × 10 9

Basophils

0–0.3 × 10 9

0–0.3 × 10 9

150–450 × 10 9

150–450 × 10 9

Thrombocytes (platelets)

 

% 100

25.7– 27.6 a 8.6 a

  48.6– 66.7 a

1.4–4.8 a 0–0.3 a  

aPercentage

of leukocytes.

PLASMA Although blood cells are of major interest in histology, a brief examination of plasma is also useful. The composition of is summarized in Table 10.2. More than 90% of plasma by weight is water, which serves as the solvent for a variety of , including proteins, dissolved gases, electrolytes, nutrients, regulatory substances, and waste materials. The solutes in the plasma help maintain , a steady state that provides optimal pH and osmolarity for cellular metabolism.

plasma solutes

homeostasis

TABLE 10.2 Composition of Blood Plasma Component Water

% 91–92

Protein (albumin, globulins, fibrinogen)

7–8

Other solutes:

1–2

Electrolytes (Na +, K +, Ca 2+, Mg 2+, Cl −, HCO 3−, PO 4 3−, SO 4 2−) Nonprotein nitrogen substances (urea, uric acid, creatine, creatinine, ammonium salts) Nutrients (glucose, lipids, amino acids) Blood gases (oxygen, carbon dioxide, nitrogen) Regulatory substances (hormones, enzymes)

Plasma proteins consist primarily of albumin, globulins, and fibrinogen. Albumin is the main protein constituent of plasma, accounting

for approximately one-half of the total plasma proteins. It is the smallest plasma protein (about 70 kDa) and is made in the liver. Albumin is responsible for exerting the concentration gradient between blood and extracellular tissue fluid. This major osmotic pressure on the blood vessel wall, called the

colloid osmotic pressure,

maintains the correct proportion of blood to tissue fluid volume. If a significant amount of albumin leaks out of the blood vessels into the loose connective tissue or is lost from the blood to urine in the kidneys, the colloid osmotic pressure of the blood decreases and fluid accumulates in the tissues. This increase in tissue fluid is most readily noted by swelling of the ankles at the end of the day. Albumin also acts as a carrier protein; it binds and transports hormones (thyroxine), metabolites (bilirubin), and drugs (barbiturates). include , the largest component of the globulin fraction, and ( and ). Immunoglobulins are antibodies, a class of functional immune system molecules secreted by plasma cells. (Antibodies are discussed in Chapter 14, Immune System and Lymphatic Tissues and Organs, pages 488490). are secreted by the liver. They help maintain the osmotic pressure within the vascular system and also serve as carrier proteins for various substances, such as copper (by ceruloplasmin), iron (by transferrin), and the protein (by haptoglobin). Nonimmune globulins also include fibronectin, lipoproteins, coagulation factors, and other molecules that are transferred between the blood and the extravascular connective tissue. , the largest plasma protein (340 kDa), is made in the liver. In a series of cascade reactions with other coagulation factors, soluble fibrinogen is transformed into insoluble protein (323 kDa). During the conversion of fibrinogen to fibrin, fibrinogen chains are broken to produce fibrin monomers that rapidly polymerize to form long fibers. These fibers become cross-linked to form an impermeable net at the site of damaged blood vessels, thereby preventing further blood loss. With the exception of these large plasma proteins and regulatory substances, which are small proteins or polypeptides, most plasma constituents are small enough to pass through the blood vessel wall into the extracellular spaces of the adjacent connective tissue.

Globulins immunoglobulins (γ-globulins) nonimmune globulins α-globulin β-globulin Nonimmune globulins hemoglobin

Fibrinogen

fibrin

In general, plasma proteins react with common fixatives; they are often retained within the blood vessels in tissue sections. Plasma proteins do not possess a structural form above the molecular level; thus, when they are retained in blood vessels in the tissue block, they appear as a homogeneous substance that stains evenly with eosin in hematoxylin and eosin (H&E)-stained sections.

Serum is the same as blood plasma, except that clotting factors have been removed. For laboratory purposes, samples of blood are often drawn from a vein (a procedure called venipuncture). When blood is removed from the circulation, it immediately clots. A blood clot consists mostly of erythrocytes entangled in a network of fine fibers composed of fibrin. To prevent clotting of a blood sample, an such as citrate or heparin is added to the blood specimen as it is obtained. Citrate binds calcium ions, which are essential for triggering the cascade of coagulation reactions; heparin deactivates the clotting factors in the plasma. Plasma that lacks coagulation factors is called . For many biochemical laboratory tests, plasma and blood serum can be used interchangeably. Serum is preferred for several specific tests because the anticoagulants in plasma can interfere with the results. However, tests of clotting ability require that all coagulation factors be preserved; therefore, serum is inappropriate for these tests.

anticoagulant

serum

The interstitial fluid of connective tissues is derived from blood plasma. The fluid that surrounds tissue cells, called interstitial fluid, has an electrolyte composition that reflects that of blood plasma, from which it is derived. The composition of interstitial fluid in nonconnective tissues, however, is subject to considerable modification by the absorptive and secretory activities of epithelia. Epithelia may create special microenvironments conducive to their function. For example, a blood–brain barrier exists between the blood and the nerve tissue. Barriers also exist between the blood and the

parenchymal tissue in the testis, thymus gland, eye, and other epithelial compartments. Fluids, barriers, and their functions are discussed in subsequent chapters that describe these particular organs.

Examination of blood cells requires special preparation and staining. The preparation method that best displays the cell types of peripheral blood is the blood smear. This method differs from usual histologic preparations in that the specimen is not embedded in paraffin and sectioned. Rather, a drop of blood is placed directly on a slide and spread thinly over the surface of the slide (i.e., “pulled” with the edge of another slide) to produce a monolayer of cells (Fig. 10.2a). The preparation is then air dried and stained. Another difference in the preparation of a blood smear is that instead of H&E, special mixtures of dyes are used to stain the blood cells. The resulting preparation may then be examined with a high-power oil-immersion lens, with or without a coverslip (Fig. 10.2b and Plate 10.1, page 336).

FIGURE 10.2. Blood smear: preparation technique and overview photomicrograph. a. Photograph showing the method of producing a blood smear. A drop of blood is placed directly on a glass slide and spread over its surface with the edge of another slide. b. Photomicrograph of smear from peripheral blood stained with Wright stain showing the cells evenly distributed. The cells are mainly erythrocytes. Three leukocytes are present. Platelets are indicated by . ×350.

arrows

The modified Romanowsky-type stain commonly used for blood smears consists of a mixture of methylene blue (a basic dye), related azures (also basic dyes), and eosin (an acidic dye). On the basis of their appearance after staining, leukocytes are traditionally divided into (neutrophils, eosinophils, and basophils) and (lymphocytes and monocytes). Although both cell types may contain granules, granulocytes possess obvious, specifically stained granules in their cytoplasm. In general, the basic dyes stain nuclei, granules of basophils, and RNA in the cytoplasm, whereas the acidic dye stains the erythrocytes and the granules of eosinophils. Scientists originally thought that the fine neutrophil granules were stained by a “neutral dye” that formed when methylene blue and its related azures were combined with eosin. The mechanism by which the specific neutrophil granules are stained is still not clearly understood. Some of the basic dyes (the azures) are metachromatic and may impart a violet to red color to the material they stain.

granulocytes agranulocytes

ERYTHROCYTES

Erythrocytes are anucleate, biconcave discs. Erythrocytes or red blood cells are anucleate cells devoid of

typical organelles. They function only within the bloodstream to bind oxygen for delivery to the tissues and, in exchange, bind carbon dioxide for removal from the tissues. Their shape is that of a biconcave disc with a 𝛍 , an edge thickness of 2.6 μm, and a central thickness of 0.8 μm (Fig. 10.3). This shape maximizes the cell’s surface area (~140 μm2 ), an important attribute in gas exchange.

diameter of 7.8 m

120 days

The life span of erythrocytes is approximately . In a healthy individual, approximately 1% of erythrocytes are removed from the circulation each day because of senescence (aging); however, the bone marrow continuously produces new erythrocytes to replace those lost. The majority of aged erythrocytes (~90%) are phagocytosed by macrophages in the spleen, bone marrow, and liver. The remaining aged erythrocytes (~10%) break down intravascularly, releasing insignificant amounts of hemoglobin (Hgb) into the blood.

FIGURE 10.3. Erythrocyte.

The erythrocyte is an anucleated cell in a shape of a biconcave disc containing hemoglobin. The surface area of an erythrocyte is about 140 μm 2, and its mean corpuscular (cell) volume ranges from 80 to 99 fL (1 fL = 10 −15 L).

In H&E-stained sections, erythrocytes are usually 7–8 μm in diameter. Because their size is relatively consistent in fixed tissue, they can be used to estimate the size of other cells and structures in tissue sections; in this role, erythrocytes are appropriately referred to as the “ .” Because both living and preserved erythrocytes usually appear as biconcave discs, they can give the impression that their form is rigid and inelastic (Fig. 10.4). They are, in fact, extremely deformable. They pass easily through the

ruler

histologic

narrowest capillaries by folding over on themselves. They stain uniformly with eosin. In thin sections viewed with the transmission electron microscope (TEM), the contents of an erythrocyte appear as a dense, finely granular material.

FIGURE 10.4. Erythrocyte morphology. a. Photomicrograph of three capillaries ( Cap ) joining to form a venule ( V ), as observed in adipose

tissue within a full-thickness mesentery spread. The erythrocytes appear in a single file in one of the capillaries (the other two are empty). The area of some of the erythrocytes results from their biconcave shape. Erythrocytes are highly plastic and can fold on themselves when passing through very narrow capillaries. The large round structures are adipose cells ( ). ×470. Scanning electron micrograph of erythrocytes collected in a blood tube. Note the biconcave shape of the cells. The stacks of erythrocytes in these preparations are not unusual and are referred to as . Such formations in vivo indicate an increased level of plasma immunoglobulin. ×2,800.

light center

A

b.

rouleau

The shape of the erythrocyte is maintained by a specialized cytoskeleton that provides the mechanical stability and flexibility necessary to withstand forces experienced during circulation. As erythrocytes in circulation navigate through a small network of capillaries, they are exposed to high amounts of shear force that cause them to undergo rapid and reversible deformations.

To cope with this stress, the erythrocyte cell membrane has a unique cytoskeletal structure. In addition to a typical lipid bilayer, it contains two functionally significant groups of proteins.

Integral membrane proteins represent most of the proteins in

the lipid bilayer. They consist of two major families: glycophorins and band 3 proteins. The extracellular domains of these integral membrane proteins are glycosylated and express specific blood group antigens. , a member of the glycophorin family of transmembrane proteins, assists in attaching the underlying cytoskeletal protein network to the cell membrane. is the most abundant transmembrane protein in the erythrocyte cell membrane. It binds Hgb and acts as an anchoring site for cytoskeletal proteins (Fig. 10.5).

Glycophorin C

Band 3 protein

FIGURE 10.5. Erythrocyte membrane organization.

This diagram shows the arrangement of peripheral and integral membrane proteins. The integral membrane protein glycophorin C associates with the peripheral membrane band 4.1 protein complex. Similarly, band 3 integral membrane protein binds to the ankyrin protein complex.

These peripheral complexes interact with spectrin to form a cytoskeletal hexagonal lattice immediately adjacent to the cytoplasmic surface of the plasma membrane. The spectrin lattice with peripheral membrane protein complexes is anchored to the plasma membrane by glycophorin C and band 3 proteins, which, on the extracellular surface, are glycosylated and support the majority of carbohydrate-defined blood group antigens.

Peripheral membrane proteins

reside on the inner surface of the cell membrane. They are organized into a two-dimensional hexagonal lattice network that laminates the inner layer of the membrane. The lattice itself, which is positioned parallel to the membrane, is composed mainly of cytoskeletal proteins, including and molecules. They assemble to form an antiparallel heterodimer held together by multiple lateral bonds. Dimers then associate in a head-to-head formation to produce long and flexible tetramers. The spectrin filaments are anchored to the lipid bilayer by two large protein complexes. The first is the containing band 4.1, actin, tropomyosin, tropomodulin, adducin, and dematin (see Fig. 10.5); this complex interacts with glycophorin C and other transmembrane proteins. The second complex is the containing ankyrin and band 4.2 protein; this complex interacts with band 3 and other integral membrane proteins (see Fig. 10.5).

α-spectrin

4.1 protein complex

β-spectrin

band

ankyrin protein complex

This unique cytoskeletal arrangement contributes to the shape of the erythrocyte and imparts elastic properties and stability to the membrane. The cytoskeleton is not static. For example, molecular bonds along the length of spectrin molecules dissociate and reassociate as the erythrocyte undergoes deformation in response to various physical factors and chemical stimuli. Therefore, flexible interactions within spectrin dimers, ankyrin, and band 4.1 complexes are key regulators of membrane elasticity and mechanical stability. Any change in the expression of genes that encode these cytoskeleton proteins can result in abnormally shaped and fragile erythrocytes. For example, is an autosomal dominant genetic disorder affecting ankyrin

hereditary spherocytosis

complex proteins (band 3, band 4.2, spectrin, and other erythrocyte integral membrane proteins) that function in anchoring the erythrocyte plasma membrane to the cytoplasm. Defects in these anchoring proteins cause the erythrocyte’s plasma membrane to detach and peel off from the cytoplasm and result in spherical erythrocytes. Another erythrocyte membrane abnormality, , is caused by one of several autosomal dominant mutations affecting spectrin molecules. In this mutation, spectrin-to-spectrin lateral bonds and spectrin–ankyrin–band 4.1 protein junctions are defective. The plasma membrane in the affected cells fails to rebound from deformations and progressively elongates, resulting in the formation of elliptical erythrocytes. In both conditions, erythrocytes are unable to adapt to changes in the environment (e.g., osmotic pressure and mechanical deformations), which results in premature destruction of the cells or .

hereditary elliptocytosis

hemolysis

FOLDER 10.1

CLINICAL CORRELATION: ABO AND RH BLOOD GROUP SYSTEMS ABO Blood Group System An important factor in blood transfusion is the ABO blood group system , which essentially involves three antigens called A , B , and O (Table F10.1.1). These antigens are glycoproteins and glycolipids and differ only slightly in their composition. They are present on the surface of erythrocytes and are attached to the extracellular domains of integral membrane proteins called and . The presence of A, B, or O antigens determines the four primary , and . All humans have enzymes that catalyze the synthesis of the O antigen. Blood is described as a specific type depending on the presence or absence of AB group antigens. Individuals with type A blood have an additional enzyme ( or NAcetylglucosamine transferase) that adds -acetylgalactosamine to the O antigen. Individuals with type B blood have an enzyme ( or B-glycosyltransferase) that adds galactose to the O antigen (Fig. F10.1.1). Individuals with type AB blood express both enzymes, whereas individuals who lack both enzymes have type O blood. In humans, consist of at

glycophorins band 3 proteins blood groups: A, B, AB O N-acetylgalactosamineN transferase galactose transferase ABO genes

least seven exons, and they are located on chromosome 9. The O allele is recessive, whereas A and B alleles are codominant.

TABLE F10.1.1 ABO Blood Group System

FIGURE F10.1.1. ABO blood group antigens.

The ABO antigens are not primary gene products but are instead products of enzymatic reactions (glycosylations). This schematic drawing shows the differences between the three major antigens responsible for the ABO blood group system. The immunodominant structure of antigen O is depicted as it attaches to an extracellular domain of glycophorins, the integral membrane proteins of erythrocyte cell membranes. Note that differences between O antigen and A antigen are due to the presence of an additional sugar molecule, acetylgalactosamine ( ), which is added by

blue arrow middle

N

N

genetically encoded functional -acetylgalactosamine transferase expressed in individuals with type A blood. Similarly, individuals with type B blood have a galactose molecule ( ) attached by the enzyme galactose transferase. Individuals with type AB blood express both enzymes (thus, both A and B antigens are present) and individuals with type O blood lack both functional enzymes, thus possessing only the immunodominant core structure of antigen O.

blue arrow right

The differences in the carbohydrate molecules of these antigens are detected by specific antibodies against either A or B antigen. Individuals with type A blood possess serum anti-B antibodies that are directed against the B antigen. Individuals with type B blood possess serum anti-A antibodies that are directed against the A antigen. Individuals with type AB blood do not have antibodies directed against A or B antigen. Thus, they are of any blood type. Individuals with type O blood have both anti-A and anti-B antibodies in their serum and neither A nor B antigen on their erythrocytes. Thus, these individuals are . If an individual is transfused with blood of an incompatible type, the recipient’s antibodies will attack the donor erythrocytes, causing a or destruction of the transfused erythrocytes. To prevent such a lifethreatening complication, blood for transfusion must be always cross-matched to the blood of a recipient. In this procedure, serum from the recipient is tested against the donor’s erythrocytes. If there is no reaction to this cross-match test, then the donor’s blood can be used for the transfusion.

universal

acceptors universal blood donors hemolytic transfusion reaction

Rh Blood Group System The other important blood group system, the Rh system, is based on the Rhesus (Rh) antigen. In humans, this system is represented by a 40-kDa transmembrane nonglycosylated Rh30 polypeptide that shares antigenic sites with rhesus monkey erythrocytes. Rh30 polypeptide is a component of a larger (90-kDa) erythrocyte integral membrane protein complex that includes Rh50 glycoprotein. Although the Rh30 polypeptide expresses many antigen sites on its extracellular domain, only five of them—D, C, c, E , and e antigens—have clinical significance. Interactions between Rh30 and Rh50 molecules are essential for the expression of D, C, c, E, and e antigens. Only one D antigen determines Rh status. Therefore, an individual who possesses D antigen is referred to as and an individual who lacks expressed D antigen is referred to as

Rh positive (Rh+),

Rh negative

(Rh−). All antigens stimulate the production of anti-Rh antibodies in individuals without the same antigens. Rh incompatibility—in which a pregnant individual is Rh(D−)

and the fetus is Rh(D+)—can become a major clinical issue in pregnancy if it is not detected early and preventive measures are taken. If an individual is Rh(D−) and becomes pregnant with an Rh(D+) fetus, anti-D antibodies will be produced in response to the D antigen expressed on fetal erythrocytes. This is called . Fetal erythrocytes can leak into the circulation during pregnancy, childbirth, or following a miscarriage or abortion. Usually, there are not enough antibodies produced during a first pregnancy to cause a reaction in the fetus. However, if a sensitized individual becomes pregnant again with an Rh(D+) fetus, the antibodies may cross the placenta and attack the Rh(D+) antigens on the fetal red blood cells, triggering a hemolytic transfusion reaction in the fetus called . Administration of to any Rh(D−) person during and following each pregnancy or following a miscarriage destroys any circulating Rh(D+) fetal erythrocytes that persist in the person’s blood, thus preventing Rhincompatibility reactions in future pregnancies.

sensitization

fetalis

Rh(D+)

erythroblastosis anti-D immunoglobulin (RhoGAM)

Erythrocytes contain hemoglobin, a protein specialized for the transport of oxygen and carbon dioxide. Erythrocytes transport oxygen and carbon dioxide bound to the protein hemoglobin (68 kDa). Hgb binds to oxygen molecules in

the lung (which requires a high oxygen affinity) and, after transporting it through the circulatory system, unloads oxygen in the tissues (which requires a low oxygen affinity). A monomer of Hgb is similar in composition and structure to myoglobin, the oxygen-binding protein found in striated muscle. The disc shape of the erythrocyte facilitates gas exchange because more Hgb molecules fit more closely to the plasma membrane than they would be in a spherical cell. Thus, gases have less distance to diffuse within the cell to reach a binding site on the Hgb. A high concentration of Hgb is present within erythrocytes and is responsible for their uniform staining with eosin and the cytoplasmic granularity seen with the TEM. consists of four polypeptide chains of globin: α, β, δ, and γ. Each polypeptide chain is bound to an iron-

Hemoglobin

containing heme group (Fig. 10.6). During oxygenation, each of the four iron-containing heme groups can reversibly bind one oxygen molecule. During gestational and postnatal periods, the synthesis of Hgb polypeptide chains varies, resulting in different Hgb types (Fig. 10.7). Depending on the activation of different globin genes and the particular globin chain being synthesized, the following types of Hgb can be distinguished:

FIGURE 10.6. Structural diagram of the hemoglobin molecule.

Each hemoglobin molecule is composed of four subunits. Each subunit contains a heme, the iron-containing portion of hemoglobin, embedded in a hydrophobic cleft of a globin chain. The folding of the globin chain places the heme near the surface of the molecule, where it is readily accessible to oxygen. There are four different types of globin chains: α, β, δ, and γ, occurring in pairs. The types of globin chains present in the molecules determine the type of hemoglobin. The figure illustrates hemoglobin A (HbA), which is composed of two αchains and two β-chains.

FIGURE 10.7. Major globin chain synthesis and hemoglobin composition in prenatal and postnatal periods. The type of

hemoglobin differs in the gestational and postnatal periods. This diagram represents a timeline for the synthesis of the four major globin chains (α, β, δ, and γ) and for hemoglobin composition. In the early stages of development, α- and γ-chains form fetal hemoglobin (HbF), which predominates at birth. In the second month of gestation, synthesis of β-chains gradually increases. After birth, it drastically escalates to form with α-chains, predominately adult hemoglobin (HbA). During this time, γ-chain synthesis declines. During the eighth month of prenatal development, δ-chain production is initiated to form hemoglobin containing two δ-chains and two αchains (HbA 2 ). Adult hemoglobin types HbA (96%) and HbA 2 (20% of the volume). In the cortex, two types of interstitial cells are recognized: , found between the basement membrane of the tubules and the adjacent peritubular capillaries, and occasional . In their intimate relationship with the base of the tubular epithelial cells, the fibroblasts resemble the subepithelial fibroblasts of the intestine. These cells synthesize and secrete the collagen and glycosaminoglycans of the extracellular matrix of the interstitium.

interstitial tissue

cells that resemble fibroblasts macrophages

In the medulla, the principal interstitial cells resemble . They are oriented to the long axes of the tubular structures and may have a role in compressing these structures. The cells contain prominent bundles of actin filaments, abundant rough endoplasmic reticulum (rER), a welldeveloped Golgi complex, and lysosomes. Prominent lipid droplets in the cytoplasm appear to increase and decrease in relation to the diuretic state.

myofibroblasts

FOLDER 20.5

FUNCTIONAL CONSIDERATIONS: STRUCTURE AND FUNCTION OF AQUAPORIN WATER CHANNELS Aquaporins (AQPs) are small, hydrophobic, transmembrane proteins that mediate water transport in the kidney and other organs (i.e., liver, gallbladder). Thirteen AQPs have been characterized and cloned. The molecular size of AQPs ranges from 26 to 34 kDa. Each protein consists of six transmembrane domains arranged to form a distinct pore. The sites where AQPs are expressed implicate their role in water transport, such as renal tubules (water reabsorption), brain and spinal cord (cerebrospinal fluid reabsorption), pancreatic acinar cells (secretion of pancreatic fluids), lacrimal apparatus (secretion and resorption of tears), and eye (aqueous humor secretion and reabsorption). Most AQPs are selective for the passage of water (AQP-1, AQP-2, AQP-4, AQP-5, AQP-6, and AQP-8), whereas others, such as AQP-3, AQP-7, and AQP-9, called , also transport glycerol and other larger molecules in addition to water. Prominent members of the AQP family include:

aquaglyceroporins

AQP-1 , expressed in kidney (proximal convoluted tubules) and other cell types such as hepatocytes and red blood cells. AQP-1 is also expressed in the lymph nodes, endothelial cells lining lymphatic sinuses, and on the vascular endothelium of high endothelial venules as well as in the endothelial cells of intestinal lacteals. , present in the terminal portion of the distal convoluted tubules, connecting tubules, and the epithelium of collecting ducts. AQP-2 is regulated by antidiuretic hormone (ADH) and is thus known as an . Mutation of the gene has been linked to . and have also been detected in the basolateral cell surface of the light (principal) cells of kidney collecting ducts as well in the gastrointestinal epithelium (AQP-3), pancreatic acinar cells (AQP-12), and the brain and spinal cord (AQP-4).

AQP-2 2

insipidus AQP-3 AQP-4

ADH-regulated water channel

congenital nephrogenic diabetes

AQP-

Research into the function and structure of the AQP proteins may lead to the development of water channel blockers that could be used to treat hypertension, congestive heart failure, and brain swelling and to regulate intracranial or intraocular pressure.

Most fibroblasts originate within the interstitial tissue through a mechanism called . The EMT is a process in which epithelial cells lose their characteristics and acquire features of mesenchymal cells. During this transition, epithelial cells lose cell-to-cell junctions, apical–basal domain polarity, and epithelial cell markers and acquire cell motility, a spindle cell shape, and mesenchymal cell markers. The reprogramming and conversion of tubular epithelial cells into a mesenchymal phenotype is initiated by an alteration in the balance of local cytokine concentrations (i.e., TGF-β, FGF, EGF) that upregulate specific transcription factors called EMT-TFs. During persistent injury and chronic inflammation of the kidney parenchyma, fibroblasts increase their numbers and, by secreting excess extracellular matrix, destroy the normal interstitial architecture of the kidney. Research studies suggest that in , more than one-third of all diseaserelated fibroblasts originate from tubular epithelial cells at the site of injury. Proliferation of fibroblasts in response to local mitogens usually leads to irreversible renal failure characterized by . Recent therapeutic interventions in renal fibrosis are directed toward inhibiting fibroblast formation by shifting local cytokine balance in favor of .

epithelial–mesenchymal transition

(EMT)

renal fibrosis

tubulointerstitial nephritis

transition

reversal mesenchymal–epithelial

HISTOPHYSIOLOGY OF THE KIDNEY

The countercurrent multiplier system creates hyperosmotic urine. The term countercurrent indicates a flow of fluid in adjacent structures in opposite directions. The ability to excrete hyperosmotic urine depends on the countercurrent multiplier system that involves three structures:

Loop of Henle,

which acts as a countercurrent multiplier. The ultrafiltrate moves within the descending limb of the thin segment of the loop toward the renal papilla and moves back toward the corticomedullary junction within the ascending limb of the thin segment. The osmotic gradients of the medulla are established along the axis of the loop of Henle. form loops parallel to the loop of Henle. They act as countercurrent exchangers of water and solutes between the descending part (arteriolae rectae) and ascending part (venulae rectae) of the vasa recta. The vasa recta help maintain the osmotic gradient of the medulla. in the medulla acts as an . Modified ultrafiltrate in the collecting ducts can be further equilibrated with the hyperosmotic medullary interstitium. The level of equilibration depends on activation of ADH-dependent water channels (AQP-2).

Vasa recta

Collecting duct equilibrating device

osmotic

A standing gradient of ion concentration produces hyperosmotic urine by a countercurrent multiplier effect. The loop of Henle creates and maintains a gradient of ion concentration in the medullary interstitium that increases from the corticomedullary junction to the renal papilla. As noted earlier, the thin descending limb of the loop of Henle is freely permeable to water, whereas the ascending limb of the loop of Henle is impermeable to water. Furthermore, the thin ascending limb cells add Na+ and Cl− to the interstitium. Because water cannot leave the thin ascending limb, the interstitium becomes hyperosmotic relative to the luminal contents. Although some of the Cl− and Na+ of the interstitium diffuse back into the nephron at the thin descending limb, the ions are transported out again in the thin ascending limb and distal straight tubule (thick ascending limb). This produces the . Thus, the concentration of NaCl in the interstitium gradually increases down the length of the loop of Henle and, consequently, through the thickness of the medulla from the corticomedullary junction to the papilla.

countercurrent multiplier effect FOLDER 20.6

FUNCTIONAL CONSIDERATIONS: ANTIDIURETIC HORMONE REGULATION OF COLLECTING DUCT FUNCTION Water permeability of the epithelium of the collecting ducts is regulated by antidiuretic hormone (ADH, vasopressin), a hormone produced in the hypothalamus and released from the posterior lobe of the pituitary gland. ADH increases the permeability of the collecting duct to water, thereby producing more concentrated urine. At the molecular level, ADH acts on the ADH-regulated water channels aquaporin-2 (AQP-2), which is located in the epithelium of the terminal portion of the distal convoluted tubule, connecting tubules, and the epithelium of the collecting ducts. However, the action of ADH is more significant in the collecting ducts. ADH binds to receptors on the cells of these ducts and triggers the following actions:

Translocation of the AQP-2–containing intracytoplasmic vesicles into the apical cell surface—a short-term effect. This results in an increased number of available AQP-2 channels at the cell surface, thus increasing water permeability of the epithelium. and their insertion into the apical cell membrane—a long-term effect

Synthesis of AQP-2

An increase in plasma osmolality or a decrease in blood volume stimulates the release of ADH, as does nicotine. In the absence of ADH, copious, dilute urine is produced. This condition is called . Mutation of two genes encoding AQP-2 and ADH receptors is responsible for a form of CDI called . Because of the loss of normal function of the AQP-2 and ADH receptor proteins synthesized by the collecting duct epithelial cells, the kidney does not respond to ADH. Excess water consumption can also inhibit ADH release, thereby promoting the production of a large volume of hyposmotic urine. Increased secretion of ADH can produce an extremely hyperosmotic urine, thereby conserving water in the body. Inadequate consumption of water or loss of water because of sweating, vomiting, or diarrhea stimulates the release of ADH. This leads to an increase in the permeability of the epithelium of the distal convoluted tubules and collecting ducts and promotes the production of a small volume of hyperosmotic urine.

central diabetes insipidus (CDI) nephrogenic diabetes insipidus

Vasa recta containing descending arterioles and ascending venules act as countercurrent exchangers. To understand the countercurrent exchange mechanism, it is necessary to resume the description of the renal circulation at

the point at which the efferent arteriole leaves the renal corpuscle. The of the renal corpuscles of most of the cortex branch to form the capillary network that surrounds the tubular portions of the nephron in the cortex, the . The efferent arterioles of the juxtamedullary renal corpuscles form several unbranched arterioles that descend into the medullary pyramid. These make a hairpin turn deep in the medullary pyramid and ascend as the . Together, the descending arterioles and the ascending venules are called the . The arteriolae rectae form capillary plexuses lined by fenestrated endothelium that supply the tubular structures at the various levels of the medullary pyramid.

efferent arterioles

peritubular capillary network arteriolae rectae vasa recta

venulae rectae

Interaction between collecting ducts, loops of Henle, and vasa recta is required for concentrating urine by the countercurrent exchange mechanism. Because the thick ascending limb of the loop of Henle has a high level of transport activity and because it is impermeable to water, the modified ultrafiltrate that ultimately reaches the distal convoluted tubule is . When ADH is present, the distal convoluted tubules and the collecting ducts are highly permeable to water. Therefore, within the cortex, in which the interstitium is isosmotic with blood, the modified ultrafiltrate within the distal convoluted tubule equilibrates and becomes isosmotic, partly by loss of water to the interstitium and partly by addition of ions other than Na+ and Cl− to the ultrafiltrate. In the medulla, increasing amounts of water leave the ultrafiltrate as the collecting ducts pass through the increasingly hyperosmotic interstitium on their course to the papillae. As noted previously, the vasa recta also form loops in the medulla that parallel the loop of Henle. This arrangement ensures that the vessels provide circulation to the medulla without disturbing the osmotic gradient established by transport of Cl− in the epithelium of the ascending limb of the loop of Henle. The vasa recta form a in the following manner: Both the arterial and venous sides of the loop

hyposmotic

countercurrent exchange system

are thin-walled vessels that form plexuses of fenestrated capillaries at all levels in the medulla. As the arterial vessels descend through the medulla, the blood loses water to the interstitium and gains salt from the interstitium so that at the tip of the loop, deep in the medulla, the blood is essentially in equilibrium with the hyperosmotic interstitial fluid. As the venous vessels ascend toward the corticomedullary junction, the process is reversed (i.e., the hyperosmotic blood loses salt to the interstitium and gains water from the interstitium). This passive countercurrent exchange of water and salt between the blood and the interstitium occurs by the endothelial cells. The energy that drives this system is the same energy that drives the multiplier system, namely, the movement of Na+ and Cl− out of the cells of the water-impermeable ascending limb of the loop of Henle. The countercurrent exchange system and other movements of molecules in different parts of the nephron are shown in Figure 20.24.

expenditure of energy

without

FIGURE 20.24. Diagram showing the movement of substances into and out of the nephron and collecting system. The symbols indicate the

mode of transport as well as specific molecule-dependent transporters that act on the nephron and collecting ducts (as noted in the key).

BLOOD SUPPLY

blood supply

Some aspects of the of the kidney have been described in relation to specific functions (i.e., glomerular filtration, control of blood pressure, and countercurrent exchange). It remains, however, to provide an overall description of the blood supply of the kidney. Each kidney receives a large branch from the abdominal aorta, called the . The renal artery branches within the renal sinus and sends into the substance of the kidney (Fig. 20.25). These arteries travel between the pyramids as far as the cortex and then turn to follow an arched course along the base of the pyramid between the medulla and the cortex. Thus, these interlobar arteries are designated .

renal artery interlobar arteries

arteries

arcuate

FIGURE 20.25. Schematic diagram of the renal blood supply.

The renal artery gives rise to interlobar arteries that branch into arcuate arteries at the border between the medulla and the cortex. Interlobular arteries branch from the arcuate arteries and travel toward the renal capsule, giving off afferent arterioles that contribute to the glomerular capillaries. Glomeruli in the outer part of the cortex send efferent arterioles to the peritubular cortical capillaries that surround the tubules in the cortex. Glomeruli near the medulla, the juxtamedullary glomeruli, send efferent arterioles almost entirely into the medullary network of capillaries that contains the descending vasa recta. Blood returns from the medulla by the ascending vasa recta and the capillary network via veins that enter the arcuate veins. Stellate veins near the capsule drain the capsular network, and the peritubular cortical plexus drains to both the interlobular and arcuate veins.

Interlobular arteries

branch from the arcuate arteries and ascend through the cortex toward the capsule. Although the boundaries between lobules are not distinct, the interlobular arteries, when included in a section cut perpendicular to the vessel, are located midway between adjacent medullary rays, traveling in the cortical labyrinth. As they traverse the cortex toward the capsule, the interlobular arteries give off branches, the , one to each glomerulus (Fig. 20.26). A single afferent arteriole may spring directly from the interlobular artery, or a common stem from the interlobular artery may branch to form several afferent arterioles. Some interlobular arteries terminate near the periphery of the cortex, whereas others enter the kidney capsule to provide its arterial supply.

afferent arterioles

FIGURE 20.26. Photomicrograph of the cortical labyrinth in the kidney from an adult monkey. This high-magnification photomicrograph

of a hematoxylin and eosin (H&E)-stained kidney shows the glomerulus with associated tubules and blood vessels. The prominent glomerulus is surrounded by the Bowman capsule and is supplied by an afferent arteriole ( ) that originates from the interlobular artery ( ) visible on the side of the image. The afferent arteriole supplying blood to the glomerulus has a distinct circular layer of smooth muscle cells ( ) that was sectioned perpendicularly to the long axis of the arteriole. The modified smooth muscle cells in the terminal portion of the afferent arteriole are termed ( ). In addition to a contractile apparatus, these cells exhibit prominent

AA

SMC

ILA

left

juxtaglomerular cells JgC

endoplasmic reticulum, Golgi apparatus, and secretory vesicles containing renin. Macula densa and extraglomerular mesangial cells are not easily discernable. The grazing section of an interlobular artery ( ) reveals a much thicker layer of circular smooth muscle cells with visible nuclei crossing the lumen of the vessel. The elongated endothelial cells ( ) are clearly visible. Note the accompanying intralobular vein ( ) and several profiles of proximal convoluted ( ) and distal convoluted ( ) tubules of the nephron. ×380.

ILA

EnC ILV PC DC Source: Kent Christensen, PhD; J. Matthew Velkey, PhD; Lloyd M. Stoolman, MD; Laura Hessler; Diedra Mosley-Brower; and Michael Hortsch, PhD; University of Michigan Medical School. © 2010 The Regents of the University of Michigan. For more information, questions, or permissions requests please contact: Michael Hortsch, PhD, University of Michigan Medical School, [email protected].)

Afferent arterioles give rise to the capillaries that form the glomerulus. The glomerular capillaries reunite to form an efferent arteriole that, in turn, gives rise to a second network of capillaries, the . The arrangement of these capillaries differs according to whether they originate from cortical or juxtamedullary glomeruli:

peritubular capillaries

Efferent arterioles from cortical glomeruli peritubular capillary network that uriniferous tubules (see Fig. 20.25).

lead into a surrounds the local

Efferent arterioles from juxtamedullary glomeruli

descend into the medulla alongside the loop of Henle; they break up into smaller vessels that continue toward the apex of the pyramid but make hairpin turns at various levels to return as straight vessels toward the base of the pyramid (see Fig. 20.25). Thus, the efferent arterioles from the juxtamedullary glomeruli give rise to the , which, together with the , are involved in the countercurrent exchange system. They drain via a peritubular medullary capillary network into the arcuate veins. These vessels are described in the explanation of the countercurrent exchange system (page 792).

descending vasa recta ascending vasa recta

Generally, venous flow in the kidney follows a reverse course to arterial flow, with the veins running in parallel with the corresponding arteries (see Fig. 20.25). Thus, the venous flow is as follows:

Peritubular cortical capillaries drain into interlobular veins, which, in turn, drain into arcuate veins, interlobar veins, and the renal vein. The medullary vascular network drains into arcuate veins and so forth. Peritubular capillaries near the kidney surface and capillaries of the capsule drain into stellate veins (socalled for their pattern of distribution when viewed from the kidney surface), which drain into interlobular veins, and so forth.

LYMPHATIC VESSELS The kidneys contain two major networks of lymphatic vessels. These networks are not usually visible in routine histologic sections but can be demonstrated by experimental methods. One network is located in the outer regions of the cortex and drains into larger lymphatic vessels in the capsule. The other network is located more deeply in the substance of the kidney and drains into large lymphatic vessels in the renal sinus. There are numerous anastomoses between the two lymphatic networks.

NERVE SUPPLY

renal plexus sympathetic division

The fibers that form the are unmyelinated, derived mostly from the of the autonomic nervous system. There is no evidence for parasympathetic innervation of the kidney. The sympathetic postsynaptic nerves that form the renal plexus are adrenergic (secrete at their nerve terminals). They travel along the renal artery and vein and are distributed to all segments of the renal vasculature. The highest concentrations of nerves are observed along the afferent arterioles, followed by the efferent arterioles. They cause contraction of vascular smooth muscle and consequent vasoconstriction that regulate urine production as follows:

norepinephrine

Constriction of the afferent arterioles to the glomeruli reduces the filtration rate and decreases the production of urine.

Constriction of the efferent arterioles from the glomeruli increases the filtration rate and increases the production of urine. Loss of sympathetic innervation leads to increased urinary output. It has been proposed that chronic hyperstimulation of sympathetic nerves in the renal plexus is an important factor in . Therefore, minimally invasive techniques targeting the renal plexus are being progressively developed, including intraluminal denervation by radiofrequency ablation, denervation by high-intensity–focused ultrasound, or computed tomography–guided injection of agents (i.e., ethanol or vincristine) that obliterate nerve fibers. Based on evidence collected from patients who received a , it is evident that the extrinsic nerve supply is not necessary for normal renal function. Although the nerve fibers to the kidney are severed during renal transplantation, transplanted kidneys subsequently function normally.

resistant hypertension

kidney transplant

URETER, URINARY BLADDER, AND URETHRA

All excretory passages, except the urethra, have the same general organization. On leaving the collecting ducts at the area cribrosa, the urine enters a series of structures that do not modify it but are specialized to store and pass the urine to the exterior of the body. The urine flows sequentially to a , a , and the and leaves each kidney through the to the , where it is stored. The urine is finally voided through the urethra. All of these excretory passages, except the urethra, have the same general structures, namely, a mucosa (lined by transitional epithelium), muscularis, and adventitia (or, in some regions, a serosa).

calyx ureter

renal pelvis urinary bladder

minor calyx

major

Transitional epithelium lines the calyces, ureters, bladder, and the initial segment of the urethra.

Transitional epithelium (urothelium)

lines the excretory passages leading from the kidney and forms the interface between the urinary space and underlying blood vessels, nerves, connective tissue, and smooth muscle cells (Figs. 20.27 and 20.28). This stratified epithelium is essentially impermeable to salts and water. The cells in the transitional epithelium are composed of at least three layers:

FIGURE 20.27. Photomicrograph of transitional epithelium (urothelium). This hematoxylin and eosin (H&E)-stained specimen shows the four- to five-cell layer thickness of the epithelium in the relaxed ureter. The surface cells exhibit a rounded or dome-shaped profile. The

TEp

connective tissue (lamina propria) below the epithelium ( ) is relatively cellular and contains a number of lymphocytes. Blood vessels ( ) are also abundant in this area. ×450.

BV

FIGURE 20.28. Transmission electron micrograph of urinary bladder epithelium. The mucous membrane of the urinary bladder consists of

TEp SupL

LP

transitional epithelium ( ) with an underlying lamina propria ( ). The superficial layer ( ) contains dome-shaped cells with unique fusiform vesicles ( ), which are evident here at this relatively low magnification. These are seen at higher magnification in Figure 20.29b. The intermediate layer ( ) of variable thickness contains cells that can differentiate and replace lost dome-shaped cells. The basal layer ( ) contains stem cells of the transitional epithelium. ×5,000.

FV

BasL

IntL

superficial layer

The contains single or multinucleated large, polyhedral cells (25–250 μm in diameter) that bulge into the lumen. They are frequently described as or because of their apical surface curvature (see Fig. 20.28). The shape of these epithelial cells depends on the filling state of the excretory passage. For instance, in an empty urinary bladder, dome-shaped cells are roughly cuboidal; however, when the bladder is filled, they are highly stretched and appear flat and squamous. Edges of the cells exhibit ridges, which are formed by the interdigitations of apical surface membranes from adjacent cells. These interdigitations resemble a closed zipper line and contribute to the highresistance paracellular barrier that reinforces tight junctions. The contains pear-shaped cells that are connected to each other and the overlying dome-shaped cells by desmosomes. The thickness of this layer varies with the state of the urinary tract expansion, which, in humans, may reach up to five layers thick. When the overlying dome-shaped cell is lost, the population of intermediate cells rapidly differentiates and replaces the lost surface cell. The consists of small cells containing a single nucleus that rests on the basement membrane. This layer contains for the urothelium.

umbrella cells

dome-shaped

intermediate cell layer

basal cell layer stem cells

The epithelium begins in the minor calyces as two cell layers and increases to four to five layers in the ureter and as many as six or more layers in the empty bladder. However, when the bladder is distended, as few as three layers are seen (see Fig. 20.28). This change reflects the ability of the cells to accommodate distension. The cells in the distended bladder, particularly the large, dome-shaped surface cells, flatten and those in intermediate layers slide past one another to

accommodate the increasing surface area. As the individual cells reorganize in the distended bladder, the resulting appearance is the “true” three layers.

The luminal surface of the transitional epithelium is covered by rigid urothelial plaques containing crystalline protein uroplakins, which play an important role in the permeability barrier. When the wall of a nondistended urinary bladder is examined with the TEM, the apical plasma membrane of dome-shaped cells

exhibits an unusual scallop-shaped appearance. Most of the apical plasma membrane is covered by the rigid-looking concave separated by intervening narrow (Fig. 20.29). In cross sections, the outer leaflet of the lipid bilayer is twice as thick as the inner leaflet; thus, the region of the urothelial plaque appears asymmetrical, hence the name . The thicker outer leaflet of the urothelial plaque contains a crystalline array of hexagonally arranged 16-nm protein particles composed of a family of five transmembrane proteins called (UPIa, UPIb, UPII, UPIIIa, and UPIIIb; Fig. 20.30). The crystalline arrangement of the uroplakin particles makes the plaque impermeable to small molecules (water, urea, and protons). In conjunction with tight junctions, urothelial plaques play an important role in the urothelial permeability barrier. The hinge areas of the plasma membrane contain all other nonplaque proteins typically found on the apical cell domain, such as receptors and channels. Approximately 85% of are caused by , which colonize the transitional epithelium. The initial adhesion to the epithelium allows the bacteria to gain a foothold on the epithelial surface, thus preventing them from being removed during micturition. This binding is mediated by located at the tip of a filamentous attachment apparatus of , which interacts with the in the asymmetric unit membrane of the urothelial plaques. In addition, the interaction with uroplakins triggers a cascade of events that lead to bacterial invasion into cells of the transitional epithelium.

urothelial plaques

hinge regions

asymmetric unit membrane (AUM)

uroplakins

urinary tract infections uropathogenic Escherichia coli uroplakins

FimH adhesins E. coli

FIGURE 20.29. Transmission electron micrograph of the apical portion of a dome-shaped cell. a. The cytoplasm displays small

vesicles, filaments, and mitochondria, but the most distinctive feature of the cell is its fusiform vesicles ( ). Note that the apical plasma membrane is covered by the rigid-looking concave urothelial plaques ( ) separated by intervening narrow hinge regions ( ). ×27,000. Higher magnification shows that the membrane forming the fusiform vesicles ( ) is similar to the apical plasma membrane of the urothelial plaque ( ). Both membranes are thickened and represent an asymmetric unit membrane ( ) in which the outer leaflet of the lipid bilayer is twice as thick as the inner leaflet. Uroplakins, the specific proteins of the urothelial plaque, are produced in the rough endoplasmic reticulum ( ) and then transported to the Golgi apparatus, where they undergo oligomerization into 16-nm particles with the final assembly into a crystalline array. The -Golgi network packages into the fusiform vesicles for delivery to the apical cell membrane. ×60,000.

FV

arrow

UP

HR

b.

AUM

rER

trans

AUMs

UP

FIGURE 20.30. Diagrams of the luminal surface of dome-shaped cells. a. This drawing depicts a luminal surface of dome-shaped cells

in a relaxed bladder. Note the apical plasma membrane of each cell is covered by the ridged concave urothelial plaques that are separated by intervening narrow hinge regions. The fusiform vesicles ( ) containing additional plaque membranes accumulate in the upper part of the cell. Most of them are vertically oriented, and some are attached to hinge regions of the apical cell membrane. This diagram depicts the same cell shown in as it would appear in a stretched bladder. Note the additional plaques that have been added to the surface from the fusiform vesicles. The remaining vesicles in this stage are visible in a more horizontal position. The urothelial plaque in cross section exhibits features of the asymmetric unit membrane ( ) in which the outer leaflet of the lipid bilayer is twice as thick as the inner leaflet. The is present in both urothelial plaques and fusiform vesicles. The thicker outer leaflet of the urothelial plaque contains a crystalline array of hexagonally arranged, 16-nm in diameter proteins that is composed of transmembrane proteins called uroplakins.

drawn in

different color

b.

a

AUM

c.

d.

AUM

Transitional epithelium maintains a urothelial permeability barrier despite dynamic changes in the wall of the urinary bladder and other urine-containing organs. As the bladder or other urine-containing organs distend, the folded surface of the mucosa becomes stretched and expands. Domeshaped cells also undergo changes in their apical membrane that are associated with the presence of . When observed with the TEM, the fusiform vesicles are oriented perpendicular and positioned in close proximity to the apical plasma membrane. They are formed by similar to those in the urothelial plaques. In response to the distention of the bladder, the apical membrane expands as a result of exocytosis of the fusiform vesicles that become part of the cell surface (see Fig. 20.28). Most of the fusiform vesicles fuse at the hinge regions to the apical cell surface, whereas the remaining vesicles assume a more parallel position in relation to the apical membrane. During micturition, the process is reversed as the added apical membrane is recovered by endocytosis and the apical membrane of the dome-shaped cells shortens.

fusiform vesicles

asymmetric unit membranes

Urothelium is an active participant in signaling mechanisms between the external environment facing the urine, nerve fibers, and smooth muscles of the urinary passages.

Experimental studies have shown that urothelium does not act only as a passive permeability barrier but also plays an active role in the signaling mechanisms of the urinary organs. The urothelium responds to chemical and mechanical stimuli and is engaged in autocrine or paracrine communication with nerve fibers and smooth muscles of the urinary passages. Expression of a variety of receptors on its surface (i.e., nicotinic, muscarinic, and adrenergic) and ion channels allows urothelial cells to respond to neurotransmitters released from nerve fibers. In addition, urothelial cells are able to produce and secrete acetylcholine (ACh), nitric oxide (NO), and nerve growth factor (NGF), which regulate the activity of underlying nerve fibers and smooth muscles.

Smooth muscle of the urinary passages is arranged in bundles.

A dense collagenous lamina propria underlies the urothelium throughout the excretory passages. Neither a muscularis mucosae nor a submucosal layer is present in their walls. In the tubular portions (ureters and urethra), usually two layers of lie beneath the lamina propria:

muscle Longitudinal layer, loose spiral pattern Circular layer, the

smooth

the inner layer that is arranged in a outer layer that is arranged in a tight

spiral pattern

Note that this arrangement of the smooth muscle is opposite to that of the muscularis externa of the intestinal tract. The smooth muscle of the urinary passages is mixed with connective tissue so that it forms parallel bundles rather than pure muscular sheets. Peristaltic contractions of the smooth muscle move the urine from the minor calyces through the ureter to the bladder.

Ureters

ureter

Each conducts urine from the renal pelvis to the urinary bladder and is approximately 24–34 cm long. The distal part of the ureter enters the urinary bladder and follows an oblique path through the wall of the bladder.

Transitional epithelium

(urothelium)

lines the luminal surface of the wall of the ureter. The remainder of the wall is composed of smooth muscle and connective tissue. The smooth muscle is arranged in three layers: an inner longitudinal layer, a middle circular layer, and an outer longitudinal layer (Plate 20.5, page 810). However, the outer longitudinal layer is present only at the distal end of the ureter. Usually, the ureter is embedded in the retroperitoneal adipose tissue. The adipose tissue, vessels, and nerves form the adventitia of the ureter. As the bladder distends with urine, the openings of the ureters are compressed, reducing the possibility of reflux of urine into the ureters. Contraction of the smooth muscle of the bladder wall also compresses the openings of the ureters into the bladder. This action helps prevent the spread of infection from the bladder and urethra, frequent sites of chronic infection (particularly in females), to the kidney. In the terminal portion of the ureters, a thick outer layer of longitudinal muscle is present in addition to the two listed earlier, particularly in the portion of the ureter that passes through the bladder wall. Most descriptions of the bladder musculature indicate that this longitudinal layer continues into the wall of the bladder to form a principal component of its wall. The smooth muscle of the bladder, however, is not as clearly separated into distinctive layers.

Urinary Bladder

urinary bladder

The is a distensible reservoir for urine, located in the pelvis, posterior to the pubic symphysis; its size and shape change as it fills. It contains three openings, two for the ureters ( ) and one for the urethra ( ). The triangular region defined by these three openings, the , is relatively smooth and constant in thickness, whereas the rest of the bladder wall is thick and folded when the bladder is empty and thin and smooth when the bladder is distended. These differences reflect the embryologic origins of the trigone and the rest of the bladder wall. Because the trigone is derived from the embryonic mesonephric ducts, neither a muscularis mucosae nor a submucosal layer is present. Because the major portion of the bladder wall

ureteric orifices internal urethral orifice trigone

originates from the cloaca (a part of the hindgut), its wall possesses layers comparable to those found in the gastrointestinal tract. The smooth muscle of the bladder wall forms the . Toward the opening of the urethra, the muscle fibers form the involuntary , a ring-like arrangement of muscle around the opening of the urethra. The smooth muscle bundles of the detrusor muscle are less regularly arranged than that of the tubular portions of the excretory passages; thus, the muscle and collagen bundles are randomly mixed (Plate 20.6, page 812). Contraction of the detrusor muscle of the bladder compresses the entire organ and forces the urine into the urethra. The by both sympathetic and parasympathetic divisions of the autonomic nervous system:

muscle

internal urethral sphincter

detrusor

bladder is innervated

Sympathetic fibers

form a plexus in the adventitia of the bladder wall. These sympathetic postsynaptic fibers originate from the hypogastric plexus and release . NA activates both to relax the detrusor 3 muscle and to contract smooth muscle 1 fibers of the internal urethral sphincter. In this way, the sympathetic nervous system relaxes the bladder and contracts the internal urethral sphincter simultaneously. originate from the S2 to S4 segments of the spinal cord and travel with pelvic splanchnic nerves into the terminal ganglia located in the muscle bundles and adventitia of the bladder. Increased stimulation of parasympathetic postsynaptic nerve fibers releases , which causes bladder contraction by stimulating 3 in the smooth fibers of the detrusor muscle. These nerves also release , which relaxes smooth muscles of the internal urethral sphincter. Therefore, the parasympathetic system provides efferent fibers for the . from the bladder to the sacral portion of the spinal cord are the afferent fibers of the micturition reflex.

β -adrenergic receptors α -adrenergic receptors

noradrenaline (NA)

Parasympathetic fibers

acetylcholine (ACh) M muscarinic receptors (NO) micturition reflex Sensory fibers

nitric oxide

Voluntary control over micturition is provided by the somatic nerve fibers from the pudendal nerve (S2–S4), which innervate

the skeletal (striated) muscle of the external sphincter of the urethra. Somatic axon terminals at the neuromuscular junctions release , which stimulates contraction of the external sphincter striated muscle by activating . This sphincter surrounds the membranous urethra as it passes through the deep perineal pouch. Innervation from pudendal nerve fibers maintains constant tonic contraction of the skeletal muscle fibers of the external sphincter. During micturition, these fibers are inhibited, causing relaxation of the external sphincter and voiding of urine.

acetylcholine (ACh) cholinergic receptors

nicotinic

Urethra

urethra orifice In the male

The is the fibromuscular tube that conveys urine from the urinary bladder to the exterior through the . The size, structure, and functions of the urethra differ in males and females. , the urethra serves as the terminal duct for both the urinary and genital systems. It is about 20 cm long and has three distinct segments:

external urethral

Prostatic urethra

extends for 3–4 cm from the neck of the bladder through the prostate gland (see pages 885-889). It is lined with transitional epithelium (urothelium). The ejaculatory ducts of the genital system enter the posterior wall of this segment, and many small prostatic ducts also empty into this segment. extends for about 1 cm from the apex of the prostate gland to the bulb of the penis. It passes through the of the pelvic floor as it enters the perineum. Skeletal muscle of the deep perineal pouch surrounding the membranous urethra forms the . Transitional epithelium ends in the membranous urethra. This segment is lined with a stratified or pseudostratified columnar epithelium that resembles the epithelium of the genital duct system more than it resembles the epithelium of the more proximal portions of the urinary duct system. extends for about 15 cm through the length of the penis and opens on the body surface at the

Membranous urethra deep perineal pouch

(voluntary) sphincter of the urethra Penile (spongy) urethra

external

glans

penis. The penile urethra is surrounded by the corpus spongiosum as it passes through the length of the penis. It is

lined with pseudostratified columnar epithelium, except at its distal end, where it is lined with stratified squamous epithelium continuous with that of the skin of the penis. and mucussecreting empty into the penile urethra.

of the bulbourethral glands (Cowper glands) urethral glands (glands of Littré) In the female,

Ducts

the urethra is short, measuring 3–5 cm in length from the bladder to the vestibule of the vagina, where it normally terminates just posterior to the clitoris (see Chapter 23, Female Reproductive System, page 944). The mucosa is traditionally described as having longitudinal folds. As in the male urethra, the lining is initially transitional epithelium, a continuation of the bladder epithelium, but changes to stratified squamous epithelium before its termination. Some investigators have reported the presence of stratified columnar and pseudostratified columnar epithelium in the midportion of the female urethra. Numerous small urethral glands, particularly in the proximal part of the urethra, open into the urethral lumen. Other glands, the , which are homologous to the prostate gland in the male, secrete into the common . These ducts open on each side of the external urethral orifice. They produce an alkaline secretion. The lamina propria is a highly vascularized layer of connective tissue that resembles the corpus spongiosum in the male. Where the urethra penetrates the urogenital diaphragm (membranous part of the urethra), the striated muscle of this structure forms the external (voluntary) urethral sphincter.

paraurethral glands (Skene glands) paraurethral ducts

URINARY SYSTEM

OVERVIEW OF THE URINARY SYSTEM urinary system

The includes the kidneys, ureters, bladder, and urethra. Essential functions of the kidneys include via control of electrolyte and water balance, plasma pH, tissue osmolality, and blood pressure; and of metabolic waste products; and , such as secretion of hormones to regulate bone marrow erythropoiesis (EPO), blood pressure (renin), and Ca2+ metabolism (activation of vitamin D).

homeostasis filtration endocrine

excretion activities

GENERAL STRUCTURE OF THE KIDNEY

capsule

Each kidney is surrounded by a connective tissue and contains an outer and inner , which is divided into 8–12 renal . The cortex extends into the medulla to form that separate the renal pyramids from each other. The is characterized by renal corpuscles and their associated and . Aggregation of straight tubules and collecting ducts in the cortex forms the . A renal includes the renal pyramid and its associated cortical tissue. The base of each renal faces the cortex, and the apical portion ( ) projects into the minor calyx, a branch of the that, in turn, is a division of the renal pelvis. At the , the renal pelvis extends into the ureter, which carries urine into the urinary bladder. Each kidney receives blood from the , which branches into the (run between pyramids) that then turn along the base of the pyramid ( ) and further branch into smaller that supply the cortex. In the cortex, the interlobular artery gives off the (one to each glomerulus), which give

cortex pyramids renal columns

cortex

convoluted medullary rays lobe

medulla

straight tubules

pyramid papilla major calyx

hilum

renal artery interlobar arteries

arcuate arteries interlobular arteries afferent arterioles

rise to the capillaries that form the glomerulus. The glomerular capillaries reunite to form a single that, in turn, gives rise to a second network of capillaries, the . Some of the peritubular capillaries form long loops called the , which accompany the thin segments of the nephrons. The peritubular capillaries drain into the , which, in turn, drain into the , , and the .

efferent

arteriole

peritubular capillaries

vasa recta

veins interlobar veins

renal vein

interlobular arcuate veins

STRUCTURE AND FUNCTION OF NEPHRONS nephron

The is the structural and functional unit of the kidney. The nephron consists of the and a long tubular part that includes a proximal thick segment ( and ), thin segment (thin part of the ), and distal thick segment ( and ). The distal convoluted tubule connects to the that opens at the renal papilla. The contains the surrounded by a double layer of the . The of the kidney consists of the , , and the Bowman capsule . The negatively charged , which contains type IV and XVIII collagens, sialoglycoproteins, noncollagenous glycoproteins, proteoglycans, and glycosaminoglycans, acts as a physical barrier and an ion-selective filter. extend their processes around the capillaries and develop numerous primary and secondary processes that give rise to , that interdigitate with other foot processes of the neighboring podocytes. The spaces between the interdigitating foot processes form that are covered by the .

renal corpuscle proximal convoluted tubule proximal straight tubule loop of Henle distal straight tubule distal convoluted tubule collecting tubule renal corpuscle glomerulus Bowman capsule filtration apparatus glomerular endothelium glomerular basement membrane (GBM) podocytes GBM Podocytes

pedicels (foot processes)

filtration slits diaphragm

filtration slit

Podocalyxin

nephrin

and play a key role in maintaining the structural and functional integrity of the kidney’s filtration apparatus. The GBM in the renal corpuscle is shared among several capillaries to create a space for and their extracellular matrix. are involved in phagocytosis and endocytosis of residues trapped in the filtration slits, secretion of paracrine substances, structural support for podocytes, and modulation of glomerular distention. The includes the + (monitors Na concentration in tubular fluid), (secrete renin), and . It regulates blood pressure by activating the .

mesangial cells

Mesangial cells

juxtaglomerular apparatus macula densa juxtaglomerular cells extraglomerular mesangial cells renin–angiotensin–aldosterone system (RAAS)

KIDNEY TUBULE FUNCTION

glomerular ultrafiltrate

The from the Bowman capsule passes through a series of tubules and collecting ducts lined by epithelial cells that secrete and absorb various substances to produce the final urine. The receives the glomerular ultrafiltrate from the Bowman capsule. This tubule is the initial and major site for of glucose, amino acids, polypeptides, water, and electrolytes. Reabsorption of the ultrafiltrate continues as it flows from the proximal convoluted into the (the thick descending limb of the loop of Henle) that descends into the medulla. The , with both the descending limb (highly permeable to water) and ascending limb (highly permeable to Na+ and Cl− ), concentrates the ultrafiltrate. The (thick ascending limb) ascends back into the cortex to reach the vicinity of its renal corpuscle, where it makes contact with the afferent arteriole. In this area, the epithelial cells of the tubule form the .

proximal convoluted tubule reabsorption

tubule loop of Henle

distal straight tubule

macula densa

proximal straight

distal convoluted tubule cortical collecting duct

The empties the ultrafiltrate into the that lies in the medullary ray, which further adjusts the concentration of Na+ and K+ in the ultrafiltrate. The medullary is lined by cuboidal cells, with a transition to columnar cells as the duct increases in size. The collecting ducts possess and that regulate water reabsorption. The open at the renal papilla, and the modified ultrafiltrate, now called , flows sequentially via the excretory passages.

collecting duct

aquaporins antidiuretic hormone (ADH)-regulated water channels collecting ducts urine

URETER, URINARY BLADDER, AND URETHRA All excretory passages for urine, except the urethra, have the same general organization: They are lined by a mucosa containing and have a smooth muscle layer and a connective tissue adventitia (or serosa). is a specialized stratified epithelium with large that bulge into the lumen. The dome-shaped cells have a modified apical membrane containing and that accommodate the invaginated excess of the plasma membrane, which is needed for the extension of the apical surface when the organ is stretched. Transitional epithelium creates a and is actively involved in molecular signaling mechanisms. The conducts urine from the renal pelvis to the urinary bladder. It is lined by transitional epithelium, underlying smooth muscle arranged in three distinct layers, and connective tissue adventitia. The is also lined by transitional epithelium and possesses many mucosal folds, except in the region. Its muscular wall is thick and well developed and forms the .

transitional epithelium (urothelium)

Transitional epithelium dome-shaped (umbrella) cells plaques

fusiform vesicles

permeability barrier

ureter

urinary bladder trigone

detrusor muscle

urethra conveys urine from the external urethral orifice . The female urethra is short and The

urinary bladder to the

lined by transitional epithelium (upper half), pseudostratified columnar epithelium (lower half), and stratified squamous epithelium (before its termination). The is much longer than the female’s and is divided into three regions: the (lined by transitional epithelium), a short that pierces the external urethral sphincter (lined by stratified or pseudostratified columnar epithelium), and a long (lined by pseudostratified columnar epithelium).

male urethra

prostatic urethra membranous urethra

penile urethra

PLATE 20.1 KIDNEY I

kidneys urinary bladder

The urinary system consists of the paired ; the paired , which lead from the kidneys to the ; and the , which leads from the bladder to the exterior of the body. The kidneys conserve body fluid and electrolytes and remove metabolic wastes, such as urea, uric acid, creatinine, and breakdown products of various substances. They produce , initially an ultrafiltrate of blood that is modified by selective resorption and specific secretion by kidney tubule cells. The kidneys also function as endocrine organs, producing , a growth factor that regulates red blood cell formation, and , a hormone involved in blood pressure and blood volume control. They also hydroxylate vitamin D, a steroid prohormone, to produce its active form. Each kidney is a flattened, bean-shaped structure ~10 cm long, 6.5 cm wide (from convex to concave border), and 3 cm thick. The concave medial border of each kidney contains a hilum, an indented region through which blood vessels, nerves, and lymphatic vessels enter and leave the kidney. The funnel-shaped origin of the ureter, the , also leaves the kidney at the hilum. A cut, hemisected fresh kidney reveals two distinct regions: a , the reddish brown outer region, and a , a much lighter inner part continuous with the renal pelvis. The cortex is characterized by and their tubules, including the convoluted and straight tubules of the , the , and an extensive vascular supply.

ureters urethra

urine

erythropoietin

renin

renal pelvis

collecting ducts

renal corpuscles

medulla

nephron

cortex

cortical

Kidney

, human, fresh specimen ×3.

A frontal section through the cortex and medulla of an unembalmed kidney obtained from autopsy is shown here. The visible consists of minor calyces (gray/white) surrounded by in appearance adipose tissue. The outer part of the kidney has a appearance; this is the . It is easily distinguished from the inner portion, the medulla, which is further divided into an outer portion ( ), identified here by the presence of straight blood vessels, the vasa recta ( ), and an inner portion ( ), which has a lighter and more homogeneous appearance. The consists of renal pyramids, which have their base facing the cortex and their apex in the form of a papilla ( ) directed toward the hilum. The are separated, sometimes only partially, as in this figure, by cortical material that is designated the ( ). The majority of the outer part of the pyramid on the has not been included in the plane of section. The papillae are free tips of the pyramids that project into the first of a series of large urine– collecting spaces referred to as the ( ); the inner surface of the calyx is white. The minor calyces drain into , and in turn, these open into the , which funnels urine into the ureter. An interesting feature in this specimen is that the blood has been retained in many of the vessels, thereby allowing for visualization of several renal vessels in their geographic location. Among the vessels that can be identified in the cut face of the kidney shown here are the interlobular vessels ( ) within the cortex; the arcuate veins ( ) and the arcuate arteries ( ) at the base of the pyramids; the interlobar arteries ( ) and veins ( ) between renal pyramids; and, in the medulla, the vessels going to and from the capillary network of the pyramid. The latter vessels, both arterioles and venules, are relatively straight and are designated collectively as the vasa recta ( ). (Specimen courtesy of Dr. Eric A. Pfeifer.)

hilar yellow region

reddish brown

medulla RCol

IM

cortex

OM

VR

P

pyramids

renal left columns

minor calyces MC renal pelvis

calyces

ILA

IV AA

major

AV

ILV

VR

Cortex and medulla and eosin (H&E) ×20.

medulla

, kidney, human, hematoxylin

cortex

A histologic section including the and part of the is shown here. Located at the boundary between the two (partly marked by the ) are numerous profiles of arcuate arteries ( ) and arcuate veins ( ). The most distinctive feature of the renal cortex, regardless of the plane of section, is the presence of the renal corpuscles ( ). These are spherical structures composed of a glomerulus (glomerular vascular tuft) surrounded by the visceral and parietal epithelium of the Bowman capsule. Also seen in the cortex are

AA

RC

dashed line AV

groups of tubules that are more or less straight and disposed in a radial direction from the base of the medulla ( ); these are the medullary rays. In contrast, the medulla presents profiles of tubular structures that are arranged as gentle curves in the outer part of the medulla, turning slightly to become straight in the inner part of the medulla. The disposition of the tubules (and blood vessels) gives the cut face of the pyramid a slightly striated appearance that is also evident in the gross specimen (see the previous figure).

arrows

AA, arcuate arteries AV, arcuate veins ILA, interlobar artery ILV, interlobar vein IM, inner medulla IV, interlobular vessels MC, minor calyx OM, outer medulla P, papilla RCol, renal column RC, renal corpuscles VR, vasa recta arrows, medullary rays dashed line, boundary between cortex and medulla

PLATE 20.2 KIDNEY II nephron

The is the functional unit of the kidney. There are about 2 million nephrons in each human kidney. They are responsible for the production of urine and correspond to the secretory part of other glands. The , responsible for the final concentration of the urine, are analogous to the ducts of exocrine glands. The nephron is made up of the and the . The renal corpuscle consists of the , a tuft of 10–20 capillary loops, surrounded by a double-layered epithelial cup, the . The glomerular capillaries are supplied at the vascular pole of the Bowman capsule by an and drained by an that leaves the Bowman capsule at the vascular pole and then branches to form a new capillary network to supply the kidney tubules. The opposite pole of the Bowman capsule, the urinary pole, is where the filtrate leaves the renal capsule. The tubular parts of the nephron are the (consisting of the and the ); the , which constitutes the ; and the , consisting of the and the . The loop of Henle is the U-shaped portion of the nephron consisting of the thick straight portions of the proximal and distal tubules and the thin segment between them. The distal convoluted tubule joins the via either the connecting tubule or arched connecting tubule. The nephron and the connecting tubule constitute the .

collecting ducts

renal corpuscle renal tubule glomerulus renal or Bowman capsule afferent arteriole efferent arteriole proximal thick segment proximal convoluted tubule proximal straight tubule thin segment thin limb of the loop of Henle distal thick segment distal straight tubule distal convoluted tubule cortical collecting duct uriniferous tubule

Cortex ×60.

, kidney, human, hematoxylin and eosin (H&E)

renal cortex cortical labyrinth CL medullary rays renal corpuscles RC

The can be divided into regions referred to as the ( ) and the ( ). The cortical labyrinth contains the ( ), which appear as relatively large spherical structures. Surrounding each renal corpuscle are the proximal and distal convoluted tubules. They are also part of the cortical labyrinth. The convoluted tubules, particularly the proximal, because of their tortuosity, present a variety of profiles, most of which are oval or circular; others, more elongated, are in the shape of a letter J, a C, or even an S. The medullary rays are composed of groups of straight tubules oriented in the same direction and appear to radiate from the base of the pyramid. When the medullary rays are cut longitudinally, as they are in this figure, the tubules present

MR

elongated profiles. The medullary rays contain proximal straight tubules (thick segments; descending limb of loop of Henle), distal straight tubules (thick segments; ascending limbs of loop of Henle), and cortical collecting ducts.

Cortex

, kidney, human, H&E ×120.

cortex This

renal

figure presents another profile of the , at a somewhat higher magnification, cut in a plane at a right angle to the section in the previous figure. The peripheral part of the micrograph shows the in which the tubules display chiefly not only round and oval profiles but also some that are more elongated and curved. The appearance is the same as the cortical labyrinth areas of the previous figure. A ( ) is also present in the cortical labyrinth. In contrast, the profiles presented by the tubules of the medullary ray in this figure are quite different from those seen in the previous figure. All of the tubules bounded by the belong to the medullary ray ( ), and all are cut in cross section. A general survey of the tubules within the medullary ray reveals that several distinct types can be recognized on the basis of the size of the tubule, shape of the lumen, and size of the tubule cells. These features as well as those of the cortical labyrinth are considered in Plate 20.3 (page 806).

cortical labyrinth renal

corpuscle RC MR

dashed line

CL, cortical labyrinth MR, medullary ray RC, renal corpuscle dashed line, approximate boundary of the medullary ray

PLATE 20.3 KIDNEY III

Proximal and distal convoluted tubules display features that aid in their identification in hematoxylin and eosin (H&E)-stained paraffin sections. generally have a larger diameter than distal tubules have; cross sections of the lumen often appear stellate. A brush border (apical microvilli) is often visible on the proximal tubule cells. Also, the proximal convoluted tubule is more than twice as long as the ; thus, the majority of tubular profiles in the cortical labyrinth will be of proximal tubules. and their extracellular matrix constitute the of the renal corpuscle. They underlie the endothelium of the capillaries of the glomerular tuft and extend to the vascular pole, where they become part of the . The terminal portion of the distal thick segment of the nephron lies close to the afferent arteriole. Tubule epithelial cells closest to the arteriole are thinner, taller, and more closely packed than other tubule cells and constitute the . Arterial smooth muscle cells opposite the macula densa are modified into that secrete in response to decreased blood NaCl concentration.

Proximal convoluted tubules

convoluted tubule Mesangial cells mesangium

juxtaglomerular cells

distal

juxtaglomerular apparatus renin

macula densa

Proximal and distal convoluted tubules

,

kidney, human, H&E ×240.

cortical labyrinth distal convoluted tubule DC proximal convoluted tubules

In this figure, an area of , there are six ( ) profiles. The (unlabeled) have a slightly larger outside diameter than the distal tubules have. The proximal tubules have a brush border, whereas the distal tubules have a cleaner, sharper luminal surface. The lumen of the proximal tubules is often star shaped; this is not the case with distal tubules. Typically, fewer nuclei appear in a cross section of a proximal tubule than in an equivalent segment of a distal tubule. Most of the abovementioned points can also be utilized in distinguishing the straight portions of the proximal and distal thick segments in the medullary rays, as shown in the figure on the .

right

Proximal and distal straight tubules

,

kidney, human, H&E ×240.

proximal

In this figure, all of the tubular profiles within the medullary ray are rounded, except for a ( ) included in the of the figure (it belongs to the adjacent cortical labyrinth). Second, the

convoluted tubule PC

lower right corner

P

D

number of proximal straight ( ) and distal straight ( ) tubular profiles are about equal in the medullary ray, as is shown by the labeling of each tubule in this figure. Note that, in contrast to the , the display a brush border and have a larger outside diameter, with many displaying a starshaped lumen. The medullary ray also contains ( ).

straight tubules

distal

proximal straight tubules cortical collecting

ducts CCD

Renal corpuscles

, kidney, human, H&E ×360.

renal corpuscle

The appears as a spherical structure whose periphery is composed of a thin capsule that encloses a narrow clear-appearing space, the ( ), and a capillary tuft or glomerulus that appears as a large cellular mass. The capsule of the renal corpuscle, known as the or , actually has two parts: a parietal layer, which is marked ( ), and a visceral layer. The consists of simple squamous epithelial cells. The consists of cells called ( ) that lie on the outer surface of the glomerular capillary. Except where they clearly line the urinary space, as the labeled cells do in the figure on the , podocytes may be difficult to distinguish from the capillary endothelial cells. To complicate matters, the mesangial cells are also a component of the glomerulus. In general, nuclei of podocytes are larger and stain less intensely than do the endothelial and mesangial cell nuclei. A distal ( ) and two proximal ( ) convoluted tubules are marked in the figure on the . The cells of the distal tubule are more crowded on one side. These crowded cells constitute the ( ) that lies adjacent to the afferent arteriole. In the figure on the , both the vascular pole and the urinary pole of the renal corpuscle are evident. The is characterized by the presence of arterioles ( ), one of which is entering or leaving ( ) the corpuscle. The afferent arteriole possesses modified smooth muscle cells with granules, the juxtaglomerular cells (not evident in this figure). At the , the parietal layer of the Bowman capsule is continuous with the beginning of the proximal convoluted tubule ( ). Here, the urinary space of the renal corpuscle continues into the lumen of the proximal tubule, and the lining cells change from simple squamous to simple cuboidal or low columnar with a brush border.

asterisks

Bowman capsule BC

podocytes Pod DC

urinary space renal parietal layer visceral layer left

PC

left

right

double-headed arrow

pole

A, arteriole BC, Bowman capsule (parietal layer)

macula densa MD A

PC

vascular pole

urinary

CCD, cortical collecting duct D, distal straight tubule DC, distal convoluted tubule MD, macula densa P, proximal (straight tubule) PC, proximal convoluted tubule Pod, podocyte (visceral layer of Bowman capsule) asterisks, urinary space double-headed arrow, blood vessel at vascular pole of renal corpuscle

PLATE 20.4 KIDNEY IV Renal corpuscles are restricted to the cortical labyrinth. The contains the thick straight segments of proximal and distal tubules, along with their thin segments, the collecting ducts, and the blood vessels that run in parallel with them. These structures function as the and systems that, ultimately, produce hypertonic urine. The final urine drains from the papillary ducts (of Bellini) into calyces that then empty into the renal pelvis.

medulla

countercurrent multiplier

countercurrent exchange

Medulla

, kidney, human, hematoxylin and eosin (H&E)

×240.

outer portion of the medulla

A section through the is shown in this figure. This region contains proximal and distal thick segments, thin segments, and medullary collecting ducts. All of the tubules are parallel, and all are cut in cross section; thus, they present circular profiles. The ( ) display typical star-shaped lumina and a brush border (or the fragmented apical cell surface from which the brush border has been partially broken). These tubules have outside diameters that are generally larger than those of the ( ). As mentioned previously and as shown here, the distal straight tubules display a larger number of nuclei than do comparable segments of proximal straight tubule cells. Note, also, that the lumen of the distal tubule is more rounded and the apical surface of the cells is sharper. The ( ) have outer diameters that are about the same as those of the proximal tubules and larger than those of distal tubules. The cells forming the collecting ducts are cuboidal and smaller than those of proximal tubules; thus, they also display a relatively larger number of nuclei than do comparable segments of proximal tubule cells. Count them. Finally, boundaries between the cells that constitute the collecting ducts are usually evident ( ); this serves as one of the most dependable features for the identification of collecting ducts. The thin segments ( ) have the thinnest walls of all renal tubules seen in the medulla. They are formed by a low cuboidal or simple squamous epithelium, as seen here, and the lumina are relatively large. Occasionally, a section includes the region of transition from a thick to a thin segment and can be recognized even in a cross section through the tubule. One such junction is evident in this figure (the tubule with in the lumen). On one side, the tubule cell ( ) is characteristic of the proximal segment; it possesses a

proximal straight tubules P

distal straight tubules D

collecting

ducts CD

T

two arrows arrow

asterisks

left-pointing

right-pointing

distinctive brush border. The other side of the tubule ( ) is composed of low cuboidal cells that resemble the cells forming the thin segments. In addition to the uriniferous tubules and collecting ducts, there are many other small tubular structures in this figure. Thin walled and lined by endothelium, they are small blood vessels.

arrow

Renal pyramid

, kidney, human, H&E ×20.

renal pyramid

This figure shows a at low magnification. The pyramid is a conical structure composed principally of medullary straight tubules, ducts, and the straight blood vessels (vasa recta). The at the of the micrograph is placed at the junction between cortex and medulla; thus, it marks the base of the pyramid. Note the ( ) that lie at the boundary of cortex and medulla. They define the boundary line. The few renal corpuscles ( ), , belong to the renal column of the medulla. They are referred to as . The pyramid is somewhat distorted in this specimen, as evidenced by regions of longitudinally sectioned tubules, , and crosssectioned and obliquely sectioned tubules in other regions. In effect, part of the pyramid was bent, thus the change in the plane of section of the tubules.

dashed line

left

arcuate vessels AV

RC upper left

corpuscles

juxtamedullary

lower left

arrowhead

papilla calyx

renal

The apical portion of the pyramid ( ), known as the , is lodged in a cup- or funnel-like structure referred to as the . It collects the urine that leaves the tip of the papilla from the papillary ducts (of Bellini). (The actual tip of the papilla is not seen within the plane of section, nor are the openings of the ducts at this low magnification.) The surface of the papilla that faces the lumen of the is simple columnar or cuboidal epithelium ( ). (In places, this epithelium has separated from the surface of the papilla and appears as a thin strand of tissue.) The calyx is lined by transitional epithelium ( ). Although not evident at the low magnification shown here, the boundary between the columnar epithelium covering the papilla and the transitional epithelium covering the inner surface of the calyx is marked by the .

minor calyx

SCEp

TEp

diamonds

AV, arcuate vessels CD, collecting duct D, distal straight tubule P, proximal straight tubule RC, renal corpuscle SCEp, simple columnar epithelium T, thin segment TEp, transitional epithelium

left-pointing arrow (upper panel), proximal tubule cell right-pointing arrow (upper panel), thin segment cell arrowhead, location of apex of pyramid asterisks, boundaries between cells of a collecting duct diamonds, boundary between a transitional and a columnar epithelium

PLATE 20.5 URETER

ureters are paired tubular structures that convey urine kidneys to the urinary bladder. They are lined transitional epithelium (urothelium) , an impervious layer The the

from with that lines the urinary excretory passages from the renal calyces through the urethra. The ability of this epithelium to become thinner and flatter allows all of these passages to accommodate to distension by the urine. The epithelium rests on a dense collagenous lamina propria, which, in turn, rests on an inner longitudinal and an outer circular layer of smooth muscle. Regular peristaltic contractions of this muscle contribute to the flow of urine from the kidney to the urinary bladder. As shown in this low-power orientation micrograph, the wall of the ureter consists of a ( ), a ( ), and an ( ). Note that the ureters are located behind the peritoneum of the abdominal cavity in their course to the bladder. Thus, a ( ) may be found covering a portion of the circumference of the tube. Also, because of contraction of the smooth muscle of the muscularis, the luminal surface is characteristically folded, thus creating a starshaped lumen.

ORIENTATION MICROGRAPH: mucosa Muc muscularis Mus

adventitia Adv serosa Ser

Ureter

, monkey, hematoxylin and eosin (H&E) ×160.

rectangular area

The wall of the ureter from the in the orientation micrograph is examined at higher magnification in this figure. One can immediately recognize the thick epithelial lining, which appears distinct and sharply delineated from the remainder of the wall. This is the ( ). The remainder of the wall is made up of connective tissue ( ) and smooth muscle. The latter can be recognized as the darker staining layer. The section also shows some adipose tissue ( ), a component of the adventitia. The transitional epithelium and its supporting connective tissue constitute the ( ). A distinct submucosa is not present, although the term is sometimes applied to the connective tissue that is closest to the muscle.

transitional epithelium (urothelium) CT Ep AT

mucosa Muc

muscularis Mus

The ( ) is arranged as an inner longitudinal layer ( ), a middle circular layer ( ), and an outer longitudinal layer ( ). However, the outer longitudinal layer is present only at the lower end of the ureter. In a cross section through the ureter, the inner and outer smooth muscle layers are cut in cross section, whereas the middle circular layer of the muscle cells is cut longitudinally. This is as they appear in this figure.

SM[l]

SM[c]

SM[l]

Transitional epithelium ×400.

muscle This

, ureter, monkey, H&E

inner longitudinal smooth

figure shows the layer ( ) at higher magnification. Note that the nuclei appear as round profiles, indicating that the muscle cells have been cross-sectioned. This figure also shows the ( ) to advantage. The surface cells of the transitional epithelium (urothelium) are characteristically the largest, and some are binucleate ( ). The basal cells are the smallest, and typically, the nuclei appear crowded because of the minimal cytoplasm of each cell. The intermediate cells appear to consist of several layers and are composed of cells larger in size than the basal cells but smaller than the surface cells.

SM[l]

epithelium Ep

arrow

Adv, adventitia AT, adipose tissue BV, blood vessels CT, connective tissue Ep, transitional epithelium Muc, mucosa Mus, muscularis Ser, serosa

transitional

SM(c), SM(l), arrow,

circular layer of smooth muscle longitudinal layer of smooth muscle binucleate surface cell

PLATE 20.6 URINARY BLADDER urinary bladder

The receives the urine from the two ureters and stores it until neural stimulation causes it to contract and expel the urine via the urethra. It, too, is lined with . Beneath the epithelium and its underlying connective tissue, the wall of the urinary bladder contains that is usually described as being arranged as an inner longitudinal layer, a middle circular layer, and an outer longitudinal layer. As in most distensible hollow viscera that empty their contents through a narrow aperture, the smooth muscle in the wall of the urinary bladder is less regularly arranged than the description indicates, allowing contraction to reduce the volume relatively evenly throughout the bladder. This orientation micrograph of the urinary bladder reveals the full thickness of the bladder wall. The luminal surface epithelium is at the of the micrograph. One of the ureters can be seen as it passes through the bladder wall to empty its contents into the bladder lumen. Most of the tissue to the sides and below the ureteral profile is smooth muscle.

transitional

epithelium (urothelium) smooth muscle

ORIENTATION MICROGRAPH:

top

Urinary bladder

, human, hematoxylin and eosin

(H&E) ×60.

This micrograph shows most of the entire thickness of the urinary bladder. An unusual feature is the presence of one of the ureters ( ) as it is passing through the bladder wall to empty its contents into the bladder lumen. The ( ) lining the bladder is seen on the . Beneath the epithelium is a relatively thick layer of connective tissue ( ) containing blood vessels ( ) of various sizes. Note that the connective tissue stains more eosinophilic than the smooth muscle of the underlying

epithelium Ep

U

transitional

BV

right

CT

muscularis ( M ). The epithelium and connective tissue constitute the mucosa of the bladder. The muscularis consists of smooth muscle arranged in three indistinct layers. It should be noted that as the ureter passes through the bladder wall, it carries with it a layer of longitudinally oriented smooth muscle ( ). Medium-sized arteries ( ) and veins ( ) are occasionally seen in the muscularis.

SM[L]

A

Transitional epithelium

V

, urinary bladder,

human, H&E ×250.

left rectangle

transitional epithelium Ep

This higher magnification of the of the previous figure shows the ( ) and the underlying connective tissue ( ) that represent the mucosa of the ureter. Adjacent to the mucosa are bundles of longitudinally sectioned smooth muscle ( ) that belong to the ureter. A small lymphatic vessel ( ) is present in the connective tissue adjacent to the smooth muscle. Note the lymphocytes, identified by their small round densely stained nuclei, within the lumen of the vessel.

Lym

SM[L]

CT

Transitional epithelium

, urinary bladder,

human, H&E ×250.

right rectangle

transitional epithelium

This higher magnification of the of the previous figure shows the bladder ( ) and the underlying connective tissue ( ) of the bladder wall. The transitional epithelium is often characterized by the presence of surface cells that are dome shaped. In addition, many of these cells are binucleate ( ). The thickness of transitional epithelium is variable. When the bladder is fully distended, as few as three cell layers are seen. Here, in the contracted bladder, it appears that there are as many as 10 cell layers, a result of the cells folding over one another as the smooth muscle contracts and the lining surface is reduced. The connective tissue consists of bundles of collagen fibers interspersed with varying numbers of lymphocytes identified by their densely stained round nuclei. A vein ( ) filled with red blood cells is also evident in the mucosal connective tissue.

Ep

CT

arrows

V

A, artery BV, blood vessel CT, connective tissue Ep, transitional epithelium Lym, lymphatic vessel M, muscularis SM(L), longitudinally cut smooth muscle

U, ureter V, vein arrows, binucleate cells

21

ENDOCRINE ORGANS

OVERVIEW OF THE ENDOCRINE SYSTEM

Hormones and Their Receptors Regulation of Hormone Secretion and Feedback Mechanism

PITUITARY GLAND (HYPOPHYSIS)

Gross Structure and Development Blood Supply Nerve Supply Anterior Lobe of the Pituitary Gland (Adenohypophysis) Posterior Lobe of the Pituitary Gland (Neurohypophysis)

HYPOTHALAMUS PINEAL GLAND

THYROID GLAND PARATHYROID GLANDS ADRENAL GLANDS

Blood Supply Cells of the Adrenal Medulla Zonation of the Adrenal Cortex Zona Glomerulosa Zona Fasciculata Zona Reticularis Fetal Adrenal Gland Functional Considerations: Regulation of Pituitary Gland Secretion Clinical Correlation: Principles of Endocrine Diseases Clinical Correlation: Pathologies Associated With Antidiuretic Hormone Secretion Clinical Correlation: Abnormal Thyroid Function Clinical Correlation: Chromaffin Cells and Pheochromocytoma Functional Considerations: Biosynthesis of Adrenal Hormones

Folder 21.1 Folder 21.2 Folder 21.3 Folder 21.4 Folder 21.5 Folder 21.6 HISTOLOGY

OVERVIEW OF THE ENDOCRINE SYSTEM

endocrine system produces various secretions called hormones [Gr. hormaein, to excite, to set in motion] that serve as effectors to The

regulate the activities of various cells, tissues, and organs in the body. Its functions are essential in maintaining homeostasis and coordinating body growth and development and are similar to that of the : Both communicate information to peripheral cells and organs. Communication in the nervous system is through transmission of neural impulses along nerve cell processes and the discharge of neurotransmitter. Communication in the endocrine system is through hormones, which are carried to their destination via connective tissue

nervous system

spaces and the vascular system. These two systems are functionally interrelated. The endocrine system produces a slower and more prolonged response than the nervous system. Both systems may act simultaneously on the same target cells and tissues, and some nerve cells secrete hormones.

Endocrine glands possess no excretory ducts, and their secretions are carried to specific destinations via the extracellular matrix of connective tissue and the vascular system. In general, endocrine glands are aggregates of epithelioid cells (epithelial cells that lack a free surface) that are embedded within connective tissue. Despite the fact that the endocrine glands vary in size, shape, and location in the body (Fig. 21.1), they still have several common characteristics. Endocrine glands do not possess excretory ducts; therefore, their secretion is discharged into the extracellular matrix of connective tissue, usually near the capillaries. From here, the secretory products (i.e., hormones) are transported into the lumen of the blood (or lymphatic) vessels for body-wide distribution. These secretory products influence target organs or tissues at some distance from the gland. For this reason, endocrine glands are well vascularized and surrounded by rich vascular networks. The exception is the placenta, where hormones produced by the syncytiotrophoblast pass directly into the maternal blood surrounding the placental villi (see Chapter 23, Female Reproductive System, page 937).

FIGURE 21.1. Location of the major endocrine glands and organs containing hormone-secreting cells. This drawing shows the major endocrine

glands in which the hormone-secreting cells constitute the majority of the gland parenchyma. Note that the placenta is a temporary organ developed from maternal and fetal tissues and is also a major endocrine organ that secretes steroid and protein hormones during pregnancy (see Chapter 23, Female Reproductive System, pages 933-938). Hormone-secreting cells, commonly

classified as part of the diffuse neuroendocrine system (DNES), are present in many organs to regulate their activity. In addition, adipose tissue is an important hormonally active tissue that secrets a variety of hormones, growth factors, and cytokines, collectively called (see Chapter 9, Adipose Tissue, page 280).

adipokines

As mentioned earlier, the majority of hormone-producing cells have an , either from the (i.e., posterior lobe of pituitary gland, pineal gland), (i.e., medulla of suprarenal gland), or from epithelial lining of the developing (i.e., anterior lobe of pituitary gland, thyroid and parathyroid glands). Only a few endocrine glands/cells have a mesenchymal origin and are derived from the (i.e., cortex of the adrenal gland, Leydig cells in the testis, and steroidsecreting cells of developing follicles in the ovary). This chapter primarily describes the major in which the hormone-secreting cells constitute the majority of the gland parenchyma. Secretory cells in the gland parenchyma form various arrangements, such as follicles (thyroid gland), anastomosing cords (adrenal glands), or nests (parathyroid glands). They are also present in clusters (nuclei in the hypothalamus) or layers surrounding the functional and structural elements of the organ (testis, ovaries, or placenta). These characteristics are useful in the microscopic identification of specific endocrine organs.

epithelial origin gut tube

central nervous system (CNS) neural crest urogenital ridges

endocrine glands

Individual hormone-secreting cells are present in many organs to regulate their activity. The collection of individual endocrine cells in various organs constitutes the diffuse neuroendocrine system (DNES; see page 644). In addition to their endocrine function, cells of the DNES system exercise autocrine and paracrine control of the activity of their own and adjacent epithelial cells by diffusion of peptide secretions. Other chapters discuss the endocrine function of adipose tissue and individual cells within the liver, pancreas, kidney, and the gastrointestinal, cardiovascular, respiratory, reproductive, lymphatic, and integumentary systems (see Fig. 21.1).

Hormones and Their Receptors

In general, a hormone is described as a biological substance acting on specific target cells. In the classic definition, a hormone is a secretory product of

endocrine cells and organs that passes into the circulatory system (bloodstream) for transport to target cells. For many years, this

endocrine control

of target tissues became a central part of endocrinology. However, a variety of hormones and hormonally active substances are not always discharged into the bloodstream but are released into connective tissue spaces. They may act on adjacent cells or diffuse to nearby target cells that express specific receptors for that particular hormone (Fig. 21.2). This type of hormonal action is referred to as . In addition, some cells express receptors for hormones that they secrete. This type of hormonal action is referred to as . These hormones regulate the cell’s own activity. Figure 21.2 summarizes various hormonal control mechanisms.

paracrine control autocrine control

FIGURE 21.2. Hormonal control mechanisms. This schematic diagram shows three basic types of control mechanisms. a. In endocrine control, the hormone is discharged from a cell into the bloodstream and is transported to the effector cells. b. In paracrine control, the hormone is secreted from one cell and acts on adjacent cells that express specific receptors. c. In autocrine control, the produces it.

hormone

responds

to

the

receptors

located

on

the

cell

that

New research characterizes exosomes as important components of the endocrine system that provide endocrine, paracrine, and autocrine signals to target cells in the body. Research over the past decade indicates that in addition to the nervous and endocrine systems, a third system of intercellular or interorgan communication exists that involves . As discussed in Chapter 2, Cell Cytoplasm (pages 47-48), exosomes are small, membrane-bound secreted into the extracellular space by virtually every cell in the body. They are released and detected in all bodily fluids,

vesicles

exosomes

cargo

including blood, lymph, cerebrospinal fluid, vitreous body, interstitial fluids, saliva, breast milk, amniotic fluid, semen, and urine. Most secreted exosomes are disseminated via blood circulation and travel to specific targets in the body. They transfer and from the original (parent) cells to the target destination. The exosomes and their cargo molecules either fuse directly with the plasma membrane of target cells, become endocytosed, or engage in receptor–ligand-mediated interactions (Fig. 21.3). Exosomes can carry a variety of molecules that include , such as viral DNA (vDNA), mitochondrial DNA (mtDNA), and fragments of double-stranded DNA (dsDNA) or single-stranded DNA (ssDNA); and , such as messenger RNA (mRNA), transfer RNA (tRNA), small nuclear RNA (snRNA), small nucleolar RNA (snoRNA), mitochondrial RNA (mtRNA), microRNA (miRNA), viral microRNA (vmiRNA), circular RNA (CircRNA), small noncoding RNA (Y-RNA), toxic small RNA (tsRNA), Piwiinteracting RNA (piRNA), and others. For details on exosomal contents, see Figure 2.21 (page 47).

cargo molecules

messages

growth factors; lipids; proteins; several forms of DNA molecules several types of RNA molecules

FIGURE 21.3. Exosomal intercellular signaling.

This drawing summarizes exosomal signaling, an intercellular communication pathway in which proteins, metabolites, and nucleic acids are delivered to recipient cells using exosomes as transfer vehicles. Exosomes are very small, membrane-bound cargo vesicles derived from multivesicular bodies (MVB) and secreted into the extracellular space. Most exosomes use the blood circulation to reach specific target cells in the body. Exosomes transfer messages in the form of cargo molecules, membrane-associated proteins, and nucleic acids from donor cells to the target destination. Exosomes and their cargo molecules either fuse directly with the plasma membrane of target cells, become endocytosed, or engage in receptor– ligand-mediated interactions. Exosomes then release their contents directly into the cytoplasm of the recipient cell, initiating downstream signaling

cascades. Note specific transmembrane proteins (tetraspanins) that serve as exosomal biomarkers.

viruses

SARS-CoV-2

In disease states, exosomes also carry such as and . Their cargo can be transmitted from cell to cell (often in distant locations) to alter cells’ metabolism, influence immune cell responses, promote differentiation, and facilitate disease development (e.g., viral diseases and cancer). For example, the actin-binding protein (pGSN) transported in exosomes promotes ovarian cancer cell survival through autocrine and paracrine mechanisms that transform chemosensitive cancer cells into resistant forms. Thus, exosomal pGSN is a determinant of in .

(COVID-19)

HIV-1 (AIDS)

gelsolin

chemoresistance ovarian cancer Hormones include three classes of compounds. Cells of the endocrine system release more than 100 hormones and hormonally active substances that are chemically divided into three classes of compounds:

Peptides

(small peptides, polypeptides, and proteins) form the largest group of hormones. They are synthesized and secreted by cells of the hypothalamus, pituitary gland, thyroid gland, parathyroid gland, pancreas, and scattered enteroendocrine cells of the gastrointestinal tract and respiratory system. This group of hormones ( , , , , , , , , and ), when released into the circulation, dissolve readily in the blood and generally do not require special transport proteins. However, most, if not all, polypeptides and proteins have specific carrier proteins (e.g., ). , cholesterol-derived compounds, are synthesized and secreted by cells of the ovaries, testes, and adrenal cortex. These hormones ( and ) are released into the bloodstream and transported to target cells with the help of plasma proteins or specialized carrier proteins, such as . Hormone-binding carrier proteins protect the hormone from degradation during transport to the target tissue. When needed, the hormone is released from the carrier protein to become active. and and their derivatives, including the (norepinephrine and epinephrine– phenylalanine/tyrosine derivatives) and , , and (arachidonic acid derivatives), are

insulin glucagon growth hormone [GH] adrenocorticotropic hormone [ACTH] follicle-stimulating hormone [FSH] luteinizing hormone [LH] antidiuretic hormone [ADH] oxytocin, interleukins various growth factors factor–binding protein [IGFBP] Steroids gonadal adrenocortical steroids protein

Amino acids

arachidonic acid analogs catecholamines prostacyclins leukotrienes

insulin-like growth

androgen-binding prostaglandins

synthesized and secreted by many neurons as well as a variety of cells, including cells of the adrenal medulla. Also included in this group of compounds are , the iodinated derivatives of the amino acid tyrosine that are synthesized and secreted by the thyroid gland. When released into the circulation, catecholamines dissolve readily in the blood, in contrast to thyroid hormones, the majority of which are bound to three carrier proteins: a specialized , prealbumin fraction of serum proteins ( ), and a nonspecific fraction of albumins.

thyroid hormones

thyroxine-binding globulin (TBG) transthyretin Hormones interact with specific hormone receptors to alter biological activity of the target cells. The first step in hormone action on a target cell is its binding to a specific hormone receptor. However, recent studies suggest that some

hormones are involved in non–receptor-mediated responses. Hormones interact with their receptors exposed on the surface of the target cell or within its cytoplasm or nucleus. In general, two groups of hormone receptors have been identified:

Cell surface receptors

interact with peptide hormones or catecholamines that are unable to penetrate the cell membrane. Activation of these receptors as a result of hormone binding rapidly generates large quantities of small intracellular molecules called (Fig. 21.4a). These molecules amplify the signal initiated by hormone–receptor interaction and are produced by activation of membrane-associated (named for the ability to hydrolyze guanosine triphosphate [GTP]). Examples of such systems include the (for most protein hormones and catecholamines), the (an antagonistic system for action of cAMP in some protein hormones), the (for insulin and epidermal growth factor [EGF]), the (for certain hormones such as oxytocin, gonadotropin-releasing hormone [GnRH], angiotensin II, and neurotransmitters such as epinephrine), and (as with most neurotransmitters). The majority of second messenger molecules exert a stimulatory function on cell metabolism. Examples of second messenger molecules include , , 3 , and 2+ . An inhibitory response is mainly achieved by , which interferes with the production of cAMP. The second messenger molecules produced in the cascade reactions of these systems alter the cell’s metabolism and produce hormone-specific responses (see Fig. 21.4a).

second messengers

G-proteins adenylate cyclase/cyclic adenosine monophosphate (cAMP) system guanylyl cyclase/cyclic guanosine monophosphate (cGMP) system tyrosine kinase system phosphatidylinositol system activation of ion channels cAMP 1,2diacylglycerol (DAG) inositol 1,4,5-triphosphate (IP ) Ca cGMP

FIGURE 21.4. General mechanisms of protein and steroid hormone actions. a. This schematic diagram shows the basis for protein hormone action involving cell surface receptors. Hormone molecules bind to the cell surface receptors ( orange glow indicates activated receptor) and initiate a

cascade of intracellular signaling reactions that may include G-protein and various protein kinases resulting in the synthesis of second messenger molecules. These molecules, in turn, elicit hormone-specific responses in the stimulated cell that may influence channel proteins, nuclear transcription, and protein synthesis or degradation. This diagram shows two mechanisms of action for steroid hormones, which include nuclear-initiated steroid signaling (involves intracellular receptors) and membrane-initiated steroid signaling. In the nuclear-initiated steroid signaling ( ), some steroid hormones (e.g., glucocorticoids, androgens) cross the plasma membrane and bind to specific cytoplasmic receptors. This binding of the hormone causes allosteric transformation of the receptor, and the resulting complex travels to the nucleus guided by the nuclear localization signal ( ), where it binds to DNA and regulates the transcription of specific genes ( ). Other steroid hormones (e.g., estrogens, progestogens) bind to their specific receptors directly in the nucleus ( ). This binding of hormone to the nuclear receptor transforms this complex into the DNA transcription factor ( ) and leads to mRNA transcription and the subsequent production of new proteins responsible for hormone-specific responses of the stimulated cell. In membrane-initiated steroid signaling ( ), the steroid receptors are expressed on the cell membrane, usually in the caveolae, and their pathway is similar to that of the cell surface receptor signaling mechanism.

b.

green arrows NLS

glow

orange glow

blue arrows

orange

red arrows

Intracellular receptors, which are localized within the cell, are used by steroid hormones, thyroid hormones, and vitamins A and D (Fig. 21.4b). Steroid hormones and vitamins A and D can easily penetrate both plasma and nuclear membranes. In the absence of hormone, steroid receptors for glucocorticoids and gonadocorticoids (adrenal androgens) reside in the cytoplasm, whereas estrogen and

progesterone receptors are located in the nucleus. Unoccupied inactive receptors for thyroid hormones and vitamins A and D also reside in the nucleus. Intracellular receptors consist of large multiprotein complexes containing three binding domains: a hormoneor ligand-binding region at the COOH-terminus, a DNA-binding region, and the NH2-terminus containing the gene regulatory region. Because the receptor–ligand complex must enter the nucleus to regulate transcription, intracellular receptors contain a nuclear localization signal (NLS) for trafficking into the nucleus (see Fig. 21.4b). Binding of the hormone to the receptor causes the allosteric transformation of the receptor into a form that binds to the chromosomal DNA and activates . This, in turn, increases transcription of mRNA, resulting in the production of new proteins that regulate cell metabolism. Therefore, hormones acting on the intracellular receptors influence gene expression directly, without the help of a second messenger (see Fig. 21.4b). This type of signaling is often described as .

RNA polymerase activity

nuclear-initiated steroid signaling

Action of steroid hormones on the cell’s genome to induce a biological response in the form of new protein synthesis takes time (hours or days). However, some cells react more rapidly (in seconds or minutes) to steroid hormone stimulation by increasing intracellular Ca+ concentration and activating several intracellular proteins. This finding led to the discovery of , which have a similar structure to intracellular receptors but are localized on the plasma membrane most often within the caveolae. Binding to steroid membrane receptors activates the signaling cascade of G-protein, which, in turn, activates protein kinases, causing a rapid change in cell activity (see Fig. 21.4b). This type of signaling is known as . Both nuclear- and membrane-initiated steroid signaling pathways converge to provide a full biological response of the target cell to the steroid hormone stimulation.

steroid membrane receptors

initiated steroid signaling

membrane-

Regulation of Hormone Secretion and Feedback Mechanism

Regulation of hormonal function is controlled by feedback mechanisms. Hormonal production is often controlled through feedback mechanisms

from the target organ. In general, feedback occurs when the response to a stimulus (action of a hormone) has an effect on the original stimulus (hormone-secreting cell). The nature of this response determines the type of feedback. Two types of feedback are recognized: occurs when the response diminishes the original stimulus and

feedback

Negative

positive feedback

is much more common than , which occurs when the response enhances the original stimulus. To better understand the function of feedback mechanisms, one can point to an air conditioning system, which also uses a simple negative feedback system. When the compressor produces enough cold air to lower the temperature below the set point of the thermostat, the thermostat is triggered and shuts off the compressor. In this negative feedback system, the lower temperature is then fed back to the compressor and diminishes its response (it shuts off its production of cold air). When the temperature rises back above the set point, the negative feedback is abolished and the compressor comes back on (for more information on negative feedback, see Folder 21.1).

FOLDER 21.1

FUNCTIONAL CONSIDERATIONS: REGULATION OF PITUITARY GLAND SECRETION

release of hormones from the anterior lobe of the pituitary gland is carefully regulated by three tiers of control mechanisms that include the following: Tier I: Hypothalamic secretion of hypothalamic-regulating hormones . The pituitary gland is under significant control by the The

hypothalamus, which regulates the release of hypothalamic-regulating hormones into the hypophyseal portal veins. The hypothalamic-regulating hormones are produced by the cells of the hypothalamus in response to circulating levels of systemic hormones and impulses from the central nervous system (CNS). These hormones act directly on the highly specific G-protein–linked receptors on the plasma membranes of cells residing in the anterior lobe of the pituitary gland. Activation of receptors elicits positive or negative signals that affect gene transcription and lead to stimulation or inhibition of pituitary hormone secretion. Most of the tropic hormones produced by the anterior lobe of the pituitary gland are regulated by polypeptide-releasing hormones, with the notable exception of dopamine. Prolactin (PRL) production is primarily regulated by the inhibitory effect of dopamine (i.e., PRL secretion is tonically inhibited by the release of dopamine from the hypothalamus).

Tier II: Paracrine and autocrine secretions of the pituitary cells . Release of hormones from the pituitary gland is also regulated by soluble growth factors and cytokines produced by the cells residing in the pituitary gland. . The level of hormones in the systemic circulation regulates the secretion of cells in the anterior lobe of the pituitary gland. This is primarily achieved by negative feedback regulation of hormones secreted by the pituitary gland by target hormones. For instance, secretion of thyroid-stimulating hormone (TSH) is inhibited by thyroid hormones produced in the thyroid gland under TSH influence.

Tier III: Feedback effect of circulating hormones

To better understand the mechanism of negative regulation, consider a simple that controls the synthesis and discharge of T 3 and T 4 thyroid hormones (see Fig. 21.18). The secretion of thyroid

negative feedback system

hormones is controlled by the release of TSH from the anterior lobe of the pituitary gland into the bloodstream. If blood levels of T 3 and T 4 are high, TRH is not produced or released. If blood levels of T 3 and T 4 are low, the hypothalamus discharges TRH into the hypothalamohypophyseal portal system. Release of TRH stimulates specific cells within the anterior lobe of the pituitary gland to produce TSH, which, in turn, stimulates the thyroid to produce and release more thyroid hormones. As the thyroid hormone levels rise, the negative feedback system stops the hypothalamus from discharging thyrotropin-releasing hormone (TRH). Using the same mechanism of negative feedback regulation, thyroid hormones also act on the thyrotropes in the anterior lobe of the pituitary gland to inhibit their secretion of TSH.

Activities of hormones are constantly monitored on many levels, beginning with molecular biosynthetic processes to the final end points of hormonal action. Several examples of feedback mechanisms are discussed in the sections on the pituitary, hypothalamus, and thyroid glands.

PITUITARY GLAND (HYPOPHYSIS) pituitary gland

hypothalamus

The and the , the portion of the brain to which the pituitary gland is attached, are morphologically and functionally linked in the endocrine and neuroendocrine control of other endocrine glands. Because they play central roles in a number of regulatory feedback systems, they are often called the of the endocrine system. In the past, the control of pituitary hormone secretion by the hypothalamus was classically regarded as the major function of the . However, the field of neuroendocrinology today has expanded to encompass multiple reciprocal interactions between the CNS, autonomic nervous system (ANS), endocrine system, and immune system in the regulation of homeostasis and behavioral responses to environmental stimuli. For example, the neuroendocrine axis’s role in maintaining energy homeostasis is discussed in Chapter 9, Adipose Tissue (pages 283-285).

master organs

neuroendocrine system

Gross Structure and Development

The pituitary gland is composed of glandular epithelial tissue and neural (secretory) tissue. The pituitary gland [Lat. pituta, phlegm—reflecting its nasopharyngeal origin] is a pea-sized, compound endocrine gland that weighs 0.5 g in males and 1.5 g in multiparous women (i.e., a woman who has given birth ≥2 times). It is centrally located at the base of the brain, where it lies in a saddle-shaped depression of the sphenoid bone called the

sella turcica. A short stalk, the infundibulum, and a vascular network connect the pituitary gland to the hypothalamus. The pituitary gland has two functional components (Fig. 21.5):

FIGURE 21.5. Pituitary gland. a.

Photomicrograph of a pituitary gland. Lobes of the pituitary gland can be identified on the basis of their appearance, location, and relation to each other. ×7. This drawing shows parts of the pituitary gland and related regions of the hypothalamus. The anterior lobe of the pituitary gland consists of the pars distalis, pars tuberalis, and pars intermedia; the posterior lobe consists of the infundibulum and pars nervosa. Note the distribution of the neurosecretory nuclei in the hypothalamus. The paraventricular nuclei produce oxytocin, and the supraoptic nuclei produce antidiuretic hormone ( ). These hormones are released in the pars nervosa of the posterior lobe. Neurosecretory cells in the ventral nuclei of the hypothalamus secrete releasing and inhibitory hormones that are discharged into capillaries (located in the median eminence and infundibulum) of the hypophyseal portal system to reach pars distalis of the anterior lobe.

b.

ADH

Anterior lobe (adenohypophysis), the glandular epithelial tissue Posterior lobe (neurohypophysis), the neural secretory tissue These two portions are of different embryologic origin. The anterior lobe of the pituitary gland is derived from an evagination of the toward the brain . The posterior lobe of the pituitary gland is derived from a downgrowth (the future infundibulum) of (the diencephalon) of the developing brain (Fig. 21.6).

ectoderm of the oropharynx (Rathke pouch) neuroectoderm of the floor of the third ventricle

FIGURE 21.6. Development of the pituitary gland. a.

The pituitary gland develops from two different structures: an ectodermal diverticulum of the roof of the oropharynx (Rathke pouch) and a downward extension of the neuroectoderm at the floor of diencephalon. This drawing shows the relationship between these two structures in a 6-week-old embryo. The pituitary gland at 10 weeks of development shows ectodermal tissue from the oropharynx in close proximity to neural tissue. The Rathke pouch is about to lose connection with the oropharynx. Cells from the Rathke pouch divide and differentiate rapidly into the pars distalis and encircle the infundibulum, which with pars nervosa forms the neuroectodermally derived posterior lobe of the pituitary gland.

b.

c.

anterior lobe of the pituitary gland

The derivatives of the Rathke pouch:

consists

of

three

Pars distalis,

which comprises the bulk of the anterior lobe of the pituitary gland and arises from the thickened anterior wall of the pouch , a thin remnant of the posterior wall of the pouch that abuts the pars distalis , which develops from the thickened lateral walls of the pouch and forms a collar or sheath around the infundibulum

Pars intermedia Pars tuberalis

The embryonic infundibulum gives rise to the posterior lobe of the pituitary gland. The consists of the following:

posterior lobe of the pituitary gland

Pars nervosa, which contains neurosecretory axons and their endings Infundibulum, which is continuous with the median eminence and contains the neurosecretory axons forming the hypothalamohypophyseal tracts (see Fig. 21.5)

Blood Supply

Knowledge of the unusual blood supply of the pituitary gland is important to understanding its functions. The pituitary blood supply is

derived from two sets of vessels (Fig. 21.7):

FIGURE 21.7. Diagram of the blood supply and hypothalamohypophyseal portal system of the pituitary gland. The superior and inferior hypophyseal

arteries originate from branches of the internal carotid arteries. The superior hypophyseal artery supplies the pars tuberalis, median eminence, and infundibulum of the hypothalamus, where it gives rise to a capillary network that drains into the hypophyseal portal veins. These veins give rise to a second capillary network in the pars distalis, where the neuroendocrine secretions produced in the hypothalamus and collected in the median eminence and infundibulum are released. The inferior hypophyseal artery provides the blood supply to the pars nervosa and has very few (if any) connections with the hypothalamohypophyseal portal system. The blood from the pituitary gland drains into the cavernous sinus and leaves the cranial cavity via the internal jugular veins.

Superior hypophyseal arteries

supply the pars tuberalis, median eminence, and infundibulum. These vessels arise from the internal carotid arteries and posterior communicating artery of the circle of Willis.

Inferior hypophyseal arteries

primarily supply the pars nervosa. These vessels arise solely from the internal carotid arteries. An important functional observation is that most of the anterior lobe of the pituitary gland has no direct arterial supply.

The hypothalamohypophyseal portal system provides the crucial link between the hypothalamus and the pituitary gland. The arteries that supply the pars tuberalis, median eminence, and infundibulum give rise to fenestrated capillaries (the primary capillary plexus). These capillaries drain into portal veins, called the , which run along the pars tuberalis and give rise to a second fenestrated sinusoidal capillary network (the secondary capillary plexus). This system of vessels carries the neuroendocrine secretions of hypothalamic neurons from their sites of release in the median eminence and infundibulum directly to the cells of the pars distalis. Most of the blood from the pituitary gland drains into the cavernous sinus at the base of the diencephalon and then into the systemic circulation. Some evidence suggests, however, that blood can flow via short portal veins from the pars distalis to the pars nervosa and that blood from the pars nervosa may flow toward the hypothalamus. These short pathways provide a route by which the hormones of the anterior lobe of the pituitary gland could provide feedback directly to the brain without making the full circuit of the systemic circulation.

hypophyseal portal veins

Nerve Supply The nerves that enter the infundibulum and pars nervosa from the hypothalamic nuclei are components of the posterior lobe of the pituitary gland (see the section that follows on the posterior lobe). The nerves that enter the anterior lobe of the pituitary gland are postsynaptic fibers of the ANS and have vasomotor function.

Anterior Lobe of the Pituitary Gland (Adenohypophysis)

The anterior lobe of the pituitary gland regulates other endocrine glands and some nonendocrine tissues. Most of the anterior lobe of the pituitary gland has the typical organization of endocrine tissue. The cells are organized in clumps and cords separated by fenestrated sinusoidal capillaries of relatively large diameter. These cells respond to signals from the hypothalamus

and synthesize and secrete a number of pituitary hormones. Four hormones of the anterior lobe— , , , and —are called because they regulate the activity of cells in other endocrine glands throughout the body. The two remaining hormones of the anterior lobe, and , are not considered tropic because they act directly on target organs that are not endocrine. The general character and effects of the pituitary hormones of the anterior lobe are summarized in Table 21.1.

adrenocorticotropic hormone (ACTH) thyroid-stimulating (thyrotropic) hormone (TSH; thyrotropin) follicle-stimulating hormone (FSH) luteinizing hormone (LH) tropic hormones growth hormone (GH) prolactin (PRL) TABLE 21.1 Hormones of the Anterior Lobe of the Pituitary Gland MW Hormone Composition (kDa) Major Functions Growth hormone Straight-chain 21,700 Stimulates liver and other (somatotropin, GH) protein (191 organs to synthesize and aa)

Prolactin (PRL)

Adrenocorticotropic hormone (ACTH)

Follicle-stimulating hormone (FSH) Luteinizing hormone (LH)

secrete insulin-like growth factor I (IGF-I), which, in turn, stimulates division of progenitor cells located in growth plates and skeletal muscles, resulting in body growth

Straight-chain protein (198 aa)

22,500

Promotes mammary gland development; initiates milk formation; stimulates and maintains secretion of casein, lactalbumin, lipids, and carbohydrates into the milk

Small polypeptide (39 aa)

4,000

Maintains structure and stimulates secretion of glucocorticoids and gonadocorticoids (adrenal androgens) by the zona fasciculata and zona reticularis of the adrenal cortex

2-chain glycoprotein a (α, 92 aa; β, 111 aa)

28,000

Stimulates follicular development in the ovary and spermatogenesis in the testis

2-chain glycoprotein a (α, 92 aa; β, 116 aa)

28,300

Regulates final maturation of ovarian follicle, ovulation, and corpus luteum formation;

Thyroid-stimulating hormone (TSH)

stimulates steroid secretion by follicle and corpus luteum; in males, essential for maintenance of and androgen secretion by the Leydig (interstitial) cells of the testis 2-chain glycoprotein a (α, 92 aa; β, 112 aa)

aa, amino acids.

28,000

Stimulates growth of thyroid epithelial cells; stimulates production and release of thyroglobulin and thyroid hormones

aThe

α-chains of FSH, LH, and TSH are identical and encoded by a single gene; the β-chains are specific for each hormone.

Pars distalis The cells within the pars distalis vary in size, shape, and staining properties. The cells within the pars distalis are arranged in cords and nests with interweaving capillaries. Early descriptions of the cells within the pars distalis were based solely on the staining properties of secretory vesicles within the cells. Using mixtures of acidic and basic dyes (Fig. 21.8), histologists identified three types of cells according to their staining reaction, namely, , , and . However, this classification provides no information regarding the hormonal secretory activity or functional role of these cells.

chromophobes (50%)

basophils (10%) acidophils (40%)

FIGURE 21.8. Pars distalis.

This specimen of the pars distalis is stained with brilliant crystal scarlet, aniline blue, and Martius yellow to distinguish the various cell types and connective tissue stroma. The cords of cells are surrounded by a delicate connective tissue stroma stained . The sinusoidal capillaries are seen in close association with the parenchyma and contain erythrocytes stained . In the region shown here, the acidophils ( ) are the most numerous cell type present. Their cytoplasm stains . The basophils ( ) stain . The chromophobes ( ), although few in number in this particular region, are virtually unstained. ×640.

Bas

yellow blue

blue

Ch

Ac cherry red

Five functional cell types are identified in the pars distalis on the basis of immunocytochemical reactions. All known hormones of the anterior lobe of the pituitary gland are small proteins or glycoproteins. This important fact has led to definitive identification of specific cell types by immunocytochemistry (Table 21.2). These studies have classified cells of the anterior lobe of the pituitary gland into five cell types:

Characteristics of Cells Found in the Anterior TABLE 21.2 Staining Lobe of the Pituitary Gland

PAS, periodic acid–Schiff.

Somatotropes (GH cells)

are most commonly found within the pars distalis and constitute approximately 50% of the parenchymal cells in the anterior lobe of the pituitary gland. These medium-sized, oval cells exhibit round, centrally located nuclei and produce . The presence of eosinophilic vesicles in their cytoplasm classifies them as acidophils. Three hormones regulate the release of GH from somatotropes. Two of these hormones are opposing hypothalamic-releasing hormones: , which stimulates GH release from the somatotropes, and , which inhibits GH release from the somatotropes. A third hormone, a 28-amino-acid peptide called , was isolated from the stomach in 1999. It is a potent stimulator of GH secretion and appears to coordinate food intake with GH secretion. Hormonally active tumors that originate from somatotropes are associated with hypersecretion of GH and cause gigantism in children and acromegaly in adults. constitute 15%–20% of the parenchymal cells in the anterior lobe of the pituitary gland. These are large, polygonal cells with oval nuclei. They produce . In their storage phase, lactotropes exhibit numerous

hormone (GH; somatotropin)

releasing hormone (GHRH) somatostatin ghrelin

Lactotropes (PRL cells, mammotropes) (PRL)

growth

growth hormone–

prolactin

acidophilic vesicles (the histologic feature of an acidophil). When the contents of these vesicles are released, the cytoplasm of the lactotrope does not stain (the histologic feature of a chromophobe). Secretion of PRL is under inhibitory control by , the catecholamine produced by the hypothalamus. However, and are known to stimulate the synthesis and secretion of PRL. During pregnancy and lactation, these cells undergo hypertrophy and hyperplasia, causing the pituitary gland to increase in size. These processes account for the larger size of the pituitary gland in multiparous women. also constitute 15%–20% of the parenchymal cells in the anterior lobe of the pituitary gland. These polygonal, medium-sized cells with round and eccentric nuclei produce a precursor molecule of known as . Corticotropes stain as basophils and also exhibit a strong positive reaction with periodic acid–Schiff (PAS) reagent because of the carbohydrate moieties associated with POMC. POMC is further cleaved by proteolytic enzymes within the corticotrope into several fragments, namely, ACTH, β-lipotropic hormone (β-LPH), melanocyte-stimulating hormone (MSH), β-endorphin, and enkephalin. ACTH release is regulated by produced by the hypothalamus. constitute about 10% of the parenchymal cells in the anterior lobe of the pituitary gland. These small, oval cells with round and eccentric nuclei produce both and . They are scattered throughout the pars distalis and stain intensely with both basic stains (thus classifying them as the basophil cell type) and PAS reagent. Many gonadotropes are capable of producing both FSH and LH. However, immunocytochemical studies indicate that some gonadotropes may produce only one hormone or the other. The release of FSH and LH is regulated by produced by the hypothalamus. Both FSH and LH play an important role in male and female reproduction, which is discussed in Chapter 22, Male Reproductive System (see pages 869-870 and 879-880), and Chapter 23, Female Reproductive System (see Folder 23.3, pages 926-927). constitute about 5% of the parenchymal cells in the anterior lobe of the pituitary gland. These large, polygonal cells with round and eccentric nuclei produce thyrotropic hormone called , which acts on the follicular cells of the thyroid gland to stimulate the production of thyroglobulin and thyroid hormones. Thyrotropes exhibit cytoplasmic

releasing hormone (TRH)

dopamine thyrotropinvasoactive inhibitory peptide (VIP)

Corticotropes (ACTH cells)

adrenocorticotropic hormone (ACTH) proopiomelanocortin (POMC)

corticotropin-releasing

hormone (CRH) Gonadotropes (FSH and LH cells) luteinizing hormone (LH)

follicle-stimulating hormone (FSH)

gonadotropin-releasing hormone

(GnRH)

Thyrotropes (TSH cells)

thyroid-stimulating hormone (TSH)

basophilia (basophils) and stain positively with PAS reagent. Release of TSH is under the hypothalamic control of , which also stimulates secretion of PRL. has an inhibitory effect on thyrotropes and decreases secretion of the TSH.

hormone (TRH)

thyrotropin-releasing Somatostatin

Distinctive characteristics of the five cell types of the anterior lobe of the pituitary gland are readily seen with transmission electron microscopy (TEM). These characteristics are summarized in Table 21.3.

Microscopic Characteristics of Cells Found in the TABLE 21.3 Electron Anterior Lobe of the Pituitary Gland

rER, rough-surfaced endoplasmic reticulum.

In addition to the five types of hormone-producing cells, the anterior lobe of the pituitary gland contains folliculostellate cells. Folliculostellate cells present in the anterior lobe of the pituitary gland are characterized by a star-like appearance with their cytoplasmic processes encircling hormone-producing cells. They are primarily non–hormone-secreting cells that have the ability to form cell clusters or small follicles and make up about 5%–10% of cells of the anterior lobe of the pituitary gland. Folliculostellate cells are interconnected by gap junctions containing protein. Based on immunocytochemical and electrophysiologic studies, it is hypothesized that the network of folliculostellate cells interconnected by gap junctions transmits signals from the pars tuberalis to pars distalis. These signals coordinate and modulate hormone release throughout the anterior lobe of the pituitary gland. Discovery of gap junctions interconnecting folliculostellate cells with hormoneproducing cells supports this proposed signaling mechanism in the anterior lobe of the pituitary gland. Thus, the

connexin-43

folliculostellate

network appears to have a regulatory function in addition to the hypophyseal portal vein system. Pars intermedia The pars intermedia surrounds a series of small cystic cavities that represent the residual lumen of the Rathke pouch. The parenchymal cells of the pars intermedia surround colloid-filled

follicles. The cells lining these follicles appear to be derived either from folliculostellate cells or various hormone-secreting cells. The TEM reveals that these cells form apical junctional complexes and have vesicles larger than those found in the pars distalis. The nature of this follicular colloid is yet to be determined; however, often, cell debris is found within it. The pars intermedia contains and (Fig. 21.9). Frequently, the basophils and cystic cavities extend into the pars nervosa.

chromophobes

basophils

FIGURE 21.9. Photomicrograph of the pars intermedia of an adult human pituitary gland. This photomicrograph of a toluidine blue–stained specimen shows the pars intermedia located between the pars distalis ( on the left ) and the pars nervosa ( on the right ). In humans, this portion of the gland is somewhat rudimentary. However, a characteristic feature of the pars intermedia is the presence of different-sized follicles filled with colloid ( CF ) and small groups of cells consisting of chromophobes and basophils. ×120.

The function of the pars intermedia cells in humans remains unclear. From studies of other species, however, it is known that basophils have scattered vesicles in their cytoplasm that contain either (a morphine-related compound). In frogs, the basophils produce , which stimulate pigment production in melanocytes and pigment dispersion in melanophores. In humans, MSH is not a distinct, functional hormone but is a by-product of β-LPH post-translational processing. Because MSH is found in the

endorphin melanocyte-stimulating hormones (MSH)

α- or β-

human pars intermedia in small amounts, the basophils of the pars intermedia are assumed to be .

corticotropes

Pars tuberalis The pars tuberalis is an extension of the anterior lobe along the stalk-like infundibulum. The pars tuberalis is a highly vascular region containing veins of the hypothalamohypophyseal system. The parenchymal cells are arranged in small clusters or cords in association with the blood vessels. Nests of squamous cells and small follicles lined with cuboidal cells are scattered in this region. These cells often show immunoreactivity for ACTH, FSH, and LH.

Posterior Lobe of the Pituitary Gland (Neurohypophysis)

The posterior lobe of the pituitary gland is an extension of the central nervous system (CNS) that stores and releases secretory products from the hypothalamus. The posterior lobe of the pituitary gland, also known as the neurohypophysis, consists of the pars nervosa and the infundibulum

that connects it to the hypothalamus. The pars nervosa, the neural lobe of the pituitary, contains the unmyelinated axons and their nerve endings of approximately 100,000 whose cell bodies lie in the and of the hypothalamus. The axons form the and are unique in two respects. First, they do not terminate on other neurons or target cells but end in close proximity to the fenestrated capillary network of the pars nervosa. Second, they contain secretory vesicles in all parts of the cells (i.e., the cell body, axon, and axon terminal). Because of their intense secretory activity, the neurons have welldeveloped Nissl bodies and, in this respect, resemble ventral horn and ganglion cells. The posterior lobe of the pituitary gland is . Rather, it is a of the neurons of the supraoptic and paraventricular nuclei of the hypothalamus. The unmyelinated axons convey neurosecretory products to the pars nervosa. Other neurons from the hypothalamic nuclei (described later in this chapter) also release their secretory products into the fenestrated capillary network of the infundibulum, the first capillary bed of the hypothalamohypophyseal portal system.

neurosecretory neurons supraoptic nuclei paraventricular nuclei hypothalamohypophyseal tract

not an endocrine gland storage site for neurosecretions

Electron microscopy reveals three morphologically distinct neurosecretory vesicles in the nerve endings of the pars nervosa. Three sizes of membrane-bound vesicles are present in the pars nervosa:

Neurosecretory vesicles

with diameters ranging between 10 and 30 nm accumulate in the axon terminals. They also form accumulations that dilate portions of the axon near the terminals (Fig. 21.10). These dilations, called , are visible in the light microscope (Plate 21.1, page 850). In the electron microscope, Herring bodies, in addition to abundant neurosecretory vesicles, contain mitochondria, few microtubules, and profiles of smooth-surfaced endoplasmic reticulum (sER) (Fig. 21.11).

Herring bodies

FIGURE 21.10. Electron micrograph of Herring bodies of rat posterior lobe. Dilated portions of axons near their terminals called Herring bodies ( HB ) contain numerous neurosecretory vesicles filled with either oxytocin or

antidiuretic hormone (ADH). They are surrounded by the specialized glial cells called pituicytes ( ). Note that Herring bodies reside in close proximity to blood vessels ( ), mainly fenestrated capillaries, lined by endothelial cells ( ). ×6,000. (Courtesy of Dr. Holger Jastrow.)

En

P

BV

FIGURE 21.11. Electron micrograph of rat posterior lobe.

Neurosecretory granules and small vesicles are present in the terminal portions of the

axonal processes of the hypothalamohypophyseal tract fibers. Capillaries with fenestrated endothelium are present in close proximity to the nerve endings. ×20,000. (Courtesy of Drs. Sanford L. Palay and P. Orkland.)

Nerve terminals also contain 30-nm vesicles that contain acetylcholine. These vesicles may play a specific role in the release of neurosecretory vesicles. Larger 50- to 80-nm vesicles that resemble the dense-core vesicles of the adrenal medulla and adrenergic nerve endings are present in the same terminal as the other membrane-bound vesicles. The membranebound neurosecretory vesicles that aggregate to form Herring bodies contain either or ( ; also called ; Table 21.4). Each hormone is a small peptide of nine amino acid residues. The two hormones differ in only two of these residues. Each vesicle also contains and a , a protein that binds to the hormone by noncovalent bonds. Oxytocin and ADH are synthesized as part of a large molecule that includes the hormone and its specific neurophysin. The large molecule is proteolytically cleaved into the hormone and neurophysin as it travels from the nerve cell body to the axon terminal. Immunocytochemical staining demonstrates that oxytocin and ADH are secreted by different cells in the hypothalamic nuclei.

vasopressin

oxytocin

antidiuretic hormone ADH ATP

neurophysin

TABLE 21.4 Hormones of the Posterior Lobe of the Pituitary Gland Hormone Oxytocin

Antidiuretic hormone (ADH; vasopressin)

Composition

Source

Major Functions

Polypeptide containing nine amino acids

Cell bodies of neurons located in the supraoptic and paraventricular nuclei of the hypothalamus a

Stimulates activity of the contractile cells around the ducts of the mammary glands to eject milk from the glands; stimulates contraction of smooth muscle cells in the pregnant uterus

Polypeptide containing nine amino acids; two forms: arginineADH (most common in humans) and lysine-ADH

Cell bodies of neurons located in the supraoptic and paraventricular nuclei of the hypothalamus a

Decreases urine volume by increasing reabsorption of water by collecting ducts of the kidney; decreases the rate of perspiration in response to

dehydration; increases blood pressure by stimulating contractions of smooth muscle cells in the wall of arterioles

aImmunocytochemical

studies indicate that oxytocin and ADH are produced by separate sets of neurons within the supraoptic and paraventricular nuclei of the hypothalamus. Biochemical studies have demonstrated that the supraoptic nucleus contains equal amounts of both hormones, whereas the paraventricular nucleus contains more oxytocin than ADH but less than the amount found in the supraoptic nucleus.

ADH facilitates reabsorption of water from the distal tubules and collecting ducts of the kidney by altering the permeability of the cells to water. ADH’s original name, vasopressin, was derived from the observation that large nonphysiologic doses increase blood pressure by promoting the contraction of smooth muscle in small arteries and arterioles. However, physiologic levels of ADH have only minimal effects on blood pressure. is the main hormone involved in the regulation of and . The primary physiologic effect of ADH on the kidney is the insertion of water channels ( ) into cells of the distal convoluted tubules and collecting ducts, which increases their permeability for water. Insertion of aquaporin-2 (AQP-2) into the apical domain and AQP-3 into the basolateral domain of these cells is responsible for rapid resorption of water across the tubule epithelium. ADH acts through its specific V2 receptor on the basolateral domain of cells lining the distal convoluted tubules and collecting ducts; mutation of this receptor is responsible for (Folder 21.3). and blood volume are monitored by specialized receptors of the cardiovascular system (e.g., carotid bodies and juxtaglomerular apparatus). An increase in osmolality or a decrease in blood volume stimulates ADH release. In addition, the cell bodies of the hypothalamic secretory neurons may also serve as osmoreceptors, initiating ADH release. Pain, trauma, emotional stress, and drugs such as nicotine also stimulate the release of ADH.

ADH homeostasis aquaporins

osmolarity of body fluids

water

nephrogenic diabetes insipidus Plasma osmolality

Oxytocin promotes contraction of smooth muscle of the uterus and myoepithelial cells of the breast. Oxytocin is a more potent promoter of smooth muscle contraction than ADH. Its primary effect includes promotion of contraction of the

following cells and tissues:

Uterine smooth muscle

during orgasm, menstruation, and parturition. As parturition approaches, uterine smooth muscle cells demonstrate about a 200-fold increase in responsiveness to oxytocin. This is accompanied by increased formation of gap junctions between smooth muscle cells and increased density of oxytocin receptors. of the secretory alveoli and alveolar ducts of the mammary gland. Oxytocin secretion is triggered by neural stimuli that reach the hypothalamus. These stimuli initiate a neurohumoral reflex that resembles a simple sensorimotor reflex. In the uterus, the is initiated by distension of the vagina and cervix. In the breast, the reflex is initiated by . Contraction of the myoepithelial cells that surround the base of the alveolar secretory cells and cells of the larger ducts causes milk to be released and passes through the ducts that open onto the nipple (i.e., milk ejection; see page 947). Synthetic are often used in intravenous infusion pumps to initiate and strengthen uterine contractions during active .

Myoepithelial cells

neurohumoral reflex (suckling)

breastfeeding

analogs of oxytocin labor and delivery The pituicyte is the only cell specific to the posterior lobe of the pituitary gland.

In addition to the numerous axons and terminals of the hypothalamic neurosecretory neurons, the posterior lobe of the pituitary gland contains fibroblasts, mast cells, and specialized glial cells called associated with the fenestrated capillaries (Plate 21.2, page 852). These cells are irregular in shape, with many branches, and resemble astroglial cells. Their nuclei are round or oval, and pigment vesicles are present in the cytoplasm. Like astroglia, they possess specific intermediate filaments assembled from . Pituicytes often have processes that terminate in the perivascular space. Because of their many processes and relationships to the blood, the pituicyte serves a supporting role similar to that of astrocytes in the rest of the CNS (see pages 410-412).

pituicytes

proteins (GFAP)

glial fibrillary acidic

HYPOTHALAMUS

The hypothalamus regulates pituitary gland activity. The hypothalamus is located in the middle of the base of the brain, and it encapsulates the ventral portion of the third ventricle. It coordinates most endocrine functions of the body and serves as one of

the major controlling centers of the ANS. Some of the functions that it regulates include blood pressure, body temperature, fluid and electrolyte balance, body weight, and appetite. The hypothalamus produces numerous neurosecretory products. In addition to and , hypothalamic neurons secrete polypeptides that promote and inhibit the secretion and release of hormones from the anterior lobe of the pituitary gland (Table 21.5). These also accumulate in nerve endings near the median eminence and infundibulum and are released into the capillary bed of the for transport to the pars distalis of the pituitary gland.

oxytocin

ADH

hypothalamic polypeptides hypothalamohypophyseal

portal system

TABLE 21.5 Hypothalamic-Regulating Hormones Hormone Composition Source Growth hormone Two forms in Cell bodies of –releasing humans: neurons located hormone (GHRH) polypeptides in the arcuate Somatostatin

containing 40 and 44 amino acids

nucleus of hypothalamus

Major Functions Stimulates secretion and gene expression of GH by somatotropes

Two forms in humans: polypeptides containing 14 and 28 amino acids

Cell bodies of neurons located in the periventricular, paraventricular, and arcuate nuclei of the hypothalamus

Inhibits secretion of GH by somatotropes and TSH by thyrotropes; inhibits insulin secretion by B cells of pancreatic islets

Catecholamine (amino acid derivative)

Inhibits secretion of PRL by lactotropes

Corticotropinreleasing hormone (CRH)

Cell bodies of neurons located in the arcuate nucleus of hypothalamus

Polypeptide containing 41 amino acids

Stimulates secretion of ACTH by corticotropes; stimulates gene expression for POMC in corticotropes

Gonadotropinreleasing hormone (GnRH)

Cell bodies of neurons located in the arcuate, periventricular, and medial paraventricular nuclei of hypothalamus

Polypeptide containing 10 amino acids

Cell bodies of neurons located in the arcuate, ventromedial, dorsal, and paraventricular

Stimulates secretion of LH and FSH by gonadotropes

Dopamine

Thyrotropinreleasing hormone (TRH)

nuclei of hypothalamus Polypeptide containing 3 amino acids

Cell bodies of neurons located by the ventromedial, dorsal, and paraventricular nuclei of hypothalamus

Stimulates secretion and gene expression of TSH by thyrotropes; stimulates synthesis and secretion of PRL

ACTH, adrenocorticotropic hormone; FSH, follicle-stimulating hormone; GH, growth hormone; LH , luteinizing hormone; POMC , proopiomelanocortin; PRL , prolactin; TSH , thyroid-stimulating hormone. FOLDER 21.2

CLINICAL CORRELATION: PRINCIPLES OF ENDOCRINE DISEASES

Abnormalities in the signaling mechanisms that coordinate and control the function of multiple organs and biological processes are the bases of many endocrine diseases. Classic biochemistry, physiology, and advances in cell and molecular biology and genetics combined with clinical observations are able to explain the mechanisms of hormonal action and endocrine diseases. Endocrine diseases can be classified into four major categories:

Hormone overproduction . The most common cause of hormone overproduction is an increase in the total number of cells producing a specific hormone. An example of this mechanism is hyperthyroidism (Graves disease; see Folder 21.4). Briefly, the presence of abnormal antibodies that mimic the action of thyroid-stimulating hormone (TSH) stimulates a dramatic increase in the number of thyroid cells. In some instances, increased hormone secretion is related to genetic abnormality that affects the regulation of hormone synthesis and release. In addition, mutation in tumor suppressor genes and proto-oncogenes may lead to proliferation of mutant cells that produce the specific hormone. This commonly occurs in cells of the anterior lobe of the pituitary gland. . Underproduction of hormones may result from destruction of an endocrine organ by a disease process (e.g., tuberculosis of the adrenal glands) or autoimmunity (e.g., Hashimoto disease in which abnormal antibodies target and destroy thyroid hormone– producing cells). Also, genetic abnormalities that lead to abnormal development of endocrine glands (e.g., hypogonadotropic hypogonadism), abnormal hormone synthesis (e.g., deletion of the GH gene), or abnormal regulation of hormone secretion (e.g., hypoparathyroidism associated with mutation of the calcium-sensing receptor expressed on parathyroid cells) can cause decreased serum levels or lack of active hormones. Iatrogenic injury to endocrine glands such as occurs when the parathyroid gland is removed during thyroidectomy (thyroid gland removal) may also be responsible. . This category of endocrine disease is often caused by a variety of genetic mutation in hormone receptors (e.g., TSH, luteinizing hormone [LH], and parathyroid hormone [PTH]). In diabetic patients, the resistance to insulin in muscles and the liver is mainly caused by signals originating from adipose tissue (see Chapter 9, Adipose Tissue, page 280).

Hormone underproduction

Altered tissue responses to hormones

Tumors of endocrine glands . Most of the tumors of endocrine gland are

hormonally active and are responsible for hormone overproduction. However, some tumors of endocrine glands do not produce hormones but compress neighboring organs or cause destructions of other organs due to metastasis. An example of such a tumor is thyroid cancer that can metastasize throughout the body without presenting signs of thyroid hormone overproduction (hyperthyroidism). Hormones are used to treat endocrine diseases. A common use is as when a specific endocrine gland is not developed or ceases to produce the required hormone. Hormones and their synthetic analogs can be used to suppress the effects of other hormones. In general, thyroid and steroid hormones can be administered orally, whereas protein hormones (e.g., insulin, growth hormone [GH]) need to be injected. Recent technological innovations, including computerized mini-pumps and depot intramuscular injections, have made therapy more manageable for patients.

hormone replacement therapies

A feedback system regulates endocrine function at two levels: hormone production in the pituitary gland and hypothalamic-releasing hormone production in the hypothalamus. The circulating level of a specific secretory product of a target organ, a hormone or its metabolite, may act directly on the cells of the anterior lobe of the pituitary gland or the hypothalamus to regulate the secretion of hypothalamic-releasing hormones (see Fig. 21.19 for a diagram of the hypothalamus–pituitary–thyroid feedback loop). The two levels of feedback allow exquisite sensitivity in the control of secretory function. The hormone itself normally regulates the secretory activity of the cells in the hypothalamus and pituitary gland that regulate its secretion. In addition, information from most physiologic and psychological stimuli that reach the brain also reaches the hypothalamus. The provides a regulatory pathway whereby general information from the CNS contributes to the regulation of the anterior lobe of the pituitary gland and, consequently, to the regulation of the entire endocrine system. The secretion of hypothalamic regulatory peptides is the primary mechanism by which changes in emotional state are translated into changes in the physiologic homeostatic state.

hypothalamohypophyseal feedback loop

PINEAL GLAND pineal gland

The (pineal body, epiphysis cerebri) is an endocrine or neuroendocrine gland that regulates daily body rhythm. It develops from neuroectoderm of the posterior portion of the roof of the diencephalon and remains attached to the brain by a short stalk. In humans, it is

located at the posterior wall of the third ventricle near the center of the brain. The pineal gland is a flattened, pine cone–shaped structure, hence its name (Fig. 21.12). It measures 5–8 mm high and 3 –5 mm in diameter and weighs between 100 and 200 mg.

FIGURE 21.12. Photomicrograph of infant pineal gland.

This hematoxylin and eosin (H&E)-stained section is obtained from a median cut through the pine cone –shaped gland. The conical anterior end of the gland is at the of the micrograph. The indicate the part of the gland that connects with the posterior commissure. The gland is formed by an evagination of the posterior portion of the roof of the third ventricle (diencephalon). The dark areas indicated by are caused by bleeding within the gland. ×25.

arrows

top

asterisks FOLDER 21.3

CLINICAL CORRELATION: PATHOLOGIES ASSOCIATED WITH ANTIDIURETIC HORMONE SECRETION

The absence or reduced production of antidiuretic hormone (ADH) leads to a condition known as , which is characterized by polyuria (production of large volumes of diluted urine—up to 20 L/d) with hypotonic and tasteless (insipid) urine. Individuals with this condition have extreme thirst, which allows them to counteract the loss of water by drinking large amounts of fluids. This disease commonly results from head injuries, tumors, or other lesions that can damage the hypothalamus or posterior lobe of the pituitary gland. This form of the disease is classified as in contrast to , in which secretion of the ADH is normal or elevated, but there is a lack of renal response to circulating levels of ADH. Nephrogenic diabetes is usually a congenital disorder related to the mutation of the aquaporin-2 (AQP-2) water channel gene or different ADH V2 receptor mutations in kidney tubules. Hypothalamic diabetes insipidus is usually treated by administration of synthetic analogs of ADH (desmopressin), whereas the treatment of the nephrogenic type of this disease is aimed at reducing the volume of urine output. Abnormally high levels of ADH are found in the , which is characterized by hyponatremia (low serum levels of sodium), decreased serum osmolality associated with excessive urine sodium excretion and elevated urine osmolality. In SIADH, the elevated level of ADH increases the absorption of water, thereby leading to the production of concentrated urine, inability to excrete water, and hyponatremia that results from excess water rather than sodium deficiency. The increase in ADH secretion may be related to central nervous system (CNS) disorders (tumors, injuries, infections, or cerebrovascular accidents), pulmonary diseases (pneumonia, chronic obstructive pulmonary disease, a lung abscess, or tuberculosis), tumors that secrete ADH (small cell carcinoma of the lung, tumors of the pancreas, thymoma, or lymphomas), and certain drugs (anti-inflammatories, nicotine, diuretics, and many others). Treatment of SIADH depends on the underlying etiology and includes fluid restrictions as well as pharmacologic treatment. An ADH V2–receptor antagonist (conivaptan) is now available to improve hyponatremia and to increase the free water diuresis without loss of other ions in the urine of patients with SIADH.

diabetes insipidus

diabetes insipidus

hypothalamic nephrogenic diabetes insipidus

syndrome of inappropriate antidiuretic hormone secretion (SIADH)

The pineal gland contains two types of parenchymal cells: pinealocytes and interstitial (glial) cells. Pinealocytes are the chief cells of the pineal gland. They are arranged in clumps or cords within lobules formed by connective tissue septa

that extend into the gland from the pia mater that covers its surface. These cells have a large, deeply infolded nucleus with one or more prominent nucleoli and contain lipid droplets within their cytoplasm. When examined with the TEM, pinealocytes show typical cytoplasmic organelles along with numerous, dense-core, membrane-bound vesicles in their elaborate, elongated cytoplasmic processes. The processes also contain numerous parallel bundles of microtubules. The expanded, clublike endings of the processes are associated with the blood capillaries. This feature strongly suggests neuroendocrine activity. The constitute about 5% of the cells in the gland. They have staining and ultrastructural features that closely resemble those of astrocytes and are reminiscent of the pituicytes of the posterior lobe of the pituitary gland. In addition to the two cell types, the human pineal gland is characterized by the presence of calcified concretions, called or (Fig. 21.13 and Plate 21.3, page 854). These concretions appear to be derived from precipitation of calcium phosphates and carbonates on carrier proteins that are released into the cytoplasm when the pineal secretions are exocytosed. The are recognizable in childhood and increase in number with age. Because they are opaque to x-rays and located in the midline of the brain, they serve as convenient in radiographic and .

interstitial (glial) cells

arenacea

corpora

brain sand

concretions

computed tomography (CT) studies

markers

FIGURE 21.13. Photomicrograph of human pineal gland. This higher magnification photomicrograph shows the characteristic concretions called brain sand or corpora arenacea. Pinealocytes (chief cells of the pineal gland) account for the majority of the cells seen in the specimen. They are arranged in clumps or cords. Those blood vessels ( BV ) that contain red blood cells are

readily apparent; numerous other blood vessels are also present but are not recognized at this magnification without evidence of the blood cells. ×250.

The human pineal gland relates light intensity and duration to endocrine activity. The pineal gland is a photosensitive organ and an important timekeeper and regulator of the day/night cycle (circadian rhythm). It obtains information about light and dark cycles from the retina via the , which connects the suprachiasmatic nucleus with sympathetic neural tracts traveling into the pineal gland. During the day, light impulses inhibit the production of the major pineal gland hormone, . Therefore, pineal activity, as measured by changes in the plasma level of melatonin, increases during darkness and decreases during light. In humans, these circadian changes of melatonin secretion play an important role in regulating . Melatonin is released in the dark and regulates reproductive function in mammals by inhibiting the steroidogenic activity of the gonads (Table 21.6). Production of is decreased by the inhibitory action of melatonin on neurosecretory neurons located in the hypothalamus (arcuate nucleus) that produce GnRH. Inhibition of GnRH causes a decrease in the release of FSH and LH from the anterior lobe of the pituitary gland. In addition to melatonin, extracts of pineal glands from many animals contain numerous neurotransmitters, such as , , , and , and hypothalamicregulating hormones, such as and . Clinically, tumors that destroy the pineal gland are associated with .

retinohypothalamic tract melatonin

daily body (circadian)

rhythms

gonadal steroids

serotonin norepinephrine dopamine histamine somatostatin TRH precocious (earlyonset) puberty TABLE 21.6 Hormones of the Pineal Gland

Hormone Composition Melatonin Indolamine (N-

Source

Major Functions

Pinealocytes

Regulates daily body rhythms and day/night cycle (circadian rhythms); inhibits secretion of GnRH and regulates steroidogenic activity of the gonads particularly as related to the menstrual cycle; in animals, influences seasonal sexual activity

acetyl-5methoxytryptamine)

GnRH, gonadotropin-releasing hormone.

Animal studies demonstrate that information relating to the length of daylight reaches the pineal gland from photoreceptors in the retina. The pineal gland thus influences seasonal sexual activity. The pineal

gland has a role in adjusting to sudden changes in day length, such as those experienced by travelers who suffer from . In addition, the pineal gland plays a role in to the reduced length of day during winter in temperate and subarctic zones known as .

jet lag altering emotional responses seasonal affective disorder (SAD)

THYROID GLAND

The thyroid gland is located in the anterior neck region adjacent to the larynx and trachea. The thyroid gland is a bilobate endocrine gland located in the anterior neck region and consists of two large lateral lobes connected by an isthmus, a thin band of thyroid tissue. The two lobes, each approximately 5 cm in length, 2.5 cm in width, and 20–30 g in weight, lie on either side of the larynx and upper trachea. The isthmus crosses the anterior surface of the second and third tracheal cartilages. A often extends upward from the isthmus. A thin connective tissue capsule surrounds the gland (Fig. 21.14). It sends trabeculae into the parenchyma that partially outlines irregular lobes and lobules. constitute the functional units of the gland.

pyramidal lobe

Thyroid follicles

FIGURE 21.14. Topography and blood supply of the thyroid gland.

This drawing shows the location of the thyroid gland in the anterior region of the neck in close proximity to the trachea and laryngeal cartilages. The gland consists of two lateral lobes connected by an isthmus. In about 40% of cases, the thyroid gland exhibits a pyramidal lobe, which is a remnant of the thyroglossal duct, a developmental connection with the base of the tongue. The thyroid gland is supplied by blood from the superior and inferior thyroid arteries, and blood from the gland is drained by the superior, middle, and inferior thyroid veins. On the posterior (deep) surface of the lateral lobes, there are two pairs of small ovoid structures that are designated as superior and inferior parathyroid glands. Note the outlined areas of the parathyroid glands’ location visible from the anterior view.

The thyroid gland develops from the endodermal lining of the floor of the primitive pharynx. The thyroid gland begins to develop during the fourth week of gestation from a primordium originating as an endodermal thickening of the floor of the primitive pharynx. The primordium grows caudally and forms a duct-like invagination known as the . The thyroglossal duct descends through the tissue of the neck to its final destination in front of the trachea, where it divides into two lobes. During this downward migration, the thyroglossal duct undergoes atrophy, leaving an embryologic remnant, the pyramidal lobe of the thyroid, which is present in about 40% of the population. At about the ninth week of gestation, endodermal cells differentiate into plates of that become arranged into follicles. By week 14, well-developed follicles lined by the follicular cells contain colloid in their lumens. During week 7, parallel to the development of thyroid follicles, epithelial cells lining the invagination of the fourth pharyngeal (branchial) pouches, known as the , start their migration toward the developing thyroid gland and become incorporated into the lateral lobes. After fusing with the thyroid, ultimobranchial body cells disperse among the follicles, giving rise to that become incorporated into the follicular epithelium.

thyroglossal duct

follicular cells

bodies

ultimobranchial

parafollicular cells The thyroid follicle is the structural and functional unit of the thyroid gland. A thyroid follicle is a roughly spherical cyst-like compartment with a wall formed by a simple cuboidal or low columnar epithelium, the follicular epithelium. Hundreds of thousands of follicles that vary in diameter from about 0.2 to 1.0 mm constitute nearly the entire mass of the human thyroid gland. The follicles contain a gel-like mass called (Fig. 21.15 and Plate 21.4, page 856). The apical surfaces of the follicular cells are in contact with the colloid, and the basal surfaces rest on a typical basal lamina.

colloid

FIGURE 21.15. Thyroid gland.

This photomicrograph of a human thyroid is from a section stained with hematoxylin and eosin (H&E). It shows the colloidcontaining follicles of the gland. Each follicle consists of a single layer of epithelial cells surrounding a central mass of colloid. The indicate some of the blood capillaries between the follicles. ×500.

arrows

Follicular epithelium contains two types of cells: follicular and parafollicular cells. The parenchyma of the thyroid containing two types of cells:

gland

is

composed

of

epithelium

Follicular cells (principal cells)

are responsible for the production of the thyroid hormones T4 and T3. These cells vary in shape and size according to the functional state of the gland. In routine hematoxylin and eosin (H&E) preparations, follicular cells exhibit a slightly basophilic basal cytoplasm with spherical nuclei containing one or more prominent nucleoli. The Golgi apparatus has a supranuclear position. Lipid droplets and PAS-positive droplets can be identified with appropriate staining. At the ultrastructural level, the follicle cells reveal organelles commonly associated with both secretory and absorptive cells (Fig. 21.16), including typical junctional complexes at the apical end of the cell and short microvilli on the apical cell surface. Numerous profiles of roughsurfaced endoplasmic reticulum (rER) are present in the basal region. Small vesicles present in the apical cytoplasm are morphologically similar to vesicles associated with the Golgi apparatus. Abundant endocytic vesicles, identified as , and lysosomes are also present in the apical cytoplasm.

colloidal resorption droplets

FIGURE 21.16. Electron micrograph of follicular cells in rat thyroid gland. This electron micrograph shows a single layer of epithelium containing low columnar follicular cells. The apical surfaces with visible microvilli ( Mv ) are in contact with the colloid, whereas basal surfaces of follicular cells rest on the basal lamina ( FBL ). A narrow extracellular connective tissue space separates the follicular cells from the lumen of the capillary. Note that the fenestrated endothelial cells ( En ) lining the capillary lumen rest on the basal lamina ( EBL ). Accumulation of lysosomes ( L ) and colloid resorption droplets ( CRD ), extensive Golgi apparatus ( G ), roughsurfaced endoplasmic reticulum ( rER ), and the presence of enlarged intercellular spaces are indicative of intensive activity of follicular cells. JC , junctional complex; N , nucleus. ×14,000. (Courtesy of Dr. Holger Jastrow.)

Parafollicular cells (C cells)

are located in the periphery of the follicular epithelium and lie within the follicle basal lamina. These cells have no exposure to the follicle lumen. They secrete , a hormone that regulates calcium metabolism. In routine

calcitonin

H&E preparations, C cells are pale staining and occur as solitary cells or small clusters of cells. Human parafollicular cells are difficult to identify with light microscopy. At the electron microscope level, the parafollicular cells reveal numerous small secretory vesicles, which range in diameter from 60 to 550 nm, and a prominent Golgi apparatus (Fig. 21.17).

FIGURE 21.17. Electron micrograph of a parafollicular cell. Cytoplasmic processes of follicular cells ( arrows ) partially surround the parafollicular cell ( PC ), which contains numerous electron-dense granules and a prominent Golgi apparatus ( G ). A basal lamina ( BL ) is associated with the follicular cells ( FC ). A portion of the central mass of colloidal material ( C ) in two adjacent follicles can be seen in the left corners of the micrograph. ×12,000. (Courtesy of Dr. Emmanuel-Adrien Nunez.)

An extensive network of fenestrated capillaries derived from the superior and inferior thyroid arteries surrounds the follicles. Blindended lymphatic capillaries are present in the interfollicular connective tissue and may also provide a second route for conveying the hormones from the gland.

Thyroid gland function is essential to normal growth and development.

The thyroid gland produces three hormones, each of which is essential to normal metabolism and homeostasis (Table 21.7):

TABLE 21.7 Hormones of the Thyroid Gland Hormone Composition Source Thyroxine Iodinated Follicular (tetraiodothyronine, tyrosine cells T 4 and derivatives (principal cells) triiodothyronine, a T3)

Calcitonin (thyrocalcitonin)

Polypeptide containing 32 amino acids

aThyroid

Parafollicular cells (C cells)

Major Functions Regulates tissue basal metabolism (increases rate of carbohydrate use, protein synthesis and degradation, and fat synthesis and degradation), regulates heat production, influences body and tissue growth and development of the nervous system in the fetus and young child, b increases absorption of carbohydrates from the intestine Decreases blood calcium levels by inhibiting bone resorption and stimulating absorption of calcium by the bones

gland secretes substantially more T 4 than T 3 ; however, about 40% of T 4 is peripherally converted to T 3 , which acts more rapidly and is a more potent hormone.

bDeficiency

of T 3 and T 4 during development results in fewer and smaller neurons, defective myelination, and severe intellectual disability.

Thyroxine (3,3′,5,5′-tetraiodothyronine, T4 ) triiodothyronine (T3 ) are synthesized and secreted cells. Both hormones regulate cell and tissue basal

3,3′,5follicular

and by metabolism and heat production and influence body growth and development. Secretion of these hormones is regulated by TSH released from the anterior lobe of the pituitary gland. is synthesized by the and is a physiologic antagonist to parathyroid hormone (PTH). Calcitonin has an important role in regulating serum calcium levels in lower animals; however, its physiologic role in humans remains elusive. Calcitonin lowers blood calcium levels by

Calcitonin (thyrocalcitonin) cells (C cells)

parafollicular

suppressing the resorptive action of osteoclasts and promotes calcium deposition in bones by increasing the rate of osteoid calcification. Secretion of calcitonin is regulated directly by blood calcium levels. High levels of calcium stimulate secretion; low levels inhibit it. Secretion of calcitonin is unaffected by the hypothalamus and pituitary gland. Calcitonin is secreted by several endocrine tumors (e.g., ); therefore, it is used as a tumor marker to monitor the progress of recovery after surgical resection of the tumor. Although calcitonin is used to treat patients with several disorders associated with excess bone resorption (e.g., and ), no clinical disease has been associated with its deficiency or even its absence after total thyroidectomy.

medullary carcinoma of the thyroid osteoporosis

Paget disease

The principal component of colloid is thyroglobulin, an inactive storage form of thyroid hormones. The principal component of colloid is a large (660 kDa), iodinated glycoprotein called thyroglobulin containing about 120 tyrosine residues. Colloid also contains several enzymes and other glycoproteins. It stains with both basic and acidic dyes and is strongly PAS positive. Thyroglobulin is not a hormone. It is an inactive storage form of thyroid hormones. Active thyroid hormones are liberated from thyroglobulin and released into the fenestrated blood capillaries that surround the follicles only after further cellular processing. The thyroid is unique among endocrine glands because it stores large amounts of its secretory product extracellularly.

Synthesis of thyroid hormone involves several steps. The synthesis of the two major thyroid hormones, thyroxine (T4 ) and triiodothyronine (T3 ), takes place in the thyroid follicle in a series of discrete steps (Fig. 21.18):

FIGURE 21.18. Diagram of steps in thyroid hormone synthesis. This diagram depicts two follicular cells: one in the process of thyroglobulin synthesis ( on the left with red pathways) and the other in the process of thyroglobulin resorption ( on the right with blue pathways ). The numbers , which are described more fully in the text, indicate the sequential steps that occur: 1 , synthesis and secretion of thyroglobulin; 2 , uptake and concentration of iodide from the blood by sodium/iodide symporters ( NIS ), release of iodide into the colloid via iodide/chloride (pendrin) transporters, and oxidation of iodide to iodine by thyroid peroxidase; 3 , iodination of thyroglobulin in the colloid; 4 , formation of T 3 and T 4 hormones in the colloid by oxidative coupling reactions; 5L , resorption of colloid via lysosomal pathway (major pathway); 5TE , resorption of colloid via megalin receptor–mediated transepithelial pathway; and 6 , release of T 4 and T 3 from the cell into the circulation. DIT , diiodotyrosine; MIT , monoiodotyrosine; rER , rough-surfaced endoplasmic reticulum.

1. Synthesis of thyroglobulin.

The precursor of thyroglobulin is synthesized in the rER of the follicular epithelial cells. Thyroglobulin is post-translationally glycosylated in the rER and the Golgi apparatus before it is packaged into vesicles and secreted by exocytosis into the lumen of the follicle. . Follicular epithelial cells actively transport from the blood into their cytoplasm using ATPase-dependent . The NIS is the 87-kDa transmembrane protein that mediates active iodide uptake in the basolateral membrane of the follicular epithelial cells. These cells are capable of establishing an intracellular concentration of iodide that is 30–40 times greater than that of the serum. Iodide ions then diffuse rapidly toward the apical cell membrane. From here, iodide ions are transported to the lumen of the follicle by the 86-kDa called located in the apical cell membrane. Iodide is then immediately oxidized to , the active form of iodide. This process occurs in the colloid and is catalyzed by membrane-bound . . One or two iodine atoms are then added to the specific tyrosine residues of thyroglobulin. This process occurs in the colloid at the microvillar surface of the follicular cells and is also catalyzed by . Addition of one iodine atom to a single tyrosine residue forms . Addition of a second iodine atom to the MIT residue forms a . 3 4 by oxidative coupling reactions. Thyroid hormones are formed by oxidative coupling reactions of two iodinated tyrosine residues in close proximity. For example, when neighboring DIT and MIT residues undergo a coupling reaction, T3 is formed; when two DIT residues react with each other, T4 is formed. After iodination, T4 and T3 as well as the DIT and MIT residues that are still linked to a thyroglobulin molecule are stored as the colloid within the lumen of the follicle. . In response to TSH, follicular cells take up thyroglobulin from the colloid by a process of receptor-mediated endocytosis. If the levels of TSH remain high, the amount of colloid in the follicle is reduced because it is synthesized, secreted, iodinated, and resorbed too rapidly to accumulate. After endocytosis, thyroglobulin follows at least two different intracellular pathways. In the , thyroglobulin is internalized and transported within endocytic vesicles to early endosomes. They

2. Resorption, diffusion, and oxidation of iodide iodide sodium/iodide symporters (NIS)

pendrin

iodine

iodide/chloride transporter

thyroid peroxidase (TPO) 3. Iodination of thyroglobulin (MIT) diiodotyrosine (DIT) 4. Formation of T and T

5. Resorption of colloid

lysosomal pathway

thyroid peroxidase (TPO) monoiodotyrosine

eventually mature into lysosomes or fuse with existing lysosomes. Resorption of thyroglobulin at this stage can be confirmed by the presence of large endocytic vesicles called in the apical region of the follicular cells. Thyroglobulin is then degraded by lysosomal proteases into constituent amino acids and carbohydrates, leaving free T4, T3, DIT, and MIT molecules (see 5L labeled pathway in Fig. 21.18). Under physiologic conditions, this is the major pathway of colloid resorption. In the , thyroglobulin is transported intact from the apical to the basolateral surface of follicular cells. To enter this pathway, thyroglobulin binds to its receptor, , a 330-kDa member of the low-density lipoprotein (LDL) endocytic receptor family. Megalin is a transmembrane protein expressed at the apical surface of follicular epithelial cells directly facing colloid. Thyroglobulin internalized by megalin avoids the lysosomal pathway, and endocytic vesicles are delivered to the basolateral membrane of follicular cells (see 5TE labeled pathway in Fig. 21.18). In pathologic conditions of high TSH or TSH-like stimulation, expression is increased, and large amounts of thyroglobulin follow the transepithelial pathway. This pathway may reduce the extent of T4 and T3 release by diverting thyroglobulin away from the lysosomal pathway. Individuals with and other thyroid diseases have detectable amounts of circulating thyroglobulin that contains portions of megalin receptor. 4 3 from follicular cells into the circulation. Follicular cells predominately produce T4 in a T4-to-T3 ratio of 20:1. Most of the T4 and T3 produced is liberated from thyroglobulin in the lysosomal pathway, and only negligible amounts of T4 and T3 are released bound to thyroglobulin. Both T4 and T3 cross the basal membrane and enter the blood and lymphatic capillaries. Most of the released hormones are immediately bound to a specific plasma protein (54 kDa), (~70%), or a prealbumin fraction of serum protein called (~20%). T4 has a stronger bond to TBG, whereas T3 has a stronger bond to transthyretin. Less than approximately 10% of released hormones are bound to a nonspecific fraction of , leaving only small amounts (~1%) of free circulating hormones that are metabolically active. The free circulating hormones also function in the feedback system that regulates the secretory activity of the thyroid (Fig. 21.19). One-third of circulating T4 is converted to T3 in peripheral

colloidal resorption

droplets

transepithelial pathway

megalin

megalin

Graves disease

6. Release of T and T

thyroxine-binding globulin (TBG) transthyretin albumin

organs, such as the kidney, liver, and heart. T3 is five times more potent than T4 and is mainly responsible for biological activity by binding to the thyroid nuclear receptors in the target cells.

FIGURE 21.19. Production, transport, and regulation of thyroid hormones. Production of T4 and T3 is regulated through a negative feedback

system. The follicular cells of the thyroid gland predominately produce about 20 times more T 4 than T 3 ; however, T 4 is converted in the peripheral organs (e.g., liver, kidney) to a more active form of T 3 . Approximately 99% of T 4 and T 3 released from the thyroid gland into the circulation bind to specific plasma proteins. The remaining free (unbound) T 4 and T 3 exert negative feedback on the system and inhibit the further release of T 4 and T 3 . This inhibition occurs at the level of the anterior lobe of the pituitary gland and the hypothalamus. At the pituitary level, T 4 and T 3 inhibit secretion of TSH by thyrotropes. To elicit an inhibitory effect on the hypothalamus, both T 4 and T 3 need to cross the blood–brain barrier by utilizing the OATP thyroid hormone transporter expressed on the membrane of the endothelial cells. Increased concentration of T 4 and T 3 reduces expression of OATP transporters as part of the negative feedback loop, thus decreasing the amount of available thyroid hormones in the brain. After crossing the blood–brain barrier, T 4 and T 3 are transferred into neighboring astrocytes, where T 4 is converted to T 3 . Note that T 3 is the predominant hormone that enters the neurons. T 4 and T 3 are also secreted into the cerebrospinal fluid and are taken up by the tanycytes (specialized

ependymal cells) and astrocytes, where T 4 is converted to T 3 . In addition to TRH, which also stimulates the production of prolactin in lactotropes, the hypothalamus secretes somatostatin that has an inhibitory effect on TSH production by thyrotropes. The feedback system is activated in response to low thyroid hormone levels in the blood or metabolic needs. In addition to chemical control mechanisms, a variety of nerve endings in the hypothalamus regulate secretion of TRH. For example, cold stress increases secretion of TRH, whereas increased body temperature inhibits TRH secretion. , central nervous system; , thyrotropin-releasing hormone; , thyroid-stimulating hormone (thyrotropin); , organic anion–transporting polypeptides.

TRH

TSH

OATP

CNS

Transport across the cell membrane is essential for thyroid hormone action and metabolism. Based on the biochemical structure of thyroid hormones, it was long thought that thyroid hormones can enter the cell by simple diffusion. However, it is now well established that thyroid hormones are transported across cell membranes by several . Within the CNS, T3 and T4 are transported via the blood–brain barrier to the nerve and glial cells by and as well as a family of . For example, the OATP1C1 transporter is exclusively expressed on the endothelial cells forming the blood–brain barrier and is responsible for T4 uptake into the brain. The MCT8 is also found in the heart, kidney, liver, and skeletal muscle. Mutations in the MCT8 gene cause severe psychomotor and intellectual disability associated with high serum T3 levels in affected male individuals, a condition known as . Defective MCT8 transporters are unable to transport T3 into nerve cells, which disrupts normal brain development. Because T3 is not utilized by nerve cells, excessive amounts of this hormone continue to circulate in the blood, causing signs and symptoms of .

transporter molecules monocarboxylate transporter-8 (MCT8) MCT10 organic anion–transporting polypeptides (OATPs)

thyroid hormone

Allan–Herndon–Dudley

syndrome

thyroid hormone toxicity The triiodothyronine (T3 ) hormone is more biologically active than thyroxine (T4 ).

Once T3 and T4 molecules enter the cell, they interact with a specific that is similar to the nuclear-initiated steroid signaling pathway (see Fig. 21.4b). T3 binds to nuclear receptors much faster and with higher affinity than T4; thus, T3 is more rapidly and biologically active than T4. In addition, T3 binds to mitochondria, increasing the production of adenosine triphosphate (ATP). Therefore, and metabolic effect of the

thyroid nuclear receptor

biological activity

thyroid hormone are largely determined by the intracellular concentration of T3. Several factors impact the intracellular concentration of T3. These include serum concentration of circulating T3, which depends on the conversion rate of T4 to T3 in the peripheral organs; transport of thyroid hormones across the cell membrane by specialized thyroid hormone transporters; and the presence of enzymes, which activate or inactivate thyroid hormones. For instance, two deiodinase enzymes called 1 and 2 convert T4 to the more active T3, whereas the third enzyme called 3 degrades T4 to the inactive form of rT3 (reverse T3) and DIT. Both T3 and T4 are deiodinated and deaminated in the target tissues, conjugated in the liver, and then passed into the bile, where they are excreted into the intestine. Conjugated and free hormones are also excreted by the kidney.

D

D

D

iodothyronine deiodinase

Thyroid hormones play an essential role in normal fetal development. In humans, thyroid hormones are essential to normal growth and

development. In normal pregnancy, both T3 and T4 cross the placental barrier and are critical in the early stages of brain development. In addition, the fetal thyroid gland begins to function during the 14th week of gestation and also contributes additional thyroid hormones. Thyroid hormone deficiency during results in irreversible damage to the central nervous system (CNS), causing reduced numbers of neurons, defective myelination, and intellectual disability. If maternal thyroid deficiency is present before the development of the fetal thyroid gland, intellectual disability can be severe. Recent studies reveal that thyroid hormones also stimulate gene expression for GH in the somatotropes. Therefore, in addition to neural abnormalities, a generalized stunted body growth is typical. The combination of these two abnormalities is called .

fetal development

congenital

hypothyroidism FOLDER 21.4

CLINICAL CORRELATION: ABNORMAL THYROID FUNCTION goiter

The most common symptom of thyroid disease is , the enlargement of the thyroid gland. It may indicate either hypothyroidism or hyperthyroidism. can be caused by insufficient dietary iodine ( ) or by one of several inherited autoimmune diseases, such as . Autoimmune thyroiditis is characterized by the presence of abnormal autoimmunoglobulins directed against thyroglobulin (TgAb), thyroid peroxidase (TPOAb), and the thyroid-stimulating hormone (TSH) receptor (TSHAb). The results are thyroid cell apoptosis and follicular destruction. The low levels of circulating thyroid hormone stimulate release of excessive

Hypothyroidism iodinedeficiency goiter, endemic goiter autoimmune thyroiditis (Hashimoto thyroiditis)

amounts of TSH, which cause hypertrophy of the thyroid through synthesis of more thyroglobulin. Adult , formerly called (due to the puffy appearance of the skin), is characterized by mental and physical sluggishness. The edema that occurs in the severe stages of hypothyroidism is caused by the accumulation of large amounts of hyaluronan in the extracellular matrix of the connective tissue of the dermis. In , excessive amounts of thyroid hormones are released into the circulation. Individuals with Graves disease have detectable levels of autoantibodies. These abnormal immunoglobulins G (IgG) bind to the TSH receptors on the follicular cells and stimulate adenylate cyclase activity. As a result, increased levels of cyclic adenosine monophosphate (cAMP) in follicular cells lead to continuous stimulation of the cells and increased thyroid hormone secretion. Because of negative feedback, the levels of TSH in the circulation are usually normal. However, under such stimulation, the thyroid gland undergoes hypertrophy, and thyroid hormone is secreted at abnormally high rates, causing increased metabolism. Most of the clinical features are associated with increased metabolic rate and increased sympathetic nerve activities. These include weight loss, excessive sweating, tachycardia, and nervousness. Noticeable signs include protrusion of the eyeballs and retraction of the eyelids, resulting from increased sympathetic activity and increased deposition of extracellular matrix in the adipose tissue located behind the eyeball (Fig. F21.4.1a). The thyroid gland is enlarged. Microscopic features include the presence of columnar follicular cells lining the thyroid follicles. Because of the high utilization of colloid, the colloid tends to be depleted in the areas of contact with the apical surface of follicular cells (Fig. F21.4.1b). Treatment for Graves disease is either surgery to remove the thyroid gland or radiotherapy by ingestion of radioactive iodine ( 131I), which destroys most active follicular cells.

hypothyroidism

myxedema

hyperthyroidism (toxic goiter or Graves disease)

FIGURE F21.4.1. Hyperthyroidism. a. A young woman with signs of hyperthyroidism. Note the enlarged mass on the neck and the typical ocular symptoms known as exophthalmos . b. Photomicrograph of a thyroid gland

specimen from an individual with Graves disease. Owing to the increased utilization of colloid, there is a lack of staining at the periphery of the colloid near the apical surface of the follicular cell. Note that the majority of the cells are columnar in shape. (Reprinted with permission from Rubin E, Gorstein F, Rubin R, et al. . 4th ed. Lippincott Williams & Wilkins; 2005.)

Foundations of Medicine

Rubin’s Pathology, Clinicopathologic

PARATHYROID GLANDS parathyroid glands

The are small endocrine glands closely associated with the thyroid. They are ovoid, a few millimeters in diameter, and arranged in two pairs, constituting the and . They are usually located in the connective tissue on the posterior surface of the lateral lobes of the thyroid gland (see Fig. 21.14 and Plate 21.4, page 856). However, the number and location may vary. In 2%–10% of individuals, additional glands are associated with the thymus. Structurally, each parathyroid gland is surrounded by a thin connective tissue capsule that separates it from the thyroid. Septa extend from the capsule into the gland to divide it into poorly defined lobules and to separate the densely packed cords of cells. The connective tissue is more evident in the adult, with the development of fat cells that increase with age and ultimately constitute as much as 60%–70% of the glandular mass. The glands receive their blood supply from the inferior thyroid arteries or anastomoses between the superior and inferior thyroid arteries. Typical of endocrine glands, rich networks of fenestrated blood capillaries and lymphatic capillaries surround the parenchyma of the parathyroids.

parathyroid glands

superior

inferior

Parathyroid glands develop from the endodermal cells derived from the third and fourth pharyngeal pouches. Embryologically, the inferior parathyroid glands (and the thymus) are derived from the superiorly located third pharyngeal pouch; the superior parathyroid glands (and ultimobranchial body) are derived from the fourth pharyngeal pouch. Initially, the inferior parathyroid glands descend with the thymus. Later, the inferior parathyroid glands separate from the thymus and come to lie below the superior parathyroid glands. Failure of these structures to separate results in the atypical association of the parathyroid glands with the thymus in the adult. The principal (chief) cells differentiate during embryonic development and are functionally active in regulating fetal calcium metabolism. The oxyphil cells differentiate later at puberty.

Principal cells and oxyphil cells constitute the epithelial cells of the parathyroid gland. Principal (chief) cells, the more numerous of the parenchymal cells of the parathyroid (Fig. 21.20), are responsible for regulating the synthesis, storage, and secretion of large amounts of PTH. They are small, polygonal cells, with a diameter of 7–10 μm and a centrally located nucleus. The pale-staining, slightly acidophilic cytoplasm contains lipofuscin-containing vesicles, large accumulations of glycogen, and lipid droplets. Small, dense, membrane-limited vesicles seen with the TEM or after using special stains with the light microscope are thought to be the storage form of PTH. Principal cells can replicate when they are chronically stimulated by changes in blood calcium levels.

FIGURE 21.20. Photomicrograph of human parathyroid gland. This hematoxylin and eosin (H&E)-stained specimen shows the gland with part of its connective tissue capsule ( Cap ). The blood vessels ( BV ) are located in the connective tissue septum between the lobes of the gland. The principal cells are arranged in two masses ( top and bottom ) and are separated by a large cluster of oxyphil cells ( center ). The oxyphil cells are the larger cell type

with prominent eosinophilic cytoplasm. They may occur in small groups or larger masses, as seen here. The principal cells are more numerous. They are smaller, have less cytoplasm, and consequently exhibit closer proximity to

their nuclei. Adipose cells ( numbers. ×175.

AC)

are present in variable, although limited,

Oxyphil cells constitute a minor portion of the parenchymal cells and are not known to have a secretory role. They are found either singly or in clusters; the cells are more rounded, considerably larger than the principal cells, and have a distinctly acidophilic cytoplasm (see Fig. 21.20). Mitochondria, often with bizarre shapes and sizes, almost fill the cytoplasm and are responsible for the strong acidophilia of these cells. No secretory vesicles and little, if any, rER are present. Cytoplasmic inclusion bodies consist of occasional lysosomes, lipid droplets, and glycogen distributed among the mitochondria.

Parathyroid hormone regulates calcium and phosphate levels in the blood. The parathyroids function in the regulation of calcium and phosphate levels. Parathyroid hormone (PTH) is essential for life. Therefore, care must be taken during thyroidectomy to leave some functioning

parathyroid tissue. If the glands are totally removed, death will ensue because muscles, including the laryngeal and other respiratory muscles, go into as the blood calcium level falls. PTH is an 84-amino-acid linear peptide (Table 21.8). It binds to a specific PTH receptor on target cells that interacts with G-protein to activate a second messenger system. PTH release causes the . Simultaneously, it reduces the concentration of serum phosphate. Secretion of PTH is regulated by the serum calcium level through a simple feedback system. When parathyroid calcium-sensing receptors on principal cells detect low serum calcium levels, they stimulate secretion of PTH; high levels of serum calcium inhibit its secretion.

tetanic contraction

level of

calcium in the blood to increase

TABLE 21.8 Parathyroid Hormone Hormone Parathyroid hormone (PTH)

Composition Source

Major Functions

Polypeptide containing 84 amino acids

Increases blood calcium level in three ways: (1) promotes calcium release from bone (acting on osteoblasts via RANK-RANKL signaling system, it increases the relative number of osteoclasts); (2) acts on the kidney to stimulate calcium reabsorption by the distal

Principal (chief) cells a

aSome

tubule while inhibiting phosphate reabsorption in the proximal tubule; and (3) increases formation of hormonally active 1,25dihydroxycholecalciferol (1,25[OH] 2 vitamin D 3 ) in the kidney, which promotes tubular reabsorption of calcium

evidence suggests that oxyphil cells, which first appear in the parathyroid gland at about 4–7 years of age and increase in number after puberty, may also produce PTH.

RANK-RANKL, receptor activator of NF-κB–receptor activator NF-κB ligand. PTH functions at several sites:

Action on bone tissue.

Until recently, bone resorption had been considered the major effect of PTH action on bone. However, the actions of PTH on bone are more complicated. PTH acts directly and indirectly on several cell types. Receptors for PTH are found on osteoprogenitor cells, osteoblasts, osteocytes, and bone-lining cells. Surprisingly, the bone-resorbing osteoclasts do not have PTH receptors; thus, they are indirectly activated by the RANK-RANKL signaling mechanism of osteoblasts (pages 250-251). The prolonged continuous exposure of PTH increases local RANK production in the osteoblasts and decreases osteoprotegerin (OPG) secretion. These changes then stimulate osteoclast differentiation, which leads to increased bone resorption and release of calcium and phosphates into the extracellular fluid. Briefly, intermittent exposure to PTH increases bone mass through the cAMP/IGF-I pathway in osteocytes and osteoblasts. This anabolic effect of increasing bone mass by intermittent dosing of PTH is utilized in the treatment of osteoporosis (see Folder 8.2 in Chapter 8, Bone, pages 263-264). is decreased by PTH stimulation of tubular reabsorption, thus conserving calcium. is increased by PTH secretion, thus lowering phosphate concentration in the blood and extracellular fluids. 3 to hormonally active 2 3 is regulated primarily by PTH, which stimulates the activity of 1α-hydroxylase and increases the production of active hormone. is increased under the influence of PTH. Vitamin D3, however, has a greater effect than PTH on the intestinal absorption of calcium.

Kidney excretion of calcium Urinary phosphate excretion

Kidney conversion of 25-OH vitamin D (OH) vitamin D Intestinal absorption of calcium

1,25-

PTH and calcitonin have reciprocal effects in the regulation of blood calcium levels. Although PTH increases blood calcium levels, the peak increase after its release is not reached for several hours. PTH appears to have a rather slow, long-term homeostatic action. Calcitonin, however, rapidly lowers blood calcium levels and has its peak effect in about 1 hour; therefore, it has a rapid, acute homeostatic action.

ADRENAL GLANDS

adrenal (suprarenal) glands

The retroperitoneal space of flattened and triangular, They are both embedded in the kidneys (Fig. 21.21). and catecholamines.

the and the The

are paired organs located in the abdominal cavity. The right gland is the left gland is semilunar in shape. perirenal fat at the superior poles of adrenal glands secrete steroid hormones

FIGURE 21.21. Topography and blood supply of the adrenal (suprarenal) gland. This drawing shows the location of the left adrenal gland at the

superior pole of the left kidney. The perirenal fat has been removed from this image to show blood supply to the organ. Note that the adrenal gland is supplied by three arteries. The middle suprarenal artery originates directly from the aorta, whereas the superior and inferior suprarenal arteries originate from the left inferior phrenic and left renal arteries, respectively. Blood drains into the suprarenal vein, which, on the side, empties into the left renal vein and, on the side, directly into the inferior vena cava.

right

left

The adrenal glands are covered with a thick connective tissue from which trabeculae extend into the parenchyma, carrying blood vessels and nerves. The secretory parenchymal tissue is organized into two distinct regions (Fig. 21.22 and Plate 21.5, page 858):

capsule

FIGURE 21.22. Photomicrograph of the adrenal gland.

This low-power micrograph of a hematoxylin and eosin (H&E)-stained specimen shows the full thickness of the adrenal gland with the cortex seen on both surfaces and a central region containing the medulla. Within the medulla are profiles of the central vein. Note that the deeper portion of the cortex stains darker than the outer portion, a reflection of the washed-out lipid in the zona glomerulosa and outer region of the zona fasciculata. This section also includes a cross section of the adrenal vein, which is characterized by the longitudinally arranged bundles of smooth muscle in its wall. ×20.

cortex medulla

The is the steroid-secreting portion. It lies beneath the capsule and constitutes nearly 90% of the gland by weight. The is the catecholamine-secreting portion. It lies deep into the cortex and forms the center of the gland.

Parenchymal cells of the cortex and medulla are of different embryologic origin. Embryologically, the cortical cells originate from mesodermal mesenchyme, whereas the medulla originates from neural crest cells that migrate into the developing gland (Fig. 21.23). Although embryologically distinct, the two portions of the adrenal gland are functionally related (see later). The parenchymal cells of the adrenal cortex are controlled in part by the anterior lobe of the pituitary gland and function in regulating metabolism and maintaining normal electrolyte balance (Table 21.9).

TABLE 21.9 Hormones of the Adrenal Glands Hormone Adrenal Cortex Mineralocorticoids: aldosterone (95% of

mineralocorticoid activity in aldosterone)

Glucocorticoids: corticosterone and cortisol (95% of

glucocorticoid activity is in cortisol)

Composition

Source

Major Functions

Steroid hormones (cholesterol derivatives)

Parenchymal cells of the zona glomerulosa

Aid in controlling electrolyte homeostasis (act on distal tubule of kidney to increase sodium reabsorption and decrease potassium reabsorption); function in maintaining the osmotic balance in the urine and in preventing serum acidosis

Steroid hormones (cholesterol derivatives)

Parenchymal cells of the zona fasciculata (and, to a lesser extent, of

Promote normal metabolism, particularly carbohydrate metabolism (increase rate of amino acid transport to

Gonadocorticoids (adrenal androgens): dehydroepiandrosterone (DHEA), dehydroepiandrosterone sulfate (DHEAS), and androstenedione

the zona reticularis)

live, promote removal of protein from skeletal muscle and its transport to liver, reduce rate of glucose metabolism by cells and stimulate glycogen synthesis by liver, stimulate mobilization of fats from storage deposits for energy use); provide resistance to stress; suppress inflammatory response and some allergic reactions

Steroid hormones (cholesterol derivatives)

Parenchymal cells of the zona reticularis (and, to a lesser, extent of the zona fasciculata)

As weak androgens, they induce development of axillary and pubic hair at puberty in women; cause masculinizing effect; at normal serum levels, usually their function is insignificant

Catecholamines (amino acid derivatives)

Chromaffin cells

Sympathomimetic (produce effects similar to those induced by the sympathetic division of the autonomic nervous system) a;

(produced in both men and women)

Adrenal Medulla Norepinephrine and epinephrine (in humans, 80% epinephrine)

aThe

increase heart rate, increase blood pressure, reduce blood flow to viscera and skin; stimulate conversion of glycogen to glucose; increase sweating; induce dilation of bronchioles; increase rate of respiration; decrease digestion; decrease enzyme production by digestive system glands; decrease urine production

catecholamines influence the activity of glandular epithelium, cardiac muscle, and smooth muscle located in the walls of blood vessels and viscera.

FIGURE 21.23. Development of the adrenal gland. a.

In this early stage, the cortex is shown developing from cells of the intermediate mesoderm, and the medulla is shown differentiating from cells in the neural crest and migrating from the neighboring sympathetic ganglion. The cells that form the fetal cortex originate from mesothelial cells located between the root of the dorsal mesentery and the developing urogenital ridges (future gonads). They divide and differentiate into fetal cortex cells. Mesodermal cells from the fetal

b.

cortex surround the cells of the developing medulla. Later, more mesenchymal cells arrive from the mesothelium of the posterior abdominal wall. They surround the original mass of cells containing the fetal cortex cells and chromaffin cells. These cells later give rise to the permanent cortex. At this stage (about 7 months of development), the fetal cortex occupies about 70% of the cortex. The permanent cortex develops outside the fetal cortex. The fully developed adrenal cortex is visible at the age of 4 months. The permanent cortex replaces the fetal cortex, which at this age has completely disappeared. Note the fully developed zonation of the permanent cortex.

c. d.

Blood Supply

adrenal gland is supplied with blood by the superior, middle, and inferior suprarenal arteries and drained by the suprarenal veins (see Each

Fig. 21.21). On the left side, the suprarenal vein drains into the left renal vein, whereas on the right side, the suprarenal vein drains directly into the inferior vena cava. These vessels branch before entering the capsule to produce many small arteries that penetrate the capsule. In the capsule, the arteries branch to give rise to three principal patterns of blood distribution (Fig. 21.24). The vessels form a system that consists of the following:

FIGURE 21.24. Organization and blood supply of the human adrenal gland.

This diagram shows the blood supply to the adrenal cortex and medulla. The cortical arterioles form a cortical network of capillaries, which drain into a second capillary network in the medulla. The medullary capillary network is formed primarily by the medullary arterioles and drains into the central medullary vein. Adrenal medulla, zones of the cortex, and features of basic cell types and their secretory products are noted. , dehydroepiandrosterone; , dehydroepiandrosterone sulfate.

DHEAS

Capsular capillaries that supply the capsule

DHEA

Fenestrated cortical sinusoidal capillaries that supply the cortex and then drain into the fenestrated medullary capillary sinusoids Medullary arterioles that traverse the cortex, traveling within the trabeculae, and bring arterial blood to the medullary capillary sinusoids The medulla thus has a dual blood supply: arterial blood from the medullary arterioles and venous blood from the cortical sinusoidal capillaries that have already supplied the cortex. The venules that arise from the cortical and medullary sinusoids drain into the small adrenomedullary collecting veins that join to form the large , which then drains directly as the suprarenal vein into the inferior vena cava on the right side and into the left renal vein on the left side (see Fig. 21.21). In humans, the central adrenomedullary vein and its tributaries are unusual in that they have a tunica media containing conspicuous, longitudinally oriented bundles of smooth muscle cells (Fig. 21.25). Synchronous contraction of longitudinal smooth muscle bundles along the central adrenomedullary vein and its tributaries causes the volume of the adrenal gland to decrease. This decreased volume enhances the efflux of hormones from the adrenal medulla into the circulation, an action comparable to squeezing a wet sponge.

adrenomedullary vein

central

FIGURE 21.25. Photomicrograph of the central adrenomedullary vein. This photomicrograph shows the center of the adrenal gland with a central adrenomedullary vein in the middle . The wall of the vein has a highly irregular appearance, containing several prominent smooth muscle ( SM ) projections (also called muscle cushions ) into the lumen. These projections represent longitudinal bundles of smooth muscles of the tunica media. In areas where muscle bundles are absent, cells of the adrenal medulla ( lower part of the

image)

upper part of the image

or sometimes adrenal cortex ( ) are separated from the lumen only by a thin layer of tunica intima. Note the close proximity of zona reticularis to the lumen of the vein. ×180.

Lymphatic vessels

are present in the capsule and the connective tissue around the larger blood vessels in the gland. They also have been found in the parenchyma of the adrenal medulla. The lymphatic vessels have an important role in distributing , a secretory product of chromaffin cells. Chromogranin A is a 48-kDa intracellular storage protein complex for epinephrine and norepinephrine and is also a precursor molecule for several regulatory peptides, including vasostatin 1 and 2 (VST I, VST II), pancreastatin (PST), catestatin (CST), and parastatin (PARA). These peptides negatively modulate the neuroendocrine function of the chromaffin cells (autocrine effect) and other hormone-producing cells in distant organs.

chromogranin A

Cells of the Adrenal Medulla

Chromaffin cells located in the adrenal medulla are innervated by presynaptic sympathetic neurons. The central portion of the adrenal gland, the medulla, is composed of a parenchyma of large, pale-staining epithelioid cells called chromaffin cells (medullary cells), connective tissue, numerous sinusoidal blood capillaries, and nerves. The chromaffin cells are, in effect, modified neurons (Folder 21.5). Numerous myelinated, presynaptic sympathetic nerve fibers pass directly to the chromaffin cells of the medulla (see Chapter 12, Nerve Tissue, page 421). When nerve impulses carried by the sympathetic fibers reach the catecholamine-secreting chromaffin cells, they release their secretory products. Therefore, chromaffin cells are considered the equivalent of postsynaptic neurons. However, they lack axonal processes. Experimental studies reveal that when chromaffin cells are grown in culture, they extend axon-like processes. However, axonal growth can be inhibited by glucocorticoids—hormones secreted by the adrenal cortex. Thus, the hormones of the adrenal cortex exert control over the morphology of the chromaffin cells and prevent them from forming neural processes. Chromaffin cells, therefore, more closely resemble typical endocrine cells, in that their secretory product enters the bloodstream via the fenestrated capillaries.

FOLDER 21.5

CLINICAL CORRELATION: CHROMAFFIN CELLS AND PHEOCHROMOCYTOMA

Chromaffin cells

(so named because they react with chromate salts) of the adrenal medulla are part of the amine precursor uptake and decarboxylation

(APUD) system of cells. The chromaffin reaction is thought to involve oxidation and polymerization of the catecholamines contained within the secretory vesicles of these cells. Classically, chromaffin cells have been defined as being derived from neuroectoderm, innervated by presynaptic sympathetic nerve fibers, and capable of synthesizing and secreting catecholamines. A rare tumor derived from chromaffin cells, called , produces excessive amounts of catecholamines. Because chromaffin cells are also found outside the adrenal medulla in paravertebral and prevertebral sympathetic ganglia and other locations, tumors may arise from outside the adrenal gland. These extra-adrenal pheochromocytomas are called because scattered groups of chromaffin cells located among or near the components of the autonomic nervous system (ANS) are called . Pheochromocytomas may cause episodic symptoms related to the pharmacologic effects of excessive catecholamine secretion and are often described according to the “ ”:

pheochromocytoma

paragangliomas paraganglia 10% the 10% 10% 10% 10% 10% 10% 10%

rule of 10s

are extra-adrenal (paragangliomas), and of those, 10% reside outside abdomen. occur in children. are multiple or bilateral. are not associated with hypertension. are malignant. are familial. recur after surgical removal. are found incidentally during unrelated imaging studies.

Pheochromocytomas may precipitate life-threatening hypertension, cardiac arrhythmias, anxiety, and fear of impending death. Most pheochromocytomas contain predominantly chromaffin cells that secrete norepinephrine compared with the normal adrenal medulla that comprises about 85% epinephrinesecreting cells. Stimulation of α-adrenergic receptors results in elevated blood pressure, increased cardiac contractility, glycogenolysis, gluconeogenesis, and intestinal relaxation. Stimulation of β-adrenergic receptors results in an increased heart rate and contractility. Surgical resection of the tumor is the treatment of choice. Careful monitoring with α- and β-blockers is required during surgery to prevent hypertensive crises.

Ganglion cells

are also present in the medulla. Their axons extend peripherally to the parenchyma of the adrenal cortex to modulate its secretory activity and innervate blood vessels and extend outside the gland to the splanchnic nerves innervating abdominal organs.

Chromaffin cells of the adrenal medulla have a secretory function. Chromaffin cells are organized in ovoid clusters and short

interconnecting cords (Plate 21.6, page 860). The blood capillaries are arranged in intimate relation to the parenchyma. They originate either from the cortical capillaries or, as branches, from the cortical arterioles. Ultrastructurally, the chromaffin cells are characterized by numerous secretory vesicles with diameters of 100–300 nm, profiles of

rER, and a well-developed Golgi apparatus. The secretory material in the vesicles can be stained specifically to demonstrate histochemically that the catecholamines epinephrine and norepinephrine secreted by the chromaffin cells are produced by different cell types (Fig. 21.26). The TEM also reveals two populations of chromaffin cells distinguished by the nature of their membrane-bound vesicles:

FIGURE 21.26. Electron micrograph of medullary cells.

Two types of medullary cells are present. The norepinephrine-secreting cells ( ) are identified by their vesicles, which contain a very dense core. The epinephrinesecreting cells ( ) possess vesicles with less intensely staining granules. ×15,000.

E

NE

dense-core vesicles contains vesicles

One population of cells contains only large . These cells secrete norepinephrine. The other population of cells that are smaller, more homogeneous, and less dense. These cells secrete epinephrine.

Exocytosis of the secretory vesicles is triggered by the release of acetylcholine from presynaptic sympathetic axons that synapse with each chromaffin cell. Epinephrine and norepinephrine account for less than 20% of the contents of the medullary secretory vesicles. The vesicles also contain large amounts of soluble 48-kDa proteins, called chromogranins, that

appear to impart the density of the vesicles’ contents. These proteins, along with ATP and Ca2+ , may help bind the low-molecularweight catecholamines and are released with the hormones during exocytosis. The catecholamines, synthesized in the cytosol, are transported into the vesicles through the action of a magnesiumactivated ATPase in the membrane of the vesicle. Drugs such as , which cause depletion of catecholamines from the vesicles, may act by inhibiting this transport mechanism.

reserpine Glucocorticoids secreted in the cortex induce the conversion of norepinephrine to epinephrine in chromaffin cells. Glucocorticoids produced in the adrenal cortex reach the medulla directly through the continuity of the cortical and medullary sinusoidal capillaries. They induce the enzyme that catalyzes the methylation of norepinephrine to produce epinephrine. The nature of the blood flow correlates with regional differences in the distribution of norepinephrine- and epinephrine-containing chromaffin cells. The epinephrine-containing cells are more numerous in areas of the medulla supplied with blood that has passed through the cortical sinusoids and thus contains secreted glucocorticoids. In some species, the norepinephrine-containing cells are more numerous in those regions of the medulla supplied by capillaries derived from the cortical arterioles.

The catecholamines, in concert with the glucocorticoids, prepare the body for the “fight-or-flight” response. The sudden release of catecholamines establishes conditions for maximum use of energy and thus maximum physical effort. Both epinephrine and norepinephrine stimulate glycogenolysis (release glucose into the bloodstream) and mobilization of free fatty acids from adipose tissue. The release of catecholamines also causes an increase in blood pressure, dilation of the coronary blood vessels, vasodilation

of vessels supplying skeletal muscle, vasoconstriction of vessels conveying blood to the skin and gut, an increase in heart rate and output, and an increase in the rate and depth of breathing.

Zonation of the Adrenal Cortex adrenal cortex

The is divided into three zones on the basis of the arrangement of its cells (Fig. 21.27):

FIGURE 21.27. Photomicrographs of the cortex and medulla of the human adrenal gland. a. This photomicrograph shows a hematoxylin and eosin (H&E)-

stained specimen of the outer cortex. It includes the connective tissue capsule, the zona glomerulosa, and the zona fasciculata. Continuous with the zona glomerulosa are the straight cords of cells that characterize the zona fasciculata. Between the cords are the capillaries and the less numerous arterioles. The linear stripes represent capillaries that are engorged with red blood cells. ×120. The deep parts of the zona fasciculata, zona reticularis, and medulla are shown here. Note that the linear arrays of the cords in the zona fasciculata give way to irregular groups of cells of the zona reticularis. The medulla, in contrast, consists of ovoid groups of cells and short interconnecting cords of cells. A central adrenomedullary vein is also seen here. Note a cross section of the thick longitudinally arranged smooth muscle bundle in part of its wall. ×120.

red

b.

Zona glomerulosa, the narrow outer zone that constitutes up to 15% of the cortical volume Zona fasciculata, the thick middle zone that constitutes nearly 80% of the cortical volume Zona reticularis, the inner zone that constitutes only 5%–7% of the

cortical volume but is thicker than the glomerulosa because of its

more central location

Zona Glomerulosa

zona glomerulosa [Lat. glomus ball]

The cells of the , are arranged in closely packed ovoid clusters and curved columns that are continuous with the cellular cords in the zona fasciculata (see Fig. 21.27a). Cells of the zona glomerulosa are relatively small and columnar or pyramidal (see Fig. 21.24). Their spherical nuclei appear closely packed and stain densely. In humans, some areas of the cortex may lack a recognizable zona glomerulosa. A rich network of fenestrated sinusoidal capillaries surrounds each cell cluster. The cells have abundant sER, multiple Golgi complexes, large mitochondria with shelflike cristae, free ribosomes, and some rER. Lipid droplets are sparse.

The zona glomerulosa secretes aldosterone, which functions in the control of blood pressure. The cells of the zona glomerulosa secrete the primary mineralocorticoid called aldosterone, a compound that functions in the regulation of

sodium and potassium homeostasis and water balance. Aldosterone acts on the principal cells in the distal tubules of the nephron in the kidney, the gastric mucosa, and the salivary and sweat glands to stimulate resorption of sodium at these sites as well as to stimulate excretion of potassium by the kidney. Aldosterone is produced from by a series of enzymatic reactions controlled by angiotensin II (see later). The final step of aldosterone biosynthesis is facilitated by , which is exclusively expressed in cells of the zona glomerulosa. Cells of the zona glomerulosa lack the enzyme 17αhydrolase and, therefore, are unable to produce other adrenal steroid hormones, such as cortisol or adrenal androgens.

cholesterol

aldosterone synthase

The renin–angiotensin–aldosterone system provides feedback control for the zona glomerulosa. The zona glomerulosa is under feedback control of the renin– angiotensin–aldosterone system (RAAS). The juxtaglomerular cells in the kidney release renin in response to a decrease in blood pressure or a low blood sodium level. Circulating renin catalyzes the conversion of circulating angiotensinogen to angiotensin I, which, in turn, is converted by angiotensin-converting enzyme (ACE) in the lung to angiotensin II. Angiotensin II then stimulates the cells of the zona glomerulosa to produce and secrete aldosterone. As the blood pressure, sodium concentration, and blood volume increase in response to aldosterone, the release of renin from the juxtaglomerular cells is

inhibited. Drugs that inhibit ACE in the lung are effective in the treatment of .

chronic essential hypertension

Zona Fasciculata

zona fasciculata [Lat. fascis bundle]

The cells of the , are large and polyhedral. They are arranged in long straight cords, one or two cells thick, that are separated by sinusoidal capillaries (see Fig. 21.27a). The cells of the zona fasciculata have a lightly staining spherical nucleus. Binucleate cells are common in this zone. TEM studies reveal characteristics typical of steroid-secreting cells, that is, a highly developed sER (more so than cells of the zona glomerulosa) and mitochondria with tubular cristae. They also have a well-developed Golgi apparatus and numerous profiles of rER that may give a slight basophilia to some parts of the cytoplasm (Fig. 21.28). In general, however, the cytoplasm is acidophilic and contains numerous lipid droplets, although it usually appears vacuolated in routine histologic sections because of the extraction of lipid during dehydration. The lipid droplets contain neutral fats, fatty acids, cholesterol, and phospholipids that are precursors for the steroid hormones secreted by these cells.

FIGURE 21.28. Electron micrograph of cells in the zona fasciculata. The boundary between adjacent cells of the cord is indicated by the arrowheads . Lipid droplets ( L ) are numerous (the lipid has been partially extracted). ×15,000. Inset. A higher magnification of an area in the cell at the top of the micrograph reveals the extensive sER that is characteristic of steroidsecreting cells. Portions of the Golgi apparatus are also evident. ×40,000. , smooth-surfaced endoplasmic reticulum.

sER

The principal secretion of the zona fasciculata is glucocorticoids that regulate glucose and fatty acid metabolism. Cells in the zona fasciculata cannot produce aldosterone because they lack the enzyme aldosterone synthase. However, they do possess two other important enzymes, 17α-hydrolase and 17,20-lyase, to produce and small amounts of . Glucocorticoids are named for their role in regulating (glucose synthesis) and (glycogen polymerization). One of the major glucocorticoids secreted by the zona fasciculata, , acts on many different cells and tissues to increase the metabolic availability of glucose and fatty acids, both of which are immediate sources of energy. The other glucocorticoid, , is secreted and circulates in the blood at 10- to 20fold lower levels than cortisol. Within this broad function,

glucocorticoids androgens) gluconeogenesis cortisol corticosterone

gonadocorticoids (adrenal glycogenesis

glucocorticoids may have different, even opposite, effects in different tissues:

liver

In the , glucocorticoids stimulate conversion of amino acids to glucose, stimulate the polymerization of glucose to glycogen, and promote the uptake of amino acids and fatty acids. In , glucocorticoids stimulate the breakdown of lipids to glycerol and free fatty acids. In , they reduce the rate of glucose use and promote the oxidation of fatty acids. In such as fibroblasts, they inhibit protein synthesis and even promote protein catabolism to provide amino acids for conversion to glucose in the liver.

adipose tissue other tissues cells

Glucocorticoids

also depress the immune and inflammatory responses and, as a result of the latter, inhibit wound healing. , a synthetic form of cortisol, is used in the treatment of and . It depresses the inflammatory response by suppressing interleukin-1 (IL-1) and IL-2 production by lymphocytes and macrophages. Glucocorticoids also stimulate destruction of lymphocytes in lymph nodes and inhibit mitosis in transformed lymphoblasts.

Hydrocortisone allergies

inflammation

ACTH regulates secretion of the zona fasciculata.

The secretion and production of glucocorticoids and sex steroids by the zona fasciculata is under feedback control of the . ACTH is necessary for cell growth and maintenance and also stimulates steroid synthesis and increases blood flow through the adrenal gland. In animals, administration of ACTH causes hypertrophy of the zona fasciculata. Circulating glucocorticoids may act directly on the pituitary gland, but they most commonly exert their feedback control on neurons in the arcuate nucleus of the hypothalamus, causing the release of corticotropin-releasing hormone (CRH) into the hypothalamohypophyseal portal circulation. Evidence also suggests that circulating glucocorticoids and the physiologic effects that they produce stimulate higher brain centers that, in turn, cause the hypothalamic neurons to release CRH.

CRH–ACTH system

Zona Reticularis

zona reticularis [Lat. rete, net]

The cells of the are noticeably smaller than those of the zona fasciculata, and their nuclei are more deeply stained (see Fig. 21.24). They are arranged in anastomosing cords separated by fenestrated capillaries. The cells have relatively

few lipid droplets. Both light and dark cells are seen. Dark cells have abundant large lipofuscin pigment granules, and deeply staining nuclei are evident. The cells in this zone are small because they have less cytoplasm than the cells in the zona fasciculata; thus, the nuclei appear more closely packed. They exhibit features of steroid-secreting cells, namely, a well-developed sER and numerous elongated mitochondria with tubular cristae, but they have little rER.

The principal secretion of the zona reticularis is gonadocorticoids (adrenal androgens). The principal secretion of the cells in the zona reticularis consists of gonadocorticoids (adrenal androgens), mostly dehydroepiandrosterone (DHEA), dehydroepiandrosterone sulfate (DHEAS), and androstenedione. The cells also secrete some glucocorticoids but in much smaller amounts than those of the zona fasciculata. Here, too, the principal glucocorticoid secreted is . DHEA and DHEAS are less potent than androgens produced by the gonads, but they do have an effect on the development of . In men, adrenal androgens have negligible importance because testosterone produced by the testis is a much more powerful androgen. However, in women, adrenal androgens stimulate the growth of axillary and pubic hair during puberty and adolescence. DHEA can be converted into androstenedione and then more potent androgens, such as testosterone and estrogens in peripheral tissues. The key enzyme that facilitates the conversion of androstenedione to testosterone is , and this reaction represents a major pathway in testosterone production in women. The zona reticularis is also regulated by the feedback control of the CRH–ACTH system and atrophies after . Exogenous ACTH maintains the structure and function of the zona reticularis after hypophysectomy.

cortisol

secondary sex

characteristics

17-

ketosteroid reductase (17KSR)

hypophysectomy

Fetal Adrenal Gland

The fetal adrenal gland consists of an outer narrow permanent cortex and an inner thick fetal cortex or fetal zone. Once fully established, the fetal adrenal gland is unusual in terms of its organization and its large size relative to other developing organs. The gland arises from mesothelial cells of mesodermal origin located between the root of the mesentery and the developing urogenital ridges (see Fig. 21.23a). The mesodermal cells penetrate the underlying mesenchyme and give rise to a large eosinophilic cell mass that will

become the functional fetal cortex (see Fig. 21.23b). Later, a second wave of cells derived from the mesothelium of the posterior abdominal wall surrounds the primary cell mass (see Fig. 21.23b). By the fourth fetal month, the adrenal gland reaches its maximum mass in terms of body weight and is only slightly smaller than the adjacent kidney (see Fig. 21.23c). At term, the adrenal glands are equivalent in size and weight to those of the adult and produce 100–200 mg of steroid compounds per day, about twice that of the adult glands. The histologic appearance of the fetal adrenal gland is superficially similar to that of the adult adrenal gland. During late fetal life, most of the gland consists of cords of large eosinophilic cells that constitute approximately 80% of its mass. This portion of the gland, referred to as the (also called ), arises from the initial mesodermal cell migration. The remainder of the gland is composed of the peripheral layer of small cells with scanty cytoplasm. This portion, referred to as the , arises from the secondary mesodermal cell migration. The narrow permanent cortex, when fully established in the embryo, appears similar to the adult zona glomerulosa. The cells are arranged in arched groups that extend into short cords. They, in turn, become continuous with the cords of the underlying fetal zone (Fig. 21.29). In H&E preparations, the cytoplasm of the cells in the permanent cortex exhibits some basophilia; in combination with the closely packed nuclei, this gives this part of the gland a blue appearance, in contrast to the eosinophilic staining of the fetal zone.

fetal cortex

fetal zone

permanent cortex

FIGURE 21.29. Photomicrographs of a human fetal adrenal gland. a. Lowpower micrograph of a hematoxylin and eosin (H&E)-strained section of a fetal adrenal gland. The permanent cortex ( PC ) is indicated in the upper portion of the micrograph. Below is the fetal zone ( FZ ) in which the cells are arranged in anastomosing linear cords. Some of the capillaries ( C ) are engorged with red blood cells, thereby making them more apparent. ×100. b. Higher power micrograph of the same specimen showing the capsule ( Cap ) and the underlying

permanent cortex. The cells are arranged in arched groups that extend into short cords. Note the close proximity of the nuclei and the small amount of cytoplasm in these cells. ×200. This micrograph shows the cells of the fetal zone at the same magnification as in . Note the slightly larger size of the nuclei and the considerable amount of cytoplasm in each of the fetal zone cells. Also note the eosinophilia of the cytoplasm, compared with the more basophilic cytoplasm of the cells of the permanent cortex. ×200. (Original specimen courtesy of Dr. William H. Donnelly.)

c.

b

FOLDER 21.6

FUNCTIONAL CONSIDERATIONS: BIOSYNTHESIS OF ADRENAL HORMONES

Cholesterol

is the basic precursor of several steroid hormones, namely, corticosteroids, sex hormones, bile acids, and vitamin D. About one-half of the cholesterol in the body comes from the diet, and the other one-half derives from de novo biosynthesis. Cholesterol synthesis occurs in the cytoplasm and organelles from acetyl-CoA. Biosynthesis in the liver accounts for ~10%, and in the intestines, ~15% of the amount is produced each day. In addition, a small portion of cholesterol is synthesized by the adrenal cortical cells. Both dietary cholesterol and that synthesized de novo are transported within . Cholesterol is stored in lipid droplets within the cytoplasm of adrenal cortical cells in the form of cholesterol esters. Steroid hormones in adrenal glands are synthesized from cholesterol esters by removal of part of the side chain and modifications at specific sites on the remainder of the molecule. The enzymes catalyzing these modifications are located in different zones of the cortex as well as in different cytoplasmic sites within the cells. For instance, cleavage of the cholesterol side chain is catalyzed by or desmolase, which is found only in the mitochondria of steroid-producing cells. This enzyme is induced by angiotensin II in the zona glomerulosa and by ACTH in the zona fasciculata and zona reticularis. The other enzymes necessary for steroid production are located within smooth-surfaced endoplasmic reticulum (sER), cytosol, and mitochondria. Thus, a precursor molecule may move from the sER to a mitochondrion and back again several times before the definitive molecular structure of a given corticosteroid is obtained. Cholesterol esters removed from cytoplasmic lipid droplets and used in steroid hormone synthesis are quickly replenished from the cholesterol esters contained within LDL carried in the bloodstream. These esters are the primary source of the cholesterol used in corticosteroid synthesis. Under conditions of short-term or prolonged ACTH stimulation, the lipid stores in adrenal cortical cells are recruited for corticosteroid synthesis.

low-density lipoproteins (LDLs)

enzyme (P450scc)

P450-linked side chain cleavage

With the TEM, the cells of the permanent cortex exhibit small mitochondria with shelf-like cristae, abundant ribosomes, and small

Golgi profiles. The cells of the fetal zone, in contrast, are considerably larger and are arranged in irregular cords of varying width. With the TEM, these cells exhibit spherical mitochondria with tubular cristae, small lipid droplets, an extensive sER that accounts for the eosinophilia of the cytoplasm, and multiple Golgi profiles. Collectively, these features are characteristic of steroid-secreting cells.

The development of the fetal adrenal gland is part of a complex process of maturation and preparation of the fetus for extrauterine life. The fetal adrenal gland lacks a definitive medulla. Chromaffin cells

are present but are scattered among the cells of the fetal zone and are difficult to recognize in H&E preparations. The chromaffin cells originate from the neural crest (see Fig. 21.23a) and invade the fetal zone at the time of its formation (see Fig. 21.23b). They remain in this location in small, scattered cell clusters during fetal life (see Fig. 21.23c). The blood supply to both the permanent cortex and the fetal zone is through sinusoidal capillaries that course between the cords and join to form larger venous channels in the center of the gland. Unlike the postnatal adrenal, arterioles are absent in the parenchyma of the fetal adrenal gland. Functionally, the fetal adrenal gland is under the control of the CRH–ACTH feedback system through the fetal pituitary. It interacts with the placenta to function as a steroid-secreting organ because it lacks certain enzymes necessary for steroid synthesis that are present in the placenta. Similarly, the placenta lacks certain enzymes necessary for steroid synthesis that are present in the fetal adrenal gland. Thus, the fetal adrenal gland is part of a . Precursor molecules are transported back and forth between the two organs to enable the synthesis of glucocorticoids, aldosterone, androgens, and estrogens. At birth, the fetal cortex undergoes a rapid involution that reduces the gland within the first postnatal month to about a quarter of its previous size. The permanent cortex grows and matures to form the characteristic zonation of the adult cortex. With the involution and disappearance of the fetal zone cells, the chromaffin cells aggregate to form the medulla. If the adrenal glands fail to develop properly, may result. CAH represents a group of autosomal recessive disorders characterized by a deficiency of an enzyme involved in the synthesis of cortisol and/or aldosterone. Deficiency of involved in the aldosterone

fetal–placental

unit

congenital adrenal hyperplasia (CAH) enzyme 21-hydroxylase

synthesis pathway is the most common form of CAH, accounting for more than 90% of cases.

ENDOCRINE ORGANS

OVERVIEW OF THE ENDOCRINE SYSTEM endocrine system

hormones

The produces various secretions called and hormonally active substances that enter the circulatory system for transport to target cells. Hormones and hormonally active substances are divided into three classes of compounds: (e.g., insulin, GH, ACTH), (gonadal and adrenocortical steroids), and and (e.g., catecholamines, prostaglandins). Hormones interact with specific (peptide hormones or catecholamines) or (steroids and thyroid hormones). Regulation of hormonal function is controlled by from the target organs.

steroids arachidonic

mechanisms

peptides amino acids acid analogs cell surface receptors intracellular receptors feedback

PITUITARY GLAND (HYPOPHYSIS)

pituitary gland is composed of two parts: the anterior lobe (adenohypophysis) , which is composed of glandular epithelial tissues, and the posterior lobe (neurohypophysis) , which is The

composed of neural secretory tissue that developed from the neuroectoderm of the CNS. The of the pituitary gland consists of three parts: , , and , which surrounds the infundibulum. The provides the blood supply to the pituitary gland and serves as the link between the hypothalamus and the pituitary gland. involves a network of fenestrated capillaries in the infundibulum and median eminence of the hypothalamus as well as hypophyseal portal veins and a secondary network of

anterior lobe pars distalis pars intermedia pars tuberalis hypothalamohypophyseal portal system

Portal circulation

releasing

capillaries in the pars distalis. Circulation carries from the hypothalamic neurons to the cells in the pars distalis, where cell secretion is controlled. Based on staining reactions of endocrine cell secretory granules, cells in the pars distalis are identified as (10%), (40%), and (50%). Based on immunocytochemical reactions, five functional cell types are identified in the pars distalis: produce ; produce ; produce , a precursor molecule of ; produce both and ; and produce . The of the pituitary gland (pars nervosa and the infundibulum) is an extension of the CNS. It releases hormones produced in the ( or ) and ( ) of the hypothalamus. The delivers ADH and oxytocin to the posterior lobe, where they are stored in axon terminals ( ) and released into circulation.

hormones

acidophils

chromophobes

basophils

somatotropes (GH cells) growth hormone (GH; somatotropin) lactotropes (PRL cells, mammotropes) prolactin (PRL) corticotropes (ACTH cells) proopiomelanocortin (POMC) adrenocorticotropic hormone (ACTH) gonadotropes (FSH and LH cells) luteinizing hormone (LH) follicle-stimulating hormone (FSH) thyrotropes (TSH cells) thyroid-stimulating hormone (TSH) posterior lobe supraoptic nuclei antidiuretic hormone [ADH] vasopressin paraventricular nuclei oxytocin hypothalamohypophyseal tract Herring bodies

PINEAL GLAND

pineal gland

The is a neuroendocrine gland that develops from the neuroectoderm and remains attached to the brain. Because the pineal gland has connections with the eye via the retinohypothalamic tract, it is an important regulator of . The pineal gland contains two types of parenchymal cells: that secrete and supporting . It also possesses characteristic calcified concretions called or .

circadian rhythm pinealocytes (glial) cells

melatonin corpora arenacea brain sand

interstitial

THYROID GLAND

thyroid gland

The is located in the neck and develops from the endodermal lining of the floor of the primitive pharynx.

thyroid follicles follicular epithelium

The thyroid gland consists mainly of , which generally are formed from simple cuboidal . The lumen of the follicles is filled with a gel-like mass called , which contains thyroglobulin, an inactive storage form of thyroid hormones. Follicular epithelium contains two types of cells: that produce thyroid hormones T4 and T3 and that produce calcitonin. of T4 and T3 takes place in follicular cells and the lumen of the follicle. It involves a series of steps, from the synthesis of thyroglobulin, to the uptake and oxidation of iodide, to iodination of the thyroglobulin to form T4 and T3. In response to , follicular cells resorb colloid and transport T4 and T3 into circulation.

colloid

follicular parafollicular

cells cells Synthesis

TSH stimulation

PARATHYROID GLANDS

parathyroid glands

The (two pairs) are located on the posterior surface of the thyroid gland. They develop from the third and fourth pharyngeal pouches. Parathyroid glands consist of two major epithelial cells: , which are the most numerous and secrete , and . regulates calcium and phosphate levels in the blood. It binds to the PTH receptors on target cells and increases the Ca2+ level in the blood.

principal cells parathyroid hormone (PTH) PTH

oxyphil cells

ADRENAL (SUPRARENAL) GLANDS adrenal glands

The are paired triangular organs embedded in the perirenal fat at the upper poles of the kidneys. Adrenal glands are organized into two distinct regions: the , a steroid-secreting portion that developed from the mesoderm, and the , a catecholamine-secreting portion that developed from neural crest cells. During development, the is composed of the fetal cortex without a definitive medulla. The contains chromaffin cells that synthesize epinephrine and norepinephrine to prepare the body for a “fight-or-flight” response.

cortex

adrenal medulla

medulla fetal adrenal gland

adrenal cortex zona fasciculata Zona glomerulosa mineralocorticoids aldosterone system Zona fasciculata glucocorticoids

zona glomerulosa zona reticularis

The is divided into three zones: (outer); (thick middle); and (inner), which communicates with the medulla. cells form ovoid clusters and produce (e.g., aldosterone). The provides the feedback mechanism to control secretion of the zona glomerulosa cells. cells are arranged in long straight cords and produce (e.g., cortisol) that regulate gluconeogenesis (glucose synthesis) and glycogenesis (glycogen polymerization). ACTH regulates secretion of the zona fasciculata cells. cells are arranged in anastomosing cords separated by fenestrated capillaries and produce weak (mostly DHEA). ACTH regulates secretion of the zona reticularis cells.

renin–angiotensin–

Zona reticularis

androgens

PLATE 21.1 PITUITARY I pituitary gland

The is located at the base of the brain and rests in a depression of the sphenoid bone called the on the floor of the middle cranial fossa. It is connected by a short stalk to the hypothalamus. Although joined to the brain, only the of the gland, the , develops from neuroectoderm. The larger of the gland, the , develops from oropharyngeal ectoderm as a diverticulum of the buccal epithelium, called the . The anterior lobe regulates other endocrine glands. It is composed of clumps and cords of epithelioid cells, separated by large-diameter fenestrated capillaries. The posterior lobe is a nerve tract whose terminals store and release secretory products synthesized by their cell bodies located in the and . The secretions contain either or . Other neurons from the hypothalamus release secretions into the fenestrated capillaries of the infundibulum, the first capillary bed of the that carries blood to the fenestrated capillaries of the anterior lobe. These hypothalamic secretions regulate the activity of the anterior lobe of the pituitary gland.

sella turcica posterior lobe adenohypophysis

neurohypophysis anterior lobe Rathke pouch

supraoptic oxytocin

paraventricular nuclei antidiuretic hormone (ADH)

hypophyseal portal system

Pituitary lobe

, human, hematoxylin and eosin (H&E) ×50.

This specimen is a sagittal section of the pituitary gland. The of the gland is delineated by the (indicated by

dashed line

posterior

arrows)

that

pars nervosa PN pars tuberalis PT anterior lobe pars intermedia PI pars distalis PD Cl

separates it from the anterior lobe. The ( ) is the expanded portion of the posterior lobe that is continuous with the infundibulum. The ( ) of the is located around the infundibular stem but may cover the pars nervosa to a variable extent. The ( ) is a narrow band of tissue that lies between the ( ) and the pars nervosa. It borders a small cleft ( ) that constitutes the remains of the lumen of the Rathke pouch. The pars distalis, the anterior lobe of the gland, is its largest part. It contains a variety of cell types that are not uniformly distributed. This accounts for differences in staining (light- and darkstaining areas) that are seen throughout the pars distalis. The pituitary gland contains a connective tissue capsule ( ) that separates the gland from the surrounding meninges. Each of the components of the anterior lobe (i.e., the pars distalis, pars tuberalis, and pars intermedia), when examined at higher magnification, exhibit features at the cellular level that aid in their identification. These features are described in the following figures as well as those in Plate 21.2 (page 852).

Caps

Pars distalis

, pituitary, human, H&E ×375.

pars distalis acidophils A Basophils B Chromophobes

This photomicrograph shows a region of the of the anterior lobe that is rich in ( ). ( ) are present in this area in lesser numbers. The acidophils are readily identified by the acidophilic staining of their cytoplasm, in contrast to the basophils whose cytoplasm is clearly basophilic. ( ) are also very numerous in this field. The cytoplasm stains poorly in contrast to that of the acidophils and basophils. The cells are arranged in cords and clumps, between which are capillaries ( ), some of which can be recognized, but most are in a collapsed state and difficult to visualize at this magnification.

C

Cap

Pars distalis

, pituitary, human, H&E ×375.

pars distalis

This photomicrograph shows a region of the of the anterior lobe that is rich in basophils ( ). At this particular site, there are no recognizable acidophils (at other sites, it is possible to find a more equal distribution of acidophils and basophils, although, typically, one cell type outnumbers the other in a given region). ( ) are also relatively numerous at this site. In this particular region, the chromophobe nuclei are readily apparent, but the cytoplasm of the cells is difficult to discern.

B

Chromophobes C

Pars intermedia

, pituitary, human, periodic acid–Schiff (PAS)/aniline blue black ×380.

pars distalis pars intermedia (PI) of the

This photomicrograph shows a small portion of the ( ); the remainder reveals the

PD

anterior lobe. The pars distalis shown here contains numerous capillaries filled with red blood cells, thus producing a bright red appearance. The pars intermedia contains a number of small cysts ( ). The cells that make up the pars intermedia, which is relatively small in humans, consist of small basophils and chromophobes. The basophils have taken up the blue stain, thus making them prominent. To the extreme is a less cellular area, the ( ).

Cy

nervosa PN

A, acidophils B, basophils C, chromophobes Cap, capillaries Caps, capsule Cl, cleft Cy, cysts PD, pars distalis PI, pars intermedia PN, pars nervosa PT, pars tuberalis

right

pars

PLATE 21.2 PITUITARY II

pars distalis consists chromophils . Chromophobes

The parenchyma of the and

chromophobes

of two general cell types: stain poorly; chromophils

stain

acidophils

well.

Chromophils are further subdivided into and Basophils stain with basic dyes or hematoxylin, whereas the cytoplasm of the acidophil stains with acid dyes such as eosin. The cytoplasm of basophils also stains with the periodic acid–Schiff (PAS) reaction because of the glycoprotein in its secretory granules. Acidophils can be further subdivided into two groups on the basis of special cytochemical and ultrastructural features. One group, called , produces the growth hormone, (STH); the other group of acidophils, called , produces (PRL). The groups of basophils can also be distinguished with the electron microscope and with special cytochemical procedures. One group of produces (TSH); another group of produces the gonadotropic hormones (FSH), and (LH); and a third group of produces (ACTH) and (LPH). Chromophobes are also a heterogeneous group of cells. Many are considered to be depleted acidophils or basophils.

basophils .

somatotropes

somatotropic hormone lactotropes prolactin

thyrotropes gonadotropes hormone corticotropes hormone

thyroid-stimulating hormone follicle-stimulating luteinizing hormone adrenocorticotropic hormone lipotropic

Pars distalis

, pituitary, human, Mallory trichrome ×360;

inset ×1,200.

acidophils A

This photomicrograph of the pars distalis is from an area where there is an almost equal distribution of ( ) and ( ). The clumps and cords of cells are delineated by strands of connective tissue ( ) that surround them. A number of engorged capillaries ( ) containing red blood cells ( ) are also seen. The acidophil’s cytoplasm in this preparation stains a reddish or rust color. The basophils stain a , and the ( ) exhibit a color. The shows the three general cell types at higher magnification. The secretory granules of the acidophils ( ) and basophils ( ) are just discernible. It is the granules that stain and provide the overall coloration to the two cell types. In contrast, the chromophobe ( ) lacks granules and simply reveals a background color.

basophils B Cap

C

B

pale blue

stained blue

reddish blue to deep blue inset

stained yellow

chromophobes A

pale blue

Pars nervosa

C

, pituitary, human, hematoxylin and eosin

(H&E) ×325.

pars nervosa pituicytes,

The of the posterior lobe seen here contains cells called and unmyelinated nerve fibers form the supraoptic and paraventricular nuclei of the hypothalamus. The ( ) are comparable with neuroglial cells of the central nervous system. The nuclei are round to oval; the cytoplasm extends from the nuclear region of the cell as long processes. In H&E preparations such as this, the cytoplasm of the pituicyte cannot be distinguished from the unmyelinated nerve fibers. The hormones of the posterior lobe, oxytocin, and antidiuretic hormone (ADH; also called ) are formed in the hypothalamic nuclei and pass via the fibers of the hypothalamohypophyseal tract to the posterior lobe, where they

vasopressin

pituicytes P

Herring bodies HB

are stored in the expanded nerve terminal portion of the nerve fibers. The stored neurosecretory material appears as ( ). In H&E preparations, the Herring bodies simply appear as small islands of eosinstained substance. Interspersed among the nerve fibers are capillaries ( ).

Cap

Pars nervosa

, pituitary, human, periodic acid–Schiff (PAS)/aniline blue black ×250; inset ×700.

pituicytes light P blue Herring bodies bottom HB

aniline blue

In this specimen from the pars nervosa, the has stained the nuclei of the ( ); the nerve fibers have taken up some of the stains to give a background. With this staining technique, the ( ) appear as . The shows a Herring body near the of the micrograph at high magnification. The granular texture of the Herring body as seen here is a reflection of the accumulated secretory granules in the nerve terminals. Also of note in this specimen are the capillaries ( ), which are prominent as a result of the contrasting red staining of the red blood cells within them.

islands

inset

Cap

A, acidophils B, basophils C, chromophobes Cap, capillaries HB, Herring bodies P, pituicytes

dark black

PLATE 21.3 PINEAL GLAND

pineal gland pineal body, epiphysis cerebri

The ( ) is located in the brain above the superior colliculi. It develops from neuroectoderm but, in

the adult, bears little resemblance to nerve tissue. Two cell types have been described within the pineal gland: and . The full extent of these cells cannot be appreciated without the application of special staining methods. These would show that the glial cells and the pinealocytes have processes that are expanded at their periphery. The pinealocytes are more numerous. In a hematoxylin and eosin (H&E) preparation, the nuclei of the pinealocytes are pale staining. The nuclei of the glial cells, on the other hand, are smaller and stain more intensely. The secretion of the pineal gland has an antigonadal effect: It decreases the production of gonadal steroids. For example, hypogenitalism has been reported with pineal tumors that consist chiefly of pinealocytes, whereas sexual precocity (puberty that occurs at an unusually early age) is associated with glial cell tumors (presumably, the pinealocytes have been destroyed). In addition, experiments with animals indicate that the pineal gland has a neuroendocrine function, whereby the pineal gland serves as an intermediary that relates endocrine function (particularly gonadal function) to cycles of light and dark. The external photic stimuli reach the pineal gland via optical pathways that connect with the superior cervical ganglion. In turn, the superior cervical ganglion sends postganglionic nerve fibers to the pineal gland. The pineal gland has a role in adjusting to sudden changes in day length, such as those experienced by travelers who suffer from jet lag, and a role in regulating emotional responses to reduced day length during winter in temperate and subarctic zones (seasonal affective disorder [SAD]).

pinealocytes (parenchymal) cells

glial cells

Pineal gland

, human, H&E ×180.

capsule CTCap

The pineal gland is surrounded by a very thin ( ) that is formed by the pia mater. Connective tissue trabeculae ( ) extend from the capsule into the substance of the gland dividing it into lobules. The ( ) appear often as indistinct groups of cells of varying size surrounded by the connective tissue. Blood vessels, generally small arteries ( ) and veins ( ), course through the connective tissue. The arteries give rise to capillaries that surround and penetrate the lobules to supply the parenchyma of the gland. In this specimen and even at this low magnification, the capillaries ( ) are prominent as a consequence of the red blood cells present in their lumina.

A

lobules L

V

C

Pineal gland

, human, H&E ×360; inset ×700.

brain sand BS

This micrograph shows at higher magnification the parenchyma of the pineal gland as well as a component called ( ) or . When viewed at even higher magnification, the corpora arenacea are seen to have an indistinct lamellated structure. Typically, they stain heavily with hematoxylin. The presence of these structures is an identifying feature of the pineal gland. A careful examination of the cells within the gland at the light microscopic level reveals two specific cell types. One cell type represents the pinealocytes (or chief cells of the pineal gland), which are by far the most numerous. Pinealocytes are modified neurons. Their nuclei are spherical and are relatively lightly stained because of the amount of euchromatin that they contain. The second cell type is the interstitial cell or glial cell that constitutes a relatively small percentage of the cells in the gland. Their nuclei are smaller and more elongated than those of the pinealocytes. The reveals several ( ) that can be identified by their more densely staining nuclei. The majority of the nuclei of the other cells seen here belong to pinealocytes. Also seen in the are several fibroblasts ( ) that are present within a trabecula.

corpora arenacea

glial cells G

A, artery BS, brain sand C, capillary Cap, capsule CT, connective tissue F, fibroblast G, glial cell L, lobule V, vein

inset

inset

F

PLATE 21.4 PARATHYROID AND THYROID GLANDS parathyroid glands

The are usually four in number. Each is surrounded by a capsule and lies on or is partially embedded in the thyroid gland. Connective tissue trabeculae extend from the capsule into the substance of the gland. The elaborate a hormone that influences calcium and bone metabolism. Injection of parathyroid hormone into laboratory

parathyroid glands

animals results in the release of calcium from bone by the action of osteocytes (osteocytic osteolysis) and osteoclasts. Removal of the parathyroid glands results in a rapid drop in blood calcium levels. The is located in the neck in close relation to the upper part of the trachea and the lower part of the larynx. It consists of two lateral lobes that are joined by a narrow isthmus. The follicle, which consists of a single layer of cuboidal or low columnar epithelium surrounding a colloid-filled space, is the functional unit of the thyroid gland. A rich capillary network is present in the connective tissue that separates the follicles. The connective tissue also contains lymphatic capillaries.

thyroid gland

Parathyroid gland

, human, hematoxylin and eosin (H&E)

×320.

As seen here, the larger blood vessels are associated with the trabeculae ( ) and, occasionally, adipose cells ( ). The parenchyma of the parathyroid glands appears as cords or sheets of cells separated by capillaries and delicate connective tissue septa. Two parenchymal cell types can be distinguished in routine H&E sections: chief cells (principal cells) and oxyphil cells. The ( ) are more numerous. They contain a spherical nucleus surrounded by a small amount of cytoplasm. ( ) are less numerous. They are conspicuously larger than chief cells but have a slightly smaller and more intensely staining nucleus. Their cytoplasm stains with eosin, and the boundaries between the cells are usually well marked. Moreover, the oxyphils are arranged in groups of variable size that appear scattered about in a much larger field of chief cells. Even with low magnification, it is often possible to identify clusters of oxyphil cells because a unit area contains fewer nuclei than a comparable unit area of chief cells, as is clearly evident in this figure. Oxyphil cells appear during the end of the first decade of life and become more numerous around puberty. A subsequent increase may be seen in older individuals.

BV

A

Oxyphil cells OC

Thyroid gland

chief cells CC

, human, H&E ×240.

thyroid follicles ( F ) vary somewhat in size and shape and appear closely packed. The homogeneous mass in the center of each follicle A histologic section of the thyroid gland is shown here. The

is the colloid. The follicular cells of the gland appear to form a simple cuboidal epithelium enclosing the colloid. Careful examination of the apical surface of follicular cells reveals small vacuoles, an indication of colloid resorption. Although the individual cells are difficult to distinguish at this magnification, the nuclei of the cells serve as an indication of their location and arrangement. The thyroid is well vascularized: Larger ( ) are found in the connective tissue ( ), and the capillary network surrounds follicles. This specimen has few areas of large groups of cells with nuclei that are of the same size, shape, and staining characteristics as follicular cells. These areas represent ( ).

vessels BV

CT

tangentially sectioned follicles tsF

blood

A, adipose cells BV, blood vessels CC, chief cells CT, connective tissue F, follicles OC, oxyphil cells tsF, tangentially sectioned follicle

PLATE 21.5 ADRENAL GLAND I adrenal glands

There are two , one at the upper pole of each kidney. The gland is a composite of two distinct structural and functional components:

cortex and a medulla . The cortex develops from mesoderm and secretes steroid hormones; the medulla develops from neuroectoderm of the neural crest and secretes catecholamines . The adrenal cortex is divided into three zones according to the type and arrangement of its parenchymal cells. These are designated as zona glomerulosa, zona fasciculata , and zona reticularis . The zona glomerulosa constitutes 15% of the cortical volume. It secretes mineralocorticoid (aldosterone). The zona fasciculata constitutes nearly 80% of the cortical volume. It secretes glucocorticoids ( cortisol and corticosterone) and a small amount of adrenal androgens. The zona a

reticularis (5%–7% of cortical volume) produces most of the adrenal androgens. The zona fasciculata and the zona reticularis are regulated by (ACTH) secreted by the anterior lobe of the pituitary gland in response to (CRF) produced by the hypothalamus. The zona glomerulosa is not regulated by ACTH but by angiotensin II, which is part of feedback control of the that also regulates blood pressure.

adrenocorticotropic hormone

corticotropin-releasing factor

–angiotensin–aldosterone system

Adrenal gland

renin

, human, hematoxylin and eosin (H&E) ×45.

This low-magnification micrograph of a section through the partial thickness of an adrenal gland shows the outer capsule ( ), the ( ) from one surface of the gland, the underlying ( ), and a very small portion of the cortex from the other surface of the gland ( ). The cortex has a distinctly different appearance in both structural organization and staining characteristics. From the inner portion, the medulla, note the lighter appearance of the medullary tissue. A small amount of adipose tissue ( ) in which the gland is partially embedded is seen at the of the micrograph. The corticomedullary boundary ( ) has a wave-like contour, a reflection of the irregular shape of the gland. Within the medulla are a number of relatively large blood vessels ( ). These are the adrenomedullary collecting veins that drain both the cortex and the medulla.

cortex Cort medulla Med Cort, bottom center

upper center dashed lines BV

Cortex

Cap

AT

, adrenal gland, human, H&E ×180.

cortex zona glomerulosa ZG

This is a higher magnification of a portion of the capsule and the full thickness of the from an area in the previous figure. The capsule consists of dense connective tissue in which the larger arteries ( ) travel to give rise to smaller vessels that will supply the cortex and medulla. The ( ) is located at the outer part of the cortex, immediately under the capsule. The parenchyma of this zone consists of small cells that appear as arching cords or as oval groups of cells. The ( ) consists of radially oriented cords and sheets of cells, usually two cells in width, that extend toward the medulla. The cells of the outer part of the zona fasciculata are generally larger than those of the inner portion of this zone and typically stain poorly because of the large

A

zona fasciculata ZF

zona reticularis

number of lipid droplets that they contain. The cells of the ( ) are relatively small and contain little or no lipid droplets and, consequently, stain prominently with eosin. Because of their small size, the nuclei are in close proximity to one another, much like the cells of the zona glomerulosa.

ZR

Cortex

, adrenal gland, human, H&E ×245.

This is a higher magnification of the area inscribed by the left rectangle in the previous figure. It shows the zona glomerulosa ( ZG ) and the outer portion of the zona fasciculata ( ZF ). Note the

smaller size of the cells in the zona glomerulosa than those in the zona fasciculata. In addition, cells of the zona glomerulosa contain fewer lipid droplets than those of the zona fasciculata. Typically, the cells in this part of the zona fasciculata are filled with lipid droplets, thus the very poor staining characteristic of their cytoplasm. Delicate connective tissue trabeculae ( ) extend from the capsule to surround the glomerular groups of cells and extend between the cords of cells in the zona fasciculata. Capillaries and arterioles are located within the connective tissue trabeculae. Usually, the capillaries are collapsed and, without the presence of red blood cells in their lumina, are thus difficult to identify.

arrows

Cortex

, adrenal gland, human, H&E ×245.

right

zona

This is a higher magnification of the area inscribed by the in the previous figure. This deep portion of the ( ) reveals smaller cells, although they are still arranged in cords and contain lipid droplets, although in lesser amounts. The cells of the ( ) are arranged in irregular anastomosing cords and contain only a small amount of lipid and, consequently, their cytoplasm stains with eosin.

rectangle

fasciculata ZF zona reticularis ZR

A, arteries AT, adipose tissue BV, blood vessels Cap, capsule Cort, cortex Med, medulla ZF, zona fasciculate ZG, zona glomerulosa ZR, zona reticularis arrows, connective tissue trabeculae dashed line, corticomedullary boundary

PLATE 21.6 ADRENAL GLAND II adrenal medulla

The cells of the develop from the same source as the postganglionic cells of the sympathetic nervous system. They are directly innervated by preganglionic cells of the sympathetic system and may be regarded as modified postganglionic cells that are specialized to secrete. These cells produce the catecholamines and .

epinephrine

norepinephrine

The adrenal medulla receives its blood supply via two routes: by arterioles that pass through the cortex and by capillaries that continue from the cortex, a type of portal circulation. Thus, some of the blood supplying the medulla contains cortical secretions that regulate medullary function. Blood leaves the medulla via the central adrenomedullary vein. Its structure is unusual in that the tunica media of the vessel contains prominent bundles of longitudinally oriented smooth muscle, the contraction of which facilitates rapid outflow of blood when medullary catecholamines are released.

Medulla

, adrenal gland, human, hematoxylin and eosin (H&E) ×175; inset ×250.

adrenal medulla . The medullary cells are organized in ovoid groups and short interconnecting cords. The cytoplasm of the medullary This moderately low-power photomicrograph shows the cells of the

cells may stain with different intensity. The cytoplasm of some cells is very poorly stained, appearing almost clear, whereas others show greater intensity of eosin staining. In this photomicrograph, a portion of the wall, namely, the tunica media ( ) of the central adrenomedullary vein, can be seen. The nature of the central adrenomedullary vein is described in the figure. The shows the ovoid groups of medullary cells at a higher magnification. Between these groups of cells are capillaries ( ) that, as in the cortex, can be identified when they contain red blood cells as shown here.

TM

inset

lower left

Cap

Medulla

, adrenal gland, human, H&E ×125.

central adrenomedullary vein AMV TM

This micrograph shows a ( ) that drains the adrenal medulla. The tunica media ( ) is unusually thick. The smooth muscle that constitutes this part of the vessel wall is in the form of bundles that are arranged longitudinally, that is, in the same direction as the vessel. Thus, the muscle seen here is cut in cross section, as is the vein. Whereas the central adrenomedullary vein occupies most of the micrograph, ( ) can be seen in several locations surrounding the vein. The portion of the figure outlined by the is seen at higher magnification in the figure.

medullary cells MC

rectangle

bottom right

Central adrenomedullary vein H&E ×350.

, adrenal gland, human,

rectangle L

bottom

central

This higher magnification view of the in the figure shows part of the lumen ( ) of the ( ) at the bottom of the field. The tunica intima ( ) of the vessel is relatively thin but may contain a variable amount of connective tissue. The smooth muscle ( ) of the tunica media ( ) is readily seen here as being arranged in bundles and appears in cross section. There is no discrete tunica adventitia in this vein. Instead, its connective tissue blends in with surrounding structures. ( ) are

TI

left

adrenomedullary vein AMV

SM

TM

Ganglion cells GC

frequently found in proximity to the wall of the central adrenomedullary vein. They are large cells with a moderately basophilic cytoplasm. Because of the large size of the cell, the nucleus is often missed in the section, and only the cell cytoplasm is seen.

AMV, central adrenomedullary vein Cap, capillary GC, ganglion cells L, lumen of central adrenomedullary vein MC, medullary cells SM, smooth muscle TI, tunica intima TM, tunica media

2 2

MALE REPRODUCTIVE SYSTEM

OVERVIEW OF THE MALE REPRODUCTIVE SYSTEM TESTIS

Sex Determination and Development of the Testis

Structure of the Testis Leydig Cells

SPERMATOGENESIS

Spermatogonial Phase Spermatocyte Phase (Meiosis) Spermatid Phase (Spermiogenesis) Structure of the Mature Sperm

SEMINIFEROUS TUBULES

Cycle of the Seminiferous Epithelium Waves of the Seminiferous Epithelium Sertoli Cells

INTRATESTICULAR DUCTS EXCURRENT DUCT SYSTEM Epididymis Ductus Deferens

ACCESSORY SEX GLANDS PROSTATE GLAND Bulbourethral Glands SEMEN PENIS AND SCROTUM Folder 22.1 Functional Considerations: Hormonal Regulation of Spermatogenesis Folder 22.2 Clinical Correlation: Factors Affecting Spermatogenesis Folder 22.3 Clinical Correlation: Sperm-Specific Antigens and the Immune Response Folder 22.4 Clinical Correlation: Benign Prostatic Hypertrophy and Cancer of the Prostate Folder 22.5 Clinical Correlation: Mechanism of Erection and Erectile Dysfunction

HISTOLOGY

OVERVIEW OF THE MALE REPRODUCTIVE SYSTEM male reproductive system

The consists of the testes, genital excurrent ducts, accessory sex glands, and external genitalia containing the penis and scrotum (Fig. 22.1). The accessory sex glands include the seminal vesicles, the prostate, and bulbourethral glands. The two primary functions of the testis are (the production of sperm, called ) and (synthesis of androgens, also called ). Androgens, mainly testosterone, are essential for spermatogenesis. They also play an important role in embryonic development of the male embryo into the phenotypic male fetus and are responsible for sexual dimorphism (male physical and behavioral characteristics). The events of cell division that occur during the production of male gametes, as well as those of the female (the ova), involve both normal division (mitosis) and reduction division (meiosis).

spermatogenesis steroidogenesis hormones

male gametes sex

FIGURE 22.1. Schematic diagram demonstrating the components of the male reproductive system. Midline structures are depicted in

sagittal section; bilateral structures, including the testis, epididymis, ductus deferens, and seminal vesicle, are shown intact.

A brief description of mitosis and meiosis is included in Chapter 3, The Cell Nucleus (pages 99-103). A basic understanding of these processes is essential to understanding the production of gametes in both sexes.

TESTIS

testes

The adult are paired ovoid organs that lie within the , located outside the body. Each testis is suspended within the end of an elongated musculofascial pouch, which is continuous with the layers of the anterior abdominal wall that projects into the scrotum. Testes are connected by the spermatic cords to the abdominal wall and tethered to the scrotum by scrotal ligaments, the remnants of the gubernaculum (see discussion later in this chapter).

scrotum

Sex Determination and Development of the Testis

Sex differentiation is accomplished through a cascade of gene activations. Genetic sex is determined at fertilization by the presence or absence of the Y chromosome. The testes, however, do not form until the seventh week of development. Gonadal sex is determined by the presence of the SRY gene located in the sex-determining region Y of the short arm of the Y chromosome. SRY gene

expression in early embryonic development triggers the differentiation of the gonads into the testis. Mutations in this gene give rise to , a condition known as . Females with Swyer syndrome have an X and Y chromosome and functional female genitalia, including the vagina, uterus, and uterine tubes. However, they lack female sex gonads (ovaries). Females with this syndrome do not produce sex hormones. Treatment with hormone therapy is necessary to induce puberty. The genetic information encoded in the Y chromosome itself is not sufficient to guide the complex development of the male gonads. Instead, the SRY gene operates as a that controls the cascade of several gene activations on autosomes 9, 11, 17, and 19 and the X chromosome. A transcription factor called the , encoded by the SRY gene, contains a molecular domain that binds to a specific region of DNA and alters its structure. The affected DNA forms a loop that permits binding of other transcription factors. These factors, in turn, cause the expression of other genes that initiate male sex determination (formation of the testes and other male sex organs). These genes include the following:

XY female embryo with gonadal dysgenesis Swyer syndrome

master switch

testis-determining factor (TDF)

WT-1 gene

(Wilms tumor 1 gene), which is required for the development of the urogenital system and regulation of the SRY transcription. Mutations of the WT-1 gene are present in children with and in children with accompanied . (SRY [sex-determining region Y]-box 9 gene) activates the (anti-Müllerian hormone gene) that is responsible for Müllerian-inhibiting factor (MIF) synthesis. Mutation of the SOX9 gene is linked the formation of ambiguous or female sex organs in a genetically male individual (46,XY).

familial Wilms tumor genitourinary malformations SOX-9 gene AMH gene

SF-1 gene (steroidogenic factor-1 gene) that regulates the expression of a number of steroidogenic genes DAX-1 gene (dosage-sensitive sex reversal, adrenal hypoplasia critical region, on chromosome X, gene 1) that encodes nuclear receptor DAX-1. Activation of this receptor suppresses the SRY gene during gonadal sex differentiation, and its mutation is responsible for .

congenital adrenal hypoplasia The testes develop on the posterior wall of the abdomen and later descend into the scrotum. The testes develop in close association with the urinary system retroperitoneally on the posterior wall of the abdominal cavity. Testes (like ovaries) are derived from three sources:

Intermediate mesoderm

forms the urogenital ridges on the posterior abdominal wall, giving rise to Leydig cells (interstitial cells) and myoid cells (peritubular contractile cells). lines the urogenital ridges and gives rise to finger-like epithelial cords called . These cords grow into the underlying intermediate mesoderm and become colonized by primordial germ cells. The primary sex cords also give rise to Sertoli cells. migrate from the yolk sac into developing gonads, where they are incorporated into the . They differentiate into , which are the precursors of the definitive germ cells called . At this stage, the cords are composed of primordial germ cells, pre-Sertoli cells, and a surrounding layer of myoid cells. Later, primary sex cords differentiate into the , which give rise to the seminiferous tubules, straight tubules, and rete testis (Fig. 22.2).

Mesodermal epithelium (coelomic mesothelium) primary sex cords Primordial germ cells sex cords cords

primary gonocytes spermatogonia

seminiferous

FIGURE 22.2. Schematic diagram of the stages of testicular development. a. This diagram shows the 5-week embryo in the stage of

indifferent gonads. The gonadal ridges visible on the posterior abdominal wall are being infiltrated by primordial germ cells ( ) that migrate from the yolk sac. Most of the developing gonad is formed by mesenchyme derived from the coelomic epithelium. The primordial germ cells become incorporated into the primary sex cords. At a later stage, under hormonal influence of testis-determining factor (TDF), the developing gonad initiates the production of testosterone. This is followed by differentiation of the primary sex cords into seminiferous cords. At the same time, the developing gonad produces Müllerianinhibiting factor (MIF), which causes regression of the paramesonephric duct and those structures derived from it. Note that the mesonephric tubules come in close contact with the developing rete testis. Final stages of testicular development. The tunica albuginea surrounding the testis contributes to the development of the testicular septa. The rete testis connects with the seminiferous cords and with the excurrent duct system that develops from the mesonephric duct and tubules.

green

b.

c.

SRY expression is responsible for the development of male sex organs in the indifferent embryo. In the first stage of development, the testes develop on the posterior abdominal wall from indifferent primordia of that are identical in both sexes. During this , an embryo has the potential to develop into either a male or female. However, expression of the SRY gene, which occurs exclusively in the pre-Sertoli cells, orchestrates male development of the embryo. Early in male development, mesenchyme separating the seminiferous cords gives rise to that produce to stimulate the development of the indifferent primordium into a testis. Testosterone is also responsible for the growth and differentiation of the mesonephric (Wolffian) ducts that develop into the male genital excurrent ducts. Also during this early stage, the that develop within the seminiferous cords produce another important hormonal substance, called . The molecular structure of MIF is similar to that of transforming growth factor-β (TGF-β). It is a large glycoprotein that inhibits cell division of the paramesonephric (Müllerian) ducts, which, in turn, inhibits the development of female reproductive organs (Fig. 22.3).

urogenital ridges indifferent stage

testosterone

cells (MIF)

Leydig (interstitial) cells

Sertoli (sustentacular) Müllerian-inhibiting factor

FIGURE 22.3. Schematic diagram of male sex development and hormonal influence on developing reproductive organs. This diagram

illustrates three levels at which the sex of the developing embryo is determined. Genetic sex is determined at the time of fertilization; gonadal sex is determined by activation of the sex-determining region Y ( ) gene located on the short arm of chromosome Y; and hormonal sex is determined by a hormone secreted by the developing gonad. The diagram shows the influence of Müllerian-inhibiting factor ( ), testosterone, and dihydrotestosterone ( ) on the developing structures. , testisdetermining factor.

SRY

DHT

MIF

TDF

Development and differentiation of the external genitalia (also from the sexually indifferent stage) occur at the same time and result from the action of , a product of the conversion of testosterone by 5α-reductase. Without DHT, regardless of the genetic or gonadal sex, the external genitalia will develop along the female template. The appearance of testosterone, MIF, and DHT is the trigger that determines the development of male sex organs in the embryo (Folder 22.1).

dihydrotestosterone (DHT)

FOLDER 22.1

FUNCTIONAL CONSIDERATIONS: HORMONAL REGULATION OF SPERMATOGENESIS

Normal function of the testis is dependent on hormones acting through endocrine and paracrine pathways. The endocrine function of the testis resides primarily in the Leydig cell population that synthesizes and secretes the principal circulating androgen, . Nearly all of the testosterone is produced by the testis; less than 5% is produced by the adrenal glands. It is estimated in humans that the total Leydig cell population produces about 7 mg of testosterone per day. As testosterone leaves the Leydig cells, it enters the bloodstream and lymphatic capillaries and crosses into the peritubular tissue to reach the seminiferous epithelium. High local levels of testosterone within the testis (estimated to be as much as 200 times the circulating levels) are necessary for the proliferation and differentiation of spermatogenic cells. This high testicular level of testosterone can be significantly decreased by negative feedback from exogenous testosterone. Intensive research in this area has focused on the development of the male testosterone-based contraceptive drugs. In early clinical studies, these drugs have been shown to cause a significant decrease in the testicular testosterone concentration and inhibition of spermatogenesis. Recovery of spermatogenesis occurs after discontinuation of contraceptive use. However, in some individuals, this type of contraceptive does not cause spermatogenic suppression.

testosterone

It also can cause significant side effects, including acne and depression. Peripheral testosterone has the following effects:

central nervous system secondary sexual characteristics accessory sex glands

Differentiation of the (CNS) and the genital apparatus and genital excurrent duct system Growth and maintenance of (such as the beard, male distribution of pubic hair, and lowpitched voice) Growth and maintenance of the (seminal vesicles and prostate and bulbourethral glands), genital excurrent duct system, and the external genitalia (mainly byproducts of testosterone conversion to dihydrotestosterone [DHT]) , including skeletal growth, skeletal muscle growth, distribution of subcutaneous fat, and kidney function , including libido

Anabolic and general metabolic processes Behavioral effects

The steroidogenic and spermatogenic activities of the testis are regulated by hormonal interaction between the hypothalamus, anterior lobe of the pituitary gland, and gonadal cells (i.e., Sertoli, spermatogenic, and Leydig cells). The anterior lobe of the pituitary gland produces three hormones involved in this process: luteinizing hormone (LH), which, in the male, is sometimes referred to as interstitial cell–stimulating hormone (ICSH); follicle-stimulating hormone (FSH); and prolactin (PRL). In response to LH release by the pituitary, Leydig cells produce increasing amounts of testosterone. PRL acts in combination with LH to increase the steroidogenic activity of Leydig cells. Because FSH and testosterone receptors are found in Sertoli cells, these cells are the primary regulators of spermatogenesis.

The testes descend from the abdomen into the scrotum along the inguinal canal at approximately 26 weeks of gestation. At approximately 26 weeks of gestation, the testes descend from the abdomen into the scrotum. This migration of the testes is caused by differential growth of the abdominal cavity combined with the action of testosterone that causes shortening of the , the testosterone-sensitive ligament connecting the inferior pole of each testis to the developing scrotum. The testes descend into the scrotum by passing through the inguinal canal, the narrow passage between the abdominal cavity and the scrotum. During descent, the testes carry their blood vessels, lymphatic vessels, and nerves as well as their principal excurrent duct system, the ductus deferens, with them. Descent of

gubernaculum

cryptorchidism

the testis is sometimes obstructed, resulting in , or . This condition is common (30%) in premature newborns and approximately 1% of full-term newborns. Cryptorchidism can lead to irreversible histologic changes in the testis and increases the risk of testicular cancer. An undescended testis requires surgical correction. (placement in the scrotal sac) should preferably be performed before histologic changes become irreversible at approximately 2 years of age.

undescended testes

Orchiopexy

Spermatogenesis requires that the testes be maintained below normal body temperature. As the testes descend from the abdominal cavity into the

scrotum, they carry blood vessels, lymphatic vessels, autonomic nerves, and an extension of the abdominal peritoneum called the , which covers their anterolateral surface. Within the scrotum, the temperature of the testes is 2°C–3°C below body temperature. This lower temperature is essential for spermatogenesis but is not required for hormone production (steroidogenesis), which can occur at normal body temperature. An increase in the testicular temperature of more than 37°C can induce testicular that alters spermatogenesis, especially the differentiation and maturation of spermatocytes and spermatids. Heat stress decreases DNA synthesis, causes DNA damage, and increases the degradation of messenger RNAs (mRNAs) and denaturation of cytoplasmic proteins in spermatocytes and spermatids, triggering their apoptosis and autophagy. Several factors that increase the temperature of the testis, such as fever, prolonged exposure to high temperatures (e.g., saunas), obesity, varicocele (enlargement of veins within the scrotum), and cryptorchidism (failure of the testis to descend into the scrotum) may lead to infertility. Each testis receives blood through a , a direct branch of the abdominal aorta. It is highly convoluted near the testis, where it is surrounded by the , which carries blood from the testis to the abdominal veins. This arrangement allows heat exchange between the blood vessels and helps maintain the testes at a lower temperature. The cooler venous blood returning from the testis

tunica vaginalis

heat stress

venous plexus

testicular artery pampiniform

cools the arterial blood before it enters the testis through a . In addition, the , whose fibers originate in the internal abdominal oblique muscle of the anterior abdominal wall, responds to changes in ambient temperature. Its contraction moves the testes closer to the abdominal wall, and its relaxation lowers the testes within the scrotum. Cold temperatures also cause contraction of a thin sheet of smooth muscle ( ) in the superficial fascia of the scrotum. Contraction of the dartos muscles causes the scrotum to wrinkle when cold to help regulate heat loss (Folder 22.2).

countercurrent heat exchange mechanism cremaster muscle

dartos muscle

FOLDER 22.2

CLINICAL CORRELATION: FACTORS AFFECTING SPERMATOGENESIS Spermatogenic cells are sensitive to noxious agents. Degenerative changes, such as apoptosis, premature sloughing of cells, or formation of multinucleated giant cells, are readily apparent after exposure to such agents. Factors that negatively affect spermatogenesis include the following:

Dietary deficiencies. Reduced dietary intake is known to impair spermatogenesis. Vitamins, coenzymes, and microelements such as vitamin A, vitamin B 12 , vitamin C, vitamin E, β-carotenes, zinc, and selenium have been shown to affect sperm formation. A recent study conducted in Denmark compared the sperm count in two groups of young men from rural and urban populations. A higher median sperm count (24%) was found in the men from the rural group compared with those from the urban group. Cryptorchidism, hypospadias, and factors such as low birth weight have been found to be important risk factors for testicular cancer associated with reduced semen quality and reduced fertility. Infections involving the testis (orchitis) may have an effect on spermatogenesis. Systemic diseases that can impair spermatogenesis include fever, kidney diseases, HIV and other viral infections, and metabolic disorders. A sedentary lifestyle may impair the ability to maintain the lower temperature in the scrotum necessary for sperm production. A higher-than-average scrotal temperature has been linked to the failure of spermatogenesis.

Environmental/lifestyle factors. Developmental disorders.

Systemic diseases or local infections. Elevated testicular temperature.

Steroid hormones and related medications. Exposure to synthetic estrogen (diethylstilbestrol) and other sex steroids can exert negative feedback on follicle-stimulating hormone (FSH) secretion, resulting in decreased spermatogenesis. Prenatal exposure to estrogens can potentially inhibit fetal gonadotropin secretion and inhibit Sertoli cell proliferation. Mutagenic agents, antimetabolites, and some pesticides, such as dibromochloropropane (DBCP), can drastically affect spermatogenesis and production of normal sperm. DBCP is a nematocide pesticide that is still used in some developing countries. It has been shown to cause a major decrease in sperm count and infertility after human exposure. Other agents that may affect fertility include chemicals in plastics (e.g., phthalates), pesticides (e.g., DDT), products of combustion (e.g., dioxins), polychlorinated biphenyls (PCBs), and others. Most of these chemicals possess weak estrogen properties and are known as . Direct toxicity to the spermatogonia is linked to changes in sperm quality. Nitrogen mustard gas and procarbazine have been found to have toxic effects on spermatogonia. and also affect sperm count and motility.

Toxic agents.

endocrine-disrupting chemicals (EDCs) Ionizing radiation and alkylating agents. Electromagnetic microwave radiation

Proliferating cells are particularly sensitive to mutagenic agents and the absence of essential metabolites. Therefore, nondividing Sertoli cells, Leydig cells, and reserve stem cells, which demonstrate low mitotic activity, are much less vulnerable than actively dividing, differentiating spermatogenic cells.

Structure of the Testis

The testes have an unusually thick connective tissue capsule, the tunica albuginea. An unusually thick, dense connective tissue capsule, the tunica albuginea, covers each testis (Fig. 22.4). The inner part of this capsule, the tunica vasculosa, is a loose connective tissue layer that contains blood and lymphatic vessels. Each testis is divided into approximately 250 lobules by incomplete connective tissue septa that project from the capsule. Along the posterior surface of the testis, the tunica albuginea thickens and projects inward as the mediastinum testis. Blood vessels, lymphatic vessels, and the genital excurrent ducts pass through the mediastinum as they enter or leave the testis.

FIGURE 22.4. Sagittal section of the human testis. a.

This schematic diagram shows a midsagittal section of the human testis. The genital duct system, which includes the tubuli recti, rete testis, efferent ducts, duct of the epididymis, and ductus deferens, is also shown. Note the thick connective tissue covering, the tunica albuginea, and the surrounding tunica vaginalis. (Modified from Dym M. In: Weiss L, ed. . 6th ed. Urban & Schwarzenberg; 1988.) Sagittal section of a hematoxylin and eosin (H&E)-stained section of the testis and the head and body of the epididymis. Again, note the surrounding tunica albuginea and tunica vaginalis. Only a small portion of the rete testis is visible in this section. Its connection with the excurrent duct system is not evident in the plane of this section. ×3.

Cell and Tissue Biology: A Textbook of Histology b.

Each lobule consists of several highly convoluted seminiferous tubules. Each lobule of the testis consists of one to four seminiferous tubules, in which sperm are produced, and a connective tissue stroma, in which testosterone-producing Leydig (interstitial) cells are contained (Fig. 22.5 and Plate 22.1, page 896). Each tubule within the lobule forms a loop and, because of its considerable length, is highly convoluted, actually folding on itself within the lobule. The ends of the loop are located near the of the testis, where they assume a short

mediastinum

straight course. This part of the seminiferous tubule is called the . It becomes continuous with the , an anastomosing channel system within the mediastinum.

straight tubule (tubulus rectus) rete testis

FIGURE 22.5. Photomicrographs of human testis. a.

This lowmagnification photomicrograph of a hematoxylin and eosin (H&E)-stained section of human testis shows seminiferous tubules and the tunica albuginea. The larger blood vessels are present in the inner aspect of the tunica albuginea. The seminiferous tubules are highly convoluted; thus, the profiles that they present in the section are variable in appearance. ×30. A higher magnification of the previous specimen shows several seminiferous tubules. Note the population of Leydig (interstitial) cells that occur in small clusters in the space between adjoining tubules. ×250.

b.

The seminiferous tubules consist of a seminiferous epithelium surrounded by a tunica propria. Each seminiferous tubule is approximately 50 cm long (range, 30 –80 cm) and 150–250 μm in diameter. The seminiferous epithelium is an unusual and complex stratified epithelium composed of two basic cell populations:

Sertoli cells, also known cells. These cells do not

supporting

sustentacular

as , or , replicate after puberty. Sertoli cells are columnar cells with extensive apical and lateral processes that surround the adjacent spermatogenic cells and occupy the spaces between them. However, this elaborate configuration of the Sertoli cells cannot be seen distinctly in routine hematoxylin and eosin (H&E) preparations. Sertoli cells give structural organization to the tubules as they extend through the full thickness of the seminiferous epithelium. , which regularly replicate and differentiate into mature sperm. These cells are derived from primordial germ cells originating in the yolk sac that colonize the gonadal ridges during the early development of the testis. Spermatogenic cells are organized in poorly defined layers of progressive development between adjacent Sertoli cells (Fig. 22.6). The most immature spermatogenic cells, called , rest on the basal lamina. The most mature cells, called , are attached to the apical portion of the Sertoli cell, where they border the lumen of the tubule.

Spermatogenic cells

spermatogonia

spermatids

FIGURE 22.6. Schematic drawing of human seminiferous epithelium.

This drawing shows the relationship of the Sertoli cells to the spermatogenic cells. The seminiferous epithelium rests on a basal lamina, and a layer of peritubular cells surrounds the seminiferous tubule. The spermatogonia—type A pale, type A dark, and type B—and preleptotene spermatocytes are located in the basal compartment of the seminiferous epithelium below the junctional complex, between adjacent Sertoli cells. Pachytene primary spermatocytes, early spermatids, and late spermatids, with partitioning residual cytoplasm that becomes the residual body, are seen above the junctional complex in the abluminal compartment. (Redrawn from Clermont Y. The cycle of the seminiferous epithelium in man. . 1963;112:35.)

Am J Anat

tunica (lamina) propria

peritubular tissue

The , also called , is a multilayered connective tissue that lacks typical fibroblasts. In humans, it consists of three to five layers of (peritubular contractile cells) and collagen fibrils, external to the basal lamina of the seminiferous epithelium (see Fig. 22.6). At the ultrastructural level, myoid cells demonstrate features associated with smooth muscle cells, including a basal lamina and large numbers of actin filaments. They also exhibit a significant amount of rough endoplasmic reticulum (rER), a feature indicating their role in collagen synthesis in the absence of typical fibroblasts. Rhythmic contractions of the myoid cells create peristaltic waves that help move spermatozoa and testicular fluid through the seminiferous tubules to the excurrent duct system. Blood vessels and extensive lymphatic vasculature as well as Leydig cells are present external to the myoid layer. As a normal consequence of aging, the tunica propria increases in thickness. This thickening is accompanied by a decreased rate of sperm production and an overall reduction in the size of the seminiferous tubules. Excessive thickening of the tunica propria earlier in life is associated with infertility.

myoid cells

Leydig Cells

Leydig cells (interstitial cells)

are large, polygonal, eosinophilic cells that typically contain lipid droplets (Fig. 22.7). Lipofuscin pigment is also frequently present in these cells as well as distinctive, rod-shaped cytoplasmic crystals, the (Fig. 22.8). In routine histologic

crystals of Reinke

preparations, these crystals are refractile and measure approximately 3 × 20 μm. Although their exact nature and function remain unknown, they probably represent a protein product of the cell.

FIGURE 22.7. Electron micrograph of Leydig cells. micrograph

shows

portions

of

several

Leydig

cells.

This electron The cytoplasm

contains an abundance of smooth endoplasmic reticulum (sER), a characteristic of Leydig cells. Other features characteristic of the Leydig cell seen in the lower power micrograph are the numerous lipid droplets ( ), the segmented profiles of the Golgi apparatus ( ), and the presence of variable numbers of lysosomes ( ). Occasional profiles of rough endoplasmic reticulum ( ) are also seen. Note also the microvilli along portions of the cell surface ( ). , cytoplasm of adjacent macrophage. ×10,000. sER at higher magnification. The very dense particles are glycogen. ×60,000.

L

rER Inset.

Ly

G

arrows M

FIGURE 22.8. Electron micrograph of a Reinke crystal.

This electron micrograph shows the internal structure of a Reinke crystal in

the cytoplasm of a human Leydig cell. Also note the smooth endoplasmic reticulum (sER) ( ) and a lipid droplet ( ) in the cytoplasm. ×16,000. (Courtesy of Dr. Don F. Cameron.)

arrows

L

Like other steroid-secreting cells, Leydig cells have an elaborate smooth endoplasmic reticulum (sER), a feature that accounts for their eosinophilia (see Fig. 22.7). The enzymes necessary for the synthesis of testosterone from cholesterol are associated with the sER. Mitochondria with tubulovesicular cristae, another characteristic of steroid-secreting cells, are also present in Leydig cells. Leydig cells differentiate and secrete during early fetal life. Secretion of testosterone is required during embryonic development, sexual maturation, and reproductive function:

testosterone

embryo

In the , secretion of testosterone and other androgens is essential for the normal development of the gonads in the male fetus. In addition to testosterone, Leydig cells secrete that stimulates the descent of the testis during development. At , secretion of testosterone is responsible for the initiation of sperm production, accessory sex gland secretion, and development of secondary sex characteristics. Secretion of INSL3 also promotes meiotic divisions in the seminiferous tubules. In the , secretion of testosterone is essential for the maintenance of spermatogenesis and secondary sex characteristics, genital excurrent ducts, and accessory sex glands. in the adult testes are the chief source of circulating . Measurement of INSL3 is utilized in clinical tests to establish the Leydig cell . In addition to the secretion of INSL3, Leydig cells produce and secrete . Testicular oxytocin stimulates contraction of myoid cells that surround the seminiferous tubules, moving the spermatozoa toward the efferent ductules.

insulin-like protein 3 (INSL3) puberty adult

Leydig cells insulin-like factor-3 (INSL3) protein steroidogenetic capacity index oxytocin

The Leydig cells are active in the early differentiation of the male fetus and then undergo a period of inactivity beginning

at about 5 months of fetal life. Inactive Leydig cells are difficult to distinguish from fibroblasts. When Leydig cells are exposed to gonadotropic stimulation at puberty, they again become androgen-secreting cells and remain active throughout life. are usually benign tumors and occur during two distinct periods (in childhood and adults between 20 and 60 years old). They are hormonally active and secrete androgens or a combination of androgens and estrogens. Commonly, they are composed of uniform cells that have all of the characteristics of steroid hormone–secreting cells, including Reinke crystals. Initial symptoms of these benign tumors, besides testicular enlargement, are usually related to an abnormal level of hormone production. In prepubertal individuals, this leads to (unexpected pubertal changes in early age), whereas in adults, it may be observed as (development of female sexual characteristics) and (development of breast in males).

Leydig cell tumors

precocity

feminization gynecomastia

sexual

SPERMATOGENESIS

Spermatogenesis is the process by which spermatogonia develop into sperm. Spermatogenesis, the process by which sperm are produced,

involves a complex and unique series of events. It begins shortly before puberty, under the influence of rising levels of pituitary gonadotropins, and continues throughout life. For descriptive purposes, spermatogenesis is divided into three distinct phases:

Spermatogonial phase, in which spermatogonia divide by mitosis to replace themselves as well as provide a population of committed spermatogonia that will eventually give rise to primary spermatocytes , in which primary spermatocytes undergo two meiotic divisions to reduce both the chromosome number and amount of DNA to produce haploid cells called

Spermatocyte phase (meiosis)

spermatids Spermatid phase (spermiogenesis), differentiate into mature sperm cells

in

which

spermatids

At the conclusion of spermatogenesis, spermatids undergo their final maturation and are released during a process called from the supporting Sertoli cells into the lumen of the seminiferous tubule.

spermiation

Spermatogonial Phase

In the spermatogonial phase, stem cells divide to replace themselves and provide a population of committed spermatogonia. Spermatogonial stem cells undergo multiple divisions and produce spermatogonia that can be differentiated into three types based on the appearance of their nuclei in routine H&E preparations:

Type A dark (Ad) spermatogonia

have ovoid nuclei with intensely basophilic, finely granular chromatin. These spermatogonia are thought to be the stem cells of the seminiferous epithelium. They divide at irregular intervals to give rise to either a pair of type Ad spermatogonia that remain as or to a pair of type Ap spermatogonia. have ovoid nuclei with lightly staining, finely granular chromatin. Ap spermatogonia are committed to the differentiation process that produces the sperm. They undergo several successive mitotic divisions, thereby increasing their number. Type Ap spermatogonia are also called . have generally spherical nuclei with chromatin that is condensed into large clumps along the nuclear envelope and around a central nucleolus (see Fig. 22.6).

reserve stem cells Type A pale (Ap) spermatogonia renewing stem cells Type B spermatogonia

An unusual feature of the division of an Ad spermatogonium into two types Ap spermatogonia is that the daughter cells remain connected by a thin cytoplasmic bridge. This same phenomenon occurs with each subsequent mitotic and meiotic division of the progeny of the original pair of Ap spermatogonia (Fig. 22.9). Thus, all of the progeny of an initial pair of Ap spermatogonia are connected, much like a strand of pearls. These cytoplasmic connections remain intact until the last stages of spermatid maturation and are essential for the synchronous development of each clone from an original pair of Ap cells.

FIGURE 22.9. Schematic diagram illustrating the generations of spermatogenic cells. This diagram shows the clonal nature of the

successive generations of spermatogenic cells. Type A dark spermatogonia are recognized as the reserve stem cells in the testis, whereas type A pale spermatogonia are the renewing stem cells. Type A pale spermatogonia undergo a series of synchronized cell divisions to either produce new type A pale cells or form more differentiated type B

spermatogonia that undergo further divisions into primary spermatocytes. Note that cytoplasmic division is complete only in the type A dark spermatogonia. All other spermatogenic cells remain connected by intercellular bridges as they undergo mitotic and meiotic division and differentiation of the spermatids. Note also that primary spermatocytes undergo meiosis I and secondary spermatocytes undergo meiosis II. The cells separate into individual spermatozoa as they are released from the seminiferous epithelium. The residual bodies remain connected and are phagocytosed by the Sertoli cells. (Based on Dym M, Fawcett DW. Further observations on the numbers of spermatogonia, spermatocytes, and spermatids connected by intercellular bridges in the mammalian testis. . 1971;4:195–215.)

Biol Reprod

After several divisions, type A spermatogonia differentiate into type B spermatogonia. The appearance of type B spermatogonia represents the last event in the spermatogonial phase.

Spermatocyte Phase (Meiosis)

In the spermatocyte phase, primary spermatocytes undergo meiosis to reduce both the chromosome number and the amount of DNA. The mitotic division of type B spermatogonia produces primary spermatocytes. They replicate their DNA shortly after they form

and before meiosis begins so that each primary spermatocyte contains the normal chromosomal number (2n). Because each chromosome consists of two sister chromatids, primary spermatocytes contain double the amount of DNA (4d). results in reduction of both the number of chromosomes (from 2n to 1n) and the amount of DNA to the haploid number (from 4d to 2d); thus, secondary spermatocytes have a haploid number of chromosomes (1n) and 2d amount of DNA. Because no DNA replication precedes , the spermatids, which are formed after this division, will also have the haploid (1n) number of chromosomes, each containing a single chromatid (1d). Meiosis is described in detail in Chapter 3, The Cell Nucleus (pages 102-103); a brief description of spermatocyte meiosis follows.

Meiosis I

meiosis II

Each spermatocyte undergoes meiosis to form four haploid spermatids.

Prophase of the first meiotic division, during which the chromatin condenses into visible chromosomes, lasts up to 22 days in human primary spermatocytes. At the end of prophase, 44 autosomes and an X and a Y chromosome, each having two chromatin strands (chromatids), can be identified. Homologous chromosomes are paired as they line up on the metaphase plate. The , called because they consist of four chromatids, exchange genetic material in a process called . During this exchange, the four chromatids rearrange into a tripartite structure called a . This process ensures genetic diversity. Through genetic exchange, the four spermatids produced from each spermatocyte differ from each other and from every other spermatid. After crossing-over is complete, the homologous chromosomes separate and move to the opposite poles of the meiotic spindle. Thus, the tetrads, which have been modified by crossing-over, separate and become dyads again. The two chromatids of each original chromosome (although modified by crossing-over) remain together. The movement of a particular chromosome of a homologous pair to either pole of the spindle is random (i.e., maternally derived chromosomes and paternally derived chromosomes do not sort themselves out at the metaphase plate). This random sorting is another source of genetic diversity in the resulting sperm. The cells derived from the first meiotic division are called . These cells immediately enter the prophase of the second meiotic division (i.e., without passing through an S phase; see pages 102103). The second meiotic division is short and lasts only several hours. Each secondary spermatocyte has a reduced number of chromosomes (1n), which is represented by 22 autosomes, and an X or a Y chromosome. Each of these chromosomes consists of two sister chromatids. The secondary spermatocyte has the (2d) diploid amount of DNA. During the metaphase of the second meiotic division, the chromosomes line up at the metaphase plate, and the sister chromatids separate and move to opposite poles of the spindle. As the second meiotic division is completed and the nuclear membranes reform, two haploid , each

paired homologous chromosomes crossing-over synaptonemal complex

secondary spermatocytes DNA

tetrads

without synthesizing new

spermatids

containing 23 single-stranded chromosomes (1n) and the 1d amount of DNA, are formed from each secondary spermatocyte (Fig. 22.10).

FIGURE 22.10. Comparison of mitosis and meiosis in spermatogenesis and oogenesis. The two pairs of chromosomes (2n) of

red

blue

maternal and paternal origin are depicted in and , respectively. Mitotic division produces daughter cells that are genetically identical to the parental (2n) cell. Meiotic division, which has two components, a reductional division and an equatorial division, produces a cell that has only half the number of chromosomes (n). In addition, during the chromosome pairing in prophase I of meiosis, chromosome segments are exchanged, a process called , creating genetic diversity. In humans, the first polar body does not divide, but it does so in other species.

crossing-over

Spermatid Phase (Spermiogenesis)

In the spermatid phase, spermatids undergo extensive cell remodeling as they differentiate into mature sperm. Each spermatid that results from the second meiotic division is haploid in DNA content (1d) and chromosome number (1n) represented by 22 autosomes and an X or Y chromosome. No further division occurs. The haploid spermatids undergo a differentiation process that produces mature sperm, which are also haploid. The 2d condition is restored when a sperm fertilizes an oocyte. The extensive cell remodeling that occurs during differentiation of the spermatid population into mature sperm (spermiogenesis) consists of four phases: the Golgi phase, cap phase, acrosome phase, and maturation phase. These phases occur while the spermatids are physically attached to the Sertoli cell’s plasma membrane by specialized junctions. The morphologic changes in all four phases that occur during spermiogenesis are described here and summarized in Figure 22.11.

FIGURE 22.11. Schematic diagram of spermiogenesis in the human.

The basic changes in the structure of the key organelles of the spermatid are illustrated (see text for detailed explanation). (Modified from Dym M. In: Weiss L, ed. . 6th ed. Urban & Schwarzenberg; 1988.)

Histology

Cell and Tissue Biology: A Textbook of

Golgi phase This phase is characterized by the presence of periodic acid– Schiff (PAS)-positive granules that accumulate in the numerous Golgi complexes of the spermatid. These ,

proacrosomal granules

rich in glycoproteins, coalesce into a membrane-bound vesicle, the , adjacent to the nuclear envelope. The vesicle enlarges and its contents increase during this phase. The position of the acrosomal vesicle determines the anterior pole of the developing sperm. Also during this phase, the centrioles migrate from the juxtanuclear region to the posterior pole of the spermatid, where the mature centriole aligns at right angles to the plasma membrane. The centriole initiates the assembly of the nine peripheral microtubule doublets and two central microtubules that constitute the of the sperm tail.

acrosomal vesicle

Cap phase

axoneme

In this phase, the acrosomal vesicle spreads over the anterior half of the nucleus. This reshaped structure is called the . The portion of the nuclear envelope beneath the acrosomal cap loses its pores and becomes thicker. The nuclear content also condenses. DNA in the spermatid is about 6 times smaller than the DNA in mitotic chromosomes. This high condensation of nuclear DNA is achieved by the presence of small, highly basic proteins called that become incorporated into the chromatin during spermiogenesis, replacing the core histones.

acrosomal cap

protamines

Acrosome phase

In this phase, the spermatid reorients itself so that the head becomes deeply embedded in the Sertoli cell and points toward the basal lamina. The developing flagellum extends into the lumen of the seminiferous tubule. The condensed nucleus of the spermatid flattens and elongates, the nucleus and its overlying acrosome also move to a position immediately adjacent to the anterior plasma membrane, and the cytoplasm is displaced posteriorly. The cytoplasmic microtubules become organized into a cylindrical sheath, the , which extends from the posterior rim of the acrosome toward the posterior pole of the spermatid. The , which had earlier initiated the development of the flagellum, now move back to the posterior surface of the nucleus where the immature centriole becomes attached to a shallow groove in the nucleus. They are then modified to form the connecting piece, or neck region, of the developing sperm. Nine coarse fibers develop from the centrioles attached to the nucleus

manchette centrioles

and extend into the tail, becoming the outer dense fibers peripheral to the microtubules of the axoneme. These fibers unite the nucleus with the flagellum; hence, the name . As the plasma membrane moves posteriorly to cover the growing , the manchette disappears, and the mitochondria migrate from the rest of the cytoplasm to form a tight, helically wrapped sheath around the coarse fibers in the neck region and its immediate posterior extension (Fig. 22.12). This region is the of the tail of the sperm. Distal to the middle piece, a consisting of two longitudinal columns and numerous connecting ribs surrounds the nine longitudinal fibers of the and extends nearly to the end of the flagellum. This short segment of the tail distal to the fibrous sheath is called the .

piece flagellum

middle piece fibrous sheath principal piece end piece

connecting

FIGURE 22.12. Diagram of a human spermatozoon. spermatozoon are indicated on the top . Key structural

Regions of the features of the

head (viewed in frontal and sagittal planes), the middle piece, and the principal piece of the spermatozoon are illustrated on the . (Modified from Pederson PL, Fawcett DW. In: Hafez ESE, ed . CV Mosby; 1976.)

and Fertility Regulation in the Male

bottom . Human Semen

Maturation phase

This last phase of spermatid remodeling reduces excess cytoplasm from around the flagella to form mature . The Sertoli cells then phagocytose this excess cytoplasm, also termed the . The intercellular bridges have characterized the developing gametes because the prespermatocyte stages remain with the residual bodies. Spermatids are no longer attached to each other and are released from the Sertoli cells.

residual body

spermatozoon

Spermatids are released into the lumen of the seminiferous tubules during the process called spermiation.

Toward the end of the maturation phase of spermatogenesis, elongated spermatids are released from Sertoli cells into the lumen of seminiferous tubule. This complex process called involves the progressive removal of specialized Sertoli-to-spermatid junctional complexes and disengagement of spermatids from the Sertoli cell. The presence of β1-integrins in the Sertoli-to-spermatid junctions, as well as increased activity of integrin-linked kinase at the time of spermiation, suggests enzymatic control of spermatid release. The rate of spermiation in the testis determines the number of sperm cells in the ejaculate of semen. Various pharmacologic treatments, toxic agents, and gonadotropin suppression result in , in which spermatids are not released but instead are retained and phagocytosed by the Sertoli cell.

spermiation

failure

Structure of the Mature Sperm

spermiation

The events of spermiogenesis result in a structurally unique cell. The mature human sperm is about 60 μm long. The sperm head is flattened and pointed and measures 4.5 μm long by 3 μm wide by 1 μm thick (see Fig. 22.12). The acrosomal cap that covers the anterior two-thirds of the nucleus contains hyaluronidase,

neuraminidase, acid phosphatase, and a trypsin-like protease called acrosin. These acrosomal enzymes are essential for

penetration of the zona pellucida of the ovum. The release of acrosomal enzymes as the sperm makes contact with the egg is the first step in the . This complex process facilitates sperm penetration and subsequent fertilization and prevents the entry of additional sperm into the ovum. The is subdivided into the neck, middle piece, principal piece, and end piece. The short neck contains the centrioles and the origin of the coarse fibers. The middle piece is approximately 7 μm long and contains mitochondria, helically wrapped around the coarse fibers and the axonemal complex. These mitochondria provide the energy for movement of the tail and thus are responsible for the motility of the sperm. The principal piece is approximately 40 μm long and contains the fibrous sheath external to the coarse fibers and the axonemal complex. The end piece, approximately the last 5 μm of the flagellum in the mature sperm, contains only the axonemal complex.

acrosome reaction

sperm tail

Newly released sperm cells are processed in the epididymis where they acquire motility and undergo further maturation. Newly released sperm cells are nonmotile and are transported from the seminiferous tubules in a fluid secreted by the Sertoli cells. The fluid and sperm flow through the seminiferous tubules, facilitated by peristaltic contractions of the peritubular contractile cells of the lamina propria. They then enter the straight tubules, a short segment of the seminiferous tubule where the epithelium consists only of Sertoli cells. At the mediastinum testis, the fluid and sperm enter the rete testis, an anastomosing system of ducts lined by simple cuboidal epithelium (Plate 22.2, page 898). From the rete testis, they move into the extratesticular portion of the efferent ductules (ductuli efferentes), the first part of the excurrent duct system, and then into the proximal portion of the duct of the epididymis (ductus epididymis). As the sperm cells move through 4–5 m of the highly coiled duct of the epididymis, they acquire motility and undergo several maturational changes. These changes include the following:

Further condensation of nuclear DNA due to a series of chromatin remodeling events, leading to replacement of histones by protamines. The head of the sperm decreases in size. Additional reduction of cytoplasm. The sperm cells become more slender. Changes in plasma membrane lipids, proteins, and glycosylation Alterations in the outer acrosomal membrane (decapacitation). The surface-associated decapacitation factor is added to inhibit the fertilizing ability of the sperm cells (page 883).

Initiation of sperm cell motility during cell transit through

the epididymis is most likely related to changes in the intracellular levels of cyclic adenosine monophosphate (cAMP), calcium ions (Ca2+ ), and intracellular pH. These factors regulate flagellar activity through changes in , resulting from activities of and . For instance, pharmacologic stimulation of activity increases motility of sperm cells, whereas inhibition of activity may initiate or stimulate such motility. This suggests that phosphatases have an important role in the regulation of . Contractions of the smooth muscle that surrounds the progressively distal and larger ducts continue to move the sperm by peristaltic action until they reach the distal portion of the duct of the epididymis, where they are stored before ejaculation. Sperm can for several weeks in the male excurrent duct system, but they will only 2–3 days in the female reproductive tract. They acquire the ability to fertilize the ovum only after some time in the female tract. This process, which involves removal and replacement of glycocalyx components (glycoconjugates) on the sperm membrane, is called . Capacitation of spermatozoa is described in detail in Chapter 23, The Female Reproductive System (pages 919-920).

protein phosphorylation protein kinases protein phosphatases protein kinase A protein phosphatase sperm kinetic activity live

survive

capacitation

SEMINIFEROUS TUBULES Cycle of the Seminiferous Epithelium

Differentiating spermatogenic cells

are not arranged at random in the seminiferous epithelium; specific cell types are grouped together. These groupings or associations occur because intercellular bridges are present between the progeny of each pair of and because the synchronized cells spend specific amounts of time in each stage of maturation. All phases of differentiation occur sequentially at any given site in a seminiferous tubule as the progeny of stem cells remain connected by cytoplasmic bridges and undergo synchronous mitotic and meiotic divisions and maturation (see Fig. 22.10). Each recognizable grouping, or , is considered a in a cyclic process. The series of stages that appears between two successive occurrences of the same cell association pattern at any given site in the seminiferous tubule constitutes a . The cycle of the seminiferous epithelium has been most thoroughly studied in rats, in which 14 successive stages occur in a linear sequence along the tubule. In humans, six stages or cell associations are defined in the cycle of the seminiferous epithelium (Fig. 22.13). These stages are not as clearly delineated as those in rodents because in humans, the cellular associations occur in irregular patches that form a mosaic pattern.

type Ap spermatogonia

stage

cell association

cycle of the seminiferous epithelium

FIGURE 22.13. Schematic drawing of the stages of the human seminiferous epithelium. This diagram shows each of the six

recognizable cell associations (stages) that occur in the cycle of the human seminiferous epithelium. These stages of spermatogenesis are artificially defined according to changes observed in the spermatids during their various steps of differentiation. In 1952, six stages of human seminiferous epithelium were first described by Leblond and Clermont and, since then, have been adopted by most researchers. Stages are labeled with Roman numerals I to VI. (Cell associations depicted in this drawing are based on Clermont Y. The cycle of the seminiferous epithelium in man. 1963;112:35–51.)

Am J Anat.

Duration of spermatogenesis in humans is approximately 74 days. After injecting a pulse of tritiated thymidine, a specific generation of cells can be followed by sequential biopsies of the seminiferous tubules. In this way, the time required for the labeled cells to go through the various stages can be determined. Several generations of developing cells may be present in the

thickness of the seminiferous epithelium at any given site and at any given time, which produces the characteristic cell associations. Autoradiographic studies have revealed that the duration of the is constant, lasting about 16 days in humans. In humans, it would require about 4.6 cycles (each 16 days long), or , for a spermatogonium produced by a stem cell to complete the process of spermatogenesis. It would then require approximately for the spermatozoon to . Approximately 300 million sperm cells are produced daily in the human testis. The length of the cycle and the time required for spermatogenesis are constant and specific in each species. Therefore, in any pharmacologic intervention (e.g., therapy for male infertility), if a drug is given that affects the initial phases of spermatogenesis, approximately are required to see the effect of that compound on sperm production.

74 days

epididymis

cycle of the seminiferous epithelium approximately

12 days

pass through the 86 days

Waves of the Seminiferous Epithelium As indicated earlier, the cycle of the seminiferous epithelium describes changes that occur with time at any given site in the tubule. In addition, the describes the distribution of patterns of cellular association ( ) along the length of the tubule. In rodents and other mammals that have been studied, including subhuman primates, each stage occupies a significant length of the seminiferous tubule, and the stages appear to occur sequentially along the length of the tubule. In the rat, there are approximately 12 waves in each tubule. A transverse section through the tubule usually reveals only one pattern of cell associations. There are no waves in the human seminiferous epithelium, and the arrangement of spermatogenic stages along the seminiferous tubule is random. Each pattern of cellular associations (spermatogenic stage) has a seminiferous tubule (Fig. 22.14). Patches do not extend around the circumference of the tubule, nor are they in sequence. Therefore, a transverse section through a human seminiferous tubule may reveal as many as six different stages of the cycle arranged in a pie-wedge manner around the circumference of the tubule.

spermatogenic stages

in the human

wave of the seminiferous epithelium

patch-like distribution

FIGURE 22.14. Diagram of organization of seminiferous epithelium in humans and other species. a. In mice and other rodent species, a specific cellular association occupies varying lengths along the tubule. Therefore, in a typical cross section, only a single cellular association is observed. In humans, cellular associations occur in irregularly shaped areas along the tubule, and therefore, a cross section typically shows two or more cellular associations.

b.

Sertoli Cells

Sertoli cells constitute the true epithelium of the seminiferous epithelium. Sertoli cells (sustentacular cells) are tall, columnar, nonreplicating epithelial cells that rest on the thick, multilayered basal lamina of the seminiferous epithelium (Fig. 22.15). They are the supporting cells for the developing spermatozoa that attach to their surface after meiosis. Sertoli cells contain an extensive sER, a well-developed rER, and stacks of annulate lamellae. They have numerous spherical and elongated mitochondria, a well-developed Golgi apparatus, and varying numbers of lysosomes, lipid droplets, vesicles, and glycogen granules.

FIGURE 22.15. Electron micrograph of a human Sertoli cell.

This electron micrograph shows characteristic crystalloid inclusion bodies of

Charcot–Böttcher in the basal cytoplasm of the Sertoli cell. The basal lamina ( ) is indicated for orientation. ×9,000. This higher magnification shows filaments of the crystalloid. ×27,000. (Courtesy of Dr. Don F. Cameron.)

BL

Inset.

The cytoskeleton of the Sertoli cell is one of the most elaborate in the human body and contains the following components:

Microtubules

that are abundant and predominately oriented parallel to the long axis of the cell. Unlike in many other cells, microtubules are nucleated at the periphery of the Sertoli cell and not from the microtubule-organizing center (MTOC). They are all oriented with their minus (−) ends toward the apex and plus (+) ends toward the base of the cell. In addition to their role in vesicular transport, recent evidence suggests that microtubules and microtubule-associated motor proteins are responsible for repositioning of the embedded elongated spermatids in the Sertoli cell’s cytoplasm. that are a major component of the Sertoli cell’s cytoskeleton and consist mainly of (class III of intermediate filament proteins). They form a perinuclear sheath that surrounds and separates the nucleus from other cytoplasmic organelles. Intermediate filaments extend from the perinuclear sheath to the desmosome-like junctions between the adjacent Sertoli cells and the hemidesmosomes. that are concentrated beneath the plasma membrane near the intercellular junctions. Actin filaments reinforce and stabilize the Sertoli intercellular junction specializations of plasma membrane.

Intermediate filaments

vimentin

Actin filaments

The euchromatic Sertoli cell nucleus, a reflection of this very active cell, is generally ovoid or triangular and may have one or more deep infoldings. Its shape and location vary. It may be flattened, lying in the basal portion of the cell near and parallel to the base of the cell, or it may be triangular or ovoid, lying near or some distance from the base of the cell. In some species, the Sertoli cell’s nucleus contains a unique tripartite structure that consists of an RNA-containing nucleolus

flanked by a pair of DNA-containing bodies called (Fig. 22.16).

karyosomes

FIGURE 22.16. Schematic drawing of the Sertoli cell and its relationship to adjacent spermatogenic cells. This drawing shows

the Sertoli-to-Sertoli junctional specialization between adjacent Sertoli cells and the Sertoli-to-spermatid junctional specialization between the Sertoli cell and late spermatids. The Sertoli-to-Sertoli junctional complex is an adhesion device that includes a tight junction that contributes to the blood–testis barrier. The junctional specialization between the Sertoli cell and late spermatids residing in deep recesses within the apical cytoplasm is an adhesion device only. Lateral processes of the Sertoli cells extend over the surface of the spermatocytes and spermatids. Note the ultrastructural features of the Sertoli cell, including the microtubule arrays and characteristic shape of the nucleus and its karyosome. , rough endoplasmic reticulum; , smooth endoplasmic reticulum. (Reprinted with permission from Bloom W, Fawcett DW. . WB Saunders; 1975.)

rER A Textbook of Histology

In

sER

inclusion bodies of Charcot–

humans, characteristic are found in the basal cytoplasm. These slender fusiform crystalloids measure 10–25 μm long by 1 μm wide and are visible in routine histologic preparations. With transmission electron microscopy, they are resolved as bundles of poorly ordered, parallel or converging, straight, dense 15-nm-diameter filaments (see Fig. 22.15). Their chemical composition and function are unknown; however, recent studies detect an accumulation of lipoprotein receptor (CLA-1) proteins. This

Böttcher

suggests that inclusion bodies may be involved in lipid transport and their utilization by the Sertoli cells.

The Sertoli cell-to-Sertoli cell junctional complex consists of a structurally unique combination of membrane and cytoplasmic specializations. Sertoli cells are bound to one another by an unusual Sertoli cell-to-Sertoli cell junctional complex (Fig. 22.17). This complex is characterized, in part, by an exceedingly tight junction (zonula occludens) that includes more than 50 parallel fusion lines in the adjacent membranes. In addition, two cytoplasmic components characterize this unique junctional complex:

FIGURE 22.17. Electron micrograph of Sertoli cell junctions.

This electron micrograph demonstrates a Sertoli-to-Sertoli junctional complex and, in close proximity, a Sertoli-to-spermatid junctional specialization. Condensation and shaping of the spermatid nucleus ( ) are well advanced. The acrosome ( ) of the spermatid appears as a Vshaped profile, and in close association with it is the Sertoli cell

A

N

junctional specialization characterized by bundles of microfilaments that are cut in cross section ( ). The associated profile of endoplasmic reticulum resides immediately adjacent to the microfilament bundles. The Sertoli-to-Sertoli junction lies below, joining one Sertoli cell ( 1) to the adjacent Sertoli cell ( 2). The indicate the limits of the junction. Note that the junction here reveals the same elements, the microfilament bundles ( ) and a profile of endoplasmic reticulum, as are seen in the Sertoli-to-spermatid junctional specialization. Not evident at this magnification is the tight junction associated with the Sertoli-to-Sertoli junctional complex. ×30,000.

arrows

S

S

arrows

arrowheads

flattened cisterna of sER Actin filament bundles

A lies parallel to the plasma membrane in the region of the junction in each cell. , hexagonally packed, are interposed between the sER cisternae and the plasma membranes. A similar-appearing junctional complex in the Sertoli cell is also present at the site where the spermatids are attached. However, no tight junction is present, and the spermatid lacks flattened cisternae of sER and actin filament bundles (see Figs. 22.16 and 22.17). Other junctional specializations of the Sertoli cells include gap junctions between Sertoli cells, desmosome-like junctions between Sertoli cells and early-stage spermatogenic cells, and hemidesmosomes at the Sertoli cell–basal lamina interface.

The Sertoli cell-to-Sertoli cell junctional complex divides the seminiferous epithelium into basal and luminal compartments, segregating postmeiotic germ cell development and differentiation from the systemic circulation. The Sertoli cell-to-Sertoli cell junctions establish two epithelial compartments, a basal epithelial compartment and a luminal compartment (see Fig. 22.16). Spermatogonia and early primary spermatocytes are restricted to the basal compartment (i.e., between the Sertoli cell-to-Sertoli cell junctions and the basal lamina). More mature spermatocytes and spermatids are restricted to the luminal side of the Sertoli cell-to-Sertoli cell junctions. produced by mitotic division of type B spermatogonia to move from the basal compartment to the luminal compartment. This movement occurs via the formation of a new junctional

Early spermatocytes must pass through the junctional complex

complex between Sertoli cell processes that extends beneath the newly formed spermatocytes, followed by the breakdown of the junction above them. Thus, in the differentiation of the spermatogenic cells, meiosis and spermiogenesis take place in the luminal compartment. In both compartments, spermatogenic cells are surrounded by complex processes of the Sertoli cells. Because of the unusually close relationships between Sertoli cells and differentiating spermatogenic cells, it has been suggested that Sertoli cells serve as “nurse,” or supporting, cells (i.e., they function in the exchange of metabolic substrates and wastes between the developing spermatogenic cells and the circulatory system). In addition, Sertoli cells phagocytose and break down the residual bodies formed in the last stage of spermiogenesis. They also phagocytose any spermatogenic cells that fail to differentiate completely.

The Sertoli cell-to-Sertoli cell junctional complex is the site of the blood–testis barrier. In addition to the physical compartmentalization described earlier, the Sertoli cell-to-Sertoli cell junctional complex also creates a permeability barrier called the . This barrier is essential in creating a physiologic compartmentalization within the seminiferous epithelium with respect to ionic, amino acid, carbohydrate, and protein composition. Therefore, the composition of the fluid in the seminiferous tubules and excurrent ducts differs considerably from the composition of the blood plasma and testicular lymph. Plasma proteins and circulating antibodies are excluded from the lumen of the seminiferous tubules. The exocrine secretory products of the Sertoli cells (particularly , which has a high binding affinity for testosterone and DHT) are highly concentrated in the lumen of the seminiferous tubules and maintain a high concentration of testosterone, which provides a favorable microenvironment for the differentiating spermatogenic cells. Most importantly, the blood–testis barrier isolates the genetically different and, therefore, antigenic haploid germ cells (secondary spermatocytes, spermatids, and sperm) from the

blood–testis barrier

protein [ABP]

androgen-binding

immune system of the adult male. Antigens produced by, or specific to, the sperm are prevented from reaching the systemic circulation. Conversely, γ-globulins and specific sperm antibodies found in some individuals are prevented from reaching the developing spermatogenic cells in the seminiferous tubule (Folder 22.3). Therefore, the serves an essential role in isolating the spermatogenic cells from the immune system.

blood–testis barrier

FOLDER 22.3

CLINICAL CORRELATION: SPERM-SPECIFIC ANTIGENS AND THE IMMUNE RESPONSE Two basic facts are well established about the immunologic importance of the blood–testis barrier:

Spermatozoa and spermatogenic cells possess molecules that are unique to these cells and are recognized as “foreign” (not self) by the immune system. Spermatozoa are first produced at puberty, long after the individual has become immunocompetent (i.e., capable of recognizing foreign molecules and producing antibodies against them).

Failure of the spermatogenic cells and spermatozoa to remain isolated results in the production of sperm-specific antibodies. Such an immune response is sometimes seen after and in some cases of . After vasectomy, sperm-specific antibodies are produced as the cells of the immune system are exposed to the spermatozoa that may leak from the severed ductus deferens. Thus, sperm no longer remain isolated from the immune system within the reproductive tract. In some cases of infertility, sperm-specific antibodies have been found in the semen. These antibodies cause the sperm to agglutinate, preventing movement and interaction with the ovum.

infertility

vasectomy

Sertoli cells have both exocrine and endocrine secretory functions. In addition to secreting fluid that facilitates passage of the maturing sperm along the seminiferous tubules to the intratesticular ducts, produce critical factors necessary for the successful progression of spermatogonia into spermatozoa. They secrete a 90-kDa . ABP concentrates testosterone in the luminal compartment

Sertoli cells

(ABP)

androgen-binding protein

of the seminiferous tubule, where high concentrations of testosterone are essential for the normal maturation of the developing sperm. Follicle-stimulating hormone (FSH) receptors and testosterone receptors are present in Sertoli cells; therefore, their secretory function is regulated by both FSH and testosterone (Fig. 22.18). Sertoli cells secrete several endocrine substances, such as , a 32-kDa glycoprotein hormone involved in the feedback loop that inhibits FSH release from the anterior pituitary gland. In addition, Sertoli cells synthesize , which converts plasminogen to the active proteolytic hormone plasmin, (an iron-transporting protein), and (a copper-transporting protein). Furthermore, the Sertoli cells secrete other glycoproteins that function as growth factors or paracrine factors, such as MIF, stem cell factor (SCF), and glial cell line–derived neurotrophic factor (GDNF).

inhibin

plasminogen activator transferrin ceruloplasmin

FIGURE 22.18. Diagram depicting the hormonal regulation of male reproductive function. Blue arrows indicate stimulatory action on the system; red arrows indicate inhibitory feedback. See text for explanation. CNS , central nervous system; DHT , dihydrotestosterone; FSH , follicle-stimulating hormone; GnRH , gonadotropin-releasing hormone; LH , luteinizing hormone.

INTRATESTICULAR DUCTS

At the end of each seminiferous tubule, there is an abrupt transition to the , or . This short terminal section of the seminiferous tubule is lined only by Sertoli cells (Plate 22.2, page 898). Near their termination, the straight tubules narrow, and their lining changes to a simple cuboidal epithelium. The straight tubules empty into the , a complex series of interconnecting channels within the highly vascular connective tissue of the mediastinum (Fig. 22.19). A simple cuboidal or low columnar epithelium lines the channels of the rete testis. These cells have a single apical cilium and relatively few short apical microvilli.

straight tubules

tubuli recti

rete testis

FIGURE 22.19. Photomicrograph of human testis. a. This hematoxylin and eosin (H&E)-stained specimen shows the site that includes the mediastinum of the testis. On the right are seminiferous tubules, and on the left are the anastomosing channels of the rete testis. The arrow

indicates termination of a straight tubule that is lined only by Sertoli cells. It is at this site that the tubule contents enter the rete testis and the channels are then lined by a simple cuboidal epithelium. ×70. This higher magnification from a slightly deeper section of the same specimen shows the rete testis ( ), a cross section of a seminiferous tubule ( ), and a terminating straight tubule ( ) where it is entering the rete testis. Note the abrupt change in the epithelial lining at this site. As noted, the lining epithelium of the rete testis is simple cuboidal. ×275.

b.

upper right

left

asterisk

EXCURRENT DUCT SYSTEM

The excurrent duct system develops from the mesonephric (Wolffian) duct and mesonephric tubules. The initial development of Leydig cells and initiation of testosterone secretion stimulate the mesonephric (Wolffian) duct to differentiate into the excretory duct system for the developing testis (Fig. 22.20). The portion of the mesonephric duct adjacent to the developing testis becomes convoluted and differentiates into the . In addition, a number (about 20) of the remaining mesonephric tubules in this region make contact with the developing seminiferous cords and finally develop into the (Fig. 22.21 and Plate 22.3, page 900). They connect the developing rete testis with the duct of the epididymis. The distal part of the mesonephric duct acquires a thick, smooth muscle coat and becomes the . The end of the distal mesonephric duct gives rise to the and .

duct of the epididymis efferent ductules

deferens ejaculatory duct

seminal vesicles

ductus

FIGURE 22.20. Schematic diagram of development of intratesticular and excurrent duct systems. a. This diagram shows the testis in the

seventh week of development before it descends into the scrotal sac.

Note that the mesonephric duct and its tubules give rise to the excurrent duct system for the developing testis. Sagittal section of a fully developed testis positioned within the scrotum. Note that the seminal vesicles, ejaculatory ducts, ductus deferens, and epididymis are all developed from the mesonephric duct. Efferent ductules originated from mesonephric tubules. The seminiferous tubules, straight tubules, and rete testis develop from the indifferent gonads. The prostate gland develops from the multiple outgrowths that originate from the pelvic urethra (a urogenital sinus derivative).

b.

FIGURE 22.21. Photomicrograph of efferent ductules.

The specimen in this photomicrograph was stained with picric acid and hematoxylin to better visualize the epithelial components of the efferent ductules. The efferent ductules are lined by pseudostratified columnar epithelium. The luminal surface has an uneven or wavy appearance because of the presence of alternating groups of tall columnar cells and cuboidal cells. The ductules are surrounded by several layers of circularly arranged smooth

SM asterisks

muscle ( ). Within the ductule lumina are clumped spermatozoa ( ). Connective tissue ( ) makes up the stroma of the organ and contains blood vessels ( ) of various sizes. ×120. This higher magnification of the pseudostratified epithelium shows columnar and cuboidal cells that contain sparse cilia. ×500.

BV

CT

Inset.

The efferent ductules are lined with pseudostratified columnar epithelium. In humans, approximately 20 efferent ductules connect the channels of the rete testis at the superior end of the mediastinum to the proximal portion of the duct of the epididymis. As the efferent ductules exit the testis, they become highly coiled and form 6–10 conical masses, the coni vasculosi, whose bases form part of the head of the epididymis.

The coni vasculosi, each about 10 mm in length, contain highly convoluted ducts that measure 15–20 cm in length. At the base of the cones, the efferent ductules open into a single channel, the duct of the epididymis (see Fig. 22.4). The efferent ductules are lined with a pseudostratified columnar epithelium that contains clumps of tall and short cells, giving the luminal surface a saw-tooth appearance (see Fig. 22.21). Interspersed among the columnar cells are occasional basal cells that serve as epithelial stem cells. The tall columnar cells are ciliated. The short nonciliated cells have numerous microvilli and canalicular invaginations of the apical surface as well as numerous pinocytotic vesicles, membrane-bound dense bodies, lysosomes, and other cytoplasmic structures associated with endocytic activity. Most of the fluid secreted in the seminiferous tubules is reabsorbed in the efferent ductules. A in the excurrent ducts first appears at the beginning of the efferent ductules. The smooth muscle cells form a layer of several cells thick in which the cells are arrayed as a circular sheath in the wall of the ductule. Interspersed among the muscle cells are elastic fibers. Transport of the sperm in the efferent ductules is affected largely by both ciliary action and contraction of this fibromuscular layer.

smooth muscle layer

Epididymis

The epididymis is an organ that contains the efferent ductules and the duct of the epididymis. The epididymis is a crescent-shaped structure that lies along the superior and posterior surfaces of the testis. It measures about 7.5 cm in length and consists of the efferent ductules and the duct of the epididymis and associated vessels, smooth

muscles, and connective tissue coverings (Fig. 22.22 and Plate 22.3, page 900). The duct of the epididymis is a highly coiled tube measuring 4–6 m in length. The epididymis is divided into a , a , and a (see Fig. 22.4). The efferent ductules occupy the head, and the duct of the epididymis occupies the body and tail. Newly produced sperm, which enter the epididymis from the testis, mature during their passage through the duct of the epididymis, acquiring motility and the ability to fertilize an oocyte. During this androgen-dependent maturation process, the head of the sperm is modified by the addition of containing epididymal fluid glycoconjugates. This process, called , inhibits the fertilizing ability of the sperm in a reversible manner. The surface-associated decapacitation factor is later released during that occurs in the female reproductive tract just before fertilization. After maturation in the epididymis, sperm can transport their haploid content of DNA to the ovum, and after capacitation, they can bind to sperm receptors on the zona pellucida of the ovum. This binding triggers the acrosome reaction in which the sperm uses its acrosomal enzymes to penetrate the outer covering of the oocyte.

head

body

tail

associated decapacitation factor capacitation

decapacitation

surface-

FIGURE 22.22. Photomicrograph of human epididymis.

This photomicrograph of a hematoxylin and eosin (H&E)-stained section shows the highly coiled ductus epididymis. Its coiled nature is reflected in the variously shaped profiles of the duct. Within the connective tissue are numerous profiles of blood vessels ( ). The vessels tend to follow the duct; thus, they too reflect multiple profiles of several vessels.

BV

The section of the duct within the magnification in Figure 22.23. ×30.

rectangle

is

shown

at

higher

The principal cells in the pseudostratified epithelium of the epididymis are characterized by stereocilia. Like most of the excurrent duct system, the duct of the epididymis is also lined with a pseudostratified columnar epithelium (Fig. 22.23). In general, it contains two types of cells:

FIGURE 22.23. Photomicrograph of human ductus epididymis. This higher magnification of the rectangular area in Figure 22.22 reveals the two cell types of the epididymal epithelium, the principal cells and the basal cells. Stereocilia ( arrows ) extend from the apical surface of the

principal cells. The nuclei of the basal cells are spherical and are located in close proximity to the basement membrane, whereas the nuclei of the principal cells are cylindrical and conform to the columnar shape

of the cell. Surrounding the duct epithelium is a layer of circularly arranged smooth muscle cells. The duct lumen contains numerous sperm. ×250.

Principal cells

that vary from approximately 80 μm in height in the head of the epididymis to approximately 40 μm in height in the tail. Numerous long, modified microvilli called extend from the luminal surface of the principal cells (Plate 22.3, page 900). The stereocilia vary in height from 25 μm in the head to approximately 10 μm in the tail. that represent small, round cells resting on the basal lamina. They are the stem cells of the duct epithelium.

stereocilia

Basal cells

migrating lymphocytes

halo cells

In addition, called are often found within the epithelium. Under normal conditions, the epithelium of the epididymis represents the most proximal level of the excurrent duct system in which lymphocytes are present.

Epididymal cells function in both absorption and secretion.

Most of the fluid that is not reabsorbed by the efferent ductules is reabsorbed in the proximal portion of the epididymis. The epithelial cells also phagocytose any residual bodies not removed by the Sertoli cells as well as sperm that degenerate in the duct. The apical cytoplasm of the principal cells contains numerous invaginations at the bases of the stereocilia, along with coated vesicles, multivesicular bodies, and lysosomes (Fig. 22.24).

FIGURE 22.24. Electron micrograph of epididymis. a. Electron micrograph of the epididymal epithelium showing principal cells ( PC ) extending to the lumen and a basal cell ( BC ) limited to the basal portion of the epithelium. Profiles of sperm ( S ) are seen in the lumen. The apical cytoplasm of the principal cells exhibits numerous long microvilli (stereocilia). ×3,000. b. Apical surface of the epithelial cell with its numerous long microvilli (stereocilia). The middle piece of a sperm ( S ) is evident in the lumen. The small, light circular profiles ( arrowheads ) are endocytotic vesicles. ×13,000. The principal cells secrete glycerophosphocholine, sialic acid, and glycoproteins, which, in addition to the glycocalyx and steroids, aid in the . They have numerous cisternae of rER surrounding the basally located nucleus and a remarkably large supranuclear Golgi apparatus. Profiles of sER and rER are also present in the apical cytoplasm.

maturation of the sperm

The smooth muscle coat of the duct of the epididymis gradually increases in thickness to become three layered in the tail.

In the head of the epididymis and most of the body, the smooth muscle coat consists of a thin layer of circular smooth muscle, resembling that of the efferent ductules. In the tail, inner and outer longitudinal layers are added. These three layers are then continuous with the three smooth muscle layers of the ductus

deferens, the next component of the excurrent duct system (Plate 22.4, page 902). Differences in smooth muscle function parallel these morphologic differences. In the head and body of the epididymis, spontaneous, rhythmic peristaltic contractions serve to move the sperm along the duct. Few peristaltic contractions occur in the tail of the epididymis, which serves as the principal reservoir for mature sperm. These sperm are forced into the ductus deferens by intense contractions of the three smooth muscle layers after appropriate neural stimulation associated with ejaculation.

Ductus Deferens

The ductus deferens is the longest part of the excurrent duct system. The ductus deferens (vas deferens) is a direct continuation of the tail of the epididymis (see Fig. 22.1). In the scrotum, the ductus deferens ascends along the posterior border of the testis, close to the testicular vessels and nerves. It then enters the abdomen as a component of the spermatic cord by passing through the inguinal canal. The contains all of the structures that pass to and from the testis. In addition to the ductus deferens, the spermatic cord contains the testicular artery, small arteries to the ductus deferens and cremaster muscle, the pampiniform plexus, lymphatic vessels, sympathetic nerve fibers, and the genital branch of the genitofemoral nerve. All of these structures are surrounded by fascial coverings derived from the anterior abdominal wall. After leaving the spermatic cord, the ductus deferens descends into the pelvis to the level of the bladder, where its distal end enlarges to form the . The ampulla is joined there by the and continues through the prostate gland to the prostatic urethra as the . The ductus deferens is lined with a pseudostratified columnar epithelium that closely resembles that of the epididymis (Plate 22.4, page 902). The tall columnar cells also have stereocilia that extend into the lumen. The rounded basal cells rest on the basal lamina. Unlike the epididymis, however, the lumen of the duct does not appear smooth. In histologic preparations (Fig.

spermatic cord

ampulla of ductus deferens duct of the seminal vesicle

ejaculatory duct

22.25), it appears to be thrown into deep longitudinal folds throughout most of its length, probably because of the contraction of the thick (1–1.5 mm) muscular coat of the duct during fixation.

FIGURE 22.25. Photomicrograph of human spermatic cord. a.

This low-magnification photomicrograph shows a cross section of the spermatic cord containing several structures. These include the ductus deferens, the accompanying testicular artery and vein, and veins of the pampiniform plexus. ×15. A higher magnification of a pampiniform vein. Note the bundles of longitudinal smooth muscles (cut in cross section) in the tunica adventitia and tunica intima. ×55. This cross section of the ductus deferens shows the thick muscular wall organized in three distinct smooth muscle layers: an inner longitudinal ( ), middle circular ( ), and outer longitudinal ( ). ×100. A higher magnification shows the pseudostratified epithelium lining the ductus deferens. The tall principal cells possess long microvilli (stereocilia; ). The basal cells are in close proximity to the basement membrane and possess spherical nuclei. ×215.

Inset.

SM[C]

b.

SM[L]

SM[L] Inset.

arrows

vas deferens

As the passes through the spermatic cord, it can be easily palpable during physical examination of the scrotum. The easy access to this structure is advantageous during , a male option for sterilization. Vasectomy is a safe and relatively simple method of sterilization and has lower rates of morbidity and mortality than female sterilization (uterine

vasectomy

tube occlusion). During a vasectomy, small incisions in the skin of the scrotum are made and the vas deferens from each testis is exposed, clamped, and severed, preventing passage of the sperm cells into the ejaculatory duct and prostatic urethra. Following vasectomy, both testes continue to produce sperm cells. In 10%– 30% of vasectomy cases, increased intraluminal pressure causes distension of the epididymis and pressure-mediated damage to the seminiferous epithelium, followed by formation at the vasectomy site, epididymis, or rete testis. A sperm granuloma is a site of spermatozoa phagocytosis by large, activated macrophages. Degradation products of macrophages are absorbed by the epididymal epithelium. Sperm granulomas are rarely symptomatic. In addition, owing to the constant leak of spermatozoal antigens into the tissues, mechanisms of humoral immunity may be activated, resulting in the production of . Although these antibodies develop in a significant proportion of patients postvasectomy, they do not increase the risk of immune complex or atherosclerotic heart disease. About 500,000 vasectomies are performed each year in the United States, with up to 6% of patients requesting in the form of vasovasostomy or vasoepididymostomy for various reasons. Research indicates that after vasectomy reversal microsurgery, the patency of vas deference can be achieved at about 89% and successful pregnancy rates follow at approximately 73%. The has taller, branched mucosal folds that often show glandular diverticula. The muscle coat surrounding the ampulla is thinner than that of the rest of the ductus deferens, and the longitudinal layers disappear near the origin of the ejaculatory duct. The epithelium of the ampulla and ejaculatory duct appears to have a secretory function. The cells contain large numbers of yellow pigment granules. The wall of the ejaculatory duct does not have a muscularis layer; the fibromuscular tissue of the prostate substitutes for it.

sperm granuloma

antisperm antibodies reversal

IgA

vasectomy

ampulla of ductus deferens

ACCESSORY SEX GLANDS

The paired seminal vesicles secrete a fluid rich in fructose.

seminal vesicles

The are paired, elongated, and highly folded tubular glands located on the posterior wall of the urinary bladder, parallel to the ampulla of the ductus deferens. A short excretory duct from each seminal vesicle combines with the ampulla of the ductus deferens to form the . Seminal vesicles develop as evaginations of the mesonephric (Wolffian) ducts in the region of future ampullae. The wall of the seminal vesicles contains a mucosa, a thin layer of smooth muscle, and a fibrous coat (Fig. 22.26). The mucosa is thrown into numerous primary, secondary, and tertiary folds that increase the secretory surface area (Plate 22.6, page 906). All of the irregular spaces thus formed, however, communicate with the lumen.

ejaculatory duct

FIGURE 22.26. Photomicrograph of human seminal vesicle. a.

This low-magnification photomicrograph shows part of a hematoxylin and eosin (H&E)-stained section of a human seminal vesicle. This gland is a tortuous tubular structure and in a section exhibits what appear to be a number of isolated lumina. In actuality, there is only one lumen. The mucosa is characterized by extensive folding ( ). It rests on a thick smooth muscle ( ) investment that is organized in two layers: an inner circular layer and an outer longitudinal layer. ×20. This higher magnification shows the mucosal folds surfaced by a pseudostratified epithelium. indicate the basal cells. ×500.

arrows

SM

Arrows

b.

The pseudostratified columnar epithelium contains tall, nonciliated columnar cells and short, round cells that rest on the basal lamina. The short cells appear identical to those of the rest of the excurrent duct system. They are the stem cells from which the columnar cells are derived. The columnar cells have the morphology of protein-secreting cells, with a welldeveloped rER and large secretory vacuoles in the apical cytoplasm. The is a whitish yellow, viscous material. It contains fructose, which is the principal metabolic substrate for sperm, along with other simple sugars, amino acids, ascorbic acid, and prostaglandins. Although prostaglandins were first isolated from the prostate gland (hence the name), they are actually synthesized in large amounts in the seminal vesicles. Contraction of the smooth muscle coat of the seminal vesicles during ejaculation discharges their secretion into the ejaculatory ducts and helps to flush sperm out of the urethra. The secretory function and morphology of the seminal vesicles are under the control of testosterone.

secretion of the seminal vesicles

PROSTATE GLAND

The prostate, the largest accessory sex gland, is divided into several morphologic and functional zones. The prostate is the largest accessory sex gland of the male

reproductive system. Its size and shape are commonly compared to those of a walnut. The main function of the prostate gland is to secrete a clear, slightly alkaline (pH 7.29) fluid that contributes to the formation of seminal fluid. The gland is located in the pelvis, inferior to the bladder, where it surrounds the prostatic part of the urethra. It consists of 30– 50 tubuloalveolar glands arranged in three concentric layers: an inner , an intermediate submucosal layer, and a peripheral layer containing the main prostatic glands (Fig. 22.27). The glands of the mucosal layer secrete directly into the urethra; the other two layers have ducts that open into the prostatic sinuses located on either side of the urethral crest on the posterior wall of the urethra.

mucosal layer

FIGURE 22.27. Schematic drawing of the zones of the human prostate gland. This drawing illustrates the relative location, by color, of the four zones of fibromuscular stroma of the gland.

the

prostate

adult prostatic parenchyma

The is anatomically and clinically distinct zones:

central zone

gland

divided

and

anterior

into

four

The surrounds the ejaculatory ducts as they pierce the prostate gland. This zone comprises approximately 25% of the glandular tissue of the prostate gland and is resistant to both carcinoma and inflammation. In comparison to the other zones, cells in the central zone have distinctive morphologic features (a more prominent and slightly basophilic cytoplasm and larger nuclei displaced at different levels in adjacent cells). Recent findings suggest that this zone originates embryologically from the inclusion of mesonephric duct cells into the developing prostate. The comprises 70% of the glandular tissue of the prostate. It surrounds the central zone and occupies the

peripheral zone

prostatic

posterior and lateral parts of the gland. Most arise from the of the prostate gland. The peripheral zone is palpable during digital examination of the rectum. This zone is also the most susceptible to inflammation. The surrounds the prostatic urethra; it comprises about 5% of the prostatic glandular tissue and contains the mucosal glands. In older individuals, the parenchymal cells of this zone frequently undergo extensive division (hyperplasia) and form nodular masses of epithelial cells. Because the is proximate to the prostatic urethra, these nodules can compress the prostatic urethra, causing difficult urination. This condition is known as and is discussed in Folder 22.4 (pages 887–888).

carcinomas

peripheral zone

transitional zone

transitional zone

benign prostatic hyperplasia (BPH)

FOLDER 22.4

CLINICAL CORRELATION: BENIGN PROSTATIC HYPERTROPHY AND CANCER OF THE PROSTATE Benign prostatic hypertrophy (nodular hyperplasia, BPH) occurs almost exclusively in the transitional and periurethral zones, leading to partial or total obstruction of the urethra (Fig. F22.4.1a). A widely accepted theory of the pathogenesis of BPH is related to the action of dihydrotestosterone (DHT). DHT is synthesized in the stromal cells by conversion from circulating testosterone in the presence of 5α-reductase. Once synthesized, DHT acts as an autocrine agent on the stromal cells and as a paracrine hormone on the glandular epithelial cells, causing them to proliferate (Fig. F22.4.1b). BHP is believed to occur to some extent in all male individuals by age 80.

FIGURE F22.4.1. Benign prostatic hyperplasia (BPH). a.

This photograph shows a cross section of the prostate gland surgically removed from an individual with BPH. The cut section shows an enlarged transitional zone with numerous well-defined hyperplastic nodules that compress the prostatic urethra. Note the normal appearance of tissue in the peripheral zone. (Courtesy of Jodi L. Hilderbrand, PA.) Photomicrograph of prostatic glands shows the hypertrophy of epithelium lining the glands. Note that the cells form folds that protrude into the lumen of the glands. ×200. (Reprinted with permission from Rubin E, Gorstein F, Schwarting R, et al. . 4th ed. Lippincott Williams & Wilkins; 2004; Fig. 17–40.)

b.

Rubin’s Pathology

Noninvasive

treatment

Several options are available to treat BHP. includes medications (α-receptor blockers) to relax the prostate smooth muscles and relieve pressure on the compressed urethra. Clinical trials have shown that inhibitors of 5αreductase reduce the DHT concentration and thus decrease the size of the prostate and reduce urethral obstruction. options use laser, microwave, or radiofrequency energy to destroy the prostate tissue causing urethral obstruction. These include interstitial laser coagulation (ILC), microwave hyperthermia, and transurethral needle ablation (TUNA). Finally, a variety of are used to

Minimally invasive treatment

surgical procedures

remove hypertrophied regions of the prostate gland. They include transurethral incision of the prostate (TUIP), a more extensive transurethral resection of the prostate (TURP), and most recently, a modification of the TURP procedure that uses laser energy to vaporize the prostate tissue called Greenlight PVP. is one of the most common cancers in the male: The lifetime risk of developing prostate cancer is 16.7% (one in six men). The incidence of prostatic cancer increases with age, and it is estimated that 70% of men between the ages of 70 and 80 years will develop this disease. Tumors usually develop in the peripheral zone of the gland. In the past, early detection was uncommon because the abnormal growth of the tumor did not impinge on the urethra to produce symptoms that demanded prompt attention. Therefore, prostatic cancer was often inoperable by the time it was discovered. In the late 1980s, universal screening for prostate-specific antigen (PSA) for prostate cancer was introduced. Its use with annual digital rectal examination in prostate cancer screening programs has significantly increased the early detection of the disease.

Cancer of the prostate

Screening with PSA to detect prostate cancer is debatable . Recently, large epidemiologic studies revealed that the proportion of men who are diagnosed with prostate cancer but

never develop associated clinical problems may range from 23% to 66%. The current view is that the value of screening for prostate cancer in most cases is low because in most cases, the chances of harm from screening (repeated testing, aggressive therapy, and patient anxiety) outweigh the chances of benefit. Therefore, screening for prostate cancer using the PSA test is among health professionals and organizations who publish screening guidelines and recommendations for health care professionals. The most common prostate cancer grading system known as the is used to predict tumor behavior and patient survival rate. Tissue obtained from two biopsies from the largest areas of prostate cancer is evaluated and grades ranging from 1 to 5 are assigned. A ranking of 1 indicates well-differentiated cells, which form the slowest growing and the least aggressive form of cancer. The ranking 5 is given to poorly differentiated cells characteristic of the fastest growing cancers. These grades, when added together, represent a Gleason score or sum between 2 and 10. The higher the score, the more likely the cancer will grow and spread rapidly. Treatment of the cancer is by surgery, radiotherapy, or both for patients with localized disease. Hormonal therapy is the treatment of choice for advanced cancer with metastases. Because prostatic cancer cells depend on androgens, the goal of therapy is to deprive the cells of testosterone by performing orchiectomy (removal of the testis) or by administration of estrogens or gonadotropin-releasing hormone (GnRH) agonists to suppress

regarded as controversial Gleason score

currently

testosterone production. Despite treatment, patients with metastasis have a poor prognosis.

periurethral zone

The contains mucosal and submucosal glands. In the later stages of BPH, this zone may undergo pathologic growth but mainly from the stromal components. Together with the glandular nodules of the transitional zone, this growth causes increased and further retention of urine in the bladder.

urethral compression

fibromuscular stroma

In addition, occupies the anterior surface of the prostate gland anterior to the urethra and is composed of dense irregular connective tissue with a large amount of smooth muscle fibers.

The growth of the prostatic glandular epithelium is regulated by the hormone dihydrotestosterone. Within each prostate zone, the glandular epithelium is generally

simple columnar, but there may be patches that are simple cuboidal, squamous, or occasionally pseudostratified (Fig. 22.28). The alveoli of the prostatic glands, especially those in older individuals, often contain of varied shape and size, often up to 2 mm in diameter (see Fig. 22.28 and Plate 22.5, page 904). They appear in sections as concentric lamellated bodies and are believed to be formed by precipitation of secretory material around cell fragments. They may become partially calcified.

amylacea)

prostatic concretions (corpora

FIGURE 22.28. Photomicrograph of human prostate gland. a. This Mallory-Azan–stained specimen shows the tubuloalveolar glands ( Gl ) and

the fibromuscular tissue that forms the septa between glandular tissues. Within the lumina, various-sized prostatic concretions can be seen. The stain used for this specimen readily distinguishes the smooth muscle component (stained ) from the dense connective tissue component (stained ) of the stroma. ×60. This higher magnification shows an area where the glandular epithelium is pseudostratified. The round nuclei adjacent to the connective tissue ( ) belong to the basal cells. Those nuclei that are more elongated and further removed from the base of the epithelium belong to the secretory cells. Note the terminal bars ( ) that are evident at the apical region of these cells. The red-stained sites within the dense connective tissue represent smooth muscle cells. ×635.

blue

red

b.

arrowheads

arrows

glandular epithelium

The is influenced by sex hormones, such as testosterone and adrenal androgens. These hormones enter the secretory cells of the glandular epithelium and are converted to by the enzyme . DHT is approximately 30 times more potent than testosterone. Binding of DHT to the results in a conformational

dihydrotestosterone (DHT) androgen receptor (AR)

5α-reductase

change of the receptor and its relocation from the cytosol to the cell nucleus. Here, the phosphorylated dimers of the AR complex bind to a specific sequence of DNA known as a residing in the promoter regions of target genes. The primary function of AR is direct upregulation or downregulation of specific genes. DHT stimulation is a factor in both proliferation and growth of BPH and androgen-dependent prostate cancer.

hormone-response

element

The prostate gland secretes prostatic acid phosphatase (PAP), fibrinolysin, citric acid, and prostate-specific antigen (PSA).

The epithelial cells in the prostate gland produce several enzymes, particularly prostate-specific antigen (PSA), prostatic acid phosphatase (PAP), fibrinolysin, and citric acid.

Prostate-specific antigen (PSA), a 33-kDa serine protease, is

one of the most clinically important tumor markers. In normal conditions, PSA is secreted into prostatic gland alveoli and ultimately incorporated into seminal fluid. The alveolar secretion from the prostate gland is pumped into the prostatic urethra during ejaculation by contraction of the fibromuscular tissue of the prostate. Because PSA is predominately released into the prostatic secretion, only a very small amount of PSA (usually 30% per embryo transfer. Further improvements in pregnancy rates may be achieved by the introduction of new drugs, such as recombinant FSH or gonadotropinreleasing hormone (GnRH) antagonists that provide individualized hormonal treatment. In addition, the occurrence of multiple pregnancies, which is the main complication of IVF, may be limited by reducing the number of transferred embryos.

The oocyte undergoes typical changes associated with degeneration and autolysis, and the remnants are phagocytosed by invading macrophages. The zona pellucida which is resistant to the autolytic changes occurring in the cells associated with it, becomes folded and collapses as it is slowly broken down within the cavity of the follicle. Macrophages in the connective tissue are involved in the phagocytosis of the zona pellucida and the remnants of the degenerating cells. The basement membrane between the follicle cells from the theca interna may separate from the follicle cells and increase in thickness, forming a wavy hyaline layer called the . This structure is characteristic of follicles in the late stages of atresia. Enlargement of the cells of the theca interna occurs in some atretic follicles. These cells are similar to theca lutein cells and become organized into radially arranged strands

membrane

glassy

separated by connective tissue. A rich capillary network develops in the connective tissue. These atretic follicles, which resemble an old corpus luteum, are called .

corpora lutea atretica The interstitial gland arises from the theca interna of the atretic follicle. As atretic follicles continue to degenerate, a scar with

hyaline streaks develops in the center of the cell mass, giving it the appearance of a small corpus albicans. This structure eventually disappears as the ovarian stroma invades the degenerating follicle. In the ovaries of a number of mammals, the strands of luteal cells do not degenerate immediately but become broken up and scattered in the stroma. These cords of cells contribute to the of the ovary and produce steroid hormones. The development of the interstitial gland is most extensive in animal species that have large litters. In the human ovary, there are relatively few interstitial cells. They occur in the largest numbers in the first year of life and during the early phases of puberty, corresponding to times of increased follicular atresia. At menarche, involution of the interstitial cells occurs; therefore, few are present during the reproductive life span and menopause. It has been suggested that in humans, the interstitial cells are an important source of the estrogens that influence the growth and development of the secondary sex organs during the early phases of puberty. In other species, the interstitial cells have been shown to produce progesterone. In humans, cells called are found in the hilum of the ovary in association with vascular spaces and unmyelinated nerve fibers. These cells, which appear to be structurally related to the interstitial cells of the testis, contain . The hilar cells appear to respond to hormonal changes during pregnancy and at the onset of menopause. Research suggests that the hilar cells secrete androgens; hyperplasia or tumors associated with these cells usually lead to masculinization.

interstitial gland

ovarian hilar cells

Reinke crystalloids

Blood Supply and Lymphatics

Blood supply to the ovaries comes from two different sources: ovarian and uterine arteries. The ovarian arteries are the branches of the abdominal aorta

that pass to the ovaries through the suspensory ligaments and provide the principal arterial supply to the ovaries and uterine tubes. These arteries anastomose with the second blood source to the ovary, the , which arise from the internal iliac arteries. Relatively large vessels arising from this region of anastomosis pass through the mesovarium and enter the hilum of the ovary. These large arteries are called because they branch and become highly coiled as they pass into the ovarian medulla (see Fig. 23.2). Veins accompany the arteries and form a plexus called the as they emerge from the hilum. The ovarian vein is formed from the plexus. In the cortical region of the ovary, networks of lymphatic vessels in the thecal layers surround the large developing and atretic follicles and corpora lutea. The lymphatic vessels follow the course of the ovarian arteries as they ascend to para-aortic lymph nodes in the lumbar region.

arteries

ovarian branches of the uterine spiral arteries

pampiniform plexus

Innervation

Ovaries are innervated by the autonomic ovarian plexus. Autonomic nerve fibers that supply the ovary are conveyed mainly by the ovarian plexus. Although it is clear that the ovary receives both sympathetic and parasympathetic fibers, little is known about their actual distribution. Groups of parasympathetic ganglion cells are scattered in the medulla. Nerve fibers follow the arteries, supplying the smooth muscle in the walls of these vessels, as they pass into the medulla and cortex of the ovary. Nerve fibers associated with the follicles do not penetrate the basal lamina. Sensory nerve endings are scattered in the stroma. The sensory fibers convey impulses via the ovarian plexus and reach the dorsal root

ganglia of the first lumbar spinal nerves. Therefore, ovarian pain is referred over the cutaneous distribution of these spinal nerves. At ovulation, about 45% of females experience (“mittelschmerz”). It is usually described as a sharp, lower abdominal pain that lasts from a few minutes to as long as 24 hours and may be accompanied by a small amount of bleeding from the uterus. It is believed that this pain is related to smooth muscle cell contraction in the ovary and its ligaments. These contractions are in response to an increased level of prostaglandin F2α mediated by the surge of LH.

midcycle pain

UTERINE TUBES uterine tubes

The are paired tubes that extend bilaterally from the uterus toward the ovaries (see Fig. 23.1). Also commonly referred to as the , the uterine tubes transport the ovum from the ovary to the uterus and provide the necessary environment for fertilization and initial development of the zygote to the morula stage. One end of the tube is adjacent to the ovary and opens into the peritoneal cavity; the other end communicates with the uterine cavity. Each is approximately 10–12 cm long and can be divided into four segments by gross inspection:

fallopian tubes

uterine tube

infundibulum

The is the funnel-shaped segment of the tube adjacent to the ovary. At the distal end, it opens into the peritoneal cavity. The proximal end communicates with the ampulla. Fringed extensions, or , extend from the mouth of the infundibulum toward the ovary. The is the longest segment of the tube, constituting about two-thirds of the total length, and is the site of fertilization. The is the narrow, medial segment of the uterine tube adjacent to the uterus. The or part, measuring about 1 cm in length, lies within the uterine wall and opens into the cavity of the uterus.

fimbriae

ampulla

isthmus uterine

intramural

The wall of the uterine tube is composed of three layers. The uterine tube wall resembles the wall of other hollow viscera, consisting of an external serosal layer, an intermediate muscular layer, and an internal mucosal layer. However, there is no submucosa.

serosa

The or peritoneum is the outermost layer of the uterine tube and is composed of mesothelium and a thin layer of connective tissue. The , throughout most of its length, is organized into an inner, relatively thick circular layer and an outer, thinner longitudinal layer. The boundary between these layers is often indistinct. The , the inner lining of the uterine tube, exhibits relatively thin longitudinal folds that project into the lumen of the uterine tube throughout its length. The folds are most numerous and complex in the ampulla (Fig. 23.15 and Plate 23.4, page 962) and become smaller in the isthmus.

muscularis mucosa

FIGURE 23.15. Photomicrograph of a human uterine tube. a.

This cross section is near the ampulla region of the uterine tube. The mucosa is thrown into extensive folds that project into the lumen of the tube. The muscularis is composed of a thick inner layer of circularly arranged fibers and an outer layer of longitudinal fibers. Note several branches of the uterine and ovarian arteries ( ) that travel along the uterine tube. ×16. The lumen of the tube is lined by a simple columnar epithelium composed of ciliated cells (above the point of the ) and nonciliated cells (below the point of the ). ×640.

arrowhead

arrowhead

b.

BV

mucosal lining

The is simple columnar epithelium composed of two kinds of cells—ciliated and nonciliated (Fig. 23.15b). They represent different functional states of a single cell type.

Ciliated cells are most numerous in the infundibulum and ampulla. The wave of the cilia is directed toward the uterus. Nonciliated, peg cells are secretory cells that produce the fluid that provides nutritive material for the ovum.

The epithelial cells undergo cyclic hypertrophy during the follicular phase and atrophy during the luteal phase in response to changes in hormonal levels, particularly estrogens. Also, the ratio of ciliated to nonciliated cells changes during the hormonal cycle. Estrogen stimulates ciliogenesis, and progesterone increases the number of secretory cells. At about the time of ovulation, the epithelium reaches a height of approximately 30 μm and is then reduced to about one-half that height just before the onset of menstruation.

Bidirectional transport occurs in the uterine tube. The uterine tube demonstrates active movements just

before ovulation as the fimbriae become closely apposed to the ovary and localize over the region of the ovarian surface where rupture will occur. As the oocyte is released, the ciliated cells in the infundibulum sweep it toward the opening of the uterine tube and thus prevent it from entering the peritoneal cavity. The oocyte is transported along the uterine tube by peristaltic contractions. The mechanisms by which spermatozoa and the oocyte are transported from opposite ends of the

uterine tube are not fully understood. Research suggests that both and are involved in the movements of the oocyte. The movement of the spermatozoa is much too rapid, however, to be accounted for by intrinsic motility. , near its junction with the isthmus. The ovum remains in the uterine tube for about 3 days before it enters the uterine cavity. Several conditions that may alter the integrity of the tubal transport system (e.g., inflammation, use of intrauterine devices, surgical manipulation, tubal ligation) may cause a tubal . Most ectopic pregnancies (98%) occur in the (tubal pregnancies). An ectopic pregnancy that occurs in the uterine tube can cause lifethreatening bleeding if the tube ruptures. Prompt treatment with surgery to remove the tube or medication to stop the growth of the fertilized ovum is needed.

ciliary movements

ampulla

peristaltic muscular activity

Fertilization usually occurs in the

ectopic pregnancy uterine tube

UTERUS The uterus receives the rapidly developing morula from the uterine tube. All subsequent embryonic and fetal development occurs within the uterus, which undergoes dramatic increases in size and development. The human uterus is a hollow, pear-shaped organ located in the pelvis between the bladder and the rectum. In a nulliparous female, it weighs 30–40 g and measures 7.5 cm in length, 5 cm in width at its superior aspect, and 2.5 cm in thickness. Its lumen, which is also flattened, is continuous with the uterine tubes and the vagina. Anatomically, the is divided into two regions:

body

uterus

The is the large upper portion of the uterus. The anterior surface is almost flat; the posterior surface is convex. The upper, rounded part of the body that expands above the attachment of the uterine tubes is called the . The is the lower, barrel-shaped part of the uterus separated from the body by the (see Fig. 23.1). The lumen of the cervix, the , has a constricted

fundus cervix

isthmus cervical canal

opening at each end. The cavity of the uterus; the

internal os communicates with external os with the vagina.

the

Organization of the Uterine Wall

The uterine wall is composed of three layers (Fig. 23.16). From the lumen outward, they are as follows:

FIGURE 23.16. Photomicrograph of a sagittal section of a human uterus. This section shows the three layers of the uterine wall: the

endometrium, the innermost layer that lines the uterine cavity; the myometrium, the middle layer of smooth muscle; and the perimetrium, the very thin layer of peritoneum that covers the exterior surface of the uterus. The deep portion of the myometrium contains the larger blood vessels ( ) that supply the uterus. ×8.

BV

endometrium myometrium

The is the mucosa of the uterus. The is the thick muscular layer. It is continuous with the muscle layer of the uterine tube and vagina. The smooth muscle fibers also extend into the ligaments connected to the uterus. The , the outer serous layer or visceral peritoneal covering of the uterus, is continuous with the pelvic and abdominal peritoneum and consists of a mesothelium and a thin layer of loose connective tissue. Beneath the mesothelium, a layer of elastic tissue is usually prominent. The perimetrium covers the entire posterior surface of the uterus but only part of the anterior surface. The remaining part of the anterior surface consists of connective tissue or adventitia.

perimetrium

Both myometrium and endometrium undergo cyclic changes each month to prepare the uterus for implantation of an embryo. These changes constitute the menstrual cycle. If an embryo implants, the cycle stops, and both layers undergo considerable growth and differentiation during pregnancy (described in the next section).

The myometrium forms a structural and functional syncytium. The myometrium is the thickest layer of the uterine wall. It is composed of three indistinctly defined layers of smooth muscle:

The middle muscle layer contains numerous large blood vessels (venous plexuses) and lymphatics and is called the . It is the thickest layer and has interlaced smooth muscle bundles oriented in a circular or spiral pattern. The smooth muscle bundles in the are predominantly oriented parallel to the long axis of the

vasculare

stratum

inner and outer layers

uterus. As in most bulb-shaped hollow organs, such as the gallbladder and urinary bladder, muscular orientation is not distinctive. The muscle bundles seen in routine histologic sections appear to be randomly arrayed. During uterine contraction, all three layers of the myometrium work together as a functional syncytium expelling the contents of the lumen through a narrow orifice. In the nonpregnant uterus, the smooth muscle cells are about 50 μm long. During pregnancy, the uterus undergoes enormous enlargement. The growth is primarily owing to the hypertrophy of existing smooth muscle cells, which may reach more than 500 μm in length, and secondarily attributable to the development of new fibers through the division of existing muscle cells and the differentiation of undifferentiated mesenchymal cells. The amount of connective tissue also increases. As proceeds, the uterine wall becomes progressively thinner as it stretches because of the growth of the fetus. After parturition, although some muscle fibers degenerate, the uterus returns to almost its original size. The collagen produced during pregnancy to strengthen the myometrium is then enzymatically degraded by the cells that secreted it. The uterine cavity remains larger and the muscular wall remains thicker than before pregnancy. Compared with the body of the uterus, the cervix has more connective tissue and less smooth muscle. Elastic fibers are abundant in the cervix but are found in appreciable quantities only in the outer layer of the myometrium of the body of the uterus.

pregnancy

The endometrium proliferates and then degenerates during a menstrual cycle. Throughout the reproductive life span, the endometrium undergoes cyclic changes each month that prepare it for the implantation of the embryo and the subsequent events of embryonic and fetal development. Changes in the secretory activity of the endometrium during the cycle are correlated with the maturation of the ovarian follicles (see Folder 23.3).

The end of each cycle is characterized by the partial destruction and sloughing of the endometrium, accompanied by bleeding from the mucosal vessels. The discharge of tissue and blood from the vagina, which usually continues for 3–5 days, is referred to as or . The is defined as beginning on the day when menstrual flow begins.

menstrual cycle

menstruation

menstrual flow

FOLDER 23.3

FUNCTIONAL CONSIDERATIONS: SUMMARY OF HORMONAL REGULATION OF THE OVARIAN CYCLE During each menstrual cycle, the ovary undergoes cyclic changes that involve two phases: Follicular phase Luteal phase Ovulation occurs between the two phases (Fig. F23.3.1).

FIGURE F23.3.1. Relationship of morphologic and physiologic events that occur in the menstrual cycle. This diagram illustrates the relationship of the morphologic changes in the endometrium and ovary to the pituitary and ovarian blood hormone levels that occur during the menstrual cycle. The pituitary and ovarian hormones and their plasma concentrations are indicated in arbitrary units. , follicle-stimulating hormone; , luteinizing hormone.

FSH

LH

follicular phase

The begins with the development of a small number of primary follicles (10–20) under the influence of follicle-stimulating hormone (FSH) and luteinizing hormone (LH). Selection of dominant follicles occurs by days 5–7 of the menstrual cycle. During the first 8–10 days of the cycle, FSH is the principal hormone influencing the growth of the follicles. It stimulates the granulosa and thecal cells, which begin to secrete steroid hormones, principally estrogens, into the follicular lumen. As estrogen production from the dominant follicle increases, FSH production is inhibited by a negative feedback loop from the pituitary gland. Estrogens continue to accumulate in the follicular lumen, finally reaching a level that allows the follicle to be independent of FSH for its continued growth and development. Late in the follicular phase, before ovulation, progesterone levels begin to increase under the influence of LH. The amount of estrogens in the circulating blood inhibits further production of FSH by the adenohypophysis. Ovulation is induced by a surge in the LH level, which occurs concomitantly with a smaller increase in the FSH level. It occurs ~34–36 hours after the start of the LH surge or about 10–12 hours after the peak of the LH surge. The begins immediately after ovulation as the granulosa and thecal cells of the ruptured follicle undergo rapid morphologic transformation to form the corpus luteum. Estrogens and large amounts of progesterone are secreted by the corpus luteum. Under the influence of both hormones, but primarily progesterone, the endometrium begins its secretory phase, which is essential for the preparation of the uterus for implantation in the event that the egg is fertilized. LH appears to be responsible for the development and maintenance of the corpus luteum during the menstrual cycle. If fertilization does not occur, the corpus luteum degenerates within a few days as hormone levels drop. If fertilization does occur, the corpus luteum is maintained and continues to secrete progesterone and estrogens. Human chorionic gonadotropin (hCG), which is initially produced by the embryo and later by the placenta, stimulates the corpus luteum and is responsible for its maintenance during pregnancy.

luteal phase

During reproductive life, the endometrium consists of two layers or zones that differ in structure and function (Fig. 23.17 and Plate 23.5, page 964):

FIGURE 23.17. Schematic diagram illustrating arterial blood supply to the endometrium of the uterus. The two layers of the endometrium, the stratum basale and stratum functionale, are supplied by branches of the uterine artery. The spiral arteries located at the interface between these two layers degenerate and regenerate during the menstrual cycle under the influence of estrogens and progesterone. (Based on Weiss L, ed. . 6th ed. Urban & Schwarzenberg; 1988.)

Cell and Tissue Biology: A Textbook of

Histology

stratum functionale

functional layer

The or is the thick part of the endometrium, which is sloughed off at menstruation. The or is retained during menstruation and serves as the source for the regeneration of the stratum functionale.

stratum basale

basal layer

The stratum functionale is the layer that proliferates and degenerates during the menstrual cycle. During the phases of the menstrual cycle, the endometrium

varies from 1 to 6 mm in thickness. It is lined by a simple columnar epithelium with a mixture of secretory and ciliated cells. The surface epithelium invaginates into the underlying lamina propria, the , forming the uterine glands. These simple tubular glands, containing fewer ciliated cells, occasionally branch into the deeper aspect of the endometrium. The endometrial stroma, which resembles mesenchyme, is highly cellular and contains abundant intercellular ground substance. As in the uterine tube, no submucosa separates the endometrium from the myometrium.

endometrial stroma

The vasculature of the endometrium also proliferates and degenerates during each menstrual cycle.

The endometrium contains a unique system of blood vessels (see Fig. 23.17). The uterine artery gives off 6–10 arcuate arteries that anastomose in the myometrium. Branches from these arteries, the , enter the basal layer of the endometrium where they give off small straight arteries that supply this region of the endometrium. The main branch of the radial artery continues upward and becomes highly coiled; it is, therefore, called the . Spiral arteries give

radial arteries

spiral artery

off numerous arterioles that often anastomose as they supply a rich capillary bed. The capillary bed includes thin-walled dilated segments called . Lacunae may also occur in the venous system that drains the endometrium. The straight arteries and the proximal part of the spiral arteries do not change during the menstrual cycle. The distal portion of the spiral arteries, under the influence of estrogens and progesterone, undergoes degeneration and regeneration with each menstrual cycle.

lacunae

Cyclic Changes During the Menstrual Cycle

Cyclic changes of the endometrium during the menstrual cycle are represented by the proliferative, secretory, and menstrual phases. The menstrual cycle is a continuum of developmental stages in the functional layer of the endometrium. It is ultimately controlled by gonadotropins secreted by the pars distalis of the pituitary gland that regulate the steroid secretions of the ovary. The cycle normally repeats every 28 days, during which the endometrium passes through a sequence of morphologic and functional changes. It is convenient to describe the cycle as having three successive phases:

proliferative phase secretory phase

The occurs concurrently with follicular maturation and is influenced by ovarian estrogen secretion. The coincides with the functional activity of the corpus luteum and is primarily influenced by progesterone secretion. The commences as hormone production by the ovary declines with the degeneration of the corpus luteum (see Folder 23.3).

menstrual phase

The phases are part of a continuous process; there is no abrupt change from one to the next.

The proliferative phase of the menstrual cycle is regulated by estrogens.

At the end of the menstrual phase, the endometrium consists of a thin band of connective tissue, about 1 mm thick, containing the basal portions of the uterine glands and the lower portions of the spiral arteries (see Fig. 23.17). This layer is the stratum basale; the layer that was sloughed off was the stratum functionale. Under the , the is initiated. Stromal, endothelial, and epithelial cells in the stratum basale proliferate rapidly, and the following changes can be seen:

proliferative phase

influence of estrogens

Epithelial cells in the basal portion of the glands reconstitute the glands and migrate to cover the denuded endometrial surface. Stromal cells proliferate and secrete collagen and ground substance. Spiral arteries lengthen as the endometrium is reestablished; these arteries are only slightly coiled and do not extend into the upper third of the endometrium. The proliferative phase continues until 1 day after ovulation, which occurs on day 14 of a 28-day cycle. At the end of this phase, the endometrium has reached a thickness of about 3 mm. The glands have narrow lumina and are relatively straight but have a slightly wavy appearance (Fig. 23.18a). Accumulations of glycogen are present in the basal portions of the epithelial cells. In routine histologic preparations, extraction of the glycogen gives an empty appearance to the basal cytoplasm.

FIGURE 23.18. Photomicrographs of the uterine lining in proliferative, secretory, and menstrual phases of the menstrual cycle. a. The upper panel shows the endometrium at the proliferative phase of the cycle. During this phase, the stratum functionale (separated by the dashed line from the stratum basale) greatly thickens. ×15. The lower panel shows at higher magnification the endometrial glands that extend from the stratum basale to the surface. ×55. b. The upper panel shows the endometrium at the secretory phase

of the cycle. The glands have acquired a corkscrew shape as the endometrium increases further in thickness. The stratum basale (below the ) exhibits less dramatic changes in morphology. ×20. The shows uterine glands that have been cut in a plane that is close to their long axes. Note the pronounced corkscrew shape of the glands and mucus secretion ( ). ×60. The shows the stratum functionale (above the ). Much of the

dashed line lower panel

arrows

c. dashed line

upper panel

lower

stratum functionale has degenerated and sloughed away. ×15. The shows the extravasated blood and necrosis of the stratum functionale. ×55.

panel

The secretory phase of the menstrual cycle is regulated by progesterone. Under the influence of progesterone, dramatic changes occur in

the stratum functionale, beginning a day or 2 after ovulation. The endometrium becomes edematous and may eventually reach a thickness of 5–6 mm. The glands enlarge and become corkscrew shaped, and their lumina become sacculated as they fill with secretory products (Fig. 23.18b). The mucoid fluid produced by the gland epithelium is rich in nutrients, particularly glycogen, required to support development if implantation occurs. Mitoses are now rare. The growth seen at this stage results from hypertrophy of the epithelial cells, an increase in vascularity, and edema of the endometrium. The spiral arteries, however, lengthen and become more coiled. They extend nearly to the surface of the endometrium (Plate 23.6, page 966). The sequential influence of estrogens and progesterone on the enables their transformation into . The stimulus for transformation is the implantation of the blastocyst. Large, pale cells rich in glycogen result from this transformation. Although the precise function of these cells is not known, it is clear that they provide a favorable environment for the nourishment of the embryo and that they create a specialized layer that facilitates the separation of the placenta from the uterine wall following parturition.

stromal cells cells

decidual

The menstrual phase results from a decline in the ovarian secretion of progesterone and estrogen. The corpus luteum actively produces hormones for about 10 days if fertilization does not occur. As hormone levels rapidly decline, changes occur in the blood supply to the stratum functionale. Initially, periodic contractions of the walls of the spiral arteries, lasting for several hours, cause the to become . The glands stop

stratum functionale

ischemic

secreting, and the endometrium shrinks in height as the stroma becomes less edematous. After about 2 days, extended periods of arterial contraction, with only brief periods of blood flow, cause disruption of the surface epithelium and rupture of the blood vessels. When the spiral arteries close off, blood flows into the stratum basale, but not into the stratum functionale. Blood, uterine fluid, and stromal and epithelial cells from the stratum functionale constitute the menstrual discharge. As patches of tissue separate from the endometrium, the torn ends of veins, arteries, and glands are exposed (Fig. 23.18c). The desquamation continues until only the stratum basale remains. Clotting of blood is inhibited during this period of menstrual flow. Arterial blood flow is restricted, except for the brief periods of relaxation of the walls of the spiral arteries. Blood continually seeps from the open ends of the veins. The period of normally lasts about 5 days. The average blood loss in the menstrual phase is 35–50 mL. Blood flow through the straight arteries maintains the stratum basale. As noted, this process is cyclic. Figure F23.3.1 in Folder 23.3 shows a single cycle of the endometrium. In the absence of fertilization, cessation of bleeding accompanies the growth and maturation of new ovarian follicles. The epithelial cells rapidly proliferate and migrate to restore the surface epithelium as the proliferative phase of the next cycle begins. In the absence of ovulation (a cycle referred to as an ), a corpus luteum does not form, and progesterone is not produced. In the absence of progesterone, the endometrium does not enter the secretory phase and continues in the proliferative phase until menstruation. In cases of , biopsies of the endometrium can be used to diagnose as well as other disorders of the ovary and endometrium.

menstrual flow

anovulatory cycle

infertility anovulatory cycles

Implantation

If fertilization and implantation occur, a gravid phase replaces the menstrual phase of the cycle.

implantation

If fertilization and subsequent occur, decline of the endometrium is delayed until after parturition. As the blastocyst becomes embedded in the uterine mucosa in the early part of the second week, cells in the chorion of the developing placenta begin to secrete and other luteotropins. These hormones maintain the corpus luteum and stimulate it to continue the production of progesterone and estrogens. Thus, the decline of the endometrium is prevented, and the endometrium undergoes further development during the first few weeks of pregnancy.

hCG

Implantation is the process by which the blastocyst settles into the endometrium. The fertilized human ovum (zygote) undergoes a series of changes as it passes through the uterine tube into the uterine cavity in preparation for becoming embedded in the uterine mucosa. The developing embryo initiates an embryo–maternal dialogue, which is critical for further implantation and development. Shortly after fertilization, the viable embryo secretes , an embryo-specific 15amino-acid peptide (MVRIKPGSANKPSDD) that promotes adhesion of the embryo to the endometrium. At the time of implantation, PIF stimulates proliferation and invasion of the trophoblast into the decidua basalis (see page 931). The zygote undergoes cleavage, followed by a series of mitotic divisions without cell growth, resulting in a rapid increase in the number of cells in the embryo. Initially, the embryo is under the control of maternal informational macromolecules that have accumulated in the cytoplasm of the ovum during oogenesis. Later development depends on activation of the embryonic genome, which encodes various growth factors, cell junction components, and other macromolecules required for normal progression to the blastocyst stage. The cell mass resulting from the series of mitotic divisions is known as a , and the individual cells are known as . During the third day after fertilization, the morula, which now contains 12–16 cells is still surrounded by the zona pellucida enters the uterine cavity. The morula

preimplantation factor (PIF)

morula [L. morum, mulberry] blastomeres

remains free in the uterus for about a day while continued cell division and development occur. The early embryo gives rise to a blastocyst, a hollow sphere of cells with a centrally located clump of cells. This will give rise to the tissues of the embryo proper; the surrounding layer of cells, the , will form the trophoblast and then the placenta (Fig. 23.19).

outer cell mass

inner cell mass

FIGURE 23.19. Schematic diagrams of sectioned blastocysts. a. A human blastocyst at about 4.5 days of development showing the formation of the inner cell mass. b. A monkey blastocyst at about 9 days of development. The trophoblastic cells of the monkey blastocyst have begun to invade the epithelial cells of the endometrium. In humans, the blastocyst begins to invade the endometrium at about the fifth or sixth day of development. A human blastocyst at 14 days after implantation. At this stage, the trophoblast cells have differentiated into syncytiotrophoblasts and cytotrophoblasts.

c.

Fluid passes inward through the zona pellucida during this process, forming a fluid-filled cavity, the . This event defines the beginning of the . As the blastocyst remains free in the uterine lumen for 1 or 2 days and undergoes further mitotic divisions, the zona pellucida disappears. The outer cell mass is now called the , and the inner cell mass is referred to as the .

blastocyst cavity blastocyst

trophoblast embryoblast Implantation occurs during a short period known as the implantation window.

The attachment of the blastocyst to the endometrial epithelium occurs during the , the period when the uterus is receptive for implantation of the blastocyst. This short period results from a series of programmed actions of progesterone and estrogens on the endometrium. such as mifepristone (RU-486) and its derivatives compete for the receptors in the endometrial epithelium, thus blocking hormone binding. The failure of progesterone to gain access to its receptors prevents implantation, thus effectively closing the window. In human, the implantation window begins on day 6 after the LH surge and is completed by day 10. As contact is made with the uterine wall by the over the embryoblast pole, the trophoblast rapidly proliferates and begins to invade the endometrium. The invading differentiates into the syncytiotrophoblast and the cytotrophoblast.

implantation window

Antiprogesterone

drugs

trophoblastic cells trophoblast cytotrophoblast

The is a mitotically active inner cell layer producing cells that fuse with the syncytiotrophoblast, the outer erosive layer. The fusion of the cytotrophoblast with the overlying multinucleated syncytiotrophoblast may be triggered by programmed cell death (apoptosis). The is not mitotically active and consists of a multinucleate cytoplasmic mass; it actively invades the epithelium and underlying stroma of the endometrium.

syncytiotrophoblast

Through the activity of the trophoblast, the blastocyst is entirely embedded within the endometrium on about the 11th day of development. (Further development of the syncytiotrophoblast and cytotrophoblast is described in the section on the placenta.) The syncytiotrophoblast has well-developed Golgi complexes, abundant sER and rER, numerous mitochondria, and relatively large numbers of lipid droplets. These features are consistent with the secretion of progesterone, estrogens, hCG, and lactogens by this layer. Recent evidence indicates that cytotrophoblast cells may also be a source of steroid hormones and hCG.

After implantation, the endometrium undergoes decidualization. During pregnancy, the portion of the endometrium that undergoes morphologic changes is called the decidua or decidua graviditatis. As its name implies, this layer is shed with the placenta at parturition. The decidua includes all but the deepest layer of the endometrium. During the decidualization process, which typically takes at least 8–10 days, the stromal cells differentiate into large, rounded decidual cells in response to the elevated progesterone levels (see page 935). The uterine glands enlarge and become more coiled during the early part of pregnancy and then become thin and flattened as the growing fetus fills the uterine lumen. are identified by their relationship to the site of implantation (Fig. 23.20):

Three different regions of the decidua

FIGURE 23.20. Development of the placenta.

This schematic drawing shows growth of the uterus during human pregnancy and development of the placenta and its membranes. Note that there is a gradual obliteration of the uterine lumen and disappearance of the decidua capsularis as the definitive placenta is established. (Modified from Williams J. Placenta circumvallata. . 1927;13:1– 16.)

Am J Obstet Gynecol

decidua basalis

The is the portion of the endometrium that underlies the implantation site.

decidua capsularis decidua parietalis

The is a thin portion of endometrium that lies between the implantation site and the uterine lumen. The includes the remaining endometrium of the uterus. By the end of the third month of gestation, the fetus grows to the point that the overlying decidua capsularis fuses with the decidua parietalis of the opposite wall, thereby obliterating the uterine cavity. By the 13th day of development, an extraembryonic space, the , has been established (Fig. 23.19c). The cell layers that form the outer boundary of this cavity (i.e., the syncytiotrophoblast, cytotrophoblast, and extraembryonic somatic mesoderm) are collectively referred to as the . The innermost membranes enveloping the embryo are called the (see Fig. 23.20).

chorionic cavity amnion

chorion

Cervix

The endometrium of the cervix differs from the rest of the uterus. The cervical mucosa measures about 2–3 mm in thickness and differs dramatically from the rest of the uterine endometrium in that it contains large, branched glands (Fig. 23.21 and Plate 23.7, page 968). It also lacks spiral arteries. The cervical mucosa undergoes little change in thickness during the menstrual cycle and is not sloughed during the period of menstruation. During each menstrual cycle, however, the undergo important functional changes that are related to the transport of spermatozoa within the cervical canal. The amount and properties of the mucus secreted by the gland cells vary during the menstrual cycle under the influence of ovarian hormones. At midcycle, the amount of mucus produced increases 10-fold. This mucus is less viscous and appears to provide a more favorable environment for sperm migration. The cervical mucus at other times in the cycle restricts the passage of sperm into the uterus. Thus, hormonal mechanisms ensure that ovulation and changes in the cervical mucus are

cervical glands

coordinated, thereby increasing the possibility that fertilization will occur if spermatozoa and the ovum arrive simultaneously at the site of fertilization in the uterine tube.

FIGURE 23.21. Photomicrograph of a human cervix.

This hematoxylin and eosin (H&E)-stained specimen is from a postmenopausal female. Its lower portion projects into the upper vagina where an opening, the external os, leads to the uterus through the cervical canal. The surface of the cervix is covered by stratified squamous epithelium ( ) that is continuous with the epithelial lining of the vagina. An abrupt transition from stratified squamous epithelium to simple columnar epithelium ( ) occurs at the entry to the cervical canal. In this specimen, the stratified epithelium has extended into the canal, an event that occurs with aging. Mucus-secreting cervical glands are seen along the cervical canal. These are simple branched tubular glands that arise as invaginations of the epithelium lining the canal. Frequently, the glands develop into nabothian cysts as a result of retention of mucus secretion by blockage of the gland opening. The material marked by the is mucus secreted from the cervical glands. ×10.

SSE

SCE

X

Blockage of the openings of the mucosal glands results in the retention of their secretions, leading to the formation of dilated cysts within the cervix called . Nabothian cysts develop frequently but are clinically important only if numerous cysts produce marked enlargement of the cervix.

nabothian cysts

The transformation zone is the site of transition between vaginal stratified squamous epithelium and cervical simple columnar epithelium. The portion of the cervix that projects into the vagina, the vaginal part or ectocervix, is covered with a stratified squamous epithelium (Fig. 23.22). An abrupt transition between this squamous epithelium and the mucus-secreting columnar epithelium of the , the , occurs in the that during reproductive age is located just outside the (Plate 23.7, page 968). Before puberty and after menopause, the transformation zone resides in the cervical canal (Fig. 23.23). in this transformation zone constitute precancerous lesions of the cervix. Metaplasia represents an adoptive and reversible response to persistent injury of the epithelium caused by chronic infection. It results from a reprogramming of epithelial stem cells that begin to differentiate into new cell lineage. Within the cervical canal (endocervix), it is

cervical canal transformation zone external os

endocervix

Metaplastic changes [Gr. change in form]

manifested as a replacement of the simple columnar epithelium with fully mature stratified squamous epithelium (Fig. 23.24). The cervical epithelial cells are constantly exfoliated into the vagina. Samples of the cervical cells may be stained and examined for morphologic changes ( or tested for the presence of high-risk strains of human papillomavirus (HPV), the leading cause of cervical cancer. These tests are used routinely to screen for precancerous and cancerous lesions of the cervix.

Papanicolaou [Pap] test)

FIGURE 23.22. Stratified squamous epithelium of the ectocervix. The stratified squamous epithelium and underlying fibrous connective tissue within the lower rectangle in Figure 23.21 are shown here at

higher magnification. The more mature epithelial cells have a clear cytoplasm ( ), a reflection of their high glycogen content. Also, note the connective tissue papillae protruding into the epithelium ( ). The bulk of the cervix is made up of dense, fibrous connective tissue with relatively little smooth muscle. ×120.

arrowheads arrows

FIGURE 23.23. Transformation zone of the cervix. The site of the squamocolumnar junction from the upper rectangle in Figure 23.21 is shown here at higher magnification. Note the abrupt change from stratified squamous epithelium to simple columnar epithelium ( arrow ). Neoplastic changes leading to the development of cervical cancer most frequently begin in this transformation zone. Within the connective tissue are the branched, mucus-secreting cervical glands ( ) composed

CG

of a simple columnar epithelium that is continuous with the lining epithelium of the cervical canal. ×120.

FIGURE 23.24. Metaplastic stratified squamous epithelium of the cervical canal. This photomicrograph shows an island of the fully mature stratified squamous epithelium surrounded by the simple columnar epithelium normally found in the cervical canal. ×450. (Courtesy of Dr. Fabiola Medeiros.)

PLACENTA

The developing fetus is maintained by the placenta, which develops from fetal and maternal tissues. The placenta consists of a fetal portion, formed by the chorion, and a maternal portion, formed by the decidua basalis. The two parts are involved in physiologic exchange of substances between the maternal and fetal circulations. The begins to develop around day 9 after fertilization, with the development of

uteroplacental circulatory system

trophoblastic lacunae

vascular spaces called within the syncytiotrophoblast. Maternal sinusoids, which develop from capillaries of the maternal side, anastomose with the trophoblastic lacunae (Fig. 23.25). The differential pressure between the arterial and venous channels that communicate with the lacunae establishes directional flow from the arteries into the veins, thereby establishing a primitive uteroplacental circulation. Numerous pinocytotic vesicles present in the syncytiotrophoblast indicate the transfer of nutrients from the maternal vessels to the embryo.

FIGURE 23.25. Schematic diagrams of sections through a developing human embryo. a. This drawing shows the chorionic sac and placenta at 16 days of development. b. The same embryo at 21 days of development. The diagrams illustrate the separation of the fetal and maternal blood vessels by the placental membrane, which is composed of the endothelium of the capillaries, mesenchyme, cytotrophoblast, and syncytiotrophoblast.

cytotrophoblast

chorionic

Proliferation of the , growth of , and blood vessel development successively give rise to the chorionic villi (Fig. 23.26). They undergo the following changes:

mesoderm

FIGURE 23.26. Schematic diagram of chorionic villi in various stages of development. This drawing shows the developmental stages

of chorionic villi. Primary villi represent the first stage of development in which the syncytiotrophoblast and cytotrophoblast form finger-like extensions into the maternal decidua. In secondary chorionic villi, the extraembryonic connective tissue (mesenchyme) grows into the villi and is surrounded by a layer of cytotrophoblast. In tertiary chorionic villi, blood vessels and supportive cells differentiate within the mesenchymal core. In early pregnancy, villi are large and edematous with few blood vessels surrounded by many cells of connective tissue. They are covered by a thick layer of syncytiotrophoblast and a continuous layer of cytotrophoblast cells. In late pregnancy, the layer of cytotrophoblast appears to be discontinuous, and nuclei of the syncytiotrophoblast aggregate to form irregularly dispersed projections called . More fetal blood vessels are found in the connective tissue core, which becomes less cellular and contains fewer placental macrophages.

syncytial knots

Primary chorionic villi

are formed by the rapidly proliferating cytotrophoblast. They send cords or masses of cells into the blood-filled trophoblastic lacunae within the syncytiotrophoblast (see Figs. 23.19b and 23.27). The primary villi appear between days 11 and 13 of development.

FIGURE 23.27. Photomicrographs of a human placenta. a. This hematoxylin and eosin (H&E)-stained specimen shows the amniotic surface ( A ), the chorionic plate ( CP ), and, below, the various-sized profiles of the chorionic villi ( CV ). These villi emerge from the chorionic plate as large stem villi and branch into increasingly smaller villi. Blood vessels ( BV ) are evident in the larger villi. The smallest villi contain capillaries where exchange takes place. ×60. Upper inset. This higher magnification shows the simple cuboidal epithelium of the amnion and the underlying connective tissue. ×200. Lower inset. This higher magnification shows a cross-sectioned villus containing several larger blood vessels and its thin surface syncytiotrophoblast layer. ×200. b. This H&Estained specimen shows the maternal side of the placenta. The basal plate ( BP ), the part of the uterus to which some of the chorionic villi ( CV ) anchor, is seen at the bottom of the micrograph. Also evident is a stromal connective tissue ( CT ) component, part of the

basal plate, to which many of the chorionic villi are also attached. Within the basal plate and the connective tissue stroma are clusters of cells, the decidual cells ( ), which arose from connective tissue cells. ×60. Decidual cells seen at higher magnification. ×200.

Inset.

Secondary chorionic villi

arrows

are composed of a central core of mesenchyme surrounded by an inner layer of cytotrophoblast and an outer layer of syncytiotrophoblast (see Fig. 23.26).

They develop at about day 16 when the primary chorionic villi become invaded by loose connective tissue from chorionic mesenchyme. The secondary villi cover the entire surface of the chorionic sac. are formed by the end of the third week as the secondary villi become vascularized by blood vessels that have developed in their connective tissue cores (see Fig. 23.25b and Plate 23.9, page 972).

Tertiary chorionic villi

As the tertiary villi are forming, cytotrophoblastic cells in the villi continue to grow out through the syncytiotrophoblast. When they meet the maternal endometrium, they grow laterally and meet similar processes growing from neighboring villi. Thus, a thin layer of cytotrophoblastic cells called the is formed around the syncytiotrophoblast. The trophoblastic shell is interrupted only at sites where maternal vessels communicate with the intervillous spaces. Chorionic villi continuously form out of the trophoblastic sprouts throughout pregnancy. The chorionic villi can remain either free ( ) in the intervillous space or grow into the maternal side of the placenta (basal plate) to form or . Future growth of the placenta is accomplished by interstitial growth of the trophoblastic shell. During pregnancy, the villi mature and become smaller in diameter. The layer of cytotrophoblast appears to be discontinuous, and in some areas, nuclei of the syncytiotrophoblast are gathered in clusters to form irregularly dispersed (see Fig. 23.26 and Plate 23.9, page 972). The number of syncytial knots increases with gestational age of the placenta and can be used to evaluate . An increased number of syncytial knots is also associated with some pathologic conditions, such as . Several types of cells are recognized in the connective tissue stroma of the villi: mesenchymal cells, reticular cells, fibroblasts, myofibroblasts, smooth muscle cells, and , historically also known as (Plate 23.9, page

trophoblastic shell

floating villi main stem villi anchoring villi syncytial knots

villous maturity uteroplacental malperfusion

fetal placental antigen–presenting cells (placental macrophages) Hofbauer cells

972). Fetal placental antigen–presenting cells are the specific villous macrophages of fetal origin that participate in placental innate immune reactions. In response to an antigen, they proliferate and upregulate specific surface receptors that recognize and bind to a variety of pathogens. Like other antigen-presenting cells, if stimulated, they increase the number of major histocompatibility complex II (MHC II) molecules on their surface. They are more common in the early placenta. The vacuoles in these cells contain lipids, glycosaminoglycans, and glycoproteins. Studies of indicate that HIV is primarily localized within the fetal placental antigen–presenting cells and in the syncytiotrophoblast.

HIV-infected

placentas

Early in development, the blood vessels of the villi become connected with vessels from the embryo.

Blood begins to circulate through the embryonic cardiovascular system and the villi at about 21 days. The intervillous spaces provide the site for exchange of nutrients, metabolic products and intermediates, and wastes between the maternal and fetal circulatory systems. During the first 8 weeks, villi cover the entire chorionic surface, but as growth continues, villi on the decidua capsularis begin to degenerate, producing a smooth, relatively avascular surface called the . The villi adjacent to the decidua basalis rapidly increase in size and number and become highly branched. This region of the chorion, which is the fetal component of the placenta, is called the or . The layer of the placenta from which the villi project is called the (Plate 23.8, page 970). During the period of rapid growth of the chorion frondosum, at about the fourth to fifth month of gestation, the fetal part of the placenta is divided by the into 15–25 areas called . Wedge-like placental septa form the boundaries of the cotyledons, and because they do not fuse with the chorionic plate, maternal blood can circulate

chorion laeve

frondosum

villous chorion

cotyledons

chorionic plate

chorion

placental (decidual) septa

easily between them. Cotyledons are visible as the bulging areas on the maternal side of the basal plate. The forms a compact layer that is the maternal component of the placenta (see Fig. 23.27). The , the outer part of the placenta that is in contact with the uterine wall, consists of embryonic tissues (trophoblastic shell containing a thin layer of syncytiotrophoblast and cytotrophoblast) and maternal tissues (decidua basalis). Vessels within this part of the endometrium supply blood to the intervillous spaces. With rare exceptions, fetal blood and maternal blood do not mix.

plate

decidua basalis

basal

Fetal and maternal blood are separated by the placental barrier. Separation of the fetal and maternal blood, referred to as the placental barrier, is maintained primarily by the layers of fetal tissue (Fig. 23.28). Starting at the fourth month, these layers become very thin to facilitate the exchange of products across the placental barrier. The thinning of the wall of the villus is caused in part by surface and volume expansion of the villi as well as by the degeneration of the inner cytotrophoblast layer (see Fig. 23.27). However, although the cytotrophoblast layer indeed becomes much thinner, it does not become discontinuous.

FIGURE 23.28. Human placental barrier in the third trimester of pregnancy. This high-magnification electron micrograph shows the thinnest layer of a fully developed placental barrier (section does not include cytotrophoblast cells that form a thin [or discontinuous] layer in the human placenta). The lumen of intervillous space containing maternal erythrocytes ( ) ( ) is separated from the fetal capillary space containing fetal erythrocytes ( ) ( ). The intervillous space is lined by the multinucleated syncytiotrophoblast ( ). Its surface contains microvilli projecting into maternal blood space. The cytoplasm of the syncytiotrophoblast contains multiple nuclei ( ) and has an abundance of transport vesicles, rER, sER, mitochondria, and occasional lipid droplets. The syncytiotrophoblast rests on the basal lamina ( ), which is separated by a thin layer of the connective tissue ( ) from the basal lamina ( ) of the fetal endothelial cells ( ). ×11,000. , rough-surfaced endoplasmic reticulum; , smooth-surfaced endoplasmic reticulum. (Courtesy of Dr. Holger Jastrow.)

ME to the left

right

Syn

EBL

At its thinnest, the

N

sER

FEn

FE to the

TBL CT

rER

placental barrier consists of

syncytiotrophoblast, thin (or discontinuous) inner cytotrophoblast layer, basal lamina of the trophoblast, connective (mesenchymal) tissue of the villus, basal lamina of the endothelium, and endothelium of the fetal placental capillary in the tertiary villus. This barrier bears a strong resemblance to the air–blood barrier of the lung, with which it has an important parallel function, namely, the exchange of oxygen and carbon dioxide—in this case, between the maternal blood and the fetal blood. It also resembles the air–blood barrier by having a particular type of macrophage in its connective tissue—in this instance, the fetal placental antigen–presenting cells (Hofbauer cell).

The placenta is the site of exchange of gases and metabolites between the maternal and fetal circulation. Fetal blood enters the placenta through a pair of umbilical arteries (Fig. 23.29). As they pass into the placenta, these arteries branch into several radially disposed vessels that give numerous branches in the chorionic plate. Branches from

these vessels pass into the villi, forming extensive capillary networks in close association with the intervillous spaces. Gases and metabolic products are exchanged across the thin fetal layers that separate the two bloodstreams at this level. Antibodies can also cross this layer and enter the fetal circulation to provide passive immunity against a variety of infectious agents—for example, those of diphtheria, smallpox, and measles. Fetal blood returns through a system of veins that parallel the arteries, except that they converge on a single .

umbilical vein

FIGURE 23.29. Schematic diagram of mature human placenta. The sagittal section of the uterus ( above ) with the developing embryo shows the most common location of the placenta. The mature placenta ( below ) is divided into cotyledons by placental septa that are formed

by outgrowths of the decidua basalis. Maternal blood enters the placenta through numerous endometrial spiral arteries that penetrate the basal plate. As the blood enters the cotyledon, it is directed deep into the intervillous spaces ( ). It then passes over the surface of the villi, where exchange of gases and metabolic products occurs. The maternal blood finally leaves the intervillous space ( ) through endometrial veins. The fetal blood enters the placenta through the umbilical arteries that divide into a series of radially disposed arteries within the chorionic plate. Branches from the vessels pass into the main stem villi and there form extensive capillary networks. The veins within the villi then carry the blood back through a system of veins that parallels that of the fetal arteries.

red arrows

blue arrows

Maternal blood is supplied to the placenta through 80–100 spiral endometrial arteries that penetrate the basal plate. Blood from these spiral arteries flows into the base of the intervillous spaces, which contain about 150 mL of maternal blood that is exchanged 3–4 times per minute. The blood pressure in the spiral arteries is much higher than that in the intervillous spaces. As blood is injected into these spaces at each pulse, it is directed deep into the spaces. As the pressure decreases, the blood flows back over the surfaces of the villi and eventually enters endometrial veins also located in the base of the spaces. Exchange of gases and metabolic products occurs as the blood passes over the villi. Normally, water, carbon dioxide, metabolic waste products, and hormones are transferred from the fetal blood to the maternal blood; water, oxygen, metabolites, electrolytes, vitamins, hormones, and some antibodies pass in the opposite direction. The placental barrier does not exclude many potentially dangerous agents, such as alcohol, nicotine, viruses, drugs, exogenous hormones, and heavy metals. Therefore, during pregnancy, exposure to or ingestion of such agents should be avoided to reduce the risk of injury to the embryo or fetus. Before the establishment of blood flow through the placenta, the growth of the embryo is supported in part by metabolic

products that are synthesized by or transported through the trophoblast. The syncytiotrophoblast synthesizes glycogen, cholesterol, and fatty acids as well as other nutrients used by the embryo.

FOLDER 23.4

CLINICAL CORRELATION: THE PLACENTA The placenta is a temporary organ made by the body only during pregnancy. The mature placenta measures about 15–20 cm in diameter and 2–3 cm in thickness, covers 25%–30% of the uterine surface, and weighs 500–600 g at term. The surface area of the villi of human placenta is estimated to be about 10 m 2. The microvilli on the syncytiotrophoblast increase the effective area for metabolic exchange to >90 m 2. After childbirth, the uterus continues to contract, reducing the luminal surface and inducing placental separation from the uterine wall. The entire fetal portion of the placenta, fetal membranes, and the intervening projections of decidual tissue are released. During uncomplicated labor, the placenta separates from the uterine wall and is delivered ~30 minutes after birth. One of the most severe complications of labor results from (abnormal attachment of the placenta on uterine wall). If decidual tissue during implantation is disrupted, the placenta invades deep into the uterine wall. This may cause one of the three clinical conditions: placenta accreta, placenta increta, or placenta percreta. Classification depends on the severity and deepness of the placental attachment. , accounting for ~75% of all cases, occurs when the placenta attaches too deeply into the uterine wall but does not penetrate the myometrium. (about 15% of all cases) occurs when the placental villi penetrate deep into the muscular layer of the myometrium. In the remaining 10% of all cases, penetrates through the entire uterine wall and attaches to another organ, such as the bladder, rectum, intestines, or large blood vessels. It is the most serious complication of placentation and may cause rupture of the uterus and other complications related to its attachment. A retained abnormal placenta or placental fragments may cause massive postpartum bleeding and need to be manually removed. Placenta increta and percreta may need to be treated by emergency hysterectomy. After physiologic delivery of the placenta, the endometrial glands and stroma of the decidua basalis regenerate. Endometrial regeneration is completed by the end of the third week postpartum, except at the placental site, where regeneration usually extends

abnormal placentation accreta

Placenta increta placenta percreta

Placenta

for another 3 weeks. During the first week after delivery, remnants of the decidua are shed and constitute the red-brown uterine discharge known as

lochia rubra. The placenta is a major endocrine organ producing steroid and protein hormones. The placenta also functions as an endocrine organ, producing steroid and peptide hormones as well as prostaglandins that play an important role in the onset of labor. Immunocytochemical studies indicate that the is the site of synthesis of these hormones. Hormones produced by syncytiotrophoblast are secreted directly into the maternal blood surrounding the placental villi. The progesterone and estrogen have essential roles in the maintenance of pregnancy. As pregnancy proceeds, the placenta takes over the major role in the secretion of these steroids from the corpus luteum. The placenta produces enough progesterone by the end of the eighth week to maintain pregnancy if the corpus luteum is surgically removed or fails to function. In the production of placental estrogen, the fetal adrenal cortex plays an essential role, providing the precursors needed for estrogen synthesis. Because the placenta lacks the enzymes needed for the production of estrogen precursors, a cooperative is established. Clinically, monitoring of estrogen production during pregnancy can be used as an in certain situations. The following are secreted by the placenta:

syncytiotrophoblast

steroid hormones

unit development

fetoplacental (endocrine) index of fetal

peptide hormones Human chorionic gonadotropin (hCG)

is required for implantation and maintenance of the pregnancy. Its synthesis begins around day 6 after fertilization, even before the syncytiotrophoblast is formed. hCG exhibits extensive (about 85%) sequence homology to LH, which is required for ovulation and maintenance of the corpus luteum during the menstrual cycle. Similar to the function of LH during the menstrual cycle, hCG maintains the corpus luteum during early pregnancy. hCG also possesses marked homology to thyroid-

stimulating

hormone

(TSH),

which may account for by stimulating the maternal thyroid gland to increase the secretion of tetraiodothyronine (T4). Measurement of hCG is used to detect early pregnancy and assess pregnancy viability. Two other clinical conditions that increase the blood levels of hCG include and . , also known as , is closely related to human growth hormone. Synthesized in the syncytiotrophoblast, it promotes general growth, regulates glucose metabolism, and stimulates mammary duct proliferation in the maternal breast. The effects of hCS on maternal metabolism are significant, but the role of this hormone in fetal development remains unknown. and are produced by and stimulate proliferation and differentiation of the cytotrophoblast. exhibits an age-dependent dual action on the early placenta. In the 4- to 5-week-old placenta, EGF is synthesized by the cytotrophoblast and stimulates proliferation of the trophoblast. In the 6- to 12week-old placenta, synthesis of EGF is shifted to the syncytiotrophoblast; it then stimulates and maintains the function of the differentiated trophoblast. is synthesized by decidual cells and is involved in the “softening” of the cervix and the pelvic ligaments in preparation for parturition. is synthesized by the syncytiotrophoblast, particularly during the last month of gestation. Leptin appears to regulate maternal nutrient storage to the nutrient requirements of the fetus. It is also involved in transporting nutrients across the placental barrier from the mother to the fetus. stimulate cytotrophoblastic growth (e.g., fibroblast growth factor [FGF], colony-stimulating factor [CSF-1], platelet-derived growth factor, and interleukins [IL-1 and IL-3]) or inhibit trophoblast growth and proliferation (e.g., tumor necrosis factor).

hyperthyroidism in pregnancy

diseases ectopic pregnancies Human chorionic somatomammotropin (hCS) placental lactogen (hPL)

IGF-I IGF-II Endothelial growth factor (EGF)

Relaxin Leptin

Other growth factors

trophoblastic human

VAGINA

The vagina is a fibromuscular tube that joins internal reproductive organs to the external environment. The vagina is a fibromuscular sheath extending from the cervix to the vestibule, which is the area between the labia minora. The opening into the vagina may be surrounded by the hymen, folds of mucous membrane extending into the vaginal lumen. The hymen or its remnants are derived from the endodermal membrane that separated the developing vagina from the cavity of the definitive urogenital sinus in the embryo. The (Fig. 23.30) consists of the following:

vaginal wall

FIGURE 23.30. Photomicrograph of a human vagina.

This lowmagnification hematoxylin and eosin (H&E)-stained specimen of the vaginal wall shows two of three layers of the vagina: the mucosal layer and the muscular layer (the outer layer, the adventitia, is not included). The mucosal layer consists of a stratified squamous epithelium and the underlying connective tissue. The epithelial–

connective tissue boundary is typically very irregular, with prominent papillae projecting into the undersurface of the epithelium. The muscular layer is seen only in part; it consists of irregularly arranged bundles of smooth muscle cells. Also, the deep region of the connective tissue contains a rich supply of blood vessels that supply the various layers of the vaginal wall. ×40.

mucosal layer

An inner has numerous transverse folds or rugae (see Fig. 23.1) and is lined with stratified squamous epithelium (Fig. 23.31). Connective tissue papillae from the underlying lamina propria project into the epithelial layer. In humans and other primates, keratohyalin granules may be present in the epithelial cells, but under normal conditions, keratinization does not occur. Therefore, nuclei can be seen in epithelial cells throughout the thickness of the epithelium.

FIGURE 23.31. Photomicrograph of the vaginal mucosa.

This micrograph, a higher magnification of Figure 23.30, shows the stratified squamous epithelium and mature cells with small pyknotic nuclei. Note a single layer of basal cells and two or three layers of cells undergoing differentiation (with eosinophilic cytoplasm). Projections of the connective tissue papillae into the epithelium

give the connective tissue–epithelial junction an uneven appearance. The tips of these projections often appear as isolated structures surrounded by epithelium ( ). ×180.

arrows

muscular layer

An intermediate is organized into two sometimes indistinct, intermingling smooth muscle layers, an outer longitudinal layer and an inner circular layer. The outer layer is continuous with the corresponding layer in the uterus and is much thicker than the inner layer. Striated muscle fibers of the bulbospongiosus muscle are present at the vaginal opening (Plate 23.10, page 974). An outer is organized into an inner dense connective tissue layer adjacent to the muscularis and an outer loose connective tissue layer that blends with the adventitia of the surrounding structures. The inner layer contains numerous elastic fibers that contribute to the elasticity and strength of the vaginal wall. The outer layer contains numerous blood and lymphatic vessels and nerves.

adventitial layer

The vagina possesses a stratified, squamous nonkeratinized epithelium and lacks glands. The lumen of the vagina is lined by stratified squamous, nonkeratinized epithelium. Its surface is lubricated mainly by mucus produced by the cervical glands. The greater and lesser vestibular glands located in the wall of the vaginal vestibule produce additional mucus that lubricates the vagina. Glands are not present in the wall of the vagina. The epithelium of the vagina undergoes during the menstrual cycle. Under the influence of estrogens, during the follicular phase, the epithelial cells synthesize and accumulate glycogen as they migrate toward the surface. Cells are continuously desquamated, but near or during the menstrual phase, the superficial layer of the vaginal epithelium may be shed. The exhibits two distinct regions. The outer region immediately below the epithelium is a highly cellular loose connective tissue. The deeper region, adjacent to the muscular layer, is denser and may be considered a submucosa. The deeper region contains many thin-walled veins that simulate erectile tissue during sexual arousal. Numerous elastic fibers

cyclic changes

lamina propria

are present immediately below the epithelium, and some of the fibers extend into the muscular layer. Many lymphocytes and leukocytes (particularly neutrophils) are found in the lamina propria and migrate into the epithelium. Solitary lymphatic nodules may also be present. The number of lymphocytes and leukocytes in the mucosa and vaginal lumen dramatically increases around the time of menstrual flow. The vagina has few general sensory nerve endings. The sensory nerve endings that are more plentiful in the lower third of the vagina are probably associated primarily with pain and stretch sensations.

EXTERNAL GENITALIA

female external genitalia

The consist of the structures located within the anterior (urogenital) triangle of the female perineum. These structures are collectively referred to as the [ ]. As the etymology of the word “vulva” implies, this region appears to be wrapped by skin folds. Structures included in the vulva consist of the , , , , and , (or hymenal caruncles after it is ruptured), and (Fig. 23.32). The vulva and its components extend from the mons pubis anteriorly to the posteriorly, which is a fibromuscular structure located in the midline of the perineum (anterior to the anus).

vulva Lat., volva, womb, female sexual organ; Lat., volvere, to turn, twist, roll, revolve, wrap mons pubis labia majora labia minora clitoral complex vestibule opening of the vagina hymen external urethral orifice perineal body

FIGURE 23.32. Schematic diagram illustrating the components of the female external genitalia (vulva). The left side of the vulva

shows intact structures that consist of the mons pubis, labium majus, labium minus, glans of the clitoris with associated prepuce and frenulum, vestibule and opening of the vagina, hymen, and external urethral orifice. The right side of the vulva is dissected and shows structures after the removal of skin and superficial fascia. Note the deep structures of the clitoral complex (body, crus of the clitoris, and the bulb of vestibule). The major vestibular (of Bartholin) gland is in close proximity to the distal ends of the .

vestibule

bulbs of the

Mons Pubis

Mons pubis is the rounded prominence over the pubic symphysis. The mons pubis is the rounded prominence over the pubic symphysis formed by an accumulation of subcutaneous adipose tissue. The mons pubis becomes more prominent with the onset of

puberty. Hair in the pubic area exhibits a characteristic growth pattern during puberty that can be used to evaluate the developmental stages of external genitalia. , also known as sexual maturity rating (SMR), is an objective system to track the development of secondary sex characteristics during puberty. follicle depth in the mons pubis is the greatest compared with other parts of the vulva. The skin in this area is rich in and that secret pheromone-like chemicals. In addition, the skin of the mons pubis contains an abundance of , including Meissner and Merkel corpuscles, free nerve endings, and Pacinian corpuscles.

Tanner staging

glands

Pubic hair sebaceous apocrine nerve endings

Labia Majora and Labia Minora

The labia majora and labia minora represent two folds of skin that form the lateral boundaries of the vestibule of the vagina. As mentioned earlier, during clinical examination, the vulva appears to be wrapped by two paired skin folds called the labia majora and labia minora. The labia majora and labia minora develop around cloacal membrane from the proliferating mesenchyme that forms the cloacal folds. These folds unite in the cranial portion to produce the (future clitoris). With the division of the cloaca into urogenital sinus and anorectal canal, the cloacal folds are likewise subdivided into and anal folds. At the same time, develop on both sides of the urogenital folds. Labioscrotal swellings in the female embryo develop into labia majora, and the urogenital folds do not fuse (as they do in the male embryo) and instead develop into the

genital tubercle

urogenital folds labioscrotal swellings

labia minora that border urogenital cleft (the vertical fissure of the vulva that develops into the vestibule of vagina). The development of external genitalia is a complex process driven by the sequential expression of regulatory encoding growth factors, especially .

Wnt genes fibroblast growth factor (FGF) labia majora [sing. labium majus] are two prominent

The longitudinal folds of skin that extend from the mons pubis anteriorly and to the perineal body posteriorly. The anterior parts of the labia majora unite beneath the mons pubis to form the . The posterior parts of the labia majora gradually merge with the surrounding skin of the perineum at their (see Fig. 23.32). The labia majora form the outer lateral boundaries of the urogenital cleft and are separated by the interlabial sulci from the labia minora. The size and contour of the labia are related to the amount of underlying adipose tissue deposits. During puberty, the labia majora increase in size, and after menopause, the labia atrophy due to decreased estrogen and loss of adipose tissue.

anterior labial commissure posterior labial commissure

stratified outer hair

The skin of the labia majora is covered by . Sebaceous, eccrine, and apocrine sweat glands are present on both surfaces. The of the labium majus is darker than the inner surface and, like that of the mons pubis, has deeply imbedded (Fig. 23.33a). Sebaceous glands associated with hair follicles are prominent, and they discharge their holocrine secretion into pilosebaceous canals. An abundance of apocrine sweat glands in the skin of labia majora discharge their secretion directly into the hair follicles (Fig 23.33b). In addition to sebaceous and apocrine sweat glands, the labia majora have many eccrine sweat glands. The thin skin of the is smooth and devoid of hair. An abundance of is found on the inner surface of labia majora. Their ducts open directly on the surface of the epithelium (Fig. 23.34). The skin of the labia majora is rich in , including Markel and Meissner corpuscles for touch, Ruffini corpuscles for stretch

squamous keratinized epithelium surface follicles

inner surface sebaceous glands

sensory receptors

and torque, Pacinian corpuscles for pressure, and free nerve endings for pain. Below the skin surface, the labia majora contain a thin layer of smooth muscle (dartos tunic) resembling the dartos muscle of the scrotum (see Fig. 23.33a) and a large amount of subcutaneous adipose tissue. The lie deep into the adipose tissue beneath the labia majora (page 942). They are homologous to the bulb of the penis in males and represent erectile tissue. They are covered by the bulbospongiosus muscle, a striated muscle that belongs to superficial muscles of the perineum.

bulbs

of the vestibule

FIGURE 23.33. Photomicrograph of the labium majus. a.

This low-magnification hematoxylin and eosin (H&E)-stained specimen shows tissue excised from the midportion of the outer surface of the labium majus. This surface shows the stratified squamous keratinized epithelium with two deeply embedded hair follicles. Sebaceous glands associated with the hair follicles are prominent, and they discharge their holocrine secretion into pilosebaceous canals. Below the skin surface, the labia majora contain a thin layer of smooth muscle resembling the dartos muscle of the scrotum. At this low magnification, dispersed smooth muscles bundle are difficult to identify in this layer. Note accumulations of subcutaneous adipose tissue in the hypodermis. ×25. In addition to sebaceous glands, the labia majora have many eccrine sweat glands and an abundance of apocrine sweat glands that discharge their secretion directly into the hair follicles. ×260. (Reprinted with permission from Hanley KZ. Vulva. In: Mills SM, ed. . Wolters Kluwer; 2020:1031–1046.)

b.

Histology for Pathologists

FIGURE 23.34. Photomicrograph of the inner surface of the labia majora. This low-power hematoxylin and eosin (H&E)-stained specimen of the labia majora’s inner surface shows its stratified squamous epithelium ( Ep ) with no visible signs of keratinization and abundant sebaceous glands ( SG ). Two sebaceous ducts ( SD ) are also evident.

Note

the

continuity

of

the

duct

epithelium

with

the

epithelium of the skin and the sebaceous gland epithelium. At this magnification, several smooth muscle bundles can just barely be discerned ( ).

arrows

major vestibular (Bartholin) glands

The oval are in close proximity to the distal ends of the bulbs of the vestibule. This paired tubuloalveolar gland corresponds to the male bulbourethral (Cowper glands). The secretory acini are lined by simple columnar cells that produce mucus secretions (Fig. 23.35) that lubricate the vestibulum and distal end of the vagina during sexual intercourse. The basal domain of secretory acini is ensheathed by myoepithelial cells. The secretion of the gland is discharged into the long (~2.5 cm) that opens into the 4 o’clock and 8 o’clock positions (posterior lateral aspect) of the vestibule of the vagina. The epithelium of the duct transitions from simple cuboidal (near the acini) to stratified cuboidal and stratified squamous epithelium at the opening into the vestibule (see Fig. 23.35). is a condition in which the major vestibular glands become infected and inflamed, and it may lead to the development of bacterial cysts or abscesses. If the duct of the Bartholin gland becomes obstructed, it usually dilates and fills with a secretion produced by the gland that may form a Symptoms include severe pain, redness, swelling of the involved labium majus, and, sometimes, fever. On physical examination, the affected labium majus appears red and enlarged. A large abscess requires surgical incision with drainage or complete excision.

Bartholin gland duct

Bartholinitis

cyst.

Bartholin

FIGURE 23.35. Photomicrograph of the major vestibular (Bartholin) gland. This low-magnification hematoxylin and eosin

(H&E)-stained specimen shows mucus acini lined by simple columnar cells that produce mucus secretions. Note that nuclei of secretory mucus cells are positioned at their basal domain ( ). The basal domain of secretory acini is ensheathed by myoepithelial cells (not visible at this magnification). The gland’s secretion is discharged into the Bartholin gland duct, visible in the of the image. The epithelium of the duct transitions from simple cuboidal (near the acini) to stratified cuboidal and stratified squamous epithelium at the opening into the vestibule. The gland is embedded in the dense irregular connective tissue ( ) of the labia majora. ×60. (Reprinted with permission from Hanley KZ. Vulva. In: Mills SM, ed. . Wolters Kluwer; 2020:1031 –1046.)

arrowheads

center

Histology for Pathologists

labia minora sing. labium minus

CT

The [ ] are paired, hairless folds of skin that border the vestibule. They lie immediately lateral to the vaginal vestibule and medial to the labia majora. Upon reaching the clitoris, the anterior end of the labia minora splits into two parts. The more anterior part travels above (anterior) to the body of the clitoris, forming the , whereas the posterior

prepuce (hood) of the clitoris

frenulum of the frenulum of the labia

part crossing below the clitoris forms the . The posterior ends of the labia minora terminate as they fuse into a skin fold called the , or fourchette. In most cases, the labia minora are not symmetrical and may protrude beyond the labia majora. The skin of the labia minora contains that exhibits a small degree of keratinization (Fig. 23.36). The epithelium lacks skin appendages; however, the lateral surface of the labia minora may contain sweat glands. In some individuals, large sebaceous glands are present in the stroma; their secretion is directly released on the surface of the labia minora. Abundant is present in the deep cells of the epithelium. The core of connective tissue within each fold is devoid of adipose tissue but does contain numerous blood vessels and fine . The labia minora are highly innervated structures that directly connect to the glans of the clitoris and the erectile tissues of the bulb of the vestibule located deep into the labia majora. The labia minora have the highest concentration of Meissner and Merkel corpuscles for sensitivity to light touch of the entire vulva (see Fig. 23.36). A high density of Pacinian corpuscles for pressure reception and free nerve endings are also found in the labia minora. Enlarged can cause functional problems related to menstrual hygiene and irritation from clothing. In specific conditions, labial alteration procedures (e.g., ) for medical indications (e.g., treatment of labial hypertrophy or asymmetrical labial growth or chronic irritation) can be performed. These procedures are sometimes performed for cosmetic purposes despite the lack of highquality data about their safety and long-term outcomes. Potential complications of labiaplasty include pain, infection, and scarring. Patients who are dissatisfied with the appearance of their labia minora can be counseled about normal variations in the color, size, and shape of external genitalia.

clitoris minora

stratified squamous

epithelium

melanin pigment

elastic fibers

labia minora

labiaplasty

FIGURE 23.36. Photomicrograph of a labium minus.

This lowmagnification hematoxylin and eosin (H&E)-stained specimen shows a section through the labium minus that includes both the lateral and medial surfaces. Each labium minus represents a fold of thin skin with a stratified squamous epithelium demonstrating a small degree of keratinization on the surface. The epithelium lacks skin appendages. Abundant melanin pigment is present in the cells in the basal layer of the epithelium; however, it is difficult to discern them at this low magnification. The core of dense regular connective tissue within the labia minora is devoid of adipose tissue but does contain numerous blood vessels ( ), elastic fibers, and nerves. Note a Meissner corpuscle residing in the dermal papilla. ×60. (Reprinted with permission from Hanley KZ. Vulva. In: Mills SM, ed. . Wolters Kluwer; 2020:1031–1046.)

BV

Pathologists

Clitoral Complex

Histology for

The clitoral complex, composed of the glans, body, crura of the clitoris, and bulbs of the vestibule, is a female erectile organ that is sexually responsive. The clitoral complex is composed of the glans of the clitoris, body of the clitoris, left and right crus of the clitoris, and two bulbs of the vestibule. In recent descriptions, the

clitoral complex is often compared to the “tip of the iceberg” because the glans of the clitoris, the only visible part of this complex, represents only 10% of the complex’s total size; the remainder is embedded deep into the vulva. The clitoral complex is well innervated, containing many nerve endings, especially Pacinian corpuscles. Other touch receptors (Meissner and Merkel corpuscles) are present in reduced numbers compared with the mons pubis or labia majora. Free nerve endings (pain receptors) are present in relatively high concentrations. The clitoral complex comprises two histologically distinct types of that consist of:

specialized vascular tissue

Erectile tissue, which is found in the body and crura of the clitoris and bulbs of the vestibule. The body of the clitoris (as seen in cross section in Fig. 23.37) contains numerous wide, irregularly shaped vascular spaces lined with vascular endothelium. These spaces are surrounded by connective tissue trabeculae with a thin layer of smooth muscle. Irregular smooth muscle bundles are frequently observed as “subendothelial cushions” surrounding irregular vascular spaces. During sexual arousal, the erectile tissue allows for distension with blood and volume expansion. The vascular spaces increase in size and rigidity by filling with blood, principally derived from the divisions of internal pudendal arteries. These include deep dorsal artery of the clitoris that is positioned at the dorsal aspect of the body of the clitoris, deep artery of the clitoris running within the erectile tissue of the body of the clitoris, and artery to the bulb of the vestibule that enters the deep surface of the bulb.

FIGURE 23.37. Schematic diagram and a photomicrograph of the body of the clitoris in cross section. a. The diagram shows a

midsection through the body of the clitoris. The body of the clitoris is not completely surrounded by the skin; instead, it has contact with divisions originating from the proximal ends of the labia minora. On the dorsal and lateral surfaces, the body is covered by the prepuce of the clitoris (hood), which emerged from the anterior division of the labia minora. The ventral part of the body is in close proximity to the frenulum that originated from the posterior division of the labia minora. Corpora cavernosa, the erectile tissue of the clitoris, is surrounded by a thick fibrous layer of tunica albuginea. Note that the corpora cavernosa are separated by the incomplete septum, which is closely associated with deep arteries of the clitoris; they are visible on both sides of the septum. The fascia of the clitoris surrounds the corpora cavernosa and the neurovascular bundle located at the dorsal aspect of the clitoris. Note the arrangement of deep dorsal arteries, deep dorsal veins, and dorsal nerves of the clitoris, which lie between the tunica albuginea and the fascia of the clitoris. The superficial dorsal vein of the clitoris resides in the superficial fascia. . This low-power photomicrograph hematoxylin and eosin (H&E)-stained specimen shows the body of the clitoris that corresponds to the drawing. The specimen has been removed through the incision of the prepuce and separated from the skin and fascial layers. Note the well-defined tunica albuginea and erectile vascular spaces incompletely separated by the septum. Vascular spaces contain numerous irregularly shaped channels lined by vascular endothelium. These spaces are defined by the connective tissue trabeculae with a thin layer of smooth muscle. The neurovascular bundle is visible on the dorsal aspect of the body of the clitoris. ×10. (Courtesy of Dr. Karen Pinder from the University of British Columbia, Vancouver, Canada.)

b

Nonerectile vascular tissue, which is found in the glans of

the clitoris. This tissue is characterized by a high density of blood vessels dispersed within a dense irregular connective tissue stroma containing a minimal amount of smooth muscle. During sexual arousal, increased blood flow is observed; however, the tissue does not undergo physical expansion. This type of nonerectile, sexually responsive vascular tissue also surrounds the urethral orifice and is present within the labia minora and the adventitia of the vaginal wall.

body of the clitoris is composed of paired conjoined erectile bodies, which originate from extensions of the crura of the clitoris. These bodies are surrounded by tunica albuginea, a thin fibroelastic connective tissue sheath (see The

Fig. 23.37). A connective tissue septum between the paired erectile bodies is incomplete, and vascular spaces communicate with each other at the dorsal aspect of the clitoris. The adventitia of the blends on both sides with the connective tissue of the septum. The body of the clitoris with investing tunica albuginea is surrounded by the inner layer of the fascia, called the , that is continuous with the deep perineal fascia investing the perineal muscles, vestibule of vagina, and crura of the clitoris. , and course between the tunica albuginea and the fascia of the clitoris (see Fig. 23.37). The outer layer is a part of the superficial (Colles) fascia of the perineum and may contain the superficial dorsal vein of the clitoris. The body of the clitoris is attached to the pubic symphysis by the connective tissue . They attach to the clitoral complex at the angle of the clitoris, a transition point (>90 degree) between the crura and the body of the clitoris. As mentioned in the section on the labia minora (see page 941), the body of the clitoris does not have circumferential skin coverage; instead, the skin of the prepuce and the frenulum of the clitoris flare laterally to the labia minora (see Fig. 23.37).

deep arteries of the clitoris

fascia of the clitoris

Deep dorsal arteries, deep dorsal veins nerves of the clitoris suspensory ligaments

dorsal

glans clitoris

The , an oval tubercle at the tip of the corpora cavernosa (average length 8 mm and width 4 mm), is formed by nonerectile vascular tissue. The skin over the glans is very thin, forms the prepuce of the clitoris, and as discussed earlier, contains numerous sensory nerve endings (see page 941). The two are extensions of erectile tissues of the body of the clitoris that firmly anchor the clitoris to the pubic rami. They are about 36 mm in length and 7 mm in width (as measured in magnetic resonance imaging [MRI] scans) and are composed of erectile tissue surrounded by tunica albuginea, consistent with that of the body of the clitoris. At the place of bony attachment, the tunica albuginea fuses with the fibrous layer of the periosteum of pubic rami. The unattached surface of the crura is invested by a thin layer of ischiocavernosus muscle (a superficial striated muscle of the perineum) that is covered by a deep layer of the perineal fascia. The are paired structures formed from the erectile tissue similar to that described in the body of the clitoris with some modifications. The erectile tissue of a bulb has more pronounced fibromuscular trabeculae and more prominent bundles of smooth muscles located just under the epithelium of the vascular spaces. In addition, a distinct tunica albuginea is often missing in the bulbs. They have a single origin from the inferior aspect of the body of the clitoris. After splitting into right and left structures, they travel posteriorly on the lateral side of the urethra and vagina and medially to the crura of the clitoris. They are firmly attached to the perineal membrane just deep into the labia majora and are covered by the thin layer of the bulbospongiosus muscle (a superficial striated muscle of the perineum). During sexual excitation, the bulb of the vestibule becomes engorged with blood. This significant enlargement of the bulbs of vestibule during sexual arousal is a result of the tunica albuginea absence.

crura of the clitoris

bulbs of the vestibule

Vestibule of the Vagina

The vestibule of the vagina is the middle part of the vulva that contains the external urethral orifice, opening of the vagina, and multiple openings of a variety of secretory glands. The vestibule of the vagina is a somewhat concave space

positioned in the middle part of the vulva that extends from the frenulum of the clitoris anteriorly to the frenulum of the labia minora posteriorly. Its lateral borders are formed by the labia minora and majora. The vestibule is lined with endodermally derived , which, on the lateral sides, blends with ectodermally derived stratified squamous keratinized epithelium of the labia minora and majora, prepuce of the clitoris, and frenulum of the labia minora. The epithelium of the vestibule is rich in and is similar to that found in the vagina. The vestibule of the vagina contains the vaginal opening, which may be partially covered by the hymen, and the external urethral meatus. Numerous small mucous glands, the (often incorrectly called Skene glands), open directly into the mucosa of the vestibule. They are simple tubular glands lined by mucous-secreting columnar cells that merge with the stratified squamous nonkeratinized epithelium of the vestibule. In addition, as discussed earlier, the ducts of the major vestibular (Bartholin) glands (see page 940 and Fig. 23.35) and glands surrounding the urethra with prominent paraurethral (Skene) glands (see page 944) discharge their secretions into the vestibule. The mucosa of the vestibule has multiple sensory and pain receptors similar to those found in other regions of the vulva. The may undergo similar to that described for the cervix (see page 126). Metaplastic epithelium may completely replace the glandular epithelium, forming . Obstruction of the glands may also produce a due to the accumulation of the mucous secretion.

stratified squamous nonkeratinized

epithelium

glycogen

vestibular glands

squamous metaplasia

lesser

lesser vestibular glands

vestibular clefts vulvar mucous cyst The hymen is a thin mucous membrane at the entrance to the vagina.

hymen

The is the thin, fibrous tissue plate that surrounds and partially covers the entrance to the vagina, leaving an opening for vaginal and menstrual discharge. When it is ruptured, small tag–like elevations known as are present at the opening of the vagina. Embryologically, the hymen is derived from the that separates the developing vagina from the cavity of the in the embryo. Thus, it marks the inferior boundary of the vagina and the superior boundary of the vestibule. Both vaginal and vestibular surfaces of the hymen are covered by a containing stratified squamous nonkeratinized epithelium, which is similar to that of the vaginal epithelium (Fig. 23.38). The lighter staining of epithelium on the vaginal surface reflects larger amounts of glycogen stored in the epithelial cells. The border between epithelium and underlying lamina propria is clearly defined by the closely packed small cells of the stratum basale (see Fig.23.38). Deep connective tissue papillae are present on both vaginal and vestibular surfaces and are formed by invaginations of underlying lamina propria into the epithelium. The vaginal surface appears smooth; however, the vestibular surface may contain small projections, known as (see Fig. 23.38). Dense irregular connective tissue containing a rich network of blood vessels with a few nerve fibers is present in the core of the hymen. The hymen has a limited number of nerve receptors. Only Merkel corpuscles and free nerve endings are present in the hymenal ring. The size, shape, and degree of the hymenal opening vary greatly among individuals. The hymen may be imperforate, round, annular, septate, or cribriform. In cases of , the hymen lacks the opening between the vagina and the vestibulum. If this condition is diagnosed during puberty, it may lead to progressive accumulation of menstrual discharge that results in distension of the vagina ( ).

sinus

hymenal caruncles endodermal membrane definitive urogenital

mucosa

vestibular papillae

imperforate hymen

hematocolpos

FIGURE 23.38. Photomicrograph of the hymen.

This low-magnification hematoxylin and eosin (H&E)-stained specimen shows the section of the hymen that includes the free edge of the hymenal ring. Note a gradual transition of the stratified squamous nonkeratinized epithelium from the vaginal surface (vaginal mucosa) to the vestibular surface (vestibular mucosa). The lighter staining of epithelium on the vaginal surface reflects larger amounts of glycogen stored in the epithelial cells. The vaginal surface also appears smooth. The vestibular surface stains darker (less glycogen content) and may contain vestibular papillae, small surface projections caused by projections of underlying lamina propria into the epithelium. The border between epithelium and underlying lamina propria is clearly defined by closely packed small cells of the stratum basale ( ). Dense irregular connective tissue containing blood vessels is present in the core of the hymen. ×45. (Reprinted with permission from Hanley KZ. Vulva. In: Mills SM, ed. . Wolters Kluwer; 2020:1031– 1046.)

arrows

Histology for Pathologists

Glandular tissue surrounding the female urethra shares immunohistochemical characteristics with the prostate gland

in males. The external urethral orifice is part of the vestibulum of the

vagina and is located between the glans of the clitoris and the opening of the vagina. In most cases, the lumen of the urethra is lined by transitional epithelium (urothelium) that changes at the external urethra orifice into stratified squamous nonkeratinized epithelium of the vaginal vestibule. However, stratified columnar and pseudostratified epithelium have also been reported in the female urethra. The glandular tissue around the urethra is represented by small . These glands are present in the wall of the urethra and open with the short ducts throughout the entire length of the urethral lumen being more numerous at the distal half of the urethra. Glandular tissue associated with the urethra has been given various names, such as glands of Littré, glands of Huffman, glands of Morgani, intermural glans of the urethra, and periurethral or paraurethral glans. These names are used interchangeably, reflecting the lack of consensus among researchers. However, two larger accumulations of glandular tissue often described in the literature, known as the , are located near the distal end of the urethra. They have longer and more prominent ducts (1.5 cm in length) that empty in the distal part of the urethra. These ducts are lined by the stratified (two layers thick) cuboidal epithelium. In multiparas, where the urethral meatus can be considerably distended, the duct of Skene glands appears visible on the side and deep into the urethral orifice. The entire glandular tissue complex surrounding the urethra exhibits a similar histologic appearance. All glands are of mixed branched tubuloalveolar type lined by the simple columnar epithelium with occasional nests of pseudocolumnar epithelium with basal cells. A prominent fibromuscular stroma separates glandular profiles. Immunohistochemical analysis reveals that all glandular tissue surrounding the lumen of the urethra stain positively for prostate-specific antigen (PSA), prostatic acid phosphatase (PAP), prostate-specific alkaline phosphatase (PSAkP), and androgen receptor (AR), leading some researchers to characterize the periurethral glandular tissue as the

periurethral glands

paraurethral glands (of Skene)

female

prostate. Also, physiologic studies determined that the urethra is the source of secretions that can occur during sexual excitement. Biochemical analysis suggests that this secretion is similar in composition to male ejaculate.

MAMMARY GLANDS mammary glands

The , or breasts, are a distinguishing feature of mammals. They are structurally dynamic organs, varying with age, menstrual cycle, and reproductive status of the female. During embryologic development, growth and development of breast tissue occur in both sexes. Multiple glands develop along paired epidermal thickenings called that extend from the developing axilla to the developing inguinal region. In humans, normally, only one group of cells develops into a breast on each side. An extra breast ( ) or nipple ( ) may occur as an inheritable condition in about 1% of the female population. These relatively rare conditions may also occur in men.

mammary ridges (milk

lines)

polymastia

polythelia

In females, mammary glands develop under the influence of sex hormones.

Until puberty, both females’ and males’ mammary glands develop in similar manner. At the onset of puberty in males, testosterone acts on the mesenchymal cells to inhibit further growth of the mammary gland. At the same time, the mammary glands undergo further development under hormonal influence of estrogen and progesterone. Estrogen stimulates further development of mesenchymal cells. The mammary gland increases in size, mainly due to the growth of interlobular adipose tissue. The ducts extend and branch into the expanding connective tissue stroma. Proliferation of epithelial cells is controlled by interactions between the epithelium and the specialized intralobular hormone–sensitive loose connective tissue stroma. By adulthood, the complete ductal architecture of the gland has been established.

FOLDER 23.5

CLINICAL CORRELATION: CERVICAL CYTOLOGY: THE PAP TEST The examination of samples of cervical cells is a valuable diagnostic tool in evaluating the vaginal and cervical mucosae (Fig. F23.5.1). Superficial epithelial cells are removed from the mucosa, added to a liquid medium, and sent to a laboratory for microscopic examination. Before the advent of liquid-based cervical cytology, cervical cell samples were spread on glass slides and stained with the Papanicolaou (Pap) stain (a combination of hematoxylin, orange G, and eosin azure). Cervical cytology provides valuable diagnostic information about the epithelium regarding pathologic changes, response to hormonal changes during the menstrual cycle, and the microbial environment of the vagina.

FIGURE F23.5.1. Photomicrographs of cervical cytology. a.

Negative cervical cytology. The surface squamous cells reveal small pyknotic nuclei and abundant cytoplasm. Other cells in the micrograph include red blood cells and neutrophils. ×600. Abnormal cytology. Many of the cells in this specimen contain large nuclei with no evidence of pyknosis ( ). The cytoplasm is relatively scant. Other cells exhibit a more normal appearance with pyknotic nuclei and more surrounding cytoplasm ( ). Neutrophils are also present. ×600.

arrows

b.

arrowheads

Cervical cytology results are reported using the Bethesda system. This system stratifies results into three major categories:

Negative for intraepithelial lesions or malignancy : This

category includes both normal results and results that indicate infectious organisms or other nonneoplastic changes. : This category is used to report the presence of benignappearing endometrial cells in a female aged 45 years or older. This finding may indicate an endometrial abnormality. : This category is used to report both squamous and glandular cell abnormalities.

Other

Epithelial cell abnormalities

Nonneoplastic Changes

The synthesis and release of glycogen by the epithelial cells of the uterus and vagina are directly related to changes in the pH of vaginal fluid. The pH of the fluid, which is normally low, around pH 4, becomes more acid near midcycle as , a lactic acid–forming bacterium in the vagina, metabolizes the secreted glycogen. An alkaline environment can favor the growth of infectious agents such as , , , and , causing an abnormal increase in vaginal transexudates and inflammation of the vaginal mucosa and vulvar skin known as . These pathologic conditions are readily diagnosed with cervical cytology. Specific antimicrobial agents (antibiotics, sulfonamides, and antifungals) may be used together with nonspecific therapy (acidified 0.1% hexetidine gel) to restore the normal low pH in the vagina and thus prevent the growth of these agents.

Lactobacillus acidophilus

Trichomonas vaginalis

Staphylococci Corynebacterium vaginale Candida albicans

vulvovaginitis

Epithelial Cell Abnormalities

Cervical cytology is widely used to screen for precancer stages of the cervix. Because precancerous cervical lesions may exist for as long as 20 years, the abnormal cells shed from the epithelium are easily detected with cervical cytology. Microscopic examination of these cells permits differentiation between normal and abnormal cells, determines their site of origin, and allows classifying cellular changes related to the spread of the disease. In the Bethesda system, epithelial lesions are differentiated as either squamous or glandular into the following categories: Squamous cells Atypical squamous cells Low-grade squamous intraepithelial lesion High-grade squamous intraepithelial lesion Squamous cell carcinoma Glandular cells Atypical (endocervical, endometrial, or glandular cells) Endocervical adenocarcinoma in situ Adenocarcinoma The Pap test is an extremely effective and inexpensive screening method for preventing cervical cancer. Because most of the cell abnormalities detected by Pap tests are in the precancerous stage, it allows prompt treatment and follow-up that is able to prevent the onset of invasive cervical cancer. Since the 1950s, when cervical cytology was first introduced, deaths from cervical cancer in the United States have declined ~70%. However, in areas where cervical cancer screening is not as widely used, cervical cancer deaths remain high.

inactive state

The mammary glands remain in an until pregnancy, during which the mammary glands assume their complete morphologic and functional maturation. This occurs in response to estrogens and progesterone initially secreted from the corpus luteum and later from placenta, prolactin (PRL) from pituitary gland, and gonadocorticoids produced by the adrenal cortex. By the end of pregnancy, secretory vesicles are found in the epithelial cells, but milk production is inhibited by high levels of progesterone. The actual initiation of occurs immediately after birth and is induced by secreted by the adenohypophysis. The ejection of milk from the breast is stimulated by released from the neurohypophysis. With the change in the hormonal environment at menopause, the glandular component of the breast regresses or involutes and is replaced by fat and connective tissue. In men, some additional development of the mammary glands normally occurs after puberty, and the glands remain rudimentary. Hormonal exposure and genetic predisposition are the major risk factors for the development of . It is the most common malignancy in women in the United States. Each year, an estimated nearly 270,000 women (and also 2,500 men) are diagnosed with breast cancer. Most breast cancers are linked to hormonal exposure (which increases with age, early menarche, late menopause, and with older age of a first fullterm pregnancy). About 5%–10% of all breast cancers are attributable to mutation in autosomal dominant .

milk

secretion prolactin (PRL)

oxytocin

breast cancer

breast cancer genes (BRCA1 and BRCA2) Mammary glands are modified tubuloalveolar apocrine sweat glands. The tubuloalveolar mammary glands, derived from modified sweat

glands in the epidermis, lie in the subcutaneous tissue. The inactive adult mammary gland is composed of 15–20 irregular lobes separated by fibrous bands of connective tissue. They radiate from the , or , and are further subdivided into numerous lobules known as (Fig. 23.39). Some of the fibrous bands, called

units (TDLUs)

mammary papilla

nipple terminal duct lobular

suspensory

Cooper ligaments

or , connect with the dermis. Abundant adipose tissue is present in the dense connective tissue of the interlobular spaces.

FIGURE 23.39. Schematic drawing of the human breast as seen during lactation. The breast is composed largely of terminal duct lobular units (TDLUs) containing branched tubuloalveolar glands. TDLUs are contained within an extensive connective tissue stroma and variable amounts of adipose tissue.

lactiferous duct nipple lactiferous sinus

Each gland ends in a that opens through a constricted orifice into the nipple. Beneath the , the pigmented area surrounding the , each duct has a dilated portion, the . Near their openings, the lactiferous ducts are lined with stratified squamous keratinized epithelium. The epithelial lining of the duct shows a gradual transition from stratified squamous to two layers of cuboidal cells in the lactiferous sinus and finally to a single layer of columnar or cuboidal cells through the remainder of the duct system. The epidermis of the adult nipple and areola is highly pigmented and somewhat wrinkled and has long dermal papillae invading its deep surface (Fig. 23.40). It is covered by keratinized stratified squamous epithelium. The pigmentation of the nipple increases at puberty, and the nipple becomes more prominent. During pregnancy, the areola becomes larger, and the degree of pigmentation increases further. Deep to the areola and nipple, bundles of smooth muscle fibers are arranged radially and circumferentially in the dense connective tissue and longitudinally along the lactiferous ducts. These muscle fibers allow the nipple to become erect in response to various stimuli.

areola

FIGURE 23.40. Photomicrographs of a section through the female nipple. a. This low-magnification micrograph of a hematoxylin and

eosin (H&E)-stained sagittal section through the nipple shows the wrinkled surface contour, a thin stratified squamous epithelium, and associated sebaceous glands ( ). The core of the nipple consists of dense connective tissue, smooth muscle bundles, and the lactiferous ducts that open at the nipple surface. ×6. The wall of one of the lactiferous ducts is shown here at higher magnification. Its epithelium is stratified cuboidal, consisting of two-cell layers. As it approaches the tip of the nipple, it changes to a stratified squamous epithelium and becomes continuous with the epidermis. ×175. A higher magnification of the sebaceous gland from the in . Note how the glandular epithelium is continuous with the epidermis ( ), and the sebum is being secreted onto the epidermal surface. ×90. A higher magnification showing bundles of smooth muscle in longitudinal and cross-sectional profiles. ×350.

arrows

b.

c. a arrows d.

rectangle

areola

The contains sebaceous glands, sweat glands, and modified mammary glands ( ). These glands have a structure intermediate between that of sweat glands and true mammary glands, and they produce small elevations on the surface of the areola. It is believed that Montgomery glands produce a lubricating and protective secretion that changes the skin’s pH and discourages microbial growth. Numerous sensory nerve endings are present in the nipple; the areola contains fewer sensory nerve endings.

Montgomery glands

The terminal duct lobular unit (TDLU) of the mammary gland represents a cluster of small secretory alveoli (in a lactating gland) or terminal ductules (in an inactive gland) surrounded by intralobular stroma. Successive branching of lactiferous ducts leads to the terminal duct lobular unit (TDLU). Each TDLU represents a grape-like cluster of small alveoli that forms a lobule (Fig. 23.41) and consists of the following:

FIGURE 23.41. Terminal duct lobular unit. a.

This schematic diagram shows components of the terminal duct lobular unit ( ). Terminal ductules and the intralobular collecting duct are surrounded by a specialized hormonally sensitive loose connective tissue called . are separated from each other by interlobular stroma containing a variable amount of dense irregular connective tissue and adipose tissue. In active mammary glands, terminal ductules differentiate into milk-producing alveoli. This photomicrograph shows the from an inactive mammary gland. The clear area in the of the image represents adipose cells. ×120.

TDLU

intralobular stroma TDLUs

TDLU upper part

b.

Terminal ductules

are present in the inactive gland. During pregnancy and after childbirth, the epithelium of the terminal ductules, which is lined by secretory cells, differentiates into fully functional secretory alveoli that produce milk. The carries alveolar secretions into the lactiferous duct. The is a specialized, hormonally sensitive loose connective tissue that surrounds the terminal ductules and alveoli. The intralobular connective tissue contains few adipose cells.

intralobular collecting duct intralobular stroma

Glandular epithelial and myoepithelial cells are the most important cells associated with mammary ducts and lobules.

Glandular epithelial cells line the duct system, whereas myoepithelial cells lie deep within the epithelium between the

epithelial cells and the basal lamina. These cells, arranged in a basket-like network, are present in the secretory portions of the gland. In a routine hematoxylin and eosin (H&E) preparation, the myoepithelial cells are more apparent in the larger ducts. However, in an immunocytochemical preparation, their discontinuous, basket-like arrangement is better visualized within the alveoli (Fig. 23.42). Contraction of myoepithelial cells assists in during lactation. Recent immunofluorescence studies have proven that breast progenitor cells found in the ductular epithelium give rise to both glandular cells of the alveoli and myoepithelial cells.

milk ejection

FIGURE 23.42. Myoepithelial cells in the mammary gland.

This immunofluorescence image is obtained from the mammary gland of a lactating mouse 2 days postparturition. The mouse carries a transgene composed of the smooth muscle α-actin promoter conjugated to enhance the green fluorescent protein (GFP) reaction. Three-dimensional organization of myoepithelial cells is visualized in due to the expression of the promoter transgene in myoepithelial cells. The tissue was also stained with antibody against smooth muscle α-actin conjugated directly with CY3 fluorescent dye. The staining results from overlapping of the and staining. The cells on the surface of the terminal duct lobular unit are stained , whereas those deeper in the tissue stained only because the antibody did not penetrate deep into the tissue. Note a small intralobular duct that merges into the larger lactiferous duct. ×600. (Courtesy Dr. James J. Tomasek, University of Oklahoma Health Science Center.)

color orange

red

green color

green

red green

orange

The morphology of the secretory portion of the mammary gland varies with the menstrual cycle. In the inactive gland, the glandular component is sparse and consists chiefly of duct elements (Fig. 23.43 and Plate 23.11, page 976). During the menstrual cycle, the inactive breast undergoes slight cyclic changes. Early in the follicular phase, the intralobular stroma is less dense, and terminal ductules appear as cords formed by the cuboidal-shaped epithelial cells with little or no lumen. During the luteal phase, the epithelial cells increase in height, and lumina appear in the ducts as small amounts of secretions accumulate. Also, fluid accumulates in the connective tissue. This is followed by abrupt involution and apoptosis during the last few days of the menstrual cycle before the onset of menstruation.

FIGURE 23.43. Photomicrograph of an inactive mammary gland. a.

This low-magnification hematoxylin and eosin (H&E)-stained specimen shows several lobules within the dense connective tissue of the breast. The epithelial component consists of a branching duct system that makes up the lobule. The clear areas ( ) are adipose cells. ×60. A higher magnification of the area in the of . The epithelial cells of the ducts are columnar and exhibit interspersed lymphocytes ( ) that have entered the epithelium. The surrounding

b.

arrows

arrows

rectangle

a

arrowheads

stained material ( ) represents the myoepithelial cells ( and collagen bundles in the adjacent connective tissue. ×700.

MEp)

Mammary glands undergo dramatic proliferation and development during pregnancy. The mammary glands exhibit a number of changes in preparation for lactation. These can be examined during the trimester of pregnancy.

First trimester

is characterized by elongation and branching of the terminal ductules. The lining epithelial and myoepithelial cells proliferate and differentiate from breast progenitor cells found in the epithelium of terminal ductules. Myoepithelial cells proliferate between the base of the epithelial cells and the basal lamina in both the alveolar and ductal portions of the gland. is characterized by differentiation of alveoli from the growing ends of the terminal ductules. The development of the glandular tissue is not uniform, and variation in the degree of development is seen even within a single lobule. The cells vary in shape from flattened to low columnar. Plasma cells, lymphocytes, and eosinophils infiltrate the intralobular connective tissue stroma as the breast develops (Plate 23.12, page 978). At this stage, the amount of glandular tissue and mass of the breast increase, mainly due to the growth of the alveoli (Fig. 23.44).

Second trimester

FIGURE 23.44. Photomicrograph of an active mammary gland during late pregnancy. a. This low-magnification hematoxylin and

eosin (H&E)-stained specimen shows the marked proliferation of the duct system giving rise to the secretory alveoli that constitute the major portion of the lobules. The intralobular ducts are difficult to identify because their epithelium also secretes. Outside the lobules is a large excretory duct. ×60. A higher magnification of an area in . The secretory alveolar cells are mostly cuboidal here. A myoepithelial cell ( ) as well as a number of plasma cells ( ) can be identified in the adjacent loose connective tissue. ×700.

arrows

a

mEp

b.

Third trimester

commences maturation of the alveoli. The epithelial glandular cells become cuboidal, with nuclei positioned at the basal cell surface. They develop an extensive rER; secretory vesicles and lipid droplets appear in the cytoplasm. The actual proliferation of the interlobular stromal cells declines, and subsequent enlargement of the breast occurs through hypertrophy of the secretory cells and accumulation of secretory product in the alveoli. The changes in glandular tissue during pregnancy are accompanied by a decrease in the amount of connective tissue

and adipose tissue.

Both merocrine and apocrine secretion are involved in the production of milk. The secreting cells contain abundant granular endoplasmic reticulum, a moderate number of large mitochondria, a supranuclear Golgi apparatus, and a number of dense lysosomes (Fig. 23.45). Depending on the secretory state, large lipid droplets and secretory vesicles may be present in the apical cytoplasm. The secretory cells produce two distinct products that are released by different mechanisms.

FIGURE 23.45. Photomicrographs and diagram of a lactating mammary gland. a. Low-magnification micrograph of a fast-green

osmium–stained section of a lactating mammary gland. Portions of several large lobules and an excretory duct are seen. Many of the alveoli exhibit a prominent lumen, even at this magnification. ×60. A higher magnification of an area in shows lipid droplets ( circular profiles) within the secretory cells of the alveoli as well as in the alveolar lumina. The indicate plasma cells within the interstitial spaces. ×480. Diagram of a lactating mammary gland epithelial cell. , rough-surfaced endoplasmic reticulum. (Redrawn after Bloom W, Fawcett DW. . 10th ed. WB Saunders; 1975.)

b.

rER

arrows

a

c. A Textbook of Histology

black

Merocrine secretion.

The protein component of the milk is synthesized in the rER, packaged into membrane-limited secretory vesicles for transport in the Golgi apparatus, and released from the cell by fusion of the vesicle’s limiting membrane with the plasma membrane. . The fatty or lipid component of the milk arises as lipid droplets that are free in the cytoplasm. The lipid coalesces to form large droplets that pass to the apical region of the cell and project into the lumen of the acinus. The droplets are invested with an envelope of plasma membrane as they are released. A thin layer of cytoplasm is trapped between the plasma membrane and lipid droplet and is released with the lipid, but the cytoplasmic loss in this process is minimal.

Apocrine secretion

The secretion released in the first few days after childbirth is known as . This premilk is an alkaline, yellowish secretion with a higher protein, vitamin A, sodium, and chloride content and a lower lipid, carbohydrate, and potassium content than milk. It contains considerable amounts of antibodies (mainly ) that provide the newborn with some degree of passive immunity. The antibodies in the colostrum are believed to be produced by the lymphocytes and plasma cells that infiltrate the loose connective tissue of the breast during its proliferation and development and are secreted across the glandular cells as in salivary glands and intestine. As these wandering cells decrease in number after parturition, the production of colostrum stops, and lipid-rich milk is produced.

colostrum

secretory IgA

Hormonal Regulation of the Mammary Gland The initial growth and development of the mammary gland at puberty occur under the influence of estrogens and progesterone produced by the maturing ovary. Under hormonal influence, the TDLUs develop and differentiate into dynamic functional units. Subsequent to this initial development, slight changes in the morphology of the glandular tissue occur during each ovarian cycle. During the follicular phase of the menstrual cycle,

estrogen in the circulation stimulates proliferation of the lactiferous duct components. After ovulation in the luteal phase, progesterone stimulates the growth of alveoli; intralobular stroma becomes edematous. Clinically, during the luteal phase, tenderness and a progressive increase in breast tissue mass may be felt. During pregnancy, the corpus luteum and placenta continuously produce estrogens and progesterone, causing a massive increase in TDLUs. It is now believed that the growth of the mammary glands also depends on the presence of PRL, which is produced by the anterior lobe of the pituitary gland (adenohypophysis); hCS, which is produced by the placenta; and adrenal glucocorticoids.

FOLDER 23.6

CLINICAL CORRELATION: CERVICAL CANCER AND HUMAN PAPILLOMAVIRUS INFECTION Human papillomavirus (HPV) is the most common sexually transmitted virus in the United States. Most individuals will become infected with HPV during their lifetime. More than 30 HPV types are known to infect the urogenital and anal regions of both sexes, targeting the stratified squamous epithelium of the perineal skin or mucous membranes. About 12 types of HPV, called HPV types, cause . Most cases of genital warts are caused by HPV types 6 and 11. Approximately 13 types of HPV, called HPV types, can cause cancer of the cervix, anus, vagina, vulva, penis, and head and neck. Most cases of HPV-associated cancer are caused by HPV types 16 and 18. HPV infection usually resolves on its own. Persistent infection develops in only a small percentage (5%–10%) of cases. Risk factors for persistent infection include older age and smoking. Persistent infection with high-risk HPV types is associated with an increased risk of cervical cancer. Most HPV-associated lesions can be diagnosed by cervical cytology. In difficult cases, ancillary techniques such as in situ hybridization can help confirm the diagnosis (Fig. F23.6.1). An HPV vaccine, called , is available for the prevention of HPV. Gardasil 9 protects against types 6, 11, 16, and 18 and five other high-risk HPV types. Vaccinations against HPV virus are recommended for both sexes between the ages of 9 and 26 years.

high-risk

genital warts

Gardasil 9

low-risk

FIGURE F23.6.1. Photomicrograph of in situ hybridization of a human cervical biopsy showing human papillomavirus (HPV) infection. a. This low-magnification photomicrograph shows stratified squamous epithelium of the cervix hybridized with DNA probes to HPV types 6 and 11 and counterstained with nuclear fast red. Note that the majority of infected cells are mature cells located in the upper layers of the stratified squamous epithelium of the ectocervix. ×120. This higher magnification photomicrograph shows viral particles stained within the nuclei of infected cells. ×225. (Courtesy of Dr. Fabiola Medeiros.)

b.

purple

None of the vaccines are therapeutic (i.e., they do not clear prior infection), but both lead to the development of specific immunity against HPV infections. The vaccines are most effective in those who have had no prior HPV exposure and who are immunized before initiation of sexual activity. Cervical cancer screening now includes HPV testing in addition to cervical cytology for certain age groups. The following are the most current for people who have no other risks factors for cervical cancer from the American College of Obstetricians and Gynecologists:

cervical cancer screening recommendations

Age 21 years and younger: no screening Ages 21–29 years: cervical cytology (Pap test) every 3 years Ages 30–65 years: primary HPV testing alone every 5 years or HPV testing plus cervical cytology every 5 years OR cervical cytology every 3 years The increased interval between screening tests (as opposed to annual testing) and the avoidance of HPV testing in younger

individuals are recommended to decrease the number of falsepositive results and unnecessary diagnostic procedures. HPV infection usually resolves spontaneously in younger patients. When infections do persist, they usually do so for many years before precancerous changes can be detected. Diagnosis of precancer of the cervix includes , in which a colposcope (a specifically designed microscope to magnify the view of the cervix) is used to examine the cervix for lesions and to perform cervical biopsy. Treatment for precancerous lesions is accomplished by removal of the precancerous cells by , a procedure in which a cone-shaped tissue of the cervix containing part of ectocervix, transformation zone, and cervical canal is excised or destroyed. Conization can be performed with a scalpel (cold-knife conization), laser, electrosurgical loop, or freezing (cryoconization).

colposcopy

biopsy (conization)

cone

Lactation is under the neurohormonal control of the adenohypophysis and hypothalamus. Although estrogen and progesterone are essential for the physical development of the breast during pregnancy, both of these hormones also suppress the effects of PRL and hCS, the levels of which increase as pregnancy progresses. Immediately after childbirth, however, the sudden loss of estrogen and progesterone secretion from the placenta and corpus luteum allows PRL to assume its lactogenic role. Production of milk also requires adequate secretion of growth hormones, adrenal glucocorticoids, and parathyroid hormones. The act of during breastfeeding initiates sensory impulses from receptors in the nipple to the hypothalamus. The impulses inhibit the release of prolactin-inhibiting factor, and is then released from the adenohypophysis. The sensory impulses also cause the release of in the neurohypophysis. Oxytocin stimulates the myoepithelial cells that surround the base of the alveolar secretory cells and the base of the cells in the larger ducts, causing them to contract and eject the milk from the alveoli and the ducts. In the , secretion of milk ceases, and the mammary glands begin to regress and atrophy. The glandular tissue then returns to an inactive condition.

suckling

prolactin

absence of suckling

oxytocin

Involution of the Mammary Gland After menopause, the specialized involutes, causing the gland to

stroma of the mammary gland atrophy. In the absence of ovarian hormone stimulation, the secretory cells of the TDLUs degenerate and disappear, leaving only ducts to create a histologic pattern that resembles that of the male breast. The connective tissue also demonstrates degenerative changes, marked by a decrease in the number of fibroblasts and collagen fibers and loss of elastic fibers.

FOLDER 23.7

FUNCTIONAL CONSIDERATIONS: LACTATION AND INFERTILITY Almost 50% of females who fully breastfeed exhibit lactational amenorrhea (lack of menstruation during lactation) and infertility. This effect is caused by high levels of serum prolactin, which inhibit secretion of pulsatile gonadotropinreleasing hormone (GnRH), thus suppressing secretion of the luteinizing hormone (LH). Ovulation usually resumes after 6 months or earlier with a decrease in suckling frequency.

Blood Supply and Lymphatics The arteries that supply the breast are derived from the thoracic branches of the , the , and anterior intercostal arteries. Branches of the vessels pass primarily along the path of the alveolar ducts as they reach capillary beds surrounding the alveoli. In general, veins basically follow the path of the arteries as they return to the axillary and internal thoracic veins. Lymphatic capillaries are located in the connective tissue surrounding the alveoli. The larger lymphatic vessels drain into , , or .

thoracic artery

axillary artery

axillary supraclavicular

Innervation

internal

parasternal lymph nodes

The nerves that supply the breast are anterior and lateral cutaneous branches from the second to sixth . The nerves convey afferent and sympathetic fibers to and from

intercostal nerves

the breast. The secretory function is primarily under hormonal control, but afferent impulses associated with suckling are involved in the reflex secretion of PRL and oxytocin.

FEMALE REPRODUCTIVE SYSTEM

OVERVIEW OF THE FEMALE REPRODUCTIVE SYSTEM

internal

The female reproductive system consists of (ovaries, uterine tubes, uterus, and vagina) and (vulva). Internal female reproductive organs undergo regular cyclic changes during each , from to , that reflect changes in hormone levels.

female reproductive organs external genitalia menstrual cycle menopause

puberty

OVARY The major functions of the ovaries are the production of gametes ( ) and the production of steroid hormones (estrogen and progesterone; ). Ovaries have a in their center that contains loose connective tissue, nerves, blood and lymphatic vessels, and a on their periphery that contains a large number of that provide a microenvironment for developing oocytes. The surface of the ovary is covered by , which is a single cuboidal epithelium that overlies a dense layer of connective tissue called .

oogenesis steroidogenesis medulla cortex ovarian follicles germinal

epithelium tunica albuginea

OVARIAN FOLLICLE DEVELOPMENT

ovarian

There are three basic developmental stages of an : , (both primary and secondary), and . Before puberty, the cortex of an ovary is occupied only by . They contain a single primary oocyte that is arrested in the first meiotic prophase and is surrounded by a single layer of . After puberty following cyclic hormonal changes, a selected cohort of primary follicles develops into . Follicle cells surrounding the oocyte become cuboidal and undergo further stratification to form the . Cells of the growing follicle develop into ; connective tissue surrounding the follicle differentiates into the and ; and the oocyte grows and produces the (ZP) that contains specific ZP glycoproteins involved in the fertilization process. As proliferate, they become involved in steroid hormone metabolism (conversion of androgens produced by the theca interna into estrogens) and actively secrete follicular fluid that accumulates in cavities between the granulosa cells. A growing follicle that contains a single fluid cavity ( ) is called the . It still contains the primary oocyte arrested in the first prophase of meiotic division. As the enlarges and undergoes further maturation, the thin layer of granulosa cells that is associated with the oocyte forms the and . The has a large antrum and a prominent, steroid-producing theca interna layer. Triggered by an LH surge just before ovulation, the

follicle

primordial growing mature (Graafian) follicle primordial follicles squamous follicle cells growing follicles primary follicle cells theca interna

granulosa theca externa zona pellucida

granulosa cells

antrum

secondary (antral) follicle

secondary follicle

corona radiata mature (Graafian) follicle

cumulus oophorus

oocyte resumes its first meiotic division and becomes the .

secondary oocyte

OVULATION During the ovarian cycle, usually, only one Graafian follicle undergoes . All other follicles in the developing cohort undergo , a process of degeneration involving apoptosis. During ovulation, a is released from the ruptured Graafian follicle. The released oocyte is arrested in the metaphase of the second meiotic division. At ovulation, the , composed of the remaining granulosa and thecal cells, is transformed into the . Under the influence of LH in the process of , the (produce estrogen) and the (produce progesterone) are formed. The is formed in the absence of fertilization; it degenerates 10–12 days after ovulation to become the . The is formed after fertilization and implantation; it is a major source of and (estrogen, insulin-like growth factor) during the first 8 weeks of pregnancy, after which it degenerates and leaves a permanent scar in the ovary.

ovulation follicular atresia secondary oocyte

follicular wall corpus luteum luteinization granulosa lutein cells theca lutein cells corpus luteum of menstruation corpus albicans corpus luteum of pregnancy progesterone luteotropins

CAPACITATION AND FERTILIZATION capacitation

During , the mature spermatozoa acquire the ability to fertilize the oocyte within the female reproductive tract. normally occurs in the ampulla of the uterine tube; it involves the capacitation of

Fertilization

spermatozoa and their penetration of the corona radiata to reach the oocyte. After capacitation, the spermatozoa bind to the , which trigger the . Enzymes released from the acrosome allow a single spermatozoon to penetrate the zona pellucida and . During impregnation, the entire spermatozoon, except for the tail plasma membrane, becomes incorporated into the ooplasm, which triggers resumption of the (transforms the secondary oocyte into a mature oocyte). At least three types of prevent other spermatozoa from entering the oocyte: fast depolarization of the oolemma, the cortical reaction (changes of polarity of the oolemma), and the zona reaction (which forms a by crosslinking proteins on the oocyte surface and degrading ZP receptors). The sperm head within the oocyte cytoplasm undergoes changes to form the . Nuclear membranes of both break down without fusion to form a diploid . The zygote immediately enters its first mitotic division.

zona acrosome

pellucida receptors reaction impregnate the oocyte

second meiotic

division

postfusion reactions

perivitelline barrier

male pronucleus male and female pronuclei zygote

UTERINE TUBES

uterine tubes

The are paired bilateral structures that connect the uterus with the ovaries. Each uterine tube has four segments: (a funnel-shaped end surrounded by adjacent to the ovary), (common site of fertilization), (narrow segment adjacent to the uterus), and part (traversing the uterine wall). The uterine tube wall consists of three layers: external , thick , and highly folded .

ampulla

serosa

muscularis

infundibulum fimbriae isthmus intramural mucosa

mucosal lining (peg) cells

The is simple columnar epithelium composed of two cell types: and . The oocyte (and zygote after fertilization) is propelled into the uterine cavity by a coordinated movement of cilia on the surface of mucosa and peristaltic muscular contractions of the uterine tube.

ciliated

UTERUS

uterus

nonciliated

body

The is divided into the (upper portion containing ) and the (lower portion that projects into the vagina). The uterine wall is composed of (lining mucosa of the uterus), (smooth muscular layer), and (a serous layer of visceral peritoneum). The is lined by simple columnar epithelium that invaginates into the underlying lamina propria ( ), forming . The endometrium consists of and , which undergoes cyclic changes due to fluctuating levels of estrogens and progesterone during the menstrual cycle. The thickness of the endometrium, its glandular activity, and its vascular pattern are unique for each of the three phases ( , , and ) of the , which lasts an average of 28 days. The is influenced by estrogens produced by the growing follicles; the is influenced by progesterone secreted from the corpus luteum; and if no implantation occurs, the represents ischemia of the stratum functionale that is being sloughed off during menses. If the embryo implants successfully, the undergoes (the process of conversion to

fundus

perimetrium endometrium endometrial stroma functionale

cervix

endometrium myometrium uterine glands stratum basale

stratum

proliferative secretory menstrual menstrual cycle proliferative phase secretory phase menstrual phase endometrium decidualization

decidua )

and, together with the trophoblastic cells from the embryo, initiates the development of the . The endometrium of the differs from the rest of the uterus in that it is not sloughed off during menstruation. modify the viscosity of the secreted mucus during each menstrual cycle. The part of the cervix projecting into the vagina has a where simple columnar epithelium of the cervix changes abruptly into stratified squamous epithelium of the vagina.

cervix Cervical glands

placenta

transformation zone

PLACENTA

placenta

The allows for exchange of gases and metabolites between the maternal and fetal circulations; it consists of a ( ) and a ( ). After implantation, the invading differentiates into the (multinucleate cytoplasmic mass that actively invades the decidua) and the (a mitotically active layer producing cells that fuse with the syncytiotrophoblast). Fetal and maternal blood remain separated by the , which develops in the (projections of chorion containing syncytiotrophoblast, occasional cytotrophoblast cells, mesenchymal connective tissue, and fetal blood vessels). Villi are immersed in the maternal blood that fills vascular spaces in the placenta ( ). The placenta is a major that supports the development of the fetus; it produces both (mainly progesterone) and (e.g., hCG, hCS, relaxin, and leptin).

fetal portion chorion maternal portion decidua basalis trophoblast syncytiotrophoblast cytotrophoblast placental barrier chorionic villi

hormones

tertiary

cotyledons endocrine organ steroid protein hormones

VAGINA AND EXTERNAL GENITALIA

vagina stratified squamous nonkeratinized epithelium Female external genitalia vulva mons pubis labia majora labia minora clitoral complex vestibule opening of the vagina hymen external urethral orifice clitoral complex glans of the clitoris body of the clitoris left and right crus of the clitoris bulbs of the vestibule

The extends from the cervix to the vestibule; it is lined with and lacks glands. ( ) consist of the (formed by underlying adipose tissue), (longitudinal folds of skin containing adipose tissue, a thin layer of smooth muscle, and sebaceous and sweat glands), (core of connective tissue devoid of adipose tissue but contains large sebaceous glands), , (lined with stratified squamous epithelium with numerous small mucous glands), , , and . The is composed of the , , , and two . They possess erectile and nonerectile vascular tissue homologous to the penis.

MAMMARY GLANDS

mammary glands develop in ridges in the embryo, but The

mammary

both sexes from they undergo further development after puberty due to the hormonal influence of estrogen and progesterone. are modified tubuloalveolar apocrine sweat glands consisting of . Each TDLU is connected to collecting duct systems, which form the that open at the . The TDLU of the mammary gland represents a cluster of small (in active lactating gland) or (in inactive gland) surrounded by a hormonally sensitive .

Mammary glands terminal duct lobular units (TDLUs) lactiferous ducts nipple secretory alveoli terminal ductules intralobular stroma

inactive

The morphology of the secretory portion of the varies with the menstrual cycle. Mammary glands undergo dramatic proliferation and development during pregnancy in preparation for under the influence of estrogen (proliferation of duct components) and progesterone (growth of alveoli). The protein component of milk is released by alveolar cells using , whereas the lipid component of milk is released by .

mammary gland lactation

merocrine secretion apocrine secretion

PLATE 23.1 OVARY I

ovaries are small, paired, ovoid structures cortex and medulla when sectioned. On one side The

that exhibit a is a hilum for the transit of neurovascular structures; on this same side is a mesovarium that joins the ovary to the broad ligament. The functions of the ovary are the production of and the synthesis and secretion of and . In the cortex are numerous that are present at the time of birth and that remain unchanged until sexual maturation. The oogonia in these follicles are arrested in prophase of the first meiotic division. At puberty, under the influence of pituitary gonadotropins, the ovaries begin to undergo the cyclical changes designated the . During each cycle, the ovaries normally produce a single oocyte that is ready for fertilization. At the beginning of the ovarian cycle, under the influence of pituitary follicle-stimulating hormone (FSH), some of the primordial follicles begin to undergo changes that lead to the development of a . These changes include proliferation of follicle cells and enlargement of the follicle. Although several primordial follicles begin these developmental changes, usually, only one reaches maturity and yields an oocyte. Occasionally, two follicles will mature and ovulate, leading to the possibility of dizygotic twin development. The discharge of the oocyte and its adherent cells is called . At ovulation, the oocyte completes the first meiotic division. Only if fertilization occurs does the oocyte complete the second meiotic division. Whether or not fertilization occurs, the other follicles that began to

ova estrogen progesterone primordial follicles

ovarian cycle

mature (Graafian) follicle

ovulation

proliferate in the same cycle degenerate, a process referred to as .

atresia

Cortex

, ovary, monkey, hematoxylin and eosin (H&E) ×120.

cortex germinal epithelium GEp

The of an ovary from a sexually mature individual is shown here. On the surface, there is a single layer of epithelial cells designated the ( ). This epithelium is continuous with the serosa (peritoneum) of the mesovarium. Contrary to its name, the epithelium does not give rise to the germ cells. The germinal epithelium covers a dense fibrous connective tissue layer, the ( ); under the tunica albuginea are the ( ). It is not unusual to see follicles at various stages of development or atresia in the ovary. In this figure, along with the large number of primordial follicles, there are four ( ), with a clearly visible eosinophilic layer of the zona pellucida ( ), an atretic follicle ( ), and part of a large follicle on the . The region of the large follicle shown in the figure includes the theca interna ( ), granulosa cells ( ), and part of the antrum ( ).

tunica albuginea TA primordial follicles PF growing follicles ZPGF A

right

TI

AF

Early primary follicles ×450.

GC

, ovary, monkey, H&E

primordial follicle

When a begins the changes leading to the formation of a mature follicle, the layer of squamous follicle cells becomes cuboidal ( ), as in this figure. In addition, the follicle cells proliferate and become multilayered. A follicle undergoing these early changes is called a . Thus, an early primary follicle may still be unilaminar, but it is surrounded by cuboidal cells, and this distinguishes it from the more numerous unilaminar primordial follicles that are surrounded by squamous cells. Note the large nucleus ( ) of the oocyte in the primary follicle. Some oocytes may not have visible nuclei (see cell label ) due to the plane of section.

primary follicle

N

Primordial follicles

X

, ovary, monkey, H&E ×450.

FC

primordial follicles

This figure shows several at higher magnification. Each follicle consists of an oocyte surrounded by a single layer of squamous follicle cells ( ). The nucleus ( ) of the oocyte is typically large, but the oocyte itself is so large that the nucleus is often not included in the plane of section, as in the oocyte marked . The group of epithelioidappearing cells ( ) are follicle cells of a primordial follicle that has been sectioned in a plane that just grazes the follicular surface. In this case, the follicle cells are seen en face.

F

N

X arrowhead

Late primary follicle ×450.

, ovary, monkey, H&E

primary follicle follicle cells FC

The in this figure shows a multilayered mass of ( ) surrounding the oocyte with a clearly visible large nucleus ( ). The innermost layer of follicle cells is adjacent to a thick eosinophilic layer of extracellular homogeneous material called the ( ). At this stage of development, the oocyte has also enlarged slightly. The entire structure surrounded by the zona pellucida is actually the oocyte. Surrounding the follicles are elongate cells of the highly cellular connective tissue, referred to as . The stromal cells surrounding a secondary follicle become disposed into two layers designated the theca interna and the theca externa. As seen in the previous figure, stromal cells become epithelioid in the cell-rich theca interna ( ).

zona pellucida ZP

stromal cells

TI

A, antrum AF, atretic follicle F, follicle cells, primordial FC, follicle cells GC, granulosa cells GEp, germinal epithelium GF, growing follicles N, nucleus of oocyte PF, primordial follicles TA, tunica albuginea TI, theca interna X, oocyte showing only cytoplasm ZP, zona pellucida arrowhead, follicle cells seen en face

N

PLATE 23.2 OVARY II Atresia

of follicles is a regular event in the ovary, beginning in embryonic life. In any section through the postpubertal ovary, follicles of various stages can be seen undergoing atresia. In atresia, the initial changes involve pyknosis of the nuclei of the follicle cells and dissolution of their cytoplasm. The follicle is then invaded by macrophages and other connective tissue cells. The oocyte degenerates, leaving behind the prominent zona pellucida. This may fold inward or collapse, but it usually retains its thickness and staining characteristics. When included in the plane of section, a distorted zona pellucida serves as a reliable diagnostic feature of an . In atresia of large, nearly mature follicles, cells of the theca interna remain to form clusters of epithelioid cells in the ovarian cortex. These are referred to collectively as and continue to secrete steroid hormones.

atretic follicle

interstitial glands

Secondary follicles

, ovary, monkey, hematoxylin and eosin (H&E) ×120. Two follicles growing under the influence of follicle-stimulating hormone (FSH) are shown in the figure on the . The more advanced follicle is a . The oocyte in this follicle is surrounded by several layers of ( ) that, at this stage, are identified as granulosa cells. At a slightly earlier time, small lakes of fluid formed between the follicle cells, and these lakes have now fused into a well-defined larger cavity called the ( ), which is evident in the figure. The antrum is also filled with fluid and stains with the periodic acid–Schiff (PAS) reaction, although only lightly. The substance that stains with the PAS reaction has been retained as an eosinophilic precipitate in the antra of the secondary follicles shown here and in the figure on the . Immediately above the obvious secondary follicle is a slightly smaller follicle. Because no antral spaces are evident between the follicle cells, it is appropriate to classify it as a . In both follicles, but particularly in the larger follicle with the antrum, the surrounding stromal cells have become altered to form two distinctive layers designated ( ) and ( ). The theca interna is a more cellular layer, and the cells are epithelioid. When

left

secondary follicle

follicle cells FC

follicular antrum FA right

primary follicle theca interna TI theca externa TE

seen with the electron microscope, they display the characteristics of endocrine cells, particularly steroid-secreting cells. In contrast, the theca externa is a connective tissue layer. Its cells are more or less spindle shaped. In the figure on the , a later stage in the growth of the secondary follicle is shown. The antrum ( ) is larger, and the oocyte is off to one side, surrounded by a mound of follicle cells called the . The remaining follicle cells that surround the antral cavity are referred to as the ( ), or simply granulosa cells.

right

FA

cumulus oophorus

membrana granulosa MG

Atretic follicle

, ovary, monkey, H&E ×65.

Atretic follicles AF

( ) are shown here and at higher magnification in the adjacent figure on the . The two smaller atretic follicles can be identified by virtue of the retained zona pellucida ( ) labeled on the adjacent figure to the . The two larger, more advanced follicles do not display the remains of a zona pellucida, but they do display other features of follicular atresia.  

right ZP

right

 

Atretic follicles

, ovary, monkey, H&E ×120.

During atresia of a more advanced follicle, the follicle cells tend to degenerate more rapidly than the cells of the theca interna, and the basement membrane separating the two becomes thickened to form a hyalinized membrane, the glassy membrane. Thus, the ( ) separates an outer layer of remaining theca interna cells from the degenerating inner follicle cells. The remaining theca interna cells may show cytologic integrity ( ); these intact theca cells remain temporarily functional in steroid secretion.  

glassy membrane arrows RTI

Atretic follicles

, ovary, monkey, H&E ×120.

atretic follicles AF

Additional ( ) are shown here. Again, some show remnants of a zona pellucida ( ), and two show a glassy membrane ( ). Note that although the atresia in these follicles is well advanced, some of the cells external to one of the glassy membranes still retain their epithelioid character ( ). These are persisting theca interna cells.  

arrows

arrowhead

AF, atretic follicle FA, antrum of follicle FC, follicle cells MG, membrana granulosa RTI, remaining theca interna cells TE, theca externa TI, theca interna ZP, zona pellucida arrowhead, persisting theca interna cells arrows, glassy membrane

ZP

PLATE 23.3 CORPUS LUTEUM After the oocyte and its immediately surrounding cells (i.e., the cells of the cumulus oophorus) are discharged from the mature ovarian follicle (ovulation), the remaining follicle cells (membrana granulosa) and the adjacent theca interna cells differentiate into a new functional unit, the . The cells of the corpus luteum, luteal cells, rapidly increase in size and become filled with lipid droplets. A lipid-soluble pigment in the cytoplasm of the cells, lipochrome, gives them their yellow appearance in fresh tissue. Electron micrographs of the luteal cells demonstrate that they have features typical of steroid-secreting cells, namely, abundant smooth-surfaced endoplasmic reticulum and mitochondria with tubular cristae. Two types of luteal cells are identified: Large, centrally located are derived from the granulosa cells; smaller, peripherally located are derived from the theca interna. A rich vascular network is established in the corpus luteum into which progesterone and estrogen are secreted by the lutein cells. These hormones stimulate the growth and differentiation of the uterine endometrium to prepare it for implantation of a fertilized ovum.

corpus luteum

granulosa lutein cells

theca lutein cells

Corpus luteum

, ovary, human, hematoxylin and

eosin (H&E) ×20.

cortex

This figure shows ovarian shortly after ovulation. The points toward the surface of the ovary at the site of ovulation. The cavity ( ) of the former follicle has been invaded by connective tissue ( ). The membrana granulosa has become plicated, and the granulosa cells, now transforming into cells of the corpus luteum, are called ( ). The plication of the membrana granulosa begins just before ovulation and continues as the corpus luteum develops. As the corpus luteum becomes more plicated, the former follicular cavity becomes reduced in size. At the same time, blood vessels ( ) from the theca of the follicle invade the former cavity and the transforming membrana granulosa cells. Cells of the theca interna follow the blood vessels into the outermost depressions of the plicated structure. These theca interna cells become transformed into cells of the corpus luteum called .

arrowhead

CT

FC

granulosa lutein cells TC BV

cells

theca lutein

Corpus luteum

, ovary, human, H&E ×20.

corpus luteum granulosa lutein

A portion of a fully formed is shown here. Most endocrine cells are the ( ). These form a folded cell mass that surrounds the remains of the former follicular cavity ( ). External to the corpus luteum is the connective tissue of the ovary ( ) with a large quantity of blood vessels ( ). Keep in mind that the theca interna is derived from the connective tissue stroma of the ovary. The location of ( ) reflects this origin, and these cells can be found in the deep outer recesses of the glandular mass, adjacent to the surrounding connective tissue.

cells GLC FC

CT

theca lutein cells

BV TLC

Corpus luteum

, ovary, human, H&E ×65 (on left) and ×240 (on right).

corpus luteum

A segment of the plicated is shown in the figure on the at slightly higher magnification. As noted earlier, the main cell mass is composed of ( ). On one side of this cell mass is the connective tissue ( ) within the former follicular cavity; on the other side are the theca lutein cells. The same arrangement of cells is shown in the figure on the at much higher magnification. The granulosa lutein cells contain a large, spherical nucleus (see also , in the figure on that ) and a large amount of cytoplasm. The cytoplasm contains a yellow pigment (usually not evident in routine H&E sections), hence the name, corpus luteum. ( ) also contain a spherical nucleus, but the cells are smaller than the granulosa lutein cells. Thus, when identifying the two cell types, aside from location, note that the nuclei of adjacent theca lutein cells generally appear to be closer to each other than nuclei of adjacent granulosa lutein cells. The connective tissue ( ) and small blood vessels that invaded the mass of granulosa lutein cells can be identified as the flattened and elongated components between the granulosa lutein cells. The changes whereby the ruptured ovarian follicle is transformed into a corpus luteum occur under the influence of pituitary luteinizing hormone. In turn, the corpus luteum itself secretes progesterone, which has a profound effect on the estrogen-primed uterus. If pregnancy occurs, the corpus luteum remains functional; if pregnancy does not occur, the corpus luteum regresses after having reached a point of peak development, roughly 2 weeks after ovulation. The regressing cellular components of the corpus luteum are replaced

left

granulosa lutein cells GLC CT GLC

Theca lutein cells TLC CT

right right

by fibrous connective tissue, and the structure is then called a .

corpus albicans

BV, blood vessels CT, connective tissue FC, former follicular cavity GLC, granulosa lutein cells TC, granulosa cells transforming into corpus luteum cells TLC, theca lutein cells

PLATE 23.4 UTERINE TUBE uterine tubes

The (oviducts, fallopian tubes) are joined to the uterus and extend to the ovaries, where they present an open flared end (abdominal ostium) for entry of the ovum at ovulation. The oviduct undergoes cyclical changes along with those of the uterus, but these are not nearly as pronounced. The epithelial cells increase in height during the middle of the cycle, just about the time the ovum will be passing through the tube and become reduced during the premenstrual period. Some of the epithelial cells are ciliated. The epithelial cells depend on the ovaries for their viability. Not only does the number of ciliated cells increase during the follicular phase of the ovarian cycle, but removal of the ovaries leads to atrophy of the epithelium and loss of ciliated cells. The uterine tube varies in size and degree of mucosal folding along its length. The mucosal folds are evident in its distal portion, the infundibulum, as it nears the open end. Near the opening, the tube flares outward and is called the . It has fringed folded edges called . The infundibulum leads proximally to the , which constitutes about two-thirds of the length of the oviduct, has the most numerous and complex mucosal folds, and is the site of fertilization. Mucosal folds are least numerous at the proximal end of the oviduct, near the uterus, where the tube is narrow and referred to as the . A or portion measures about 1 cm in length and passes through the uterine wall to empty into the uterine cavity. Fertilization of the ovum usually occurs in the distal portion of the ampulla. For the first several days of development, as it navigates the complex pathway created by the mucosal folds, the embryo is transported proximally by the beating of the cilia of the ciliated epithelial cells and by peristaltic contractions of the well-developed muscularis layer that underlies the mucosa.

infundibulum

fimbria

ampulla

isthmus

Uterine tube

uterine

intramural

, human, hematoxylin and eosin

(H&E) ×40.

ampulla

A cross section through the of the uterine tube is shown here. Many mucosal folds project into the lumen ( ), and the complicated nature of the folds is evident by the variety of profiles that are seen. In addition to the ( ), the remainder of the wall consists of a ( ) and connective tissue.

L

mucosa Muc

muscularis Mus

The muscularis consists of smooth muscle that forms a relatively thick layer of circular fibers and a thinner outer layer of longitudinal fibers. The layers are not clearly delineated, and no sharp boundary separates them.

Mucosal fold

, uterine tube, human, H&E ×160;

inset ×320.

rectangle

The area enclosed by the in the previous figure is shown here at higher magnification. The specimen shows a longitudinal section through a lymphatic vessel ( ). In other planes of section, the lymphatic vessels are difficult to identify. The fortuitously sectioned lymphatic vessel is seen in the core of the , along with a highly cellular connective tissue ( ) and the blood vessels ( ) within the connective tissue. The epithelium ( ) lining the mucosa is shown in the . The ciliated cells are readily identified by the presence of well-formed cilia ( ). Nonciliated cells, also called ( ), are readily identified by the absence of cilia; moreover, they have elongate nuclei and sometimes appear to be squeezed between the ciliated cells. The connective tissue ( ) contains cells whose nuclei are arranged typically in a random manner. They vary in shape, being elongated, oval, or round. Their cytoplasm cannot be distinguished from the intercellular material ( ). The character of the connective tissue is essentially the same from the epithelium to the muscularis, and for this reason, no submucosa is described.

Lym

mucosal fold Ep

BV

inset peg cells PC

CT

inset

BV, blood vessels C, cilia CT, connective tissue Ep, epithelium L, lumen Lym, lymphatic vessel Muc, mucosa Mus, muscularis PC, peg cells

CT

C

PLATE 23.5 UTERUS I uterus

The is a hollow, pear-shaped organ with a thick wall and, in the nonpregnant state, a narrow cavity. The uterine wall is composed of a mucosa, referred to as the ; a muscularis, referred to as the ; and, externally, a serosal cover, the . The myometrium consists of smooth muscle and connective tissue and contains the large blood vessels that give rise to the vessels that supply the endometrium. The uterus undergoes cyclical changes that are largely manifested by changes that occur in the endometrium. If implantation of a fertilized ovum does not occur after preparation for this event, the state of readiness is not maintained, and much of the endometrium degenerates and is sloughed off, constituting the menstrual flow. The part of the endometrium that is lost is referred to as the ; the part that is retained is called the . The stratum basale is the deeper part of the endometrium and adjoins the myometrium. The myometrium also undergoes changes associated with implantation of a zygote. In the nonpregnant uterus, the smooth muscle cells are about 50 μm in length; during pregnancy, they undergo enormous hypertrophy, often reaching >500 μm in length. In addition, new muscle fibers develop after division of existing muscle cells and division and differentiation of undifferentiated mesenchymal cells. The connective tissue also increases to strengthen the uterine wall. Fibroblasts increase by division and secrete additional collagen and elastic fibers. After parturition, the uterus nearly returns to its normal size. Most muscle fibers return to their normal size, and some degenerate. Collagen secreted during pregnancy is digested by the very cells that secreted it, the fibroblasts. Similar, but less pronounced, proliferation and degeneration of fibroblasts and collagen occur in each menstrual cycle.

myometrium perimetrium

functionale basale

endometrium

stratum stratum

Uterus

, human, hematoxylin and eosin (H&E) ×25; inset ×120.

stratum functionale SF stratum basale SB

After the ( ) is sloughed off, resurfacing of endometrium occurs. The epithelial resurfacing comes from the glands that remain in the ( ). The gland epithelium simply proliferates and grows over the surface. This figure shows the endometrium as it appears when resurfacing is complete. The area

upper small rectangle inset right Gl

inscribed in the is shown at higher magnification in the on the . Note the simple columnar epithelium ( ) that covers the endometrial surface and its similarity to the glandular epithelium ( ). The endometrium is relatively thin at this phase, and over half of it consists of the stratum basale. The area inscribed by the , located in the region of the stratum basale, is shown at higher magnification in the in the next figure. The glandular epithelium of the deep portion of the glands is similar to that of the endometrial surface. Below the endometrium is the myometrium ( ), in which a number of large blood vessels ( ) are present.

SEp

inset

lower small rectangle M

BV

Endometrium, proliferative phase

,

uterus, human, H&E ×25; inset ×120.

Under the influence of estrogen, the various components of the endometrium proliferate (proliferative phase) so that the total thickness of the endometrium is increased. As shown in this figure, the endometrial glands ( ) become rather long and follow a fairly straight course within the ( ) to reach the surface. Several profiles of blood vessels ( ) that represent spiral arteries are visible in the lower two-thirds of the stratum functionale. The ( ) remains essentially unaffected by the estrogen and appears much the same as in the previous figure. Below the stratum basale is the myometrium ( ). In this figure, the stratum functionale ( ), on the other hand, has increased in thickness and constitutes about four-fifths of the endometrial thickness.

Gl

stratum functionale SF BV stratum basale SB SF

BV, blood vessels Gl, endometrial glands M, myometrium SB, stratum basale SEp, surface epithelium SF, stratum functionale

M

PLATE 23.6 UTERUS II estrogen

After brings about the uterine events designated the proliferative phase, another hormone, progesterone, influences uterine changes that constitute the secretory phase of the uterine cycle. This hormone brings the endometrium to a state of readiness for implantation, and as a consequence of its actions, the thickness of the endometrium increases further. There are conspicuous changes in the glands, primarily in the stratum functionale, where the glands take on a more pronounced corkscrew shape and secrete mucus that accumulates in sacculations along their length. The of the endometrium also proliferates and degenerates in each menstrual cycle. enter the stratum basale of the endometrium from the myometrium and give rise to small, straight arteries that supply the stratum basale and continue into the endometrium to become the highly coiled . Arterioles derived from the spiral arteries supply the stratum functionale. The distal portion of the spiral arteries and the arterioles are sloughed with the stratum functionale during menstruation. Alternating contraction and relaxation of the basal portions of the spiral arteries prevent excessive blood loss during menstruation.

vasculature

Radial arteries

spiral arteries

Uterus

, human, hematoxylin and eosin (H&E) ×25.

stratum functionale SF

stratum lower left myometrium M

This view of the endometrium in the secretory phase shows the ( ), the ( ), and, in the of the photomicrograph, a small amount of the ( ). The uterine glands have been cut in a plane that is close to their long axes, and one gland ( ) is seen opening at the uterine surface. Except for a few glands of the figure that resemble those of the proliferative phase, most of the glands ( ) in this figure, including those that are labeled, show numerous shallow sacculations that give the profile of the glandular epithelium a serrated appearance. This is one of the distinctive features of the secretory phase. It is seen most advantageously in areas where the plane of section is close to the long axis of the gland. In contrast to the characteristic sinuous course of the glands in the stratum functionale, glands of the stratum basale more closely resemble those in the proliferative phase. They are not oriented in any noticeable relationship to the uterine surface, and many of their long profiles are even parallel to the plane of the surface.

basale SB

Gl

arrow near the center

Endometrium, secretory phase

, uterus,

human, H&E ×30; inset ×120.

stratum functionale This

slightly

higher

magnification view of the shows essentially the same characteristics of the ( ) described earlier; it also shows other modifications that occur during the secretory phase. One of these is that the endometrium becomes edematous. The increase in endometrial thickness because of edema is reflected by the presence of empty spaces between cells and other formed elements. Thus, many areas of this figure, especially the area within and near the , show histologic signs of edema. In addition, in this phase, the glandular epithelial cells begin to secrete a mucoid fluid that is rich in glycogen. This product is secreted into the lumen of the glands, causing them to dilate. Typically, the glands of the secretory endometrium are more dilated than those of the proliferative endometrium. The in this figure highlights two glands that are shown at higher magnification in the . Each of these glands contains some substance within the lumen. The mucoid character of the substance within one of the glands can be surmised from its blue staining. Although not evident in routine H&E paraffin sections, the epithelial cells also contain glycogen during the secretory phase, and as mentioned earlier, this becomes part of the secretion. The indicate stromal cells; some of these cells undergo enlargement late in the secretory phase. These modified stromal cells, called , play a role in implantation.

endometrial glands Gl rectangle

rectangle

inset

cells

Gl, endometrial glands M, myometrium SB, stratum basale SF, stratum functionale arrow, glandular opening at uterine surface arrowheads, stromal cells

arrowheads decidual

PLATE 23.7 CERVIX cervix

The is the narrow or constricted inferior portion of the uterus, part of which projects into the vagina. The cervical canal traverses the cervix and provides a channel connecting the vagina and the uterine cavity. The structure of the cervix resembles the rest of the uterus in that it consists of a mucosa (endometrium) and a myometrium. There are, however, some important differences in the mucosa. The of the cervix does not undergo the cyclical growth and loss of tissue that is characteristic of the body and fundus of the uterus. Rather, the amount and character of the mucus secretion of its simple columnar epithelium vary at different times in the uterine cycle under the influence of ovarian hormones. At midcycle, there is a 10-fold increase in the amount of mucus produced; this mucus is thin and provides a favorable environment for sperm migration. At other times in the cycle, the mucus is thick and restricts the passage of sperm into the uterus. The forms the major thickness of the cervix. It consists of interweaving bundles of smooth muscle cells in an extensive, continuous network of fibrous connective tissue.

endometrium

myometrium

Cervix

, uterus, human, hematoxylin and eosin (H&E) ×15.

vaginal part ectocervix top cervical canal CCCC lower internal

The portion of the cervix that projects into the vagina, the or , is represented by the upper two-thirds of the figure. The lower third of the micrograph reveals the portion of the ( ). The figure shows the continuation of the cervical canal ( ). The plane of section in both figures passes through the long axis of the cervical canal, which is narrowed and cone shaped at its two ends. The upper end, the , communicates with the uterine cavity, and the lower end, the ( ), communicates with the vagina. (For purposes of orientation, realize that only one side of the longitudinal section of the cervix is shown in these figures and that the actual specimen, as seen in a section, would present a similar image on the other side of the cervical canal.)

os external os Os

mucosa Muc two rectangles

The ( ) of the cervix differs according to the cavity it faces. The in the figure delineate representative areas of the mucosa that are shown at higher magnification in the and figures, respectively. The figure emphasizes the nature of the ( ). The glands differ from those of the uterus in that they branch extensively. They secrete mucus into the cervical canal that serves to lubricate the vagina.

upper

upper right middle right bottom Gl

Ectocervix

cervical glands

, uterus, human, H&E ×240.

ectocervix SSEp

vaginal part of the cervix

The surface of the , the , is covered by stratified squamous epithelium ( ). The epithelium–connective tissue junction presents a relatively even contour in contrast to the irregular profile seen in the vagina. In other respects, the epithelium has the same general features as the vaginal epithelium. Another similarity is that the epithelial surface of the ectocervix undergoes cyclical changes similar to those of the vagina in response to ovarian hormones. The mucosa of the ectocervix, like that of the vagina, is devoid of glands.

Transformation zone

, cervix, uterus, human,

H&E ×240.

stratified simple columnar transformation

The mucosa of the cervical canal is covered with columnar epithelium. An abrupt change from ( ) to ( ) occurs within the ( ) at the vaginal opening of the cervical canal (external os). The in the figure marks this site, known as the , which is shown at a higher magnification here. Note the abrupt change in the epithelium at the transformation zone as well as the large number of lymphocytes and blood vessels ( ) present in this region.

squamous epithelium SSEp epithelium CEp zone lower TZ rectangle top left transformation zone BV

Cervical glands gland

, cervix, uterus, human, H&E ×500.

cervical

This figure shows, at high magnification, portions of the identified in the in the figure on the . Note the tall epithelial cells and the lightly staining supranuclear cytoplasm, a reflection of the mucin that dissolved out of the cell during tissue

rectangle

left

preparation. The crowding and the change in shape of the nuclei ( ) seen at the of one of the glands in this figure are due to a tangential cut through the wall of the gland as it passed out of the plane of section. (It is not uncommon for cervical glands to develop into cysts as a result of obstruction in the duct. Such cysts are referred to as .)

asterisk

upper part

cysts

BV, blood vessels CC, cervical canal CEp, columnar epithelium Gl, cervical glands Muc, mucosa Os, ostium of the uterus SSEp, stratified squamous epithelium TZ, transformation zone asterisk, tangential cut of the epithelial surface

nabothian

PLATE 23.8 PLACENTA I placenta

The is a disc-shaped organ that serves for the exchange of materials between the fetal and maternal circulations during pregnancy. It develops primarily from embryonic tissue, the . One side of the placenta is embedded in the uterine wall at the basal plate. The other side faces the amniotic cavity that contains the fetus. After childbirth, the placenta separates from the wall of the uterus and is discharged along with the contiguous membranes of the amniotic cavity. The connects the fetus to the placenta. It contains two arteries that carry blood from the fetus to the placenta and a vein that returns blood from the placenta to the fetus. The umbilical arteries have thick muscular walls. These are arranged as two layers, an inner longitudinal layer and an outer circular layer. Elastic lamellae are poorly developed in these vessels and, indeed, may be absent. The umbilical vein is similar to the arteries, also having a thick muscular wall arranged as an inner longitudinal and an outer circular layer.

chorion frondosum

umbilical cord

Placenta

, human, hematoxylin and eosin (H&E)

×16.

A section extending from the amniotic surface into the substance of the placenta is shown here. This includes the ( ), the ( ), and the ( ). The amnion consists of a layer of simple cuboidal epithelium and an underlying layer of connective tissue. The connective tissue of the amnion is continuous with the connective tissue of the chorionic plate as a result of their fusion at an earlier time. The plane of fusion, however, is not evident in H&E sections; the separation ( ) in parts of this figure in the vicinity of the fusion is an artifact. The chorionic plate is a thick connective tissue mass that contains the ramifications of the umbilical arteries and vein. These vessels ( ) do not have the distinct organizational features characteristic of arteries and veins; rather, they resemble the vessels of the umbilical cord. Although their identification as blood vessels is relatively simple, it is difficult to distinguish which vessels are branches of an umbilical artery and which are tributaries of the vein. The main substance of the placenta consists of chorionic villi of different sizes (see Plate 23.9, page 972). These emerge from the chorionic plate as large stem villi that branch into increasingly smaller villi. Branches of the umbilical arteries and vein ( , in

amnion A chorionic plate CP chorionic villi CV asterisks

BVp

BVv

the next figure) enter the stem villi and ramify through the branching villous network. Some villi extend from the chorionic plate to the maternal side of the placenta and make contact with the maternal tissue; these are called . Other villi, the , simply arborize within the substance of the placenta without anchoring onto the maternal side.

anchoring villi

villi

Placenta

free

, human, H&E ×70; inset ×370.

basal plate BP

The maternal side of the placenta is shown in this figure. The ( ) is on the of the illustration. This is the part of the uterus to which the chorionic villi anchor. Along with the usual connective tissue elements, the basal plate contains specialized cells called ( ). The same cells are shown at higher magnification in the . Decidual cells are usually found in clusters and have an epithelial appearance. Because of these features, they are easily identified. Septa from the basal plate extend into the portion of the placenta that contains the chorionic villi. The septa do not contain the branches of the umbilical vessels and, on this basis, can frequently be distinguished from stem villi or their branches.

decidual cells DC inset

A, amnion BP, basal plate BVp, blood vessels in chorionic plate BVv, blood vessels in chorionic villi CP, chorionic plate CV, chorionic villi DC, decidual cells asterisks, separation that is actually an artifact

right side

PLATE 23.9 PLACENTA II As the embryo develops, the invasive activity of the syncytiotrophoblast erodes the maternal capillaries and anastomoses them with the trophoblast lacunae, forming the maternal blood sinusoids. These communicate with each other and form a single blood compartment, lined by syncytiotrophoblasts, called the . At the end of the second week of development, cytotrophoblast cells form . They project into the maternal blood space. In the third week of development, invasion of the extraembryonic mesenchyme into the primary chorionic villi creates . At the end of the third week, core mesenchyme differentiates into connective tissue and blood vessels that connect with the embryonic circulation. These constitute functional units for exchange of gases, nutrients, and waste products between maternal and fetal circulation without direct contact with each other. This separation of fetal and maternal blood is referred to as the . Each tertiary villus consists of a connective tissue core surrounded by two distinct layers of trophoblastderived cells. The outermost layer consists of the syncytiotrophoblast; immediately beneath it is a layer of cytotrophoblast cells. Starting at the fourth month, these layers become very thin to facilitate the exchange of products across the placental barrier. The thinning of the wall of the villus is due to the loss of the inner, cytotrophoblastic layer. At this stage, the syncytiotrophoblast forms numerous trophoblastic buds that resemble the primary chorionic villi; however, the cytotrophoblast and the connective tissue grow very rapidly into these structures, transforming them into tertiary villi. At term, the consists of the syncytiotrophoblasts; a spare, thin (or discontinuous), inner cytotrophoblast layer; the basal lamina of the trophoblast; the connective tissue of the villus; the basal lamina of the endothelium; and the endothelium of the fetal placental capillary in the tertiary villus.

villi

intervillous space

primary chorionic

secondary chorionic

villi

chorionic villi

tertiary

placental barrier

placental barrier

Tertiary chorionic villi

, placenta, fullterm, human, hematoxylin and eosin (H&E) ×280. This photomicrograph shows a section through the intervillous space of the placenta at term. It includes ( ) of different sizes and the surrounding ( ). The connective tissue of the villi contains branches and tributaries of the umbilical

chorionic villi CV intervillous space IS

UV

vein ( ) and arteries. The intervillous space usually contains maternal blood (only a few maternal blood cells are seen here). The outermost layer of each chorionic villus derives from the fusion of cytotrophoblast cells. This layer, known as the ( ), has no intercellular boundaries, and its nuclei are rather evenly distributed, giving this layer an appearance similar to that of cuboidal epithelium. In some areas, nuclei are gathered in clusters forming ( ); in other regions, the syncytiotrophoblast layer appears relatively free of nuclei ( ). These stretches of the syncytiotrophoblast may be so attenuated in places that the villous surface appears devoid of a covering. The syncytiotrophoblast contains microvilli that project into the intervillous space. In well-preserved specimens, they may appear as a striated border (see ). The cytotrophoblast consists of an irregular layer of mononucleated cells that lies beneath the syncytiotrophoblast. In immature placentas, the cytotrophoblasts form an almost complete layer of cells. In this full-term placenta, only occasional ( ) can be discerned. Most of the cells within the core of the villus are typical connective tissue fibroblasts and endothelial cells. Other cells have a visible amount of cytoplasm that surrounds the nucleus. These are considered to be fetal placental antigen–presenting cells or placental macrophages ( ) historically known as .

syncytiotrophoblast

S

syncytial knots

SK

arrows

inset below

cytotrophoblast cells C

PM

Hofbauer cells

Secondary chorionic villi

, placenta, midterm, human, H&E ×320; inset ×640.

villi

This

secondary chorionic mesenchymal core MC cytotrophoblast cells syncytiotrophoblast S

micrograph shows the in the third week of embryonic development. These villi are composed of a ( ) surrounded by two distinct layers of the trophoblast. Secondary villi have a much larger number of ( ) than the mature tertiary villi and form an almost complete layer of cells immediately deep into the ( ) (see ). The syncytiotrophoblast not only covers the surface of the chorionic villi but also extends into the chorionic plate. Maternal red blood cells are present in the intervillous space.

C inset

Tertiary chorionic villi

, placenta, full-term, human,

H&E ×320.

immature ISchorionic tertiary villi

This higher magnification photomicrograph shows a cross section through surrounded by the intervillous space ( ). At this stage, chorionic villi are growing by

proliferation of their core mesenchyme, syncytiotrophoblast ( ), and fetal endothelial cells. Note a discontinued layer of cytotrophoblast cells ( ). The syncytiotrophoblast surrounding the chorionic villus ( ) forms ( ), which are present in the full-term mature placenta. They represent aggregation of syncytiotrophoblast nuclei on the surface of mature terminal villi. In addition to fibroblasts, several fetal placental ( ) can be identified by the amount of cytoplasm surrounding their nuclei.

S

center of the image

(placental macrophages) PM C, cytotrophoblast cells CV, chorionic villi IS, intervillous space MC, mesenchymal core PM, placental macrophages S, syncytiotrophoblast SK, syncytial knot UV, umbilical vein

syncytial knots

C SK

antigen–presenting cells

PLATE 23.10 VAGINA vagina

The is the fibromuscular tube of the female reproductive tract that leads to the exterior of the body. The wall of the vagina consists of three layers: a , a , and an . The epithelium of the mucosa is nonkeratinized stratified squamous. It undergoes changes that correspond to the ovarian cycle. The amount of glycogen stored in the epithelial cells increases under the influence of , whereas the rate of desquamation increases under the influence of . The glycogen liberated from the desquamated cells is fermented by , producing lactic acid that acidifies the vaginal surface and inhibits colonization by yeasts and potentially harmful bacteria. The vagina has certain histologic similarities to the proximal portion of the alimentary canal but is distinguished by the following features: The epithelium does not keratinize, and except for the deepest layers, the cells appear to be empty in routine hematoxylin and eosin (H&E) sections; the mucosa contains neither glands nor a muscularis mucosae; and the muscle is smooth and not well ordered. This can be contrasted with the oral cavity, pharynx, and upper part of the esophagus in which the muscle is striated. The more distal portion of the esophagus, which contains smooth muscle, can be distinguished easily from the vagina because it has a muscularis mucosae.

mucosa

adventitia

progesterone

muscularis

estrogen

lactobacilli vaginalis

Vagina

, human, H&E ×90.

squamous epithelium Ep CT

stratified

The mucosa of the vagina consists of a ( ) and an underlying fibrous connective tissue ( ) that often appears more cellular than other fibrous connective tissue. The boundary between the two is readily identified because of the conspicuous staining of the closely packed small cells of the basal layer ( ) of the epithelium. Connective tissue papillae project into the underside of the epithelium, giving the epithelial– connective tissue junction an uneven appearance. The papillae may be cut obliquely or in cross section and thus may appear as connective tissue islands ( ) within the lower portion of the epithelium. The epithelium is characteristically thick, and although keratohyalin granules may be found in the superficial cells, keratinization does not occur in the human vaginal epithelium. Thus, nuclei can be observed throughout the entire thickness of the epithelium despite the fact that the cytoplasm of most of the cells above the basal layers appears empty. These cells are normally filled with large deposits of

B

arrows

glycogen that are lost in the processes of fixation and embedding of the tissue. The outlines a portion of the epithelium and connective tissue papillae that is examined at higher magnification (next figure). The muscular layer of the vaginal wall consists of smooth muscle arranged in two ill-defined layers. The outer layer is generally said to be longitudinally arranged ( ), and the inner layer is generally said to be circularly arranged ( ), but the fibers are more usually organized as interlacing bundles surrounded by connective tissue. Many blood vessels ( ) are seen in the connective tissue.

rectangle

SML

BV

Mucosa

SMC

, vagina, human, H&E ×110.

Ep

This is a higher magnification of the epithelium ( ) that includes the area outlined by the in the figure (turned 90 degree). The obliquely cut and cross-sectioned portions of connective tissue papillae that appear as connective tissue islands in the epithelium are more clearly seen here ( ), in some instances, outlined by the surrounding closely packed cells of the basal epithelial cell layer. Note, again, that the epithelial cells even at the surface still retain their nuclei and there is no evidence of keratinization.

rectangle

upper

arrows

Mucosa

, vagina, human, H&E ×225.

This is a higher magnification micrograph of the basal portion of the epithelium ( ) between connective tissue papillae. Note the regularity and dense packing of the basal epithelial cells. They are the stem cells for the stratified squamous epithelium. Daughter cells of these cells migrate toward the surface and begin to accumulate glycogen and become less regularly arranged as they move toward the surface. The highly cellular connective tissue ( ) immediately beneath the basal layer ( ) of the epithelium typically contains many lymphocytes ( ). The number of lymphocytes varies with the stage of the ovarian cycle. Lymphocytes invade the epithelium around the time of menstruation and appear along with the epithelial cells in vaginal smears.

Ep

L

Muscularis

B

CT

, vagina, human, H&E ×125.

This higher magnification micrograph of the smooth muscle of the vaginal wall emphasizes the irregularity of the arrangement of the muscle bundles. At the of the figure is a bundle of smooth

right edge

SML

muscle cut in a longitudinal section ( ). Adjacent to this is a bundle of smooth muscle cut in cross section ( ). This bundle abuts on a longitudinally sectioned lymphatic vessel ( ). To the left of the lymphatic vessel is another longitudinal bundle of smooth muscle ( ). A valve ( ) is seen in the lymphatic vessel. A small vein ( ) is present in the circular smooth muscle close to the lymphatic vessel.

SMC SML

V

Va

LV

B, basal layer of vaginal epithelium BV, blood vessels CT, connective tissue Ep, epithelium L, lymphocytes LV, lymphatic vessel SMC, smooth muscle, cross section SML, smooth muscle, longitudinal section V, vein Va, valve in lymphatic vessel arrows, connective tissue islands in epithelium

PLATE 23.11 MAMMARY GLAND INACTIVE STAGE mammary glands

The are branched tubuloalveolar glands that develop from epidermis and come to lie in the subcutaneous tissue (superficial fascia). They begin to develop at puberty in the female but do not reach a fully functional state until after pregnancy. The glands also develop in the male at puberty; the development is limited, however, and the glands usually remain in a stabilized state.

Mammary gland

, inactive stage, human, hematoxylin and eosin (H&E) ×80.

inactive gland

This figure is a section through an . The parenchyma is sparse and consists mainly of duct elements. Several ducts ( ) are shown in the of the field. A small lumen can be seen in each. The ducts are surrounded by loose connective tissue (see , in the next figure), and together, the ducts and surrounding connective tissue constitute a lobule. Two terminal duct lobular units ( ) are bracketed in this figure. Beyond the , the connective tissue is more dense ( ) and contains adipocytes ( ). The two types of connective tissues can be distinguished at the low magnification of this figure.

D

CT[D]

lobular unit

A

center CT[L] TDLU

Mammary gland

, inactive stage, human, H&E ×200; inset ×400. Additional details are evident at higher magnification. In distinguishing between the loose and dense connective tissue, recall that both extracellular and cellular features show differences that are evident in both the figure and the . Note the thicker collagenous fibers in the dense connective tissue in contrast to the much thinner fibers of the loose connective tissue. The loose connective tissue ( ) contains far more cells per unit area and a greater variety of cell types. This figure shows a cluster of ( ) and, at still higher magnification ( ), ( ) and individual lymphocytes ( ). Both plasma cells and lymphocytes are cells with a rounded shape, but plasma cells are larger and show more cytoplasm. In addition, regions of plasma cell cytoplasm display basophilia. Elongate nuclei in spindle-shaped cells belong to fibroblasts. In contrast, although the cell types in the

inset

lymphocytes L cells P

CT[L]

L

inset

plasma

dense connective tissue may also be diverse, a simple examination of equal areas of loose and dense connective tissue will, by far, show fewer cells in the dense connective tissue. Characteristically, the dense connective tissue contains numerous aggregates of adipocytes ( ). The epithelial cells within the resting lobular units are regarded as being chiefly duct elements ( ). Usually, alveoli are not found; their precursors, however, are represented as cellular thickenings of the duct wall. The epithelium of the resting lobule is cuboidal; in addition, myoepithelial cells are present. Reexamination of the shows a thickening of the epithelium in one location, presumably the precursor of an alveolus, and ( ) at the base of the epithelium. As elsewhere, the myoepithelial cells are on the epithelial side of the basement membrane. During pregnancy, the glands begin to proliferate. This can be thought of as a dual process in which ducts proliferate and alveoli grow from the ducts.

A

D

myoepithelial cells M

A, adipocytes CT(D), dense connective tissue CT(L), loose connective tissue D, ducts L, lymphocytes M, myoepithelial cells P, plasma cells TDLU, terminal duct lobular unit

inset

PLATE 23.12 MAMMARY GLAND, LATE PROLIFERATIVE AND LACTATING STAGES Mammary glands

exhibit a number of changes during pregnancy in preparation for lactation. Lymphocytes and plasma cells infiltrate the loose connective tissue as the glandular tissue develops. As the cells of the glandular portion proliferate by mitotic division, the ducts branch and alveoli begin to develop at their growing ends. Alveolar development becomes most prominent in the later stages of pregnancy, and accumulation of secretory product takes place in the alveoli. At the same time, lymphocytes and plasma cells become prominent in the loose connective tissue of the developing lobules. Myoepithelial cells proliferate between the base of the epithelial cells and the basal lamina in both the alveolar and the ductal portion of the glands. They are most prominent in the larger ducts. Both and are involved in the production of milk. The protein component is synthesized, concentrated, and secreted by exocytosis in a manner typical for protein secretion. The lipid component begins as droplets in the cytoplasm that coalesce into large droplets in the apical cytoplasm of the alveolar cells and cause the apical plasma membrane to bulge into the alveolar lumen. The droplets are surrounded by a thin layer of cytoplasm and are enveloped in plasma membrane as they are released. The initial secretion in the first days after birth is called . This premilk is an alkaline secretion with a higher protein, vitamin A, sodium, and chloride content than milk and a lower lipid, carbohydrate, and potassium content. Considerable amounts of antibodies are contained in colostrum, and these provide the newborn with passive immunity to many antigens. The antibodies are produced by the plasma cells in the stroma of the breast and are carried across the glandular cells in a manner similar to that for secretory immunoglobulin A (IgA) in the salivary glands and intestine. A few days after parturition, the secretion of colostrum stops and lipid-rich milk is produced.

merocrine

apocrine secretion

colostrum

Mammary gland

, late proliferative stage, human, hematoxylin and eosin (H&E) ×90; inset ×560.

early proliferative

Whereas the development of the duct elements in the mammary gland occurs during the , the development of the alveolar elements becomes

stage

terminal duct lobular units TDLU connective tissue septa S

conspicuous in the late proliferative stage. This figure shows the ( ) at the late proliferative stage. Individual lobular units are separated by narrow, dense ( ). The connective tissue within the lobular unit is a typical loose connective tissue that is now more cellular, containing mostly plasma cells and lymphocytes. The alveoli are well developed, and many exhibit precipitated secretory product. Each of the alveoli is joined to a duct, although that relationship can be difficult to identify. The epithelium of the intralobular ducts is similar in appearance to the alveolar epithelium. The cells of both components are secretory. The alveoli as well as the intralobular ducts consist of a single layer of cuboidal epithelial cells subtended by myoepithelial cells. Often, what appear to be several alveoli are seen merging with one another ( ). Such profiles represent alveolar units opening into a duct. ( ) are easy to identify as they are surrounded by dense connective tissue. In one instance, an intralobular duct can be seen emptying into an interlobular duct ( ). The shows the secretory epithelium at a much higher magnification. Note that it is a simple columnar epithelium. The nucleus of a ( ) is seen at the base of the epithelium. Generally, these cells are difficult to recognize. Also, as noted earlier, numerous plasma cells ( ) and lymphocytes ( ) are present in the loose connective tissue of the lobule.

asterisks

Interlobular ducts D

arrow

inset

myoepithelial cell M

Ly

P

Mammary gland

, lactating stage, human, methyl green–osmium ×90; inset ×700.

gland

lactating mammary

The specimen shown here is from a . It is similar in appearance to the gland at the late proliferative stage but differs mainly to the extent that the alveoli are more uniform in appearance and their lumina are larger. As in the late proliferative stage, several alveoli can be seen merging with one another ( ). The use of osmium in this specimen stains the lipid component of the secretion. The reveals the lipid droplets within the epithelial cell cytoplasm as well as lipid that has been secreted into the lumen of the alveolus. The lipid first appears as small droplets within the epithelial cells. These droplets become larger and ultimately are secreted into the alveolar lumen along with milk proteins. The milk proteins are present in small vacuoles in the apical part of the cell but cannot be seen by light microscopic methods. They are secreted by exocytosis. The lipid droplets, in contrast, are large and surrounded by the apical cell membrane as they are pinched off to enter the lumen; thus, it is an apocrine secretion. Several

inset

asterisks

interlobular ducts

D

( ) are evident. One of these ducts reveals a small branch, an ending intralobular duct ( ) joining the interlobular duct.

arrows

D, interlobular duct Ly, lymphocyte M, myoepithelial cell P, plasma cell S, connective tissue septa TDLU, terminal duct lobular unit arrows, union of intralobular duct with interlobular duct asterisks, sites of merging alveoli

24 OVERVIEW OF THE EYE GENERAL STRUCTURE OF THE EYE Layers of the Eye

EYE

Chambers of the Eye Development of the Eye

MICROSCOPIC STRUCTURE OF THE EYE Corneoscleral Coat Vascular Coat (Uvea) Retina Crystalline Lens Vitreous Body

ACCESSORY STRUCTURES OF THE EYE Folder 24.1 Clinical Correlation: Glaucoma Folder 24.2 Clinical Correlation: Retinal Detachment Folder 24.3 Clinical Correlation: Age-related Macular Degeneration Folder 24.4 Clinical Correlation: Clinical Imaging of the Retina Folder 24.5 Clinical Correlation: Color Blindness Folder 24.6 Clinical Correlation: Conjunctivitis HISTOLOGY

OVERVIEW OF THE EYE eye

The is a complex sensory organ that provides the sense of sight. In many ways, the eye is similar to a digital camera. Like the optical system of a camera, the and of the eye capture and automatically focus light, whereas the iris automatically adjusts the diameter of the pupil to differences in illumination. The light detector in a digital camera, the chargecoupled device (CCD), consists of closely spaced photodiodes that capture, collect, and convert the light image into a series of electrical impulses. Similarly, the in the of the eye detect light intensity and color (wavelengths

retina

cornea

lens

photoreceptor cells

of visible light that are reflected by different objects) and encode these parameters into electrical impulses for transmission to the brain via the . The retina has other capabilities beyond those of a CCD: It can extract and modify specific impulses from the visual image before sending them to the central nervous system (CNS). In other ways, the optical system of the eye is far more elaborate and complex than a camera. For example, the eye is able to track moving objects with coordinated eye movements. The eye can also protect, maintain, self-repair, and clean its transparent optical system. Because the eyes are paired and spatially separated, two slightly different and overlapping views (visual fields) are sent to the brain. The brain integrates these two slightly different images from each eye into a single in a process called . The primary visual cortex located in the occipital lobes processes the differences between the two images to create the perception of depth. The final image is then projected onto the visual cortex. In addition, other complex neural mechanisms coordinate eye movements, enabling refinements in the perception of depth and distance. Therefore, the way in which we see the world around us largely depends on impulses processed within the retina and the analysis and interpretation of these impulses by the CNS.

optic nerve

stereopsis

three-dimensional (3D) image

GENERAL STRUCTURE OF THE EYE The eye measures approximately 25 mm in diameter. It is suspended in the bony orbital socket by six extrinsic muscles that control its movement. A thick layer of adipose tissue partially surrounds and cushions the eye as it moves within the orbit. The extraocular muscles are coordinated so that the eyes move symmetrically around their own central axes.

Layers of the Eye

The wall of the eye consists of three concentric layers or coats.

The eyeball is composed of three concentric structural layers (Fig. 24.1):

FIGURE 24.1. Schematic diagram of the layers of the eye.

The wall of the eyeball is organized in three separate concentric layers: an outer supporting fibrous layer, the corneoscleral coat; a middle vascular coat or uvea; and an inner layer consisting of the retina. Note that the retina has two layers: a neural retina ( ) and a retinal pigment epithelium ( ). The photosensitive and nonphotosensitive parts of the neural retina occupy different regions of the eye. The photosensitive part of the retina is found in the posterior part of the eye and terminates anteriorly along the ora serrata. The nonphotosensitive region of the retina is located anterior to the ora serrata and lines the inner aspect of the ciliary body and the posterior surface of the iris. The vitreous body ( ) occupies considerable space within the eyeball.

orange

yellow

partially removed

corneoscleral coat, the outer or fibrous layer, includes sclera, the white portion, and the cornea, the transparent vascular coat, the middle layer, or uvea, includes the choroid and the stroma of the ciliary body and iris. The retina, the inner layer, includes an outer pigment The the portion. The

epithelium, the inner neural retina, and the epithelium of the

ciliary body and iris. The neural retina is continuous with the CNS through the .

optic nerve The corneoscleral coat consists of the transparent cornea and the white opaque sclera. The cornea covers the anterior one-sixth of the eye (see Fig. 24.1). In this window-like region, the surface of the eye has a prominence or convexity. The cornea is continuous with the sclera [Gr. skleros, hard]. The sclera is composed of dense fibrous

connective tissue that provides attachment for the extrinsic muscles of the eye. The corneoscleral coat encloses the inner two layers, except where it is penetrated by the optic nerve. The sclera constitutes the “white” of the eye. In children, it has a slightly blue tint because of its thinness; in elderly people, it is yellowish because of the accumulation of lipofuscin in its stromal cells. A noticeable feature of patients with is a yellow discoloration of the sclera ( ) caused by a high level of circulating bilirubin.

jaundice scleral icterus The uvea consists principally of the choroid, the vascular layer that provides nutrients to the retina. Blood vessels and melanin pigment give the choroid an intense

dark brown color. The pigment absorbs scattered and reflected light to minimize glare within the eye. The choroid contains numerous venous plexuses and layers of capillaries and is firmly attached to the retina (see Fig. 24.1). The anterior rim of the uveal layer continues forward, where it forms the stroma of the and . The is a ring-like thickening that extends inward just posterior to the level of the corneoscleral junction. Within the ciliary body is the , a smooth muscle that is responsible for lens . Contraction of the ciliary muscle changes the shape of the lens, which enables it to bring light rays from different distances to focus on the retina. The is a contractile diaphragm that extends over the anterior surface of the lens. It also contains smooth muscle and melanin-containing pigment cells scattered in the connective tissue. The is the central circular aperture of the iris. It appears black because one looks through the lens toward the

ciliary body iris ciliary body iris

pupil

ciliary muscle accommodation

adaptation

heavily pigmented back of the eye. In the process of , the iris contracts or expands, changing the size of the pupil in response to the amount of light that passes through the lens to reach the retina.

The retina consists of two components: the neural retina and pigment epithelium. The retina is a thin, delicate layer (see Fig. 24.1) consisting of two components:

neural retina retinal pigment epithelium (RPE)

The is the inner layer that contains lightsensitive receptors and complex neuronal networks. The is the outer layer composed of simple cuboidal melanin-containing cells. Externally, the retina rests on the choroid; internally, it is associated with the vitreous body. The neural retina consists largely of , called retinal and , and interneurons. Visual information encoded by the rods and cones is sent to the brain via impulses conveyed along the optic nerve.

photoreceptor cells

rods

cones

Chambers of the Eye

The layers of the eye and the lens serve as boundaries for three chambers within the eye. The chambers of the eye are as follows:

anterior chamber posterior chamber vitreous chamber

The is the space between the cornea and the iris. The is the space between the posterior surface of the iris and the anterior surface of the lens. The is the space between the posterior surface of the lens and the neural retina (Fig. 24.2). The cornea, the anterior and posterior chambers, and their contents constitute the anterior segment of the eye. The vitreous chamber, visual retina, RPE, posterior sclera, and uvea constitute the posterior segment.

FIGURE 24.2. Schematic diagram illustrating the internal structures of the human eye. This diagram shows the relationship

between the layers of the eye and internal structures. The lens is suspended between the edges of the ciliary body. Note the posterior chamber of the eye, which is a narrow space between the anterior surface of the lens and the posterior surface of the iris. It communicates through the pupil with the larger anterior chamber that is bordered by the iris and the cornea. These spaces are filled with the aqueous humor produced by the ciliary body. The large cavity posterior to the lens, the vitreous chamber, is filled with a transparent jelly-like substance called the . In this figure, most of the vitreous body has been removed to illustrate the distribution of the central retinal vessels on the surface of the retina. The other layers of the eyeball and the attachment of two of the extraocular muscles to the sclera are also shown.

vitreous body

The refractile media components of the eye alter the light path to focus it on the retina. As light rays pass through the components of the eye, they are refracted. Refraction focuses the light rays on the photoreceptor cells of the retina. Four transparent components of the eye,

called the light rays:

refractile (or dioptric) media, alter the path of the

cornea aqueous humor lens

The is the anterior window of the eye. The is the watery fluid located in the anterior and posterior chambers. The is a transparent, crystalline, biconvex structure suspended from the inner surface of the ciliary body by a ring of radially oriented fibers, the . The is composed of a transparent gel-like substance that fills the vitreous chamber. It acts as a “shock absorber” that protects the fragile retina during rapid eye movement and helps maintain the shape of the eye. The vitreous body is almost 99% water with soluble proteins, hyaluronan, glycoproteins, widely dispersed collagen fibrils, and traces of other insoluble proteins. The fluid component of the vitreous body is called the .

vitreous body

cornea

zonule of Zinn

vitreous humor

The is the chief refractive element of the eye. It is the single most powerful focusing element of the eye and has a refractive index of 1.376 (air has a refractive index of 1.0). The cornea provides about 80% of the eye’s refractive power and is almost twice as powerful as the lens. The lens is second in importance to the cornea in refracting light rays. It is responsible for fine-tuning and focusing light onto the retina. Because of its elasticity, the shape of the can undergo slight changes in response to the tension of the ciliary muscle. These changes are important in for proper focusing on near objects. The aqueous humor and vitreous body have only minor roles in refraction. However, the aqueous humor plays an important role in providing nutrients to two avascular structures, the lens and cornea. In addition to transmitting light, the vitreous body helps maintain the position of the lens and helps keep the neural retina in contact with the RPE.

accommodation

lens

Development of the Eye To appreciate the unusual structural and functional relationships in the eye, it is helpful to understand how it forms in the embryo.

The tissues of the eye are derived from neuroectoderm, surface ectoderm, and mesoderm. By the 22nd day of development, the eyes are evident as shallow grooves—the optic sulci or optic grooves—located in the neural folds at the cranial end of the embryo. As the neural tube closes, the paired grooves form outpocketings called optic vesicles (Fig. 24.3a). As each optic vesicle grows laterally, the connection to the forebrain becomes constricted into an optic stalk, and the overlying surface ectoderm thickens and forms a . These events are followed by concomitant invagination of the optic vesicles and the lens placodes. The invagination of the optic vesicle results in the formation of a double-layered (Fig. 24.3b). The inner layer becomes the . The outer layer becomes the . The mesenchyme surrounding the optic cup gives rise to the .

lens placode

optic cup neural retina

RPE sclera

FIGURE 24.3. Schematic drawing illustrating the development of the eye. a. Forebrain and developing optic vesicles as seen in a 4-mm

b. c.

embryo. Bilayered optic cup and invaginating lens vesicle as seen in a 7.5-mm embryo. The optic stalk connects the developing eye to the brain. The eye as seen in a 15-week fetus. All the layers of the eye are established, and the hyaloid artery traverses the vitreous body from the optic disc to the posterior surface of the lens.

lens placode

Invagination of the central region of each results in the formation of the . By the fifth week of development, the lens vesicle loses contact with the surface ectoderm and comes to lie in the mouth of the optic cup. After the lens vesicle detaches from the surface ectoderm, this same site again thickens to form the corneal epithelium. from the periphery then give rise to the and the . Grooves containing blood vessels derived from mesenchyme develop along the inferior surface of each optic cup and stalk. Called the , the grooves enable the hyaloid artery to reach the inner chamber of the eye. This artery and its branches supply the inner chamber of the optic cup, lens vesicle, and mesenchyme within the optic cup. The hyaloid vein returns blood from these structures. The distal portions of the hyaloid vessels degenerate, but the proximal portions remain as the and . By the end of the seventh week, the edges of the choroid fissure fuse, and a round opening, the future pupil, forms over the lens vesicle. The forms a single layer of pigmented cells (Fig. 24.3c). Pigmentation begins at the end of the fifth week. The undergoes a complex differentiation into the nine layers of the . The photoreceptor cells (rods and cones) as well as the bipolar, amacrine, and ganglion cells and nerve fibers are present by the seventh month. The macular depression, a future site of fovea centralis, begins to develop during the eighth month and is not complete until about 6 months after birth. During the third month, the growth of the optic cup gives rise to the and the future , which forms a double row of epithelium in front of the lens. The mesoderm located external to this region becomes the stroma of the ciliary body and iris. Both epithelial layers of the iris become pigmented. In the ciliary body, however, only the outer layer is pigmented. At birth, the iris is light blue in fair-skinned people because the

lens vesicle

cells endothelium

Mesenchymal corneal

corneal stroma

choroid fissures

central retinal artery

central retinal vein

outer layer of the optic cup inner layer

ciliary body

iris

neural retina

pigment is usually not present. The dilator and sphincter pupillary muscles develop during the sixth month as derivatives of the neuroectoderm of the outer layer of the optic cup. The embryonic origins of the individual eye structures are summarized in Table 24.1.

Origins of the Individual Structures of TABLE 24.1 Embryonic the Eye Source

Derivative

Surface ectoderm

Lens Epithelium of the cornea, conjunctiva, and lacrimal gland and its drainage system

Neural ectoderm

Vitreous body (derived partly from neural ectoderm of the optic cup and partly from mesenchyme) Epithelium of the retina, iris, and ciliary body Sphincter pupillae and dilator papillae muscles Optic nerve

Mesoderm

Sclera Stroma of the cornea, ciliary body, iris, and choroids Extraocular muscles Eyelids (except epithelium and conjunctiva) Hyaloid system (most of which degenerates before birth) Coverings of the optic nerve Connective tissue and blood vessels of the eye, bony orbit, and vitreous body

MICROSCOPIC STRUCTURE OF THE EYE The

three

vascular coat molecular functions.

corneoscleral coat

layers of the eye—the , the , and the —are in turn composed of complex layers and structures that reflect their various

retina

Corneoscleral Coat The cornea is a unique tissue and the most powerful focusing element of the eye. It forms part of the anterior segment of the eye, protecting structures within the eye from the external environment. The most important characteristics of the cornea

include its mechanical strength and transparency to incoming light.

The cornea consists of five layers: three cellular layers and two noncellular layers. The transparent cornea (see Figs. 24.1 and 24.2) is only 0.5 mm thick at its center and about 1 mm thick peripherally. It consists of three cellular layers that are distinct in both appearance and origin. These layers are separated by two important membranes that appear homogeneous when viewed in the light microscope. Thus, the seen in a transverse section are the following:

five layers of the cornea

Corneal epithelium Bowman membrane (anterior basement membrane) Corneal stroma Descemet membrane (posterior basement membrane) Corneal endothelium The corneal epithelium is a nonkeratinized stratified squamous epithelium. The corneal epithelium (Fig. 24.4) represents nonkeratinized stratified squamous epithelium that consists of approximately five layers of cells and measures about 50 μm in average thickness. It is continuous with the conjunctival epithelium that overlies the adjacent sclera. The epithelial cells adhere to neighboring cells via desmosomes that are present in short interdigitating processes. Like other stratified epithelia, such as that of the skin, the cells proliferate from a basal layer and become squamous at the surface. The basal cells are low columnar with round, ovoid nuclei; the surface cells acquire a squamous or discoid shape, and their nuclei are flattened and pyknotic (Fig. 24.4b). As the cells migrate to the surface, the cytoplasmic organelles gradually disappear, indicating a progressive decline in metabolic activity. The corneal epithelium has a remarkable regenerative capacity with a turnover time of approximately 7 days.

FIGURE 24.4. Photomicrograph of the cornea. a.

This photomicrograph of a section through the full thickness of the cornea shows the corneal stroma and the two corneal surfaces covered by different types of epithelia. The corneal stroma does not contain blood or lymphatic vessels. ×140. A higher magnification of the anterior surface of the cornea showing the covered by a stratified squamous (corneal) . The basal cells that rest on , which is a homogeneous condensed layer of corneal stroma, are low columnar in contrast to the squamous surface cells. Note that one of the surface cells is in the process of desquamation ( ). ×280. A higher magnification photomicrograph of the posterior surface of the cornea covered by a thin layer of simple squamous epithelium (corneal ). These cells are in direct contact with the aqueous humor of the anterior chamber of the eye. Note the very thick (basal lamina) of the corneal endothelial cells. ×280.

b. corneal stroma epithelium

Bowman membrane

arrow

endothelium membrane

c.

Descemet

The actual stem cells for the corneal epithelium, called , reside at the , the junction of the cornea and sclera. The microenvironment of this stem cell niche is important in maintaining the stem cell population. It also acts as a barrier that prevents migration of conjunctival epithelial cells to the corneal surface. The may be partially or totally depleted by disease or extensive injury, resulting in abnormalities of the corneal surface that lead to of the cornea, which is characterized by vascularization, appearance of goblet

corneolimbal stem cells corneolimbal stem cells

corneoscleral limbus

conjunctivalization

cells, and an irregular and unstable epithelium. These changes cause ocular discomfort and reduced vision. Minor injuries of the corneal surface heal rapidly by inducing stem cell proliferation and migration of cells from the corneoscleral limbus to fill the defect. Numerous free nerve endings in the corneal epithelium provide it with extreme sensitivity to touch. Stimulation of these nerves (e.g., by small foreign bodies) elicits blinking of the eyelids, flow of tears, and, sometimes, severe pain. Microvilli present on the surface epithelial cells help retain the tear film over the entire corneal surface. Drying of the corneal surface may cause ulceration.

DNA in the corneal epithelial cells is protected from UV light damage by nuclear ferritin.

Despite constant exposure of the corneal epithelium to ultraviolet (UV) light, cancer of the corneal epithelium is extremely rare. Unlike the epidermis, which is also exposed to UV light, melanin is not present as a defense mechanism in the corneal epithelium. The presence of melanin in the cornea would diminish light transmission. Instead, it has recently been shown that corneal epithelial cell nuclei contain , an ironstorage protein. Experimental studies with avian corneas have shown that protects the DNA in the corneal epithelial cells from free radical damage caused by UV light exposure.

nuclear ferritin

ferritin

Bowman membrane is a homogeneous-appearing layer on which the corneal epithelium rests. Bowman membrane (anterior basement membrane) is a homogeneous, faintly fibrillar lamina that is approximately 8–10 μm thick. It lies between the corneal epithelium and the underlying corneal stroma and ends abruptly at the corneoscleral limbus. The collagen fibrils of Bowman membrane have a diameter of about 18 nm and are randomly oriented. Bowman membrane imparts some strength to the cornea, but more significantly, it acts as a barrier to the spread of infections. It does not regenerate. Therefore, if damaged, an opaque scar forms that may impair

vision. In addition, changes in Bowman membrane are associated with .

recurrent corneal erosions The corneal stroma constitutes 90% of the corneal thickness. The corneal stroma, also called substantia propria, is composed

of about 60 thin lamellae. Each lamella consists of parallel bundles of collagen fibrils. Located between the lamellae are nearly complete sheets of slender, flattened fibroblasts. The collagen fibrils are very uniform, measuring approximately 23 nm in diameter and as long as 1 cm in length, and are arranged at approximately right angles to those in adjacent lamellae (Fig. 24.5). The ground substance of cornea contains , which comprise sulfated glycosaminoglycans—chiefly, keratan sulfate proteoglycan ( ) and chondroitin sulfate proteoglycan ( ). They are responsible for the 3D organization of collagen fibrils. Lumican regulates the normal collagen fibril assembly in the cornea and is critical to the development of a highly organized collagenous matrix.

rich proteoglycans (SLRPs) lumican

small leucinedecorin

FIGURE 24.5. Electron micrograph of the corneal stroma. This electron micrograph shows parts of three lamellae and a portion of a corneal fibroblast ( CF ) between two of the lamellae. Note that the collagen fibrils in adjacent lamellae are oriented at right angles to one another. ×16,700.

Corneal transparency is achieved by the regular arrangement of small collagen fibrils and the spaces between them that are smaller than one-half of a wavelength of visible light. The transparency of the cornea is directly related to the spaces between collagen fibrils containing glycosaminoglycans and the size of the collagen fibrils. If these spaces are smaller than one-half of a wavelength of visible light, the cornea is clear and transparent. The uniform spacing of type I collagen fibrils and lamellae, as well as the of the lamellae

orthogonal array

(alternating layers at right angles), helps maintain corneal transparency. Proteoglycans ( ), along with , regulate the precise diameter and spacing of the type I collagen fibrils, maintaining corneal clarity. The necessity for uniformity of collagen fibrils explains the ratio of type V to type I collagen, which is much higher in the corneal stroma than in other tissues. after injury to the epithelium or endothelium disrupts this precise array and leads to translucency or opacity of the cornea. The hazy appearance of the cornea is related to the enlargement of the spaces between collagen fibers. Lumican is overexpressed during the wound healing process following corneal injury. Normally, the cornea contains no blood vessels or pigments. During an inflammatory response involving the cornea, large numbers of neutrophils and lymphocytes migrate from the blood vessels of the corneoscleral limbus and penetrate the stromal lamellae.

collagen

lumican

type V

Corneal swelling

Descemet membrane is an unusually thick basal lamina. Descemet membrane (posterior basement membrane) is the

basal lamina of corneal endothelial cells. It is intensely positive to periodic acid–Schiff (PAS) and can be as thick as 10 μm. Descemet membrane has a felt-like appearance and consists of an interwoven meshwork of fibers and pores. It separates the corneal endothelium from the adjacent corneal stroma. Unlike Bowman membrane, Descemet membrane readily regenerates after injury. It is produced continuously but slowly thickens with age. Descemet membrane also contributes to the diagnosis of , a rare inherited disorder of copper metabolism that causes excessive deposition of copper in organs and other tissues. A common ophthalmologic finding in individuals with Wilson disease is the presence of . These are caused by increased depositions of copper within Descemet membrane. A Kayser–Fleischer ring usually appears as a gold brown ring located in the periphery of the cornea. Descemet membrane extends peripherally beneath the sclera as a trabecular meshwork forming the . Strands from the pectinate ligament penetrate the ciliary muscle and sclera and may help maintain the normal curvature of the cornea by exerting tension on Descemet membrane.

Wilson disease

Kayser–Fleischer rings

pectinate ligament

The corneal endothelium provides for metabolic exchange between the cornea and the aqueous humor. The corneal endothelium is a single layer of squamous cells

covering the surface of the cornea that faces the anterior chamber (Fig. 24.4c). The cells are joined by well-developed zonulae adherentes, relatively leaky zonulae occludentes, and desmosomes. Virtually, all of the metabolic exchanges of the cornea occur across the endothelium. The endothelial cells contain many mitochondria and vesicles and an extensive roughsurfaced endoplasmic reticulum (rER) and Golgi apparatus. They demonstrate endocytotic activity and are engaged in active transport. Na+ /K+ -activated ATPase is located on the lateral plasma membrane. Transparency of the cornea requires precise regulation of the water content of the stroma. Physical or metabolic damage to the endothelium leads to rapid and, if the damage is severe, corneal opacity. Restoration of endothelial integrity is usually followed by deturgescence (dehydration necessary to maintain the transparency), although corneas can swell beyond their ability for self-repair. Such swelling can result in permanent focal opacities caused by aggregation of collagen fibrils in the swollen cornea. Essential sulfated glycosaminoglycans that normally separate the corneal collagen fibers are extracted from the swollen cornea. Human has a . Severely damaged endothelium can be repaired only by transplantation of a donor cornea. Recent studies indicate that the periphery of the cornea represents a regenerative zone of the corneal endothelial cells. However, soon after , endothelial cells exhibit contact inhibition when exposed to the extracellular matrix of Descemet membrane. The discovery that inhibitory factors released by Descemet membrane prevent proliferation of endothelial cells has focused current corneal research on the reversal or prevention of this inhibition with exogenous growth factors.

corneal swelling

capacity

corneal endothelium

transplantation

limited proliferative corneal

The sclera is an opaque layer that consists predominantly of dense connective tissue.

sclera

The is a thick fibrous layer containing flat collagen bundles that pass in various directions and in planes parallel to its surface. Both the collagen bundles and the fibrils that form them are irregular in diameter and arrangement. Interspersed between the collagen bundles are fine networks of elastic fibers and a moderate amount of ground substance. Fibroblasts are scattered among these fibers (Plate 24.4, page 1016). The opacity of the sclera, like that of other dense connective tissues, is primarily attributable to the irregularity of its structure. The sclera is pierced by blood vessels, nerves, and the optic nerve (see Fig. 24.2). It is 1 mm thick posteriorly, 0.3–0.4 mm thick at its equator, and 0.7 mm thick at the corneoscleral margin or limbus. The sclera is divided into three rather ill-defined layers:

episcleral layer (episclera) substantia propria sclera proper capsule suprachoroid lamina (lamina fusca)

The , the external layer, is the loose connective tissue adjacent to the periorbital fat. The ( , also called ) is the investing fascia of the eye and is composed of a dense network of thick collagen fibers. The , the inner aspect of the sclera, is located adjacent to the choroid and contains thinner collagen fibers and elastic fibers as well as fibroblasts, melanocytes, macrophages, and other connective tissue cells.

Tenon

episcleral space (Tenon space)

In addition, the is located between the episcleral layer and substantia propria of the sclera. This space and the surrounding periorbital fat allow the eye to rotate freely within the orbit. The tendons of the extraocular muscles attach to the substantia propria of the sclera.

The corneoscleral limbus is the transitional zone between the cornea and the sclera that contains corneolimbal stem cells. At the junction of the cornea and sclera (Fig. 24.6 and Plate

24.4, page 1016), Bowman membrane ends abruptly. The overlying epithelium at this site thickens from the 5 cell layers of the cornea to the 10–12 cell layers of the conjunctiva. The surface of the limbus is composed of two distinct types of epithelial

cells: One type constitutes the conjunctival cells, and the other constitutes the corneal epithelial cells. The basal layer of the limbus contains the that generate and maintain the corneal epithelium. These cells proliferate, differentiate, and migrate to the surface of the limbus and then toward the center of the cornea to replace damaged epithelial cells. As mentioned previously, this movement of cells at the corneoscleral limbus also creates a barrier that prevents conjunctival epithelium from migrating onto the cornea. At this junction, the corneal lamellae become less regular as they merge with the oblique bundles of collagen fibers of the sclera. An abrupt transition from the avascular cornea to the wellvascularized sclera also occurs here.

corneolimbal stem cells

FIGURE 24.6. Schematic diagram of the structure of the eye. This drawing shows a horizontal section of the eyeball with color-coded layers of its wall. Upper inset. Enlargement of the anterior and posterior chambers is shown in more detail. Note the location of the iridocorneal angle and canal of Schlemm (scleral venous sinus), which

Lower

drains the aqueous humor from the anterior chamber of the eye. Typical organization of the cells and nerve fibers of the fovea.

inset.

The limbus region, contains the apparatus 24.7). In the stromal the

iridocorneal angle

specifically, the , for the outflow of aqueous humor (Fig. layer, endothelium-lined channels called (or ) merge to form the . This sinus encircles the eye (see Figs. 24.6 and 24.7). The aqueous humor is produced by the ciliary processes that border the lens in the posterior chamber of the eye. The fluid passes from the posterior chamber into the anterior chamber through the valve-like potential opening between the iris and lens. The fluid then passes through the openings in the trabecular meshwork in the limbus region as it continues its course to enter the scleral venous sinus. Collecting vessels in the sclera, called (of Ascher) because they convey aqueous humor instead of blood, transport the aqueous humor to episcleral and conjunctival (blood) veins located in the sclera. Changes in the may lead to blockage in the drainage of aqueous humor, causing (see Folder 24.1, page 990). The iridocorneal angle can be visualized during eye examination using a , a specialized optical device that uses mirrors or prisms to reflect the light from the iridocorneal angle into the direction of the observer. In conjunction with a slit lamp or operating microscope, the ophthalmologist can examine this region to monitor various eye conditions associated with glaucoma. The iridocorneal angle can be also visualized using the . This high-resolution imaging technique utilizes a high-frequency ultrasound transducer to visualize the narrowed iridocorneal angle in primary angle-closure glaucoma.

trabecular meshwork spaces of Fontana scleral venous sinus (canal of Schlemm)

aqueous veins

angle

glaucoma gonioscope

biomicroscopy (UBM)

iridocorneal

ultrasound

FIGURE 24.7. Photomicrograph of the ciliary body and iridocorneal angle. This photomicrograph of the human eye shows the anterior portion of the ciliary body and parts of the iris and sclera . The inner surface of the ciliary body forms radially arranged, ridge-shaped elevations, the ciliary processes , to which the zonular fibers are anchored. The ciliary body contains the ciliary muscle , connective tissue with blood

vessels of the vascular coat, and the ciliary epithelium, which is responsible for the production of aqueous humor. Anterior to the ciliary body, between the iris and the cornea, is the . The scleral venous sinus ( ) is located in close proximity to this angle and drains the aqueous humor to regulate intraocular pressure. ×120. The shows that the ciliary epithelium consists of two layers, the outer pigmented layer and the inner nonpigmented layer. ×480.

canal of Schlemm inset

iridocorneal angle

FOLDER 24.1

CLINICAL CORRELATION: GLAUCOMA Glaucoma is a clinical condition resulting from increased intraocular pressure over a sustained period of time. It can be caused by excessive secretion of aqueous humor or impedance of the drainage of aqueous humor from the anterior chamber. The internal tissues of the eye, particularly the retina, are nourished by the diffusion of oxygen and nutrients from the intraocular vessels. Blood flows normally through these vessels (including the capillaries and veins) when the hydrostatic pressure within the vessels exceeds the intraocular pressure. If the drainage of the aqueous humor is impeded, the intraocular pressure increases because

the layers of the eye do not allow the wall to expand. This increased pressure interferes with normal retinal nourishment and function, causing the retinal nerve fiber layer to atrophy (Fig. F24.1.1).

FIGURE F24.1.1. Glaucoma.

This image shows a view of the fundus of the left eye in a patient with advanced glaucoma. As a result of the increased intraocular pressure, retinal nerve fibers undergo atrophy and shrink in size. Note a pale optic disc in the of the image with a less pronounced rim due to atrophy of nerve fibers. Enlargement of the optic nerve cup (central area of the optic disc) is also visible and a characteristic finding for glaucoma. Compare this image to a normal retina in Figure 24.15. (Courtesy of Dr. Renzo A. Zaldivar.)

center

There are two major types of glaucoma:

Open-angle glaucoma is the most common type of glaucoma and the leading cause of blindness among adults. The removal of aqueous humor is obstructed because of reduced flow through the trabecular

meshwork of the iridocorneal angle into the scleral venous sinus (canal of Schlemm). is less common and is characterized by a narrowed iridocorneal angle that obstructs the inflow of the aqueous humor into the scleral venous sinus. Usually, it is associated with a sudden, painful, complete blockage of the scleral venous sinus and can result in permanent blindness if not treated promptly.

Angle-closure glaucoma (acute glaucoma)

Visual deficits associated with glaucoma include blurring of vision and impaired dark adaptation (symptoms that indicate loss of normal retinal function) and halos around lights (a symptom indicating corneal endothelial damage). If the condition is not treated, the retina will be permanently damaged, and blindness will occur. Treatment is directed toward lowering the intraocular pressure by decreasing the rate of production of aqueous humor or eliminating the cause of the obstruction of normal drainage. Topical (i.e., latanoprost, bimatoprost, travoprost) are the first line of treatment for open-angle glaucoma. They are very effective in reducing intraocular pressure by increasing the drainage of aqueous humor into the canal of Schlemm. , which were used in the past to decrease the production of aqueous humor, have largely been replaced by prostaglandin analogs that have fewer systemic side effects. There are two main types of laser surgery to treat glaucoma. They facilitate drainage of aqueous humor from the iridocorneal angle. Laser utilizes a laser beam to induce focal scarring of the trabecular meshwork. This results in mechanical stretching of the surrounding untreated regions of the meshwork, which facilitates drainage of the aqueous humor. Trabeculoplasty is often used in open-angle glaucoma when medications are not effective or cause intolerable side effects. is used in patients with angle-closure glaucoma. The laser beam incises a small opening at the base of the iris, which widens the iridocorneal angle to allow better drainage of aqueous humor.

prostaglandin analogs

Carbonic anhydrase inhibitors trabeculoplasty

Iridotomy

Vascular Coat (Uvea)

The iris, the most anterior part of the vascular coat, forms a contractile diaphragm in front of the lens. The iris arises from the anterior border of the ciliary body (see Fig. 24.7) and is attached to the sclera about 2 mm posterior to the corneoscleral junction. The pupil is the central aperture of this thin disc. The iris is pushed slightly forward as it changes in size in response to light intensity. It consists of a highly vascularized connective tissue stroma that is covered on its

posterior

posterior

surface

by highly pigmented cells, the (Fig. 24.8). The basal lamina of these cells faces the posterior chamber of the eye. The degree of pigmentation is so great that neither the nucleus nor the character of the cytoplasm can be seen in the light microscope. Located beneath this layer is a layer of myoepithelial cells, the . The apical (posterior) portions of these myoepithelial cells are laden with melanin granules, which effectively obscure their boundaries with the adjacent posterior pigment epithelial cells. The basal (anterior) portions of myoepithelial cells possess processes containing contractile elements that extend radially and collectively make up the of the iris. The contractile processes are enclosed by a basal lamina that separates them from the adjacent stroma.

pigment epithelium

anterior pigment myoepithelium dilator pupillae muscle

FIGURE 24.8. Structure of the iris. a.

This schematic diagram shows the layers of the iris. Note that the pigmented epithelial cells are reflected as occurs at the pupillary margin of the iris. The two layers of pigmented epithelial cells are in contact with the dilator pupillae muscle. The incomplete layer of fibroblasts and stromal melanocytes is indicated on the anterior surface of the iris. Photomicrograph of the iris showing the histologic features of this structure. The , which

b.

lens

lies posterior to the iris, is included for orientation. The iris is composed of a stroma covered on its posterior surface by the posterior pigment epithelium. The basal lamina (not visible) faces the posterior chamber of the eye. Because of intense pigmentation, the histologic features of these cells are not discernible. Just anterior to these cells is the anterior pigment myoepithelium layer (the separates the two layers). Note that the posterior portion of the myoepithelial cells contains melanin, whereas the anterior portion contains contractile elements forming the dilator pupillae muscle of the iris. The sphincter pupillae muscle is evident in the stroma. The color of the iris depends on the number of scattered throughout the connective tissue stroma. At the , note the presence of the lens. ×570.

connective tissue

dashed line

stromal

melanocytes bottom

Constriction of the pupil is produced by smooth muscle cells located in the stroma of the iris near the pupillary margin of the iris. These circumferentially oriented cells collectively compose the . The anterior surface of the iris reveals numerous ridges and grooves that can be seen in clinical examination with the ophthalmoscope. When this surface is examined in the light microscope, it appears as a discontinuous layer of fibroblasts and melanocytes. The number of melanocytes in the stroma is responsible for variation in eye color. The function of these in the iris is to absorb light rays. If there are few melanocytes in the stroma, the color of the iris is derived from light reflected from the pigment present in the cells of the iris’s posterior surface, giving it a blue appearance. With increasing amounts of pigment present in the stroma, the iris color changes from blue to shades of greenish blue, gray, and, finally, brown.

sphincter pupillae muscle

pigment-containing cells

The sphincter pupillae is innervated by parasympathetic nerves; the dilator pupillae muscle is under sympathetic nerve control. The size of the pupil is controlled by contraction of the sphincter pupillae and dilator pupillae muscles. The process of adaptation (increasing or decreasing the size of the pupil) ensures that only the appropriate amount of light enters the eye. Two muscles are actively involved in adaptation:

sphincter pupillae muscle,

The muscle

cells

(Plate

24.3,

a circular band of smooth page 1014), is innervated by

parasympathetic nerves carried in the oculomotor nerve (cranial nerve III) and is responsible for reducing pupillary size in response to bright light. Failure of the pupil to respond when light is shined into the eye—“ ”—is an important clinical sign showing a lack of nerve or brain function. The is a thin sheet of radially oriented contractile processes of pigmented myoepithelial cells constituting the anterior pigment epithelium of the iris. This muscle is innervated by sympathetic nerves from the superior cervical ganglion and is responsible for increasing pupillary size in response to dim light.

pupil fixed and dilated

dilator pupillae muscle

ophthalmoscopic examination

Just before , mydriatic agents such as are given as eye drops to cause dilation of the pupil. Acetylcholine (ACh) is the neurotransmitter of the parasympathetic nervous system (it innervates the sphincter pupillae muscle); the addition of atropine blocks muscarinic acetylcholine receptors, temporally blocking the action of the sphincter muscle, and leaving the and unreactive to light originating from ophthalmoscope.

atropine

pupil wide open The ciliary body is the thickened anterior portion of the vascular coat and is located between the iris and the choroid. The ciliary body extends about 6 mm from the root of the iris posterolaterally to the ora serrata (see Fig. 24.2). As seen

from behind, the lateral edge of the ora serrata bears 17–34 grooves or crenulations. These grooves mark the anterior limit of both the retina and the choroid. The anterior third of the ciliary body has approximately 75 radial ridges or (see Fig. 24.7). The fibers of the zonule arise from the grooves between the ciliary processes. The layers of the ciliary body are similar to those of the iris and consist of a stroma and an epithelium. The stroma is divided into two layers:

ciliary

processes

outer layer inner vascular region

ciliary muscle

An of smooth muscle, the , makes up the bulk of the ciliary body. An extends into the ciliary processes.

The epithelial layer covering the internal surface of the ciliary body is a direct continuation of the two layers of the retinal epithelium (see Fig. 24.1).

The ciliary muscle is organized into three functional portions or groups of smooth muscle fibers.

The smooth muscle of the ciliary body has its origin in the scleral spur, a ridge-like projection on the inner surface of the sclera at the corneoscleral junction. The muscle fibers spread out in several directions and are classified into three functional groups on the basis of their direction and insertion:

meridional (or longitudinal) portion

The consists of the outer muscle fibers that pass posteriorly into the stroma of the choroid. These fibers function chiefly in stretching the choroid. It also may help open the iridocorneal angle and facilitate drainage of the aqueous humor. The consists of deeper muscle fiber bundles that radiate in a fan-like manner to insert into the ciliary body. Its contraction causes the lens to flatten and thus focus on distant vision. The consists of inner muscle fiber bundles oriented in a circular pattern that forms a sphincter. It reduces the tension on the lens, causing the lens to accommodate for near vision.

radial (or oblique) portion

circular (or sphincteric) portion

Examination of a histologic preparation does not clearly reveal the arrangement of the muscle fibers. Rather, the organizational grouping is based on microdissection techniques.

Ciliary processes are ridge-like extensions of the ciliary body from which zonular fibers emerge and extend to the lens. Ciliary processes are thickenings of the inner vascular region of the ciliary body. They are continuous with the vascular layers of the choroid. Scattered macrophages containing melanin pigment granules and elastic fibers are present in these processes (Plate 24.3, page 1014). The processes and the ciliary body are covered by a double layer of columnar epithelial cells, the , which was originally derived from the two layers of

epithelium

ciliary

the optic functions:

cup.

The

ciliary

epithelium

has

three

principal

aqueous humor blood–aqueous barrier (part of the blood –ocular barrier zonular fibers that form the suspensory ligament of the lens Secretion of Participation in the ) Secretion and anchoring of the

The inner cell layer of the ciliary epithelium has a basal lamina facing the posterior and vitreous chambers. The cells in this layer are nonpigmented. The cell layer that has its basal lamina facing the connective tissue stroma of the ciliary body is heavily pigmented and is directly continuous with the pigmented epithelial layer of the retina. The continues over the iris, where it becomes the posterior pigmented epithelium and anterior pigmented myoepithelium. The zonular fibers extend from the basal lamina of the nonpigmented epithelial cells of the ciliary processes and insert into the lens capsule (the thickened basal lamina of the lens).

epithelium

double-layered ciliary

The blood–aqueous barrier separates the interior environment of the eye from the blood entering the ciliary body. The cells of the nonpigmented layer have all the characteristics of a fluid-transporting epithelium, including complex cell-tocell junctions with a well-developed zonula occludens, extensive lateral and basal plications, and localization of Na+ /K+ -ATPase in the lateral plasma membrane. In addition, they have an elaborate rER and Golgi complex, consistent with their role in the secretion of zonular fibers. Tight junctions (zonulae occludentes) between the nonpigmented ciliary epithelial cells are responsible for maintaining the . This barrier restricts free diffusion across the ciliary epithelium to maintain the unique environment of the aqueous humor, which is quite different from that of blood vessels and stroma of the ciliary body. The blood–aqueous barrier contributes to the nutrition and function of the cornea and the lens. may be observed in ocular inflammation, intraocular surgery, trauma, or vascular diseases.

blood–aqueous barrier

the blood–aqueous barrier

Disruption of

The aqueous humor becomes cloudy because of the leakage of plasma proteins (fibrinogen) and migration of inflammatory cells from the stroma of the ciliary body and iris into the posterior and anterior chambers of the eye. The have a less developed junctional zone and often exhibit large, irregular lateral intercellular spaces. Both desmosomes and gap junctions hold together the apical surfaces of the two cell layers, creating discontinuous “luminal” spaces called .

cells of the pigmented layer

ciliary channels The aqueous humor is derived from plasma and maintains intraocular pressure. The aqueous humor is secreted by the double-layered ciliary epithelium and originates from blood capillaries. It is similar in ionic composition to plasma but contains less than 0.1% protein (compared to 7% protein in plasma). The main functions of the aqueous humor are to maintain and to provide nutrients and remove metabolites from the avascular tissues of the cornea and lens. The aqueous humor passes from the ciliary body toward the lens and then between the iris and the lens, before it reaches the anterior chamber of the eye (see Fig. 24.6). In the anterior chamber of the eye, the aqueous humor passes laterally to the angle formed between the cornea and the iris. Here, it penetrates the tissues of the limbus as it enters the labyrinthine spaces of the limbus’s trabecular meshwork in the iridocorneal angle and finally reaches the , which communicates with the veins of the sclera (see Folder 24.1). Normal turnover of the aqueous humor in the human eye is approximately once every 1.5–2 hours.

intraocular pressure

canal of Schlemm

The choroid is the portion of the vascular coat that lies deep into the retina. The choroid is a dark brown vascular sheet only 0.25 mm thick

posteriorly and 0.1 mm thick anteriorly. It lies between the sclera and the retina (see Fig. 24.1). Two layers can be identified in the choroid:

Choriocapillary layer, an inner vascular layer Bruch membrane, a thin, amorphous hyaline membrane

The choroid is attached firmly to the sclera at the margin of the optic nerve. A potential space, the (between the sclera and the retina), is traversed by thin, ribbon-like branching lamellae or strands that pass from the sclera to the choroid. These lamellae originate from the (lamina fusca) and consist of large, flat melanocytes scattered between connective tissue elements, including collagen and elastic fibers, fibroblasts, macrophages, lymphocytes, plasma cells, and mast cells. The lamellae pass inward to surround the vessels in the remainder of the choroid layer. Free smooth muscle cells, not associated with blood vessels, are present in this tissue. Lymphatic channels called , long and short posterior ciliary vessels, and nerves on their way to the front of the eye are also present in the suprachoroid lamina. Most of the blood vessels decrease in size as they approach the retina. The largest vessels continue forward beyond the ora serrata into the ciliary body. These vessels can be seen with an ophthalmoscope. The large vessels are mostly veins that course in whorls before passing obliquely through the sclera as vortex veins. The inner layer of vessels, arranged in a single plane, is called the . The vessels of this layer provide nutrients to the cells of the retina. The fenestrated capillaries have lumina that are large and irregular in shape. In the region of the fovea, the choriocapillary layer is thicker, and the capillary network is denser. This layer ends at the ora serrata. , also called the , measures 1–4 μm in thickness and lies between the choriocapillary layer and the pigment epithelium of the retina. It runs from the optic nerve to the ora serrata, where it undergoes modifications before continuing into the ciliary body. Bruch membrane is a thin, amorphous refractile layer. The transmission electron microscope (TEM) reveals that it consists of a multilaminar sheet containing a center layer of elastic and collagen fibers. Five different layers are identified in Bruch membrane:

perichoroidal space

suprachoroid lamina

epichoroid lymph spaces

choriocapillary layer

Bruch membrane

lamina vitrea

The basal lamina of the endothelial cells of choriocapillary layer A layer of collagen fibers approximately 0.5 μm thick

the

A layer of elastic fibers approximately 2 μm thick A second layer of collagen fibers (thus forming a “sandwich” around the intervening elastic tissue layer) The basal lamina of the RPE cells At the ora serrata, the collagenous and elastic layers disappear into the ciliary stroma, and Bruch membrane becomes continuous with the basal lamina of the RPE of the ciliary body.

Retina

The retina represents the innermost layer of the eye. The retina, derived from the inner and outer layers of the optic cup, is the innermost of the three concentric layers of the eye (see Fig. 24.1). It consists of two basic layers:

neural retina retina proper retinal pigment epithelium (RPE)

The or is the inner layer that contains the photoreceptor cells. The is the outer layer that rests on and is firmly attached through the Bruch membrane to the choriocapillary layer of the choroid. A potential space exists between the two layers of the retina. The two layers may be separated mechanically in the preparation of histologic specimens. Separation of the layers, “ ” (see Folder 24.2), also occurs in the living state because of eye disease or trauma.

retinal

detachment

FOLDER 24.2

CLINICAL CORRELATION: RETINAL DETACHMENT

A potential space exists in the retina as a vestige of the space between the apical surfaces of the two epithelial layers of the optic cup. If this space expands, the neural retina separates from the retinal pigment epithelium (RPE), which remains attached to the choroid layer. This condition is called . As a result of retinal detachment, the photoreceptor cells are no longer supplied by nutrients from the underlying vessels in the choriocapillary plexus of the choroid. Clinical symptoms of retinal detachment include visual sensations commonly described as a “shower of pepper” or floaters. These are caused by red blood cells extravasated from the capillary vessels

retinal detachment

that have been injured during the retinal tear or detachment. In addition, some individuals describe sudden flashes of light as well as a “web” or “veil” in front of the eye in conjunction with the onset of floaters. A detached retina can be observed and diagnosed during ophthalmoscopic eye examination (Fig. F24.2.1).

FIGURE F24.2.1. Retinal detachment.

This image shows a view of the fundus of the right eye in a patient with retinal detachment. The central retinal vessels emerging from the optic disc are in focus, but in the they appear to be out of focus. Because the area of retinal detachment is elevated (note multiple ridges and shadows), it is located anterior to the plane of focus of the ophthalmoscope. (Courtesy of Dr. Renzo A. Zaldivar.)

area of the retinal detachment,

Another common retinal condition occurs with aging. As the vitreous body ages (in the sixth and seventh decades of life), it tends to shrink and pull away from the neural retina, which causes single or multiple tears in the neural retina. If not repositioned quickly, the detached area of the retina will undergo necrosis, resulting in blindness. An argon laser is often used to repair retinal detachment by photocoagulating the edges of the detachment and producing scar tissue. This method prevents the

retina from further detachment and facilitates the repositioning of photoreceptor cells.

In the neural retina, two regions or portions that differ in function are recognized:

nonphotosensitive region

The (nonvisual part), located anterior to the ora serrata, lines the inner aspect of the ciliary body and the posterior surface of the iris (this portion of the retina is described in the sections on the iris and ciliary body). The (optic part) lines the inner surface of the eye posterior to the ora serrata, except where it is pierced by the optic nerve (see Fig. 24.1).

photosensitive region

The site where the optic nerve joins the retina is called the or . Because the optic disc is devoid of photoreceptor cells, it is a blind spot in the visual field. The is a shallow depression located about 2.5 mm lateral to the optic disc. It is the area of greatest visual acuity. The visual axis of the eye passes through the fovea. A yellow-pigmented zone called the surrounds the fovea. In relative terms, the fovea is the region of the retina that contains the highest concentration and most precisely ordered arrangement of visual elements. The region of the retina surrounding the macula lutea may be affected in older individuals by (see Folder 24.3).

optic disc optic papilla fovea centralis

macula lutea

age-related macular degeneration

FOLDER 24.3

CLINICAL CORRELATION: AGE-RELATED MACULAR DEGENERATION Age-related macular degeneration (ARMD) is the most common cause of blindness in older individuals. Although the cause of this disease is still unknown, evidence suggests both genetic and environmental (ultraviolet [UV] irradiation, drugs) components. The disease causes loss of central vision, although peripheral vision remains unaffected. Two forms of ARMD are recognized: a dry (atrophic, nonexudative) form and a wet (exudative, neovascular) form. The latter is considered a complication of the first. is the most common form (90% of all cases) and involves degenerative lesions localized in the area of the macula lutea. The degenerative lesions include , which are focal thickenings of

ARMD

Dry

drusen

Bruch membrane, atrophy, depigmentation of the RPE, and obliteration of capillaries in the underlying choroid layer. These changes lead to the deterioration of the overlying photosensitive retina, resulting in the formation of blind spots in the visual field (Fig. F24.3.1). is a complication of dry ARMD caused by neovascularization of blind spots of the retina in the large drusen. These newly formed, thin, fragile vessels frequently leak and produce exudates and hemorrhages in the space just beneath the retina, resulting in fibrosis and scarring. These changes are responsible for the progressive loss of central vision over a short time. The treatment of wet ARMD includes conventional therapy and pharmacologic therapy with intravitreal injection of ranibizumab, a . Other surgical methods, such as , have been recently introduced. In this procedure, the retina is detached, translocated, and reattached in a new location, away from the choroid neovascular tissue. Conventional laser treatment is then applied to destroy pathologic vessels without destroying central vision.

Wet ARMD

photocoagulation growth factor (VEGF) inhibitor macular translocation

laser vascular endothelial

FIGURE F24.3.1. Photograph depicting the visual field in individuals with age-related macular degeneration. Note that

central vision is absent because of the changes in the macula region of the retina. To maximize their remaining vision, individuals with this condition are instructed to use eccentric fixation of their eyes.

Layers of the retina Ten layers of cells and their processes constitute the retina. Before discussing the ten layers of the retina, it is important

to identify the types of cells found there. This identification will aid in understanding the functional relationships of the cells. Studies of the retina in primates have identified at least 15 types of neurons that form at least 38 different types of synapses. For convenience, neurons and supporting cells can be classified into four groups of cells (Fig. 24.9):

FIGURE 24.9. Schematic drawing and photomicrograph of the layers of the retina. On the basis of histologic features that are evident in the photomicrograph on right , the retina can be divided into 10 layers. The layers correspond to the diagram on left , which shows the

distribution of major cells of the retina. Note that light enters the retina and passes through its inner layers before reaching the photoreceptors of the rods and cones that are closely associated with

the retinal pigment epithelium. Also, the interrelationship between the bipolar neurons and ganglion cells that carry electrical impulses from the retina to the brain is clearly visible. Bruch membrane (lamina vitrea) separates the inner layer of the vascular coat (choroid) from the retinal pigment epithelium. ×440.

Photoreceptor cells—the retinal rods and cones Conducting neurons—bipolar neurons and ganglion cells Association neurons and others—horizontal, centrifugal, interplexiform, and amacrine neurons Supporting (neuroglial) cells—Müller cells, microglial cells, and astrocytes

The specific arrangement and associations of the nuclei and processes of these cells form 10 retinal layers that can be seen with the light microscope. The layers of the retina can also be imaged and examined in living individuals using spectral-domain optical coherence tomography (see Folder 24.4). The 10 layers of the retina, from outside inward, are as follows (see Fig. 24.9):

FOLDER 24.4

CLINICAL CORRELATION: CLINICAL IMAGING OF THE RETINA

The standard ophthalmoscopic examination of the eye has been recently supplemented by a new examination technique that utilizes (SD OCT). This noninvasive and noncontact examination is not only useful in visualizing the retinal surface, but it also provides a highresolution cross-sectional image of the retina in vivo. All histologic layers of the retina can be easily differentiated with SD OCT (Fig. F24.4.1), and they can be objectively measured for tissue thickness and change. SD OCT technology is based on comparisons of spectral characteristics of the reflected light beam from the retina with those of the reference beam. For this purpose, an infrared laser beam (~840 nm wavelength with 50 nm bandwidth) is used that is able to produce images at 5-μm resolution. The laser beam passes through the structures of the eye and is partially absorbed and partially reflected depending on tissue characteristics. The reflected light is detected by a multichannel spectrometer, and the interference pattern is compared to the reference beam using complex computer algorithms. The spectral differences are used to construct the cross-sectional (line) scans as shown in Figure F24.4.1 or the three-dimensional images of the retina as shown in Figure F24.4.2. Introduced in the 1990s, the SD OCT has revolutionized the management and diagnosis of many eye diseases. SD OCT established itself as an imaging modality of choice in (measurement of

spectral-domain optical coherence tomography

glaucoma

macular degeneration retinal detachment macular holes epiretinal membranes optic disc pits diabetic retinopathy cystoid macular edema central serous choroidopathy optic nerve and retinal nerve fiber layer) and retinal diseases. It is used for the early and accurate detection of , , , , and and for the detection of fluid accumulation within the retina that occurs in conditions such as , , and .

FIGURE F24.4.1. Spectral-domain optical coherence tomography (SD OCT) cross-sectional (line) image of the retina in a healthy eye. The upper image represents a normal cross-sectional image of the retina containing fovea and optic disc on the right side of the image. The optically transparent vitreous body is invisible and appears as the black region in the upper part of the image. Hyperreflective and hyporeflective bands of retinal tissue correspond to the histologic layers of the retina. Note the photoreceptor layer containing rods and cones as well the retinal pigment epithelium are well defined and are separated from the choroid layer containing blood vessels. (Courtesy of Drs. Andrew J. Barkmeier and Denise M. Lewison.)

FIGURE F24.4.2. Spectral-domain optical coherence tomography (SD OCT) three-dimensional image of the retina of a healthy right eye. The scan area is ~12 mm × 9 mm in size and includes a portion of the optic disc ( on the left ) and fovea ( on the right ). A

three-dimensional data set is acquired from four scans (two vertical and two horizontal), which is then processed with a motioncorrection technology (MCT) algorithm. The MCT algorithm analyzes and compares the vascular pattern in each of the scans and reduces artifacts and image distortions associated with eye movement. This image has two parts. The upper false-color image (optical densities are coded in different colors) shows the surface and thickness of all layers of the retina and represents a motion-corrected, threedimensional volume rendering of the entire data set. The lower grayscale vascular map image (optical densities are coded in grayscale) is a two-dimensional image created by summing all the pixels in each column. It is curved to match the curvature of the eye. The letters S (for superior) and T (for temporal) on the eye orientation icon in the provide reference to the positioning of the scan in the patient’s eye. (Image courtesy of Dr. Pravin Dugel, Phoenix, Arizona.)

lower right corner

1. Retinal pigment epithelium (RPE)—the

outer layer of the retina, actually not part of the neural retina but intimately associated with it —contains the outer and inner segments of photoreceptor cells —the apical boundary of Müller cells —contains the cell bodies (nuclei) of retinal rods and cones —contains the processes of retinal rods and cones and processes of the horizontal, amacrine, and bipolar cells that connect to them —contains the cell bodies (nuclei) of horizontal, amacrine, bipolar, and Müller cells —contains the processes of horizontal, amacrine, bipolar, and ganglion cells that connect to each other —contains the cell bodies (nuclei) of ganglion cells —contains processes of ganglion cells that lead from the retina to the brain —composed of the basal lamina of Müller cells

2. Layer of rods and cones 3. Outer limiting membrane 4. Outer nuclear layer 5. Outer plexiform layer 6. Inner nuclear layer 7. Inner plexiform layer

8. Ganglion cell layer 9. Layer of optic nerve fibers 10. Inner limiting membrane

Each of the layers is more fully described in the following sections (see corresponding numbers).

The cells of the retinal pigment epithelium (layer 1) have extensions that surround the processes of the rods and cones. The RPE is a single layer of cuboidal cells about 14 μm wide and 10–14 μm tall. The cells rest on Bruch membrane of the choroid layer. The pigment cells are tallest in the fovea and adjacent regions, which account for the darker color of this region. Adjacent RPE cells are connected by a junctional complex consisting of gap junctions and elaborate zonulae occludentes and adherentes. This junctional complex is the site of the . This barrier makes the retinal vessels impermeable to molecules larger than 20–30 kDa. The pigment cells have cylindrical sheaths on their apical surface that are associated with, but do not directly contact, the tip of the photoreceptor processes of the adjacent rod and

retina barrier

blood–

cone cells. Complex cytoplasmic processes project for a short distance between the photoreceptor cells of the rods and cones. Numerous elongated melanin granules, unlike those found elsewhere in the eye, are present in many of these processes. They aggregate on the side of the cell nearest the rods and cones and are the most prominent feature of the cells. The nucleus with its many convoluted infoldings is located near the basal plasma membrane adjacent to Bruch membrane. The cells also contain material phagocytosed from the processes of the photoreceptor cells in the form of lamellar debris (lipofuscin) contained in residual bodies or phagosomes. These lipofuscin granules reside in the basal cytoplasm of the RPE cell and are relatively difficult to detect in routine hematoxylin and eosin (H&E) preparation. Because the lipofuscin pigment is fluorescent, it can be clearly seen in the UV fluorescent microscope. A supranuclear Golgi apparatus and an extensive network of smooth-surfaced endoplasmic reticulum (sER) surround the melanin granules and residual bodies that are present in the cytoplasm. The serves several important functions, including the following:

RPE

absorbs light passing through the neural retina to prevent reflection and resultant glare. It isolates the retinal cells from blood-borne substances. It serves as a major component of the blood–retina barrier via tight junctions between RPE cells. It participates in restoring photosensitivity to visual It

pigments that were dissociated in response to light. The metabolic apparatus for visual pigment resynthesis is present in the RPE cells. It from the rods and cones of the retinal photoreceptor cells.

phagocytoses and disposes of membranous discs

The rods and cones of the photoreceptor cell (layer 2) extend from the outer layer of the neural retina to the pigment epithelium. The rods and cones are the outer segments of photoreceptor cells whose nuclei form the outer nuclear layer of the retina (Figs.

24.9 and 24.10). The light that reaches the photoreceptor cells must first pass through all of the internal layers of the neural retina. The rods and cones are arranged in a palisade manner; therefore, in the light microscope, they appear as vertical striations.

FIGURE 24.10. Schematic diagram of the ultrastructure of rod and cone cells. The outer segments of the rods and cones are closely

associated with the adjacent pigment epithelium.

120 million rods 7 highest density of cones

The retina contains approximately and . They are not distributed equally throughout the photosensitive part of the retina. The is detected in the , which corresponds to the highest visual acuity and best color vision (Fig. 24.11). The highest density of rods is outside the fovea centralis, and their density steadily decreases toward the periphery of the retina. Rods are not present in the fovea centralis nor at the optic disc, which is devoid of any photoreceptors (see Fig. 24.11). The rods are about 2 μm thick and 50 μm long (ranging from about 60 μm at the fovea to 40 μm peripherally). The cones vary in length from 85 μm at the fovea to 25 μm at the periphery of the retina.

million cones

fovea centralis

FIGURE 24.11. Distribution of rods and cones in the human eye.

This graph shows the density of rods and cones per mm 2 across the retina. The peak number of cones occurs in the fovea centralis, where it reaches ~150,000 cones/mm 2. Rod density peaks about 20 degrees from the visual axis and is roughly the same as that of cones. Rods density decreases toward the periphery of the retina. Note that there are no photoreceptors at the optic disc.

Rods are sensitive in low light and produce black-and-white images; cones are less sensitive in low light and produce color images.

rods

sensitive to light

Functionally, the are more and are the receptors used during periods of low light intensity (e.g., at dusk or at night). The rod pigments have a maximum absorption at 496 nm of visual spectrum, and the image provided is one composed of gray tones (a “black-and-white picture”). In contrast, the exist in : (long-, middle-, and short-wavelength sensitive, respectively) that cannot be distinguished morphologically. They are less sensitive to low light but more sensitive to red, green, and blue regions of the visual spectrum. Each class of cones contains a different visual pigment molecule that is activated by the absorption of light at the (420 nm), (531 nm), and (588 nm) ranges in the color spectrum. Cones provide a visual image composed of color by mixing the appropriate proportion of red, green, and blue light. For a description of different types of color blindness, see Folder 24.5.

cones

blue

three classes L, M, and S green

red

FOLDER 24.5

CLINICAL CORRELATION: COLOR BLINDNESS

In individuals with normal color vision, the three primary colors (red, green, and blue) are combined to achieve the full spectrum of color vision. These individuals are called and possess three independent channels for conveying color information that are derived from three different classes of cones (L—red sensitive; M— green sensitive; and S—blue sensitive). Approximately 90% of trichromats can apperceive any given color from impulses generated in all three classes of cones. Some individuals have an impairment of normal color vision, which occurs when one of the cones is altered in its spectral sensitivity. For example, about 6% of trichromats matches colors with an unusual proportion of red and green. These individuals are called . is a condition in which individuals are missing or have a defect in a specific class of cones. True color-blind individuals are and have a defect either in the L, M, or S cones. In this condition, the affected cones are completely missing. Dichromats can only distinguish different colors by matching the impulses generated by the two remaining normal classes of cones. Three major types of color blindness have been identified:

trichromats

Color blindness dichromats

anomalous trichromats

Protanopia is characterized as a defect affecting the longwavelength L cones responsible for red vision. The genes encoding L cone photoreceptor proteins are located on the X chromosome; therefore, protanopia is a sex-linked disorder affecting mainly

males (1% of the male population). These individuals have difficulty distinguishing between blue and green as well as red and green colors; thus, this color vision deficiency is a serious risk factor in driving (Fig. F24.5.1).

FIGURE F24.5.1. Color blindness.

This chart shows the six-color spectrum in normal color vision and in individuals with the three types of color blindness.

Deuteranopia is characterized as a defect affecting the middlewavelength M cones responsible for green vision. Deuteranopia is the most common form of color blindness, affecting about 5% of the male population. It is also a sex-linked disorder because the genes encoding M cone photoreceptor proteins are located in the same region of the X chromosome as the genes for L cones. Similar to protanopia, red and green are the main problem colors (see Fig. F24.5.1). is characterized as a defect affecting the shortwavelength S cones responsible for blue vision (see Fig. F24.5.1). The defect is autosomal and involves mutation of a single gene encoding S cone photoreceptor proteins that reside on chromosome 7. This color blindness occurs very rarely (1 in 10,000) and affects women and men equally.

Tritanopia

Each rod and cone photoreceptor consists of three parts:

outer segment

The of the photoreceptor is roughly cylindrical or conical (hence, the descriptive name or ). This portion of the photoreceptor is intimately related to microvilli projecting from the adjacent pigment epithelial cells. The contains a cilium composed of nine peripheral microtubule doublets extending from a basal body. The connecting stalk appears as the constricted region of the cell that joins the inner to the outer segment. In this region, a thin, tapering process called the extends from the distal end of the inner segment to surround the proximal portion of the outer segment (see Fig. 24.10). The is divided into an outer and an inner . This segment contains a typical complement of organelles associated with a cell that actively synthesizes proteins. A prominent Golgi apparatus, rER, and free ribosomes are concentrated in the myoid region. Mitochondria are most numerous in the ellipsoid region. Microtubules are distributed throughout the inner segment. In the outer ellipsoid portion, cross-striated fibrous rootlets may extend from the basal body among the mitochondria.

rod

cone

connecting stalk

calyceal process

inner segment myoid portion

ellipsoid

The outer segment is the site of photosensitivity, and the inner segment contains the metabolic machinery that supports the activity of the photoreceptor cells. The outer segment is considered a highly modified cilium because it is joined to the inner segment by a short connecting stalk containing a basal body (Fig. 24.12a).

FIGURE 24.12. Electron micrographs of portions of the inner and outer segments of cones and rods. a. This electron micrograph shows

the junction between the inner and outer segments of the rod cell. The outer segments contain the horizontally flattened discs. The plane of this section passes through the connecting stalk and cilium. A centriole, a cilium and its basal body, and a calyceal process are identified. ×32,000. Another electron micrograph shows a similar section of a cone cell. The interior of the discs in the outer segment of the cone is continuous with the extracellular space ( ). ×32,000. (Courtesy of Dr. Toichiro Kuwabara.)

b.

arrows

With

the

TEM, 600–1,000 regularly spaced horizontal are seen in the outer segment (Fig. 24.12). In rods, these discs are membrane-bound structures measuring about 2 μm in diameter. They are enclosed within the plasma membrane of the outer segment (see Fig. 24.12a). The parallel membranes of the discs are about 6 nm thick and are continuous at their ends. The central enclosed space is about 8 nm across. In both rods and cones, the membranous discs are formed from repetitive transverse infolding of the plasma membrane in the region of the outer segment near the cilium. Autoradiographic studies have demonstrated that rods form new discs by infolding of the plasma membrane throughout their life span. Discs are formed in cones in a similar manner but are not replaced on a regular basis. Rod discs lose their continuity with the plasma membrane from which they are derived soon after they are formed. They then pass like a stack of plates, proximally to distally, along the length of the cylindrical portion of the outer segment until they are eventually shed and phagocytosed by the pigment epithelial cells. Thus, each rod disc is a membrane-enclosed compartment within the cytoplasm. Discs within the cones retain their continuity with the plasma membrane (Fig. 24.12b).

membranous discs

Rod cells contain the visual pigment rhodopsin; cone cells contain the visual pigment iodopsin. Rhodopsin (also called visual purple) is a 39-kDa protein in rod cells that initiates the visual stimulus when it is bleached by light. Rhodopsin is present in globular form on the outer surface of the lipid bilayer (on the cytoplasmic side) of the membranous discs. In the cone cells, the visual pigment protein on the membranous discs is the photopigment . Each cone cell is specialized to respond maximally to one of three colors: red, green, or blue. Both rhodopsin and iodopsin contain a membranebound subunit called an and a second small light-absorbing component called a . The opsin of rods is ; the opsins of cones are . The chromophore of rods is a vitamin A–derived carotenoid called . Thus, an adequate intake of is essential for normal vision. Prolonged dietary deficiency of vitamin A leads to the inability to see in dim light ( ).

iodopsin

opsin chromophore photopsins retinal vitamin A night blindness

scotopsin

The interior of the discs of cones is continuous with the extracellular space. The basic difference in the structure of the rod and cone discs— that is, continuity with the plasma membrane—is correlated with the slightly different means by which the visual pigments are renewed in rods and cones. Newly synthesized rhodopsin is incorporated into the membrane of the rod disc as the disc is being formed at the base of the outer segment. It then takes several days for the disc to reach the tip of the outer segment. In contrast, although visual proteins are constantly produced in retinal cones, the proteins are incorporated into cone discs located anywhere in the outer segment.

Vision is a process by which light striking the retina is converted into electrical impulses that are transmitted to the brain. The impulses produced by light reaching the photoreceptor cells are conveyed to the brain by an elaborate network of nerves. The conversion of the incident light into electrical nerve impulses is called and involves several steps:

visual processing

A photochemical reaction occurs in the outer segment of the rods and cones. In the dark, molecules contain a chromophore called retinal in its isometric form of . When rods are exposed to light, the 11-retinal undergoes a conformational change from a bent to a more linear molecule called . The conversion of 11retinal to all-retinal activates opsin, which results in the release of all-retinal into the rod’s cytoplasm (a reaction called ). The activated interacts with a G-protein called , which subsequently activates phosphodiesterase that breaks down . In the dark, high levels of cGMP molecules produced in the photoreceptor cells by guanylyl cyclase are bound to the + cytoplasmic surface of , causing them + to stay open. Steady influx of Na into the cells results in of the plasma membrane and continuous

retinal

transducin

depolarization

rhodopsin

cis

all-trans-retinal trans trans bleaching opsin cyclic guanosine monophosphate (cGMP) cGMP-gated Na channels

11-ciscis

release

of the neurotransmitter (glutamate)

at the synaptic junction

with the bipolar neurons (Fig. 24.13).

FIGURE 24.13. Schematic diagram of visual processing in the photoreceptor cell. a. In the dark, high levels of cGMP generated

by guanylyl cyclase are present in the cytoplasm of the rod. Some of the cGMP molecules are bound to the cytoplasmic surface of cGMP-gated Na + channels, causing them to stay open and resulting in continuous influx of Na + and depolarization of the plasma membrane. This results in a steady release of glutamate, a neurotransmitter, in the synaptic junctions with bipolar neurons. Also in the dark, rhodopsin molecules that contain 11-retinal are inactive. After exposure to light, 11-retinal undergoes a conformational change to all-retinal. This conversion activates opsin (a reaction called ) and releases all-retinal into the rod’s cytoplasm. The activated opsin interacts with G-protein, which subsequently activates phosphodiesterase that breaks down cGMP, effectively lowering the concentration of cGMP in the cell. In this condition, cGMP molecules dissociate from Na + channels, resulting in their closing and hyperpolarization of the plasma membrane. This results in a decrease in glutamate secretion, which is detected by the bipolar neurons and conveyed as electrical impulses to the brain. The released retinal from opsin is converted to its original conformation in retinal pigment epithelial ( ) cells by the RPE65 enzymatic complex and is recycled to the photoreceptor cell. , cyclic guanosine

cis

b.

cis

trans bleaching

trans

RPE

cGMP

monophosphate; monophosphate;

GDP, guanosine diphosphate; GTP, guanosine triphosphate.

GMP,

guanosine

A decrease in the concentration of cGMP within the cytoplasm of the inner segment of the photoreceptor cells is due to the action of phosphodiesterase. Dissociation of cGMP from Na+ channels effectively closes the channels and reduces the influx of Na+ into the cell, resulting in of the plasma membrane. The hyperpolarization causes a at the synapses with bipolar cells, which is detected and conveyed as electrical impulses (see Fig. 24.13).

glutamate secretion

hyperpolarization decrease of

Released retinal from opsin is converted back to its original conformation in the RPE cells and Müller cells. After release, all-trans-retinal is converted to all-trans-

retinol in the cytoplasm of rods and cones and then transported to the cytoplasm of RPE cells (from rods) or both RPE cells and Müller cells (from cones). The energy for this process is provided by the mitochondria located in the inner segment of these photoreceptors. Both Müller cells and RPE cells participate in a multistep conversion of all-retinol to 11- -retinal, which is transported back to the photoreceptor cells for the resynthesis of rhodopsin. The is involved in this conversion; thus, the visual cycle can begin again. During the normal functioning of the photoreceptor cells, the membranous discs of the outer segment are shed and phagocytosed by the pigment epithelial cells (Fig. 24.14). It is estimated that each of these cells is capable of phagocytosing and disposing of about 7,500 discs per day. The discs are constantly turning over, and the production of new discs must equal the rate of disc shedding.

specific protein 65 kDa (RPE65)

trans cis retinal pigment epithelium–

FIGURE 24.14. Electron micrograph of the retinal pigment epithelium in association with the outer segments of rods and cones. Retinal pigment epithelial (RPE) cells contain numerous elongated melanin granules that are aggregated in the apical portion of the cell, where the microvilli extend from the surface toward the outer segments of the rod and cone cells. The retinal pigment epithelial cells contain numerous mitochondria and phagosomes . The arrow indicates the

location of the junctional complex between two adjacent cells. ×20,000. (Courtesy of Dr. Toichiro Kuwabara.)

Discs are shed from both rods and cones.

disc shedding

In rods, after a period of sleep, a burst of occurs as light first enters the eye. The time of disc shedding in cones is more variable. The shedding of discs in cones also enables the receptors to eliminate superfluous membrane. Although not fully understood, the shedding process in cones also alters the size of the discs so that the conical form is maintained as discs are released from the distal end of the cone.

The outer limiting membrane (layer 3) is formed by a row of zonulae adherentes between Müller cells. The outer limiting membrane is not a true membrane. It is a row of zonulae adherentes that attaches the apical ends of Müller cells (i.e., the end that faces the pigment epithelium) to each other and to the rods and cones (see Fig. 24.9). Because Müller cells end at the base of the inner segments of the receptors,

they mark the location of this layer. Thus, the supporting processes of Müller cells, on which the rods and cones rest, are pierced by the inner and outer segments of the photoreceptor cells. This layer is thought to be a metabolic barrier that restricts the passage of large molecules into the inner layers of the retina.

The outer nuclear layer (4) contains the nuclei of the retinal rods and cones.

The region of the rod cytoplasm that contains the nucleus is separated from the inner segment by a tapering process of the cytoplasm. In cones, the nuclei are located close to the outer segments, and no tapering is seen. The cone nuclei stain lightly and are larger and more oval than rod nuclei. Rod nuclei are surrounded by only a thin rim of cytoplasm. In contrast, a relatively thick investment of cytoplasm surrounds the cone nuclei (see Fig. 24.10).

The outer plexiform layer (5) is formed by the processes of the photoreceptor cells and neurons. The outer plexiform layer is formed by the processes of retinal rods and cones and the processes of horizontal, interplexiform, amacrine, and bipolar cells. The processes allow the electrical coupling of photoreceptor cells to these specialized interneurons via synapses. A thin process extends from the region of the nucleus of each rod or cone to an inner expanded portion with several lateral processes. The expanded portion is called a in a rod and a in a cone. Normally, many photoreceptor cells converge onto one bipolar cell and form interconnecting neural networks. Cones located in the fovea, however, synapse with a single bipolar cell. The fovea is also unique in that the compactness of the inner neural layers of the retina causes the photoreceptor cells to be oriented obliquely. Horizontal cell dendritic processes synapse with photoreceptor cells throughout the retina and further contribute to the elaborate neuronal connections in this layer.

spherule

pedicle

The inner nuclear layer (6) consists of the nuclei of horizontal, amacrine, bipolar, interplexiform, and Müller cells.

Müller cells

form the scaffolding for the entire retina. Their processes invest the other cells of the retina so completely that they fill most of the extracellular space. The basal and apical ends of Müller cells form the inner and outer limiting membranes, respectively. Microvilli extending from their apical border lie between the photoreceptor cells of the rods and cones. Capillaries from the retinal vessels extend only to this layer. The rods and cones carry out their metabolic exchanges with extracellular fluids transported across the blood–retina barrier of the RPE. The four types of conducting cells—bipolar, horizontal, interplexiform, and amacrine—found in this layer have distinct orientations (see Fig. 24.9):

Bipolar cells and their processes extend to both the inner and outer plexiform layers. In the peripheral regions of the retina, the axons of bipolar cells pass to the inner plexiform layer where they synapse with several ganglion cells. Through these connections, the bipolar cells establish communication with multiple cells in each layer, except in the fovea, where they may synapse only with a single ganglion cell to provide greater visual acuity in this region. and their processes extend to the outer plexiform layer where they intermingle with processes of bipolar cells. The cells have synaptic connections with rod spherules, cone pedicles, and bipolar cells. This electrical coupling of cells is thought to affect the functional threshold between rods and cones and bipolar cells. processes pass inward, contributing to a complex interconnection of cells. Their processes branch extensively to provide sites of synaptic connections with axonal endings of bipolar cells and dendrites of ganglion cells. Besides bipolar and ganglion cells, the amacrine cells synapse in the inner plexiform layer with interplexiform and other amacrine cells (see Fig. 24.9). and their processes have synapses in both inner and outer plexiform layers. These cells convey impulses from the inner plexiform to the outer plexiform layer.

Horizontal cells Amacrine cells’

Interplexiform cells

The inner plexiform layer (7) consists of a complex array of intermingled neuronal cell processes. The inner plexiform layer consists of synaptic connections between axons of the bipolar neurons and dendrites of ganglion cells. It also contains synapses between intermingling processes of amacrine cells and bipolar neurons, ganglion cells, and interplexiform neurons. The course of these processes is parallel to the inner limiting membrane, thus giving the appearance of horizontal striations to this layer (see Fig. 24.9).

The ganglion cell layer (8) consists of the cell bodies of large multipolar neurons. The cell bodies of large multipolar nerve cells, measuring as

much as 30 μm in diameter, constitute the ganglion cell layer. These nerve cells have lightly staining round nuclei with prominent nucleoli and Nissl bodies in their cytoplasm. An axonal process emerges from the rounded cell body, passes into the , and then enters . The dendrites extend from the opposite end of the cell to ramify in the inner plexiform layer. In the peripheral regions of the retina, a single ganglion cell may synapse with 100 bipolar cells. In marked contrast, in the macular region surrounding the fovea, the bipolar cells are smaller (some authors refer to them as ), and they tend to make one-to-one connections with ganglion cells. Over most of the retina, the ganglion cells are only a single layer of cells. At the macula, however, they are piled as many as eight deep, although they are absent over the fovea itself. Scattered among the ganglion cells are small neuroglial cells with densely staining nuclei (see Fig. 24.9).

fiber layer

the optic nerve

nerve

midget

bipolar cells

The layer of optic nerve fibers (9) contains axons of the ganglion cells. The axonal processes of the ganglion cells form a flattened layer running parallel to the retinal surface. This layer increases in depth as the axons converge at the (Fig. 24.15). The axons are thin, nonmyelinated processes measuring as much as 5 μm in diameter (see Fig. 24.9). The retinal vessels, including the superficial capillary network, are primarily located in this layer.

optic disc

FIGURE 24.15. Normal view of the fundus in ophthalmoscopic examination of the right eye. The site where the axons converge to form the optic nerve is called the optic disc . Because the optic disc is devoid of photoreceptor cells, it is a blind spot in the visual field. From the center of the optic nerve (clinically called the optic cup ), central retinal vessels emerge. The artery divides into upper and lower branches, each of which further divides into nasal and temporal branches (note the nasal and temporal directions on the image). Veins have a similar pattern of tributaries. Approximately 17 degree or 2.5 times optic disc diameters lateral to the disc, the slightly oval-shaped, blood vessel–free, and pigmented area represents the macula lutea. The , a shallow depression in the center of the , is also visible. (Courtesy of Dr. Renzo A. Zaldivar.)

fovea centralis

macula lutea

The inner limiting membrane (layer 10) consists of a basal lamina separating the retina from the vitreous body. The inner limiting membrane forms the innermost boundary of the

retina. It serves as the basal lamina of Müller cells (see Fig. 24.9). In younger individuals, reflections from the internal limiting membrane produce a that is seen during ophthalmoscopic examination of the eye. In older individuals, a semitranslucent sheet of cells and extracellular matrix can be formed on the inner surface of the retina in conjunction with the

retinal sheen

epiretinal

inner limiting membrane. This condition is called or and is responsible for variable clinical symptoms, including optical distortion and blurred vision. ERM is initially formed by cells from within the retina (RPE cells, Müller cells, and astrocytes) that begin proliferating and migrating onto the surface of the internal limiting membrane. Later, the membrane is infiltrated by macrophages, fibroblasts, and myofibroblasts. To prevent damage to the underlying retina, surgical removal of the ERM may be performed.

membrane (ERM)

macular pucker

Specialized regions of the retina The fovea (fovea centralis) appears

as a small (1.5 mm in diameter), shallow depression located at the posterior pole of the visual axis of the eye. Its central region, known as the , is about 200 μm in diameter (see Fig. 24.15). Except for the photoreceptor layer, most of the layers of the retina are markedly reduced or absent in this region (see Fig. 24.6). Here, the photoreceptor is composed entirely of cones (~4,000) that are longer and more slender and rod like than they are elsewhere. The fovea is the area of the retina specialized for the discrimination of details and color vision. The ratio between cones and ganglion cells is close to 1:1. Retinal vessels are absent in the fovea, allowing light to pass unobstructed into the cones’ outer segments. The adjacent pigment epithelial cells and choriocapillaris are also thickened in this region. The is the area surrounding the fovea and is approximately 5.5 mm in diameter. It is yellowish because of the presence of yellow pigment (xanthophyll). The macula lutea contains approximately 17,000 cones and gains rods at its periphery. Retinal vessels are also absent in this region. Here, the retinal cells and their processes, especially the ganglion cells, are heaped up on the sides of the fovea so that light may pass unimpeded to this most sensitive area of the retina.

foveola

macula lutea

Vessels of the retina The central retinal artery

central retinal vein

and , the vessels that can be seen and assessed with an ophthalmoscope, pass through the center of the optic nerve to enter the bulb of the eye at the optic disc (see Fig. 24.2 and pages 982-983, the

section on the development of the eye). The central retinal artery provides nutrients to the inner retinal layers. The artery branches immediately into the upper and lower branches, each of which divides again into nasal and temporal branches (see Fig. 24.15). Veins undergo a similar pattern of branching. The vessels initially lie between the vitreous body and the inner limiting membrane. As the vessels pass laterally, they also move deeper within the inner retinal layers. Branches from these vessels form a capillary plexus that reaches the inner nuclear layer and, therefore, provides nutrients to the inner retinal layers (layers 6–10; see pages 993-994). Nutrients to the remaining layers (layers 1–5) are provided by diffusion from the vascular choriocapillary layer of the choroid. The branches of the do not anastomose and, therefore, are classified as . Evaluation of the retinal vessels and appearance of the optic disc during ophthalmoscopy not only gives important information on the state of the eye but also may reveal early clinical signs of a number of conditions, including increased , , , and .

retinal artery anatomic end arteries diabetes

central

intracranial pressure hypertension glaucoma

Crystalline Lens

Like the lens in a camera, the basic function of the eye lens is to transmit and focus light onto the retina. The lens is a transparent, biconvex structure that has no vessels or nerves and is almost totally devoid of connective tissue, except for an enveloping capsule of basal lamina. It is suspended between the edges of the ciliary body by the . The pull of the zonular fibers keeps the lens in a flattened condition. Release of tension causes the lens to widen or to bend light rays originating close to the eye so that they focus on the retina. The lens has three principal components (Fig. 24.16):

zonular fibers

accommodate

FIGURE 24.16. Structure of the lens. a.

This schematic drawing of the lens suspended from ciliary processes by zonular fibers indicates its structural components. Note that the capsule of the lens is formed by the basal lamina of the lens fibers and the subcapsular epithelium located on the anterior surface of the lens. A strip of capsule was removed on this drawing to show underlying epithelium. Also note the location of the germinal zone ( ) at the lens equator, where cells divide and differentiate into the lens fiber cells. The organelle-free center of the lens is occupied by the lens nucleus. This highmagnification photomicrograph of the germinal zone of the lens (near its equator) shows the active process of lens fiber formation from the . Note the thick and the underlying layer of nuclei of lens fibers during their differentiation. The do not possess nuclei. ×570.

yellow

subcapsular epithelium lens fibers

lens capsule

b.

lens capsule

mature

The is a thick basal lamina that surrounds the outer surface of the lens. It originates as the basal lamina of the embryonic lens vesicle. The anterior part of the capsule is thick, measuring approximately 10–20 μm, and is produced by the anterior lens cells. The posterior part of the capsule is much thinner, measuring approximately 5–10 μm. The lens capsule, composed primarily of type IV collagen and proteoglycans (i.e., laminin, entactin, perlecan), is elastic. It is thickest at the equator where the zonular fibers attach to it.

subcapsular epithelium

The is derived from the epithelial cells of the anterior part of the embryonic lens vesicle. It represents a single cuboidal layer of present only on the anterior surface of the lens. The epithelial cells of the posterior part of the vesicle elongate anteriorly and form the that fill the cavity of the optic vesicle. are formed at the periphery near the . Here, epithelial cells proliferate and migrate along the posterior lens capsule to differentiate into mature lens fiber cells. In the center of the lens, epithelial cells are quiescent. As lens fiber cells differentiate, they undergo massive elongation and lose all of their organelles, including nuclei, forming the .

lens epithelial cells

primary lens fibers Secondary lens fibers (lens fiber cells) lens equator

organelle-free

zone Gap junctions

connect the cuboidal cells of the subcapsular epithelium. They have few cytoplasmic organelles and stain faintly. The apical region of the cell is directed toward the internal aspect of the lens and the , with which they form . The lens increases in size during normal growth and then continues to produce new lens fibers at an ever-decreasing rate throughout life. The new lens fibers develop from the subcapsular epithelial cells located near the equator (see Fig. 24.16) are laid down peripherally as concentric lamellae in an onion-like arrangement. Cells in this region increase in height and then differentiate into lens fibers. As the lens fibers develop, they become highly elongated and appear as thin, flattened structures. They lose their nuclei and other organelles as they become filled with proteins called . Mature lens fibers attain a length of 7–10 mm, a width of 8–10 μm, and a thickness of 2 μm. In the adult lens, only lens fibers in the outermost region maintain their nuclei and organelles. Near the center, in the , the fibers are compressed and condensed to such a degree that individual fibers are impossible to recognize. The lens nucleus is an organelle-free zone and is composed of primary lens fiber cells laid down during embryonic and fetal development. The lens fibers are joined at their apical and basal ends by specialized junctions called . Despite its density and protein

junctional complexes

lens fibers

crystallins

lens nucleus

sutures

content, the lens is normally transparent (see Fig. 24.16). The high density of lens fibers makes it difficult to obtain routine histologic sections of the lens that are free from artifacts.

Changes in the lens are associated with aging.

With increasing age, the lens gradually loses its elasticity and ability to accommodate. This condition, called , usually occurs in the fourth decade of life. It is easily corrected by wearing reading glasses or using a magnifying lens. Loss of transparency of the lens or its capsule is also a relatively common condition associated with aging. This condition, called , may be caused by conformational changes or cross-linking of proteins. The development of a cataract may also be related to disease processes, metabolic or hereditary conditions, trauma, or exposure to a deleterious agent (such as ultraviolet radiation). Cataracts that significantly impair vision can usually be corrected surgically by removing the lens and replacing it with a plastic lens implanted in the posterior chamber.

presbyopia

cataract

Vitreous Body

The vitreous body is the transparent jelly-like substance that fills the vitreous chamber in the posterior segment of the eye. The vitreous body is loosely attached to the surrounding structures, including the inner limiting membrane of the retina. The main portion of the vitreous body is a homogeneous gel containing approximately 99% water (the vitreous humor), collagen, glycosaminoglycans (principally hyaluronan), and a small population of cells called . These cells are believed to be responsible for the synthesis of collagen fibrils and glycosaminoglycans. Hyalocytes in routine H&E preparations are difficult to visualize. Often, they exhibit a well-developed rER and Golgi apparatus. Fibroblasts and tissue macrophages are sometimes seen in the periphery of the vitreous body. The (or ), which is not always visible, runs through the center of the vitreous body from the optic disc to the posterior lens capsule. It is the remnant of the pathway of the hyaloid artery of the developing eye.

hyalocytes

canal

Cloquet canal

hyaloid

ACCESSORY STRUCTURES OF THE EYE

The primary functions of the eyelids are to cover, protect, and lubricate the eyes. The eyelids represent folds of modified skin containing highly

modified epidermal appendages to cover, protect, and lubricate the anterior portions of the eyes. The anterior surface of the eyelid is covered by thin , and its posterior surface is lined by a specialized mucous membrane, the . The skin of the eyelids is loose and elastic to accommodate their movement. Within each eyelid is a flexible support, the , consisting of dense fibrous and elastic tissue. In the upper eyelid, the lower free edge of the tarsal plate extends to the lid margin, and its superior border serves for the attachment of smooth muscle fibers of the . The undersurface of the tarsal plate is covered by the conjunctiva (Fig. 24.17). The striated , a facial expression muscle, forms a thin oval sheet of circularly oriented skeletal muscle fibers overlying the tarsal plate. In addition, the connective tissue of the upper eyelid contains tendon fibers of the that open the eyelid (see Fig. 24.17). A mucocutaneous junction between eyelid skin and conjunctiva occurs near the lid margin. The emerge from the most anterior edge of the lid margin. They are short, stiff, curved hairs and may occur in double or triple rows. The lashes on the same eyelid margin may have different lengths and diameters.

skin

plate

Müller)

conjunctiva tarsal

superior tarsal muscle (of orbicularis oculi muscle

levator palpebrae superioris muscle

eyelashes

FIGURE 24.17. Structure of the eyelid. a.

This schematic drawing of the eyelid shows the skin, associated skin appendages, muscles, tendons, connective tissue, and conjunctiva. Note the distribution of multiple small glands associated with the eyelid and observe the reflection of the palpebral conjunctiva in the fornix of the lacrimal sac to become the bulbar conjunctiva. Photomicrograph of a sagittal section of the eyelid stained with picric acid for better visualization of epithelial components of the skin and the numerous glands. In this preparation, muscle tissue (i.e., ) stains , and the epithelial cells of the skin, conjunctiva, and glandular epithelium are . Note the presence of numerous glands within the eyelid. The is the largest gland, and it is located within the dense connective tissue of the tarsal plates. This sebaceous gland secretes into ducts opening onto the eyelids. ×20. Higher magnification of a tarsal gland from the showing the typical structure of a holocrine gland. ×60.

b.

orbicularis oculi muscle

green tarsal (Meibomian) gland

boxed area

yellow

Inset.

The conjunctiva lines the space between the inner surface of the eyelids and the anterior surface of the eye without covering the cornea. The conjunctiva is a thin, transparent mucous membrane that extends from the corneoscleral limbus located on the peripheral margin of the cornea across the sclera (bulbar conjunctiva) and covers the internal surface of the eyelids (palpebral conjunctiva). The palpebral conjunctiva merges with the bulbar conjunctiva at the fornices of the conjunctival sac; this part is

forniceal conjunctiva

called the (Fig. 24.18). Bulbar, palpebral, and forniceal conjunctiva form a conjunctival sac, a space between the eyelid and eyeball that opens anteriorly at the palpebral fissure. The conjunctival sac can hold fluid up to 30 µL. Because a standard eyedropper dispenses about 50 µL of suspended medicine per drop, one drop is more than enough to overfill the conjunctival sac.

FIGURE 24.18. Conjunctiva and conjunctival sac.

This photograph of the lower part of the eyeball with a reflected lower eyelid shows different regions of the conjunctiva that line the conjunctival sac. The area shown is located between the inner surface of the eyelid and the anterior surface of the eye. The bulbar conjunctiva extends from the corneoscleral limbus covering the sclera of the eye (it does not cover the cornea) to its reflections onto the internal surface of the eyelid, at which point it is called the . This photograph shows the inferior point of reflection onto the lower eyelid (called the of the conjunctival sac). The conjunctiva in these regions is recognized as the forniceal conjunctiva.

inferior fornix

palpebral conjunctiva

stratified columnar epithelium

The conjunctiva consists of a containing numerous and rests on a lamina propria composed of loose connective tissue. The goblet cells secrete a

goblet cells

component of the tears that bathe the eye. Melanocytes are present in the basal epithelial layer and, like melanocytes in the skin, transfer melanosomes into neighboring epithelial cells. Accumulation of diffuse lymphatic tissue is evident, especially deep to the forniceal conjunctiva (Fig. 24.19). These specialized collections of T and B lymphocytes underlying the conjunctiva are called (Fig. 24.20). It functions to recognize and process antigens and trigger an appropriate immune response against the microbial invasion of the ocular surface. The conjunctiva is supplied with blood by the branches of arteries of the eyelid (marginal tarsal arcades) and from the eyeball (anterior ciliary arteries). The conjunctiva receives sensory innervation from the branches of the trigeminal nerve. , an inflammation of the conjunctiva, commonly called , is characterized by redness, irritation, and watering of the eyes. For more clinical information about this condition, see Folder 24.6.

conjunctiva-associated lymphatic tissue (CALT)

Conjunctivitis pinkeye

FIGURE 24.19. Superior fornix of the conjunctival sac.

This lowmagnification hematoxylin and eosin (H&E)-stained specimen was obtained from the superior fornix of the conjunctival sac as indicated by the in the inset. The palpebral conjunctiva lines the inner surface of the eyelid, and in the superior fornix of the conjunctival sac, it reflects onto the eyeball (bulbar conjunctiva). This reflection

rectangle

is identified as the forniceal conjunctiva and is composed of stratified columnar epithelium containing numerous goblet cells. Accumulations of lymphatic tissue called ( ) are clearly visible. There are numerous blood vessels ( ) underlying the palpebral conjunctiva. ×120. (Courtesy of Dr. Nick Mamalis, University of Utah, Moran Clinical Ophthalmology Resource for Education [CORE], Salt Lake City, Utah.)

conjunctiva-associated lymphatic tissue CALT BV

FIGURE 24.20. Forniceal conjunctiva.

This high-magnification hematoxylin and eosin (H&E)-stained specimen shows the fornix of the conjunctival sac. The forniceal conjunctiva shows a typical pattern of the stratified columnar epithelium containing goblet cells that rests on a lamina propria composed of loose connective tissue. The stratified columnar epithelium farther away from the fornix may change into columnar stratified or squamous stratified nonkeratinized epithelium ( ). Note the accumulations of diffuse lymphatic tissue deep into the conjunctiva known as ( ). ×220. (Courtesy of Dr. Nick Mamalis, University of Utah, Moran Clinical Ophthalmology Resource for Education [CORE], Salt Lake City, Utah.)

lower right corner of conjunctival sac associated lymphatic tissue CALT

FOLDER 24.6

conjunctiva-

CLINICAL CORRELATION: CONJUNCTIVITIS Conjunctivitis , otherwise known as pinkeye, is an inflammation of the conjunctiva. It may be localized in either the palpebral conjunctiva or the bulbar conjunctiva. Individuals may present with relatively nonspecific symptoms and signs that include redness, irritation, and watery discharge from the eye (Fig. F24.6.1). The symptoms can also mimic a foreign-body sensation. Extended use of contact lenses can cause allergic or bacterial conjunctivitis and may be the first sign of more serious ocular disease (i.e., corneal ulcer). In general, symptoms that last 90% of patients experience a complete reversal of their hearing loss.

FOLDER 25.2

CLINICAL CORRELATION: HEARING LOSS—VESTIBULAR DYSFUNCTION

Several types of disorders can affect the auditory and vestibular system and result in deafness, dizziness (vertigo), or both. Auditory disorders are classified as either sensorineural or conductive. results when sound waves are mechanically impeded from reaching the auditory sensory receptors within the internal ear. This type of hearing loss principally involves the external ear or structures of the middle ear. Conductive hearing loss is the second most common type of loss after sensorineural hearing loss, and it usually involves a reduction in sound level or the inability to hear faint sounds. Conductive hearing loss may be caused by otitis media (ear infection); in fact, this is the most common cause of temporary hearing loss in children. Fluid that collects in the tympanic cavity can also cause significant hearing problems in children. Other common causes of conductive hearing loss include excess wax or foreign bodies in the external acoustic meatus or diseases that affect the ossicles in the middle ear (otosclerosis; see also Folder 25.1). In many cases, conductive hearing loss can be treated either medically or surgically and may not be permanent. may occur after injury to the auditory sensory hair cells within the internal ear, cochlear division of cranial nerve VIII, nerve pathways in the central nervous system (CNS), or auditory cortex. Sensorineural hearing loss accounts for about 90% of all hearing loss. It may be congenital or acquired. Causes of acquired sensorineural hearing loss include infections of the membranous labyrinth (e.g., meningitis, chronic otitis media), fractures of the temporal bone, acoustic trauma (i.e., prolonged exposure to excessive noise), and administration of certain classes of antibiotics and diuretics. Another example of sensorineural hearing loss often results from aging. Sensorineural hearing loss not only involves a reduction in sound level but also affects the ability to hear clearly or to distinguish speech. A loss of the sensory hair cells or associated nerve fibers begins in the basal turn of the cochlea and progresses apically over time. The characteristic impairment is a highfrequency hearing loss termed (see presbyopia, page 1004). In selected patients, the use of a can partially restore some hearing function. The cochlear implant is an

Conductive hearing loss

Sensorineural hearing impairment

presbycusis cochlear implant

electronic device consisting of an external microphone, amplifier, and speech processor linked to a receiver implanted under the skin of the mastoid region. The receiver is connected to the multielectrode intracochlear implant inserted along the wall of the cochlear canal. After considerable training and tuning of the speech processor, the patient’s hearing can be partially restored to various degrees ranging from recognition of critical sounds to the ability to converse.

Two muscles attach to the ossicles and affect their movement. The tensor tympani muscle lies in a bony canal above the auditory tube; its tendon inserts on the malleus. Contraction of this muscle increases tension on the tympanic membrane. The lies in a bony eminence on the posterior wall of the middle ear; its tendon inserts on the stapes. Contraction of the stapedius tends to dampen the movement of the stapes at the oval window. The stapedius is only a few millimeters long and is the smallest skeletal muscle. The two muscles of the middle ear are responsible for a protective reflex called the or . In response to intense sound, involuntary contraction of the muscles makes the chain of ossicles more rigid, thus reducing the transmission of vibrations to the internal ear. The muscles will contract on both sides, regardless of which ear is stimulated. This reflex protects the internal ear from the damaging effects of very loud sounds. In certain conditions, such as impulse noise (i.e., fireworks or gun fire), the attenuation reflex is ineffective.

stapedius muscle

reflex

attenuation reflex

acoustic

The auditory tube connects the middle ear to the nasopharynx. The auditory (Eustachian) tube is a narrow flattened channel approximately 3.5 cm long. This tube is lined with ciliated pseudostratified columnar epithelium, about one-fifth of which is composed of goblet cells. It vents the middle ear to nasopharynx, equalizing the pressure of the middle ear with atmospheric pressure. In addition, the auditory tube is responsible for draining the secretion produced by the mucous membrane of the middle ear towards the nasopharynx with the aid of the ciliated pseudostratified columnar epithelium. The

auditory tube is normally closed; its walls are pressed together but they separate during yawning, chewing, swallowing, and when individual holds the nose and blows. Children are more vulnerable to the middle ear infections, due to the immature development of their auditory tubes which are shorter, narrower, and more horizontal than in the adults. It is common for infections to spread from the pharynx to the middle ear via the auditory tube (causing ). A small mass of lymphatic tissue, the , is often found at the pharyngeal orifice of the auditory tube.

otitis media tubal tonsil The mastoid air cells extend from the middle ear into the temporal bone. A system of air cells projects into the mastoid portion of the

temporal bone from the middle ear. The epithelial lining of these air cells is continuous with that of the tympanic cavity and rests on periosteum. This continuity allows infections in the middle ear to spread into , causing . Before the development of antibiotics, repeated episodes of otitis media and mastoiditis usually led to deafness.

mastoid air cells

mastoiditis

The middle ear contributes to the amplification of mechanical forces generated by the vibration of the tympanic membrane. All three ossicles in the tympanic cavity are involved in the amplification of the mechanical force that vibrates the tympanic membrane in two ways:

differences in the surface

The main amplification comes from between the tympanic membrane and the footplate of the stapes. The has a surface area of 2 approximately , whereas the footplate of the has 2 a surface area of about . Sound waves apply force to every square millimeter of the tympanic membrane, and this energy is transferred via the chain of ossicles to the much smaller area of the footplate. Therefore, the pressure applied to the cochlear fluid by the footplate is

area

tympanic membrane 65 mm 3 mm

stapes

about 22

times

the pressure applied to the tympanic membrane (Fig.

25.7).

FIGURE 25.7. Summary of amplification of sound entering the ear. This drawing shows the external and middle ear structures and

their contributions to the amplification of sound entering the ear. Note that the largest amplification comes from the difference in the surface area between the tympanic membrane (65 mm 2) and the footplate of the stapes (3 mm 2). This surface area difference results in ~22 times the amplification of the pressure applied by the footplate of the stapes. Another source of amplification comes from the external acoustic meatus and middle ear. The external acoustic meatus acts as a resonator that increases the sound pressure acting on the tympanic membrane by ~2 times. Finally, the arrangement of auditory ossicles resembles a basic lever that multiplies the applied mechanical force acting on the footplate of the stapes by ~1.3 times. By multiplying these three amplification factors, the acoustic energy entering the ear is amplified ~60 times.

Additional

amplification comes from that act as

auditory ossicles

levers

the arrangement of that multiply the

mechanical force applied to the stapes. Because the pivot point of the ossicle chain is located farther from the tympanic membrane than from the stapes, the amplification of the mechanical force at the oval window is increased by a factor of . This lever system is adjustable by the action of muscles in the tympanic cavity and may attenuate loud sounds to protect the ear (see Fig. 25.7).

approximately 1.3

Under normal conditions, the acoustic energy entering the ear is amplified , allowing humans to detect frequencies between 2,000 and 5,000 Hz. The degree of amplification is calculated by multiplying the amplification factors contributed by the external acoustic meatus (~2 times) as described earlier on pages 1018-1019, the surface area differences between the tympanic membrane and the footplate of stapes (~22 times), and the basic lever action of ossicles (~1.3 times). However, this calculation (2 × 22 × 1.3 = 57.2) must be used with caution owing to variability in the mechanical function of the middle ear and its components, such as ossicular joints, ligaments, muscles, and air volumes, as well as varying frequencies of sound.

approximately 60 times

INTERNAL EAR

The internal ear consists of two labyrinthine compartments, one contained within the other. The bony labyrinth is a complex system of interconnected cavities and canals in the petrous part of the temporal bone. The membranous labyrinth lies within the bony labyrinth and consists of a complex system of small sacs and tubules that also form a continuous space enclosed within a wall of epithelium and connective tissue. There are three fluid-filled spaces in the internal ear:

Endolymphatic spaces are labyrinth. The endolymph

contained within the membranous of the membranous labyrinth is similar in composition to (it has a high K+ concentration and a low Na+ concentration). The endolymph

intracellular fluid

is produced in the stria vascularis, a specialized area of the cochlear duct (see pages 1032-1034). It drains via the endolymphatic duct to the endolymphatic sac, which terminates in the epidural space of the posterior cranial fossa. The lies between the wall of the bony labyrinth and the wall of the membranous labyrinth. The is similar in composition to and (it has a low K+ concentration and a high Na+ concentration). Perilymph is produced as an ultrafiltrate from the periosteal microvasculature within the bony labyrinth. It drains via a narrow channel within the temporal bone, called the , directly into the cerebrospinal fluid contained within the subarachnoid space of the cranial cavity. The lies within the tunnels of the organ of Corti of the cochlea. It is a true intercellular space. The cells surrounding the space loosely resemble an absorptive epithelium. The cortilymphatic space is filled with , which has a composition similar to that of .

perilymphatic space perilymph cerebrospinal fluid

extracellular fluid

cochlear aqueduct

cortilymphatic space

cortilymph extracellular fluid

Structures of the Bony Labyrinth

The bony labyrinth consists of three connected spaces within the temporal bone. The three spaces of the bony labyrinth, as illustrated in Figure 25.8, are the

FIGURE 25.8. Photograph of a cast of the bony labyrinth of the internal ear. The cochlear portion of the bony labyrinth appears blue green; the vestibule and semicircular canals appear orange red. (Courtesy of Dr. Merle Lawrence.)

semicircular canals, vestibule, and cochlea. The vestibule is the central space that contains the utricle and saccule of the membranous labyrinth. The vestibule is the small oval chamber located in the center of the bony labyrinth. The utricle and saccule of the membranous labyrinth lie in elliptical and spherical recesses, respectively. The semicircular canals extend from the vestibule posteriorly, and the cochlea extends from the vestibule anteriorly. The oval window into which the footplate of the stapes inserts lies in the lateral wall of the vestibule.

The semicircular canals are tubes within the temporal bone that lie at right angles to each other. Three semicircular canals, each forming about three-quarters of a circle, extend from the wall of the vestibule and return to it. The semicircular canals are identified as anterior, posterior, and lateral and lie within the temporal bone at approximately right angles to each other. They occupy three planes in space—sagittal, frontal, and horizontal. The end of each semicircular canal closest to the vestibule is expanded to form the (Fig. 25.9a and b). The three canals open into the vestibule through five orifices; the anterior and posterior semicircular canals join at one end to form the (see Fig. 25.9a).

ampulla

limb

common bony

FIGURE 25.9. Diagrams and photograph of the human internal ear. a. This lateral view of the left bony labyrinth shows its divisions: the vestibule, cochlea, and three semicircular canals. The openings of the oval window and the round window can be observed. b. This photograph of a cast obtained by injection of polyester resin into the human internal ear shows an authentic shape of the bony labyrinth.

Note that the cast material is pouring out of the cochlea through the oval and round windows. Also, in this image, the cast of the facial canal that contains the facial nerve is visible. ×5. (Courtesy of Dr. Elsa Erixon.) Diagram of a membranous labyrinth of the internal ear lying within the bony labyrinth. The cochlear duct can be seen spiraling within the bony cochlea. The saccule and utricle are positioned within the vestibule, and the three semicircular ducts are lying within their respective canals. This view of the left membranous labyrinth allows the endolymphatic duct and sac to be observed. This view of the left membranous labyrinth shows the sensory regions of the internal ear for equilibrium and hearing. These regions are the macula of the saccule and macula of the utricle, the cristae ampullares of the three semicircular ducts, and the spiral organ of Corti of the cochlear duct.

c.

d.

The cochlea is a cone-shaped helix connected to the vestibule. The spiral lumen of the cochlea, called the cochlear canal

(like the semicircular canals), is continuous with that of the vestibule. It connects to the vestibule via two openings, the and the , both of which are located on the side opposite the openings of the semicircular canals. Between its base and the apex, the cochlear canal makes approximately 2.75 turns around a central core of spongy bone called the (Plate 25.1, page 1042). A sensory ganglion, the , lies in the modiolus. A thin membrane (the secondary tympanic membrane) covers the round window, whereas the footplate of the stapes is positioned within the oval window. These two openings are located at the base of the cochlear canal.

round window

oval window

modiolus spiral ganglion

Structures of the Membranous Labyrinth

The membranous labyrinth contains the endolymph and is suspended within the bony labyrinth. The membranous labyrinth consists of a series of communicating sacs and ducts containing endolymph. It is suspended within the bony labyrinth (Fig. 25.9c), and the remaining space is filled with perilymph. The membranous labyrinth is composed of two

cochlear labyrinth

vestibular labyrinth

divisions: the and the (Fig. 25.9d). The vestibular labyrinth contains the following:

semicircular ducts utricle saccule utriculosaccular duct

Three lie within the semicircular canals and are continuous with the utricle. The and the , which are contained in recesses in the vestibule, are connected by the membranous .

cochlear duct

The cochlear labyrinth contains the , which is contained within the cochlea and is continuous with the saccule (see Fig. 25.9c and d).

Sensory cells of the membranous labyrinth Specialized sensory cells are located in six regions in the membranous labyrinth. Six sensory regions of membranous labyrinth are composed of sensory hair cells and accessory supporting cells. These regions project from the wall of the membranous labyrinth into the endolymphatic space in each internal ear (see Fig. 25.9d):

cristae ampullares (ampullary crests)

Three are located in the membranous ampullae of the semicircular ducts. They are sensitive to the angular acceleration of the head (i.e., turning the head). Two maculae, one in the utricle ( ) and the other in the saccule ( ), sense the position of the head and its linear movement. The projects into the endolymph of the cochlear duct. It functions as a sound receptor.

macula of utricle macula of saccule spiral organ of Corti

Hair cells are epithelial mechanoreceptors of the vestibular and cochlear labyrinth. The hair cells of the vestibular and cochlear labyrinths function as mechanoelectrical transducers; they convert mechanical energy into electrical energy that is then transmitted via the vestibulocochlear nerve to the brain. The

hair cells derive their name from the organized bundle of rigid projections at their apical surface. This surface holds a that is formed by rows of stereocilia called . The rows increase in height in one particular direction across the bundle (Fig. 25.10). In the vestibular system, each hair cell possesses a single true cilium called a , which is located behind the row of longest stereocilia (Fig. 25.11). In the auditory system, the hair cells lose their cilium during development but retain the . The position of the kinocilium (or basal body) behind the longest row of stereocilia defines the polarity of this asymmetric hair bundle. Therefore, movement of the stereocilia toward the kinocilium is perceived differently than movement in the opposite direction (see later).

bundle hairs

hair sensory

kinocilium

basal body

FIGURE 25.10. Electron micrographs of the kinocilium and stereocilia of a vestibular sensory hair cell. a. Scanning electron micrograph of the apical surface of a sensory hair cell from the macula of the utricle. Note the relationship of the kinocilium ( K ) to the stereocilia ( S ). ×47,500. b. Transmission electron micrograph of the kinocilium ( K ) and stereocilia ( S ) of a vestibular hair cell in cross section. The kinocilium has a larger diameter than the stereocilia. ×47,500. ( a. Reprinted with permission from Rzadzinska

AK, Schneider ME, Davies C, et al. An actin molecular treadmill and myosins maintain stereocilia functional architecture and self-renewal. . 2004;164:887–897. Reprinted with permission from Hunter-Duvar IM, Hinojosa R. Vestibule: sensory epithelia. In: Friedmann I, Ballantyne J, eds. . Butterworth; 1984.)

J Cell Biol Ear

b. Ultrastructural Atlas of the Inner

FIGURE 25.11. Diagram of two types of sensory hair cells in the sensory areas of the membranous labyrinth. The type I hair cell

has a flask-shaped structure with a rounded base. The base is enclosed in a chalice-like afferent nerve ending containing several ribbon synapses in addition to several synaptic boutons for efferent nerve endings. Note the apical surface specializations of this cell, which include a kinocilium and hair bundle. The apical cytoplasm of hair cells contains basal bodies for the attachment of the kinocilium and a terminal web for the attachment of stereocilia. The type II hair cell is cylindrical and possesses several nerve terminals at its base for both afferent and efferent nerve fibers. The apical surface specializations are identical to those of the type I cell. The molecular organization of the stereocilia is depicted in the diagram on the . The top link connects the lateral plasma membrane of the stereocilium shaft (where K + transduction channels are located) with the tip of the shorter stereocilium (where the mechanoelectrical transduction [ ] channel protein is located). Movement of the stereocilia toward the kinocilium opens the channels, causing depolarization of the hair cell, whereas movement in the opposite direction (away from the kinocilium) causes hyperpolarization. Note that the proximal end of each stereocilium is tapered and its narrow rootlets are anchored within the terminal web (cuticular plate) of the hair cell. Several other fibrillar connectors between neighboring stereocilia are also shown.

right

MET

MET

Stereocilia of hair cells are rigid structures that contain mechanoelectrical transducer channel proteins at their distal ends. The stereocilia of hair cells have a molecular structure similar to those described on pages 127-128. Tightly packed actin filaments cross-linked by fimbrin and espin (actinbundling proteins) form their internal core structure. Espins provide the most rigid cross-linking for stereocilia; mutations that alter their structure cause cochlear and vestibular dysfunction. The high density of actin filaments and the extensive cross-linking pattern impart rigidity and stiffness to the shaft of the stereocilium. The shaft tapers at its proximal end near the apical surface of the cell, where the core filaments of each stereocilium are anchored within the terminal web (cuticular plate). When stereocilia are deflected, they pivot at their proximal ends like stiff rods (see Fig. 25.11). Transmission electron microscope examination of the distal free end of the stereocilium reveals an electron-dense plaque at the cytoplasmic site of the plasma membrane. This plaque represents the . A fibrillar cross-link called the connects the tip of the stereocilium with the shaft of an adjacent longer stereocilium (see Fig. 25.11). These tip links are anchored to mechanically gated ion channels on both ends. The upper insertion of the tip link to the shaft of neighboring stereocilium contains a cluster of motor proteins (unconventional myosin VIIa) that maintains a resting tension on the tip link. The lower insertion to the distal free end of the stereocilium is connected to the MET channel complex. The tip link is composed of (CDH23) and (PCDH15); however, the molecular composition of the MET channel complex remains elusive. Recently, two transmembrane channel-like (TMC) proteins, TMC1 and TMC2, have been identified in the MET channels that are expressed in developing hair cells. Mutations in the genes encoding TMC1 cause in humans.

complex

15

mechanoelectrical transducer (MET) channel tip link

cadherin-23

protocadherin-

deafness

The tip link plays an important role in activating the MET channel complex at the tip of the stereocilia and opening additional transduction K+ channels at the site of its attachment to the shaft of neighboring stereocilium (see Fig. 25.11). The molecular structure of the transduction K+ channels is unknown. A mutation that disrupts the gene that encodes the actinbundling protein causes cochlear and vestibular symptoms in experimental mice. They lose their hearing early in life; these animals also spend most of their time walking or spinning in circles. The stereocilia of these animals do not maintain the rigidity necessary for the proper functioning of the . In humans, mutations in a gene located on chromosome 1 that encodes espin are associated with deafness without vestibular involvement.

espin

channels

MET

All hair cells use mechanically gated ion channels to generate action potentials. All hair cells of the internal ear appear to function by moving (pivoting) their rigid stereocilia. Mechanoelectrical transduction occurs in stereocilia that are deflected toward its tallest edge (toward the kinocilium, if present). This movement exerts tension on the fibrillar tip links, and the generated force is used to open near the tip of the stereocilium. This allows for an influx of K+ , causing depolarization of the receptor cell. This depolarization results in the opening of voltage-gated Ca2+ channels in the basolateral surface of the hair cells and the secretion of a neurotransmitter that generates an action potential in afferent nerve endings. Movement in the opposite direction (away from the kinocilium) closes the MET channels, causing hyperpolarization of the receptor cell. The means by which stereocilia are deflected varies from receptor to receptor; these are discussed in the sections describing each receptor area.

channels

mechanically gated ion

Hair cells communicate with afferent nerve fibers through ribbon synapses, a specialized type of chemical synapse.

Deflection of the stereocilia on hair cells generates a high rate of prolonged impulses that are quickly transmitted to the afferent nerve fibers. To ensure rapid release of the glutamate neurotransmitter from synaptic vesicles, hair cells possess specialized that contain unique organelles called . In electron microscopy, ribbons appear as ovoid, 30-nm-thick, electron-dense plates that are anchored to the presynaptic membrane by electron-dense structures (Fig. 25.12). This arrangement allows the ribbons to float just above the presynaptic plate like balloons on a short leash. The ribbons tether a large number of synaptic vesicles on their surface that are primed for fusion with the presynaptic membrane, which contains a high density of voltage-gated Ca2+ channels (see Fig. 25.12). After activation of the Ca2+ channels, the ribbon serves as a fast-moving conveyor belt, delivering the vesicles to the presynaptic membrane for fusion. The tethered pool of synaptic vesicles is approximately fivefold greater than the pool of the remaining vesicles. The ribbons contain several proteins, including the active-zone protein RIM that interacts with rab3, a GTPase enzyme expressed on the surface of synaptic vesicles. Other proteins of the ribbon complex include presynaptic matrix proteins, such as RIBEYE, Bassoon, and Piccolo. A hair cell typically contains about 10–20 ribbons. These ribbon synapses are also found in the photoreceptors and bipolar cells of the retina.

ribbon synapses ribbons

FIGURE 25.12. Diagram and electron micrograph of a ribbon synapse in a hair cell. Diagram on the left shows a type I hair cell

with several ribbon synapses that are specialized for transmitting long-lasting and high-volume impulses to the afferent nerve cell endings ( ). This schematic view of a ribbon synapse shows the ribbon protein complex that contains several presynaptic matrix proteins (RIM, RIBEYE, and Piccolo) and is anchored into the presynaptic plate by another protein called . The surface of the ribbon serves as the tethering platform for multiple synaptic vesicles. Note the presence of voltage-sensitive Ca 2+ channels in the presynaptic membrane next to the attachment of the ribbon. Upon influx of Ca 2+, the ribbon accelerates movement of the attached vesicles toward the presynaptic membrane for fusion (similar to the action of a fast-moving conveyor belt). This electron micrograph of a ribbon synapse from a mouse cochlear hair cell shows the ribbon protein complex with attached synaptic vesicles. ×27,400. (Reprinted with

yellow a.

Bassoon

b.

permission from Neef A, Khimich D, Pirih P, et al. Probing mechanism of exocytosis at the hair cell ribbon synapse. 2007;27:12933–12944.)

the

J Neurosci.

Two types of hair cells are present in the vestibular labyrinth. Both hair cell types are associated with afferent and efferent nerve endings (see Fig. 25.11). Type I hair cells are flask shaped, with a rounded base and thin neck, and are surrounded by an afferent nerve chalice and a few efferent nerve fibers. are cylindrical and have afferent and efferent bouton nerve endings at the base of the cell (see Fig. 25.11).

Type II hair cells

Sensory receptors of the membranous labyrinth The crista ampullaris senses angular movements of the head. Each ampulla of the semicircular duct contains a crista ampullaris, which is a sensory receptor for angular movements of the head (Figs. 25.13 and 25.14). The crista ampullaris is a thickened transverse epithelial ridge that is oriented perpendicularly to the long axis of the semicircular canal and consists of the epithelial hair cells and supporting cells (Plate 25.1, page 1042).

FIGURE 25.13. Diagram of function and structure of the crista ampullaris within a semicircular duct. a. As shown in this

drawing, the crista ampullaris functions as the sensor for angular movement of the head. For example, when the head of the individual shown in this diagram rotates toward the left side, the bony labyrinth also rotates at the same speed together with the head. However, the endolymph lags behind due to its own fluid inertia. Because the crista ampullaris is attached to the wall of the bony labyrinth, it will be swayed by the lagging endolymph in the opposite direction to the movement of the head. The structure of the crista ampullaris includes sensory epithelium and large cupula made of a gelatinous protein–polysaccharide mass that projects toward the nonsensory wall of the ampulla. Note that the membranous ampulla is filled with endolymph and is surrounded by perilymph. The sensory epithelium of the crista ampullaris is composed of both type I and type II hair cells and supporting cells. The stereocilia and kinocilium of each hair cell are embedded in the cupula. Their mechanical deflection opens the K + channels, causing depolarization of the cell.

b.

c.

FIGURE 25.14. Photomicrograph of the crista ampullaris and macula of the utricle of the internal ear. a. This low-

magnification view of a horizontal section of the temporal bone reveals several regions of the internal ear. The prominent contains a well-preserved cochlear duct with a emerging from the base of the modiolus. Note the cross section of the and . The central cavity of the slide represents the vestibule that contains three parts of the membranous labyrinth: the , , and . The locations of sensory receptors (macula of utricle, macula of saccule, and crista ampullaris) are enclosed within the rectangles. ×20. This high-magnification view of the crista ampullaris from the anterior semicircular canal shows a thick that contains two types of cells: the in the upper layer and the in the basal layer. Note that the sensory hair processes of the cells are barely discernable and are covered by the . The underlying loose extends to the wall of the bony labyrinth and contains nerve fibers with associated Schwann cells, fibroblasts, capillaries, and other connective tissue cells. ×380. This high-magnification view of the macula of the utricle shows similar to that of the crista ampullaris. The sensory epithelium is overlaid by the containing a darker stained layer of (otoliths) on its surface. ×380. (Copyright 2010 Regents of the University of Michigan. Reprinted with permission.)

cochlea cochlear nerve stapedius

muscle facial nerve utricle saccule ampulla of the anterior semicircular canal

b.

supporting cells cupula

c. sensory epithelium

hair cells

sensory epithelium

connective tissue

otoconia

otolithic membrane

A gelatinous protein–polysaccharide mass, known as the , is attached to the hair cells of each crista (see Fig. 25.13). The cupula projects into the lumen and is surrounded by endolymph. During rotational movement of the head, the walls of the semicircular canal and the membranous semicircular ducts move, but the endolymph contained within the ducts tends to lag behind because of inertia. The cupula, projecting into the endolymph, is swayed by the movement differential between the crista fixed to the wall of the duct and the endolymph. Deflection of the stereocilia in the narrow space between the hair cells and the cupula generates nerve impulses in the associated nerve endings.

cupula

The maculae of the saccule and utricle are sensors of gravity and linear acceleration. The maculae of the saccule and utricle are innervated sensory

thickenings of the epithelium that face the endolymph of the saccule and utricle (see Figs. 25.14 and 25.15). As in the cristae, each macula consists of and

type I

type II hair

cells, supporting cells, and nerve endings associated with the

hair cells. The maculae of the utricle and saccule are oriented at right angles to each another. When a person is standing, the macula of the utricle is in a horizontal plane and the macula of the saccule is in a vertical plane.

FIGURE 25.15. Diagram of function and structure of the macula within the utricle. a. As shown in this drawing, the macula of the

utricle (as well as macula of the saccule) functions as a sensor for gravity and linear acceleration. For example, when the head of the individual shown in this diagram is tilted forward, tiny crystals of calcium carbonate called are shifted on the surface of the otolithic membrane. This movement is detected by the underlying hair cells. The macula is composed of a sensory epithelium containing both type I and type II hair cells. The hair cell processes are embedded in the gelatinous polysaccharide otolithic membrane. The luminal surface of the membrane is covered by otoconia that are heavier than endolymph. As visible on the map below the macula, the hair cells are polarized with respect to the striola, an imaginary plane that curves through the center of each macula. Note that on each side of the striola, the kinocilia of the hair cells are oriented in opposite directions facing toward the striola (see direction of the and on the polarization map of the utricle). This arrangement is only seen in the utricle because in the macula of the saccule, the kinocilia of the hair cells are turned away from the striola.

otoconia

b.

c.

blue

green arrows

striola

Hair cells are polarized with respect to the , an imaginary plane that curves through the center of each macula (see Fig. 25.15). On each side of the striola, the kinocilia of the hair cells are oriented in opposite directions, facing toward the striola in the utricle and turning away from the striola in the saccule. Owing to polarization of the hair cells, the maculae of the saccule and utricle are sensitive to multiple directions of linear accelerations. The gelatinous polysaccharide material that overlies the maculae is called the (see Fig. 25.15). Its outer surface contains 3- to 5-μm crystalline bodies of calcium carbonate and a protein (Fig. 25.16). , also called , are heavier than endolymph. The outer surface of the otolithic membrane lies opposite the surface in which the stereocilia of the hair cells are embedded. The otolithic membrane moves on the macula in a manner analogous to that by which the cupula moves on the crista. Stereocilia of the hair cells are deflected by gravity in the stationary individual when the otolithic membrane and its otoliths pull on the stereocilia. They are also displaced during linear movement when the individual is moving in a straight line and the otolithic membrane drags on the stereocilia because of inertia. In both cases, movement of the otolithic membrane causes the stereocilia to move toward the kinocilium, activating MET channels. This depolarizes hair cells and generates an action potential. Displacement of stereocilia in the opposite direction away from the kinocilium causes hyperpolarization of hair cells and inhibits the generation of the action potential.

otolithic membrane

otoconia

Otoliths

FIGURE 25.16. Scanning electron micrograph of human otoconia.

Each otoconium has a long cylindrical body with a three-headed facet on each end. ×5,000.

The spiral organ of Corti is the sensor of sound vibrations. The cochlear duct divides the cochlear canal into three parallel compartments or scalae:

Scala media, the middle compartment in the cochlear canal Scala vestibule Scala tympani The cochlear duct itself is the scala media (Fig. 25.17).

The scala vestibuli and scala tympani are the spaces above and below, respectively, the scala media. The scala media is an

endolymph-containing space

that is continuous with the lumen of the saccule and contains the spiral organ of Corti, which rests on its lower wall (see Fig. 25.17).

FIGURE 25.17. Schematic diagram and photomicrograph of the cochlear canal. a. Cross section of the basal turn of the cochlear duct is shown in the box on the smaller orientation view. This view of

a midmodiolar section of the cochlea illustrates the position of the cochlear duct within the 2.75 turns of the bony cochlea. Observe that at the top of the cochlea, the scala vestibuli and scala tympani communicate with each other at the helicotrema. The scala media and the osseous spiral lamina divide the cochlea into the scala vestibuli and the scala tympani, which are filled with perilymph. The scala media (the space within the cochlear duct) is filled with endolymph and contains the organ of Corti. This photomicrograph shows a section of the basal turn of the cochlear canal. The osseous spiral lamina ( ) and its membranous continuation, the basilar membrane ( ) as well as the vestibular membrane ( ) are visible. Note the location of the , the scala media ( ) or cochlear duct, and the . The three walls of the scala media are

BM

OSL

b.

scala vestibuli scala tympani

VM

SM

SV

formed by the basilar membrane inferiorly, the stria vascularis ( ) and underlying spiral ligament ( ) laterally, and the vestibular membrane superiorly. The spiral organ of Corti resides on the inferior wall of the cochlear duct. Dendrites of the cochlear nerve ( ) that originate in the spiral ganglion ( ) enter the spiral organ of Corti. The axons of the spiral ganglion cells form the cochlear part of the vestibulocochlear nerve. ×65.

SL

SG

CN

scala vestibuli and the scala tympani are perilymphcontaining spaces that communicate with each other at the apex of the cochlea through a small channel called the helicotrema (see Fig. 25.17b). The scala vestibuli begins at the oval window, and the scala tympani ends at the round window. The scala media is a triangular space with its acute angle attached to the modiolus. In transverse section, the scala media appears as a triangular space with its most acute angle attached to a bony extension of the modiolus, the osseous spiral lamina (see Fig. 25.17). The upper wall of the scala media, which separates it from the scala vestibuli, is the vestibular (Reissner) membrane (Fig. 25.18). The lateral or outer wall of the scala media is bordered by a unique epithelium, the stria vascularis. It is The

responsible for the production and maintenance of endolymph. The stria vascularis encloses a complex capillary network and contains three types of cells (Fig. 25.19). The marginal cells, primarily involved in K+ transport, line the endolymphatic space of the scala media. Intermediate pigment-containing cells are scattered among capillaries. The basal cells separate stria vascularis from the underlying spiral ligament. The lower wall or floor of the scala media is formed by a relatively flaccid that increases in width and decreases in stiffness as it coils from the base to apex of the cochlea. The spiral organ of Corti rests on the basilar membrane and is overlain by the .

basilar membrane

tectorial membrane

FIGURE 25.18. Transmission electron micrograph of the vestibular (Reissner) membrane. Two cell types can be observed: a

mesothelial cell, which faces the scala vestibuli and is bathed by perilymph, and an epithelial cell, which faces the scala media and is bathed by endolymph. ×8,400.

FIGURE 25.19. Transmission electron micrograph of the stria vascularis. The apical surfaces of the marginal cells (M) of the stria are bathed by endolymph ( E ) of the scala media. Intermediate cells ( I ) are positioned between the marginal cells and the basal

B

cells ( ). The basal cells separate the other cells of the stria vascularis from the spiral ligament ( ). ×4,700.

SpL

The spiral organ of Corti is composed of hair cells, phalangeal cells, and pillar cells. The spiral organ of Corti is a complex epithelial layer on the floor of the scala media (Fig. 25.20 and Plate 25.2, page 1044). It is formed by the following:

FIGURE 25.20. Photomicrograph of the vestibular duct and spiral organ of Corti. This higher magnification photomicrograph of the cochlear duct shows the structure of the spiral organ of Corti. Relate this structure to the inset , which labels the structural features of the spiral organ. ×180. Inset. Diagram of the sensory and supporting cells of the spiral organ of Corti. The sensory cells are divided into an inner row of sensory hair cells and three rows of outer sensory hair cells. The supporting cells are the inner and outer pillar cells, inner and outer (Deiters) phalangeal cells, outer border cells (Hensen cells), inner border cells, Claudius cells, and Böttcher cells.

Inner hair cells (close to the spiral lamina) and outer hair cells (farther from the spiral lamina) Inner phalangeal (supporting) cells and outer phalangeal cells Pillar cells Several other named cell types of unknown function are also present in the spiral organ.

The hair cells are arranged in inner and outer rows of cells. The inner hair cells form a single row of cells throughout all 2.75 turns of the cochlear duct. The number of cells forming the width of the continuous row of outer hair cells is variable. Three ranks of hair cells are found in the basal part of the coil (Fig. 25.21). The width of the row gradually increases to five ranks of cells at the apex of the cochlea.

FIGURE 25.21. Scanning electron micrograph of the spiral organ of Corti. This electron micrograph illustrates the configuration of

stereocilia on the apical surfaces of the inner row and three outer rows of the cochlear sensory hair cells. ×3,250.

The phalangeal and pillar cells provide support for the hair cells. Phalangeal cells are supporting cells for both rows of hair

cells. The phalangeal cells associated with the inner hair cells surround the cells completely (Fig. 25.22a). The phalangeal cells associated with the outer hair cells surround only the basal portion of the hair cell completely and send apical processes toward the endolymphatic space (Fig. 25.22b). These processes flatten near the apical ends of the hair cells and collectively form a complete plate surrounding each hair cell (Fig. 25.23).

FIGURE 25.22. Electron micrograph of an inner and outer hair cell. a. Observe the rounded base and constricted neck of the inner hair cell. Nerve endings ( NE ) from afferent nerve fibers ( AF ) to the inner hair cells are seen basally. IP , inner pillar cell; IPH , inner phalangeal cell. ×6,300. b. Afferent ( AF ) and efferent ( EF ) nerve

fiber endings on the base of an outer sensory hair cell are evident. Outer phalangeal cells ( ) surround the outer hair cells basally. Their apical projections form the apical cuticular plate ( ). Note that the lateral domains in the middle third of the outer hair cells are not surrounded by supporting cells. ×6,300. (Reprinted with permission from Kimura RS. Sensory and accessory epithelia of the cochlea. In: Friedmann I, Ballantyne J, eds. . Butterworth; 1984.)

OPH

ACP

Ultrastructural Atlas of

the Inner Ear

FIGURE 25.23. Structure of the outer phalangeal cell. a.

This scanning electron micrograph illustrates the architecture of the outer phalangeal (Deiters) cells. Each phalangeal cell cups the basal surface of an outer hair cell and extends its phalangeal process apically to form an apical cuticular plate that supports the outer sensory hair cells. ×2,400. Schematic drawing showing the relationship of an outer phalangeal cell to an outer hair cell.

b.

The apical ends of the phalangeal cells are tightly bound to one another and to the hair cells by elaborate tight junctions. These junctions form the that separates the endolymphatic compartment from the true intercellular spaces of the organ of Corti (see Figs. 25.20 and 25.22b). The extracellular fluid in this intercellular space is . Its composition is similar to that of other extracellular fluids and to perilymph. have broad apical and basal surfaces that form plates and a narrowed cytoplasm. The inner pillar cells rest on the tympanic lip of the spiral lamina; the outer pillar cells

reticular lamina

cortilymph

Pillar cells

rest on the basilar membrane. Between them, they form a triangular tunnel, the (see Fig. 25.20).

inner spiral tunnel The tectorial membrane extends from the spiral limbus over the cells of the spiral organ of Corti. The tectorial membrane is attached medially to the modiolus. Its lateral free edge projects over and attaches to the organ of Corti by the stereocilia of the hair cells. It is formed from the radially oriented bundles of collagen types II, V, and IX embedded in a dense amorphous ground substance. Glycoproteins unique to the internal ear, called and , are associated with the collagen bundles. These proteins are also present in the otolithic membranes overlying the maculae of the utricle and saccule as well as in the cupulae of the cristae in the semicircular canals.

otogelin

tectorin

Sound Perception As described on pages 1018-1019, sound waves striking the tympanic membrane are translated into simple mechanical vibrations. The ossicles of the middle ear convey these vibrations to the cochlea.

In the internal ear, the vibrations of the ossicles are transformed into waves in the perilymph.

Movement of the stapes in the oval window of the vestibule sets up vibrations or traveling waves in the perilymph of the scala vestibuli. The vibrations are transmitted through the vestibular membrane to the scala media (cochlear duct), which contains endolymph, and are also propagated to the perilymph of the scala tympani. Pressure changes in this closed perilymphatic–endolymphatic system are reflected in movements of the membrane that covers the round window in the base of the cochlea. As a result of entering the internal ear, a traveling wave is set up in the basilar membrane (Fig. 25.24). A sound of a specified frequency causes displacement of a relatively long segment of the basilar membrane, but the region

sound vibrations

of maximal displacement is narrow. The point of maximal displacement of the basilar membrane is specified for a given frequency of sound and is the morphologic basis of frequency discrimination. High-frequency sounds cause maximal vibration of the basilar membrane near the base of the cochlea; lowfrequency sounds cause maximal displacement nearer the apex. Amplitude discrimination (i.e., perception of sound intensity or loudness) depends on the degree of displacement of the basilar membrane at any given frequency range. Thus, coding acoustic information into nerve impulses depends on the vibratory pattern of the basilar membrane.

FIGURE 25.24. Schematic diagram illustrating the dynamics of the three divisions of the ear. The cochlear duct is shown here as

if straightened. Sound waves are collected and transmitted from the external ear to the middle ear, where they are converted into mechanical vibrations. The mechanical vibrations are then converted at the oval window into fluid vibrations within the internal ear. Fluid vibrations cause displacement of the basilar membrane (traveling wave) on which rest the auditory sensory hair cells. Such displacement leads to stimulation of the hair cells and a discharge of neural impulses from them. Note that high-frequency sounds cause vibrations of the narrow, thick portion of the basilar membrane at the base of the cochlea, whereas low-frequency sounds displace basilar membrane toward the apex of the cochlea near its helicotrema.

Movement of the stereocilia of the hair cells in the cochlea initiates neuronal transduction.

Hair cells are attached through the phalangeal cells to the basilar membrane, which vibrates during sound reception. The stereocilia of these hair cells are, in turn, attached to the tectorial membrane, which also vibrates. However, the tectorial membrane and the basilar membrane are hinged at different points. Thus, a shearing effect occurs between the basilar membrane (and the cells attached to it) and the tectorial membrane when sound vibrations impinge on the internal ear. Because they are inserted into the tectorial membrane, the stereocilia of the hair cells are the only structures that connect the basilar membrane and its complex epithelial layer to the tectorial membrane. The shearing effect between the basilar membrane and the tectorial membrane deflects the stereocilia and thus the apical portion of the hair cells. This deflection activates located at the tips of stereocilia and generates action potentials that are conveyed to the brain via the (cochlear division of the vestibulocochlear nerve, cranial nerve VIII).

MET channels cochlear nerve

Innervation of the Internal Ear

The vestibular nerve originates from the sensory receptors associated with the vestibular labyrinth. The vestibulocochlear nerve (cranial nerve VIII) is a special sensory nerve and is composed of two divisions: a vestibular division called the vestibular nerve and a cochlear division called the cochlear nerve. The vestibular nerve is associated with equilibrium and carries impulses from the sensory receptors located within the vestibular labyrinth. The cochlear nerve is associated with hearing and conveys impulses from the sensory receptors within the cochlear labyrinth (Fig. 25.25).

FIGURE 25.25. Diagram illustrating the innervation of the sensory regions of the membranous labyrinth. Note that cochlear

and vestibular nerves form the vestibulocochlear nerve (cranial nerve VIII). The cochlear nerve carries the sound impulses from the spiral organ of Corti located within the cochlear duct; the vestibular nerve carries balance information from the three cristae ampullares of the semicircular canals, utricle, and saccule. The cell bodies of these sensory fibers are located in the spiral ganglion (for hearing) and vestibular ganglion (for equilibrium).

vestibular vestibular ganglion (of Scarpa)

The cell bodies of the bipolar neurons of the are located in the in the internal acoustic meatus. Dendritic processes of the vestibular ganglion cells originate in the cristae ampullares of the three semicircular ducts, the macula of the utricle, and the macula of the saccule. They synapse at the base of the vestibular sensory hair cells, either as a chalice around a type I hair cell or as a bouton associated with a type II hair cell. The axons of the vestibular nerve originate from the vestibular ganglion, enter the brainstem, and terminate in four vestibular nuclei. Some secondary neuronal fibers travel to the

nerve

cerebellum and to the nuclei of cranial nerves III, IV, and VI, which innervate the muscles of the eye.

The cochlear nerve originates from the sensory receptors of the spiral organ of Corti. Neurons of the cochlear nerve are also bipolar, and their cell bodies are located in the spiral ganglion of Corti within the modiolus. Dendritic processes of spiral ganglion cells exit the modiolus through the small openings in the bony spiral lamina and enter the spiral organ. Approximately 90% of dendrites originating from the spiral ganglion cells synapse with the inner hair cells; the remaining 10% of dendrites synapse with the outer hair cells of the spiral ganglion. The axons of the spiral ganglion cells form the cochlear nerve, which enters the bony cochlea through the modiolus to appear in the internal acoustic meatus (see Fig. 25.25). From the internal acoustic meatus, the cochlear nerve enters the brainstem and terminates in the cochlear nuclei of the medulla. Nerve fibers from these nuclei pass to the geniculate nucleus of the thalamus and then to the auditory cortex of the temporal lobe. The organ of Corti also receives a small number of efferent fibers conveying impulses from the brain that pass parallel to the afferent nerve fibers of the vestibulocochlear nerve (olivocochlear tract, cochlear efferents of Rasmussen). Efferent nerve fibers from the brainstem pass through the vestibular nerve. They synapse either on afferent endings of the inner hair cell or on the basal aspect of an outer hair cell. Efferent fibers are thought to affect the control of auditory and vestibular input to the central nervous system, presumably by enhancing some afferent signals while suppressing other signals. Damage to the organ of Corti, cochlear nerve, nerve pathways, or auditory cortex is responsible for (see Folder 25.2).

sensorineural hearing loss FOLDER 25.3

CLINICAL CORRELATION: VERTIGO The sensation of rotation without equilibrium ( dizziness, vertigo) signifies dysfunction of the vestibular system. Causes of

acoustic neuroma

vertigo include viral infections, certain drugs, and tumors such as . Acoustic neuromas develop in or near the internal acoustic meatus and exert pressure on the vestibular division of cranial nerve VIII or branches of the labyrinthine artery. Vertigo can also be produced normally in individuals by excessively stimulating the semicircular ducts. Similarly, excessive stimulation of the utricle can produce motion sickness (seasickness, carsickness, or airsickness) in some individuals. The most common vestibular disorder is . In this condition, otoconia become detached from the macula of the utricle and lodge in one of the three cristae ampullares. The anatomic position of the posterior semicircular canal (it has an opening inferior to the macula) makes it the most common site for the detached otoconia to enter (81%– 90%). The otoconia remain either free floating within the canal ( ) or are attached to the cupula ( ), causing inappropriate movement of the stereocilia at the apical surface of the receptor hair cells. Individuals with BPPV report episodes of an erroneous sensation of spinning evoked by certain movements of the head. Otoconia may detach following trauma or viral infections, but in many instances, it occurs idiopathically. Some diseases of the internal ear affect both hearing and equilibrium. For example, people with initially complain of episodes of dizziness and tinnitus (ringing in the ears) and later develop low-frequency hearing loss. The causes of Ménière disease are related to blockage of the cochlear aqueduct, which drains excess endolymph from the membranous labyrinth. Blockage of this duct causes an increase in endolymphatic pressure and distension of the membranous labyrinth (endolymphatic hydrops).

positional vertigo (BPPV)

benign paroxysmal

canalithiasis cupulolithiasis

Ménière disease

Blood Vessels of the Membranous Labyrinth

Arterial blood is supplied to the membranous labyrinth by the labyrinthine artery; venous blood drainage is to the venous dural sinuses. The blood supply to the external ear, middle ear, and bony labyrinth of the internal ear is from vessels associated with the external carotid arteries. The to tissues of the membranous labyrinth of the internal ear is from the intracranial , a common branch of the anterior inferior cerebellar or basilar artery. The labyrinthine artery is a terminal artery: It has no anastomoses

arterial blood supply labyrinthine artery

with other surrounding arteries. Branches of this artery are exactly parallel to the distribution of the superior and inferior parts of the vestibular nerve. from the cochlear labyrinth is via the posterior and anterior spiral modiolar veins that form the . The common modiolar vein and the vestibulocochlear vein form the vein of the cochlear aqueduct, which empties into the inferior petrosal sinus. Venous drainage from the vestibular labyrinth is via that join the vein of the cochlear aqueduct and by the vein of vestibular aqueduct, which drains into the sigmoid sinus.

Venous drainage common modiolar vein

vestibular veins

EAR

OVERVIEW OF THE EAR ear

The is a paired specialized sensory organ that is responsible for sound perception and balance. Tissues of the ear are derived from (epithelia lining of the membranous labyrinth) and components of the (auditory tube and middle ear cavity), (external acoustic meatus), (malleus, incus, and anterior part of the auricle), and (stapes and posterior part of the auricle).

surface ectoderm first pharyngeal pouch first pharyngeal groove first pharyngeal arch second pharyngeal arch

EXTERNAL EAR auricle

The is the external component of the ear that collects and amplifies sound.

external acoustic meatus

The extends from the auricle to the tympanic membrane. It is lined by skin that contains hair follicles as well as sebaceous and ceruminous glands (which produce , or ).

cerumen

earwax

MIDDLE EAR

middle ear

The is an air-filled space lined by a mucous membrane that contains three (malleus, incus, and stapes). It is separated from the external acoustic meatus by the tympanic membrane and is connected by the to the nasopharynx. The middle ear generated by the vibration of the tympanic membrane. The is composed of skin of the external auditory meatus, a thin core of connective tissue, and mucous membrane of the middle ear. The auditory ossicles ( , , and ) cross the space of the middle ear in series and connect the tympanic membrane to the oval window. Movement of the ossicles is modulated by the that inserts to the malleus and the that inserts to the stapes.

auditory ossicles

auditory (Eustachian) tube amplifies mechanical forces tympanic membrane malleus incus

stapes

tensor tympani muscle stapedius muscle

COMPARTMENTS OF THE INTERNAL EAR

internal ear bony labyrinth labyrinth endolymphatic space perilymphatic space

The consists of two compartments within the temporal bone: the and the , which is contained within the bony labyrinth. The internal ear has three fluid-filled spaces: the within the membranous labyrinth + (which has a high K and a low Na+ concentration), the between the wall of the bony and membranous labyrinth (which has a low K+ and a high Na+

membranous

cortilymphatic space

concentration), and the that lies within the tunnels of the organ of Corti of the cochlea. The consists of three connected spaces: , , and , each containing different parts of the membranous labyrinth. The consists of a series of communicating sacs ( , , and endolymphatic sac) and ducts ( , , utriculosaccular duct, endolymphatic duct, and ductus reuniens) that contain .

bony labyrinth semicircular canals vestibule cochlea membranous labyrinth utricle saccule three semicircular ducts cochlear duct endolymph

SENSORY RECEPTORS OF THE MEMBRANOUS LABYRINTH Specialized sensory cells are located in six regions in the membranous labyrinth: three in the ampullae of the semicircular ducts (receptors for angular acceleration of the head), two in the utricle and saccule (receptors for position of the head and its linear movements), and the (receptors for sound). Utricle and saccule maculae contain that are epithelial mechanoreceptors. These hair cells contain on their apical surfaces (formed by rows of stereocilia with a single kinocilium) and are overlaid with a gelatin-like that contains otoliths (otoconia). Movement of the is detected by the hair bundles, which activate to generate an action potential. Sensory receptors in the are also covered by a gelatin-like mass without otoliths called the . The cupula is deflected during the flow of endolymph through the semicircular canal. Movement of the cupula stimulates to generate an action potential. The is divided into three parallel compartments: or (the middle

cristae ampullares maculae spiral organ of Corti hair cells

hair bundles

otolithic membrane otoliths mechanically gated ion channels crista ampullaris

cupula

mechanically gated ion channels

cochlear canal scala media

cochlear duct

compartment filled with endolymph that contains the spiral organ of Corti), , and (both containing perilymph). The is a triangular space with its lower wall forming the on which the spiral organ of Corti resides. The upper wall ( ) separates the scala media from scala vestibuli, and the lateral wall contains the that produces endolymph. The is composed of (arranged in inner and outer rows), supportive , and . Movement of the stereocilia on hair cells during interaction with the overlying generates electrical impulses that are transmitted to the cochlear nerve. are transmitted from the vibrating tympanic membrane by the ossicles to the oval window, where they produce movement (waves) of the perilymph in the scala vestibule. This movement deflects the basilar membrane and spiral organ of Corti to generate electrical nerve impulses, which are perceived by the brain as sounds. Nerve impulses from the cristae ampullares and maculae travel with the , and the impulses from the spiral organ of Corti travel with the . These two nerves join together in the internal acoustic meatus to form the .

scala vestibuli

tympani scala media

basilar membrane

membrane vascularis spiral organ of Corti phalangeal cells pillar cells tectorial membrane Sound waves

vestibular nerve

nerve (cranial nerve VIII)

scala

vestibular stria hair cells

cochlear vestibulocochlear nerve

PLATE 25.1 EAR internal ear

The , located in the temporal bone, consists of a system of chambers and canals that contain a network of membranous channels. These are referred to, respectively, as the and . In some areas, the membranous labyrinth forms the lining of the bony labyrinth; in others, they are separated. Within the space lined by the membranous labyrinth is a watery fluid called . External to the membranous labyrinth, that is, between the

bony labyrinth

membranous labyrinth

endolymph

membranous and bony labyrinths, is an additional fluid called . The bony labyrinth is divided into three parts: , , and . The cochlea and semicircular canals contain membranous counterparts of the same shape; however, the membranous components of the vestibule are more complex in form, being composed of ducts and two chambers, the and . The cochlea contains the receptors for hearing (the ); the semicircular canals contain the receptors for movement of the head; and the utricle and saccule contain receptors for position of the head.

perilymph semicircular canals utricle

cochlea

vestibule

saccule organ of Corti

Internal ear

, ear, guinea pig, hematoxylin and eosin (H&E) ×20.

internal ear

In this section through the , bone surrounds the entire internal ear cavity. Because of its labyrinthine character, sections of the internal ear appear as a number of separate chambers and ducts. However, these structures are all interconnected (except for the perilymphatic and endolymphatic spaces, which remain separate). The largest chamber is the ( ). The left side of this chamber ( ) leads into the ( ). Just below the and to the right is the oval ligament ( ) surrounding the base of the stapes ( ). Both structures have been cut obliquely and are not seen in their entirety. The facial nerve ( ) is in an osseous tunnel to the left of the oval ligament. The communication of the vestibule with one of the semicircular canals is marked by the . Note the crista ampullaris ( ) that is projecting into the lumen of the semicircular canal. At the are cross sections of the membranous labyrinth passing through components of the semicircular duct system ( ). The cochlea is a spiral, cone-shaped structure. The specimen illustrated here makes 3½ turns (in humans, there are 2¾ turns). The section goes through the central axis of the cochlea. This consists of a bony stem called the ( ). It contains the beginning of the cochlear nerve ( ) and the spiral ganglion ( ). Because of the plane of section and the spiral arrangement of the cochlear tunnel, the tunnel is cut crosswise in seven places (note 3½ turns). A more detailed examination of the cochlea and the organ of Corti is provided in Plate 25.2 (page 1044).

vestibule V cochlea OLC

black arrow black arrow S

FN

arrow

CA

DS

CN

modiolus M

upper right

SG

white

Semicircular canal

, ear, guinea pig, H&E ×85; inset

×380.

crista ampullaris CA lower right

A higher magnification of one of the semicircular canals and of the ( ) within the canal seen in the corner of the previous figure is provided here. The receptor for movement, the crista ampullaris (note its relationships in the previous figure), is present in each of the semicircular canals. The epithelial ( ) surface of the crista consists of two cell types, supporting cells and receptor hair cells. (Two types of hair cells are distinguished with the electron microscope.) It is difficult to identify the hair and supporting cells on the basis of specific characteristics; they can, however, be distinguished on the basis of location (see ), as the ( ) are situated in a more superficial location than the supporting cells ( ). A gelatinous mass, the cupula ( ), surmounts the epithelium of the crista ampullaris. Each receptor cell sends a hairlike projection deep into the substance of the cupula. The epithelium rests on a loose, cellular connective tissue ( ) that also contains the nerve fibers associated with the receptor cells. The nerve fibers are difficult to identify because they are not organized into a discrete bundle.

EP

HC

SC

inset

Cu

hair cells CT

C, cochlea CA, crista ampullaris CN, cochlear nerve CT, connective tissue Cu, cupula DS, duct system (of membranous labyrinth) EP, epithelium FN, facial nerve HC, hair cell M, modiolus OL, oval ligament S, stapes SC, supporting cell SG, spiral ganglion V, vestibule black arrow, entry to cochlea white arrow, entry to semicircular canal

PLATE 25.2 COCHLEAR CANAL AND ORGAN OF CORTI hair cell

The , a nonneuronal mechanoreceptor, is the common receptor cell of the vestibulocochlear system. Hair cells are epithelial cells that possess numerous , modified microvilli also called . They convert mechanical energy to electrical energy that is transmitted via the vestibulocochlear nerve (cranial nerve VIII) to the brain. Hair cells are associated with afferent, as well as efferent, nerve endings. All hair cells have a common basis of receptor cell function that involves bending or flexing of their stereocilia. The specific means by which the stereocilia are bent varies from receptor to receptor, but in each case, stretching of the plasma membrane caused by the bending of the stereocilia generates transmembrane potential changes that are transmitted to the afferent nerve endings associated with each cell. Efferent nerve endings on the hair cells serve to regulate their sensitivity.

sensory hairs

stereocilia

Cochlear canal

, ear, guinea pig, hematoxylin and eosin (H&E) ×65; inset ×380. A section through one of the turns of the cochlea is shown here. The most important functional component of the cochlea is the organ of Corti, enclosed by the and shown at higher magnification in the next figure. Other structures are included in this figure. The spiral ligament ( ) is a thickening of the periosteum on the outer part of the tunnel. Two membranes, the basilar membrane ( ) and the vestibular membrane ( ), join with the spiral ligament and divide the cochlear tunnel into three parallel canals, namely, the ( ), the ( ), and the ( ). Both the scala vestibuli and the scala tympani are perilymphatic spaces; these communicate at the apex of the cochlea. The cochlear duct is the space of the membranous labyrinth and is filled with endolymph. It is thought that the endolymph is formed by the portion of the spiral ligament that faces the cochlear duct, the stria vascularis ( ). This is highly vascularized and contains specialized “secretory” cells. A shelf of bone, the osseous spiral lamina ( ), extends from the modiolus to the basilar membrane. Branches of the cochlear nerve ( ) travel along the spiral lamina to the modiolus, where the main trunk of the nerve is formed. The components of the cochlear nerve are bipolar neurons whose cell bodies constitute the spiral ganglion ( ). These cell bodies are shown at higher magnification in the ( ). The spiral lamina supports an elevation of cells, the

rectangle SL

vestibuli SV CD

StV

upper right

BM

VM

scala cochlear duct

scala tympani ST

OSL

CN

SG inset

limbus spiralis columnar cells.

(

LS).

The

surface

Organ of Corti

of

the

limbus

is

composed

of

, ear, guinea pig, H&E ×180;

inset ×380.

cochlear canal CD

The cross section of the ( ) visible in this image appears as a triangular space. The upper wall (roof) of this canal is formed by the vestibular membrane ( ) that separates it from the scala vestibuli ( ). The lateral (outer) wall of the cochlear canal is bordered the stria vascularis ( ) and underlying spiral ligament ( ). The lower wall (or floor) is formed by the basilar membrane, an extension of the osseous spiral lamina with visible branches of the cochlear nerve ( ). The basilar membrane supports the spiral . The components of the organ of Corti, beginning at the limbus spiralis ( ), are as follows: inner border cells ( ), inner phalangeal and hair cells ( ), and inner pillar cells ( ). The sequence continues, repeating itself in reverse as follows: outer pillar cells ( ), hair cells ( ) and outer phalangeal cells ( ), and outer border cells or cells of Hensen ( ). Hair cells are receptor cells; the other cells are collectively referred to as . The hair and outer phalangeal cells can be distinguished in this figure by their location (see ) and because their nuclei are well aligned. Because the hair cells rest on the phalangeal cells, it can be concluded that the upper three nuclei belong to outer hair cells, whereas the lower three nuclei belong to outer phalangeal cells. The supporting cells extend from the basilar membrane ( ) to the surface of the organ of Corti (this is not evident here but can be seen in the ), where they form a reticular membrane ( ). The free surface of the receptor cells fits into openings in the reticular membrane, and the “hairs” of these cells project toward, and make contact with, the tectorial membrane ( ). The latter is a cuticular extension from the columnar cells of the limbus spiralis. In ideal preparations, nerve fibers can be traced from the hair cells to the cochlear nerve ( ). In their course from the basilar membrane to the reticular membrane, groups of supporting cells are separated from other groups by spaces that form spiral tunnels. These tunnels are named the inner tunnel ( ), the outer tunnel ( ), and the internal spiral tunnel ( ). Beyond the supporting cells are two additional groups of cells, the cells of Claudius ( ) and the cells of Böttcher ( ).

SV

SL

IBC IPC

CH

VM

StV

CN LS

organ of Corti

IP&HC

OPC

OP supporting cells

HC

inset

BM

inset

RM

TM

CN

IT

BM,

CC

basilar membrane

OT

CB

IST

CB, cells of Böttcher CC, cells of Claudius CD, cochlear duct CH, cells of Hensen CN, cochlear nerve HC, hair cells IBC, inner border cells IPC, inner pillar cells IP&HC, inner phalangeal and hair cells IST, internal spiral tunnel IT, inner tunnel LS, limbus spiralis OP, outer phalangeal cells OPC, outer pillar cells OSL, osseous spiral lamina OT, outer tunnel RM, reticular membrane SG, spiral ganglion SL, spiral ligament ST, scala tympani StV, stria vascularis SV, scala vestibule TM, tectorial membrane VM, vestibular membrane

INDEX NOTE :

italics

Page numbers in designate figures; page numbers followed by designate folders; page numbers followed by designate plates; page numbers followed by designate tables.

f

A

p

t

f 352 t 717

A1c (glycosylated hemoglobin A1c) test, 305 A band, of sarcomere, 348, , 351, A (alpha) cells, of pancreas, 716, 716 , , 718, 726 –727 Abacus bodies, 606, Abaxonal plasma membrane, 405, ABCD rule, for melanoma, 552 Abdomen, autonomic innervation of, 421 Abetalipoproteinemia, 694 ABO blood group system, 303 –304 , 303 ABO genes, 303 Absorption by alimentary mucosa, 589, , 633–634 by epithelial tissue, 124 by kidney (renal tubules), 784–786 by oral mucosa, 590–591 by skin, 539 by small intestine, 651–652 Absorptive cells columnar, 660–661 gastroduodenal, 678 –679 large intestine, 660, 686 –687 small intestine (enterocytes), 648 –649 , 651–655, , 680 – 683 Accessory lacrimal glands, 1005 Accessory pancreatic duct (of Santorini), , , 712, 713 Accessory proteins in sarcomere, 351, Accessory sex glands, male, 862, 884–889 Accessory structures of eye, 1004–1007 Accidental cell death (necrosis), 104, 105 Accommodation, visual, 981, 982, 1003

349

606

f

f

406

f

f

p

t

589

p

p

f

p

p p

p

f

f

352

683 707 t

654

p

f

ACE inhibitors, 783 Acentriolar pathway in basal body formation, 76, , 137 Acetaminophen, 700 Acetylation, in postranslational modifications, 37, 132 Acetylcholine (ACh) in adrenal medulla, 842–843 in cardiac regulation, 449 in myasthenia gravis, 358 , 403 in neuromuscular junction, 357, 359, 403 as neurotransmitter, 400, 401, 402 in penile erection, 892 in sweat gland, 571 in urinary bladder, 798–799 Acetylcholine receptors, 358, 358 , 401, 402 , 643, 799 Acetylcholinesterase (AChE), 358, 358 , 403 Acetylcholinesterase inhibitors, 403 -Acetylgalactosamine (GaINAc), 194 -Acetylgalactosamine transferase, 303 -Acetylglucosamine (GIcNAc), , 194 ACh. Acetylcholine AChE. Acetylcholinesterase Achlorhydria, 641 Acid cytokeratin, 73, 74 Acid hydrolases, 307 Acid phosphatase, 874 Acidic dyes, 5–6, 5 Acidophil(s), 822, 850 –853 Acidophilia, 5, 57 Acinar cells, , 713, , , 726 –727 Acinar glands, 164, 165 Acini (sing., acinus), 164, of liver, 696, 697, , 722 of lung, 743 of mammary gland, , of pancreas, 712–713, , , 719, 726 –727 of salivary glands, 609–613, 626 –631 Acne vulgaris, 568 Acoustic meatus external, 1018–1019, , internal, , , 1036, 1037 Acoustic neuroma, 1039 Acquired immunodeficiency syndrome (AIDS), 504 , 550. HIV/AIDS Acquired pellicle, 617 Acquired resistance, 484 Acromegaly, 266 Acrosin, 874 Acrosomal cap, 872, 874,

f

77

f

t

f

f

N N N

185

See See

f

p

f

p

713 714 p p t 166 697 p 947 948 712 713 p p p

1019 1021 f f

f

t

t

712

t

p

1019 1022

f

874

See also

874

Acrosomal vesicle, 872, Acrosome, 872, 874, Acrosome phase, of spermiogenesis, 872 Acrosome reaction, 874, 911, 920 Acrosyringium, 569 ACTH. Adrenocorticotropic hormone Actin-binding proteins (ABPs), 70–71, 81 , 161, Actin-bundling proteins, 70, 127 Actin-capping proteins, 71 Actin cross-linking proteins, 71 Actin-dependent endocytosis, 38 Actin filament(s), 30, 71–72, 114–115 minus (pointed) end of, 69–70, plus (barbed) end of, 69–70, abnormalities in, 72, 80 in anchoring junctions, 141, 146, 148 in cardiac muscle, 362 cell adhesion molecules and, 145–146 characteristics of, 81 distribution of, 69, focal adhesions and, 161, functions of, 72 in hair cells of internal ear, 1026 in microvilli, 127, in muscle tissue, 344 in occluding junctions, 141 in osteoclast clear zone, 250 in platelet structural zone, 318, polymerization of, 70, in myoepithelial cells of salivary glands, 613 in Sertoli cells, 877–878, in skeletal muscle, 349–351, in skeletal muscle contraction, 353–355, in small intestine, 653 in smooth muscle, 198, , 202 , 367, 613 in smooth muscle contraction, 367, , in splenic sinuses, 517 in stereocilia, 127–128, in terminal web, 72, 127, , 653 treadmilling effect of, 70, 128 Actin filament-severing proteins, 70–71 Actin-independent endocytosis, 38 Actin motor proteins, 71 Action potential, 395, 414, 1028 Activated fibroblasts, 197 Activated lymphocytes, 315 Active osteoblasts, , 246 Active transport, 38,

874

See

t

71

f

t 71

71

161

128

319

71

878 349

202

130 128

245 38

f

354

367 369

161

Active zones, of synapses, 399 Actomyosin cross-bridge cycle, 353–355, , 359 Acute bacterial rhinosinusitis, 742 Acute conjunctivitis, 1004 Acute glaucoma, 990 Acute inflammatory demyelinating polyradiculoneuropathy, 406 Acute laryngitis, 737–738 Acylation-stimulating protein (ASP), 286 Acylglucosylceramide, 543 Adaptation, in vision, 981 Adaptin (clathrin adaptor protein), 39, Adaxonal plasma membrane, 405, ADCC. Antibody-dependent cell-mediated cytotoxicity Adducin (actin cross-linking protein), 71 Adenocarcinomas, 126 , 667 Adenohypophysis. Anterior lobe of pituitary gland Adenoids (pharyngeal tonsils), 505, 589 Adenomas, 617 , 664 Adenosine diphosphate (ADP), 319 Adenosine triphosphatase (ATPase), 7, , 35, 58 in bile canaliculi, 706 calcium (Ca 2+)-activated pump, 367 in ciliary movement, 132–134 copper (Wilson), 694 in skeletal muscle, 346–347, 351, 354, 356 in smooth muscle, 363 Adenosine triphosphate (ATP), 62, in fast transport, 397 in neurosecretory vesicles, 824 in skeletal muscle, 346, 354–355, in smooth muscle, 370–371 in thermogenesis, 289 Adenosine triphosphate (ATP)-dependent calcium pumps, 370 Adenosine triphosphate (ATP) synthase, 35, 63 Adenosine triphosphate (ATP)/ADP exchange protein, 63, in actin polymerization, 70 Adenylate cyclase/cyclic adenosine monophosphate (cAMP) system, 817 ADH. Antidiuretic hormone Adhesion molecules, 139, 145–149, , 155 endothelial, 307, 332, 452–453, 452 , 509–510 neutrophil, 307 Adipoblasts (lipoblasts), 282, Adipocyte(s) (adipose cell), , 196, 204, 280, 296 –297 on bone marrow smear, 338 –339 beige, 280, , 290-291, , 292 , 293 brown, , 285–288, 289–290, 296 –297 differentiation of, 282–283, , 288

f

f

354

f

f

t

41

406

See

See f f

f

f

8

63

354

63

See

146

282

282

282 177 p p 291 t p 282

f

p

p

p

external lamina of, 282 mature, 282, in obesity, 280 obesity and metabolism of, 287 structure of, 283, , synthesis and secretion by, 281, , 286 , 296 transdifferentiation of, , 292–293 white, 282, , 292–293, 296 –297 Adipocyte(s), basal lamina in, 155 Adipokines, 281, , 296 Adiponectin, 281, 286 Adipophilin, 286 Adipose tissue, 204, 280–295, 296 –297 beige, 290–291 in bone marrow, 281, 332–333 brown, 280, 285–290, , 296 –297 in dermis, 296 , 555 as endocrine organ, 280 features of, 292 glucocorticoids in, 844 in mammary glands, 281, mobilization of, 285 in obesity, 280 PET scanning and, 286, 290, 293 regulation of, 283–285, 290 structure of, 283, synthesis and secretion by, 281, , 286 , 296 transdifferentiation of, , 292–293 tumors of, 289 white, 281–285, 296 –297 Adrenal androgens, 840 , 844, 845–846 Adrenal cortex, 839–840, , , 843, 858 –859 blood supply to, 840–841, cells of, 845 development of, 117, , 840 fetal, 846–847, hormones of, 840 , 843 permanent in fetal adrenal glands, 846 zonation of, 781, , 843, , 858 –859 Adrenal glands, 769, 781, , 839–847, 858 –861 blood supply to, 838–839, , cortex of ( Adrenal cortex) development of, 116, 838, epithelioid tissue of, 123 fetal, 846–847, hormones of, 285, 840 , 843, 846 lymphatic vessels of, 841 medulla of ( Adrenal medulla)

282

283 284 282

282 281 p t t p

281 p p p

288

t

f

p

t

p

t

p

p

p

p p

p

p

p

946

283

f

281 282 f p p t 839 842 841 839 846 t 841 844 p 818 838 841 See 839 846 t f See

838

structure of, 837, Adrenal hyperplasia, congenital (CAH), 847 Adrenal hypoplasia, congenital, 863 Adrenal medulla, , 839–842, , 858 –861 blood supply to, 840–841, cells of, 841–847, 844 , 860 –861 development of, , 840 fetal, 846–847 hormones of, 840 , 841 secretory vesicles of, 842 Adrenaline. Epinephrine Adrenergic neurons, 401 Adrenergic neurotransmitters, 571 Adrenergic receptors, 402 , 798 Adrenocortical steroids, 817 Adrenocorticotropic hormone (ACTH), 816, 821 , 823, 847 Adrenocorticotropic hormone cells (corticotropes), 821 , 823 Adrenomedullary vein, central, 468, Adult stem cells, 166, 196, 205. Adventitia, 632, , 635 bronchial, 743 esophageal, 636, 670 –671 gallbladder, 710, 724 –725 tracheal, 739, 741–742 of ureters, 810 –811 vaginal, 938 Adventitial cells, 331, . Pericyte(s) Afadin, 144, 147–148, Afferent (sensory) nerve fibers, 359, Afferent (sensory) neurons, 389–390, , dorsal root ganglia, 422–423 somatic, 389–390, 415–416 visceral, 390, 415–416, 418 Afferent arterioles in kidney glomerulus, 771–772, , 793, Afferent lymphatic vessels, 501, , 505, , Age pigment, 50, 82 Age-related macular degeneration (ARMD), 992 Aggrecan, 195, , 196 , 218, , 224, 226 Aggrecan–hyaluronan aggregates, 218 Agouti signaling protein (ASIP), 143 Agranulocyte(s), 299 . Agrin, 156, 775 AIDS, 504 , 550. HIV/AIDS Air cells, mastoid, 1019, 1023 Air conduction, 730 Air filtration, 730 Air passages, 730–731, , 764 –765 Air–blood barrier, 749, , , 766 –767

838

842 p 841 f p p

839 t

See

t

633 p

t

t 469 See also specific locations

p

p

p p p 331 See also 147

360 391 392

502

195

f

p

t

507 509 f f

218

t t See also specific types See also 731 p 749 751

p p

p

774

794

133

Alar sheets in basal body, 132, Albinism, 561 Albumin, 285, 299, 692, 779–751, 834 Albuminuria, 751 Alcohol dehydrogenase, 704 Alcoholic liver cirrhosis, 80 Aldehyde groups and Schiff reagent, 6 Aldosterone, 781, 783 , 788, 840 , 843–844 Alexander disease, 80 Alignment, in light microscopy, 15 Alimentary canal, 588–589, epithelium of, 117, 633 immune functions of, 657 organization of, 632–635, Alimentary mucosa, 588–589, 632 Alkaline phosphatase (ALP), 245–246, 259–260, , 706 All-retinal, 999 Allergen, 205 , 492 Allergic reactions, 205 , 492 basophils in, 205 , 314, 492 eosinophils in, 207, 313, 492 glucocorticoids for, 847 macrophages in, 200 mast cells in, 204, 205 , 314, 492 , 742 Allergic rhinitis, 742 α-Actinin, 128, 146, , 161, in skeletal muscle, 349, 351, in smooth muscle, 368 in spleen, 517 Alpha (A) cells, of pancreas, 716, 716 , , 718, 726 –727 α-Defensins, 656 α-Endorphin, 824 α-Globulins, 299, 693 α Granules, platelets, 318, α-Internexin, 74 , 75 α-Mannosidosis, 51 α-Oxidation, 64 α-Smooth muscle actin (α-SMA), 198, , 202 , 613 α-Tocopherol, 693 α-Tubulin, 66, , 79 α1 -Antitrypsin deficiency, 56, 754 α4 β6 integrin, 162 α6 β4 integrin, 161, Alport posttransplantation disease, 775 Alport syndrome, 775 Alveolar bone proper, 607 Alveolar cells, 747–748

f

f

f f

f

trans

f

f f f

f 147

t

589

f

633

f

f

263

f

f

161 352

f

f

t 717

t

p

319

f

202

67

f

161

f

p

751 752 p p 749 752 p p 731 743 754 p 752

type I, 747–748, , , 766 –767 type II, 748, , , 766 –767 Alveolar ducts, 731, , , 747, , 764 –767 Alveolar glands, 164 Alveolar macrophages, 749–751, Alveolar pores (of Kohn), 752 Alveolar processes, 607–608 Alveolar sacs, 731, , , 747, , , 764 –767 Alveoli (sing., alveolus) of lungs, 731, , 743, 747–751, , , , 764 –767 air–blood barrier of, 749, , , 766 –767 cells of, 747–748, 749–751, , , 766 –767 epithelium of, 747–748 surfactant of, 748–749, 766 of mammary glands, 947–948, 949 of tooth, 605, 607–608 Alzheimer disease, 53, 80 , 396, 403, 420, 718 Amacrine cells, of retina, 993–994, , 1000 Ameloblast(s), 600–605, – , , Ameloblastins, 604 Amelogenesis, 576–581, Amelogenins, 604 Amenorrhea, lactational, 950 American (universal) system, for dentition, 597 –598 (anti-Müllerian hormone) gene, 863 Amiloride-sensitive Na + channels, 595–596, Amine precursor uptake and decarboxylation (APUD) cells, 644 Amino-terminus (7S) domain, of collagen IV, 156, Ammonium, 788 Amnion, , 932, Amorphous calcium phosphate, 261, Ampulla of ductus deferens, 884 hepatopancreatic, 708, 711–712, of internal ear, 1025 of uterine tube, , 920, 923, 924, 960 –961 Ampullary crests, of internal ear, 1025–1026 Amputation neuroma, 429 Amylase, in saliva 616, 616 Amylolytic enzymes, 713 Amyotrophic lateral sclerosis (ALS), 360 Anabolic action, of PTH, 262 Anagen, in hear growth, 566 Anal canal, , 116, 660, 664–665, , 690 –691 Anal columns, 664, Anal glands, 665, – Anal sinuses, 664

731 743 731

747 754 p p 747 748 754 p 750 751 p p 749 752 p p p

f 993 601 602 603 604 603 f

AMH

595

931

936

p

f

f

156

263

711

909

p

p

t

66

665 665 665

f

665

p

p

f

p

665 666 100 101 f f

p

p 665 666 f

Anal sphincter, 635, – , 690 –691 Anal transitional zone (ATZ), 665, – , 690 –691 Anaphase, 98, , , 103 Anaphylactic hypersensitivity, 492, 492 Anaphylactic shock, 205 Anaphylaxis, 205 , 314 Anatomic crown, of tooth, 599, Anatomic end arteries, 1003 Anchorin CII, 218 Anchoring fibrils, 158–159, , , 162 Anchoring filaments, 162, , 471 Anchoring junctions, 141, 145–149, 152 , 160 Anchoring plaques, 158, Anchoring villi, 933, 970 –971 Androgen(s), 566, 840 , 843, 847, 862, 915, 919 Androgen-binding protein (ABP), 817, 878–879 Androgen receptor (AR), 889 Androstenedione, 840 , 846 Anemia, 299, 306 blood islands in, 675 hemolytic, 305, 313 pernicious, 306 , 641, 641 , 644 recombinant erythropoietin for, 769 sickle cell disease, 305, 306 Aneuploid cells, 98 Angelman syndrome, 53 Anginal pain, 470 Angiogenesis, 200, 464 Angiolipoma, 289 Angiotensin-converting enzyme (ACE), 781 Angiotensin-converting enzyme (ACE) inhibitors, 783 Angiotensin I, 454, 781, 843–844 Angiotensin II, 286 , 454, 781, 783 , 844 Angiotensinogen (AGE), 281, 286 , 843–844 Angle-closure glaucoma, 990 Angulin family of proteins, 144 Anion channels, voltage-dependent, 62, 63, , 64 Anionic components, of cells and tissues, 5 Anisotropic bands of sarcomere, 348, , 351, Ankylosis, 240 Ankyrin protein complex, 302, Annexins, 260–261, Annulate lamellae, of oocytes, 911, Anoikis, 107 Anomalous trichromats, 1000 Anorectal junction, epithelial tissue of, 174 –175 Anorexia nervosa, 285 Anosmia, 733

t

160 161 465 t 160 p p

f

f

f

f

f

t

f

599

t

f

f

p

f

t

302

263

f

f

f

349

63

352

911

p

p

p

Anovulatory cycle, 930 Anterior chamber, of eye, 982, , , 1010 –1011 Anterior (ventral) horns of spinal cord, 422, 440 –441 Anterior lobe of pituitary gland, 110, 818, , 821–824, 850 – 853 cells of, 821–824, 822 , 850 –853 electron microscopic characteristics in, 822, 822 functions of, 822 hormones of, 821 , 822 lactation control by, 951 origin of, 116 staining characteristics in, 822, 822 Anterior nares, 731 Anterior pigment myoepithelium, of iris, 988, Anterograde (Wallerian) degeneration, in nerve injury, 426 Anterograde transport, 56, , 396–397 Anti-D immunoglobulin (RhoGAM), 304 Anti-Müllerian hormone gene , 863 Antibiotics, 55 Antibodies, 7–10, 483. Immunoglobulin(s) in blood group systems, 303 –304 , 303 fluorescent dye-labeled, 3 monoclonal, 8, 9 plasma cell secretion of, 483 polyclonal, 8 production of, 316, 483, 488 structure of, Antibody-dependent cell-mediated cytotoxicity (ADCC), 495–497, Antibody-mediated immunity, 206 Antibody-producing cell line, 8 Anticholinergic drugs, 617 Anticoagulants, 300, 453 Antidiuretic hormone (ADH), 791 , 816, 825, 826 , 828 and aquaporins, 761 , 788, 825 and collecting ducts, 788, 792 , 825 pathologies associated with, 828 and smooth muscle, 363 Antigen(s), 7–10, 483 in blood group systems, 303 –304 , 303 in hypersensitivity reactions, 492, 492 immune response to, 483, 492–493 presentation of, 485, 492–493, 495, , . Antigenpresenting cells sperm-specific, 879 Antigen-dependent activation, 491–492 Antigen-independenproliferation and differentiation, 491 Antigen-presenting cells (APCs), 203 , 494, , fetal placental, 934–935, , 972 –973

982 987

p

t

p

p p p p 819

p

t

t

t

56 f (AMH) See also f f

f

p

989

t

493

496

f

f

f

f

t

f

f

t f 495 500 See also

f

934

f

p

495 500 p

Langerhans cells as, 498, 549–550 macrophages as, 200, 317, 498 Antigen receptors, 485 Antigen-transporting cells, 657 Antigen–antibody complex, 495 Antigen–MHC complex, 494 Antiglomerular basement membrane (anti-GBM) antibody-induced glomerulonephritis, 782 Antihistamines, 204, 492 Antimicrobial peptides, 307, 311–312 Antiprogesterone drugs, 931 Antiretroviral therapy, 504 Antisecretory factor (AF)-6, 143 Antithrombogenic substances, 453 Antral follicle, 913–914, Antrum mastoid, 1019 ovarian follicle, , 914, Aorta, , , 476 –477 Aortic bodies, 450 Aortic stenosis, 190, 448 Aortic valve, , , 446, 448, 457 AP180 (adaptin), 41 tumor suppressor gene, 667 Apelin, 281, 286 Apical cytoplasm, 60 Apical foramen, of tooth, 607 Apical plasma membrane, 59, , 796, Apocrine glands, 560, , 569–570, , 580 –583 , 946–947 modified, mammary glands as, 946–947 Apocrine glands, of eyelashes, 568, 1005, Apocrine secretion, 163, , 949 Apolipoprotein B-100, 694 Aponeuroses, 180 Apoptosis, 63, 97, – , 104–107, 105 antibody-dependent cell-mediated cytotoxicity and, 495, cytotoxic T cells and, 496, epithelial cell differentiation and, 539 macrophages in, 200 natural killer cells and, 490 osteoclast, 251 ovarian granulosa cell, 921–922 Apoptotic bodies, 36, , 105 Apoptotic processes, 105 Appendicitis, 664 Appendix, 482, , 505, 660, 663–664, , 688 –689 Appositional growth, 224–225, 230 –231 , 254 Aquaporins (AQPs), 785, 791

f

f

f

t

913

913 p p

443 444

APC

914

443 444

f

t

61

560

163 f 100 107

797 571

p 1005

p

t

497

496

104

483

f

p

665 p

p

p

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ADH action on, 788, 791 , 825 in gallbladder, 711 in glomerular capillaries, 775 in high endothelial venules, 509 in water homeostasis of brain, 425 Aqueous channels, extracellular, 142, 145 Aqueous humor, 982, 986, 989, 991 Aqueous veins, 987 Arachidonic acid analogs, 817 Arachnoid, 423–424, , Arachnoid trabeculae, 423–424, Arched connecting tubule, 774–775 Arcuate vessels, , , 808 –809 Area cribrosa, 770, 775, 795 Areola, , 947 Areolar connective tissue. Loose connective tissue Argentaffin cells, 676 –677 Argyrophilic fibers, 187, 187 –188 Aromatase, 915 Aromatase inhibitors, 915 Arrector pili muscles, 555, , 564 Arteriolae rectae, of kidney, 791–792, Arterioles, 442, 451 , 456, 461–462, , , 480 –481 afferent, 771–772, , 793, efferent, 771–772, , 792–794, medullary, of adrenal glands, 839, metarteriole, 465 penicillar, 518, Arteriovenous (AV) anastomoses or shunts, , 465 Artery(ies), 442, 456–462 anatomic end, 1003 characteristics of, 451 general features of, 450–456 large or elastic, 451 , 456–460, , , 476 –477 layers of wall (tunics), 450–451, , 451 medium or muscular, 451 , 456, , 460–461, , 478 –479 nutrient, 189–190, 214 –215 , 242, small, 451 Arthritis, 241 gouty arthritis, 241 osteoarthritis, 217 rheumatoid arthritis, 186, 227, 241 Articular cartilage, 216–217, 217 , 221–222, , , 240, 272 – 273 Artifact, in microscopy, 14 Arylsulfatase, 313 Ascending colon, 660 Ascending limb, of loop of Henle, 773–774, , 786–787

423 424

770 794

946

p

See p f

p

t

560

774 774

521

p

f

f

p

f

794 461 465 794 794 841

p

p

450

t

t

424

t

t p

457 459 p 450 t 459 460 p 243

f

f

f

222 239

773

p

p

p

p

794

Ascending vasa recta, 794, Ascorbic acid (vitamin C), 185, 265 Aspartate, 401 Aspartoacylase deficiency, 51 Aspartylglycosaminuria, 51 Aspirin, 639 Asthma, 200, 204, 741, 745–746, 746 Asthma, chronic obstructive, 754 Astral microtubules, 76, 99 Astrocyte(s), 389, 409–414, in blood–brain barrier, 410, 425, fibrous, 410, origin of, 414 potassium regulation by, 411–412 protoplasmic, 410, in retina, 993 in water homeostasis, 425 Astrocyte-derived scar, 428 Astrocytoma, fibrous, 410 Asymmetric unit membrane (AUM), 797, Asymmetrical right–left development, 401 (autophagy-related) genes, 52 Atg1 protein-kinase autophagy-regulated complex, 52 Atheromatous plaque, 452 –453 , 460, 470 Atherosclerosis, 452 –453 , 456, 459, 470 Atomic force microscope (AFM), 1, 24–25, ATP. Adenosine triphosphate gene, 694 ATPase. Adenosine triphosphatase Atria (sing., atrium), 362, 443–444, , , , Atrial fibrillation, 467 Atrial granules, 362 Atrial natriuretic factor (ANF), 362 Atrialis, of heart valve, 446–447, Atrioventricular (AV) bundle (of His), 384 , , , 448 Atrioventricular (AV) node, 384 , , 448–449 Atrioventricular (AV) septum, 474 –475 Atrophy, 358–359 Atropine, 401, 988 Attachment epithelium, 608 Attenuation reflex, 1022 Atypical blood vessels, 467–469 Auditory (Eustachian) tubes, 117 Auditory ossicles. Ossicles of ear Auditory system, 1018 Auditory tube, 117, , 1018, 1019, , 1022 Auerbach’s plexus, 635, 636, 651, 660, Auricle, 1018,

f

f

f

412

411

f

f

425

411

797

Atg

ATP7B

See

f

See

f

f

f

f

f 24

443 444 445 446

447 p 444 445 p 445 p p

1019

See 732

1019 660

Auricular hillocks, 1018 Autocrine control, 815, Autocrine signaling, 163, Autodigestion, 104 Autoimmune disease, 482, 497, 498, 775–776 Autoimmune thyroiditis, 836 Automatic segmentation, 23 Autonomic nervous system (ANS), 116, 389, 418–421 cardiac regulation by, 449–450 enteric division of, 363, 389, 418, 420, , 634 functions of, 389, 418 innervation (distribution), 420–421 parasympathetic division of, 389, 418, 418–420, smooth muscle regulation by, 371, 389, 418 sympathetic division of, 389, 418, 418–420, Autophagosome, 52, , 107 Autophagy, 50–53, , , 107 Autophagy-related (Atg) genes, 52 Autoradiography, 1, 3, 10–11, , 21 Autosomal dominant polycystic kidney disease (ADPKD), 135 Autosomal recessive polycystic kidney disease (ARPKD), 135 Autosomes, 90 Auxiliary techniques, 1–2 Axillary artery, 951 Axillary lymph nodes, 951 Axoaxonic synapses, 397, Axodendritic synapses, , 397, , Axon(s), 115, , 390, , 392–396, , gray matter, 421 impulse conduction along, 395, 414 injury response of, 426–429 microtubules of, 395–396, muscle fiber contact with, 357, myelinated, 404–405, , 414 unmyelinated, 407, , 414 Axon hillock, , 391, 395, , 414 Axon initial segment (AIS), , 395, Axon regeneration, , 429 Axonal degeneration, 426–429, Axonal transport, 396–397 Axonemal dyneins, 69 Axonemal microtubules, 79 Axoneme, 79, 128–129, 132, 872 Axosomatic synapses, 397, Azidothymidine (AZT), 504 , 769 Azurophilic granules, 305, 306–307, 312–313

815 163 f

420

419

52 50 52

397 395 390

116

390

11

408 409

427

f

397 399 392 394

395

395 390 427

397

357

395

419

B

B7 molecules of antigen-presenting cells, 494 B (beta) cells, of pancreas, 716, 716 , 719 , 726 –727 B cell(s). B lymphocytes B-cell lymphoma 2 (Bcl-2) protein family, 64, 106, 107 B-cell receptors (BCRs), 489 B chain, insulin synthesis, 719 B Lymphocytes (B cells), 8, 206, 315–316, 484, 487 activation of, 483, 484, 485, 494, in antibody-mediated immunity, 206 antigen presentation by, 498–501 in cell-mediated immunity, 484–485 circulation of, 501 development of, , 331, 490 , 491–492 differentiation of, 490 , 491–492, 495, 510 distribution in lymph node, follicular dendritic cells and, 507 immunocompetent, 483 life spans of, 316, 488 mature, 489 memory, 488, 492, 495, , 510 migration of, 509–510, 516 naïve, 503, 509–510 origin of name, 490 in primary immune response, 492 in secondary immune response, 492 in specific (adaptive) immunity, 484–485 surface molecules of, 316 Backscattered electrons, 22 Bacterial pneumonia, 755 Bacterial ribosomes, 55 Bacteriolytic enzymes, 311–312 Bad (Bcl-2–associated death promoter), 107 Balbiani body of oocyte, 911, Baldness, 566 BALT. Bronchus-associated lymphatic tissue Band (stab) cell, , 327, 329 Band 3 protein, 302, 303 Band 4.1 protein complex, 302, Bands of Büngner, 403, 429 Bare zone, in thick (myosin) filaments, 351, Baroreceptors, 450 Barr body, 91, 306 Bartholin’s cyst, 946 Bartholin’s glands, 939–940 Basal body(ies), 77, 79, 128–129, 733, 739, , 762 –763 anchoring of cilia, 132, ,

t

See

f

322

f

p

p

496

f 509

f

496

f

f

See

f

911

324

f

t 302

133 134

351

741

p

p

77 1028

formation of, 76, , 79, 128–129, 137 in internal ear, microscopic features of, staining appearance of, 131–132 Basal body–associated structures, 132 Basal cell(s) of ductus deferens, 902 –903 of epididymis, 883, 900 –901 of nasal cavity, 732, 733, , 735–736, 758 –759 of skin, 539–540, 544–545 of taste buds, , 624 –625 of trachea, , 741, of urothelium, 796 Basal cell carcinoma, 551 Basal cell membrane infoldings, 153, 162, Basal compartment, of seminiferous epithelium, 878, Basal foot, 132, , Basal lamina, 114, , 154–161 attachment to connective tissue, 158–159, of blood vessels, 450, of blood–brain barrier, 425 of blood–nerve barrier, 389, 417 of blood–thymus barrier, 514, of capillaries, 462–465, compartmentalization by, 159 continuous, 462 correspondence to basement membrane, 155 discontinuous, 463, of elastic artery, 457–458, electron microscopy of, 155, , filtration by, 159 formation of, 157, of glia limitans, 410–411 in nonepithelial cells, 155, organ-specific molecules within, 159–160 of ovarian follicle, 911–912, , , 913, , regulation and signaling by, 160 renal, , 159 reticular fibers underlying, 157–158 splenic, 159, of splenic sinuses, 518 structure of, 155–158 terminology for, 156 of testes, tissue scaffolding by, 159–160 Basal layer, of endometrium, 926 Basal plate, of placenta, 935, , 970 –971 Basal striations, renal, 784,

76

p p

740

p p 734 p p 741 f

594

133 134 140

157

158

163

878

514

457 155 156

155 911 912

913 914

159

867

p

160

458

463

463

p

f

936 785

p

p

123

Basement membrane, 122, , 153–160 of cornea, 984, , 985, , 1016 –1018 correspondence to basal lamina, 155 of glomerulus, , 775, , , 777–779, reticular fibers of, 157–158 of splenic sinuses, 534 –535 staining properties of, 153–154, terminology for, 156 of trachea, 153, , 741–742, , 762 –763 Basement membrane–forming collagens, 183 Basic cytokeratin, 73, 74 Basic dyes, 5–6, 5 Basic thresholding segmentation, 23 Basilar membrane, 935, , 1036 Basolateral plasma membrane, 59–60, Basolateral region, of osteoclast, 251, Basophil(s), 197, 200, 204, 299 , 313–314, , 336 –337 in allergic reactions, 205 , 314, 492 development of, 200, 204, 314, , , 327–328, 329 functions of, 314 granules of, 313 immune function of, 483 in inflammatory response, 312 mast cell , 201 mature, 327 of pars distalis, 821–822, 850 –853 of pars intermedia, 824 Basophil progenitor (BaP) cells, 200, 314, 323, 327, 329 Basophilia, 5, 57, , 93 Basophilic erythroblast, , 325, , 326, 329 , 340 –341 Basophilic secretory granules, 204 Basophil–mast cell progenitor (BMCP) cell, 314, 323, 329 Bax protein (Bcl-2 family), 106 Bcl-2 protein family, 64, 106–107 gene, 561 Beaded filaments, 74 , 75 “Beads-on-a-string” chromatin, 90, Becker muscular dystrophy (BMD), 353 Beige adipocytes, 290 Beige adipose tissue, 290–291 de novo differentiation, 291 white-to-brown transdifferentiation, 291 Bell stage, of tooth, 600 Bellini, ducts of, , 775 Benign paroxysmal positional vertigo (BPPV), 1039 Benign prostatic hyperplasia (BPH), 886, 887 –888 Berger disease, 780 Beta (B) cells, of pancreas, 716, 716 , 719 , 726 –727

984 774

987 p p 775 776 781 p p 155 f 154 741 p p t t 1033 61 253 t 314 p f f 322 324

versus

t

58

Bcl2

f

t

p

324

t

p

325

t

p

90 f

773

t

f

f

f f p

p

p

t

t

p

t

β-Endorphin, 402, 402 , 793 β-Globulins, 299, 693 β-Oxidation, 62, 64, 695, 704 β-Tubulin, 66, , 79 Bethesda system, for cervical cytology, 945 Bicarbonate, 639, 714, 788 Bicellular contact, 142, Bid protein (Bcl-2 family), 106 Bilaminar embryonic disc, 129 Bile, , 692, 695, 695 , 704–708, 711 canaliculus, 705–706, , 722 –726 duct(s), 692, 704–706, , , , 722–726 duct occlusion, 697, 698 ductule, intrahepatic, , 707 salts, 708 stasis, 697, 698 Biliary tree, 706–708 Bilirubin, 313 , 326, 521, 561 , 708 Bilirubin glucuronide, 326, 708 Bim protein (Bcl-2 family), 106 Binucleated cells, 366 Biologic mineralization, 243, , 259–261 Bipolar cells, of retina, , 993–994, , 1001 Bipolar neurons, 390, Birbeck granules, 550 Birefringence, 20 Birefringent bands, 348 Bisphosphonates, 264 Bitter taste, 593, Bladder. Gallbladder; Urinary bladder Blastocyst, 930, Blastocyst cavity, 930 Blastomeres, 930 Bleaching, 999 Blebs, membrane, 36, 104, Blepharospasm, 44 Block endocytosis, 39 Block exocytosis, 39 Blood, 298–335, 336 –343 circulation of, 443–444, , clotting of (coagulation), 300, 319–320, composition of, 298–299 as connective tissue, 114, 298 formed elements of, 298–299, , 299 functions of, 298 plasma of, 114, 298–300, transport via, 298, 303–305 Blood cells, 298–299, 299 .

67

f

142

589

t

706 p p 704 707 711 704

f

f

391

See

987

245

993

f 595 930

104

p

p

443 444 299

320

t

299 t See also specific types

counts of, 320–321 formation of (hemopoiesis), 321–331, preparation and staining of, 300–301, Blood clot, 202 , 300, 319–320, Blood filtrate, 442 Blood flow, arteriole control of, 462 Blood group systems, 303 –304 , 303 Blood islands, 675 Blood pressure, 454, 781–784, 783 elevated, 458 Blood smear, 300–301, , 315, 336 –339 Blood transfusions, blood typing for, 303 –304 , 303 Blood vessels, 442, 450–456. Artery(ies); Capillary(ies); Vein(s) atypical, 467–469 characteristics of, 451 constriction and dilation of, 454, , 462 endothelium of, 124, 450, 452–456, histogenesis of, 116 layers of wall (tunics), 450–451, , 451 , 474 –475 structural features of, Blood–aqueous barrier, 990–991 Blood–brain barrier, 389, 410, 424–426, Blood–follicle barrier (ovarian), 912 Blood–nerve barrier, 389, 417 Blood–ocular barrier, 990 Blood–retina barrier, 994 Blood–testis barrier, 878–879, 879 Blood–thymus barrier, 514–516, , 536 –537 BMI. Body mass index BMP-7, bone morphogenic protein, 239 Body (structure) of epididymis, 882 of pancreas, 711 of stomach, 637 of uterus, , 925 Body (circadian) rhythms, 829 Body mass index (BMI), 287 Body wall, autonomic innervation of, 421 Bombesin, 647 , 656, 741 Bone(s), 238–267 blood supply to, 242, calcium storage in, 238, 261–262 cells of, 239, 243–251, , 253 compact (dense), 239–243, – , – , 259, 272 –273 as connective tissue, 114, 238 fetal development of, 220–221, , 232 –233 formation of, 252–259

f

320

f

f

322 300

f

300

f

t

p p f See also

t

455 458 450

450

909

t

t

t

p

p

425

514

See

f

f

p

p

f

243 245 t 239 240 242 243 221 p p

p

p

221

p p 254 255 p

in endochondral ossification, 220–221, , 232 –233 , 254– 255, – , 274 –277 in intramembranous ossification, 252–254, , , 278 –279 nutritional factors in, 265 ground section, 270 –271 growth of, 252, 255–258, 266 hormonal regulation of, 266 hormone production in, 262 immature, 242–243, lamellar, 242 matrix of, 238–239, 245–246 mature, 241–242, , , mineralization of, 243, 259–261, , , nonlamellar, 242 as organ, 239–240 outer surface of, 240–241 physiologic aspects of, 261–262 remodeling of, 259, , , 267 repair of, 245, 262–264, , resorption of, 248, , 257, , 278 shape of, 239–240 spongy (cancellous), 239–240, , , 242, , , 272 – 273 structure of, 239–241, subchondral, , 223 tissue, 239, 241–243, , 272 –273 Bone-lining cells, 239, , 248–249, Bone marrow, 204, 241, 272 –273 , 331–333 adipose tissue of, 281, 332–333 B-cell differentiation in, 490 cellularity of, 332 examination and analysis of, 333, hemopoiesis in, 321–331 as primary lymphatic organ, 482, , 491 red, 241, 321, , 332–333 sinusoids of, , 332–333 yellow, 241, 333 Bone marrow aspiration, 333 Bone marrow cavity, 241, 274 –275 Bone marrow core biopsy, 333, Bone marrow phase, of fetal hemopoiesis, 321 Bone marrow reserve pool, 327 Bone marrow smear, 338 –341 Bone marrow stromal cells (BMSCs), 205 Bone marrow suppression, 769 Bone matrix proteins, 245 Bone morphogenic proteins (BMPs), 239, 254 Bone-remodeling unit, 259,

256 257

p

p

244

p

f p f f

242 243 244

p

262 263 265

259 261 266 267 251 258 p 239 240 240 222 242 p p 245 249 p p f f 333 483 331 331 p

p

p

261

333

p

243 244

p

p

Bone sialoproteins, 239 Bone-specific vitamin K-dependent proteins, 239 Bony collar, 255, , 274 –275 Bony labyrinth, , 1023–1025, Botulinum toxin, 37, 43–44, 358 Botulism, 44 Bouton terminal, 397 Boutons en passant, 372, 397 Bowman capsule, 771, , , 806 –807 parietal layer of, , 780, 806 –807 podocytes of, , 775, , , visceral layer of, 776, 806 –807 Bowman (olfactory) glands, 733, , 736, 758 –759 Bowman membrane, of cornea, 984, , 985, , 1016 –1018 Bowman (urinary) space, , , 780, 806 –807 Boyden, sphincter of, , 708 BP 230 (desmoplakin-like family proteins), , 162 Bradyarrhythmia, 449 Bradycardia, 449 Brain, 421, 436 –439 barrier protecting, 389, 410–411, 424–425, gray matter of, 421, , 436 –437 organization of, 421 stem cells in, 391–392, 414 ventricles of, 413 water homeostasis in, 425 white matter of, 421, 436 –437 Brain natriuretic factor (BNF), 362 Brain sand, 829, , 854 –855 Brain–gut–adipose axis, 283, 656 Brainstem, 421 1 and 2 genes, 946 and genes, 94 Breast(s). Mammary glands Breast cancer, 94 , 946 Breastfeeding, 826, 946, 949–951, 951 Bright-field microscope, 14, Broad ligament, of ovary, 909 Bronchi (sing., bronchus), , 742–743 development of, 730 lobar or secondary, 730, 743, primary or main, 730, 731, , 739, 742–743, segmental or tertiary, 730, 743, Bronchial buds, 730 Bronchial circulation, 752 Bronchial tree, 731, 738–752, , 762 –767 Bronchiectasis, 745 Bronchiolar unit, respiratory, 743–744

256 1019

p

p

1025

773 774 p p 775 p p 774 775 780 781 p p 734 p p 984 987 p 774 775 p p 707 161

p

p

829

BRCA BRCA1

BRCA BRCA2 See

422 p

p

p

p

425

p

p

f

f

21 741

731

f

f

754

754

754

754

p

p

p

731 754 p p 741 731 743 754 p p 741 743 744 754 p p f f f f 483 t 282 t versus f

Bronchioles, 731, , 743–744, , 764 –767 epithelial tissue of, , 745 respiratory, 731, , 743–744, , 747, , 764 –767 terminal, , 743–744, – , , 764 –767 Bronchitis, chronic, 745 , 754 Bronchodilators, 746 Bronchopulmonary segments, 730, 743 Bronchospasm, 204, 746 Bronchus-associated lymphatic tissue (BALT), , 505, 521 , 741 Brown adipocytes, , 285–288, 292–293 Brown adipose tissue features of, 292 metabolic activity of, 290 newborn adult, 285–286 PET scanning and, 286, 290, 293 regulation of, 290 thermogenic activity of, 289 transdifferentiation of, 292–293 Bruch’s membrane, 991–992, Brunner’s glands, 659, , 678 –679 , 678 –681 Brush border, 126, 784, Brush cells of alveoli, 748 of nasal cavity, 732, 733, 735 of trachea, 740 BSP-1 (osteopontin), 196, , 197 , 239, BSP-2, 239 Buccal administration, 591 Buccal glands, 608 Bud stage, of tooth, 600 Buffy coat, Bulb, hair, 561 Bulbourethral glands, 799, 862, , 889, Bullous pemphigoid, 162 Bullous pemphigoid antigen 1 (BPAG1), 162 Bullous pemphigoid antigen 2 (BPAG2), 162 Bundle bone, 242 Bundle branches, 448 Bundle, muscle, 345 Burn injuries, 544 Bursa-equivalent organs, 331, 490 , 491 Bursa of Fabricius, 490, 490

659 784

993

p

p

196

299

f

C

p

t

263

863

889

f

C cells (parafollicular cells), 831, c-fos, 250

831

p

C-jun, 429 C peptide, insulin, 719 C-type natriuretic peptide (CNP), 914 Cadherin-23, 1027 Cadherin zipper, 149 Cadherins, 107, 145–149 Calcification of heart valves, 448 of hyaline cartilage, 227, , 255, 274 –277 Calcified zone, of articular cartilage, , 223 Calcitonin, 252, 262, 741, 831, 832 , 839 Calcitriol, 769 Calcium blood level of, 838 bone mineralization, 261–262, bone storage of, 238, 261–262 in cardiac muscle contraction, 363–365, deficiency of, 265 in female reproductive system, 913 metabolism of, 261–262, 837 in saliva, 616–617 as second messenger, 817 in skeletal muscle contraction, 353, 355–356, 359, in smooth muscle contraction, 370, 371 Calcium (Ca 2+)-activated ATPase pump, 356 Calcium (Ca 2+)-calmodulin complex, 370, 454 Calcium (Ca 2+)-dependent CAMs, 145–146 Calcium (Ca 2+)-independent CAMs, 146 Calcium (Ca 2+) ions, in zonula adherens, 146–147 Calcium-binding proteins, 245 Calcium channels gated, 355–356, , 363, , 370 ligand-gated, 370 primary cilia and, 135, voltage-gated, 399, 594, voltage-sensitive, 370, 595 Calcium-dependent polymerization, 157 Calcium phosphate, 261, Calcium pumps, ATP-dependent, 370 Calcium-triggered calcium release mechanism, 363 Caldesmon, 367 Call-Exner bodies, , 914 Callus, in bone repair, 265–267, , Calmodulin, 370 Calponin, 367 Calsequestrin, 356, , Calyceal process, , 996,

f

227

f

t

p 222

263

f

359

365 135 595

263

913

359 365 994 996

266 267

p

365

359

770 772

p

p

p

p

982 987 988 p 706 p p f 1007 319 f 704 f 240 243 244 244 See also specific types f f 933 f t

p

Calyx, renal, 770, , , 795, 802 –803 , 808 –809 cAMP. Cyclic adenosine monophosphate Canada balsam, 2 Canal of Schlemm, , 987, , , 991, 1014 –1015 Canaliculi bile, 706–707, , 722 –726 bone, 239, 247, 247 lacrimal, 1007, parietal cell, 642 platelet, 319, Canalithiasis, 1039 Canals of Hering, 704, Canavan disease, 51 Cancellous (spongy) bone, , 241, 242, , , 272 –273 mature, 242, Cancer. lymphatic sinuses in, 507 metaplasia and, 126 , 745 , 933, Candidate hormones, 644 , 647 , 656 Canine tooth, 597 Cannibalism, cell, 107 Cantilever, of atomic force microscope, 24, Cap phase, of spermiogenesis, 872 Cap stage, of tooth, 600 Capacitation, 875, 911, 919–920 Capillary(ies), 442, 462–465 capsular, adrenal, 840 characteristics of, 451 classification of, 462–464, , cortical sinusoidal, adrenal, 840 efferent, 719 functional aspects of, 464–465 glomerular, 771–772, – , 775, 776–777, , lymphatic, 464, , 469–471 peritubular, 792–794, retinal, sheathed, 518, Capillary beds, 462 Capillary network, density of, 464 Capsular capillaries, 840 Capsular matrix, of cartilage, 220, Capsule of adrenal glands, , 842 of eye (Tenon’s capsule), 986 of glomerulus ( Bowman capsule) of kidney, 769, of lacuna (in hyaline cartilage matrix), 230 –231 of lens, 1003, , 1016 –1018

See

p

24

t

463 464

774 782 465 794 462 521

See 770 1003

781 794

220

841

p

p

p

p

p

of liver (Glisson’s capsule), 692, 696 of lymph node, 506, , 511, 528 –531 of pineal glands, 830, 854 –855 of spleen, 516, of thymus, , 536 –537 Capture reagent, 7 Carbohydrates, 4, 648 hepatic metabolism of, 694–695 Carbon dioxide, blood transport of, 303–305 Carbonic anhydrase (in salivary ducts), 613 Carbonic anhydrase II (in osteoclasts), 251 Carbonic anhydrase inhibitors, 990 Carboxy-terminus globular noncollagenous domain (NC1 domain), 156– 157, Carcinoid syndrome (tumor), 644 , 664 Cardiac arrest, 444 Cardiac cells, 362–365, 448–449 attachments of, 148, 149 filament arrangement in, Cardiac cirrhosis, 700 Cardiac conducting cells, 365, 384 –385 , 389, 444, 448–449, 474 –475 Cardiac glands esophageal, 636, gastric, 646, , 672 –675 Cardiac muscle, 115, , 345, 361–366, 382 –385 , 444 cell-to-cell junctions of, 362 characteristics of, 373 comparison with other muscle types, 372 –373 contraction of, 365, , 448 histogenesis of, 116 injury and repair, 365–366 muscle fibers of, 362–363, , regeneration of, 366 structure of, 362–365 Cardiac muscle cells, 474 –475 Cardiac muscle progenitor cells, 366 Cardiac region, of stomach (cardia), 637, , 672 –675 Cardiac tamponade, 445 Cardiac troponin complex, 366 Cardiac veins, 444 Cardiolipin, 62 Cardiomyopathy, 456 Cardiopulmonary resuscitation (CPR), 444 Cardiovascular receptors, 450 Cardiovascular system, 298, 442–473. of circulation pathways of, 443

512

516

507

p f

p

p

p

p

p

f

157

f

72

f

p

647

638 p 115

p

p

p

p

p

f 365

f

p

f

363 364

p

p

638

p

p

See also specific components

components of, 442 overview of, 442–443 Cargo receptors in endocytosis, 40, Caries, dental, 600, 610 , 617 Carotid bodies, 450 Carrier proteins, 38, Cartilage, 114, 216–237. calcification of, 227, cells of, 216, 219–220, 224–225 composition of, 216 development of, 224 growth of, 224–225, 230 –231 internal remodeling of, 218–219 malignant tumors of, 226 mineralization of, 259–261 skeletal development from, 220–221, , 232 –233 , 254–255, – , 274 –277 summary of features, 225 Cartilage layer, of bronchi, 743 Cartilage matrix, 216 calcification of, 255, 274 –277 elastic, 223 fibrocartilage, 224, 236 –237 hyaline, 217–223, , , 230 –231 Cartilage model, 254–255, Cartilage-specific collagen, 217, 226, 230 –231 Cartilaginous layer, of trachea, 739 Cartwheel, of plasma cell, 207 Caspase-1 enzyme, 108 Caspase(s), 105 Caspase inhibitors, 105 Casts, urinary, 784 , 787 Catabolic action, of PTH, 262 Catagen, 566 Catalase, 704 Cataracts, 153, 1004 Catechol O-methyltransferase (COMT), 403 Catecholamine(s), 401, 403, 741, 817, 842–843 Catecholaminergic neurons, 401 Catenins, 144, 146–147, Cathelicidins, 307, 311 Cathepsin(s), 313 Cathepsin K, 251 Cathepsin L2, 548 Cathode, 20 Cationic groups, 5 CatSpers, 920 Caveolae, 35, 370

38

41

f

See also specific types 227 p f

256 257

p

p

p

221

t

p

p

p p 218 220 p 256

f

f

147

p

p

p

p

p

Caveolar rafts (caveolae), 32–33 Caveolated “tuft” cell, 661 Caveolins, 32–33, 39 CBFA1 transcription factor, 252 CCD. Charge-coupled device CD (cluster of differentiation), 206, 485, 486 –487 CD1a molecules, 549–550 CD3 marker, 492, CD4 + T lymphocytes, 200, 316, 484, 492–493, , 498 CD4 +CD25 +FOXP3 + regulator (suppressor) T cells, 316, 488, 497–498 CD8 + CD45R0 + T suppressor cells, 316 CD8 + T lymphocytes, 316, 489, 495, , 496–497, CD27, 495 CD28, 486 , CD31. Platelet and endothelial cell adhesion molecule-1 (PECAM1) CD34 + cell-surface marker, 321 CD38 − cell-surface marker, 321 CD40, 487 , 495, 500 CD40L, 487 , 495, 500 CD40L protein, 313 CD90 + cell-surface marker, 3231 CD151 protein, 162 Cdc2 (Cdk-1), 98, 99 Cecum, 660, 663 Cell(s). compartments of, 28 cytoplasm of, 28–86 functions of, 28 histologic features of, locomotion of, 72 microscopic features of, 30 mitotic activity in, 96–97 nucleus of, 28, 30 , 31 , 92–109 organelles of, 28–30 functions and pathologies of, 31 membranous, 29–65 microscopic features of, 30 movement of, 69, nonmembranous, 30, 65–81 specialization of, 28, Cell accumulation, disorders of, 103, Cell adhesion molecules (CAMs), 139, 145–149, , 155 endothelial, 307, 332, 452, 452 , 509–510 neutrophil, 307 Cell association, in seminiferous tubules, 875–876, Cell body, 115, 116

See

t

493

See

495

495

t 495 t

t

497

t

t See also specific cellular components and cell types 29

t

70

t

t

t

t

29

f

103

146

876

Cell cannibalism, 107 Cell coat (glycocalyx), 32, , 318, , 634, 776–777, 875 Cell cycle, 97–103 and cancer, 94 and centrosome duplication, 78–79, checkpoints in, 97, , 105, 106 and ciliogenesis, 78–79, malfunctions in, 93, 94 , 98 phases of, 97–98, reentry of reserve stem cells, 98 regulation of, 91–93, 94 , 98–99, , 99 Cell death, 103–109 accidental (necrosis), 104, 105 mitotic catastrophe and, 98 nuclear alterations in, 92 programmed, 64, 98, 104–107, , 105 , – , 163 relationship with cell division, , 105 Cell division, 99–103. in cytokinesis, 99 in karyokinesis, 99 in meiosis, 99–103, in mitosis, 99, relationship with cell death, 103, Cell envelope, of epidermis, 543, Cell injury, 36, 104 Cell junctions, 112, 113, 122, Cell loss, disorders of, 103, Cell matrix receptors, 162 Cell-mediated immunity, 207, 484–485 Cell membrane. Plasma (cell) membrane Cell populations rapidly renewing, 97 renewing, 96, 166–167 resident, of connective tissue, 196 slowly renewing, 97 stable, 96, 167 static, 96 wandering, of connective tissue, 196 Cell process extensions, 72 Cell renewal. Cell signaling, 36–37 Cell surface receptors, 37 Cell swelling, 103 Cell-to-extracellular matrix interactions, 156 Cell volume, decrease in, 105 Cell surface receptors, 817, Cellular-signaling devices, primary cilia as, 135 Cement line, , 223

32

f

101

101

319

79

79

f

f

t

99

t

104 t 106 107 103 See also specific processes

100

100

123 103

See

See specific types

222

817

544

103

Cementoblasts, 605 Cementocytes, 605 Cementoenamel junction, 599, Cementogenesis, 605 Cementoid, 605 Cementum, 259, 260, 597, 605, Central adrenomedullary vein, 468, , 840, , 860 –861 Central artery, of spleen, 516, , 518, , 532 –535 Central axonal branch, 390, Central canal of spinal cord, 422 Central diabetes insipidus (CDI), 792 Central lymphatic organs, 482, , 491 Central nervous system (CNS), 115–116, 388, 421–426 cardiac regulation by, 450 cilia in, 135 components of, 115, 421 connective tissue of, 423–424 histogenesis of, 116 injury response in, 426–429, myelin sheath of, 412 nerve cells of ( Neuron(s)) origins of cells in, 414–415 supporting cells of, 389, 409–413, vasculature of, 389 Central neuroglia, 389, 403, 409–413, Central plug/transporter, 95 Central pore, of nuclear pore complex, , 96 Central pulp cavity, 607 Central retinal vessels, , 983, , 1002–1003 Central serous choroidopathy, 997 Central vein, of liver, 696, , 697, , 699, , 702, 722 – 726 Central zone, of prostate gland, 885–886, Centrifugal neurons, 993 Centrilobular necrosis, 697, 700 Centrin, 77, Centriolar pathway, 76, , 79, 137 Centrioles, 30, 75–79 abnormalities, and cancer, 83 basal body formation by, 76, , 79, 128–129, 137 ciliogenesis in, 78–79, , 137–139 immature, 78, , mature, 78, , mitotic function of, 77, , 79 orthogonal orientation of, 75 spermiogenesis and, 872 structure of, 77–78, triplet microtubules of, 75–79,

599

605 469 516 391 f 483

See

p

78

521

842

p

p

p

p

427

412 412 95 982 1001 f 696 697

77

79

78 79 78 79

f 77

77

78

78

886

699

p

712 101

713

p

p

Centroacinar cells, , 713, , 726 –727 Centromere, 90, 99, Centrosome (microtubule-organizing center), 30, 66–68, 75–79 abnormalities, and cancer, 83 ciliogenesis in, 78–79, , 137–139 duplication of, 78–79, mitotic function of, 77, , 99 structure of, 76, Ceramides, 543 Cerebellar cortex, 421, , 438 –439 Cerebellum, 421, 438 –439 Cerebral cortex, 421, , , 436 –437 Cerebrospinal fluid (CSF), 413, 424 Cerebrum, 421, 436 –437 Ceruloplasmin, 694, 879 Cerumen, 1019 Ceruminous glands, 568, 1019 Cervical canal, , 925, 932, , 968 –969 Cervical cancer, 950 Cervical cytology, 933, 945 , 950 Cervical glands, 932, , 968 –969 Cervix of uterus, , 925, 932–933, , 968 –969 endometrium of, 932, epithelium of, 932–933, , 968 –969 HPV infection and, 933, 950 metaplasia of, 126 metaplastic changes in, 933, Pap test of, 933, 945 cGMP. Cyclic guanosine monophosphate CGN. Golgi network A chain, insulin synthesis, 719 Chalazion, 1006 Channel(s), 34, 144, 150 Channel proteins, 37, 38, Chaperone heat shock protein 47 (hsp47), 185 Chaperone-mediated autophagy, , 53 Chaperone therapy, pharmacological, 51 Charge-coupled device (CCD), 20, 980 Checkpoints, in cell cycle, , 98, 105, 106 Chemical defenses, 483 Chemical messengers, 498 Chemical signature, sebum as, 568 Chemical synapses, 398–399, , 1028–1029, Chemiosmotic coupling, 63 Chemokines, 509–510 Chemoreceptors, 450, 645, 732 Chemotaxis, 309 Chiasmata (sing., chiasma) in meiosis, 102

76

79 79 77

f

422 p p p 422 424 p p p p p

909

See See cis-

932 p p f f 932 p p 909 932 p 932 932 p p f f 933 f f

f

38

52

97

399

f

1029

p

Chief cells of parathyroid glands, 837, 856 –857 of pineal gland, 829 of stomach, , 641–642, , 649, 674 –677 Chloride channel protein, 754 Chloride channels, in osteoclasts, 251 Chloride reabsorption, 788 Chloride–carbonate protein exchangers, 251 Chloroquine, 49 Choanae, 731, Cholangiocytes, , 705–706 Cholecalciferol (vitamin D 3 ), 693, 695, 769, 769 , 839 Cholecystokinin (CCK), 402, , 646 , 656, 714–715, 718, 719 Cholestasis, intrahepatic, 707 Cholesterol, 32, 454, 456 , 543, 708, 846 , 915 Cholinergic neurons, 401, 402 Cholinergic neurotransmitters, 571 Cholinergic receptors, 358, 358 , 401, 799 Chondro-osseous junction, , 223 Chondroblasts, 224–225, 265 Chondroclasts, 227 Chondrocyte(s), 189, 217 development of, 224–225 in elastic cartilage, , 234 –235 in endochondral ossification, 255, 274 –275 in extracellular matrix production, 219–220 in fibrocartilage, 223, , 236 –237 in hyaline cartilage, 217, 219–220, , 230 –231 , 272 –273 isogenous groups of, 219, 236 –237 Chondrogenesis, 224 Chondrogenic nodule, 224 Chondroitin sulfate, 156, 193 , 217–218, 238, 985 Chondromodulin 1, 448 Chondrosarcomas, 226 Chorda tympani, 596 Chordae tendineae, , 447, Choriocapillary layer, of choroid, 991–992 Chorion, , 932, Chorion frondosum, 935 Chorion laeve, 935 Chorionic cavity, , 932 Chorionic mesoderm, 933 Chorionic plate, 935, Chorionic villi, , 933–935, , 970 –971 Choroid, 981, – , , 991–992, , 1010 –1011 Choroid fissures, 983, Choroid plexuses, 413

640

642 f

732 704

p

646

t

t

222

f 446 936

p

f

f

p

224

p

f

t

223

931

p

p

p

p

p

p p 219

p

p 993

p

p

p

p

t

447

931

936 931 981 983 987 983

934

p

p

p

f 840 842

Choroidopathy, central serous, 997 Chromaffin cells, 840–842, , , 844 , 860 –861 Chromatids, 90, 99, Chromatin, 92–93 “beads-on-a-string,” 90, condensation of, 90, 92–98 erythrocyte, 326 forms of, 88–89, , 92 neutrophil, 306 smallest units of, 89–90 Chromatin fibers, 90, Chromatin fibril, 90, Chromatolysis, 429 Chromogranin(s), 842 Chromogranin A, 841 Chromophobes, 822, 824, 850 –853 Chromophore, 998 Chromosome(s), 90–91 diploid, 90, 99–103, 920 fertilization and, 920 formation of, 90, 92–98, 99 haploid, 90, 99–103, 871–872 homologous, 90, 99, , 102, 871–872 karyotype of, 90–91, 9292 meiosis and, 90, 99–103, 871–872, , 920 mitosis and, 90, 99 nuclear envelope and, 96 number and pairs of, 90 random assortment of, 103 segregation of, 99, , 103 sex, 90, 91, , spermatogenesis and, 871–872 telomeres of, 90 translocation of, 9292 Chromosome analysis, 9292 Chromosome scaffold, 90, Chromosome-segregation checkpoint, , 98 Chronic allergic inflammation, 205 Chronic bacterial rhinosinusitis, 742 Chronic bronchitis, 745 , 754 Chronic conjunctivitis, 1004 Chronic coughing, 741 Chronic essential hypertension, 783 , 844 Chronic granulomatous disease (CGD), 311–312, 311 Chronic inflammation, 200, 701 Chronic joint pain, 217 Chronic kidney disease (CKD), 769 Chronic laryngitis, 738

101

f

p

90

89

90 90

p

100

91 865

p

f

873

100

f f 90

f f

f

f

f

97 f

f

f

f

p

f

Chronic obstructive pulmonary disease (COPD), 746–747, 754 Chronologic aging, skin, 193 Chronotropic effect on heart rate, 449–450 Chylomicrons, 649 , 694 , 717 Chymase, 204 Chyme, 635, 651, 6137 Cilia, 128–139, 130 axonemal organization of, 129, 132, basal bodies for, formation of, 76, , 79, 128–129, 137 classification of, 129 disorders in, 135–137, 139 formation of, 78–79, , 134, 137–139 immotile, 129, 135 intraflagellar transport in, 137–139, microscopic features of, microtubules of, 132–135, , motile, 129–135, 130 basal bodies anchoring, 132, , mechanism of movement, 132–134 movement of fluids and particles, 131 synchronous pattern of, 134–135 nodal, 129, 130 , 137 primary (monocilia), 129, 130 , 135–137, , 136 , Ciliary activity, 132–134 Ciliary apparatus, 128 Ciliary body, 981, – , 983, 988–991, , 1010 –1011 , 1014 –1015 Ciliary channels of ciliary body, 991 Ciliary dynein, 132, Ciliary dyskinesia, primary, 137, 139 Ciliary epithelium of ciliary body, 990–991 Ciliary muscle, 981, – , 988–989, 1014 –1015 Ciliary processes, , 988–989, 1014 –1015 Ciliated cells of nasal cavity, 732 of trachea, 739, , of uterine tubes, 924 Ciliated epithelium of lacrimal sac, 732 Ciliogenesis, 78–79, , 134, 137–139 Circadian rhythms, 829 Circular portion, of ciliary muscle, 989 Circularly oriented layer, of muscularis externa, 634–635 Circulating pool, of lymphocytes, 487 Circulating pool, of neutrophils, 328 Circulating satiety factor, 281 Circulation. bronchial, 750 closed of spleen, 518,

f

f

f

t

79

133 77

f

76 133 134 t 133 134

t

p

t

981 983 133

987 988 987

f

p

138

135

t 137

988

p

p p

740 741 79

See also specific anatomic structures 521

p

p

p

936 521 443 444 443 444 934 242 p p 592 593 p

fetal–maternal, 935–936, open of spleen, 518, pulmonary, 443, 443–444, , , 750 splenic, 518, systemic, 443, 443–444, , uteroplacental, 933, Circumanal glands, 665 Circumferential lamellae, 242, , 270 –271 Circumvallate papillae, 592, , , 622 Circumventricular organs, 426 Cirrhosis, 80 , 701 Golgi network (CGN), 58, -SNARE complex, 43, Cisternae of Golgi apparatus, 58, of rough endoplasmic reticulum, , 54 Citric acid cycle, 62, Cl − channel protein, 754 Classic liver lobule, 696–697, , 722 –726 Clathrin, 39, 40, Clathrin adaptor proteins, 41, Clathrin-dependent endocytosis, 39, 40, 41, Clathrin-independent endocytosis, 39, Claudin-14, 142 Claudin(s), , 142, , 143 , 145 Clear cells, of eccrine sweat glands, 569, Clear zone, of osteoclast, 250, Clearing, of tissue specimen, 2 Cleavage furrow in cell division, 101 Cleavage lines of skin, 552 Climatization of air in respiratory passages, 731, 733 Clinical crown, of tooth, 574, Clitoris, 908, 939, , 940, 941-942, Clonal expansion of T cells, 497 Cloquet’s canal, of vitreous body, 1004 Closed circulation of spleen, 518, Closed enteroendocrine cells, 645, Closing cone, (resorption canal), 259, , 358 , 148 Closure of autophagosomes, 36 Cloudy mucus, 642 Club cells, 745–747, 766 –767 Club cell secretory protein (CC16), 746–747 Club hair, 566 Cluster of differentiation (CD) molecules, 485, 486 –487 .

521

ciscis

f

43 63 f

41

141

60

60

52

697 41

142

t

p

39

943

521 645

Clostridium botulinum Clostridium perfringens

f

p

f also specific types

41

570

253

599

939

p

261

p

t

t See

Cluster of differentiation (CD) proteins, 206 Coagulating factor VIII, 457 Coagulation, 300, 319–320, Coated pits, 40, Coated vesicles, 29, 41, , 43, 49 COP-I transport, COP-II transport, 56, , Coatomers (COPs), 56, Cochlea, , 1025, , 1036, 1042 –1043 Cochlear (round) window, 1019, 1025, , 1032 Cochlear canal, 1025, , 1044 –1045 Cochlear duct, 1025, , 1030–1031, , Cochlear implant, 1034 Cochlear labyrinth, 1026, Cochlear nerve, , , 1036–1038, Coelomic mesothelium, 863, Cofilin, 70 Cohesins, 99, 103 Coiled-coil dimers, 72–73, Colchicine, 80 Cold, common (viral infection), 733, 742 Cold exposure, and adipose tissue, 293 Cold receptors of skin, Colitis, ulcerative, 664 Collagen(s), 179–186 in adipose tissue, 283 in basal lamina, 154, 155–157, 158 biosynthesis of, 183–187, , 196, 702 in bone, 238, 241, 245 in cartilage, 217, 223, 224, 230 –231 in cartilage repair, 226 cartilage-specific, 217, 226, 230 –231 in chondrosarcoma, 226 classes of, 183 composition of, 182 –183 degradation of, 186–187 diseases associated with, 187 –188 functions of, 182 –183 globular structure of, 185 locations of, 182 –183 triple helix of, 185 type I, 181, 182 , 183, 185 in bones, 238, 245 in cartilage, 224, 226 in chondrosarcoma, 226 in dermis, 551–552 in liver fibrosis, 702 type II, 182 , 183, 217, 224, 226

41

320

41 57 60 57 1019 1026 1033 p 1026 f 1026 1021 1035 864 60

p p 1026 p 1033 1035, 1044p–1045p 1038

73

f

f

556 f

184

t

t

t t

f

t

t t

p

p

f

f

f

t

p

f

p

161

t

type III, 157–159, , 182 , 183, 187 in dermis, 551 in liver fibrosis, 702 secretion by smooth muscle, 372 type IV, 154, 156, , , 182 in basal lamina assembly, 156, in cornea, 985 in glomerular basement membrane, 775, 777 in glomerulonephritis, 775–776, 782 network of, 156 secretion by smooth muscle, 372 type V, 182 , 183, 238 type VI, 182 , 217 type VII, 156, 158, , 161, , 182 type VIII, 182 type IX, 182 , 183, 217 type X, 182 , 183, 217, 226 type XI, 182 , 183, 217 type XII, 182 type XIII, 182 type XIV, 182 type XV, 156, 182 , 183 type XVI, 182 type XVII, , , 162, 182 , 183 type XVIII, 156, 182 , 183 type XIX, 182 type XX, 182 type XXI, 182 type XXII, 183 type XXIII, 183 type XXIV, 183 type XXV, 183 type XXVI, 183 type XXVII, 183 type XXVIII, 183 type XXIX, 183 types of, 181, 182 –183 Collagen fibers, 180–187, , 210 –211 , 214 –215 in cementum, 605 degradation of, 186–187 in dermis, 551–552 in elastic arteries, , 458, 460 elastic fibers , , 189–190 in extracellular matrix, 192 formation of, 183–187, in heart valves, 447 in muscular arteries, , 461 in periosteum, 240

160 161

t 157

f

t

t

161

t

t t t t t 160 161 t t t t t t t t t t t t t t t 189 p

p

t t t

t

160

f

457 versus 187 184 460

p

p

180

Collagen fibrils, 180, collagen types in, 186, in cornea, 985 formation of, 185–186, in myotendinous junction, 380 –381 in peripheral nerves, 417 in reticular fibers, 187–189 in seminiferous tubules, 866 in vitreous body, 982 Collagen mineralization phase, 261 Collagen molecule, 180–181, , 185–186, , 217 Collagen scaffold of type IV collagen, 157, Collagen table, of large intestine, 662 Collagenases, 187, 313 Collagenopathies, 187 –188 Collagenous helical domain, 156, Collar, bony, 255, , 274 –275 Collecting duct, intralobular, of mammary gland, 947 Collecting ducts, 770, 774–775, , 806 –809 , 825 ADH regulation of, 788, 791 , 825 cells of, 788 cortical, 772, , 774–775, 788–789, 806 –807 function of, 789 interaction with loop of Henle and vasa recta, 792 medullary, 772, , 775, 788–789 as osmotic equilibrating device, 791 Collecting tubule, 772 Colloid in thyroid gland, 830, , 832–833, Colloid osmotic pressure, 299 Colloidal resorption droplets, 831, 833, Colon, 660–661, 662, 686 –687 . Large intestine connective tissue of, 210 –211 epithelial tissue of, 172 –173 muscularis externa of, 372 smooth muscle of, , 372 Colony-forming units–lymphoid. Common lymphoid progenitor (CLP) cells Colony-forming units–myeloid. Common myeloid progenitor (CMP) cells Colony-stimulating factors (CSFs), 327, 330 , 454 Color blindness, 1000 Color vision, 995, 1000 Colorectal cancer, 667 Colorectal zone, 665, , 690 –691 Colostrum, 949 Colposcopy, 950 Columnar absorptive cells, 660–661 Columnar cells, of epididymis, 900 –901

186 185

p

p

181

f 256

f p

157 p 789

f

773

185 157

p

p

p

773

831

366 f

f

833 p p See also p p p p

f f 665

See See

p

t

p

p

p

833

p

113

t

p

p

Columnar epithelium, 113, , 123, 125 , 172 –175 Columnar-to-squamous metaplasia, 126 , 745 Common bile duct, 692, , 708, , 722 Common bony limb of semicircular canals, 1025 Common canaliculus, lacrimal, 1007, Common cold (viral infection), 733, 742 Common hepatic duct, 707–708, , Common lymphoid progenitor (CLP) cells, 314, , 323, 330–331, 511, 4511 Common modiolar vein, 1039 Common myeloid progenitor (CMP) cells, , 323, 326–327, 329 Communicating junctions, 141, 150–153, 152 atomic force microscopy of, 151, diseases associated with, 153 formation of, 150 structure of, 150, study of, 150 Community-acquired pneumonia, 755 Compact bone, 239–242, – , – , 259, 272 –273 immature, 242–243 mature, 242, remodeling of, 265 Compartmentalization, by basal lamina, 159 Compartments, cellular, 28 Compartments of uncoupling receptors and ligands (CURLs), 44 Complement activators, 307 Complement receptors (CRs), 309 Complement system, 496 Complete blood count (CBC), 320–321 Complete heart block, 448 Compound glands, 164, 165 , Computed tomography (CT), 829 Concentric lamellae, of bone, 241, Conchae, 732, , 733 Concretions of pineal gland, 829–830, of prostate gland, 886–889, 904 –905 Condenser lens, 14, 20, Conditioning of air, 731, 733 Conducting neurons, of retina, 993 Conducting portion, of respiratory system, 730–731 Conducting system of heart, 365, 444, , 448–449, Conductive hearing loss, 1019, 1022, 1034 Cone biopsy of cervix, (conization), 950 Cone of light, in otoscopic examination, 1020 Cones (photoreceptor cells), 981, , 993, 1012 –1013 classes of, 995, 1000 color sensitivity of, 995, 1000

707

711

f

1007 f 707 711 151

p

f

322

322

t

t

151

244

f 239 240 242 243

t 166

732

21

f

829

p

p

244

p

989 f

p

445 f f

449

p

p

development of, 983 discs of, 998, 1000 distribution of, 995, nuclei of, 1000 pedicle of, 1001 structure of, , visual processing in, 998–999, Confocal scanning microscope, 8, , 19, Congenital adrenal hyperplasia (CAH), 847 Congenital adrenal hypoplasia, 863 Congenital deafness, 153 Congenital hypothyroidism, 266 , 835 Congenital muscular dystrophy (CMD), 353 Congenital nephrogenic diabetes insipidus, 791 Congestive heart failure, and liver necrosis, 700 Coni vasculosi, epididymis, 882 Conjugate gaze, 1007 Conjugation, in liver, 694 Conjunctiva, , , 1004, , 1007 Conjunctival epithelium, 1014 –1015 Conjunctivalization of cornea, 985 Conjunctivitis, 1004 , 1006 Connecting piece, of sperm, 872, Connecting stalk, in photoreceptor cells, 995–996 Connecting tubules of kidney, 770, 774–775, 788 Connective tissue, 112, 114, 176–207 basal lamina attachment to, 158–159, blood as, 114, 298 bone as, 114, 238 cells of, 176, , 196–207 central nervous system, 423–424 classification of, 176, 177 dense, 114, , 178–180, – , 210 –213 , 417 dermis attachment to, 551 embryonic, 114, 176–178, 177 , extracellular matrix of, 176, 192–196 fibers, 180–191. histogenesis of, 116 loose (areolar), 114, , , 178, , 210 –211 mast cells (MC TC ), 200–201 in membrane functional unit, 167 mucous, 177–178, overview, 176 peripheral nerve, 415, 417 primary cilia of, , 136 primitive in embryo, 177 proper, 116, 177 , 178–180

995

994 996

8

999

f

982 987 f

19 f

f

f

1004 p p 874

160

177

t 178 179 p t 178 See also specific types 114 177 179 f 178

114

t

135

t

p

p

p

f

scar, 202 septa, of mammary glands, 978 –979 septa, of testis, 855, , skeletal muscle–associated, 345–346, , 376 –377 smooth muscle–associated, 386 –387 specialized, 114, 177 stroma, of liver, 696 subendothelial layer of, 384 –385 in submucosa, 634 Connectomics, 22 Connexin-43, 823 Connexins, 150–153, Connexons, 150, Constitutive heterochromatin, 88 Constitutive pathway, 39, 41–42, , 49, , Contact mode, in atomic mode microscopy, 24, Continuous basal lamina, 462 Continuous capillaries, 462, Continuous exfoliation, 541 Continuously renewing cell populations, 166–167 Contractile ring, 101 Contractile speed, of muscle fibers, 346 Contraction, muscle. Muscle contraction Convoluted tubules, 770, 772–774, – , 788, 806 –807 Cooper ligaments, 947 COP-I–coated transport vesicles, COP-II–coated transport vesicles, , 58, Copper, 693–694, 708 Copper ATPase, 694 COPs (coatomers), 56, Core binding factor alpha-1 (CBFA1), 244 Core biopsy, bone marrow, 333, Core particles in proteasome-mediated degradation, , 53, 54 Core protein of GAGs, 218 Cornea, 980–987, – , , 1010 –1011 , 1016 –1018 conjunctivalization of, 985 development of, 116, 983 ferritin protection of, 985 junction with sclera, 985, 986–987, , 1016 –1018 layers of, 983–984 metabolic exchange with aqueous humor, 986 proliferative capacity of, 986 transparency of, 985 Corneal endothelium, 983, 984, , 986, , 1016 –1018 Corneal epithelium, 984–985, , , 1007, 1014 –1015 Corneal erosions, recurrent, 985 Corneal proteoglycans, 985 Corneal stroma, 983, 984, , 985, ,

t

151

p 866 867 p

p

p

p

346

p

p

p

151

42

50 60 24

463

See

773 774 60 57 60

56

p

333

981 984 987

p

984 987 984 987

984

52 p

p

987

985 987

p

p

p

p

p p

p p

Corneal swelling, 985, 986 Corneolimbal stem cells, 985, 986–987 Corneoscleral coat, 980–987, , 983–987 Corneoscleral limbus, 985, 986–987, , 1016 –1018 Corona, of lymphatic nodule, 503 Corona radiata in oocyte, 914, , 920 Coronary arteries, , , 467–468, , 474 –475 Coronary artery bypass graft (CABG), 468 Coronary artery thrombosis, 470 Coronary heart disease, 452 , 468, 470 Coronary vasculature, 444 Corpora amylacea of prostate gland, 886–889, 904 –905 Corpora arenacea of pineal gland, 829–830, , 854 –855 Corpora cavernosa female, 939 male, 890, Corpora lutea atretica, 923 Corporal veno-occlusive mechanism, 892 Corpus albicans, , 919, Corpus cavernosum, , Corpus hemorrhagicum, , 918 Corpus luteum, , 918–919, , 961 –962 Corpus luteum of menstruation, 919, 928–929 Corpus luteum of pregnancy, 921 Corpus spongiosum, 799, , 890, Cortex of adrenal gland, 841–842, , , 858 –859 of hair, , 564 of kidney, 770–771, , , , 802 –805 of lymph node, , 508, , 510, , 528 –531 , 530 –531 of ovary, 909, 956 –957 , 961 –962 of thymus, 512, , , 536 –537 Corti, spiral organ of, 1026, , 1031–1039, , , , 1044 –1045 Cortical (subcapsular) sinus, of lymph node, 528 –531 Cortical collecting duct, of kidney, 772, , 774–775, 788–789, 806 –807 Cortical granules, of oocyte, 911 Cortical labyrinths, of kidney, 770, 804 –805 Cortical nephron, 774 Cortical reaction in capacitation, 921 Cortical sinusoidal capillaries, adrenal, 840 Corticosterone, 840 , 844 Corticotropes, 822 , 823 Corticotropin-releasing hormone (CRH), 568, 823, 828 , 845 Cortilymph, 1025, 1035 Cortilymphatic space, 1025 Cortisol, 840 , 844, 846

981

914

444 446

f

f

987

p

468

p

f

829

891

910 919 863 891 910 910 918

p p

p

p

p

p

f

p

p

863

891 841 844 p p 560 770 771 773 p p 507 509 510 p p p p p p p p 512 513 p p 1026 1033 1035 1036 p p p p 773 p p p p t

t

t

t

Costimulatory signal in lymphocyte activation, 495 Cotyledons, 935, Coughing, chronic, 741 Countercurrent exchange system, of kidney, 770, 792 Countercurrent heat exchange mechanism, testicular, 865 Countercurrent multiplier effect, of kidney, 791 Cowper’s (bulbourethral) glands, 799, 862, , 889, CPR (cardiopulmonary resuscitation), 444 Cranial nerve(s), 390, 415 , 596 Cranial nerve ganglia, 390, 415 Creatine phosphatase, 351 Cremaster muscle, 865 Crescent, in glomerulus, 782 Crevicular epithelium in gingiva, 608 Cricoid cartilage, , Cristae ampullaris, 1026, 1030, , , 1042 –1043 Cristae, of mitochondria, 62, , 64 Cross-bridges, in skeletal muscle contraction, 353–355, Cross-linkers, in cytoplasmic matrix, 83 Cross-linking actin proteins, 71 Cross-striations, of skeletal muscle, 348 Crossing-over, 102, 871–872 Crown of tooth, 599, Crura of clitoris, 939 Cryptorchidism, 864, 870 Crypts of Lieberkühn, 652, 686 –687 Crystallins, in lens, 1003 Crystalline inclusions in Sertoli and Leydig cells, 82 Crystalloid body, of eosinophils, 313 Crystalloid core inclusion of peroxisomes, 64 Crystals of Reinke, of Leydig cells, 868 CTLA-4 (cytotoxic T lymphocyte–associated protein 4), 497 Cuboidal cells, 170 –171 Cumulus oophorus, 914, Cupula of membranous labyrinth, 1030, , Cupulolithiasis, 1039 Curare, 358, 401 Cusps of heart valves, 447–448, of tooth, 599 Cuticle, of hair, , 564 Cutis laxa, 190 Cutting cone (resorption canal), 259, , 272 –273 (cytochrome B, b subunit) gene, 311 Cyclic adenosine monophosphate (cAMP), 251–252, 262, 454, 817, 913 –914, 920 Cyclic guanosine monophosphate (cGMP), 370, 402, 817, 913–914, 999 Cyclin(s), 98–99, 99

936

863

t

732 737

599

p

f

560

CYBB

t

f

889

t

f

62

1030 1031

p

p 914

p

p

p

1030 1031

447

261 f

p

p

354

Cyclin B, 98 Cyclin-dependent kinase (Cdk), 78, 98, , 99 Cyclin E–Cdk2, Cyclin E–Cdk2 complex, 78 Cyclin–Cdk complexes, 78–79, 98–99, Cyclosporine A, 498 Cystatin, 543 Cysteine, 545 Cystic diverticulum, 692 Cystic duct, 692, , 708, Cystic fibrosis (CF), 566 , 754 –755 Cystic fibrosis transmembrane conductance regulator (CFTR), 754 Cystinosis, 51 Cystoid macular edema, 997 Cytochalasin B, 80 Cytochalasin D, 80 Cytochemistry. Histochemistry and cytochemistry Cytochrome B558, 311 Cytochrome , 62, 105 Cytochrome P450, 57 Cytocrine secretion, 546 Cytogenetic testing, 91, 9292 Cytokeratins, 73–74, 74 . Keratin(s) Cytokines, 239, 265, 281, 498 hematopoietic, 328, 330 in lymphocyte activation, 498 Cytokinesis, 98, 99 Cytokinetic abscission, 36 Cytoplasm, 28–86. component apical, 60 cell adhesion molecules of, 145–146 dendritic, 393 hepatocyte, 704 organelles of, 28–30 functions and pathologies of, 31 membranous, 29–65 microscopic features of, 30 movement of, 69, nonmembranous, 30, 65–81 perinodal, 407, perinuclear, 390–391 Schwann cell, 407, , transport to/from nucleus, 96 Cytoplasmic components, 6 Cytoplasmic densities, in smooth muscle, 367, , 370 Cytoplasmic domains, 145 Cytoplasmic dyneins, 69, 131, 137–139, Cytoplasmic filaments, 6

99

99

99

707

f

c

See

t

f f

711

f

f

f

f

f

f t See also t

See also specific cytoplasmic

70 408 408 409

t

t

138

369

s

f

Cytoplasmic inclusions, 81–82 Cytoplasmic matrix, 28, 83 Cytoplasmic processes, 540, Cytoplasmic ring, of nuclear pore complex, 95, Cytoskeletal proteins, intracellular, 4 Cytoskeleton, 28, 30, 145 elements of, 30 , 31 , 81 . functions and pathologies of, 31 microscopic features of, 30 Cytosol (cytoplasmic matrix), 83 Cytotoxic T lymphocyte(s), 104, 316, 484, 492, in HIV/AIDS, 504 thymic education and, Cytotoxic T lymphocyte–associated protein 4 (CTLA-4), 492, 495 Cytotoxicity, antibody-dependent cell-mediated, 495–497, Cytotrophoblast, , 931, 933, Cytotrophoblast cells, 972 –973

540

t

t

f

930

515 p

95

t See also specific elements t t 495

496

934 p

D

t 717

t

D (delta) cells, of pancreas, 716, 716 , , 717 , 718 δ-tubulin, in MTOC, 81 d -Amino acid oxidase, 704 Dacryocystitis, 1007 Daily body rhythms, 829 Danon disease, 51 Dark cells, of eccrine sweat gland, 569, , 788–789 Dark-field microscopy, 16 Dark umbrella. Melanosome microparasol Dartos muscle, 865 1 gene, 863 gene, 667 De novo differentiation, 291 Deafness, 1028 congenital, 153 hereditary, 142 Death by neglect, 515 Death, cell, 103–109 accidental (necrosis), 104, 105 mitotic catastrophe and, 98 nuclear alterations in, 92 programmed, 64, 97, 103–109, , 105 , , , 163 relationship with cell division, 105, Death receptors, 107, 109 Death, sudden cardiac, 444 Decapacitation, 883 Decidua (decidua graviditatis), 931

f

DAXDCC

570

See

f

t

104

t 106 107 105

931

Decidua basalis, 931, , 935 Decidua capsularis, 931, Decidua graviditatis, 931 Decidua parietalis, 931, , Decidual (placental) septa, 935, Decidual cells, of placenta, 928, 931, 970 –971 Decidualization process, 931, Deciduous teeth, 597, 597 –598 Decorin, 196 , 985 Deep (radial) zone, of articular cartilage, , 223 Deep artery of penis, 890, Deep cortex, of lymph node, , 508, 510, , 530 –531 Deep investing fascia, 345–346 Deep perineal pouch, 799 Deep venous thrombosis (DVT), 466 Defensins, 307, 656 Degeneration anterograde (Wallerian), 426–429 axonal, 426–429, retrograde, 429 traumatic, 429 Degenerative calcific aortic valve stenosis, 448 Deglutition, 736 Dehydration, of tissue specimen, 2 Dehydroepiandrosterone (DHEA), 840 , 845–846 Dehydroepiandrosterone sulfate (DHEAS), 840 , 845–846 Delayed hypersensitivity reactions, 497 Delta (D) cells, of pancreas, 716, 716 , , 718 Demilunes (half-moons), 164, , 609–610, , 630 –631 Demyelinating disease, 406, 406 , 413 Dendrites, 115, 390, , 392–396 gray matter, 421 microtubules of, 395–396, Dendritic cells (DCs), 323 epidermal, 544–545, 553 follicular, 485, 503, 507, lymphatic system, 483, 498, 501, 507 melanocytes as, 544–545 Merkel cells as, 550 Dendritic processes, 733, 1036–1038 Dendritic spines, 392–393, , Dendritic transport, 397 Dendritic trees, 392 Denosumab, 264 Dense (compact) bone, 239–242, , , , 259, 272 –273 immature, 242–243 mature, 242, remodeling of, 265

931 931 936 f

t

931 f

936

891 507

p

p

222 510

p

p

t t 717 609

p

p

427

t

390

603 f

395

508

394 395

f

244

240 243 244

p

p

369 178 179

Dense bodies, in smooth muscle, 367, 368–369, Dense connective tissue, 114, , 178–180, , , 210 –213 , 417 Dense core vesicles, 842 Dense tubular system (DTS), of platelets, 319, Dental caries, 600, 610 , 617 Dental pulp, – , , , 607 Dentin, 259, 260, 597, , 605–607, Dentin matrix protein (DMP), 239 Dentin phosphoprotein (DPP), 606 Dentin sialoprotein (DSP), 606 Dentinal tubules, , 606 Dentition, 597, 597 –598 Deoxyribonucleic acid. DNA Depolarization in smooth muscle contraction, 370 in synaptic transmission, 399 in visual processing, 999, Depression, clinical, 403 Dermal papillae, 550, , 559, , , 576 Dermal ridges, 551 Dermatan sulfate, 156, 193 Dermatoglyphics, 551 , 148 Dermis, 178, 538, , 550–552, 576 –577 adipose tissue of, 296 , 555 attachment to connective tissue, 551 attachment to epidermis, 550–551 elastic fibers of, 214 –215 , 551–552 layers of, 551–552 repair of, 572 Dermoid cyst, 118 Descemet’s membrane, of cornea, 984, , 985–986, , 1016 – 1018 Descending colon, 660 Descending limb, of loop of Henle, 745, , 786–787 Descending vasa recta, of kidney, 792, 794, Desmin, 74, 74 , 351, 367, 368 Desmocollins, 149 Desmogleins, 149 Desmolase, 846 Desmoplakin(s), 75, 149, 543 Desmoplakin-like family of proteins, 161 Desmoplakin-like proteins, 75 Desmosine, , 190 Desmosomal attachment plaque, 149 Desmosomal proteins, 543 Desmosomes, 75, 149, 152 , 362–363, 540, 706

114

999 559 560

t Dermatophagoides pteronyssinus 539 p

p

605

f See

556

f f

p

319

f 601 602 603 605 599 605 f

p

p

p

f p

p

p

984

773

t f

189

t

987

794

p

degradation of, 541–542 Merkel cells and, 550 Desquamation, of keratinocytes, 541–542 Detector aperture, of confocal scanning microscope, 19, Detoxification oxidative enzymes and, 64 smooth endoplasmic reticulum and, 57–58 Detrusor muscle, 798 Deubiquitinating (DUB) enzymes, , 53 Deuteranopia, 1000 Deuterosomes in basal body formation, 79 DHEA. Dehydroepiandrosterone 1,2-Diacylglycerol (DAG), 818 Diabetes insipidus, 791 , 792 , 825, 828 Diabetes mellitus, 718, 1003 hemoglobin in, 305 type 2, 287 , 293 Diabetic nephropathy, 780 Diabetic retinopathy, 997 Diads in cardiac myocytes, 363 Diakinesis, , 102 Diamond knives, 21 Diapedesis, 307–309, 332–333, 510 Diaphragm of fenestrated capillaries, 463 Diaphyseal bone, 274 –275 Diaphysis, 240, , 272 –273 Diastole, of heart, 457 Dichromats, 1000 Differential interference microscope, 16 Differential white blood cell count, 320 Diffuse lymphatic tissue, 482–483, 501–506, , 634 Diffuse neuroendocrine system (DNES), 644 , 815 Diffusion, 37–38, , 453 Digestive system. absorption by, 589, , 633–634 alimentary canal of, 588–589, 632–635, associated organs of, 588 functions of, 588–589, overview of, 588–589 secretions of, 588, Dihydropyridine-sensitive receptors (DHSRs), 356, 358, 359, 365 Dihydrotestosterone (DHT), 864, , , 889 Diiodotyrosine (DIT) residue, 833, 834 Dilator pupillae muscle, 988–989, Diluting segment, of nephron, 787 Dimeric IgA antibodies (dIgA), 658 Dimeric tubulin molecules, 66–67, , Dioptric media, of eye, 982

19

52

f

See

f

f

f

f

f

f

100

240 f

p

p

p

p

502 f 38 See also specific components of 589 633 589 589 865 880 989 67 68

Dipalmitoylphosphatidylcholine (DPPC), 748 Diploid chromosomes, 90, 99–103, 920 Diplotene, Direct (primary) bone healing, 262 Direct fluorescent antibody (DFA) test, 421 Direct immunofluorescence, 9, Disc shedding, in photoreceptor cells, 1000 Discontinuous (saltatory) conduction, 414 Discontinuous basal lamina, 463, Discontinuous capillaries, 463, Discontinuous sinusoidal endothelium, 699 Distal connecting fibers, of centriole, 77, Distal convoluted tubule, 772, , 774, , 788, 806 –807 Distal straight tubule, 773–774, 781, 787, 806 –809 Distal thick segment, of nephron, 772 Diverticulum hepatic, 692 laryngotracheal, 730 Dizziness, 1034 , 1039 DMP. Dentin matrix protein DNA, 10 atomic force microscopy of, crossing-over of, 102 damage checkpoint, in cell cycle, , 98, 105, 106 folding and packing of, 90–91 fragmentation, 105 meiosis and, 102–103 mismatch repair genes, 667 mitochondrial, 62 mitosis and, 99–101 probe, 92 radioactive precursors of, 10 replication of, 97–98 satellite, 99 transcription of, 54, Docking complex, 42, Docking protein, 55, Dome-shaped cells, of urothelium, 795, , Dopamine, 398 , 401, 402 , 425, 822, 828 , 830 Dorsal (posterior) horns, 440 –441 Dorsal root ganglia, 390, , 415 , 422–423, , 432 –433 Double-negative stage, of T cells, 514 Double-positive stage, of T cells, 514 Down Syndrome, 917 Drug degradation, 694 Drusen, 992 Dry age-related macular degeneration, 992 Duchenne muscular dystrophy (DMD), 351, 353

100

9

463 463

773

f

See

774

78

p

p

p

p

p

p

f

24

97

f

f

f

f

43 55

55

t

392

p

796 797 t p t 423 f

f

Duct segment of apocrine glands, 571 of eccrine glands, 569, 580 –581 Duct system, of salivary glands, 613, 626 –631 Ducts. Ducts of Luschka, 708 Ductus deferens, 127, , , , 882, 883–884, 902 –903 Ductus epididymis, 875, , 882–883, 900 –901 Duodenojejunal junction, 651 Duodenum, 634, 651, 680 –681 , . Small intestine bile flow to, 705–706 glands of, 659, , 678 –681 major papilla of, 680, , minor papilla of, , mucosa of, 678 –681 Dupuytren’s disease, , 202 Dura mater, 423, , Dural venous sinuses, 424, 468, 1039 Dust cells (alveolar macrophages), 750–753, Dwarfism, pituitary, 266 Dynamic instability of microtubules, 30, 67–68, Dynamic γ fibers, of muscle spindle, 359, Dynamin, 35, 39, 41, Dyneins, 69, , 132–134, , 137–139, , 396–397 Dynorphins, 402 Dysautonomia, familial type I, 596 Dyskinesia, primary ciliary, 137, 139 Dystonia, 44 Dystrophic epidermolysis bullosa, 159 Dystrophin, 351, , 353 Dystrophin–glycoprotein complex, 353

p p p p See specific ducts and duct types 863 866 881 881 p p p p 711 See also 659 p p 707 711 707 711 p p 202 f 423 424 752 f 68 360 41 70 133 138 f f 352

E

f

f

E11 (podoplanin), 239 E-Cadherin, 146, E-Cadherin–catenin complex, 146–148 E-face, of membrane freeze fracture, 22, 34, E-Selectin, 307–308, Ear, 1018–1045, 1042 –1043 development of, 116, 1018, divisions of, 1018, external, 1018–1019, internal, 1018, , 1023–1039, , 1042 –1043 blood vessels of, 1039 bony labyrinth of, , 1023–1025, development of, 116, 1018

147

p

308

1019

p

1019 1019 1019

p

34

1020

1026

1025

p

p

p

hearing function of, 1035–1036 innervation of, 1036–1038 membranous labyrinth of, 1023, 1025–1039 middle, 1018, 1019–1023, bones of, 1018, 1019–1023, development of, 1018 infection of (otitis media), 1023 otoscopic examination of, 1020, overview of, 1018 sensory (hair) cells of, 127, 135, 1026–1030, , –1043 hearing function of, 1036 inner, 1034–1035, , ion channels and action potentials of, 1028 outer, 1034–1035, , synapses of, 1028–1029, type I, 1029, type II, 1029, sound perception in, 1035–1036 stereocilia of, 127, 128, 1026–1027, , , 1036 supporting cells of, 1034–1035, Eardrum. Tympanic membrane Early endosomes, 44–48, Early lipoblasts, 282, Early melanosomes, 546 Early myoblasts, 360 Early-onset puberty, 830, 868 Early spermatids, Early spermatocytes, 878 Early stage, of T cells development, 514 Earwax (cerumen), 1019 Eating disorders, 284 Eccrine glands, 560, , 568, , , 571, 580 –583 ECG (electrocardiogram), 448 Ectocervix, 932, , 968 –969 Ectoderm, 116, 982–983, derivatives of, 116, neural, 982–983, 984 oropharynx, , 820 surface, 116, , 982–983, 984 , 1018, Ectomesenchyme, 177 Ectopic pregnancy, 917, 924–925, 938 Ectosomes in bone mineralization, 260 Edema cystoid macular, 997 peripheral, 464–465 Education, of T lymphocytes, 514, Effective stroke, of ciliary movement, 134

1019 1025

1021

p

1035 1037 1035 1037 1029 1029 1029

See

1037

44 282

1027 1028, 1042p

1026 1028

878

932

819 117

560

p 983 117 t

f

568 570 p t

515

p

1020

p

Effector lymphocytes, 483, 492 Efferent (motor) nerve fibers, 359, Efferent (motor) neurons, 390, , , reestablishing contact with muscle, 429 skeletal muscle, 356–359 somatic, 390, 416 ventral, 422 visceral, 390, 418, Efferent arterioles in kidney glomerulus, 771–772,

360 390 391 392

418

774, 792–794, 794 Efferent ductules, of testis, 866 , 875, 882–883, 882 , 900 p –901 p Efferent lymphatic vessels, 501, 502 , 506, 507 , 509 , 530 p –531 p Ejaculate, 890 Ejaculation, 875, 885, 891 Ejaculatory duct, , , 882, 884, 885 Elafin, 543, Elastic (large) arteries, 189–190, 214 –215 , 451 , 456–460, , 476 –477 Elastic cartilage, 217, 223, , 225 , 234 –235 laryngeal, 736 Elastic cartilage matrix, 223 Elastic fibers, 189–192, , , , 214 –215 biosynthesis of, , 192 composition of, 189–190 in dermis, 214 –215 , 551–552 in elastic arteries, 189–190, 214 –215 , 458, 460 in elastic cartilage, 223, , 234 –235 in extracellular matrix, 189–190, 192 in heart valves, 447 in muscular arteries, , 461 photoaging and, 193 Elastic lamellae, 214 –215 , 447, 457, Elastic ligaments, 180, 189–190 Elastic membrane external, of blood vessels 451, 460–461, , 478 –479 internal, of blood vessels 450, 457–459, , 460–461, tracheal, 741 Elastin, 189–190, 223, 459 Elastin gene , 190 Electric current flow, between cells, 150 Electrical synapses, 398 Electrocardiogram (ECG), 448 Electrochemical proton gradient, 63, Electrolytes active transport of, 711 reabsorption of, 660, 708 in saliva, 616 Electron microscope (EM), 1, , 20–24,

863 881

544 459 p

p

190 p p

223

t

p

p

189 190 191 223

p

p

460 f p p

p

p

p

p

t

p

p

457

460 459

(ELN)

63

11

21

p

p

460

457,

11

Electron microscopic autoradiography, , 21 Electron-probe X-ray microanalysis, 22 Electron-transport chain, 63, Elementary particles in mitochondria, 62 Ellipsoid portion, of photoreceptors, 996 Elliptocytosis, hereditary, 303 Elongation stage, of ciliogenesis, 137 Embedding medium, 2 Embryo, trilaminar, 116 Embryoblast, 930, Embryogenesis, cilia in, 135 Embryonic cilia, 137 Embryonic connective tissue, 114, 176–178, 177 , Embryonic development. Embryonic disc, bilaminar, 129 Embryonic hemopoiesis, 321, , Embryonic neural tube, 409 Emerin, 75, 94 Emery-Dreifuss muscular dystrophy (EDMD), 94 EMILIN-1, 192, 471 Emitter, 20 Emotion, pineal gland and, 830 Emotional sweating, 582 Emphysema, 56, 754 Enamel, 116, 259, 597–604, , – composition of, 599–600, production of, 600–604, – , , Enamel crystals, 599 Enamel epithelium, 600, – , 603 Enamel matrix, 600 Enamel organ, 116, 600 Enamel rods, 599–600, , Enamel tufts, 604 Enamelins, 604 Encapsulated nerve endings, 417, 555, , , 584 –585 End piece, of sperm, 872, , 875 End-stage renal disease, 769 Endemic goiter, 836 Endocardium, 124, 384 –385 , 444–446, , , 474 –475 Endocervix (cervical canal), , 925, 932, , 968 –969 Endochondral bone, 274 –277 Endochondral growth, 255–258 Endochondral ossification, 220–221, , 232 –233 , 254–255, – , 274 –277 Endocrine cells, of nasal cavity, 732 Endocrine control, 815, Endocrine disease, 828 Endocrine-disrupting chemicals (EDCs), 870

63

930

t 178 See specific anatomic structures 322 323 p

f

599 601 602 600 601 602 603 604 601 602

600 603

f

257

p

556 559

874

p

p

p

f

p

815

p

909

p

445 446 932

221

p

f

p

p p

p

p p

256

815

Endocrine gland(s), 163, 814–815, primary cilia of, 136 secretion mechanism of, 163, Endocrine gland tumors, 828 Endocrine islet, 174 –175 Endocrine signaling, 36–37 Endocrine system, 814–849 cells of, 815 components of, 816, . gastrointestinal, 644 hormones of, 816–818, . origins of, 815 overview of, 814–818 secretion regulation and feedback in, 818, 828 Endocytic vesicles, 29, 35 Endocytosis, 38–41, actin-dependent, 38 actin-independent, 38 clathrin-dependent, 39, 40, 41, clathrin-independent, 39, endosome formation in, 44–48, , , mechanisms of, 37–39 neurotoxins blocking, 37 receptor-mediated, 39–41, , , 50, , 453 renal, 780 SNARE proteins in, 39, 43 Endoderm, 117, 837 derivatives of, 117, Endodermal evagination, 692 Endolymph, 1025, 1032, Endolymphatic spaces, 1023, 1032 Endometrial stroma, 928 Endometrium, , 925–932, , 946 –965 basal layer of, 926 blood supply to, , 928 cervical, 932, cyclic changes in (menstrual cycle), 926–928, 926 –927 , 966 – 967 decidualization of, 931, functional layer of, 926–928, 964 –965 implantation in, 930–932 pregnancy changes in, 931–932 Endomitoses in megakaryoblast, 326 Endomysium, 345, , 362, 378 –379 Endoneurial tubes, 429 Endoneurium, , 416, 417, 434 –435 Endopeptidases, in pancreas, 713

t

p

p

163

f

816 See also specific components f 817 See also specific hormones

38

41 44 45 46

39

41 45

50

117 1032

909

p

926 932

925

p

f

931

392

346

p

p

p

p

p

p

p

f

p

See

Endoplasmic reticulum (ER). Rough endoplasmic reticulum; Smooth endoplasmic reticulum Endorphins, 402, 402 , 823 Endosomal sorting complex required for transport (ESCRT), 36, 45 Endosomes, 29, 30 , 31 , 44–48, 60 early, 44–48, late, 44–48 maturation model of, 44–45 sorting mechanism of, 44–45, 60, stable compartment model of, 44 Endosteal cells, 241, 245, , 249 Endosteum, 241, Endotendineum, 179, 212 –213 Endothelial activation, 452 Endothelial basal lamina, 425 Endothelial cells, , 192, 417, 425, 452–456, 456 , of capillaries, 462, 462–463, of elastic arteries, , 459 of liver, 722 –726 of splenic sinuses, 517–518 Endothelial-derived relaxing factor (EDRF), 454 Endothelial dysfunction, 452 , 456 Endothelial growth factor (EGF), 938 Endothelial lining, 457–458 Endothelial nitric oxide synthase (eNOS), 454 Endothelin(s), 455 Endothelium, 124, 384 –385 , 450, 452–456, of blood–thymus barrier, 514, of capillaries, 462–464 discontinuous sinusoidal, 699 of elastic artery, 457–459, of glomerular capillaries, 775, 775–777 of muscular artery, , 461 of small arteries, 461–462 of veins, 466–467, , of venules, 465–466 Endothelium-derived hyperpolarizing factor (EDHF), 454 Energy homeostasis, 280, 281, 283–285 Energy, mitochondrial generation of, 62, Enkephalins, 402, 402 , 647 , 656 Entactin, 775, 777 Entactin/nidogen, 156, 162, 197 Enteric division, of ANS, 371, 389, 418, 420, , 634 Enteric ganglia, 432 Enteric neuroglial cells, 389, 403 Enteroceptors, 417 Enterochromaffin cell, 717 Enterocytes, 166, 648 –649 , 652–656, , 680 –683

t

t t 44

61

245 p p

242

177

p

p

t 458

463

457

f

p

p

459 466 467

t

t

p

f

t f

458

514

459

63

t

420

653

p

p

Enteroendocrine cells, 166 closed, 645, gastric, , 641, 644–646, 644 , , 651, 674 –677 intestinal, 402, 653, 656, 659 life span of, 651 of nasal cavity, 732 nomenclature for, 644–645 open, 645, pancreatic regulation and, 714–715 secretions of, 644–645, of trachea, 740–741 Enterokinases, 648 , 713 Enteropeptidase, 648 Entosis, 107 Envoplakin, 543 Enzymatic velocity, of muscle fibers, 346 Enzyme(s). amylolytic, (α-amylase) in pancreas, 713 bacteriolytic, 311–312 detoxifying, 57 hydrolytic, 48 lipid-processing, 543 nucleolytic, (deoxyribonuclease, ribonuclease) 713 oxidative, in peroxisomes 64 pancreatic, 648 , 713 proteolytic, 713 in synaptic transmission, 400 Enzyme 21-hydroxylase, in adrenal gland, 847 Enzyme histochemistry, 7–8, Enzyme-linked receptors, 37 Enzyme-replacement therapy, 51 Eosin, 2–3, 5, 5 Eosinophil(s), 176, , 299 , 312–313, , 336 –339 activation of, 485 in alimentary canal, 634 in allergic reactions, 207, 313, 492 on bone marrow smear, 338 –339 in connective tissue, 197, 207 development of, , , 327–328, 329 in diffuse lymphatic tissue, 502 granules of, 312–313 immune function of, 485 in inflammatory response, 312 mature, 327 in parasitic infections, 207, 313 Eosinophil cationic protein (ECP), 313 Eosinophil chemotactic factor (ECF), 204, 492 Eosinophil-derived neurotoxin (EDN), 313

640

645

f 645

645

f

p

646

f

See also specific enzymes

f

8

t

177

322 324

t

p

f

312

p

p

f

t

f

p

p

Eosinophil peroxidase (EPO), 313 Eosinophil progenitors (EoP), 323, 327, 329 Eosinophilia, 313 Ependymoma, 98 Epicardium, 444–445, , Epichoroid lymph spaces, 992 Epidermal derivatives, 538 Epidermal growth factor (EGF), 46 Epidermal ridges, 550 Epidermal stem cells, 539, 564, Epidermal–dermal junction, 550 Epidermal–melanin unit, 544 Epidermis, 538, 539–541, , 576 –579 appendages of, 559–572, , 586 –587 attachment to dermis, 550–551 cancers originating in, 551 –552 cell renewal in, 167, 539–540, 544, cells of, 541–550 dynamic equilibrium in, 544 histogenesis of, 116 layers of, 539–541, 576 –577 mandibular ossification and, 278 –279 neuronal receptors in, 555 pH of, 541, 546 repair of, 572 sensory receptors of, 555–559, , 584 –585 thick or thin, 539, , , 576 –577 turnover of, 544 water barrier of, 541, 543, Epidermolysis bullosa, 159, 162 Epididymis, , , 875, 882–883, , , , 900 –901 cells of, 883, 900 –901 duct of, 875, , 882–883, 900 –901 epithelial tissue of, 172 –173 , 883, stereocilia of, 127, 883, 900 –901 structure of, 882, 900 –901 Epiglottis, , 234 –235 , , Epimysium, 345–346, , 376 –377 Epinephrine, 401, 402 , 450, 840 , 842 Epineurium, , 417, 434 –435 Epiphyseal cartilage, 232 –233 , 255–257, Epiphyseal closure, 258 Epiphyseal growth plates, 220–221, , , 257–258, 272 –273 , 274 –277 Epiphyseal line, 240, , 258 Epiphysis, 240, , 274 –277 Epiretinal membrane (ERM), 997 , 1002 Episclera (episcleral layer), 986–987

t

445 446

564

539 p p 560 p p f f 545 p

f

863 866 p 881

539 540

p

544

p

556 p

p

p

p

p

882 883 884 p p p p 883 p p p p 223 p p 592 732 346 p p t t 392 p p p p 259 221 256 p p 240 240 p p f p

p

p

p

p

Episcleral space, 986 Epitendineum, 179, 212 –213 Epithelial attachment, 608 Epithelial cells, 113, 122, apical domain (free surface) of, 113, 122, , 124, 126–139 cilia of, 128–139, 130 microvilli of, 126–127, , , 130 specialization of, 124 stereocilia of, 126, 127–128, , 130 , basal domain of, 122, , 124, 153–163 basement membrane/basal lamina of, 153–163 cell-to-extracellular matrix junctions of, 153, 160–162 basal surface of, 162 characteristics of, 122 collagen biosynthesis in, 186 corneal, 985 differentiation of, 539 epidermal, 539–540 lateral domain of, 122, , 124, 139–153 anchoring junctions of, 141, 145–149 cell adhesion molecules of, 139 cell-to-cell attachment sites of, 139–141 communicating junctions of, 141, 150–153 junctional complexes of, 140–153, , morphologic modifications of, 153, occluding junctions of, 141–145 terminal bars of, 139–140, lateral surface of, 153, lens, 1003, mammary gland, 947 parietal, of Bowman capsule, , 780, polarity of, 124–126, 141 as receptors, 122, 124 renewal of, 166–167 in large intestine, 661–662 in skin, 167, 539–540 in small intestine, 659 in stomach, 647–650 sebocytes as, 567 Epithelial metaplasia, 126 Epithelial–mesenchymal transition, 791 Epithelioid cells, 122–123, 174 –175 , 814 Epithelioid tissue, 122–123, 174 –175 Epithelioreticular cells, 485, 498, 512–513, , 536 –537 Epithelium (epithelial tissue), 112, 113–114, 122–175, 170 –175 . and locations basement membrane of, 122, cells of, 113, 122, . Epithelial cells

p

p 123 t 127 128 129 123

t

123

t 131

123

1003

153

140 141 153

140

775

f

p

p

See also specific types 123 123 See also

781

p

p

514

p

p p

p

ciliated, 732 classification of, 113–114, 123–124, 125 columnar, 113, , 123, 125 , 172 –175 crevicular, 608 cuboidal, 113, , 123, 125 , 170 –175 distribution of, 122, 125 enamel, 600, – , 603 functions of, 123, 124, 125 glandular, 163–164, 389 histogenesis of, 117 junctional or attachment, 608 keratinized, 113, 589–590, , 620 –621 in membrane functional unit, 167 mesodermal, 863, mucosal, 633, 661 nonkeratinized, 113 parakeratinized, , 620 –621 as pathogenic target, 148 primary cilia of, 136 pseudostratified, 124, 125 , 172 –173 renewal of, 166–167 as selective barrier, 123 simple, 113, , 123–124, 125 , 170 –175 squamous, 113, , 123, 125 , 170 –175 stratified, 113–114, , 123–124, 125 , 172 –175 structure of, 122–123 surface modifications of, 113 transitional, 124, 125 , 174 –175 , 795–796, , 810 –813 transport across, 124, 144–145, of urothelium, 810 –811 Epithelium, olfactory, 733, , Eponychium, 565, , 586 –587 Epoxy resin, 21 Epsilon cell, of pancreas, 717 ε-Tubulin, in MTOC, 77 Equatorial division (meiosis II), 102, 103 Erbin, , , 162 Erectile dysfunction (ED), 892 Erectile tissue, 890, , 892 , 940, 941, 942, Erection of penis, 890, 892 Ergastoplasm, 54, 57, 58 Ergocalciferol, 769 Erythroblast(s), , 325–326, , , 329 , 340 –341 Erythroblastosis fetalis, 304 Erythrocyte(s) on blood smear, 336 –337 on bone marrow smear, 338 –339 chromatin of, 326

113 113 601 602

t t

t

590

113 113

p f

t

p p

t

t

864 590

p p

p

f

p

p

t

p

t

113

p

890

f f

p

p

p t

p

p 144 p p 734 735 567 p p t

160 161

t

t

p

f 324

p

f

p p

f

p

p

796

p

943

325 326 p

t

p

p

p

322

324 325

t

p

p

development of, 321, , 323–326, , , 329 , 340 –341 diameter of, 301 hemoglobin of, 303–305 hemolysis of, 303 as histologic ruler, 301 indices of, 321 life span of, 301, 326 maturation stage of, 325–326 mature, 340 –341 nucleoli of, 326 nucleus of, 325 packed volume of (hematocrit), 298–299, , 321 polychromatophilic, , 325, , 329 , 340 –341 shape and morphology of, , 302–303, splenic filtration of, 517 staining appearance of, 326 Erythrocyte (red blood cell) count, 298–299, 299 , 301–305, 320– 321, 336 –341 Erythrocyte-committed progenitor (ErP or CFU-E) cells, 323 Erythrocyte distribution width (RDW), 321 Erythropoiesis, 321, 323–326, , , 329 , 340 –341 Erythropoietin (EPO), 326, 328, 330 , 769 Erythropoietin-sensitive erythrocyte-committed progenitors (ErPs or CFU-E), 324 , uropathogenic, 796 Esophageal cardiac glands, 636, Esophageal glands proper, 636, Esophageal sphincter, 635 Esophagogastric junction, , 672 –673 Esophagus, , 635–637, 670 –673 adventitia of, 636, 670 –671 epithelial tissue of, , 174 –175 innervation of, 637 lamina propria of, 636, 670 –671 mucosa of, 636, , , 670 –671 muscularis externa of, 636, , 670 –671 muscularis mucosae of, 636, 670 –671 striated musculature of, 637 submucosa of, 636, , 670 –671 wall, muscle of, 637 Espin, 127, 128, , , 1026–1027, 1028 Essential fatty acid deficiency (EFAD), 544 Essential hypertension, chronic, 783 , 844 Essential light chain (ELC), 350, Essential transcription factors (E2F), 97 Ester neurotransmitters, 402 Estradiol, Estrogen(s), 568, 919, 924, 928, 937

p

p

324

p

301

325

299 t p 302

p

t

p

324 325 t

t

Escherichia coli

638 637 638 p p 633 p p p p 113 p p p p 636 637 p p 636 p p p 636 p p 128 131 f 350 t 880

p

p

p

in luteal cells, 919 in mammary gland regulation, 949–951 in menstrual cycle, 928 ovarian synthesis and secretion of, 909, 914–915, placental, 937 in uterine tube physiology, 924 η-Tubulin, in MTOC, 77 Euchromatin, 88–89, , , 92 Eukaryotic ribosomes, 55 Eumelanin, 545, 561 Eustachian tube. Auditory tube Excitatory synapses, 393, 400–401 Excludes cytoplasm, 35 Excretion, 768 by skin, 539 Excretory ducts, 609, 613, 626 –629 Excurrent duct system, of male reproductive system, 862, 875, 880– 882, , Exfoliation, 541, 544 Exocrine glands, 163 multicellular, 164, 165 secretion mechanisms of, 163, unicellular, 164, Exocytosis, 38, , 41–44 in constitutive pathway, 41–42, neurotoxins blocking, 37, 43 in regulated secretory pathway, 42, SNARE proteins in, 39, 42–44, Exopeptidases, 713 Exophthalmos, 836 Exosomal signalling, 37 Exosome-based mRNA vaccines, 48 Exosomes, 36, 44, 48 Expansion microscopy, , 11, , Expansion pathology (ExPath), 13 Exportin, 96 External acoustic meatus, 1018–1019, , External anal sphincter, – , 690 –691 External capsule, of muscle spindle, 359, External ear, 1018–1019, External elastic membrane, of blood vessels, 451, 460–461, , 478 –479 External genitalia female, 908, 939–941 male, 862, 890–891 External lamina, 155, 156 of smooth muscle, 372 External nose, 731

915

f See

89 90

p

881 882 38

164

t

p

163

42

43

f

10

p

p

42

12 13

665 666 1019 f

1019 1022 p p 360

460

909

932 p p 560 564

External os of cervix, , 925, 932, , 968 –969 External root sheath, of hair follicle, , 564, External urethral orifice, 799, 908 External urethral sphincter, 799 Exteroceptors, 417 Extracellular aqueous channels, 142, 145 Extracellular matrix (ECM), 114, 146, 176, 192–196 bone, 238–239 cartilage, 217 cell interactions with, 156 degeneration, in valvular disease, 448 elastic fibers in, 189–190 endothelial cell maintenance of, 456 fibrocartilage, 224 functions of, 192 ground substance of, 193–194 junctions anchoring cells to, 153 photoaging and, 193 production of, 219–220 Extracellular matrix adhesion molecules, 250 Extrahepatic bile ducts, 707–708 Extramural digestive gland epithelium, 117 Extramural glands, , 634 Extraocular muscles, 1007 Extremities, autonomic innervation of, 421 Extrinsic laryngeal muscles, 736 Extrinsic muscles of eye, 1007 Eye(s), 980–1017, 1010 –1018 accessory structures of, 1004–1007 anterior segment of, 1014 –1015 barriers protecting, 989, 990–991, 994 chambers of, 982, color of, 988 corneoscleral coat of, 980–981, , 983–987 development of, 116, 982–983, , 984 general structure of, 980–983 innervation of, 980–981, – , , , 994, 1002, 1010 layers of, 980–981, microscopic structure of, 983–1004, movements of, 1007 muscles of, 981, 987–989, – , 1004, , 1007, 1014 – 1015 ophthalmoscopic examination of, 989, overview of, 980 pressure in (intraocular pressure), 990 , 991 refractile (dioptric) media of, 982 vascular coat of, 981, , 987–992 Eyelash glands, , 1006

t

f

633

p

982

p

1005

p

p

p

981 983 t 981 983 988 993 981 988 987 989 1004 1002 f 981

p

p

Eyelashes, 1006 Eyelids, 163, 1004–1006, Ezrin, 128

F

1005

F-actin (filamentous actin), 69, 344 F cell, of pancreas, 717 Facial expression, muscles of, 555 Facial nerve (CN VII), 592, 614 Factor VIII, (coagulating), 457 Facultative heterochromatin, 88, 91 False vocal cords, 737–738 Fas signaling pathway, 109 Fascia deep investing, 345–346 subcutaneous (superficial), 281, 538, 555 Fascia adherens, 148–149, , 362, Fascicle, muscle, 345, , , 376 –377 Fascicle, nerve, 417 Fascin, 70, 127, Fast anterograde transport, 396–397 Fast oxidative glycolytic muscle fibers, 347 Fast retrograde transport, 397 Fast-twitch, 347 Fast-twitch muscle fibers, 347 Fat absorption, 708 Fat droplets (lipid inclusions), 30 , 31 , 81–82 Fatigue-prone muscle units, 347 Fatigue-resistant motor units, 347 Fatty acid(s), 32, , 285, 350 , 544, 844–845 Fatty acid β-oxidation, 62, 64, 695, 713 Fatty liver disease, 693 Fatty streak, 452 F c receptors, 200, 204, 309, 313, 495, 521, 550 18F-FDG (radioactive glucose isotope), 293 Feedback mechanisms, 818, 827 Female gametes. Oogenesis; Ova Female reproductive system, 908–979, 956 –957 cilia in, 135 components of, 908, cyclic changes in, 908–909, 938 menopause, 908–909, 951 menstrual cycle, 908, 925, 926–930, 947, 949, 966 –967 ovarian cycle, 926 –927 external genitalia of, 908, 939–941 hormones of, 909, 926 –927

t

128

148 346 347

364 p p

t

33

t

f

f

f

See

p

909

f

f

f

p

p

f

p

908

internal organs of, 908, oogenesis in, , 102 overview of, 908–909 Female urethra, 799, 908 Feminization, 869 Fenestrae, of hepatic sinusoids, 699 Fenestrated capillaries, 462–463, , , 775, 842 Fenestrations, 462, Ferritin, 326, 701, 985 Fertilization, 90, , 919–921, 924–925 Fertilization, in vitro (IVF), 922 Fetal adrenal gland, 846–847, Fetal circulation, 935–937, Fetal cortex (adrenal), 846–847 Fetal development estrogen as index of, 937 thyroid hormones and, 835 Fetal hemoglobin, 304–305, Fetal placental antigen–presenting cells, 934–935, , 972 –973 Fetal zone, of adrenal gland, 846, Fetal–placental unit, 847 Fetoplacental (endocrine) unit, 937 Feulgen microspectrophotometry, 7 , 18 Feulgen reaction, 6 Fever, 309 FGF. Fibroblast growth factor(s) Fibers. Fibril(s). Fibril-associated collagens with interrupted triple helixes (FACITs), 183, 186 Fibrillar centers, of nucleoli, 91 Fibrillar collagens, 183 Fibrillar material, of nucleoli, 91 Fibrillin-1, 190, 471 Fibrillin gene ( ), 192 Fibrillin microfibrils, 159, , 190, 192, 193 Fibrillogenesis, 185–186, Fibrin, 299, 320 Fibrinogen, 299, 320, 693 Fibrinolysin, 889 Fibroblast(s), 176, , 197–199, , activated, 197 in bone repair, 265 collagen synthesis in, 186, 197 differentiation of, 203 , 206 in elastic arteries, 461 elastic fiber synthesis in, 190, 197 in endoneurium, 417

100

463 464

463 917

846 936

305

f

934

846

f

See

See specific types See specific types FBN1

160 185

177

f

197 198

f

p

p

in epithelial–mesenchymal transition, 791 fibrocartilage, 223, 236 –237 in inflammation, 312 in muscular arteries, , 461 reticular fiber synthesis in, 189–190, 197 in wound repair, , 202 , 206, 312 Fibroblast growth factor(s) (FGFs), 204, 254, 262, 293, 454 Fibroblast growth factor 21 (FGF-21), 293 Fibroblast growth factor 23 (FGF-23), 262 Fibrocartilage, 217, 223–224, , 225 , 236 –237 Fibrocystin/polyductin, 135 Fibrofatty plaque, 452 Fibrogenesis, hepatic, 702 Fibrolipoma, 289 Fibromuscular stroma, of prostate gland, 886, 904 –905 Fibronectin, 155, , , 195, , 197 , 218, 777 Fibronexus, 198 Fibrosa, of heart valve, 446–447, Fibrosis heart valve, 448 hepatic, 702 renal, 791 Fibrous astrocytes, 410, Fibrous astrocytomas, 410 Fibrous pericardium, Fibrous sheath, of sperm, 872, , 875 Fibrous skeleton, of heart, 444, , Fight-or-flight response, 401, 843 Filaggrin, 540–541, 543, Filamentous actin, 69 Filaments, 30. Filensin, 74 , 75 Filgrastim (Neupogen), 330 Filiform papillae, 592, , 622–623 Filopodia, 72 Filtration apparatus of kidney, 768, 775–780, Filtration, basal lamina and, 159 Filtration pores, 463 Filtration slit, , 776, 780, Filtration slit diaphragm, 776, 780, Fimbriae, of uterine tube, , 924–925, 960 –961 Fimbrin, 70, 127, , 1026 FimH adhesins, 796 Fingerprints, 551 First intention, of wound healing, 572 First polar body, 917, Fistulas, anal, 665 Fixation of tissues, 2, 5, 11,

p 460

202

f

f

224

f

160 161

445

t

p

t

p

p

p

196 t 447

p

411

874 444 446

544 See also specific types 593

775

775

909

128

917

781

781 f

12

p

p

Fixatives, 2–3 Flaccid part, of tympanic membrane, 1020 Flagella axonemal organization of, 129 basal bodies for, formation of, 76, Flagellum, of sperm, 872, , 875 Flat bones, 240 Floating villi, 933, 971 –971 Flotillins, 32, 39 Flow cytometry, 7 , 320 Fluid–mosaic model, modified, 32, Fluorescein, 8, Fluorescence in situ hybridization (FISH), 9–10, , 92 Fluorescence microscope, 17–18 Fluorescence photoactivated localization microscopy (FPALM), 20 Fluorescent dye(s) (fluorochromes), 8 Fluorescent dye–labeled antibody, 3 Fluorescent molecules, 17–18 Fluorescent probes, 10 Fluorescent proteins (FPs), 18 Fluoride, 600, 610 18-Fluorine-2-fluoro-2-deoxy-D-glucose ( 18F-FDG), 293 Fluorochromes, 8 Fluorophores, 17–18 Foam cells, 452 , 454 Focal adhesion(s), , 145, 152 , 160, , , 551 Focal adhesion kinase, 161 Focused ion beam SEM (FIBSEM), 23 Foliate papillae, 592, , , 622 , 624 –625 Folic acid deficiency, 306 Follicle(s) hair ( Hair follicles) ovarian ( Ovarian follicles) thyroid ( Thyroid follicles) Follicle-stimulating hormone (FSH), 816, 821, 821 , 823 in female reproductive system, 913, 915, 926 –927 in male reproductive system, 870 , Follicle-stimulating hormone cells (gonadotropes), 822 , 823 Follicular bulge, 544, , 561, Follicular cells of ovary, 911–912, , , 956 –957 of thyroid gland, 831, Follicular dendritic cells (FDCs), 485, 501, 507, Follicular epithelium, of thyroid gland, 831, Follicular helper CD4 + lymphocytes (TFH), 510 Follicular phase, of ovarian cycle, 926 –927 Follicular stigma, 916

8

p

f

874

77

p

33

10

f

f

See

f

f

140

t

592 593 f

160 161

p

p

p

See See

f

f 880 560 564 911 912 p p 831 f

f

831

t

f

508

t

Folliculostellate cells, 824 Fontana, spaces of, 987 Foot processes, of podocytes, , , 776, Foramen cecum, 591, Fordyce spots, 591 Foreign body giant cells, 200 Formalin, 2–3 Formative osteocytes, 248, Formed elements of blood, 298–299, , 299 Fornix of lacrimal sac, 1007 Fovea centralis, , 995, , 1002, Foveola, of retina, 1002 Foveolae (gastric pits), 638 FPALM. Fluorescence photoactivated localization microscopy Fracture healing, 244, 262–265, Fracture hematoma, 264, Free (floating) villi, 933, 970 –971 Free fatty acids, 543 Free nerve endings, 417, 555, , 584 –585 , 946 Free radical, as neurotransmitter, 401–402, 402 Free ribosomes, 54, 55, Free surfaces, 113–114, 122, Freeze fracture, 22, 33–34, Frey syndrome, 617 Frozen sections, 3, 4 Functional layer, of endometrium, 926–928, 929, 964 –965 Fundic glands, 637, , 639–646, , 647–649, 674 –677 Fundic region, of stomach (fundus), 637, , 674 Fundic segment, of gastric glands, 639 Fundocardiac junction, 674 –675 Fundus, of uterus, Fungiform papillae, 592, , , 622 –623 Fusiform vesicles, 797 Fusion pore, 400 Fuzzy plaque, 148

774 775

592

248

987

266 58

f

299 t 1002

995

See

f 638

266

p 556

p

p

p

123 34

640

p p 909 592 593

G

781

638

p

p

G-actin (globular actin), 69–70, 344, 349 G protein(s), 401, 594, , 656, 817, G protein signaling cascade, 656 G protein–coupled receptors, 37, , 400 olfactory receptors, 734 in smooth muscle contraction, 370 taste receptors, 594–595, , 735 G protein–gated ion channels, 400 G 1 (gap 1 ) phase, 97, ,

595

97 100

399

595

817

t

p

p p

p p

97

100

G 2 (gap 2 ) phase, , 98, GABA (γ-aminobutyric acid), 400, 401, 402 Galactose transferase, in blood group systems, 303 Gallbladder, 708–712, , 724 –725 bile flow to, 695, 705–707 bile storage and concentration in, 711, 6709 development of, 692, 708 epithelial tissue of, , 709, junctional complexes of, 709, mucosa of, 709–711, , 724 –725 muscularis externa of, 709–711, , 724 –725 sinuses of, 710–711, GALT. Gut-associated lymphatic tissue Gametes, 102 female. Oocyte(s); Oogenesis; Ova male. Sperm; Spermatogenesis Gametogenesis female. Oogenesis male. Spermatogenesis Gamma/delta (γ/δ) T cells, 316, 488, 733 γ-Globulins, 299 γ Nerve fibers, 359, γ-Tubulin, in nucleation, 66–67, – , 76, , 77, Ganglia, 116, , 388 cranial nerve, 390, 415 dorsal root, 390, , 415 , 422–423, , 432 –433 enteric, 432 parasympathetic, 415 , 432 paravertebral, 418, 432 peripheral, , 415, 415 prevertebral, 432 sensory, 415 , 416 sympathetic, 415 , 432 –433 terminal, 432 visceral, 419 Ganglion cells, 415 of adrenal medulla, 842, 860 –861 of retina, , 993–994, , 1002, 1012 –1013 Gap (communicating) junctions, 141, 150–153, 152 atomic force microscopy of, 151, diseases associated with, 153 formation of, 150 structure of, 150, study of, 150 Gap junctions, 247 bone cell, 246–247 cardiac muscle cell, 363,

See

711

p

p

113 709 711

710 710 p p 711

t

f

p

p

See See See See

116 p

410 p t t p

360

67 68

t

392 t

p

p

t

t p

423

78

p

p

p 993

987

76

151

364

p

151

p

t

p

p

lens cell, 1003 smooth muscle, 358, 372, 386 as synapse equivalent, 398 Gardasil 9, 950 Gas exchange (respiration), 730, 747 Gastric cytoprotection, 639 Gastric glands, 637, , 639–646, , 647–650, 672 –677 Gastric inhibitory peptide (GIP), , 646 , 656 Gastric juices, , 639–644 Gastric lamina propria, 651 Gastric mucosa, 638–647 blood flow in, 639 cells of, 641–646, 650–651 drugs damaging, 639 epithelial cell renewal in, 647–650 glands of, 37, , 639–646, , 650–651 hormones of, 644–645, , 646 physiologic barrier of, 641 protection and recovery of, 639 Gastric muscularis externa, 651 Gastric muscularis mucosae, 651 Gastric pits, 638, 639, , 672 –673 Gastric rugae, 638, Gastric serosa, 651 Gastric stem cells, 641 Gastric submucosa, 650 Gastrin, 641, 642–643, 642 , , 646 , 716, 718 Gastrin receptors, 643 Gastrin-releasing peptide (bombesin), 647 , 656, 741 Gastrinomas, 642 Gastroduodenal junction, 678 –679 Gastroenteropancreatic (GEP) neuroendocrine tumors, 644 Gastroesophageal reflux disease (GERD), 635, 637, 641 Gastrointestinal endocrine system, 644 Gastrointestinal hormones, 644–645, 644 , , 646 , 656 Gastrointestinal smooth muscle spasms, 44 Gastrointestinal tract, 588, 632–635.

p

f

638

589

638

638

f

640 646 t

646

640

p

640 t

p

p

f 646

t

p

p

t

p

f

f

f

f 646 t See also specific components

of

GATA-1 transcription factor, 323 GATA-3 transcription factor, 331 Gated Ca 2+ (calcium)-release channels, 355–356, Gated Na + (sodium) channels, 356, 358, 358 , 359 Gaucher disease, 51 Gaze, conjugate, 1007 Gelatinases, 187 Gelation, for expansion microscopy, , 13 Gelsolin, 70–71

f

f

12

359, 365, 365, 370

865

Gender determination, 863, Gene-transfer therapies, for lysosomal storage diseases, 51 Genetic code, 5 Genetic sex, 863, Genital warts, 950 Genitalia, external female, 908 male, 862, 890–891 Genitourinary malformations, 863 Genome, human, 88 Germ cells, primordial, 863–864, 910 Germ layers, 116–117, , 176–178 Germinal center, 502–503, , , 528 –535 Germinal center reaction, 503 Germinal epithelium, of ovary, 910, 956 –957 Germinative zone, of nail, 564 Ghrelin, 284, 287 , , 646 , 717 , 822 Gigantism, 266 Gingiva, 589, , , 608, Gingival mucosa, 608 Gingival sulcus, 608, GIP. Gastric inhibitory peptide Glands, 113, 163–164. and gland types autonomic regulation of, 389 mucous, 164, multicellular, 164, 165 secretion mechanisms of, 163, secretory portion (parenchyma), 122 serous, 166, swollen, 511, 518 unicellular, 164, Glandular adenocarcinomas, 126 Glans clitoris, Glans penis, 799, , 890–891 Glassy membrane of hair, , 564 of ovarian follicles, 922–923, 957 –960 Glaucoma, 403, 987, 990 , 997 , 1002 Gleason score, 888 Glia. Neuroglial cells Glia limitans, 411, Glial cell line–derived neurotrophic factor (GDNF), 879 Glial cells, 409 Glial fibrillary acidic protein (GFAP), 74, 74 , 410, 428 , 826 Glial growth factors (GGFs), 427 Glial scar, 428 Gliosis, 398 , 428 Glisson’s capsule, 692, 696

f

865 f

117

507 509

p p

p p

f 646 t t f 590 599 608 608 See See also specific glands 166 t 163 166 f 164 f 939, 942 863 560 p p f f f See 412

t

f

f

f

304

Globins, 304, , 326 Globular actin (G-actin), 69–70 Globular structure, of collagen, 185 Globulins, 299, 693 Glomerular basement membrane (GBM), , 775–779, , , Glomerular capillaries, 771–772, , 775, , 776–777, , , , Glomerular disease, thrombotic, 775 Glomerular filtration barrier, 768, 775, , 777, 780 Glomerular ultrafiltrate, 768, 771, , 784 Glomerulonephritis, 775–776, 780, 782 Glomerulus, 768–770, , , , capsule of ( Bowman capsule) crescent, in glomerulonephritis, 782 distention of, 780 filtration apparatus of, 768, 775–780, Glossopharyngeal nerve (CN IX), 592, 596 Glucagon, 718, 718 , 816 action of, 718, 719 adipose tissue regulation by, 285 hepatic action on, 695 pancreatic secretion of, 712, 716, 718 Glucagon-like peptide-1 (GLP-1), 647 Glucocorticoids, 281, 840 , 843 Gluconeogenesis, 718, 844 Glucose blood levels of, 718 digestion of, 648 metabolism and regulation of, 262, 285, 350 , 694–695, 716–717, 844–845 renal reabsorption of, 786 storage of, 717 synthesis of, 844 Glucose-6-phosphatase, 58 Glucose-6-phosphate, 694–695 Glucose transporters, 290, 716 Glucuronate, 194 Glutamate, 393, 402 , 999, 1028–1029 Glutamine, 400 Glutaraldehyde, 21 Glycated hemoglobin, 305 Glycine, 181, , 400, 402 Glycocalyx, 32, , 318, , 634, 776–777, 875 Glycogen breakdown of (glycogenolysis), 717 in cardiac muscle, 361–362 digestion of, 648 hepatic storage of, 694–695

774 774

781 794

775 f 773 774 777 781 f

See

t

t

f

f

f

775

775

t

f

t

181 32

775

t 319

775 776 781 776 777

t

t

82 319

inclusions of, 30 , 31 , 81–82, metabolism of, 717 in platelet organelle zone, 318, staining properties of, 5, 6, 82 synthesis of (glycogenesis), 717, 844 Glycogen granules, 362 Glycogenesis, 717, 844 Glycogenolysis, 695, 718 Glycolipids, 32 Glycolysis, 717 Glycophorin C, 302 Glycophorins, 303 Glycoprotein(s), in basal lamina, 154, 156 in bone matrix, 238–239, 245 collagen as, 181, in extracellular matrix, 193 hepatic production of, 693 in hyaline cartilage, 217, 218, 230 –231 in lysosomal membrane, 48 multiadhesive ( Multiadhesive glycoproteins) in myelin sheath, 412 in plasma membrane, 32, in saliva, 616 transmembrane, 146 in vitreous body, 982 zona pellucida, 911 Glycoprotein 91 (gp91), in NADPH oxidase complex, 311 Glycoprotein GP2, in M cells, 657 Glycosaminoglycans (GAGs), 5, 193, 193 in bone matrix, 238 in cartilage, 216 chondrocyte secretion of, 219 in glomerular basement membrane, 778 in heart valves, 447 heparin-like sulphated, 453–454 Glycosphingolipids, 32 Glycosylated hemoglobin, 305 Glycosylation, in posttranslational modifications, 37, 56, 58–59 G o (gap o ) phase of cell cycle, 97 Goblet cells, , , 164, 166 in conjunctiva, 1006 in large intestine, 661, , 686 –687 of nasal cavity, 732 in small intestine, 652, 655, , 659, 680 –683 of trachea, 739–740, , , 762 –763 Goiter, 836

f 196

181

See

p

p

32

f

f

t

f

154 163

f

662

740 741

655

p

p

p

p

p

p

Golgi apparatus, 29, 58–61 functions and pathologies of, 31 hepatocyte, 703, microscopic features of, 30 , 58, , of odontoblast, 606–607, protein processing in, 58–59 protein secretion from, 60, sorting signals in, 61 transport to and from rough endoplasmic reticulum, 48, , 56, Golgi-derived coated vesicle secretory pathway, 49, histogenesis of, 117 Golgi outposts, 393, Golgi phase, of spermiogenesis, 872 Golgi tendon organs, 359 Gonadal dysgenesis, 863 Gonadal sex, 863, Gonadal steroids, 817, 829 Gonadocorticoids, 840 , 844, 845–846 Gonadotropes, 822 , 823 Gonadotropin-releasing hormone (GnRH), 823, 828 , Gonioscope, 987 Gonocytes, in testis development, 863–864, 898 –899 Goodpasture syndrome, 775–776, 782 Gout (gouty arthritis), 241 gp91 deficiency, 311 Graafian follicle, 910, , 914–915 Graft rejection, 498 Granular disintegration of axonal cytoskeleton, 426 Granular layer, of cerebrum, 436 –437 Granular material, of nucleoli, 91 Granulation tissue, 202 , 265, 572 Granule cell layer, of cerebellum, 438 –439 Granule cells, 438 –439 of nasal cavity, 732 of trachea, 740–741 Granulocyte(s), 299 , 305, 336 –337 . bone marrow reserve pool of, 328 circulating pool of, 328 development of, , 327–328, 329 marginated pool of, 328 Granulocyte colony-stimulating factor (G-CSF), 327, 330, 330 , 454 Granulocyte macrophage colony-stimulating factor (GM-CSF), 327, 330, 330 , 454 Granulocyte/macrophage progenitor cells (GMP, CFU-GM), , 250,

704

t 606 61

t

59 60

50

395

865

t

t

f

t

324

251

p

f p

p

t

910

p

56

t 880 p p

f

f

50

p

f

p

p

p See also specific types t

t

245

Granulocyte/monocyte progenitor cells (GMP, CFU-GM), 321–323, 327, 329 , 413, 415 Granuloma(s), 311

t

f

322,

f

Granulomatous disease, chronic, 311–312, 311 Granulopoiesis, , 327–328, 342 –343 kinetics of, 327–328 mitotic (proliferative) phase of, 327 postmitotic phase of, 327 Granulosa cells, of ovarian follicle, 911–912, , , 914–915, , , 921–922 Granulosa layer, of primary ovarian follicle, 911–912, Granulosa lutein cells, 919, 960 –961 Granzymes (fragmentins), 489, 497, Graves disease, 836, 836 Gravity, sensors of membranous labyrinth, 1029 Gray hepatization stage, of pneumonia, 755 Gray matter brain, 421, , 436 –437 spinal cord, 422, , 440 –441 Graying, process of hair, 561 Great saphenous vein, 468, Greater vestibular glands, 939–941 Ground bone, 270 –271 Ground substance of bone, 238 of connective tissue, 176, 193–194 of cytoplasm, 83 of elastic arteries, 459 of eye, 985, 986 of heart valves, 447 of internal ear, 1035 Growing follicle, ovarian, 910, 911 Growth cone, in nerve regeneration, 429 Growth factors. adipose tissue and, 201, 281 bone, 239 endothelial, 454, 459, 938 mast cells and release of, 204 obesity and, 281 ovarian follicle, 913 placental, 938 synthesis of, 204, 454 Growth hormone (GH), 285, 695, 816, 822, 822 Growth hormone cells (somatotropes), 822, 822 Growth hormone releasing hormone (GHRH), 822, 828 Growth plates, 220–221, , 257–258, 272 –273 , 274 –277 Growth stops, in hair growth, 566 Guanosine triphosphate (GTP), 66, 96 Guanylyl cyclase/cyclic guanosine monophosphate (cGMP) system, 817 Gubernaculum, 862, 864, 909 Guillain-Barré syndrome, 406

324

p

p

912 913

914 915

p

f

422

p 423

p

p

p 497

912

f

p p f 469

p

See also specific types

t

221

f

f

p

t

t p

p

p

f

Gustatory sweating, 617 Gut-associated lymphatic tissue (GALT), 505, 521 , 634, 652, 657 , 658, 662 Gut tube, 815 Gynecomastia, 869

H

t

349

352 355 363 p

f

H band of sarcomere, 348, , 351, , , Hair, 538, 560–564, 566 , 586 –587 Hair bundle, 1026, Hair cells of internal ear, 127, 135, 1025–1028, , –1043 hearing function of, 1036 inner, 1034–1035, , ion channels and action potentials of, 1028 outer, 1034–1035, , synapses of, 1028–1029, type I, 1029, type II, 1029, Hair follicles, 538, 560–564, , 569–670, 586 –587 Hair growth, 566 Hair matrix, , 561, Half-channels, 150 Half-moons (demilunes). Demilunes Halo cells, in epididymis, 883 Haploid chromosomes, 90, 102–103, 871–872 Haptoglobin, 694 Hard callus, in bone repair, 265, Hard keratin, 73, 541, 564, 565 Hard palate, 589, , Hashimoto thyroiditis, 836 Hassall (thymic) corpuscles, 513, Hassall’s (thymic) corpuscles, 536 –537 Haustra coli, 660, Haversian canals, 241–242, , , 245, 259, 270 –271 Hay fever, 742 Head (structure) of epididymis, 882, 900 –901 of pancreas, 711 of sperm, 874, Head of body, autonomic innervation of, 420 Healing bone (fractures), 244, 262–265, skeletal muscle, 360–361 wound, 197, 200, 202 , 206, 312, 572 Hearing, 1035–1036,

1028

p

f

p

1035 1037 1035 1037 1029 1029 1029 564 f 560 564 See 590 732

f

f

p

f 1038

p

p

266

661

874

1027 1028, 1042p

513 p 242 243

p

p

266

f

p

p

f p p 443 445 446

f

Hearing loss, 1019, 1022, 1024 , 1027, 1034 , 1039 Heart, 442–450, 474 –475 chambers of, 443, , , circulation of blood through, 443–444, , conducting system of, 365, 444, , 448–449, extrinsic regulation of, 449–450 fibrous skeleton of, 444, , intrinsic regulation of, 448–449 location of, 443 wall of, layers of, 444–446, , Heart block, complete, 448 Heart disease coronary, 452 , 468, 470 hypertensive, 458 rheumatic, 448 valvular, 448 Heart failure, and liver necrosis, 700 Heart rate, 448–450 Heart valves, 443, , 446–448, , , 457, 474 –475 Heartburn, 637 Heat-shock chaperone proteins, 53 Heavy chains of myosin II, 350, of smooth muscle myosin, 367 Heavy meromyosin (HMM), , 351 Heavy metal ions, 21 Heister, spiral valve of, , 708 Helical monomers of intermediate filaments, 72, Helicine arteries, in penis, 890 , 148 , 641, 641 Helicotrema, 1032, Helper T lymphocytes, 316, 492, follicular, 503, 511 in HIV/AIDS, 504 interaction with antigen-presenting cells, 200, 316, 495 thymic education and, Hemangiopericytoma, 464 Hematocrit, 298–299, , 321 Hematoma, fracture, 260, Hematoxylin, 2–3, 5 Hematoxylin and eosin (H&E) staining, 2–3, formalin fixation in, 2–3 loss of tissue components in, 4–5 Hematuria, 779 Heme, 304, , 311, 326 Hemichannels, of osteocytes, 247 Hemidesmosomes, 75, , 145, 152 , 160–161, dermal, 551

445 444 446

443 444 449

445 446

f

f

f

f 446 447

443

p

350 350 707

Helicobacter pylori

1033

f

304

f

495

515 299 266

140

73

f

3

t

160

p

epidermal, 540 structure of, 160, transmembrane proteins of, 161 Hemochromatosis, 694 Hemoglobin, 299, 303–305, breakdown, and jaundice, 313 in diabetes mellitus, 305 disorders of, 305, 306 measurement and indices, 321 prenatal and postnatal, 304–305, Hemoglobin A (HbA), 304, , 306 Hemoglobin A1c (HbA1c), 305 Hemoglobin A 2 (HbA 2 ), 304, Hemoglobin F (HbF), 304–305, Hemoglobin H (HbH) disease, 305, Hemoglobin S (HbS), 306 Hemolysis, 303 Hemolytic anemias, 305, 313 Hemolytic disease of newborn, 304 Hemolytic transfusion reaction, 303 –304 , 313 Hemopexin, 693, 694 Hemopoiesis, 321–331, , bone marrow phase of (fetal), 321 dynamics, embryonic through adult, 321, embryonic, 321, , hepatic phase of, 321, monophyletic theory of, 321–323 transcription factors in, 328 yolk-sac phase of, 321, Hemopoietic stem cell (HSC), 200, 205, 255, 321–323, Hemopoietic stem cell, 482 Hemorrhoids, 665 Hemosiderin, 82, 326, 561 Hemosiderin granules, 694, 700 Hemostasis, 319 Hemostatic plug primary, 319 secondary, 320 Henle layer, of hair follicle, 563 Henle, loop of. Loop of Henle Heparan sulfate, 156, 193 , 313, 775, Heparin, 193 , 204, 308, 313 Heparin-like sulphated glycosaminoglycans, 453–454 Hepatic artery, 695–696, , , 699, Hepatic diverticulum, 692 Hepatic ducts, 695, 707, Hepatic fibrogenesis, 702

161

f

f

304 f f

305 f f 305 305 305 f

f

322 323

f

305

f

f

323

322 323 323

323

f

t

See

t

696 697 711

780

699

322

323 696 699

Hepatic phase, of hemopoiesis, 321, Hepatic portal system, 443 Hepatic portal vein, 443, 695–696, Hepatic stellate cells (Ito cells), , 702 Hepatic stem cells, 707 Hepatic veins, 696, 699 Hepatitis, 701 Hepatitis C, 144 Hepatocyte(s), 170 –171 , 692, 702–705, , 722 –726 Hepatopancreatic ampulla (of Vater), , 708, 711–712, Hepatopancreatic sphincter (of Oddi), , 712 Hereditary elliptocytosis, 303 Hereditary glomerulonephritis, 775 Hereditary spherocytosis, 303 Hering, canals of, , 707 Herring bodies, 825, , 852 –853 Heterochromatin, 6, 88–89, , , 91, 92 Heterotrimeric molecule, 181 Heterotypic binding, 145 Hexagonal network–forming collagens, 183 Hibernation, 290 Hibernoma, 289 High-affinity reuptake, 402–403 High-density lipoproteins (HDLs), 693, 694 High endothelial venules (HEVs), 124, 465–466, 501, , 509–510, , 528 –531 Hilar cells, ovarian, 923 Hilum of kidney, 769, , 802 –803 of lymph node, of spleen, 516 Hinge regions, of urothelium, 796, Histaminase, 313 Histamine, 204, 308, 313, 492 , 568, 647 , 830 Histamine H 2 receptor(s), 643 Histamine H 2 receptor–antagonist drugs, 641 Histiocytes. Macrophage(s) Histiocytosis X, 550 Histochemistry and cytochemistry, 1, 3–13 autoradiography, 1, 3, 10–11, chemical basis of staining, 5–6 acidic and basic dyes, 5–6, 5 aldehyde groups and Schiff reagent, 6 metachromasia, 6 chemical composition of histologic samples, 3–5 enzyme digestion, 6 enzyme histochemistry, 7–8,

p

p

707 707

704 825

p

f

p

770 507

p

p

p p 89 90

f

509

703

p

502

p

797

f

See

11 t

8

t

f

711

hybridization techniques, 9–10 immunocytochemistry, 1, 8–9 Histologic ruler, erythrocytes as, 301 Histology auxiliary techniques in, 1–2 definition of, 1 methods used in, 1–27 Histones, , 88, 90 HIV/AIDS, 504 , 550, 659, 769, 934–935 Hofbauer cells, 934–935, Holocrine secretion, 163, , 567, 582 Homeostasis cell division–cell death, , 104 energy, 280, 281, 283–285 iron, liver and, 693–694, 708 kidneys and, 768–769 loss, and necrosis, 104 plasma and, 299 skin and, 539 water, 425, 542, 825 Homogenization, mechanical, , 13 Homologous chromosomes, 90, , 102, 871–872 Homotrimeric molecule, 181 Homotypic binding, 145 Hordeolum (stye), 1006 Horizontal cells, of retina, 993–994, , 1001 Hormonal mobilization, 285 Hormonal sex, 864, Hormone(s), 163, 816–818, adrenal, 285 adrenal cortex, 840 , 842 adrenal medulla, 840 , 842 altered tissue responses to, 828 anterior pituitary, 821 , 822 autocrine, 815 autocrine control by, 815, bone cells producing, 261–262 candidate or putative, 644 , 647 , 656 circulating, 815 definition of, 816 endocrine control by, 815, endothelial production of, 454 gastrointestinal, 644–645, 644 , , 646 , 656 hepatic action on, 695 hypothalamic-regulating, 828, 828 mechanisms of action, 815, neurocrine, 647 , 656 overproduction of, 827

10

f

934 163

p

103

12 100

993

865

t

817

t

t

f

f

f

t

f

815 815

f

815

t

f 646 t

t

t

pancreatic, 715–718, 718 paracrine, 644 , 647 , 656, 815 paracrine control by, 815, parathyroid, 837–839, 837 pineal, 830, 830 placental, 937–938 posterior pituitary, 824–826, 826 regulation and feedback, 818 sex, 281 female, 909, 926 –927 , 946, 949–951 male, 862, 864, , 869 –870 , 879, skin secretion of, 539 target cells of, 815 thyroid, 285, 818, 831, 832 tropic, 821 underproduction of, 827 Hormone receptors, 817–818, Hormone replacement therapy, 264 , 827 Hormone-response element, 889 Horn cells, anterior, 422 Hospital-acquired pneumonia, 755 Howship lacuna, 249, hsc73, 53 Human adaptive thermogenesis, 289 Human chorionic gonadotropin (hCG), 921, 930, 937–938 Human chorionic somatomammotropin (hCS), 938, 950 Human genome/Human Genome Project, 88 Human immunodeficiency virus (HIV), 504 , 550, 659, 769, 934–935 Human papillomavirus (HPV), 933, 950 Human papillomavirus (HPV) vaccines, 950 Human placental lactogen (hPL), 938 Human recombinant parathyroid hormone, 264 Humoral (cell-mediated) immunity, 207 Humoral (antibody-mediated) immunity, 485 Hunger and satiety, 283–285 Hunter syndrome, 51 Huntington disease, 396 Hurler syndrome, 51 Huxley layer, 563 Hyaline cartilage, 217–223, 225 , 230 –231 articular, 210 , 211 , 217–218, 221–223, , 240, , 272 – 273 bronchial, 743 calcification of, 227, , 255, 274 –277 cells of, 219–220, composition of, 217–218, distribution of components in, , 220, general structure of,

f

t

t

t

815

f

t

f f 865 f f

250

f

880

t

817

f

f

f

f

f

f f

p

f

f

p

p

227 p 219 217 217 217

p

f

t

f

222

220

240

p

hydration of, 218–219 internal remodeling of, 218–219 laryngeal, 736 matrix of, 217–220, , microscopic appearance of, 219–220, , perichondrium of, , 221, 230 –231 , 254 proteoglycans in, 217–218, repair of, 225–226 skeletal development from, 220, , 232 –233 , 254–255, – , 274 –277 tracheal, , 739, , 741–742 Hyaline cartilage model, 254, Hyalocytes, 1004 Hyaloid canal, 1004 Hyaluronan (hyaluronic acid), 193 , 194, 217–218, 224, 238, 982 Hyaluronidase, 874 Hybridization techniques, 9–10 Hybridoma, 8 Hydrocephalus internus, 139 Hydrochloric acid (HCl), 640–644, Hydrocortisone, 845 Hydrogen peroxide (H 2 O 2 ), 64, 704–706 Hydrogen protons, in taste, 595–596 Hydrolases, 47, 58 Hydrolytic enzymes, 48 Hydrophilic polar head, 32, Hydrophobic fatty-acid chain, 32, Hydroxyapatite, 238, 260–261, , 270 , 599, 25-Hydroxycholecalciferol, 693, 695, 769, 769 , 837 Hydroxyl radicals (OH −), 311 Hydroxylysine, 181 Hydroxyproline, 181 Hymen, 938 Hyoid bone, Hyper-IgE syndrome, 488 Hypercellular bone marrow, 332 Hyperhidrosis, 572 Hypermuscular phenotypes, 360 Hyperpolarization, in visual processing, 999, Hypersensitivity reactions, 205 , 314, 492, 492 , 497, 550 Hypertension, 458 , 459 chronic essential, 783 , 844 ophthalmological signs of, 1003 portal, 665, 702 resistant, 794 systemic, 456 Hypertensive heart disease, 458

218 220 220 p 218

257

p 737

p

739

219 220 p

221

p

p

256

t

f

643

33

732

263

33

p

f

603

f

f

f

f f

999 f

256

f

Hyperthyroidism, 836 , 937–938 Hypertrophic cells, 255 Hypertrophic pyloric stenosis, 635 Hypertrophic scar, 202 Hypocellular bone marrow, 332 Hypochlorous acid (HOCl), 311 Hypodermis, 538, 555, 576 , 584 –585 Hypoglossal nerve (CN XII), 596 Hyponychium, 565, , 586 –587 Hypophyseal arteries, 819, Hypophyseal portal veins, 821, Hypophysectomy, 846 Hypophysis. Pituitary gland Hypoprothrombinemia, 693 Hypothalamic diabetes insipidus, 828 Hypothalamic polypeptides, 827 Hypothalamic regulating hormones, 828 , 830 Hypothalamic releasing hormones, 402 Hypothalamic–hypophyseal portal system, 443 Hypothalamohypophyseal feedback loop, 827 Hypothalamohypophyseal portal system, 821, Hypothalamohypophyseal tracts, 818, , 824 Hypothalamus, , 818, , 826 hormone production in, 792 , 827 hunger/satiety and, 284 lactation control by, 950–951 nuclei of, , 824 pituitary gland regulation by, 819 , 827 Hypothyroidism, 266 , 836, 836 Hypovolemic shock, 465 I band, of sarcomere, 348, , 351 Idiopathic Parkinson disease, 398

f

567

See

I

p p p p 821 821 f

815

820

p

f

818

f

821, 827

819

f

349

t

f

f

f

See

iExM. Iterative expansion microscopy Ikaros family of transcription factors, 330 Ileocecal junction, 651 Ileocecal valve, 635 Ileum, 634, 651, 684 –685 . Small intestine Image segmentation, 23 Immature bone, 242–243, Immature centriole, 78, , Immature Schwann cells, 403, Immediate hypersensitivity reaction, 205 , 492 Immotile cilia, 129, 135 Immotile cilia syndrome, 139

p

p See also 244 78 79 404 f

f

f

Immune memory, 484 Immune response. Immunity Immune system, 482–501. Immunity cells of, 206–207, 485–501 overview of, 482–485 physiologic role of, 482 Immunity, 483–484 alimentary canal and, 657 antibody-mediated (humoral), 207, 485 cell-mediated, 207, 484–485 digestive system and, 589 endothelial cells in, 454, 456 lamina propria in, 634 mucosal, 733 nonspecific (innate), 483–484 primary response, 492 respiratory system and, 730 responses to antigens, 492–493 saliva and, 617 secondary response, 492 secretory substances in, 483 skin and, 539 small intestine in, 652, 657 , 659 specific, 484–485, 492 sperm-specific antigens and, 879 Immunizations, 492 Immunocompetent cells, 314, 483 Immunocytochemistry, 1, 8–9 Immunogen, 483 Immunoglobulin(s), 6, 8, 299, 488, 490 in intestinal mucosa, , 659 superfamily, 146, , 308 Immunoglobulin A (IgA), 490 antisperm antibodies, 884 colostrum, 949 intestinal, 657 , 658–659, salivary, 591, , 617, 657 Immunoglobulin A (IgA) nephropathy, 780 Immunoglobulin D (IgD), 316, 489, 490 Immunoglobulin E (IgE), 204, 314, 490 , 492 , 742 Immunoglobulin G (IgG), 490 , 492, 495–496, 521 Immunoglobulin M (IgM), 316, 489, 490 , 492, 658 Immunologic surveillance, 487, 659 Immunoperoxidase method, 9 Immunosuppressive agents, 498 Impacted cerumen, 1019 Implantation, 930–932 Implantation window, 931

See

See also f

t

f

146

f

t

658 t

f 616

658 f

t

t t t

f

f

Importin, 96 Impregnation of oocyte, , 920 Impulse conduction cardiac, 444, nerve (action potential), 395, 414 In situ hybridization, 9–10 In vitro fertilization (IVF), 922 Inactive osteoblasts, 246 Inactive osteoclasts, , Incisors, 597, Includes Cytoplasm, 35 Inclusion(s), 28, 29, 81–82 crystalline, 82 crystalloid, 64 functions and pathologies of, 31 glycogen, 82 hemosiderin, 82 lipid, 82 lipofuscin, 81–82 microscopic features of, 30 Inclusion bodies (of Charcot-Böttcher), 877 Inclusion-cell (I-cell) disease, 51 Incus, 1018, 1020–1022, Indifferent stage, of gonad development, 864, Indirect (secondary) bone healing, 262, Indirect immunofluorescence, 9, , Induced pluripotent (iPS) cells, 167 Inferior (lower) esophageal sphincter, 635 Inferior parathyroid glands, 837 Inferior segment, of hair follicle, 561 Infertility, 879 , 930, 951 Inflammatory kidney diseases, 787 Inflammatory lymphadenitis, 518 Inflammatory response (process), 484, 492 allergic reaction, 205 in bone (fracture) repair, 264 cells of, 200, 206, 250, 312, 313, 484 hydrocortisone in, 845 mediators of, 204, 205 in necrosis, 104 obesity and, 287 resolution of, 200 wound repair, 202 , 312 Infoldings, epithelial, 153, , 162, Infundibulum of hair follicle, 560 of pituitary gland, 819, , 824 of uterine tube, , 923

917

445

f

245 251

599

t

t

f

1023

9 10

f

f

f

f

f

266

f

f

909

153

820

163

865

870

Inhibin, , 879, 919 Inhibitory synapses, 400–401 Initial segment, of axon, , 395, Innate (nonspecific) immunity, 483–484 Inner cell mass, 930 Inner collar of Schwann cell cytoplasm, 405 Inner enamel epithelium, 600, – Inner limiting membrane, of retina, , 994, 1002, 1012 –1013 Inner medulla, renal, 770, Inner mesaxon, 405, Inner mitochondrial membrane, 62, , Inner nuclear layer, of retina, , 994, 1001, 1012 –1013 Inner nuclear membrane, , 94 Inner plexiform layer, of retina, , 994, 1001–1002, 1012 –1013 Inner spiral tunnel, of ear, 1035, Inner stripe, of renal medulla, 770–771, Inositol 1,4,5-triphosphate (IP 3 ), 370, 594, 818 Inotropic effect, 450 Insoluble mucus, 642 Insulin adipose tissue regulation by, 283–285 Alzheimer disease association with, 718, 718 female reproductive action of, 921 hepatic action on, 695 pancreatic secretion of, 712, 716, 718 posttranslational processing of, 719 stimulation by, 716–717, 719 Insulin (insulin-like) growth factor(s) (IGFs), 716–719, 718 , 816 adipose secretion of, 281, 286 Alzheimer’s disease association with, 718 bone, 244, 252, 266 deficiencies of, 695 in female reproductive system, 921 placental production of, 938 production of, 695 Insulin-glucose transporter (GLUT) receptor complex, 45 Insulin-like growth factor I (IGF-I), 695, 718 , 921, 938 Insulin-like growth factor II (IGF-II), 718 , 921, 938 Insulin-like protein 3 (INSL3), 868 Insulin resistance, 287 Insulinoma, 719 Integral membrane proteins, 32–35, , , , 48, 302 Integrase strand transfer inhibitor, 504 Integrative neurons, 390, Integrin(s), 37, 107, 145–146, , 160–162, – , 308, 510 Integrin receptors, 156, 250, Integumentary system, 538–575. Hair; Nail(s); Skin

390

771

406

93

395

601 602 993

62 63 993 993 1035

p

p

771

p

p

p

p

f

f

t

f

f

f

f

391

t

33 34 35 f

146 253 See also

f

160 161

748

750 751

p

p

p

p

Interalveolar septum, 747, , 749, , , 752, 766 –767 Interatrial septum, 443, 446 Intercalated cells, of collecting ducts, 788–789 Intercalated discs, 361–362, , , 382 –385 , 474 –475 Intercalated ducts of pancreas, , 713, 713–713, , 726 –727 of salivary glands, 609, 613, 628 –631 Intercalated neurons (interneurons), 390, , 420, 993 Intercellular space, 147, 149, 153 Intercostal nerves, 951 Interdigitation of basal processes, 784, Interdigitations, epithelial, 153, Interference microscope, 16 Interferon(s), 483 Interferon γ (IFN-γ), 316, 330 , 488, 500 Interleukin-1 (IL-1), 309, 330 , 499 , 845 Interleukin-2 (IL-2), 239, 250, 330 , 488, 499 , 845 Interleukin-3 (IL-3), 309, 328, 330 , 499 Interleukin-4 (IL-4), 313, 330 , 498, 499 Interleukin-5 (IL-5), 330 , 499 Interleukin-6 (IL-6), 330 , 499 Interleukin-7 (IL-7), 330 , 499 Interleukin-8 (IL-8), 308, 330 , 499 , 816 Interleukin-9 (IL-9), 330 , 499 Interleukin-10 (IL-10), 330 , 499 Interleukin-11 (IL-11), 330 , 499 Interleukin-12 (IL-12), 330 , 499 Interleukin-13 (IL-13), 313, 330 , 499 Interleukin(s), 200, 204 in diapedesis, 308 in hemopoiesis, 328, 330 in lymphocyte activation, 498 in phagocytosis, 309 secretion/production of, 281, 309 Interlobar arteries, of kidney, 793 Interlobular arteries, of kidney, 793, Interlobular ducts bile, 707 pancreatic, 713 Interlobular mammary ducts, 978 –979 Interlobular veins, of kidney, 794, Interlobular vessels, of liver, 697–678 Intermediate (transitional) zone, of articular cartilage, , 223 Intermediate cell layer, of urothelium, 795–796 Intermediate cells, 658 Intermediate filament(s), 30, 72–75 abnormalities in, 80 in anchoring junctions, 141, 146

362 364 p 713 p p p 391

712

784

153

t

t t t t

t

t t

t

t t t

t t t t t

t

t t t

p p

t

t t

t

t

t

t

794

p

f

p 794

222

t t 75

characteristics of, 81 classes of, 73–75, 74 distribution of, 75, formation of, 72, in macula adherens, 149 microscopic features of, in nuclear lamina, 93–94 in Sertoli cell, 877 in smooth muscle, 368–369 Intermediate filament proteins, 72–73, 81 Intermediate line of desmosome, 149 Intermediate mesoderm, 863 Intermediate muscle fibers, 346 Intermediate nephrons, 774 Intermembrane space, mitochondrial, 62, , Internal acoustic meatus, , , 1036, 1037 Internal anal sphincter, 635, – , 690 –691 Internal capsule, of muscle spindle, 359, Internal ear, 1018, , 1023–1039, , 1042 –1043 blood vessels of, 1039 bony labyrinth of, , 1023–1025, development of, 116, 1018 hearing function of, 1035–1036, innervation of, 1036–1038, membranous labyrinth of, 1025, 1025–1035 sensory (hair) cells of, 127, 1026–1030, , , , 1042 –1043 hearing function of, 1036 inner, 1034–1035, , ion channels and action potentials of, 1028 outer, 1034–1035, , synapses of, 1028–1029, type I, 1029, type II, 1029, stereocilia of, 127, 1026–1027, , , 1036 supporting cells of, 1034–1035, Internal elastic membrane, 450, 457–459, , , 478 –479 Internal hemorrhoids, 665 Internal os, of cervix, , 925, 968 –969 Internal remodeling of bone, 259 of cartilage, 218–219 Internal root sheath cuticle, 563 Internal root sheath, of hair, , 563, Internal thoracic artery, 951 Internal urethral orifice, 798 Internal urethral sphincter, 798 International system, for dentition, 597 –598

73

73

t

1019 1019

1038

p

62 63 p p 360 1025 p 1025 1038

1019 1021 665 666

1035 1037 1035 1037 1029 1029 1029 909

p

1027 1028 1035

1026 1028 1037 459 460 p p

560

564

f

f

p

p

p

391

Interneurons, 390, , 420, 993 Internodal segment of myelin, 407, 412 Interphase, of cell cycle, 97, Interphase (nondividing) cell, 92 Interplexiform cells of retina, 993, 1001 Interstitial cells of kidney, 789–791 of pineal gland, 828, 854 –855 of testes, 57, 82, 123, 174 –175 , 864–865, , – valvular, 447–448 Interstitial cell–stimulating hormone (ICSH), 870 Interstitial fluid, 300 Interstitial gland, of ovary, 923 Interstitial growth, 224–225, 230 –231 , 254 Interstitial lamellae, 242, , , 270 –271 Interterritorial matrix, of cartilage, 220, Intertubular dentin, 607 Interventricular septum, 443–444, , , 446 Intervertebral disc, 223, , 236 –237 Intervillous space, 972 –973 Intestinal basement membrane, Intestinal glands, 652, , 678 –681 of duodenum, 678 –679 of pylorus, 678 –679 of small intestine, 678 –685 Intestinal microvilli, 126–127 Intestinal stem cells, 658, 659 Intestines. Large intestine; Small intestine Intracellular attachment plaque, 161, Intracellular cement, 139 Intracellular cytoskeletal proteins, 4 intracellular membranous components, 6 Intracellular microcompartments, 29 Intracellular receptors, 37, 818 Intracellular space, 113 Intracranial pressure, 1002 Intraepithelial lymphocytes, 316 Intraflagellar transport (IFT), 137–139, Intrahepatic bile ductule, , 707 Intrahepatic cholestasis, 707 Intralobular collecting duct, of mammary gland, 947, Intralobular ducts pancreatic, , 713, 726 –727 sublingual gland, 630 –631 Intralobular stroma, of mammary glands, 947, Intraluminal vesicles, of multivesicular bodies, 46, 48 Intramembranous bone, 254, 278 –279 Intramembranous ossification, 252–254, ,

97

p

p

p

p

p 242 244

865 867 868 f

p

p

443 444 224 p p p p 154 652 p p p p p p p p

See

161

138

704

712

p 220

p

p

p

p

947

p

p

947

254 255

Intramembranous particle strands, 142 Intramural part, of uterine tube, 923 Intraocular pressure, 990 , 991 Intraperiod lines of myelin sheath, 405 Intratesticular ducts, 880, Intrinsic factor, 306 , 641, 641 , 644–645 Intrinsic laryngeal muscles, 736 Involucrin, 543, Iodide, 833 Iodide/chloride transporter, 833, Iodination of thyroglobulin, 833 Iodine, 834 Iodine-deficiency goiter, 836 Iodopsin, 998 Iodothyronine deiodinase, 835 Ion(s) heavy-metal, 21 nucleocytoplasmic transport of, 96 Ion channels, 38, 400–401. activation of, 818 mechanically gated, 1028 in taste, 595–596, Ion concentration, 791 Ionotropic receptors, 401, 402 Iridocorneal angle, 987, Iridotomy, 990 Iris, 981, , , 983, , 987–988, , , , 1010 –1011 Iron homeostasis, 693–694, 708 Iron overload, 694 Iron-storage complex, 82 Irregular bones, 240 Irregular dense connective tissue, 178–179, , 210 –211 , 417 Ischemia in menstrual cycle, 929 in muscle, 350 , 358 Ischemic heart disease, 470 Islet cells, 716, 716 , , 717 , 719 , 726 –727 Islets of Langerhans, 174 –175 , 644 , 712, 715–718, , , 726 –727 Isodesmosine, , 190 Isogenous groups, 219, 236 –237 isoSTED. Isotropic stimulated emission depletion (isoSTED) microscopy Isotropic bands, of sarcomere, 348, , 351 Isotropic stimulated emission depletion (isoSTED) microscopy, 20 Isthmus of fundic gland, 639, of hair follicle, 561

f

544

881

f

f

833

f

See also specific channels

595

f 981 982

988 983

t

987 988 989 178

f

p

See

p 189

t 717 p

f

p

p

t

f

f

p

349

640

p

p

p

p

p

715 716

p

of thyroid gland, 830 of uterine tube, , 923 of uterus, 925 Iterative expansion microscopy (iExM), 13, Ito cells, 463, , 702

909

699

J

f

f

13

Jaundice, 313 , 561 , 708, 981 Jejunum, 651, 682 –683 . Small intestine Jet lag, 830 Job syndrome, 488 Joint(s), 239, 241 Joint cavities, 232 –233 , 274 –275 Joint deformity, 217 Joint diseases, 241 Joint injury, 223 Joint pain, 217 Junctional adhesion molecule (JAM), 143 , 144 Junctional complexes, 124, 140–153, , anchoring, 141, 145–149, 152 communicating, 150–153, 152 gallbladder, 709, lens, 1003 nasal cavity, occluding, 141–145, 152 pancreatic, 713 as pathogenic target, 148 renal, 784 Sertoli cell–to–Sertoli cell, , 877–879, types of, 140–141, 149–150 Junctional epidermolysis bullosa, 162 Junctional epithelium, 608 Juxtaglomerular apparatus, 780, 780–784 Juxtaglomerular cells, , 843–844 Juxtamedullary nephrons, 774 Juxtanuclear region, 362

p

f

p See also

p f f

734

p

p

t

710

t

p

t

f

t 140 141

867

774

K

K-Ras protooncogene, 667f

Kallikrein-related serine peptidases, 541 Kartagener’s syndrome, 80 , 139 Karyokinesis, 98, 99 Karyolysis, 92 Karyorrhexis, 92 Karyosomes, 88, 877,

878

f

f

878, 879

f

Karyotype, 90–91, 92 Katanin, 68 Keloid, 202 Keratan sulfate, 193 , 217–218, 238, 985 Keratin(s), 73–74, 74 , 539–541, 564 in epidermal water barrier, 543, hard, 73, 541, 564 soft, 541 Keratin-associated proteins (KAPs), 564 Keratin filaments, 540–541, Keratinization, 541, 564, 568 Keratinized epithelium, 113, 589–590, , 620 –621 Keratinocyte atrophy, 572–573 Keratinocytes, 539–540, 541–544, desquamation of, 541–542 keratin production by, 541 melanin distribution to, 544–545, water barrier formation by, 541 Keratogenous zone, 564 Keratohyalin granules, 513, , 540, 541, , 636 Ketone bodies, 695 17-Ketosteroid reductase (17KSR), 846 Kidney(s), 768–794, 802 –809 absorptive function of (reabsorption), 784–786 ADH action in, 791, 791 , 825 basal lamina of, , 159–161 blood supply to, 792–794, capsule of, 769, cilia of, 135, , 136 cortex of, 770–771, , , , 802 –805 countercurrent exchange system in, 770, 792 countercurrent multiplier system in, 791 development of, 771, endocrine function of, 769 epithelial tissue of, 170 –173 excretory function of, 768 filtration apparatus of, 768, 775–780, fundamental unit of (nephron), 771, 771–775, 804 –805 hilum of, 769, , 802 –803 histogenesis of, 117 histophysiology of, 791–792 homeostatic function of, 768 interstitial tissue and cells of, 791 lobes and lobules of, 771 lymphatic vessels of, 794 medulla of, 770–771, , , 802 –803 , 808 –809 nerve supply to, 794–795 nomenclature for structures of,

f

t

t

544

544

542

590

p

p

547

514

p

590

p

f

158 794 770 135 t 770 771 773 772

770

p

p

p

p

775

p

770 771

p

p 771

p

p

p

p

p

reabsorption in, 785–786 renin–angiotensin–aldosterone system of, 781–784, 783 , 843– 844 structure of, 769–784, , urine flow from, 795 vitamin D regulation by, 769, 769 Kidney disease chronic, 769 end-stage, 769, 769 inflammatory, 787 thrombotic glomerular, 775 Kidney failure, 346 Kidney transplantation, 795 Kinesin II, 137, Kinesins, 69, , 396–397 Kinetochore, 99, 103 Kinocilium, of internal ear, 1026, , , KLK5, 541 KLK7, 541 KLK14, 541 Köhler illumination, 15 Krabbe disease, 51 Krause, glands of, , 1007 Krause’s end bulb, Krebs cycle. Citric acid cycle Kulchitsky cells, 732, 740–741 Kupffer cells, 463, 498, 699–700, , , 722 –726

770 773

f

70

f

f

138

1026 1028 1032

f

See

f

1005 556

f

699 705

L

p

p

L-3,4-dihydroxyphenylalanine (L-DOPA), 545 L cones, of retina, 995, 1000 L-DOPA (L-3,4-dihydroxyphenylalanine), 545 l -Dopa, 425 L-selectin, 510 L-tyrosine, 545 λ granules, platelets, 318, 319–320, Labia majora, 908, 939, Labia minora, 908, 939 Labial glands, 608 Labyrinthine artery, 1039 Labyrinths, cortical, of kidney, 770, 804 –805 Labyrinths, of internal ear. Bony labyrinth; Membranous labyrinth Lacis cells, 780 Lacrimal canaliculi, 1007, Lacrimal glands, , 1007–1008,

f

319

939

p

See

105

1007

1007

p

1007 1007

Lacrimal puncta, 1007, Lacrimal sac, 1007, Lactating mammary gland, 163, 826, 946, 949, , 978 –979 Lactation, 946, 947, 950–951, 951 Lactational amenorrhea, 951 Lacteal, 652, Lactiferous duct, , 947, , 949 Lactiferous sinus, , 947 Lactose, 648 Lactotropes, 822, 822 Lacunae bone, 239, , 247, 247 cartilage, 217, 230 –231 of endometrium, , 928 trophoblastic, 933 Lamellae of bone, 241–242, , , 270 –271 of elastic arteries, 457, , 459 of heart valves, 447 lipid, 543 of myelin sheath, 405 Lamellar bodies, 543, , 748, , 886–889 Lamellar bone, 241–242 Lamellipodia, 72 Lamin(s), 74 , 75, 94 Lamin A, 74 , 75, 94 Lamin-associated proteins, 94 Lamin B, 74 , 75, 94 Lamin B receptor (LBR), 75, 94 Lamin C, 74 , 94 Lamin receptors, 94 Lamina-associated polypeptides, 75 Lamina cribrosa, 1012 –1013 Lamina densa, 154–155, 159, . Basal lamina of glomerular basement membrane, 778–779 Lamina fusca, 986 Lamina lucida, 155 Lamina propria, 167 , 178, 502, , 632, , 634 of anal canal, of esophagus, 636, 670 –671 of gallbladder, 709, , 724 –725 of large intestine, 662–663, 664 , 686 –687 of nasal cavity, 732, 733, 758 –759 of oral cavity, 590 of seminal vesicles, 904 –905 of seminiferous tubules, 866, 896 –897 of small intestine, 652, 658, 680 –683 of stomach, 651, 672 –673

652

f

242

f

946 946 t 926

951

f

947

f p

p

242 244 p 457 544

t t t

p

750

t

p

p

f 666

160 See also

p 709

p

p

p p

p

p

502

p

633

f

p p

p

p

p p p

p

p

p

of testes, 866 of trachea, 741, , 762 –763 of urothelium, , 797, 799 of vagina, 939 Lamina rara externa, 778 Lamina rara interna, 778 Lamina vitrea, 991, Laminin(s), 154, 156–157, , , 162, 197 in extracellular matrix, 195, in glomerular basement membrane, 775, 779 Laminin-332, , 162 Laminin receptors, 155 Laminopathies, 94 LAMPs. Lysosome-associated membrane proteins Langer lines, 552 Langerhans cells, 200, 485, 498, 549–550, Langerhans, islets of, 174 –175 , 644 , 712, 715–718, , , 726 –727 Langerin (CD207) molecule, 550 Large (elastic) arteries, 189–190, 214 –215 , 451 , 456–460, , 476 –477 Large granular lymphocytes (LGLs), 316, 489 Large intestine, , 660–666, 686 –691 absorption by, epithelial cell renewal in, 661–662 lamina propria of, 662–663, 664 , 686 –687 of large intestine, 686 –691 lymphatic vessels in, 663, 664 mucosa of, 660–661, muscularis externa of, 372, 663, 686 –689 muscularis mucosae of, , 686 –691 reabsorption and elimination by, 660 serosa of, 663 submucosa of, 663 Large veins, 451 , 465, 467, , Laryngeal cartilage, 736 Laryngeal epithelium, 738, , 760 –761 Laryngeal muscles, 736 Laryngitis, 737–738 Laryngopharynx, Laryngotracheal diverticulum, 730 Larynx, 731, , , 736–738, , , 760 –761 elastic fibers of, 189–190 Laser hair removal (LHR), 566 Laser photocoagulation, 992 Latch state, of smooth muscle, 371 Late endosomes, 44–48 Late lipoblasts, 282,

741 796

p

993

p

158 160 196

161

t

See

p

459

p

p

p

p

p

p

633 589

549

f

p

715 716

p

t

457,

p

f p p p p f 662 p p 662 p p

t

467 468 738 p

732 731 732

f

282

f

737 738

p

p

p

Late myoblasts, 360–361 Late-phase allergic reaction, 205 Late spermatids, Lateral incisor, 597 Lateral lobes, of thyroid gland, 830 Lateral plications of gallbladder, 709 of small intestine, 654 Lead citrate, 21 Leading edge, of cell, 72 Learning, dendritic spines in, 393 Lecithin, 708 Left atrium, , 444, , Left hepatic duct, 707 Left ventricle, , 444, , Left ventricular hypertrophy, 458 Left–right asymmetry of internal organs, 136 Lens capsule, 1003, , 1016 –1018 Lens epithelial cells, 1003, Lens equator, 1003, Lens fibers/lens fiber cells, 1003–1004, , 1016 –1018 Lens nucleus, 1003, Lens, of eye, 116, 980, 982, , 1003–1004, , 1016 –1018 Lens, of microscope, 14, 20, Lens placode, 983, Lens vesicles, 983 Leptin, 281, 283–285, 286 , 287 , 938 Leptotene, , 102 Lesser vestibular glands, 939 Leukocyte(s). buffy coat, 299, classification of, 305 development of, 321, , , 327–331, 329 granules of, 305 Leukocyte (white blood cell) count, 320, 298–299, 299 , 305–317, 336 –339 . Leukopoiesis, 321 Leukotriene(s), 204, 313, 817 Leukotriene C (LTC 4 ), 204 Leukotriene D (LTD 4 ), 204 Leukotriene E (LTE 4 ), 204 Leukotriene receptor antagonists, 204 Levator palpebrae superioris muscle, 1005, Levodopa, 425 Lewy bodies, 398 Leydig cells, 57, 82, 123, 174 –175 , 865–869, , , 896 – 897

f

878

443

100

p

p

444 446 443 444 446 f 1003 p p 1003 1003 1003 987 21 983 t f

See also specific types 299 322 324

1003

1003

t

p See also specific types

f

p

p

p

p

t

1005

p

p

867 868

p

865

development of, 863, 864, , 868 testosterone secretion by, 868 tumors of, 868–869 LGPs. Lysosomal membrane glycoproteins Ligamenta flava, 189 Ligaments, 179–180, 189–190, 212 Ligamentum nuchae, 184 Ligand(s), 36–37, 44–45 Ligand-gated Ca 2+ (calcium) channels, 370 Ligand-gated channels, 401 Ligand-gated ion channels, 38 Ligand–receptor complex, 44–45, Light cells, of collecting ducts, 788, 789 Light chains of myosin II, 350, of smooth muscle myosin, 367 Light meromyosin (LMM), , 351 Light microscope, 1, 13–14, alignment steps for, 15 artifact with, 16 examination of histologic slide with, 14–16, proper use of, 15 –16 resolving power of, 14, 14 , 15 Light sheet fluorescence microscopy (LSFM), 18, Light source, of microscope, 14, Limb girdle muscular dystrophy (LGMD), 353 Limiting membranes, of retina, , 994, 1000, 1002, 1012 –1013 LIMPs. Lysosomal integral membrane proteins − Lin cell surface marker, 321 Lineage-restricted progenitors, 323 Linear acceleration, 103 Lines of Retzius, 599, Lingual glands, 570, 608, 624 –625 Lingual muscles, 591 Lingual papillae, 591, 593, Lingual salivary glands, 592 Lingual tonsils, 505, 589, , 596 Lining mucosa, 589–590 Lining tissue, of bone, 241 Link proteins, 194 Linker proteins, 35 Lip(s), 590, , 620 –621 Lipases, 285, 713 Lipid(s) deposition of, 285 formation of, 717 hepatic metabolism of, 694–695

See

p

45

350 350 21 f

f

f

t

See

599

590

p

p 593 592

p

f 21 993

p

17

f

18

p

p

metabolism in brown adipose tissue, 289 mobilization of, 285 probarrier, in epidermis, 543 β-oxidation of, 62, 64, 695, 704 Lipid bilayer, of plasma membrane, 32, 35 Lipid droplets, 280, 282, 285–286, 717 Lipid envelope, of epidermis, 543, Lipid inclusions, 30 , 31 , 81–82, 282 Lipid lamellae, 543 Lipid metabolism, 57 Lipid oxidases, 58 Lipid-processing enzymes, 543 Lipid rafts, 32–33, , 35, 145 Lipid storage diseases, 82 Lipoblasts, 282, Lipofuscin, 50, 81–82, 868 Lipofuscin granules, 50 Lipogenesis, 717 Lipolysis, 718 Lipoma, 289 Lipoproteins, 44, 454, 456 , 692–693, 694 , 847 , 915 Liposarcoma, 289 Liquor folliculi, 881–882 , 72 Listeriosis, 72 Littré, glands of, 799 Liver, , 692–708, 722 –726 acini of, 696, 697, , 722 bile flow from, 695, , 705–707 bile production by, 692, 695, 708 blood supply to, 695–696, , 697–698, cells of, 699–705, 722 –726 . Hepatocyte(s) cirrhosis of, 80 , 702 congestive heart failure and, 700 degradation in, 694 development of, 692 endocrine functions of, 692–693 epithelial tissue of, 170 –171 exocrine functions of, 692 fetal, blood islands in, 702 glucocorticoids in, 844–845 gross anatomy of, 692, hormone modification by, 695 inflammation of, 702 innervation of, 708 iron and copper in, 693–694 lipoprotein synthesis and regulation by, 692–693, 694 lobules of, 696–698, , 722 –723

t

282

f

33

f Listeria monocytogenes 633

544

t

t

f

p 697 699

f

f

p p

696 699 p See also f

p

p

p

693

697

p

p

f

703

lymphatic pathway in, 702, metabolic conversions in, 695 necrosis of, 700 parenchyma of, 696, 697–698 perfusion of, reduced, 697 perisinusoidal spaces of, 696, , 700–701, physiology of, 692–694 primary cilia of, 136 regeneration of, 703 sinusoids of, 696, , , 699, , stem cells of, 707 structural organization of, 696–701 vitamin storage and metabolism in, 693, 702 Liver failure, 694 Liver injury, 700 Liver transplantation, 694 Lobar bronchi, 730, 743, Lobe(s) of kidney, 771 of liver, 692, of lung, 743 of pituitary gland. Anterior lobe of pituitary gland; Posterior lobe of pituitary gland of secretory acini, 609 of thyroid gland, 830 Lobule(s) of kidney, 771 of lacrimal gland, 1007 of liver, 696–698, , 722 –723 of lung, 743 of mammary glands, , 976 –977 of pancreas, of pineal gland, 828 of secretory acini, 609 of testes, 865 of thymus, 236 –237 , 512 Lochia rubra, 937 Locomotion, of cells, 72 Long bones, 239, , , blood supply to, 242, ground section, 270 –271 growth of, 255–258 Long-term weight regulation, 283–285 Longitudinally oriented layer, of muscularis externa, 634–635 Loop domains, 90, Loop of Henle, , 774, 786–787, 791–792 Loose connective tissue, 114, , , 178, , 210 –211 Loricrin, 543,

f

699

t 696 697

f

702

699 704

754

693

715

See

697 946

p p

p p

p p f 239 240 242 243 p p

773 544

90

114 177

179

p

p

f

f

Low-density lipoprotein(s) (LDLs), 454, 693, 694 , 847 , 913, 915 Low-density lipoprotein–receptor complex, 45 Low-frequency stimuli, 559 Low-resistance junctions, 150 Lower esophageal sphincter, 635 Lumican, 985 Luminal compartment, of seminiferous epithelium, 878, Lung(s), 747–753 acini of, 743 alveoli of, 730, , 743, 747–752, , , , 764 –767 air–blood barrier of, 749, , , 766 –767 cells of, 747–748, , 750–752, , , 766 –767 epithelium of, 747–748 surfactant of, 748–749, 766 blood supply to, 731, 752 bronchopulmonary segments of, 730, 743 development of, 730 epithelial tissue of, 170 –171 innervation of, 754 lobes of, 743 lobules of, 743 lymphatic vessels of, 754 Lung bud, 730 Lunula of nail, 565 Lupus nephritis, 780 Luschka, ducts of, 708 Luteal cells, 918–919, Luteal gland (corpus luteum), , 918–919, Luteal phase, of ovarian cycle, 926 –927 Lutein cells, 960 –961 Luteinization, 919 Luteinizing hormone (LH), 816, 821, 821 , 823 in female reproductive system, 913, 914, 921, 926 –927 in male reproductive system, 870 , Luteinizing hormone (LH) receptors, 913 Luteinizing hormone cells (gonadotropes), 822 , 823 Luteotropins, 921 Lymph, 469–471, 470, 505, 509, 703 Lymph nodes, 470, 482, , , 506–511, 521 , 528 –531 lymphocyte circulation in, 508–509, mammary gland drainage, 951 reticular meshwork of, , structure or architecture of, supporting elements of, 506 Lymphadenitis, 511, 518 Lymphadenopathy, 518 Lymphatic capillaries, 464, , 469–471 Lymphatic channels, , 508–509, , 528 –531

878

731

747 748 754 750 751 p p 750 752 p p

749

p

919

p

p

p

910

f

f

918

f

t f 880

483 503

f 507

p p

507 508 507 465

509

f

f

p

p

t

t

509

p

p

p

See

Lymphatic follicles. Lymphatic nodules Lymphatic nodules, 482–483, 502–504, , , 521 , 528 –531 aggregations of, 505, 589, 634, 652, , 658 in alimentary canal, 634 in appendix, 688 –689 in intestines, 505, , 634, 652, , 658, 684 –685 primary, 502 secondary, 502 single (solitary), 505 Lymphatic organs, 501 major, comparison of, 521 primary or central, 482, , 491 secondary or peripheral, , 491–492 Lymphatic sinuses, , 509, , 528 –531 Lymphatic system, 482–485, 501 cells of, 482–483 comparison of major organs of, 521 components of, 482–483, organs and tissues of, 482–483 overview of, 482–485 Lymphatic tissue, 482–485, 501–506 bronchus-associated, , 505, 521 , 741 diffuse, 482–483, 502–504, , 634 functions and features of, 521 gut-associated, 505, 521 , 634, 652, 657 , 658, 664 in lamina propria, 634 in liver, 703, mucosa-associated, , 501, 505, 521 oral cavity, 589 Lymphatic vessel hyaluronan receptor-1 (LYVE-1), 471 Lymphatic vessels, 442, 464, 469–471, 480 –481 , , 501 in adrenal glands, 741 afferent, 501, , 506, , in alimentary canal, 634 efferent, 501, , 506, , , 530 –531 in eye, 992 histogenesis of, 116 in kidney, 794 in large intestine, 664, 664 in lungs, 754 in mammary glands, 951 in ovaries, 923 Lymphoblasts, 503 Lymphocyte(s), 176, , 299 , 314–317, , 482–485.

p

503 507 653

p 506

653

t 483 483 509

507

703

502 t

t

483

502 502

p

p

p

p

p

p

t

483

483

t

t

f

t

507 509 507 509

p

p

p 483

p

f

177 specific types

t

315 See also activation of, 484, 491–492, 494–498, 494 –495 , 506 antigen-specific, 506 on blood smear, 315, 338 p –339 p

p

p

514

blood–thymus barrier protecting, 236 –237 , 514–516, circulation of, 488, 501, cluster of differentiation (CD) molecules of, 206, 485, 486 – 487 connective tissue, 197, 206–207, 210 –211 development and differentiation of, , 330–331, 482, 491–492 differences from other leukocytes, 314 distribution in lymph nodes, 508–509, effector, 483, 492 functional classification of, 487 functional types of, 315–316 heterogeneity of, 206–207 in HIV infection, 504 immune memory of, 484 immunocompetent, 483 immunologic surveillance by, 487 in inflammatory response, 206, 312 in lymph nodes, 508–509, in lymphatic nodules, 502–504 migration of, 509–510 origin of names, 490 origins of, 314 in specific (adaptive) immunity, 484–485 specificity of, 484 splenic, 517–518 structure of, 315, Lymphocytes, mammary gland, 948, 949, 976 –977 Lymphocytic infiltration, 613 Lymphoepithelial Kazal-type inhibitor (LEKT1), 541–542 Lymphoid progenitor cells, 314, , 323, 330–331 Lymphokines, 200 Lymphopoiesis, 330–331 Lysine residues, 185 Lysis, 104, 489 Lysobisphosphatidic acid, 48 Lysosomal acid hydrolases, 313 Lysosomal enzymes, 251 Lysosomal hydrolases, 251 Lysosomal integral membrane proteins (LIMPs), 48, 60 Lysosomal membrane, 48, Lysosomal membrane glycoproteins (LGPs), 48 Lysosomal pathway, of thyroid hormone synthesis, 834 Lysosomal storage diseases (LSDs), 50, 50 –51 Lysosome(s), 7, 29, 48–53, 60 autophagy in, 50–53, , biogenesis of, 48, drugs affecting, 49 endosomes becoming, 44–48

502

t

p p 322 509

f

509

f

315

p

p

322

49

49

50 52

f

f

t

entosis by, 107 functions and pathologies of, 31 hepatocyte, 703 in leukocytes, 305 in macrophages, 50, 199–200, 500 as major digestive compartment, 48 microscopic features of, 30 in neutrophils, 50 in osteoclasts, 251 pathways of delivery to, 48–49, primary, 48 secondary, 48 structure of, 48, Lysosome-associated membrane proteins (LAMPs), 48 Lysosomotropic agent, 49 Lysozyme, 616, 656

t

t

50

49

M

f

M (microfold) cells, 652, 657, 657 M cones, of retina, 995, 1000 M line, 348, , , M line proteins, of sarcomere, 351, M Phase, of cell cycle, , 98 Mitosis M-Protein, 351, Macroautophagy, 52, , 548 Macrophage(s), 176, , 196, 199–200, 500 activation of, 488, 498–501, in alimentary canal, 634 in allergic reactions, 200 alternatively activated (M2), 200, 500 alveolar, 750–752, antigen presentation by, 200, 317, 498 in apoptosis, 200 in atherosclerosis, 452 in blood–thymus barrier, , 516 classically activated (M1), 200, 500, development of, 199–200, 317, , 329 in elastic arteries, 460 in endoneurium, 417 fusion with foreign bodies, 200 immune functions of, 483, 484 in inflammatory response, 200, 202 , 312, 484, 492 lymph node, 506–507 lysosomes of, 50, 199–200, 500 microscopic features of, , 200 myelin debris cleared by, 426

349 352 363 97 352 52 177

f

352 . See also 501

752

f

514

501 t

322

f

199

origin of, 199–200 perisinusoidal (Kupffer cells), 463, 699–700, , , 722 – 723 phagocytic activity of, 199, 312, 750–752, 884 placental, 934–935, , 972 –973 renal, 790 resident, 427 septal, 750, surface proteins of, 200 thymic, 513, 516 Macrophage metalloelastases, 187 Macropinocytosis, 39, Macropinocytotic vesicles, 453 Macropinosomes, , 40 Macula adherens, , 145, 149, 152 , Macula densa, , 774, , 781, 806 –807 Macula lutea, , 1002, Macula of saccule, 1026, , 1030 Macula of utricle, 1026, , 1030, Macula pellucida, 916 Maculae adherentes, 362–363 Macular degeneration, 997 age-related, 992 Macular edema, cystoid, 997 Macular holes, 997 Macular pucker, 1002 Macular translocation, 992 Magnesium (Mg2+) in actin polymerization, 70 in microtubule polymerization, 66 MAGP-1, 192 Main (primary) bronchi, 730, 731, , 739, 742–743, Main pancreatic duct (of Wirsung), , 711–712, , 713 Main stem villi, 933, Major basic protein (MBP), 313 Major calyces, 770, , 795, 802 –803 Major dense lines, 405 Major duodenal papilla, 708 Major histocompatibility complex (MHC), 492–493, 498, antigen presentation on, 200, 485, 492, 493, , 498, dendritic cells and, 506 Langerhans cell expression of, 550 MHC I, 46, 494, MHC II, 46, 200, 316, 492, 495, , 498, recycling of, 46 Major histocompatibility gene complex, 492 Malaria, 49 Male gametes. Sperm; Spermatogenesis

699 702

p

934

p

p

754

39

39 140 773 982

t 364 p p

774 1002 1026 1026 f

f f

1031

f

f

936 770

731 707

p

711

p

495

495

See also

754

495

500

500 500

p

863

p

p

Male reproductive system, 862–907, , 896 –906 accessory sex glands of, 862, 884–889 cilia in, 135 components of, 862 excurrent duct system of, 862, 875, 880–882, , external genitalia of, 862, 890–891 hormonal regulation of, 869 –870 , 879, hormones of, 862, 864, intratesticular ducts of, 880, overview of, 862 puberty in, 868 spermatogenesis in, , 102, 862, 865, 869–875 Male urethra, 799, Malignant cells, 91–93 Malignant melanoma, 552 Malleus, 1018, 1020–1022, Mallory bodies, 80 Mallory staining technique, 5 Malpighian corpuscles (splenic nodules), 516, Mamillated areas, of stomach, 638 Mammalian target of rapamycin (mTOR), 52, 718 Mammary fat pad, 281 Mammary glands, 538, 908, 946–951 adipose tissue of, 281, blood supply to, 951 cancer of (breast cancer), 94 , 946 cells of, 826, 947, , 976 –979 connective tissue of, 210 –211 cyclic (menstrual) changes in, 947, 949–951 early proliferative stage, 978 –979 hormonal regulation of, 946, 949–951 inactive, 946, 947, , 976 –977 innervation of, 951 involution of, 951 late proliferative stage, 978 –979 lymphatics of, 951 male, development of, 869 oxytocin action in, 826, 946, 951 pregnancy changes in, 948–949, Mammary papilla (nipple), 946–947, , Mammary ridges, 946 Mammillary body, Mammotropes (lactotropes), 822, 822 Manchette of acrosome phase, 872, Mandible alveolar process of, 607–608 ossification of, 278 –279 Mandibular division, of trigeminal nerve, 596

865

863

f

f

f 881

880

881 882

100 f

1024

516

946

947

p

948

p

f

p

p p

p

p

p

p

p

948 946 947

820

t 874

p

p

Mannose-6-phosphate (M-6-P), 47, 58–59 Mantle dentin, 606 Mantle zone, of lymphatic nodule, 503 Mantoux (tuberculin) screening, 498 Marfan syndrome, 159, 192 Marginal chromatin, 88 Marginal sinuses, 518 Marginated pool, of neutrophils, 328 Maroteaux-Lamy syndrome, 51 Marrow cavity, 232 –233 , 240, , 241, 332 Mast cell(s), , 196, 200–204, activation of, 488 in allergic reactions, 204, 205 , 314, 492 , 742 basophil , 201 connective tissue (MC TC ), 200–201 in endoneurium, 417 granules of, 204 immune function of, 483, 485 mucosal (MC T ), 201 origins of, 201, 314 secretory products of, 204 staining properties of, 200 Mast cell progenitors (MCPs), 314, 200 Master switch, in sex determination, 863 Masticatory mucosa, 589–590, Mastoid air cells, 1019, 1023 Mastoid antrum, 1019 Mastoid process, 1019 Mastoiditis, 1023 Maternal sinusoids, 933, Math1 transcription factor, 166, 659 Matrilysins, 187 Matrix bone, 238–239, 245–246 cytoplasmic, 83 elastic cartilage, 223 enamel (tooth), 600 extracellular ( Extracellular matrix) fibrocartilage, 224 hair, , 561, hyaline cartilage, 217–220, , , 230 –231 , 255, 274 – 275 mitochondrial, 62, nail, 565, , 586 –587 nuclear, 90 pericentriolar, of MTOC, 75, Matrix cells, 561,

177

p

p

versus

f

t

240 201 f

f

f

p

p

590

934

560 p

See 564

567

62 p

564

218 220

p

76

p

Matrix Gla-protein (MGP), 239 Matrix granules, 62, Matrix metalloproteinases (MMPs), 186–187, 193 , 247, 251 Matrix vesicles, 245, 259–261, Maturation model, of endosomes, 44–45 Maturation phase, of spermiogenesis, 872–874 Maturation promoting factor (MPF), 98 Maturation-stage ameloblasts, 600, 601–604, Mature bone, 241–242, , Mature centriole, 78, , Mature teratoma, 118 Maxilla, alveolar process of, 607–608 Mean corpuscular hemoglobin (MCH), 321 Mean corpuscular hemoglobin concentration (MCHC), 321 Mean corpuscular volume (MCV), 321 Mean platelet volume (MPV), 321 Measles virus (MV), 147–148 Mechanical homogenization, , 13 Mechanical protection, by epithelial tissue, 124 Mechanical stress, on skin, 550 Mechanically gated ion channels, 38, 1028 Mechanoelectric transducer (MET) channel protein, 1028–1029, , 1036 Mechanoelectric transducers, 1026–1027 Mechanoreceptors, 135, 550, 559, 1026–1027 Mechanosensitivity, 161 Mechanosensors, 129 Mechanotransduction system, 198, 247 Meckel’s cartilage, 278 –279 Medial (central) incisor, 597 Medial border of kidney, 769 Medial-Golgi network, 58, Median eminence, 819, Mediastinum, location of heart in, 443 Mediastinum of testes, 865, , 898 –899 Mediators of inflammation, 204, 205 Medium (muscular) arteries, 451 , 456, , 460–461, , 478 – 479 Medium veins, 451 , 465, 466–467, 478 –479 Medulla of adrenal gland, 842–843, , , 858 –861 . Adrenal medulla of hair, , 564 of kidney, 770–771, , , 802 –803 , 808 –809 of lymph node, 507, of ovary, 909 of thymus, 236 –237 , , 513, Medullary (marrow) cavity, 232 –233 , 240, , 332

62

f

f

263

604

242 244 78 79

12

1028

p

820

p

t

560

p

p

60

866

t

f

p

p 842 844

770 771 507 p 512 p

p

513 p

p 459

460

p p

p

p See also

p

240

p

p

841

Medullary arterioles, adrenal, 840, Medullary capillary sinusoids, of adrenal glands, 840 Medullary collecting duct of kidney, 772, , 775, 788–789 Medullary cords of lymph node, 508 Medullary pyramids, of kidney, , 771, , 802 –803 Medullary rays (of Ferrein), 770, , , , 804 –805 Medullary sinuses of lymph node, , 508, , 530 –531 Medullary thyroid carcinoma, 831 Medullary vascular network, of kidney, 794 Megakaryoblast, 326–327, 329 Megakaryocyte(s), 317, , 326–327, 329 , 338 –339 , 517 Megakaryocyte-committed progenitor (MKP) cells, 323, 326–327, 329 Megakaryocyte/erythrocyte progenitor (MEP) cells, , 323, 326– 327, 329 Megalin, , 834 Meibomian (tarsal) glands, 163, , 1006 Meibomian glands, 568 Meiosis, 90, 102–103 in females, , 102 in fertilization, 920 in males, , 102 mitosis , molecular motor proteins in, 69 nuclear events of, 102 in oocyte growth and maturation, 914, 917–918, in oogenesis, phases of, , 102 in spermatogenesis, 869, 871–872, , Meiosis I, 102, 871, Meiosis II, 102, 871, Meiosis-specific cohesion complexes, 102 Meissner corpuscles, 555, Meissner’s corpuscles, , 584 –585 , 946 Meissner’s plexus, 634, 636, 650, 676 –677 Melanin, 544–548, , 561 , 578 –579 Melanocortin 1 receptor (MC1R), 547 Melanocyte(s), 544–548, , 561 , 578 –579 , 988, Melanocyte precursor cells, 544 Melanocyte stem cells, 561 Melanocyte-stimulating hormone (MSH), 824 Melanocyte-stimulating hormone, 547 Melanogenesis, 544 Melanoma, 552 Melanosome microparasol, 547 Melanosomes, 546, 561 Melatonin, 829 Membrana granulosa, 911–912 Membrane, 167 .

773 770 773 p p 770 771 773 p p 507 509 p p

318

t

833

t

t

p

p 322

1005

100 100 versus 100 100

917

873

871 871

559 556 p 547 f p 546 f f

f

871 873 p p p p

f

f See also specific membranes

p

p

989

t

104

Membrane blebbing, 36, 104, Membrane bone, 254 Membrane-bound vesicles, 163 Membrane-coating granules, 543 Membrane-initiated steroid signaling, , 818 Membrane phospholipid protein complexes, 4 Membrane proteins, 302 Membrane transport, 37–44, Membrane zone, of platelets, 319, Membranoproliferative glomerulonephritis, 780 Membranous discs, of photoreceptors, 996–997 Membranous labyrinth, 1025–1035 Membranous organelles, 29–65. Membranous urethra, 799 Memory axons in, 396 dendritic spines in, 393 Memory cells, 492, , 497, 511 Menarche, 908 Ménière’s disease, 1039 Meninges, 201, 421, 423–424, , Menopause, 908–909, 951 Menstrual cycle, 908, 925, 926–960, 926 –927 , beginning of, 926 lactation and, 951 mammary gland changes in, 949 menstrual phase of, 928–930 ovarian changes in, 908, 926 –928 , 928–930 proliferative phase of, 928 secretory phase of, 928, 966 –967 vaginal changes in, 938 Menstrual flow, 926, 929–930 Menstrual phase, of menstrual cycle, 928–930, Menstruation, 929–930 corpus luteum of, 919, 928–929 definition of, 926 Meridional portion, of ciliary muscle, 989 Merkel cell(s), 550 Merkel cell carcinoma (MCC), 550 Merkel corpuscle, 550, Merocrine secretion, 163, , 949 Meromyosin, , 351 Mesangial cells, , 780, Mesangium, 780–781, Mesaxon, 405, Mesenchymal cells in endochondral ossification, 255 in eye, 983

817

38

319

See also specific organelles

496

f

423 424

f

f p

f

f 929

f p

929

350

406

556

774

781

163 781

256

p

in intramembranous ossification, 252–254, , 278 –279 Mesenchymal stem cells, 205 adipocytes from, 282, , 288 in endochondral ossification, 255 osteoprogenitor cells from, 243–245, smooth muscle cells from, 372 undifferentiated, 463–464, 465 Wharton’s jelly, 177 Mesenchyme, 177, , 207, , 846 Mesentery, 214 –215 , , 635 epithelial tissue of, 170 –171 Mesoderm, 116–117, 176–178, 863, 982–983, 984 chorionic, 933 derivatives of, 116–117, Mesodermal epithelium, 863, Mesodermal mesenchyme, 839 Mesometrium, Mesonephric (Wolffian) duct, 880–882, , 885 Mesonephric tubules, 880–882 Mesosalpinx, Mesothelial cells, 170 –171 Mesothelioma, 98 Mesothelium, 117, 124, 167 , 170 –171 , 635 Mesovarium, 909, , Messenger RNA (mRNA), 54, MET channels, 1028–1029, 1036 Metabolic stress, 454 Metabotropic receptors, 401, 402 Metachromasia, 6, 93 Metachronal rhythm, of cilia, 134–135 Metalloelastases, macrophage, 187 Metalloproteinases, 307 matrix ( Matrix metalloproteinases) in neutrophil granules, 307 Metamyelocyte, , 327, 329 Metaphase, , , 103, 917–918 Metaphase I, Metaphase II, Metaphase plate, 101, 103 Metaphase spread, 90 Metaphysis, , 232 –233 , 240, 272 –273 Metaplasia cervical, 933, epithelial, 126 respiratory system, 745 Metarteriole, 465, Metastatic cancer, 107 Methods, 1–27

282

245

178 820 p p 633 p

p

t

117 864

909

881

909

p

909 910

p

f 55

p

p

t

See

324 100 101 100 100 39

p p 933 f f 465

t

p

p

p

auxiliary techniques, 1–2 histochemistry and cytochemistry, 1, 3–13 autoradiography, 1, 10–11, chemical basis of staining, 5–6 chemical composition of histologic samples, 3–5 enzyme digestion, 6 enzyme histochemistry, 7–8, hybridization techniques, 9–10 immunocytochemistry, 1 microscopy, 1, 13–25 artifact in, 16 atomic force, 1, 24–25, bright-field, 14, confocal scanning, 8, , 19, dark-field, 16 electron, 1, , 20–24 expansion, , , 13, fluorescence, 17–18 instrument components in, interference, 16 light, 1, 13–14, 15 –16 nonoptical, 24 phase contrast, 16 polarizing, 19 resolving power in, 14, 14 , 15 super-resolution, 1, 20 ultraviolet (UV), 18 virtual, 1, 25, tissue preparation, 2–3 frozen sections, 3, 4 hematoxylin and eosin staining with formalin fixation, 2–3, linear equivalents used in, 2 other fixatives used in, 3 Methylation, in posttranslational modifications, 36 MHC I-related proteins (MR1), 488 Microautophagy, 52, Microbodies. Peroxisome(s) Microcirculation, 442, , 465, Microcirculatory bed, 442, , 465 Microfold cells. M (microfold) cells Microglia, 389, 409, , 413, CNS scarring and, 428 phagocytosis by, 203 , 413, 428 retinal, 993 Microphthalmia-associated transcription factor (MITF), 544 Micropinocytosis, 39, Micropinocytotic vesicles, 453 Microscopic anatomy, 1

11 8

21

8

11 10 12

24

13

f

f

19

21 t

25

52

See

See

f

450

412 f f 39

f

t

450

465

413

3

Microscopy, 1, 13–25 artifact in, 16 atomic force, 1, 24–25, bright-field, 14, confocal scanning, 8, , 19, dark-field, 16 electron, 1, , 20–24, expansion, , , 13, fluorescence, 17–18 instrument components in, interference, 16 light, 1, 13–14, 15 –16 , nonoptical, 24 phase contrast, 16 polarizing, 19 resolving power in, 14, 14 , 15 super-resolution, 1, 20 ultraviolet (UV), 18 virtual, 1, 25, Microtome, 2 Microtrabecular strands, 83 Microtubule(s), 30, 65–69 abnormalities in, 80 astral, 76, 99 in axons and dendrites, 395–396, , 397 characteristics of, 81 in cilia, 132–134, , , 137 depolymerization of, 66–67, drugs targeting, 80 dynamic instability of, 67–68, functions of, 66 immunocytochemistry of, kinetochore, 99 microscopic features of, 68–69, , in mitosis, 99 mixed polarity, 397 molecular motor proteins associated with, 69, nucleation activity of, 66, polar, 99 polymerization of, 66–67, selective stabilization process of, 68 Sertoli cell, 877, structure of, 66, triplet, 75–79, , 132, visualization of, 68–69 Microtubule-associated proteins (MAPs), 67, 81 , 396 Microtubule catastrophe, 68 Microtubule-organizing center (MTOC, centrosome), 30, 65, 66–68

21

11 10 12

8

24

19

21 13 21 f f 21 t

f

25

f

t 133 134 68 f 10

878 67 78

395

68

68 69

70

67 67

133

t

f 77

abnormalities, and cancer, 83 axonal and dendritic, , 396 basal body formation by, 76, , 79, 128–129 ciliogenesis in, 78–79, , 137–139 duplication of, 78–79, mitotic function of, 77, 99 structure of, 76, Microtubule-severing protein, 68 Microvascular bed, 442, , 465 Microvesicles, 36 Microvilli, 126–128, , , 130 , 634 of epididymis, 883, 900 –901 formation of structural core, 72 of gallbladder, 709 of intestines, 126 of kidney, 126–127, 784, , 786 of liver, , 700–701, 706 of nasal cavity, of small intestine, 634, 651 Micturition control, voluntary, 799 Micturition reflex, 799 Midcortical nephron, 774 Midcycle pain, in women, 923 Middle ear, 1018, 1019–1023, bones of, 1018, 1019–1023, development of, 1018 infection of (otitis media), 1023 Middle mediastinum, 443 Middle piece, of sperm, 872, , 875 Middle plate of internal root sheath, 563 Middle stage, of T cells differentiation, 514 Midstage lipoblasts, 282, Mifepristone, 931 Milk (deciduous) teeth, 597, 597 –598 Milk ejection, 947 Milk lines, 946 Milk production, 949 Milk secretion, 946 Mineral(s), bone storage of, 238 Mineralization, 243, 259–261, , , , 606 Mineralized nodules, 261, Mineralocorticoid receptors, 789 Mineralocorticoids, 840 Minor calyces, , 796, 802 –803 , 808 –809 gene, 561 Mitochondria, 29, 61–64 apoptosis initiation by, 64 ATP generation in, 62,

395 79 79

76

450 127 128 t p p

699

784

734

1019 1025

874

282

Mitf

f

770

t

263 p

63

f

f

261 262 263 p

p

p

363

in cardiac muscle, 362, condensed configuration of, 64 evolution of, 62 functions and pathologies of, 31 in gallbladder, 709 loss of function, 104 microscopic features of, 30 , 62, , 64 morphologic changes in, 64 orthodox configuration of, 64 in smooth muscle, 358–359 in sperm, , 875, 920 structure of, 62, thermogenesis and, 289 Mitochondrial defects, 63 Mitochondrial DNA, 61 Mitochondrial porins, 62 Mitogen-activated protein kinases (MAPKs), 108 Mitosis, 95, , 98 in cardiac muscle, 366 centrioles in, 77, 79 chromosome formation in, 90, 99 in epidermis, 540, 544 in erythropoiesis, 326 in gastric mucosa, 646–649 in granulopoiesis, 327 meiosis , molecular motor proteins in, 69, in oogenesis, , 921 phases of, 99, , in spermatogenesis, , Mitotic activity, in cells, 96–97 Mitotic catastrophe, 98, 105, 106 Mitotic spindles, 76–77, , 98, 99, Mitral valve, , , 446–448, , , 474 –475 Mitral valve disease, myxomatous, 448 Mittelschmerz, 923 Mixed acini, 609, , , 626 –627 Mixed polarity microtubules, 397 Mixed spicule, 255, 276 –277 Mobilization, of adipose tissue, 285 Modified fluid–mosaic model, , 35 Modiolar vein, common, 1039 Modiolus, 1025, , 1042 –1043 Modulation, of ameloblasts, 601 Mohs, Frederic E., 556 Mohs micrographic surgery (MMS), 556 –558 Molar glands, 608 Molar teeth, 597

t

t

874

62

62

97

versus 100 873 100 101 871 873 443 444

77

70

101 446 447

609 612 p p p p 33 1033 p p f f

p

f

p

p

p

Molecular layer, of cerebellum, 438 –439 Molecular motor proteins, 69, Moll, glands of, 568, , 1007 Monoamine neurotransmitters, 402 Monoamine oxidase (MAO), 403 Monoblast, 329 Monocarboxylate transporter 10 (MCT10), 834 Monocarboxylate transporter 8 (MCT8), 834 Monocilia (primary cilia), 129, 130 , 135–137, , 136 , Monoclonal antibodies, 8, 9 , 264 Monocyte(s), 196, 299 , 317, , 338 –339 in atherosclerosis, 452 cells derived from, 199–200, 203 , 312, 317, 329 , 427–428, 699 in connective tissue, 207 development of, , 329 , 330 immune function of, 483, 484 Monocyte chemotactic protein-1 (MCP-1), 281 Monocyte colony-stimulating factor (M-CSF), 250, 328, 330 Monocyte progenitors (MoP or CFU-M), 323, 329 , 330 Monocytes, 199 Monoiodotyrosine (MIT), 834 Mononuclear phagocyte system (MPS), 38, 203 , 317, 699–700 Monophyletic theory of hemopoiesis, 321–323 Monorefringent bands in sarcomere, 348 Mons pubis, 908, 939 Montgomery glands, 946 Morning sickness, 921 Morula, 930 Motile cilia, 129–135, 130 axonemal organization of, 132 basal bodies anchoring, 132, , mechanism of movement, 132–134 movement of fluids and particles, 131 synchronous pattern of, 134–135 Motilin, , 646 , 656, 717 Motor (efferent) nerve fibers, 359, Motor (efferent) neurons, 390, , , reestablishing contact with muscle, 429 skeletal muscle, 356–359 somatic, 390, 416 ventral, 422 visceral, 390, 418, Motor domain, of myosin filaments, 350–351 Motor nerve endings, 555 Motor unit, 358 fast-twitch, fatigue-prone, 347 fast-twitch, fatigue-resistant, 347 slow-twitch, fatigue-resistant, 346–347

70

1005

t

t

f

322

f

317

t

t

f

t

f

p

135

p

t

t

f

t

646

t

133 134

t

418

360 390 391 392

t 137

t

Mounting medium, 2 Movement disorders, 44 Mucin-secreting glands, 709 Mucinogen granules, 164, 610, 638, 642 Mucociliary escalator, 739 Mucocutaneous junction, , 620 –621 Mucolipidosis II, 51 Mucosa (mucous membrane), 612, 632–634, 636 of alimentary canal, 588–589, 632 of bronchus, 743 of cervix, 968 –969 of esophagus, 633–634, , , 670 –671 of gallbladder, 709–711, , 724 –725 of gastrointestinal tract, 633–634 of large intestine, 660–661, of nasal cavity (olfactory), 730, , 733, , 742 , 758 –759 of oral cavity, 589–591 of prostate gland, 885 of rectum and anal canal, 664–665 of respiratory system, 732–733, 745 of seminal vesicles, 904 –905 of small intestine, 652–663, of stomach ( Gastric mucosa) of trachea, 739, of uterine tube, 924, 960 –961 of vagina, 938, 974 –975 Mucosa-associated invariant T (MAIT) cells, 316, 488 Mucosa-associated lymphatic tissue (MALT), , 501, 505, 521 Mucosal fold, of uterine tube, 960 –961 Mucosal glands, 634, 636–637 Mucosal immunity, 733 Mucosal inflammation, 742 Mucosal mast cells (MC T ), 201 Mucous acini, 510–511, 609, , 626 –627 , 630 –631 Mucous cells of gallbladder, 724 –725 of salivary glands, 609, , 610–611, , 630 –631 of stomach, 638–642, , , 646–650, 672 –673 , 676 –677 of trachea, 739–740, , , 762 –763 Mucous connective tissue, 177–178, Mucous glands, 164, Mucous membrane, 167 Mucous neck cells, , 641–642, 650, 676 –677 Mucous secretions, 164 Mucous surface cells, 638–639, , , 646–649, 672 –673 , 676 –677 Mucus, 636–637, 641, 655

590

f

p

See

p

p

p

636 637 p p 709 p p 662 732 734

739 p

p

p 652

p p

p

p

166 f 640

p

612

p 609 639 640 740 741

p

p

f

p

f

f

483

p

p

p 178

639 640

t

p

p

p

612 p p p p p p

p

p

p

p

p

p

insoluble or cloudy, 642 soluble, 642 visible, 639 Müller cells, 403 Müllerian-inhibiting factor (MIF), 864, , 879 Müller’s cells, 993–994, , 999–1000 Multiadhesive glycoproteins in bone matrix, 238–239, 245 common, 195, , 197 in extracellular matrix, 193 in glomerular basement membrane, 775 in hyaline cartilage, 217, 218, 230 –231 Multicellular glands, , 164, 165 Multilocular adipose tissue. Brown adipose tissue Multiple myeloma, 8 Multiple sclerosis (MS), 406 , 511 Multiplexins, 183 Multipolar neurons, 390, Multipotent adult progenitor cells (MAPCs), 205 Multivesicular bodies (MVBs), 36, 44, 46 Mumps, 616 Muscarinic ACh receptors, 401, 402 , 798 Muscle cells, 28, 71, 114–115. basal lamina in, 155, smooth muscle, 366–367, , , 372, 386 –387 Muscle contraction, 344 cardiac muscle, 365, , 448 skeletal muscle, 346, 351, 355–356 actomyosin cross-bridge cycle in, 353–355, , 359 neuromuscular transmission in, , 357–359, power stroke in, 355 recovery stroke in, 355 regulation of, 355–356 summary of events in, smooth muscle, 367–372, , 2+ Ca -mediated, 370–371 chemical stimuli of, 370 electrical depolarizations in, 370 mechanical impulses in, 370 Muscle creatine phosphatase (MM-CK), 351 Muscle cushions, 468 Muscle fascicle, 345, , , 376 –377 Muscle fibers of cardiac muscle, 362, , of myotendinous junction, 380 –381 of skeletal muscle, 345–347, , , , 376 –379 bundle or fascicle of, 345, ,

865

993

196

t

163

391

f

See

t

p

p

t See also specific types 155 367 368 p p 365 354 356 357 359 369 371

346 347 p p 363 364 p p 345 346 348 346 347

p

p

color in vivo, 346 contractile speed of, 346 enzymatic velocity of, 346 fast glycolytic (type IIb), 347 fast oxidative glycolytic (type IIa), 347 metabolic profile of, 346 slow oxidative (type I), 346–347 structural and functional subunit of, 347–351, types of, 346–347, of smooth muscle, 366, Muscle spindles, 359, , 417 Muscle tissue, 112, 114–115, , 344–375. classification of, 344–345 comparison of types, 372 –373 contraction of ( Muscle contraction) histogenesis of, 116 metabolism and ischemia of, 350 , 365–366 overview of, 344–345 primary cilia of, 136 striated, 114–116, , 344–346, 348, , 555, 622 Muscle-wasting diseases, 360 Muscular (medium) arteries, 451 , 456, , 460–461, , 478 – 479 Muscular dystrophy, 94, 351, 353 , 360 Muscular vein, 468 Muscular venules, 451 , 465, 466 Muscularis of bronchi, 743 of ureters, 810 –811 of urinary bladder, 812 –813 of uterine tubes, 924, 960 –961 of vagina, 974 –975 Muscularis externa, 632, , 634–635 anal canal, 665 contractions of, 635 esophageal, 636, , 670 –671 gallbladder, 709–711, , 724 –725 gastric, 372, 650, 674 –677 large intestine (colon), 372, 663, 686 –689 small intestine, 386 –387 , 660, 680 –685 Muscularis mucosae, 167 , 632, , 634 esophageal, 636, 670 –671 gastric, 650, 672 –673 , 674 –677 gastroduodenal junction, 678 –679 large intestine, , 686 –691 small intestine, 652, , 680 –685 Myasthenia gravis, 358 , 403 , 498, 750–752

347

347 366 360

115 f f

See

f

t 115

t

p

t

p

p

p

p

See also specific types

p

633

p

459

f

p

355

p 460

p

636

p p 709 p p p p p p p p f 633 p p p p p p p p 662 p p 653 p p f Mycobacterium tuberculosis

p

p

p

Mydriatic agents, 988 Myelin basic protein (MBP), 405 Myelin debris, clearance of, 426 Myelin oligodendrocyte glycoprotein (MOG), 412 Myelin sheath, 116, 404–406 of central nervous system, 412 diseases associated with, 406 , 412–413 of peripheral nervous system, 405–406, , , , Myelin-specific proteins, 405 Myelinated axons, 404–405, , 414 Myelinated Schwann cells, 404 Myelinating phenotype, 403 Myelinating Schwann cells, 403–404, Myelination, 404–405, , Myeloblast, , 327, 329 Myelocytes, , 327 Myeloid progenitor cells, , 323, 326–327, 329 Myeloperoxidase (MPO), 306–307, 310–311, Myenteric plexus, 635, 636, 651, 660, Myf5 negative adipocytes, 291 Myoblasts, 345, 360 Myocardial infarction (MI), 365–366, 470 Myocardial sleeves, 467 Myocardium, 384 –385 , 444–446, , , Myoclonic epilepsy with ragged red fibers (MERRF), 63 MyoD transcription factor, 360 Myoepithelial cells, 372 of apocrine glands, 569–570, 580 –581 of eccrine glands, , 569, , 580 –581 of iris, 988, of mammary glands, 826, 947, , 976 –977 , 976 –979 of salivary glands, 611–613, Myofibrils of cardiac muscle, 362, 382 –385 of skeletal muscle, , 347–351, , Myofibroblast(s), 197–199, , 202 , 372, 702, 769, 790 Myofibroblast-like cells, of cardiac valves, 448 Myofilaments, 71, 115 of cardiac muscle, 363 of skeletal muscle, 344, 348, 351 Myogenic stem cells, 360 Myoglobin, 346 Myoid cells, of testes, 372, 866, Myoid portion, of photoreceptors, 996 Myomesin, 351, Myometrium, , 925–926, , 964 –965 Myosin in platelet structural zone, 318,

f

405 406 407 408

408

324 324

p

568

346

352

310

660

t

f 445 446 447

p

989

909

404

406 407 t 322

p 198

570 949 613

p

p p

p

p 347 352 f

867

925

p

319

p

p p

p

p

rigor configuration of, 355 in skeletal muscle, 349, 350–351, , in skeletal muscle contraction, 346, 353–355, in smooth muscle contraction, 367–371, unbent confirmation of, 354–355 Myosin (thick) filaments, 71, 115, 344 of skeletal muscle, 349, 350–351, , , of smooth muscle, 367–371, , Myosin ATPase reactions, 346 Myosin-binding protein C (MyBP-C), 351, Myosin I, 71, 127, Myosin II, 71, 101, 127, , 344, 350–351, Myosin light chain kinase (MLCK), 370, 454 Myostatin, 360 Myotendinous junction, 380 –381 Myotubes, 360–361, Myxomatous mitral valve disease, 448

350 351 369

354

350 351 368 368 369 352 128 128 350 361

p

p

N

N-Acetylgalactosamine (GaINAc) transferase, 194, 303f N-Acetylglucosamine (GIcNAc) transferase, 194, 303f Na -dependent transporters, 403 Na -phosphate cotransporter protein 3 (NPT3), 261, 263 Na /K -ATPase pumps, 785 Nabothian cysts, 932 , 968 p –969 p Nail(s), 538, 586 p –587 p Nail bed, 568 Nail matrix, 567 , 586 p –587 p Nail plate, 565 Nail root, 565, 567 + + +

+

Nails, 564–566 Naïve B lymphocytes, 503, 509–510 Naïve T lymphocytes, 509–510 Nares, anterior, 731 Nasal cavities, 730, 731–736, cells of, 732–777, 758 –759 conditions affecting, 733, 742 epithelium of, 758 –759 innervation of, 733, 735 mucosa of, , 742 , 758 –759 olfactory region of, 731, 733–736 olfactory transduction in, 734, respiratory region of, 731–733 stem cells of, 732, 735–736 Nasal septum, 731, 732 Nasal vestibule, 731–732,

732

p

p

f

p

731 p f

p

732

p

735

Nasolacrimal duct, 731, 1007 Nasopharynx, 731, , , 736 Natural cytotoxicity receptors (NCRs), 489 Natural killer (NK) cells, 104, 315–316, , 331, 489 activation of, 496, connective tissue, 206 development of, 489 immune function of, 483, 489 NC1 domain of collagen IV, 156–157, , 782 gene, 311 Nebulin, 349–350, Neck of sperm, , 875 Neck of tooth, 599 Neck segment, of gastric glands, 639, Necroptosis, 109 Necrosis, 104, , 105 Necrosis, hepatic, 700 Nectin–afadin complex, 147–148 Nectins, 145, 146–148, Negative chronotropic effect, 450 Negative feedback, 818, 819 Negative inotropic effect, 450 Negative selection, of T cells, Negri bodies, 421 Neonatal line of enamel, 606 Neonatal respiratory distress syndrome (RDS), 748–749 Nephrin, 776 Nephrin gene , 776 Nephrogenic diabetes insipidus, 791 , 792 , 825, 828 Nephron(s), 770, 771–775, 804 –805 distal thick segment of, 772 general organization of, 771–772 intermediate (midcortical), 774 juxtamedullary, 774 proximal thick segment of, 744 subcapsular (cortical), 774 thin segment of, 744 tubules of, 744–746, , , 785–787, 806 –809 Nephrotic syndrome, 776 Nerve(s), 388 cranial, 390, 415 , 596 injury response of, 426–429, , peripheral, 388, 415–416, , 426–429, 434 –435 spinal, 422, Nerve endings encapsulated, 417, 555, , , 584 –585 encapsulated, 584 –585 in female genitalia, 946

731 732

322

496

NCF1

f

874

157

352

104

f

f

640

t

147

(NPHS1)

f

515

f p

p

f

f

773 774

t

416

423

p

p

426 427

556 559

p

p

p

p

p

p

556

p

p

free (nonencapsulated), 417, 555, , 584 –585 , 946 motor, 555 Nerve fascicle, 417 Nerve fibers, use of term, 415 Nerve impulse conduction, 395, 414 Nerve tissue, 112, 115–116, , 388–429. Nervous system composition of, 389 histogenesis of, 116 origins of cells in, 414–415 overview of, 388–389 Nervi vasorum (vascularis), 451, 460 Nervous system anatomic divisions of, 388 functional divisions of, 388–389 nerve cells of. Neuron(s) overview of, 388–389 response to stimuli, 389 secretions of, 389 supporting cells of, 389, 403–414. Neuroglial cells vasculature of, 389 Nestin, 74 , 75, 392 Netherton syndrome, 542 Neural apoptosis inhibitory protein (NAIP), 921 Neural crest, 116, 415, 544–545, 815, 839 Neural crest cells, 403, Neural mobilization, 285 Neural pathways, 388 Neural regeneration, 426, , 429 Neural retina, 981, , 983, , 992–993 Neural stem cells, 391–392, 414 Neural tube, 116, , 409, 414, Neuraminidase, 874 Neuregulin (Ngr1), 407 Neuregulin-1 (Nrg1), 404 Neurilemma, 434 –435 Neurilemmal (Schwann) cells, 115, 357, 389, , , 403–407, 434 –435 cytoplasm of, 407, , injury response of, 426, junction between, 407, , 434 –435 myelin sheath production by, 405–406, myelinating Schwann cells, 403–404, nonmyelinating Remak Schwann cells, 403–404, origin of, 415 regenerative function of, , 429 Neurites, 429 Neurocrine hormones, 647 , 656 Neuroectoderm, 116, 819, , 982–983, 984

116

See also

See

See also

t

404

427

981 117

p

p p

983

839

p

408 409 427 408 p t

390 392

p 407 404

427

820

t

404

117 See

derivatives of, 116, Neuroendocrine cells. Enteroendocrine cells Neuroendocrine system, 818 diffuse, 815 Neuroendocrine system, diffuse (DNES), 644 Neuroendocrine tissue, 389 Neuroendocrine tumors, 644 Neuroepithelial cells, of taste buds, , 624 –625 Neurofibrillary tangles, 80 Neurofilaments, 74–75, 74 Neuroglia, 389, 403–414 central, 389, 409–413, enteric, 389, 409, 420 functions of, 389 interdependence with neurons, 409 origins of, 415 peripheral, 389, 403 Neuroglial cells, 115 Neurohumoral reflex, 826 Neurohypophysis. Posterior lobe of pituitary gland Neuroma amputation, 429 traumatic, 429 Neuromediators, 115 Neuromuscular junction, , 357–359, , 403 Neuron(s), 115–116, 389–403 adrenergic, 401 bipolar, 390, catecholaminergic, 401 cell body of, 115, 390–392, , , 416 communication among, 397–403. Neurotransmitters; Synapse(s) communication among. Neurotransmitters; Synapse(s) development of, 414 functional components of, 390 Golgi type I, 395 Golgi type II, 395 injury response of, 426–429, , integrative, 390, intercalated (interneurons), 390, , 420 intercalated (interneurons), 993 interdependence with neuroglia, 409 life span of, 391–392 motor (efferent), 390, , , reestablishing contact with muscle, 429 of skeletal muscle, 356–359 somatic, 390, 416 ventral, 422

f

f

f

t 412

594

See

356

391

357

392 393 See also See also

391

426 427 391

390 391 392

p

p

418

visceral, 390, 418, multipolar, 390, neurosecretory, 824 nucleus of, 390 postsynaptic, 420 presynaptic, 397, 418–420 pseudounipolar, 390, renewal of, 391–392 retinal, 993 secretions of, 389 sensory (afferent), 389–390, , , 416, 417, 418 dorsal root ganglia, 422–423 somatic, 389–390, 416 visceral, 390, 416, 418 serotonergic, 401 unipolar, 390, Neuron terminals, 359 Neuronal apoptosis inhibitory protein (NAIP), 106 Neuronal transport systems, 392, 396–397 Neurophysin, 825 Neuropil, 421 Neuropsychiatric manifestations, of liver failure, 694 Neurosecretory granules, 550, Neurosecretory neurons, 824 Neurosecretory vesicles, 824 , 650 Neurotensin, 402 Neurotransmitter transport proteins, 402–403 Neurotransmitters, 400–403, 402 , 571, 999, 1028 characterizations of, 402 excitatory, 400–401 gastrointestinal, 644 inhibitory, 400–401 receptors for, 401, 402 reuptake of, 403 Neutrophil(s), , 197, 200, 207, 299 , 306–307, 336 –339 in alimentary canal, 634 bone marrow reserve pool of, 328 on bone marrow smear, 338 –339 chemotaxis of, 309 chromatin of, 306 circulating pool of, 328 development of, , , 327–328, 329 disorders of, 311–312, 311 granules of, 306–307 immune function of, 483 inflammatory response, 200, 202 , 483 lysosomes of, 50

391

391

391 392

391

550

Neurospora crassa

f

177

t

t

t

t

p

322 324

p

p

t

f

f

p

marginated pool of, 328 mature, motility (migration) of, 307–309, nucleus of, 306 phagocytosis by, 200, 309–312, , 492 polymorphonuclear, 306, 327 receptors of, 309 segmented, 327 Neutrophil chemotactic factor (NCF), 204 Neutrophil progenitors (NoP), 323, 327, 329 Neutrophil–endothelial cell recognition, 146 Nexin, 132, Nexus, 372 Nexuses. Communicating junctions NF-κB. Nuclear factor-κB Niches, of stem cells, 166, 205, 564, 658, 662 Nicotinamide adenine dinucleotide phosphate (NADPH) oxidase complex, 310–311, , 311 Nicotinamide adenine dinucleotide–tetrazolium reductase (NADH–TR), 346, Nicotine withdrawal symptoms, 539 Nicotinic ACh receptors, 358, 358 , 401, 402 , 799 Nidogen, 775 Niemann-Pick disease, 51 Night blindness, 693, 998 Nipple, 946–947, , Nissl bodies, 57, , 390–391, – , , 429, 440 –441 in nerve injury, 429 pituitary, 824 Nitric oxide (NO) in digestive system, 635, 639 in intracellular killing, 311 macrophage production of, 200 as neurotransmitter, 401–402, 402 in penile erection, 892 in renal function, 775 in respiratory system, 736 in urinary bladder function, 798 in vasodilation, 454 Nitric oxide synthase (NOS), 401–402, 454, 635 Nitric oxide–cGMP pathway, 370 Nitrosylation, in posttranslational modifications, 36 NK cells. Natural killer (NK) cells Nodal cardiac muscle cells, 448–449 Nodal cilia, 129, 130 , 137 Node of Ranvier, , , 407, , 414, 434 –435 Nodes, of cardiac conducting cells, 365 Nodular calcification, of heart valves, 448

307

309

See See

t

133

310

347

f 946 947 58

f

f

t 390 392

t

390 391 393

p

t

f

See

308

408

p

p

p

f p

Non-nucleoside reverse transcriptase inhibitor, 504 Nonalcoholic steatohepatitis (NASH), 693 Nonencapsulated (free) nerve endings, 417, , 584 –585 , 946 Nonessential amino acids, 695 Nonhistone proteins, 88 Nonimmune globulins, 299, 693 Nonkeratinized epithelium, 113 Nonlamellar bone, 242 Nonmembranous organelles, 30, 65–81. Nonmotile sperm, 875 Nonmyelinating phenotype, 403 Nonmyelinating Remak Schwann cells, 403–404, Nonoptical microscope, 24 Nonshivering thermogenesis, 289 Nonspecific (innate) immunity, 483–484 Nonsteroidal anti-inflammatory drugs (NSAIDs), 639 Nontasters, 596 Norepinephrine, 829, 840 , 841–842 in cardiac regulation, 450 in kidney, 794 as neurotransmitter, 401, 402 in sweat gland stimulation, 571 in urinary bladder, 794 in vasoconstriction, 464–465 Normoblast, , 325, , 329 , 340 –341 Normocellular bone marrow, 332 Nose, external, 731 Nosocomial pneumonia, 755 Nostrils, 731 NSF/α-SNAP protein complex, 43, Nuclear (fibrous) lamina, 93–94, Nuclear bag fiber, 359, Nuclear basket, , 96 Nuclear chain fiber, 359, Nuclear envelope, 92, 93–96 Nuclear export sequence (NES), 96 Nuclear factor-κB (NF-κB), 250 Nuclear ferritin, 985 Nuclear import receptor, 96 Nuclear-initiated steroid signaling, 818, Nuclear lamins, 74 , 75, 93–94 Nuclear layers, of retina, , 994, 1000–1001, 1012 –1013 Nuclear localization signal (NLS), 96 Nuclear matrix, 90 Nuclear pore(s), 92, 93, , 94–96, , Nuclear pore complex (NPC), 95, Nucleation of hydroxyapatite crystals, 261 Nuclei of gray matter, 421, 422

556

p

See also specific organelles 404

f

t

t

324

325

t f

f

360 360

95

t

p

43 93

818

993

93

p

95

94 95

p

p

Nucleocytoplasmic transport, 96 Nucleoid, 66 Nucleolar-associated chromatin, 88 Nucleolonema, 91 Nucleolus, 6, 30 , 31 , 91–93, , 92 morphologic regions of, 91 regulation of cell cycle, 91–93 staining properties of, 93 Nucleolytic enzymes, 713 Nucleophosmin/B23, 78 Nucleoplasm, 92, 96 Nucleoporins (Nup proteins), 95 Nucleoside reverse transcriptase inhibitors (NRTIs), 504 Nucleosomes, 89–90 Nucleostemin, 91–93 Nucleotide probe, 10 Nucleus, 28, 92–105 alterations in dying cell, 92 components of, 92–96 functions and pathologies of, 31 microscopic features of, 30 of neuron, 390 overview of, 92 relationship to rough endoplasmic reticulum, , 92, 93, transport to/from cytoplasm, 96 Nucleus–basal body connectors (NBBCs), 77, Nup (nucleoporins) proteins, 95 Nurim, 75, 94 Nutrient arteries, 242, Nutrient-deprivation antimetabolites, 106 Nutrient foramina, 242,

t

t

91

f

t

t

78

243 243

O

Ob(Lep) gene, 284

f

Obesity, 280, 284–285, 287 , 293 Obesity genes, 287 Objective lens, 14, 20, Obscurin, 351, Occludin, , 142–144, , 143 Occluding junctions, , 141–145, in arteries, 457–459 bicellular, 142 in biliary tree, 705–706 in blood–brain barrier, 410 in blood–nerve barrier, 417 in capillaries, 462,

141

352

f

140

21 142

463

t

143, 152t

88

93

lymphatic capillaries, 471 permeability of, 145 proteins of, 142–144, 143 in semipermeable membrane, 453 in small intestine, 653 strand formation, 142–144 transport across, 144–145, tricellular, 142, , 145 viruses exploiting, 142–144, 148 Octacalcium phosphate, 261, Octamer, 90 Ocular lens, 14, Oddi, sphincter of, , 712 Odontoblasts, , 600, , , 606–607, , Odorant-binding proteins (OBPs), 734, Olfactory epithelium, 733, , , 758 –759 Olfactory glands, 733, , 736, 758 –759 Olfactory mucosa, 730, , 733, , 742 , 758 –759 Olfactory nerve (CN I), 733, Olfactory receptor(s) (ORs), 734, Olfactory receptor cells, 733–735, , , 758 –759 Olfactory region, of nasal cavity, 731, 733–736 Olfactory transduction, 734, Olfactory vesicle, 733, Oligodendrocyte(s), 389, , 409, 412, , 414 Oligodendrocyte myelin glycoprotein (OMgp), 412 Oligonucleotide probes, 9 Omental appendices, 660, OnabotulinumtoxinA, 44 Oncogenes, 106 Oncovin (vincristine), 80 Oocyte(s), , , 909 fertilization of, , 919–921, 924–925 follicular development of, 910, , impregnation of, , 920 maturation of, 913 microenvironment for, 910 primary, 102, 916, release of, , 916–918 secondary, 103, 916–918 Oocyte maturation inhibitor (OMI), 914 Oocyte meiotic arrest, 914, 917 Oogenesis, , 102, , 909, 910, , Oolemma, 913, 920 Open-angle glaucoma, 990 Open canalicular system (OCS), 319, Open circulation, 518, Open enteroendocrine cells, 645,

t

144

142

599

21

707

263

f

603 605 606 607 735 734 735 p p 734 p p 732 734 f p p 734 735 734 735 p p 735 734 390 412 661

100 873 910

100

917 917

f

910 917

917

873

f 521

910 917

645

319

1002

Ophthalmoscopic examination, 988, Opioid peptides, endogenous, 402 Opportunistic pneumonia, 755 Opsin, 998–999 Optic cup, 983, Optic disc, , , , 1002, , 1012 –1013 Optic disc pits, 997 Optic grooves, 982–983 Optic nerve, 980–981, , , , , , 994, 1002, 1010 – 1011 Optic papilla (disc), , , , 1002, , 1012 –1013 Optic sulci, 982–983 Optic vesicles, 982–983, Optical coherence tomography, 994, 997 –998 Ora serrata, , , 989, 1010 –1011 Oral cavity, 589–595, 731, components of, 589 development of, 116 epithelium of, 589–590, mandibular ossification and, 278 –279 roof of, Oral cavity proper, 589 Oral mucosa, 589–591 Oral transmucosal delivery, 590–591 Orbicularis oculi muscle, 1005, Orbit, 980, 1007 Orchiopexy, 864 Orchitis, 870 Organelle(s), 28–30. functions and pathologies of, 31 membranous, 29–65 microscopic features of, 30 movement of, 69, nonmembranous, 29–81 Organelle free zone, of lens, 1003, Organelle zone, of platelets, 318, Organic anion transporting polypeptides (OATPs), 834 Oropharynx, 731, , , 736 Oropharynx ectoderm, 819, Orthochromatophilic erythroblast, , 325, , 329 , 340 –341 Orthogonal array, 180, 212 –213 , 985 Osmium tetroxide, 3, 21 Osmolarity, of body fluids, 825 Osmosensors, 129 Osmotic equilibrating device, 791 Osmotic pressure, colloid, 299 Osseous spiral lamina, 1032, Ossicles of ear, 1019–1023, ,

f

983 982 987 995 1002 f 981 982 983 987 p 981 987 995 983 f 982 987 p p 732 590

590

p

p

p

993 1002

p

p

p

f

p

1005

f

70

See also specific organelles t t

731 732

1003 319

820 324 p p

1033 1019 1024

325

t

p

p

1020

development of, 1018, diseases affecting, 1022, 1024 hearing function of, 1036 muscles moving, 1022 Ossification endochondral, 220–221, , 232 –233 , 254–255, , –277 intramembranous, 252–254, , 278 –279 Ossification centers, 252, 255 primary, 255, secondary, , 257, 274 –275 Osteoadherin, 238 Osteoarthritis, 217 Osteoblast(s), 239, 245–247, , 253 active, 246 in bone repair, 262, 265 communication via gap junctions, 246–247 differentiation of, 245 in endochondral ossification, 255, 274 –277 hormone production by, 262 inactive, 246 in intramembranous ossification, 252–254, , 278 –279 microscopic features of, 245, , , in mineralization, 259–261 staining properties of, 246 transformation to osteocyte, 245 Osteocalcin, 239, 245, 260, 262, Osteoclast(s), 203 , 227, 239, 249–252, 253 activation of, 250–251 active, , apoptosis of, 251 basolateral region of, 251, clear zone of, 250, in endochondral ossification, 276 –277 inactive, , location of, 249, origin of, 249, regulation of, 251–252 resorption function of, 249, 250, , 259, 278 ruffled border of, 250, , , , 276 –277 schematic drawing of, Osteoclast-type phenotype, of chondroclasts 227 Osteocyte(s), 176, 239, , 247–248, 253 , 270 –271 communication via gap junctions, 246–247 in endochondral ossification, 276 –277 functional states of, 247–248, hormone production by, 262 in intramembranous ossification, 254, , 278 –279

221

p

256

256

p

f

f

p

255

p

p

255 256, 274p

p

p

245

t

p

p

255 245 246 252 263

f 245 251

253

245 251 251 251

253

p

p

t

p

251 p 250 252 253 p p 253 245 t p p p p 248 254 p p

p

f

lacunae of, 247, 247 life span of, 248 osteoblast transformation to, 247 response to mechanical forces, 247 Osteocytic osteolysis, 248 Osteocytic remodeling, 247–248 Osteogenic layer, 255 Osteogenic protein-1 (OP-1), 239 Osteoid, 245–246 Osteomalacia, 265 Osteon(s), 241–242, , 259, 270 –271 Osteonal (Haversian) canal, 241–242, , , 245, 259, 270 –271 Osteonectin, 239, 245, Osteopetrosis, 252 Osteopontin, 196, , 197 , 239, Osteoporosis, 186, 252, 259, 263 –264 , 265 , 833 secondary, 263 type I primary, 263 type II primary, 263 Osteoprogenitor cells, 239, 243–245, , 252–254, Osteoprotegerin (OPG), 250 Osteosarcoma, 98 Otic placode, 1018 Otic vesicle, 1018 Otitis media, 1023 Otoconia (otoliths), 1031, , Otocyst, 1018 Otogelin, 1035 Otolith(s), 1031, , Otolithic membrane, 1031, , Otosclerosis, 1024 Otoscopic examination, 1020, Outer cell mass, 930 Outer collar of perinuclear cytoplasm, 405 Outer enamel epithelium, 600, , 603 Outer limiting membrane, of retina, , 994, 1000, 1012 –1013 Outer medulla, renal, 770, Outer mesaxon, 405, Outer mitochondrial membrane, 62, , Outer nuclear layer, of retina, , 994, 1000, 1012 –1013 Outer nuclear membrane, 93, Outer plexiform layer, of retina, , 994, 1000–1001, 1012 –1013 Outer stripe, of renal medulla, 770–771, Ova (sing., ovum), , 102, , 909, 910, 930 Oval (vestibular) window, 1019, 1020–1022, , 1025, 1032 Ovarian arteries, , 923 Ovarian cancer, 910 Ovarian cycle, 926 –927

f

f

242 263 196 t f

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p 242 243

263

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f

p

p

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245

254

1031 1032

1031 1032 1031 1032 f 1021 406

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602

993

62 63 993 93 993 771 873 1024

p

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p

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Ovarian follicles, 909, 957 –960 atretic, 910, , 921, 923, 929 –960 barrier protecting, 912 collapse and reorganization of, 918–919, cyclic growth and development of, 910 development of, 910, estrogen synthesis and secretion, 909, 914–915, growing, 910, 911, 957 –960 mature (Graafian), 910, , 914–915 microenvironment of, 910 monitoring or imaging of, 916–917 oocyte release from, , 916–918 primary, 910–911, , 957 –960 primordial, 910–911, , , 957 –958 secondary, 910, , 913–914, , 929 –960 wall of, 918 Ovarian follicular atresia, 910, , 921–923, 959 –960 Ovarian hilar cells, 923 Ovarian ligament, 909, Ovarian teratomas, 118 Ovary(ies), 909, , , 957 –960 blood supply to, 923 cortex of, 909, 957 –958 , 960 –961 cyclic changes in, 908–909 menopause, 908–909 menstrual cycle, 926 –927 , 928–930 midcycle pain, 913 endocrine function of, epithelioid tissue of, 123 estrogen synthesis in, 914–915, , 919, 921 germinal epithelium of, 910 histogenesis of, 116 innervation of, 923 lymphatic vessels in, 923 medulla of, 909 polycystic, 916 primary cilia of, 136 structure of, 909–910 Ovulation, 910, 916–918, 923 Oxidation in liver, 694 α-oxidation of fatty acids, 64 β-oxidation of fatty acids, 62, 64 β-oxidation of fatty acids, 695, 704 Oxidation reactions, 62 Oxidative enzymes, 64 Oxidative metabolism, 346 Oxidative phosphorylation, 63

910

910 p

910

p

p

918

915

p

910 910 p p 910 911 p p 910 913 p p 910 p 909 f 909 910 p p p p p p f

f

t

815

f

915

p

Oxygen, blood transport of, 303–305 Oxygen-dependent intracellular killing, 310–311 Oxygen-independent killing mechanisms, 311–312 Oxyntic cells. Parietal cells Oxyphil cells, 837, 856 –857 Oxytalan fibers of periodontal ligament, 608 Oxytocin, 372, 816, 825–826, 826 , 868, 946, 951 Oxytocin analogs, 827

See

p

p

t

P

p53-binding protein (nucleostemin), 91 p53 tumor-suppressor protein, 97 mutations, 94 tumor suppressor gene, 667 P-cells (pacemaker cells), 449 P-face, of membrane freeze fracture, 22, 34, P-selectin, 307–308, , 457 P450 aromatase, 915, P450-linked side chain cleavage enzyme (P450scc), 847 Pacemaker cells (P-cells), 448–449 Pacemaker of heart, 448–449 Pachytene, , 102 Pacinian corpuscles, , 555, , 559, , , 584 –585 , 946 Packed cell volume (PCV), 298–299, , 321 Paclitaxel, 80 Paget’s disease, 833 Paired homologous chromosomes, 871–872 Palate hard, 589, , soft, , Palatine glands, 608 Palatine raphe, 590, Palatine tonsils, 505, , 523 –527 , 589, PALM. Photoactivated localization microscopy Palmar fibromatosis, , 202 Palmer system, for dentition, 597 –598 Palpitations, 449 Pampiniform venous plexus, 865, 902 –903 , 923 Pancreas, , 711–720, , 726 –727 acinar cells of, , 713, , , 726 –727 acini of, 712–713, , , 719, 726 –727 acinus of, 166 blood supply to, 719 endocrine, 712, 715–719, 726 –727 epithelial tissue of, , 170 –175 exocrine, 712–713, 726 –727

p53 p53

f

f

34

308 915

100

f

392

f

556

590 732 590 732 590 505 p See 202 f 633

559 567

299

p

f

592

f

p p 711 p p 712 713 714 p p 712 713 p p 113 p

p p p

p

p

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hormonal and neural regulation of, 714–715 innervation of, 718 islet cells of, 716, 716 , , 717 , 719 , 726 –727 islets of, 174 –175 , 644 , 712, 715–718, , , 726 –727 secretions of, Pancreatic ducts, , 708, 711–715, , Pancreatic enzymes, 648 , 713 Pancreatic hormones, 715–718, 718 Pancreatic polypeptide, 647 , 717 , 718 , 719 Paneth cells, 166, 652, 656–657, , 661 Panniculus adiposus, 280, 555 Panniculus carnosus, 555 Papanicolaou (Pap) test, 933, 945 Papillary cells of enamel, 603 Papillary ducts (of Bellini), , 775 Papillary layer, of dermis, 551–552, 578 –579 Papillary layer, of enamel (tooth), 603 Papillary muscles, of heart, , , 447, Paracellular diffusion barrier, 141 Paracellular gap, 308 Paracellular pathway, , 145, 308, 453 Paracetamol, 700 Paracortex (deep cortex), of lymph nodes, , 508, Paracrine control, 815, Paracrine hormones, 644 , 647 , 656, 819 Paracrine luteotropins, 921 Paracrine secretion, 402 Paracrine signaling, 163, Paraffin, 2 Parafollicular cells (C cells), 831, Paragangliomas, 844 Parakeratinized epithelium, , 590, 620 –621 Paranasal sinuses, 731, 736 Paranemin, 74 , 75 Paraptosis, 108 Parasitic infections, 148 , 207, 313 Parasternal lymph nodes, 951 Parasympathetic division, of ANS, 389, 418–420, cardiac regulation by, 449 liver innervation by, 708 pancreatic innervation by, 718 penis innervation by, , 892 tongue innervation by, 596 urinary bladder innervation by, 798 Parasympathetic ganglia, 415 , 432 Parathyroid glands, 117, , 837–839, , 856 –857 Parathyroid hormone (PTH), 251, 261–262, 769, 769 , 787, 837–839, 837

p

589 707

t 717 f

p

f

t

f

711 712

t t t 656

t

773

f

p

443 446

144

f

f

815

t

f

163

f

t

831

50

p 447

507

p

510

p

f

419

891

t

p p 715 716 p

815

f

t

p

837

p

f

p

p

Parathyroid hormone receptors, 251 Paraurethral ducts, 799 Paraurethral glands, 799 Paraventricular nuclei, of hypothalamus, , 824 Paravertebral ganglia, 418, 432 Parenchyma, 122 of liver, 696, 697–698 of lung, 743 of lymph node, 508 of prostate gland, 885–886 of thymus, 512–513 Parenchymal cells of adrenal glands, 839 of pineal gland, 828–829 Parietal cells, 640–644, , , 649–650, 674 –677 Parietal epithelial cells, of Bowman capsule, 780, Parietal layer, of Bowman capsule, , 780, 806 –807 Parietal layer of serous pericardium, 445, Parkinson disease, 396, 398 , 420 Parkinsonism, secondary, 398 Parotid (Stensen) duct, 589 Parotid gland, 164, Parotid gland tumors, 617 Parotid glands, 589, 608, 614, , 628 –629 Parotid papilla, 589 Pars distalis, of adenohypophysis, 819, , 821–824, , 850 – 853 Pars fibrosa, of nucleolus, 91 Pars flaccida, of tympanic membrane, 1020 Pars granulosa, of nucleolus, 91 Pars intermedia, of pituitary gland, 819, , 824, , 850 –851 Pars nervosa, of neurohypophysis, 819, , 824, 850 –853 Pars tensa, of tympanic membrane, 1020 Pars tuberalis, of pituitary gland, 819, , 824, 850 –851 PAS cells (microphages in thymus), 512 PAS staining, 6, , 154, , , 187 Passive (simple) diffusion, 37–38, Passive transport, 38, Pathogen-associated molecular patterns (PAMPs), 40, 309 Pathology expansion, 13 telepathology, 25 Pattern recognition receptors (PRRs), 309, 483–484 3 gene, 544 Pax5 transcription factor, 331 Pax7, 361 Paxillin, 161, PDZ-domain proteins, of zonula occludens, 144

820

p

640 643 f

609

f

775

f

615

p

p

6

Pax

161

38

445

154 155

820

p

820 820 820

38

p p 781 p p

822

824 p p p p p

p

p

Pectinate ligament, 986 Pedicels, of podocytes, , , 776, Pedicle, of cone, 1001 Peg cells, of uterine tubes, 924 Pellicle, acquired, 617 Pelvis, autonomic innervation of, 421 Pendrin, 833, Penicillar arterioles, of spleen, 518, Penile urethra, 799 Penis, 862, , 892 Pentose phosphate pathway (pentose shunt), 310–311 Pepsin, 641 Pepsinogen, 641, 642 Peptic ulcer disease (PUD), 641 Peptide hormones, 45, 816, 937–938 Peptide neurotransmitters, 402, 402 Peptide YY (PYY), 284, 647 Peptides, signal, 55, Perforating canals, of Volkmann, 242, 245, 270 –271 Perforating fibers, of Sharpey, 241 Perforins, 105, 489, 497, Periarterial lymphatic sheath (PALS), 516, , , 532 –533 Periaxonal plasma membrane, 405 Periaxoplasmic plaques, 396 Pericardial cavity, 445 Pericardiocentesis, 445 Pericardium, 443, 444–446, , Pericellular matrix, of cartilage, 220, Pericentrin, 77 Pericentriolar material, 75 Pericentriolar matrix, of MTOC, 75, Perichondrium, , 221, 223, 230 –231 , 234 –235 , 254 Perichoroidal space, 992 Pericryptal fibroblast sheath, of large intestine, 662 Pericyte(s), 205, in blood–thymus barrier, , 518 in capillaries, 463–464, , in postcapillary venules, 465 as stem cells, 205, 372, 463–464 Perikaryon. Cell body, of neuron Perilymph, 1023, 1032, , 1036 Perilymphatic space, 1023, 1032 Perimetrium, , 925, Perimysium, 345, , 376 –377 Perineal pouch, deep, 799 Perineurial cells, 417 Perineurium, 372, , , 417, 434 –435 Perinodal cytoplasm, 407,

774 775

781

833

891

521

f

f

55

t

t

p

497

516 521

445 446

220

p

206

220

76

p

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514 463 464

See

1032 909 925 346 p 392 416

408

p

p

p

p

p

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Perinuclear cisternal space, of nuclear envelope, 93 Perinuclear cytoplasm, 390–391, 405 Periodic acid–Schiff (PAS) cells, 512 Periodic acid–Schiff (PAS) reaction, 6, in basement membrane, 154, , in goblet (mucus) cells, , 164 in osteoblasts, 246 in reticular fibers, 187 Periodontal disease, 607 Periodontal ligament, 605, 607–608 Periodontal ligament stem cells (PDLSCs), 608 Periodontitis, 608 Periodontium, 608 Periosteal bony collar, 255, , 274 –275 Periosteal cells, 241, 245, , 249 Periosteum, 240, Peripheral artery disease, 452 Peripheral dendritic branch, 390, , Peripheral edema, 464 Peripheral ganglia, , 415, 415 Peripheral lymphatic organs, 482–483, , 492 Peripheral membrane proteins, , 35, 302 Peripheral myelin protein of 22 kDa (PMP22), 405 Peripheral nerve(s), 388, 415–416, , 434 –435 injury response of, 426–429 regeneration of, 429 Peripheral nervous system (PNS), 115–116, 388 autonomic division of, 116. Autonomic nervous system basal lamina in, 155 cardiac regulation by, 449–450 histogenesis of, 116 injury response in, 426–429, , myelin sheath of, 405–406, , , organization of, 415–417 origins of cells in, 415 regeneration in, , 429 satellite cells of, 407, supporting cells of, 155, 389 unmyelinated axons of, 407, , 415 vasculature of, 389 Peripheral neuroglia, 389, 403 Peripheral zone of platelets, 318, of prostate gland, 886, Peripherin, 74, 74 Periportal space (of Mall), 697, 703, , 707 Perisinusoidal macrophages, 463 Perisinusoidal space (of Disse), 696, , 700–701,

6

154 155 154

256 245 f

242

410

33

p

t

p

391 392 483

416

p

p

See also

426 427 405 406 408

427

t

319

410

886

409

704 699

703

Peristalsis, 635, 660, 797, 924 Perisynaptic/terminal Schwann cells (teloglia), 403, Peritendineum, 212 –213 Peritubular capillaries, 793–794, Peritubular dentin, 607 Peritubular tissue, of testes, 866 Periurethral zone, of prostate gland, 886 Perivascular cells. Pericyte(s) Perivitelline barrier, 921 Perivitelline space, 913, 920 Perlecan, 156, 162, 775 Permanent adrenal cortex, 843 Permanent teeth, 597, 597 –598 Permount, 2 Pernicious anemia, 306 , 641, 641 , 644 Peroxins, 64 Peroxisomal proteins, 64–65 Peroxisomal targeting signal, 64 Peroxisome(s), 30, 30 , 31 , 64–65, , hepatocyte, 703–705 Peroxisome proliferator–activated receptor gamma (PPARγ), 282, Peroxisome proliferator–activated receptor gamma (PPARγ) coactivator-1 (PGC-1), 288 PET. Positron emission tomography Peyer’s patches, 505, , 634, 652, , 656, 684 –685 Phagocyte oxidase (phox) system, 310–311, , 311 Phagocyte system, mononuclear, 203 , 317, 699–700 Phagocytic degradation, of collagen, 186, 187 Phagocytosis, 38, 39, , 49, by epididymal cells, 883 inflammatory response and, 493 liver and, 699–700 lymph nodes and, 510–511 by macrophages, 199–201, 312, 884, 750–752 by mesangial cells, 780 by microglia, 203 , 413, 427 by neutrophils, 200, 309–312, oxygen-dependent killing mechanisms in, 310–311 oxygen-independent killing mechanisms in, 311–312 phagocyte oxidase (phox) system of, 310–311, , 311 respiratory burst in, 310, by retinal pigment epithelium, 994 by Sertoli cells, 872, 874, 878 Phagolysosome, , 310 Phagosome, 49 Phagosomes, 40, , 49, , , 310 Phakinin, 74 , 75 Phalangeal cells, of internal ear, 1034–1035, ,

p

p

404

794

See f

f

t

See

f

t

65 66

506

40

50

f

t

f

653

310

p f

p

310

f

309

310

309 40

f

50 309

1035 1037

282

f

Phalloidin, 80 Pharmacologic chaperone therapy, 51 Pharyngeal arches, 1018 Pharyngeal pouches, 837 Pharyngeal tonsils, 505, 589, 736 Pharyngoesophageal sphincter, 635 Pharynx, 731, , 733, 736 Phase contrast microscope, 16 Phenylthiocarbamide (PTC), 596 Pheochromocytoma, 288, 844 Pheomelanin, 545, 561 Pheromones, 571 Phonation, 737 Phosphasome, 307 Phosphatases, in neutrophil granules, 307 Phosphate, 838 bone mineralization, 260–261, bone storage of, 238 metabolism of, 262, 838 in saliva, 616–617 Phosphatidylinositol system, 818 Phospholipids, in plasma membrane, 35 Photoactivated localization microscopy (PALM), 20 Photoaging, 193 Photopsins, 998 Photoreceptor cells, 980, 981, , 993, , 1012 –1013 color sensitivity of, 995, 1000 development of, 983 discs of, 998, 1000 distribution of, 995, light sensitivity of, 995 nuclei of, 1000 spherule or pedicle of, 1001 structure of, , 995–998, visual pigment of, 703, 998–999 visual processing in, 998–999, Phox (phagocyte oxidase) system, 310–311, , 311 Physical activity, and adipose tissue, 293 Physical barriers, in immunity, 483, 539 Physiologic gastric mucosa barrier, 641 Physiologic jaundice, 313 Pia-arachnoid, 423 Pia mater, 423, , 424, , 440 –441 Pigment-containing cells, of iris, 988 Pigment donation, in epidermis, 546–547, Pigment inclusions, 81–82 Pigmentation, 548, 561 , 578 –579 Pigmented layer, of ciliary body, 991

f

732

f

f

f

265

f

987 f

993

p

995

994

996

f

423

f

999

424

p

p

p

310

p

547

f

p

1035

Pillar cells, of internal ear, 1034–1035, Pilosebaceous canal, 560, 567, 582 –583 Pilosebaceous units, 566–567 Pineal gland, , 827–829, , 854 –855 cells of, 827–829, 854 –855 concretions of, 828–829, development of, 828, hormones of, 829, 830 Pinealocytes, 828, 854 –855 Pinkeye (conjunctivitis), 1004 , 1006 Pinna (auricle), 1018 Pinocytic vesicles, 29 Pinocytosis, 39, , 50, Pinocytotic vesicles, 462–463, , Pitch, 737 Pituicytes, 829, 852 –853 Pituitary dwarfism, 266 Pituitary gland, , 818–826, 850 –853 anterior lobe of ( Anterior lobe of pituitary gland) blood supply to, 819–821, development of, 116, 819, epithelioid tissue of, 123, 174 gross structure of, 818–819, hypothalamic regulation of, 819 , 827 location of, 818 as master gland, 818–819 nerve supply of, 821 posterior lobe of ( Posterior lobe of pituitary gland) regulation of, 819 Pituitary growth hormone, 266 gene, 135 gene, 135 gene, 135 Placenta, 908, 933–939, , 937 , 970 –973 abnormalities of, 937 development of, 931, , 933–934 endocrine function of, 937–938 exchange of gases and metabolites, 936–937 fate at birth, 937 HIV-infected, 934–935 mature, , 937 Placenta accreta, 937 Placenta increta, 937 Placenta percreta, 937 Placental (decidual) septa, 935, Placental barrier, 935–936, Placental macrophages, 934–935, , 972 –973 Plakoglobins, 75, 149

815

829 p p 829 828 t p p f

39

50

p

f 815 See

f

PKD1 PKD2 PKHD1

p

See

f

p

p

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463 464

821 820

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935 f 931

936

p

f

p

p

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f f

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936

936 934

p

p

Planar lipid rafts, 35 Plaques in atherosclerosis, 452 –453 , 460, 470 in nervous tissue, 406 , 428 urothelial, 796 Plasma (cell) membrane, 29, 31–44 abaxonal, 405, adaxonal or periaxonal, 405, apical, 60, , 796, basolateral, 61, freeze fracture of, 33–34, functional domains of, 145 functions and pathologies of, 31 microdomains of (lipid rafts), 32–33, , 35, 145 microscopic features of, 30 , 31, modified fluid–mosaic model of, 32, movement or transport across, 37–44, proteins of, 32–35 structure of, 31–32, Plasma, in blood, 114, 298–300, , 300 Plasma cells, 176, , 197, 206, , 210 –211 , 482 in diffuse lymphatic tissue, 502 Golgi apparatus of, 58, in immune response, 485, 495 in mammary glands, 949 secretory function of, 40, 56 Plasma-membrane blebs, 35, 104, Plasma osmolality, 825 Plasma proteins, 299–300, 300 , 692–693, 777 Plasmablasts, 503, 511 Plasminogen, 320 Plasminogen activator, 879 Plasminogen activator inhibitor-1 (PAI-1), 281, 286 , 49 Plasticity, synaptic, 393 Platelet(s), 298–299, 299 , 317–320, 338 –339 buffy coat, 299, development of, 317, , 321, , 326–327, 329 functions of, 319–320 low level of, 327 structure of, 317–318, Platelet activation factor (PAF), 492 Platelet and endothelial cell adhesion molecule-1 (PECAM-1), 471 Platelet count, 321 Platelet demarcation channels, 317 Platelet-derived growth factor(s) (PDGFs), 239, 454, 780 Platelet-derived growth factor receptors (PDGFRs), 781 Platelet-derived growth factor β (PDGFβ), 781

f

61

406

61

f

f f

406

797

34

t

177

f

32

t

32

299 207

59

t

33

33 38 t

p

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104

t

Plasmodium falciparum

t 299 318

319

p

322

f

p

t

Platelet thromboplastin factor (PF 3 ), 320 Plectin family, 75, , 161, Pleomorphic adenoma, 617 Pleura, visceral, 754, 764 –765 Plexiform layer, of cerebrum, 436 –437 Plexiform layers, of retina, , 994, 1000–1002, 1012 –1013 Plicae, 153, , 784 Plicae circulares, 633, 651, 680 –683 Ploidy, analysis of, 7 Pluripotent stem cells, induced (iPS), 167 Pluripotent stem cell (PPSC), 321 Pluripotential progenitor cells, 225–226 Pneumocytes, 747–748, 766 –767 Pneumonia, 754 –755 Podocytes, , , 776, , Podoplanin (E11), 239 Podoplanin (PDPN), 471 Point-scanning methods, 20 Polar bodies, , 102, 917–918, Polar microtubules, 99 Polarity, epithelial cell, 124–126, 141 Polarizing microscope, 19 Polychromatophilic erythroblast, , 325, , 326, 329 , 340 – 341 Polychromatophilic erythrocytes, , 325, , , 329 , 340 – 341 Polyclonal antibodies, 8 Polycystic kidney disease (PKD), 135–136 Polycystic ovary disease, 916 Polycystin-1, 135 Polycystin-2, 135 Polymastia, 946 Polymerase chain reaction (PCR), 10 Polymeric immunoglobulin receptor (pIgR), 617, 658 Polymerization of actin filaments, 70, of intermediate filaments, 72, of laminins, 157, of microtubules, 66–67, Polymers, in expansion microscopy, 13 Polymorph(s). Neutrophil(s) Polymorphic cells, 436 –437 Polymorphic ventricular tachycardia, 365 Polymorphonuclear neutrophils, 306. Neutrophil(s) Polyploid nuclei, 366 Polyribosomes (polysomes), 54, Polyspermy, 921

160

153 f

774 775

f

161 f p p p 993 p

p

p

p

p p 779 781

f

100

917

324 324

p p

325 325 326

f

158

See

p

p

71

73

67 p

54

See also

t t

p p

Polythelia, 946 Polyubiquitin chain, 53 Polyubiquitination, 53 Pompe disease, 51 Population coding scheme, in olfactory transduction, 734 Pore domain, 38 Porins, mitochondrial, 62 Porocytosis, 399–400 Poromas, 569 Porta hepatis, 695–696, Portal canals, , 697–698, , 707, 722 –726 Portal hypertension, 665, 702 Portal lobule, 696, 697, , 722 –723 Portal system hepatic, 443 hypothalamohypophyseal, 443 hypothalamohypophyseal, 820, , 827 Portal triad, , 696, , , 722 –726 Portal vein, 443, 695–696, , , 699, , , 722 –723 Positive chronotropic effect, 450 Positive feedback, 818 Positive inotropic effect, 450 Positive selection, of T cells, 514, Positron emission tomography (PET), 286, 293, 293 Postcapillary venules, 124, 307, , 372, 442, 451 , 465–466, 501 Posterior (dorsal) horns, of spinal cord, 440 –441 Posterior chamber, of eye, 982, , , 1010 –1011 Posterior lobe of pituitary gland, 819, , 824–826, 850 –855 cells of, 827 hormones of, 824–826, 826 neurosecretion storage in, 824–826 neurosecretory vesicles of, 824–825 origin of, 824 structure of, 824 Posterior pigment epithelium, of iris, 987, Postfusion reactions, 921 Postmitotic phase, of granulopoiesis, 327 Postsynaptic density, 393, , 399 Postsynaptic fibers, 450 Postsynaptic membrane, 399–400, , Postsynaptic neurons, 397, 420 Posttranscriptional modifications, 55, Posttranslational modifications, 37, 55–56, 58–59 Posttranslational processing, of insulin, 719 Posttranslational transport, 55, Potassium (K +) in actin polymerization, 70

f

696

693

696

697

697

p

p

p

821 696 699 p 696 697

p

p 699 704

515

308 982 987

t

820

p

f

p

989

395

399 400 55 55

f

p

t p

p

p

p

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astrocyte regulation of, 411–412 in gastric mucosa, 639 Potassium channels, 414, 595, Potassium spatial buffering, 411–412 Potassium–sodium exchange, 789 Power stroke, 355 PP cell (F cell), 717 PR domain containing 16 (PRDM16), 288 Prader-Willi syndrome, 284 Prader-Willi/Angelman syndrome (PWS/AS), 92 Pre-mRNA, 54, Preadipocytes, 282, Precapillary sphincter, 462, 465, 480 Precapillary sphincters, 464 Precocious puberty, 830, 869 Predentin, , 606, Preformed mediators, 204 Pregnancy, 908 corpus luteum of, 921 ectopic, 917, 924–925, 938 endometrial changes in, 931–932 hCG levels in, 921, 937–938 hyperthyroidism in, 937–938 mammary gland changes in, 948–952, placenta in, 933–939, , 937 , 970 –973 uterine changes in, 926 Pregnancy tests, 921 Preimplantation factor (PIF), 930 Prelysosomes, 48 Premelanosomes, 546 Premolar teeth, 597 Prenatal diagnostic testing, 10 Preodontoblasts, 600 Preprocollagen molecules, 185 Preproinsulin, 719 Prepuce of clitoris, 939 Prepuce of penis, 891 Presbycusis, 996 Presbyopia, 1004 Pressure receptors, 555, 559, Presynaptic element, 398–399, Presynaptic fibers, 449–450 Presynaptic neurons, 397, 418–420 Presynaptic parasympathetic neurons, 419 Presyncope, 449 Prevertebral ganglia, 432 Prickle cell layer (stratum spinosum), 539, 540, 590

595

t

55

f

282

605

p

607

935

f

948 p

p

f

f

559 399

p

540, 576p–577p,

731

754

Primary (main) bronchi, 730, 731, , 739, 742–743, Primary (azurophilic) granules, in neutrophils, 305, 306–307, 312– 313 Primary antibody, 9 Primary cilia (monocilia), 129, 130 , 135–137, , 136 , Primary ciliary dyskinesia (PCD), 137, 139 Primary hemostatic plug, 319 Primary hyperhidrosis, 572 Primary immune response, 492 Primary lymphatic organs, 482, , 491 Primary lysosomes, 48 Primary messengers, 36–37 Primary myotubes, 360 Primary nodules, 502 Primary oocyte, 102, 917, Primary ossification center, 255, Primary ovarian follicle, 910–912, , , 957 –960 Primary sex cords, 863–864, Primary spermatocyte, 102 Primary spermatocytes, 871, Primary teeth, 597, 597 –598 Primary union, in wound healing, 572 Primitive connective tissue, 177 Primitive fat organs, 282 Primitive node, 129, 137 Primordial germ cells, 863–864, , 910 Primordial ovarian follicle, 910–911, , , 957 –958 Principal cells of collecting ducts (light cells), 788, 789 of epididymis, 883 of parathyroid glands (chief cells), 837 of thyroid gland (follicular cells), 830–831, Principal piece, of sperm, 872, , 875 Pro-α chains, of collagen, 185 Proacrosomal granules, 872 Probarrier lipids, 543 Procentrioles, 76, , 79, 137 Procollagen, 183, , Procollagen type I N-terminal propeptide (PINP), 185 Proerythroblast, 324–325, , 326, 329 , 340 –341 Progesterone, 909, 919, 921, 926 –927 , 928, 937, 949–951 Programmed cell death, 64, 97, 103–109, , 105 , – , 163 Prohydrolase, 47 Proinsulin, 719 Projector lenses, 20 Prokaryotic ribosomes, 55 Prolactin (PRL), 821, 821 , 870 , , 921, 946, 951 Prolactin cells (lactotropes), 821, 822

t

135

f

t 137

483

917

256 910 912

864 878 f

f

p

f

864

910 911

324

f

f

t

f

t

f 880

p

p

831

874

77 184 185

p

t

104

p

p t 106 107

929

Proliferative phase, of menstrual cycle, 928, Proline, 181 Proline residues, 185 Prometaphase, 99 Promonocyte, 329 Promyelinating Schwann cells, 404, Promyelinating transcription factors, 404 Promyelocyte, , 327, 329 Pronuclei, 917, , 920 Proopiomelanocortin (POMC), 823 Prophase, 99, Prophase I, 102 , 568 Proprioceptors, 359, 417 6- -Propylthiouracil (PROP), 596 Prospero-related homeobox transcription factor (Prox-1), 471 Prostacyclin (PGI 2 ), 286 , 454, 817 Prostaglandin(s), 639, 775, 817 Prostaglandin analogs, 990 Prostaglandin D 2 (PGD 2 ), 204 Prostaglandin E 2 (PGE 2 ), 639, 775 Prostaglandin F 2α, 286 Prostaglandin H 2 , 456 Prostaglandin I 2 , 286 , 454 Prostate cancer (carcinoma), 886, 887 –888 , 889 Prostate gland, 862, , 885–889, , 904 –905 adult parenchyma of, 885–886 concretions of, 886–889, 904 –905 epithelium of, 886–889 fibromuscular stroma of, 886, 904 –905 function of, 885 tumors of, 889 zones of, 885–886, Prostate-specific antigen (PSA), 888 , 889 Prostatic acid phosphatase (PAP), 889 Prostatic hyperplasia, benign, 886, 887 –888 Prostatic urethra, 799, Protamines, 872 Protanopia, 1000 Protease inhibitors, 504 Proteases, 543 Proteasome(s), 30, 30 , 31 , 53 26S proteasome complex, 53, Proteasome-mediated degradation, 53–54, Protein(s). carrier, 38,

t

324 917 100 Propionibacterium acnes N

404

t

t

f

f

t

t 863

f f 887 p p p p p

886

f

863

f

t

f

t

f

p

f

54 53 See also specific proteins and protein types 38

38 f

channel, 37, 38, digestion of, 649 extracellular, 4 fluorescent, 18 hepatic metabolism of, 694–695 intracellular cytoskeletal, 4 linker, 35 membrane, 32–35, , , , 49 molecular motor, 69, peroxisomal, 64–65 plasma, 299–300, 300 plasma, 692–693, 777 posttranslational modification of, 37, 55–56 posttranslational transport of, 55, , 58–59 processing of, 54, 58–59 receptor, 34–36. Receptor(s) renal endocytosis of, 786 secretion of, 164 structural, 35 synthesis of, 54, , 718 transport, 38, 49, 144 Protein 0 (P0), 405 Protein 22 (p22), 311 Protein 40 (p40), 311 Protein 4.1, 71 Protein 47 (p47), 311 Protein 4.9, 71 Protein 67 (p67), 311 Protein kinase(s), 37, 875 Protein kinase A, 454, 875 Protein p210, 77 Protein phosphatases, 36, 875 Protein phosphorylation, and sperm, 875 Protein S, 239 Proteinases, 180–186 Proteinuria, 784 Proteoglycan(s), 196 in basal lamina, 154, 156 in bone matrix, 238, 245 chondrocyte secretion of, 219 in cornea, 9985 diversity of, 195, in elastic arteries, 459 in extracellular matrix, 193–194, in glomerular basement membrane, 775, 778, in ground substance, 193–194 in heart valves, 447 in hyaline cartilage, 217–218, , 230 –231

33 34 35 70 t

55

See also 55 f f f f

f

t

195

195

218

780

p

p

negative charge of, 156 staining properties of, 5 structure of, 156, , 195 Proteoglycan aggregates, 194, 218, 224, 230 –231 Proteoglycan monomer, 218, Proteolipid protein (PLP), 412 Proteolytic degradation, of collagen, 186–187 Proteolytic enzymes, 713 Prothrombin, 693 Prothrombogenic agents, 454 Protocadherin-15, 1027 Protofilament, of microtubule, 66 Proton (H +) pumps, 49, 63, 251, 642, 650 Proton motive force, 63 Protoplasm, 34 Protoplasmic astrocytes, 410–411, Proximal connecting fibers, of centriole, 77, Proximal convoluted tubule, 773, , , 784–786, , , 806 –807 Proximal straight tubule, 773, 786, 806 –807 Pseudostratified epithelium, 124, 125 , 172 –173 Pseudounipolar neurons, 390, Psoriasis, 544 PU-1 transcription factor, 323 Puberty female, 921, 923, 946, 947, 949 male, 868 precocious (early-onset), 830, 869 Pudendal nerve, 799 Pulmonary acini, 743 Pulmonary circulation, 443–444, , , 750 Pulmonary embolism, 466 Pulmonary lobules, 743 Pulmonary valves, , 446, 457 Pulp chamber, of tooth, – , , , 607 Pulpal horns, 607 Pulsed electromagnetic field stimulation (PEMF), 244 Pumps. Pupil, 981, , 987 Purkinje cells, 389, , 438 –439 Purkinje fibers, 365, 384 –385 , 389, 444, , 448–449, 474 – 475 Putative (candidate) hormones, 644 , 647 , 656 Pyknosis, 92 Pyloric glands, 646, , 678 –679 Pyloric mucosa, 678 –679 Pyloric region, of stomach (pylorus), 638, , 674

194

p

218

p

391

p

410 78 773 774 784 785 p p t p p

443 444

444

601 602 603 605

See specific pumps 982 391 p p p p p f 650 p p p p

t

445

639

p

p

p

Pyloric sphincter, 635 Pyloric stenosis, 635 Pyramidal cells, 123, , 436 –437 Pyramidal lobe, of thyroid gland, 830 Pyramids, medullary (renal), 770–771, Pyrogen, 309 Pyroptosis, 108 Pyrosis, 637

391

Q

Quiescent osteocytes, 248,

R

p

p

770, 773, 802p–803p

248 43

Rab-GTPase docking complexes, 42, , 399 Rabies virus (RABV), 421 Rac-2 GTPase, 311 RAD-51 protein, 94 Radial (oblique) portion, of ciliary muscle, 989–990 Radial (deep) zone, of articular cartilage, , 223 Radial arteries, of endometrium, , 928 Radial glial cells, 410 Radial growth phase, of melanoma, 552 Radial sorting, in nerve development, 403–404, Radiation, and spermatogenesis, 870 Radiation, ultraviolet. Ultraviolet (UV) radiation Radioactive precursors of DNA, 10 Radioactive precursors of RNA, 20 Random assortment of chromosomes, 103 RANK (receptor activator of nuclear factor-κB), 250 RANKL (RANK ligand molecule), 250, 251 RANK–RANKL signaling mechanism, 250 Rapamycin, 498 Rapidly progressive glomerulonephritis, 775–776 Rapidly renewing cell populations, 97 Rathke pouch, 819 Reabsorption by kidney, 784–786 by large intestine, 660 Reactive (inflammatory) lymphadenitis, 518 Reactive gliosis, 428 Reactive microglial cells, 413, 428, 428 Reactive nitrogen intermediates (RNIs), 311 Reactive oxygen intermediates (ROIs), 64, 310, Rec8 , 102 Receptor(s), 34–36.

f

f

894

See

f

p

f

222

f

404

f

f

See also specific types

310

activation of, 37 cell matrix, 162 epithelial, 122, 124 fate of, 44–45, Receptor activator of nuclear factor-κB (RANK), 250 Receptor-mediated endocytosis, 39–41, , , 50, 453 Recombinant erythropoietin, 330 Recombinant human erythropoietin (RhEPO), 769 Recombination, 102 Recovery stroke, of myosin head, 355 Recovery stroke in ciliary movement, 134 Rectal folds, transverse, 664, Rectum, 660, 664–665, Recycling, receptor and ligand, 44–45 Red blood cell distribution width (RDW), 321 Red blood cells (RBCs). Erythrocyte(s) Red bone marrow, 241, 321, , 332–333 Red hepatization stage, of pneumonia, 755 Red margin, of lips, 590, 620 –621 Red muscle fibers, 346 Red pulp, of spleen, , , 532 –535 5α-Reductase, 889 Reductional division (meiosis I), 102 Reflex arcs, 388 Refractile media, of eye, 982 Regeneration hepatic, 703 neural, 426, , 429 Regeneration tracks, 403 Regular dense connective tissue, 178, 179–180, , 212 –213 Regulated secretory pathway, 42, , Regulatory light chain (RLC), 350, , 370 19S regulatory particles, of proteasome, 53, Regulatory proteins, 259 Regulatory T cells, 316, 497–498 Regulatory T lymphocytes, 488 Reinke crystalloids, of ovary, 923 Reinke crystals, 868–868, 869 Reissner’s (vestibular) membrane, 1032, , Relaxation, of skeletal muscle, 356 Relaxin, 938 Remak bundles, 403 Remak Schwann (nonmyelinating) cell, 389, 403, , 407 Remodeling of bone, 247–248, 259, , , 265, of cartilage, 218–219 Remodeling process of plasma membrane, Renal artery, 792–793

45

41 45

665

665

See

331 p 516 520

p p

f

p

427

179

42 60 350

54

1033 1035

259 261

36

266

404

p

p

See 770

Renal capsule. Bowman capsule Renal columns, , 771, , 802 –803 Renal corpuscles, 771, , 775–780, 804 –807 Renal cortex, 770–771, , , , 802 –805 Renal failure, 346 Renal fibrosis, 791 Renal medulla, 770–771, , , 802 –803 , 808 –809 Renal papilla, 771, , , 808 –809 Renal pelvis, 769, , , 802 –803 Renal plexus, 794 Renal pyramids, 770–771, , , 802 –803 Renal sinus, 769 Renal tubules, 770–771, . Renewing cell populations, 96, 166–167 Renewing stem cells, 870 Renin, 769, 781, 843–844 Renin–angiotensin–aldosterone system (RAAS), 454, 781–784, 783 , 843–844 Repair cell phenotype, 403 Repair Schwann cells, 403 Replication of DNA, 97–98 Replicons, 98 Reproductive system female, 908–979, 957 –979 . Female reproductive system male, 862–907, , 896 –906 . Male reproductive system Resealing of the post-mitotic nuclear envelope, 36 Reserpine, 843 Reserve stem cells, 98, 870 Resident cell population, of connective tissue, 196 Resident macrophages, 427 Residual body, 50, 872–874 Resistant hypertension, 794 Resistin, 281, 286 Resonance, 737 Resorption bone, 249, , 257, 259, , 278 colloid, 833 Resorption bay, 249, Resorption canals, 259, , 272 –273 Resorptive osteocytes, 248, Respiration (gas exchange), 730, 747 Respiratory bronchiolar unit, 743–744 Respiratory bronchioles, 731, , 743–744, , 747, , 764 – 767 Respiratory burst, in phagocytosis, 310, Respiratory chain, of mitochondria, 62 Respiratory distress syndrome (RDS), 748–749

773 p p 774 p 770 771 773 p

p

p

770 771 p p p p 772 773 p p 770 773 p p 770 773 p p 773 See also specific tubules

f

863

p

p

p See also p See also

t

250

p

250

259

261

248

p

p

731

p

310

743

754

p

Respiratory diverticulum, 730 Respiratory epithelium, 732 Respiratory mucosa, 732, 745 Respiratory passages, 730–731, , 764 –765 Respiratory portion, of respiratory system, 731 Respiratory region, of nasal cavity, 731–733 Respiratory system, 730–731 anatomy of, 730, blood supply to, 731, 752 cilia of, conducting portion of, 730–731 development of, 730 epithelial tissue of, 117 functions of, 730 innervation of, 754 lymphatic vessels of, 754 metaplasia in, 745 mucous and serous secretions of, 731 passages of, 730–731, , 764 –765 respiratory portion of, 731 Response factor (RF), serum, 372 Rest period, in hair growth, 566 Restriction checkpoint, in cell cycle, 98, Rete ridges, 550 Rete testis, , , 875, 880, , 882, 896 –889 Reticular cells, 189, 331, of lymphatic system, 485, 502–504, , , 528 –531 Reticular fibers, 187–189, , 198 of adipose tissue, 283 of basement membrane, 157–158 of lymphatic system, 485, 506, , of spleen, 518 Reticular formation, of brainstem, 421 Reticular lamina, 157–158, 1035 Reticular layer of dermis, 178, 552, 578 –579 of lamina propria, 590 Reticular meshwork, of lymph node, 506–511, , Reticulocytes, , 325, , , 329 , 340 –341 Retina, 980–982, , , , 992–1002, 1010 –1011 barrier protecting, 994 blood vessels of, 1002–1003 capillary network of, cells of, 993–1002 development of, 983, imaging of, 997 –998 layers of, 993–1002, neural, 981, , 983, , 992

f

731

p

p

p

p

731

134

f

731

f

864 866

881

331 187

101

p 507 508

p p

507 508

p

p

324 325 326 981 982 987 f 981

462 983 f 993 983

t

507 508 p p p p

p

neuron types in, 993 nonphotosensitive region of, , 993 ophthalmoscopic examination of, 988, photoreceptors of, 1012 –1013 , 980–981 , 993–1002, . Photoreceptor cells photosensitive region of, , , , 993 specialized regions of, 1002 visual processing in, 998–999, Retina proper (neural retina), 981, , 983, , 992–993 Retinal, 998–999 Retinal detachment, 991 , 993, 997 Retinal pigment epithelium (RPE), 981–983, , , , 992–994, , 999–1000, , 1012 –1013 Retinal pigment epithelium–specific protein 65 kDa (RPE65), 999– 1000 Retinal sheen, 1002 Retinitis pigmentosa, 136 Retinoblastoma susceptibility protein (pRb), 97 Retinohypothalamic tract, 829 Retinoid X receptor (RXR), 282, Retinol (vitamin A), 693, 702, 998 Retinol-binding protein (RBP), 693, 702 Retinol-binding protein 4 (RBP-4), 281, 286 Retinyl esters, 702 Retrograde degeneration, in nerve injury, 429 Retrograde signaling, 429 Retrograde transport, 56, , , 396–397 Reuptake, of neurotransmitters, 403 Reversal mesenchymal–epithelial transition, 791 Reverse transcriptase, 504 Reverse-transcription PCR (RT-PCR), 10 Rh(D−), 304 Rh blood group system, 304 Rh incompatibility, 304 Rh negative (Rh−), 304 Rh positive (Rh+), 304 Rh(D+) sensitization, 304 Rh30 polypeptide, 304 Rh50 glycoprotein, 304 Rhesus (Rh) antigen, 304 Rheumatic fever, 448 Rheumatic heart disease, 448 Rheumatoid arthritis, 186, 227, 241 Rhinitis, 742 Rhinosinusitis, 742 Rhodopsin, 702, 998–999 RhoGAM, 304

981 1002 p p , 987 994, 995 See also 981 982 987 999 981 983 f f 981 982 983 993 1001 p p 282

t

56 60 f

f

f

f

f

f

f

f f f f

f

f

f

993,

1028

Ribbon synapses, in hair cells, 398, 1028, , 1029 Riboflavin, 488 Ribonuclease (RNAse), 93 Ribonucleic acid. RNA Ribosomal RNA (rRNA), 54, 91–93 Ribosomes, 30, 30 , 31 , 54–57, , 91–93, 393 Rickets, 265 , 693, 769 Right atrium, 443–444, , , Right hepatic duct, 707 Right ventricle, , 444, , , Rigor configuration, 355 Rigor mortis, 355 Riley-Day syndrome, 596 Rima glottidis, 736, RNA, 10 DNA transcription to, 54, messenger, 54, radioactive precursors of, 10 ribosomal, 54, 91–93 small nucleolar, 91 translation of, 55, RNA polymerase, 818 RNAse (ribonuclease), 93 Rods (photoreceptor cells), 981, 993–1002, , 1012 –1013 development of, 983 discs of, 998, 1000 distribution of, 995, light sensitivity of, 995 nuclei of, 1000 spherule of, 1001 structure of, , visual pigment of, 702, 998–999 visual processing in, 998–999, Rokitansky-Aschoff sinuses, 710–711, , 724 –725 Roof of oral cavity, Root, of tooth, 599, , 605 Root sheath, of hair, , 563, 586 –587 Rootletin, 132 Rosenthal fibers, 80 Rotational movement, of nodal cilia, 129 Rouget cells. Pericyte(s) Rough endoplasmic reticulum (rER), 29, 54–57 in active secretory cells, 56 functions and pathologies of, 31 lysosomal protein synthesis in, 49 microscopic features of, 30 , 54, , in neuron, 393 nucleus relationship to, , 92, 93,

t

See

54 f 443 444 445 443 444 445 446

f

t

f 737

55

55

55

993

p

995

994 996

See

f

999

590 599 560

p

88

t

t

711

p

p

54 58 93

p

p

55 50

protein synthesis and processing in, 54–57, as quality checkpoint, 56 transport to and from Golgi apparatus, 49, , 56, Round (cochlear) window, 1019, , 1026, 1032 RU-486 (mifepristone), 931 Ruffini corpuscles, 555, , 559 Ruffled border, of ameloblasts, 601 Ruffled border, of osteoclast, 250, , , , 276 –277 Rugae, of stomach, 638, Rule of 10 sin pheochromocytoma, 844 Runt-related transcription factor 2 (RUNX2), 244 Ryanodine receptors, 355–356, , 365,

1024

556

250 252 253 f 359 365

638

S

f 101

S cones, of retina, 995, 1000 S DNA-damage checkpoint, 97, S (synthesis) phase, of cell cycle, 97–98, 99, Saccule, , 1025, 1026 Saccule, macula of, , 1026, 1030 Saliva, , 615–617, 616 Salivary antimicrobial peptides, 591 Salivary ducts, 589, 608, 613–614, 626 –631 .

1024 589

1024

56 p

p

100, 101

t

p p See also specific Salivary glands, 608–613. See also specific glands lingual, 592, 608, 624 p –625 p major, 589, 608, 614–615, 615 , 626 p –631 p minor, 589, 608 salivon of, 609, 609 secretions of, 615–616 secretory acini of, 609–613, 626 p –631 p tumors of, 617 f Salivon, 609, 609 Saltatory conduction, 413, 414 Salty taste, 593, 595 , 596 Sandhoff disease, 51 f Santorini, duct of, 707 , 711 , 712, 713 Sarafotoxin, 456 Sarcolemma, 345, 355 , 363 , 364 ducts

Sarcomere cardiac muscle, 363 myotendinous junction, 380 –381 skeletal muscle, , 348, , 351, Sarcoplasm, 344, 365 Sarcoplasmic reticulum, 57 of cardiac muscle, 363, , 365 of skeletal muscle, 348, 355–356

347

p

363

349

p

352, 378p

Sargramostim (Leukine), 330 Satellite cells of peripheral nervous system, 115, 389, 407, of skeletal muscle, 361, Satellite DNA, 99 Satiety factor (leptin), 281, 283–285, 286 , 287 Scab, 572 Scala media, 1031–1033, , , 1044 –1045 Scala tympani, 1031–1032, , 1044 –1045 Scala vestibuli, 1031–1032, SCALP mnemonic, Scanning electron microscope (SEM), 1, , 22 Scanning-transmission electron microscope (STEM), 22 Scar connective tissue, 202 hypertrophic, 202 keloid, 202 nervous system, 428, 428 Scavenger receptors (SRs), 309 Schiff reagent, 6 Schlemm, canal of, , 987, , , 991, 1014 –1015 Schmidt-Lanterman clefts, 407, , Schwann cell(s), 115, 357, 389, , , 403–407, 434 –435 cytoplasm of, 407, , injury response of, 426, junction between, 407, , 434 –435 myelin sheath production by, 403–407, origin of, 415 regenerative function of, , 429 Schwann cell precursors, 403, , 415 Schwann, sheath of, 405, 434 –435 Sclera, 980, – , 983, 986–987, , 1010 –1011 , 1016 – 1018 Sclera proper, 986 Scleral icterus (jaundice), 981 Scleral venous sinus (canal of Schlemm), , 987, , , 991, 1014 –1015 Sclerostin, 239 Scotopsin, 998 Scrotum, 862, Scurvy, 185, 265 Seasickness medication, 539 Seasonal affective disorder (SAD), 830 Sebaceous glands, 538, 559, , , 566–568, , 576 –577 , 582 –583 association with hair follicles, 566 in eyelashes, , 1006 holocrine secretion by, 163,

410

361

f

1033 1035 p p 1033 p p 1033 21

424

f

982

987 988 408 409 390 392 408 409 427 408 p p

p

981 983

p

f

f

f

f

t

427 404 p p

p

p

407

987

p

p

982

p

p p

p

987 988

863 f

p

p

560 564

1005

163

567

p

p

in oral cavity, 591 secretions of (sebum), 560, 567, 567 Sebocytes, of sebaceous glands 567, 582 –583 Sebum, 560, 567 Second messenger-dependent protein kinases, 37 Second messenger-independent protein kinases, 36 Second messenger system, 37, 838 Second messengers, 370, 371–372, 400, 818 Second polar body, , 918 Secondary (lobar) bronchi, 730, 743, Secondary (permanent) teeth, 597, 597 –598 Secondary antibody, 9 Secondary electrons, 22 Secondary granules, in neutrophils, 307, 313 Secondary hemostatic plug, 320 Secondary immune response, 492 Secondary intention of wound healing, 572 Secondary lymphatic organs, 482–483, , 491–492 Secondary lysosomes, 48 Secondary myotubes, 360–361 Secondary lymphatic nodules, 502 Secondary oocyte, 103, 916–918 Secondary ossification center, , 257, 274 –275 Secondary ovarian follicle, 910, , 913–914, , 959 –960 Secondary parkinsonism, 398 Secondary sex characteristics, 846 Secondary spermatocytes, 103, 872 Secondary union, in wound healing, 572 Secretin, , 646 , 656, 714, 717 , 718 Secretion. endocrine, 163, exocrine, 163, mechanisms of, 163, mucous, 164 Secretory acini, 609–613, 626 –631 Secretory component (SC), , 659 Secretory IgA, 46, 591, 617, 657 , , 659, 949 Secretory pathways, 39, , 49, Secretory phase of menstrual cycle, 928, , 966 –967 Secretory segment of apocrine gland, 569 of eccrine gland, , 569, 580 –581 Secretory-stage ameloblasts, 600–601, , Secretory vesicles, 30 , 31 , 60 Segmental bronchi, 730, 743, Segmentation algorithms, 23 Segmentation, in small intestine, 660 Segmented neutrophil, 327

p

917

p

754 f f 483

f

256 910

f

p

p 913

p

f

646 t t See also specific sites and substances 163 163 163 p p 658 f 658 42 50 929 p 568 t t

p

754

p 603 604

p

p

Segregation of chromosomes, 99, 103 Selectins, 145–146, , 307, , 328, 457 Selective estrogen receptor modulators (SERMs), 264 Selective permeability, 453, 456 Self-tolerance, 497 Self nonself, 483, 495, Sella turcica, 818 Semen, 885, 889–890 Semicircular canals, , , 1025, 1042 –1043 Semicircular ducts, 1026 Semilunar valves, 446 Seminal vesicles, 862, , 882, 884–885, Seminiferous cords, 864, Seminiferous tubules, 866, , , 875–879, 896 –899 barrier protecting, 878–879, 879 cell associations in, 875–876, cells of, 866, 876–879, – development of, , 865–866 epithelium of, 866, 875–876, , 896 –897 basal compartment of, 878, luminal compartment of, 878, patch-like distribution of, 876, Sertoli cells of, 876–879 stage of, 875, wave of, 875–876 Sensorineural hearing loss, 1034 , 1038 Sensory (afferent) neurons, 389–390, , dorsal root ganglia, 422–423 somatic, 389–390, 416 visceral, 390, 416, 418 Sensory cells of internal ear (hair cells), 127, 135, 1026–1030, 1042 –1043 hearing function of, 1036 inner, 1034–1035, , ion channels and action potentials of, 1028 outer, 1034–1035, , synapses of, 1028–1029, type I, 1029, type II, 1029, of taste buds, 592, , 624 –625 Sensory ganglia, 415 , 416 Sensory mechanoreceptors, 128 Sensory nerve fibers, 359, Sensory receptors, 417 in membranous labyrinth, 1030–1035 in skeletal muscle, 359 in skin, 555–559, , 584 –585

146

308 t 495

versus

f

1019 1024

p

p

863 886 864 866 867 p f 876 877 879 864 876 p p 878 878 877 875 f 391 392

p

p

p

1027, 1028,

1035 1037 1035 1037 1029 1029 1029 594 p p t 360 556

p

p

Separase, in meiosis II, 103 Septal cells, alveolar, 748, , Septal (alveolar) macrophages, 750, Septal wall, alveolar, 747, , 749, , , 752, 766 –767 Serial block-face SEM (SBFSEM), 23, Serial section TEM (SSTEM), 22–23 Serine protease inhibitor Kazal-type 5 (SPINK5), 542 Serine proteases, 204 Serosa (serous membrane), 167 , 632, , 635 of gallbladder, 710 of large intestine, 663 of small intestine, 660, 680 –683 of stomach (gastric), 651, 674 –675 of ureters, 810 –811 of uterine tube, 923 Serotonergic neurons, 401 Serotonin, 319, 400, 401, 402 , 741, 830 Serous acini, 609–610, 626 –629 Serous cells, 164 of pancreas, 712–713 of salivary glands, 608 –629 , 609–610, Serous demilunes, , 609–610, , 630 –631 Serous glands, 164, Serous membrane. Serosa Serous pericardium, 445, Serous secretions, 164 Sertoli (sustentacular) cells, 82, 866, , 876–879, – , 896 –897 development of, 864, junctional complex of, , 877–879, – phagocytosis by, 872, 874, 878 Serum, 300 Serum response factor (RF), 372 Sex (gender) determination, 864, Sex chromosomes, 90, 91, , Sex cords, primary, 863–844, Sex-determining region Y, 863 Sex hormones, 281 female, 909, 926 –927 male, 869 –870 , 879, Sexual dimorphism, 862 Sexual precocity, 830, 869 1 gene, 863 Sharpey fibers, 241, 605, Shear stress, 454 Sheath of Schwann, 405, 434 –435 Sheathed capillaries of spleen, Shock, anaphylactic, 205

749 750 754 748 750 751 23 f

p

p

p

603 166 See

p

p

p

t

p

609

445

f

f

SF-

f

p

609 p

878 879

865

880

605 p

p

867

865 867

f

p

p

91 865 864

f

p

633

p

p

p

p 521

877 879

Shock, hypovolemic, 465 Short bones, 240 Short-term weight regulation, 284 Sialoproteins, 239 Sick sinus syndrome (SSS), 449 Sickle cell disease, 305, 306 Sickle hemoglobin (HbS), 306 Side-polar myosin thick filament, 367, 370 Sigmoid colon, 660 Signal detection, 161 Signal patch, 47 Signal-recognition particle (SRP), 55, Signal sequences (signal peptides), 55, Signal transduction, 36–37, 161 Signaling molecules and pathways, 36–37, 328 Signaling platforms, lipid rafts as, 33 Silver-reactive basement membrane, 154 SIM. Structured illumination microscopy Simian virus (SV40), 98 Simple diffusion, 37–38, , 453 Simple epithelium, 73, 113, , 123–124, 125 , 170 –175 Simple glands, 164, 165 Single-molecule localization methods, 20 Sinoatrial (SA) node, , 448–449 Sinus(es). anal, 664 gallbladder, 710–711, , 724 –725 lactiferous, , 947 lymphatic, , 508, , 528 –531 paranasal, 731, 736 renal, 769 splenic, , 517–518, , , 532 –535 Sinusitis (rhinosinusitis), 742 Sinusoid(s), 463 of adrenal glands, 840–841, of bone marrow, , 332–333 of liver, 696, , , 699, , maternal, in placenta, 933, Sinusoidal endothelium, 699 Situs inversus, 137, 139 Sjögren syndrome, 613 Skeletal muscle, 115, , 344–361, 376 –379 accessory proteins of, 351, atrophy of, 359 characteristics of, 373 comparison with other muscle types, 372 –373 connective tissue of, 345–346, , 376 –377 contraction of, 351, , 355–356,

f

f

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See

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113 t 445 See also specific sinuses 711 p p 946 507 509 p p 516

t

519 520 p p f 844 331 696 697 699 704 934 f 115 p p 352 f f f 346 p p 354 359

p

p

cross-striations of, 348 development, repair, healing, and renewal of, 360–361 histogenesis of, 116 metabolism and ischemia of, 350 motor innervation of, 356–359, , muscle fibers of, 345–347, , , , 376 –379 muscle spindles of, 359, relaxation of, 356 sensory innervation of, 359 tendon attachment to, 380 –381 thick filaments of, 349, 350–351, , , thin filaments of, 349–351, traumatic injuries to, 346 Skeletal myogenic progenitor cells, 288 Skene glands, 799, 939 Skin, 538–575, 576 –579 absorption via, 539 adipose tissue of, 296 , 555 appendages of, 559–572, , 586 –587 cell renewal in, 167, 539–540, 544 cells of, 541–550 chronological aging of, 193 connective tissue of, 179 dark, 548, epidermal derivatives of, 538 epithelial tissue of, 124, 167, 174 –175 excretion by, 539 functions of, 538–539 layers of, 539–541, light, 548, mechanical stress on, 551 nerve supply of, 555–559, , 584 –585 photoaging of, 193 primary cilia of, 136 repair of, 572 secretion by, 539 stem cells in, 539, 564, thick or thin, 539, , , 576 –577 vitamin D production in, 769 Skin aging, 572–573 Skin cancer, 551 –552 , 556 –558 Skin color (pigmentation), 548, 561 , 578 –579 Sliding movement, ciliary, 132–134 Slow anterograde transport system, 396 Slow oxidative fibers, 346–347 Slow-twitch muscle fibers, 346–347 Slowly renewing cell populations, 97 SMAC/DIABLO, 105

f 356 357 345 346 348 360 p

p

p p

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p 350 351 368 349

560

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548

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539

548 f

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556

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564 539 540

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Small arteries, 451 , 456, 461–462 Small granule cells of nasal cavity, 732 of trachea, 740–741 Small intestine, , 651–660, 680 –685 absorptive cells (enterocytes) of, , 648 –649 , 652–655, 680 –683 bile flow to, 707–708 cells of, 652–658 contractions of, 660 digestive and absorptive functions of, , 651–652 divisions of, 651, 678 –685 epithelial cell renewal in, 659 glands of, 652, , 678 –681 immune functions of, 652, 657 , 658–659 lamina propria of, 652, 658, 680 –683 lymphatic nodules of, 505, , 634, 652, , 658, 684 –685 mucosa of, 651–659, , 678 –685 muscularis externa of, 386 –387 , 660, 680 –685 muscularis mucosae of, 652, , 680 –685 plicae circulares of, 633, 651, 680 –683 secretions of, serosa of, 660, 680 –683 smooth muscle of, 386 –387 stem cells in, 166–167, 205, 659 submucosa of, 659, 680 –685 tight junctions in, 654 villi of, 633, 652, , 680 –685 Small leucine-rich proteoglycans (SLRPs), 985 Small nucleolar RNAs (snoRNAs), 91 Small peptides, as neurotransmitters, 402, 402 Small proline-rich (SPR) proteins, 543, Small pyramidal cell layer, of cerebrum, 436 –437 Small veins, 451 , 465–466 Smooth border, of ameloblasts, 601 Smooth endoplasmic reticulum (sER), 29, 57–58 detoxification in, 57–58 functions and pathologies of, 31 hepatocyte, , 704 of Leydig cell, 868 lipid metabolism in, 57 microscopic features of, 30 , 57, of Sertoli cell, 877, Smooth muscle, 115, , 344–345, 366–372, 386 –387 actin filaments in, 198, , 202 autonomic regulation of, 372 , 389, 418 basal lamina in, 155, bronchial, 743

p

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633

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653

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630

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589

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878 155

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58

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cells of, characteristics of, 373 comparison with other muscle types, 372 –373 contraction of, 367–372, , dense bodies of, 367, 368–369, elastic fiber synthesis in, 192 in excurrent ducts of male reproductive system, 882 functional aspects of, 371–372 histogenesis of, 116 innervation of, 371 latch state of, 371 matrix secretion by, 372 muscle fibers of, 366, in muscularis externa, 634–635 in muscularis mucosae, 634 nerve terminals of, 372 in prostate gland, 904 –905 renewal, repair, and differentiation of, 372 spasms of, 44 spontaneous contractile activity of, 371 structure of, 367–371 thick filaments of, 367, , 370–371 thin filaments of, 367, 368–369 urinary passages, 797 uterine, 925–926 vascular arteriole, 462 artery, 456–462 vein, 466–467 Smooth muscle cells, 367, , , 372, 386 –387 Smooth muscle myosin (SMM), 367 Smooth muscle progenitor cells, 372 SNAP-25, 43, 398, SNARE proteins, 39, 42–44, , 358, 398–399, Sneeze reflex, 733 snoRNAs. Small nucleolar RNAs Sodium channels amiloride-sensitive, 594–596, cGMP-gated, 999 taste-specific, 594, transmitter-gated, 358, 358 , 400 voltage-gated, 356, 359, 400, 414 voltage-sensitive, 595, Sodium concentration, monitoring of, 781–784 Sodium-dependent transporters, 403 Sodium ions, in taste, 595–596 Sodium reabsorption, 785, 788 Sodium/iodide symporters (NIS), 833,

f

369 371 369

f

f

367

p

p

368

367 368

399

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43

See

399

595

595

595

p

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833

Sodium–potassium exchange, 789 Sodium–potassium–ATPase pumps, 785 Soft callus, in bone repair, 265, Soft keratin, 541 Soft palate, , Soluble mucus, 642 Soluble -ethylmaleimide-sensitive factor attachment protein receptor (SNARE), 39, 42–44, , 358, 398–399, Solute(s), in plasma, 299 Solvent drag, 654 Somatic afferent neurons, 389–390, 416 Somatic efferent neurons, 390, 416 Somatic nervous system (SNS), 388–389 Somatostatin, , 647 , 695, 716–719, 718 , 728 , 830 Somatotropes (GH cells), 822, 822 Somatotropin. Growth hormone Sorting mechanism, of endosomes, 44–45, 61, Sorting signals, in Golgi apparatus, 61 Sound perception, 1035–1036, Sound resonance, 737 Sound vibrations, 1036 Sour taste, 593, 595, 9 gene, 863 SOX-9 transcription factor, 224, 226 Spaces of Fontana, 987 Special sense receptors, 122 Special senses. Hearing; Vision bipolar neurons in, 390 Specialized mucosa, 589, 591–592 Specific binding of dye, 3 Specific immunity, 484–485, 492 Spectral domain optical coherence tomography, 994, 997 –998 Spectrin, 71, 127, , 302–303, Sperm. Spermatozoa capacitation of, 875, 911, 919–920 decapacitation of, 883 fertilization by, , 919–920, 924–925 hyperactivation of, 920 IgA antibodies against, 884 kinetic activity of, 875 maturation of, 872–874, 875, 883 motility of, 875 production of, 862, 865, 869–875, , 896 –897 . Spermatogenesis structure of, 872, 874–875, Sperm granuloma, 884 Sperm-specific antigens, 879 Spermatic cords, 862, 883, , 902 –903

N

266

590 732

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646 See

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399

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595

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See also

See also

128

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302

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917

f 885

871

874

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100

867 871

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Spermatid(s), , 102, 866, , , 872–874, 896 –897 differentiation of, 872–844 early, late, release of, 874 Spermatid phase, of spermatogenesis, 869, , 872–874, Spermatocyte(s), , 871–872, , 896 –897 early, 878 primary, 102, 871, secondary, 103, 872 Spermatocyte phase, of spermatogenesis, 869, 871–872, Spermatogenesis, , 102, 862, 865, 869–874, , 896 –897 duration or cycle of, 875–876, factors affecting, 871 hormonal regulation of, 869 –870 spermatid phase of, 869, , 872–874, spermatocyte phase of, 869, 871–872, spermatogonial phase of, 869, 870–871, temperature and, 865, 871 Spermatogenic cells, 866, 875, 896 –897 . Spermatid(s); Spermatogonia Spermatogenic stages, 876 Spermatogonia (sing., spermatogonium), 863–864, 866, , 870–871, , 896 –897 groupings or associations of, 875–876, type A dark (Ad), 870, , type A pale (Ap), 870–871, , 875, type B, 871–872, , Spermatogonial phase, of spermatogenesis, 869, 870–871, Spermatogonial stem cells, 870 Spermatozoa (sing., spermatozoon), , 102, capacitation of, 875, 911, 919–920 decapacitation of, 883 fertilization by, 917, 919–920, 924–925 formation of, 862, 865, 869–875, hyperactivation of, 920 structure of, 874–875, Spermiation, 869, 874 Spermiogenesis, 869, , 872–874, Spherocytosis, hereditary, 303 Spherule, of rod, 1001 Sphincter(s). Sphincter of common bile duct (of Boyden), , 708 Sphincter of hepatopancreatic ampulla (of Oddi), , 712 Sphincter pupillae muscle, 988, Sphincter spasms, 44 Sphincteric portion, of ciliary muscle, 9589 Sphingosine-1-phosphate (S1 ), 511

878 878

867

871

878

100

f

876 f

f 871 f

871

p

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100

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871

867

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871 p p

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871

874

P

874

876 876

871 876 871 871 876

871

871

707

707

P

Sphingosine-1-phosphate receptor 1 (S1 R1), 511 Spicules, 242, , , 254, 257, , 276 –278 Spinal cord, 421, 422–423, , 440 –441 presynaptic neurons of, 418–420 Spinal nerves, 422, Spindle-assembly checkpoint, 98, Spindle(s), muscle, 359, , 417 Spinous layer, of epidermis, 539, 540, , 576 –577 Spinous processes, 540, Spiral arteries, 923, , 928, Spiral ganglion, 1025, Spiral lamina, osseous, 1032, Spiral organ of Corti, , 1026, 1031–1038, , 1044 –1045 Spiral valve (of Heister), , 708 Spirochetes, 16 Spleen, 482, , 516–521, , 521 , 532 –535 basal lamina of, 159, histogenesis of, 117 primary cilia of, 136 Splenectomy, 521 Splenic artery, 516 Splenic cords, , 517, , 532 –535 Splenic nodules, , 517, 532 –535 Splenic sinuses, , 517, , , 532 –535 Spongiosa, of heart valve, 446–447, Spongy bone, 239–240, , 242, , , 272 –273 Spongy urethra, 799 SPR (small proline-rich) proteins, 543, Sprouts, 429 Squamous cell(s), 170 –171 Squamous cell carcinoma, 126 , 551 , 745 Squamous epithelium, 113, , 123, 125 , 170 –175 Squamous follicle cells, of ovary, 911 Squamous metaplasia, 126 , 745 , 933 Squamous zone, of anal canal, 665, – , 690 –691 gene, 863, 864, Stab (band) cell, , 327, 329 Stable cell populations, 96, 167 Stable compartment model, of endosomes, 44 Stage, of microscope, 14 Staggered tetramer, of intermediate filament, 72, Staining acidic and basic dyes in, 5–6, 5 chemical basis of, 5–6 loss of tissue components in, 4–5 metachromasia in, 6, 93 Stapedectomy, 1024

245 250 423

p

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483

423

257 p

101

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360 540 540 926 936 1033 1033 1024 707 516 t p 159 t

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516 516 516

SRY

519 p p p p 519 520 p p 447 239 242 244 p p 544 p p f f f 113 t p p f f 665 666 p p 865 324 t t

f

73

Stapedius muscle, 1022 Stapes, 1018, 1020–1022, Starvation, 284 Static cell populations, 96 Static cytometry, 7 Static γ fibers, 359, Steady-state of treadmilling, 70 STED. Stimulated emission depletion (STED) microscopy Stellate cells, hepatic, , 702 Stellate reticulum, 600, – , 603, Stellate veins, of kidney, 794, STEM. Scanning-transmission electron microscope Stem cell factor (SCF), 879 Stem cells, 124 adult, 166, 196, 205–206, 641 cilia of, 135 corneolimbal, 985, 986 epidermal, 539, 564, gastric, 641 hemopoietic, 200, 205, 255, 321–323, , 482 hepatic, 707 intestinal, 166–167, 205, 659, 662 melanocyte, 561 mesenchymal, 205 Wharton’s jelly, 177 smooth muscle cells from, 372 undifferentiated, 463–464, 465 adipocytes from, 282, , 288 in endochondral ossification, 255 osteoprogenitor cells from, 243–245, myogenic, 360 nasal cavity, 732, 735–736 neural, 391–392, 414 niches of, 166, 205, 659, 662, 564 pericytes as, 205, 372, 464, 465 periodontal ligament, 607–608 pluripotent, 321 renewing, 870 reserve, 98, 870 spermatogonial, 870 tissue, 205 uroepithelial, 796 Stensen (parotid) duct, 589 Stereocilia, 126, 127–128, , 130 , of epididymis, 127, 883, 900 –901 of internal ear, 127, , 1026–1027, , 1036 Stereopsis, 980 Stereovilli, 127

1025

f

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360

699 601 602 794

See

564

603

322

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282

129 p 1024

245

t 131 p

1028

818

Steroid(s), 281, 285, 818, , 830 in female reproductive system, 909 in male reproductive system, 862, 863, 868–869, 870 in placenta, 937 transdermal delivery of, 539 Steroid membrane receptors, 818, Steroidogenesis, 862, 863, 868–869, 909 Steroidogenetic capacity index, 868 Steroidogenic factor-1 gene 1 , 863 Stimulated emission depletion (STED) microscopy, 20 Stochastic optical reconstruction microscopy (STORM), 20 Stoichiometric reaction, 6 Stomach, , 637–651, 672 –677 cardiac region of, 637, , 672 –675 epithelial cell renewal in, 647–650 glands of, 637, , 639–646, , 650, 672 –677 inner surface of, 638–639, lamina propria of, 650, 672 –673 mamillated areas of, 638 mucosa of ( Gastric mucosa) muscularis externa of, 372, 650, 674 –677 muscularis mucosae of, 650, 672 –673 , 674 –677 regions of, 637, rugae of, 638, secretions of (gastric juices), , 639–644 serosa of, 651, 674 –675 stem cells in, 641 submucosa of, 650, 674 –677 Strabismus, 44 Straight tubules of kidney, 770–771, 773–774, , 781, 786, 787, 806 –809 Straight tubules of testes, 864, , 875, 880, , 896 –899 Stratified epithelium, 73, 113–114, , 123–124, 125 , 172 –175 Stratum basale, 539, , 576 –577 , 590 cell differentiation in, 539 cell renewal in, 167, 539–540 of endometrium, 926, 964 –965 melanocytes of, 544–545 Stratum corneum, 539, , 541, 576 –577 keratinocyte desquamation from, 541–542 Stratum functionale, of endometrium, 926–928, 929, 964 –965 Stratum germinativum. Stratum basale Stratum granulosum, 539, 540, , 576 –577 , 911–912, Stratum intermedium, 600, 601, – , 603, Stratum lucidum, 539, 541, 576 –577 Stratum spinosum, 539, 540, , 576 –577 , 590 Stratum superficiale, of lining mucosa, 590 Stratum vasculare, of myometrium, 925

f

818

(SF- )

633

p p 638 p 640 639 p p

638

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638 638 p

p

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p

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589

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773 881

866 113 p p

540

540

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540 p p 601 602 603 p p 540 p p

t

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912

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Streaming potential, 247 Stretch receptors, 359, 559 Stria vascularis, 1032–1034, , Striated border, 126–127 of enterocytes, 653, 682 –683 of microvilli, 651 Striated border, of ameloblasts, 601 Striated ducts, 162, 613, 626 –629 Striated muscle, 114–116, , 344–346, 348, , 555, 622 Striated rootlet, 132, , Striola, 1030, Stripes, of renal medulla, 770–771, Stromal cells, of endometrium, 928 Stromelysins, 187 Structural (hard) keratin, 73, 541, 564 Structural proteins, 35 Structural zone, of platelets, 318, Structured illumination microscopy (SIM), 20 Stye, 1006 Subarachnoid space, 423–424, Subcapsular (cortical) sinus, , 508, , 528 –531 Subcapsular epithelium, of lens, 1003, Subcapsular nephron, 774 Subchondral bone, , 223 Subcutaneous (superficial) fascia, 281, 538, 555 Subcutaneous tissue (hypodermis), 538, 555, 576 , 584 –585 Subendocardial layer, 446 Subendocardial layer of the endocardium (SELE), 384 –385 Subendothelial layer, 450, 458 Subendothelial layer of dense connective tissue (SELCT), 384 –385 Sublingual administration, 591 Sublingual caruncle, 589 Sublingual duct, 589 Sublingual gland tumors, 617 Sublingual glands, 589, 608, 614–615, , 630 –631 Sublobular veins, 696, , 699 Submandibular (Wharton) duct, 589 Submandibular gland tumors, 617 Submandibular glands, 589, 608, 614, Submucosa, 178, 632, , 634 of anal canal, 665 of bronchus, 743 of esophagus, 636, , 670 –671 of large intestine, 663 of oral cavity, 591 of small intestine, 659, 680 –685 of stomach (gastric), 674 –677 of trachea, 739, , 741–742, , 762 –763

1033 1034 p p

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1032

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355

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771

319

424 507

509 1003

222

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696

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633

636

739

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615

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Submucosal (Meissner’s) plexus, 634, 636, 650, 676 –677 Submucosal glands, 634 of small intestine, 659, , 678 –679 , 678 –681 of stomach, 636–637, Subpodocyte space, , 777, Substance P, 402, 402 , 568, 717 Substantia propria of cornea, 984, , 985, of sclera, 986 Succinic dehydrogenase, 346 Succinylcholine, 358 Suckling (breastfeeding), 825, 951 Sudden cardiac death, 444 Sulcus terminalis, 591 SUMOylation, in posttranslational modifications, 36 Sun exposure, 193 , 561 Super-resolution microscopy, 1, 20 Superficial (nodular) cortex, of lymph node, , 508, , 510, 530 –531 Superficial (subcutaneous) fascia, 281, 538, 555 Superficial (tangential) zone, of articular cartilage, 221–223,

637

775 t 984

f

p

659 779 985

p

p

p

p

t

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507

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509

222

Superior (upper) esophageal sphincter, 635 Superior parathyroid glands, 837 Superior tarsal muscle (of Müller), 1005, Superoxide anions (O 2 −), 311 Supertasters, 596 Supporting cells of CNS, 115 of PNS, 115 Suppressor T cells, 316, 488, 497 Suprachoroid lamina, 986 Supraclavicular lymph nodes, 951 Supraoptic nuclei, of hypothalamus, , 824 Suprarenal arteries, 840, , Suprarenal veins, 840, Supravalvular aortic stenosis (SVAS), 190 Surface-associated decapacitation factor, 883 Surface ectoderm, 116, , 982–983, 984 , 1018, Surface membrane ruffles, 39, Surfactant (surface-active agent), 746, 748–749 Surfactant protein A, 749 Surfactant protein B, 749 Surfactant protein C, 749 Surfactant protein D, 749 Survival factors, in apoptosis, 106 Suspensory ligament of lens, 989

1005

f

840 117

840 844 39

820

t

1020

Suspensory ligament of mammary glands, 947 Suspensory ligament of ovary, 909, Sustentacular cells, 733, 734–735. Sutures, of lens, 1004 SV40 (T-antigen of simian virus), 98 Swallowing, 736 Sweat (sweating), 560 disease and, 566 emotional, 582 excessive, 572 thermoregulatory, 569, 582 Sweat glands, 538, 560, 569, 576 –577 , 580 –585 apocrine, 560, , 569–572, 580 –583 of eyelashes, 569, 1005 pheromone production by, 571 modified, mammary glands as, 946–947 eccrine, 560, , 568, , 571–572, 580 –583 Sweet taste, 593–594, Swellable polymers, in expansion microscopy, 11 Swollen glands, 511, 518 Swyer syndrome, 863 Sympathetic division, of ANS, 389, 418, 418–420, cardiac regulation by, 449–450 kidney innervation by, 794 liver innervation by, 708 pancreatic innervation by, 718 penis innervation by, , 892 sweat gland innervation by, 569 tongue innervation by, 596 urinary bladder innervation by, 798 Sympathetic ganglia, 415 , 432 –433 Sympathetic presynaptic fibers, 449–450 Sympathetic trunk, 418–419 Symplekin, 143 Synapse(s), 115, 389, , , 397–403, 1028–1029, chemical, 398–399 components of, 398–399, electrical, 398 morphology of, 397, ribbon, 398 Synapsis, in meiosis, 102 Synaptic bouton, Synaptic cleft, 358, 399, , 403 Synaptic plasticity, 393 Synaptic transmission, 399–403 excitatory, 393, 400–401 inhibitory, 400–401 Synaptic vesicles, , 398–399,

909 See also specific types

p

f

p

560

560

595 f

p

568, 570

t

395

p

p

p

f

p

p

390 395 399 397

395

p

p

419

890

t

p

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1029

399

399

399

Synaptobrevin, 43, 398, Synaptonemal complex, 102, 872 Synaptotagmin 1, 398 Synaptotagmin binding proteins, 399 Syncoilin, 74 , 75 Syncope, 449 Syncytial knots, 934, , 972 –973 Syncytiotrophoblast, , 931, , 972 –973 Syncytium, 345 Syndecan, 195, , 196 Syndrome of inappropriate antidiuretic hormone secretion (SIADH), 828 Synemin, 74 , 75 Synovial joint, 239, 240 Syntaxin, 43, 398, Syphilis, 16 Syringomas, 569 Syrinx, 569 Systemic circulation, 443, 443–444, , Systemic hypertension, 456 Systole of heart, 457

t

f

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934 930 195 t

p

934

p

p

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399

443 444

T

T-antigen of simian virus (SV40), 98 T cell(s). T lymphocytes T-cell receptors (TCRs), 206, 315–316, 492, T lymphocytes (T cells), 206, 315–316, 485.

See

types

495 p

493, 733 See also specific

activation of, 483, 485, 494–498, , 506 blood–thymus barrier protecting, 236 –237 , 514–516, in cell-mediated immunity, 207, 484–485 circulation of, 501 development and differentiation of, 315, , 330, 490f, 491, distribution in lymph node, 508–509, education of, follicular helper, 503 in HIV infection, 504 immunocompetent, 483 migration of, 509–510, 516 mucosa-associated invariant, 316, 488 naïve, 509–510 nasal cavity, 733 origin of name, 490 regulatory, 316, 497, 497–498 in specific (adaptive) immunity, 484–485 splenic, 516

p

509

515

f

f

322

514

515

subsets of, 316 surface molecules of, 316, 485 T tubule in cardiac muscle, 363–365, , in cell remodeling, 35, in skeletal muscle, 355–356, , 359 Tachyarrhythmia, 449 Tachycardia, 365, 450 Tacrolimus, 498 Tail of epididymis, 882 of sperm, , 875 Talin, 161, , 250, Tamm-Horsfall protein, 784 , 787 Tangential (superficial) zone, of articular cartilage, 221–223,

363 364 36 355

874 161

222

253

f

f

Tanning, 561 Tanycytes, in ependyma, 413 Tapping mode, in atomic mode microscopy, 24, Target cells, of hormones, 815 Target-specific SNARE (t-SNARE), 43, , 398–399 Targeting mechanism, in exocytosis, 42, Tarsal (Meibomian) glands, 163, , 1006 Tarsal muscle, superior, 1005, Tarsal plate, of eyelid, 1005, Tartrate-resistant acid phosphatase (TRAP), 249 Tastants, 593 Taste, 593–596, , 596 Taste buds, 591, 592–594, , , 624 –625 Taste pore, 592, , 624 –625 Taste receptor(s), 594–595, , 735 Taste receptor genes, 594–595 Taste-specific Na + channels, 594, Taxol (paclitaxel), 80 Tay-Sachs disease, 50 Tears, 1007 Tectorial membrane, 1035, , 1036 Tectorin, 1035 Teeth, 597–608, , – cementum of, 597, 605 classification, 598 decay of, 610 , 617 dentin of, 597, 505–507 developing, 278 –279 enamel of, 599–604 histogenesis of, 116 saliva and, 616

1005 1005 1005

595 594

f

f

43

f 593 594 p p p 595 595

f

1035 599 601 602 f p

43

24

p

p

supporting tissues of, 607–608 Telepathology, 25 Telogen, in hear growth, 566 Teloglia, 403 Telomerase, 90 Telomere, 90 Telophase, , 101, Telophase I, , 103 Temporal bone, 1019, , , 1023, 1025 Tenascin, 196, , 197 , 218 Tendinocytes, 179–180, , 212 –213 Tendon(s), 179–180, , 212 –213 Tendon fascicles, 179, 212 –213 Tendon–muscle junction, 380 –381 Teniae coli, 635, 660, , 663, 686 –687 Tenon’s capsule, 986 Tenon’s space, 986 Tense part, of tympanic membrane, 1020 Tensor tympani muscle, 1022 Teratomas, 118 Terminal bars, 139–140, Terminal bronchioles, , 743–744, – , , 764 –767 Terminal cisternae, 355–356, , 363, Terminal differentiation, in cell cycle, 97 Terminal ductal lobular units (TDLUs), 946–947, , 978 –979 Terminal ductules, of mammary glands, 947, Terminal ganglia, 432 Terminal hair, 566 Terminal hepatic venule, , 696–699, – , , 707, 722 – 726 Terminal neuroglia, 403 Terminal ring, of nuclear pore complex, , 96 Terminal web of actin filaments, 72, 127, , 653 Territorial matrix, of cartilage, 220, Tertiary (segmental) bronchi, 730, 743, Tertiary granules, in neutrophils, 307 Testes, 862–869, , 896 –899 barrier protecting, 878–879, 879 blood supply to, 865 cells of, 123, 864, 865, 868–871, 372, 57, 82 development of, 116, 863–865, , 868 ducts of, 880, endocrine function of, epithelial (epithelioid) tissue of, 123, 174 –175 immature (prepubertal), 896 –899 lobules of, 865 primary cilia of, 136 spermatogenesis in, 862, 865, 869–875,

f

100 101 100 1019 1021 196 t 179 p p 179 p p p p p p 661 p p f

p

140 741

f

p

743 744 754 p p 363 947 p p 947

355

693

863

696 698 699

p

881

815

t

p

95 128 220 754

f

864

p

p

p

871

p

p

865 867 f

structure of, 865–866, , temperature and, 865, 870 undescended, 864, 870 Testicular artery, 865 Testicular teratomas, 118 Testis-determining factor (TDF), 863 Testosterone, 864, , 868, 869 –870 , Tetanic contraction, in hypocalcemia, 838 Tetanospasmin toxin, 44 Tetanus, 37, 44, 485 Tethering proteins, 42, Tetrads, in meiotic division, 872 Tetraiodothyronine (T 4 ), 695, 831, 832 TGN. -Golgi network TH1 cells, subset of CD4 + T cells, 488, 494 TH17 cells, subset of CD4 + T cells, 494 TH2 cells, subset of CD4 + T cells, 494 Thalassemia, 305 Theca externa, 913, , , 959 –960 Theca folliculi, 912–913, Theca interna, 912–913, , 914–915, , , 959 –960 Theca layers, of ovarian follicle, 912–913 Theca lutein cells, 919, , 960 –961 Thermogenesis, 289 Thermogenin, 288 Thermoregulation, 465, 569 Thermoregulatory sweating, 569, 582 Thick ascending limb of loop of Henle, 773–774, 787 Thick descending limb of loop of Henle, 773 Thick filaments, 71, 115, 344 of skeletal muscle, 349, 350–351, , , of smooth muscle, 367, , Thick skin, 539, , 576 –577 Thin ascending limb of loop of Henle, 773, 786–787 Thin descending limb of loop of Henle, 773, 786–787 Thin filaments, 71, 115, 344 of cardiac muscle, 362 of skeletal muscle, 349–351, of smooth muscle, 367, 368–369, Thin segment of loop of Henle, 786–787, Thin skin, 539, , 576 –577 Thoracic artery, internal, 951 Thoracic duct, 469, Thorax, autonomic innervation of, 420–421 Three-dimensional electron microscopy (3DEM), 22 Thrombocyte(s). Platelet(s) Thrombocytopenia, 327

f

865

f

f

f 880

43

t

See trans

913 914 p 912 913 919 p

p 914 915 p

p

368 369 p p

540

349

539 See

483

p

p

350 351 368

369

786

p

p

t

Thrombopoiesis, 321, 326–327, 329 Thrombopoietin, 326, 328, 330 Thrombosis, 204, 470 Thrombotic glomerular diseases, 775 Thromboxane A 2 , 319, 456 Thrombus (pl., thrombi), 453 Thymic-cell education, Thymic corpuscles, 236 –237 , 513, Thymic cortex, 512, , Thymic lobules, 236 –237 , 512 Thymic medulla, , 513, 3H-thymidine, 10 Thymocytes. T lymphocytes (T cells) Thymus, 236 –237 , 482, , 491, 511–516, 521 architecture of, , barrier from blood, 236 –237 , 514–516, development of, 511 epithelioreticular cells of, 236 –237 , 498, 512, T-cell development in, 315, 330, 490f, 513, , 516 Thymus-dependent (deep) cortex, in lymph nodules, 508 Thymus gland epithelioid tissue of, 123 histogenesis of, 117 Thyroglobulin, 833 Thyroglossal duct, 830 Thyroid carcinoma, medullary, 832–833 Thyroid cartilage, , Thyroid follicles, 830, , 856 –857 Thyroid gland, , 830–839, 856 –857 abnormal function of, 375–938, 836 blood supply to, cells of, 830–831, , development of, 117, 830–831 epithelial tissue of, 830–831, function of, 831–832 location and structure of, 830, structural and functional unit of, 830, Thyroid hormone toxicity, 834 Thyroid hormone transport molecules, 834 Thyroid hormones, 285, 818, 831, 832 . biological activity of, 834 developmental role of, 837 regulation of, release of, 834–835 synthesis of, 833–834, , transport of, 834, Thyroid nuclear receptor, 834

t

f

p

See

515 p p 512 513 p p 512 513 p

815

483 512 513 p

732 737 830

830 830 832

513

p

p p

p

p

514

t

515

514

p p f

831 831

830

t See also specific hormones

835

835

833 835

833

Thyroid peroxidase (TPO), , 834 Thyroid-stimulating hormone (TSH), 792, 821 , 823, 937–938 Thyroid-stimulating hormone cells (thyrotropes), 822 , 823 Thyroidectomy, 838 Thyroiditis, autoimmune, 836 Thyrotropes, 822 , 823 Thyrotropin. Thyroid-stimulating hormone Thyrotropin-releasing hormone (TRH), 728 , 822, 823, 830 Thyroxine, 695, 831–832, 832 Thyroxine-binding globulin (TBG), 817, 833 Tidemark, of articular cartilage, , 223 Tight junctions. Occluding junctions TIM complexes, in inner mitochondrial membrane, 62 Tissue(s), 112–119. definition of, 112 histogenesis of, 116–117 identification of, 119 types and classification of, 112–113 Tissue atrophy, 358–359 Tissue inhibitors of metalloproteinases (TIMPs), 187, 193 Tissue injury, 200 Tissue nonspecific alkaline phosphatase (TNAP), 245–246, 259–261,

See

t

t

f

t

t

t

See

222

See also specific tissues

f

265

Tissue plasminogen activator (TPA), 320 Tissue preparation, 2–3 for expansion microscopy, , 13 frozen sections, 3, 4 hematoxylin and eosin staining with formalin fixation, 2–3, linear equivalents used in, 2 other fixatives used in, 3 Tissue scaffolding, 159–160 Tissue stem cells, 205 Tissue–mordant–hematoxylin complex, 5 Titin, in sarcomere, 351, TNAP. Tissue nonspecific alkaline phosphatase TNFRs. Tumor necrosis factor receptors Toll-like receptors, 38, 309, 483–484 Toluene-based synthetic resins, 2 Toluidine blue, 5 , 6 TOM complexes, in outer mitochondrial membrane, 62 Tomes process, of ameloblast, 600–601, Tongue, 591–596, , 622 –625 dorsal surface of, 591, 622 –623 muscles of, 591 nerve supply of, 596 papillae of, 591–592, , 622 –625 taste buds of, 590, 592–594, , , 624 –625 ventral surface of, 622 –623

12

f

t

352

See See

t

591

p

p

p

p

603

593 p p 593 594 p p

p

p

3

Tonofibrils, 541 Tonofilaments (keratin filaments), 541 Tonsil(s), , 505, , 526 –527 , 589, , 596 lingual, 505, 589, , 596 palatine, 505, 505, 523 –527 , 589, 592, pharyngeal, 505, 589, 736 tubal, 1023, 589 Tonsillar crypts, 505, , 526 –527 Tonsillar ring, of Waldeyer, 526 , 589 Tonsils, 117, 589 Torque receptors, 559 Touch receptors, 559, Toxic goiter, 836 Toxins, degradation of, 694 Trabeculae of arachnoid, 423–424 of bone, 241, 242, 254, 272 –273 , 276 –277 of lymph node, 506, , 513, 528 –529 of spleen, 516, , 532 –533 of thymus, 236 –237 , Trabecular artery, of spleen, Trabecular meshwork, of eye, 987 Trabecular sinuses, of lymph node, , 508, Trabecular vein, of spleen, , 534 –535 Trabeculoplasty, 990 Trachea, 731, – , , 738–7744, – , 762 –763 adventitia of, 739, 741–742 basement membrane of, 153, , 741–742, , 762 –763 cartilage of, 230 –231 , 739, , 741–742 epithelium of, 172 –173 , 739–740, mucosa of, 739, secretions in, 741 submucosa of, 739, , 741–742, , 762 –763 Tracts, of CNS, 414, 421 Trailing edge, of cell, 72 -Golgi network (TGN), 58, , 61, -SNARE complex, 43, Transcellular diapedesis, 308, 332–333 Transcellular pathways, 144, , 308, 453 Transcellular pore, 308 Transcription, 54, Transcription factors c-jun, 429 CBFA1, 252 essential (E2F), 97 GATA-1, 323 GATA-3, 330 in hemopoiesis, 328

483

505 592

p

p

p

505

592

p

p

559

f

p

p

p

516

trans trans

521

507 p

p 521

154 p, 737 p

739

p p

60

144

p

509 p 739 740 741 739 741

741

43

55

p p

507 p p 512

f 731 732 737 p p 739

p

61

p

p p

p

p p

Ikaros family of, 330 in lymphopoiesis, 330–331 Math1, 166, 659 MyoD, 360, 361 Pax5, 331 Pax7, 361 PU-1, 323 runt-related 2 (RUNX2), 244 SOX-9, 224, 226 Transcytosis, 46, 462 Transdermal medications, 539 Transducin, 999 Transepithelial pathway, of thyroid hormone synthesis, 834 Transferrin, 45, 694, 879 Transformation zone, of cervix, 932–933, , 968 –969 Transforming growth factor β (TGF-β), 239, 286 , 454 Transfusions, 303 –304 Transient cell population, 197 Transient electrical potential, 247 Transitional cells, of conducting system of the heart, 449 Transitional epithelium, 124, 125 , 174 –175 , 795–796, , 810 –813 Transitional zone of anal canal (ATZ), 665, 665–666, 690p–691 of articular cartilage, , 223 of cilia, 132, of prostate gland, 886, Translation, 55, Translocase of the inner mitochondrial membrane (TIM complexes), 61 Translocase of the outer mitochondrial membrane (TOM complexes), 61 Translocator, in protein synthesis, 55, Transmembrane channel-like (TMC) proteins, 1027 Transmembrane channels, 150 Transmembrane collagens, 183 Transmembrane glycoproteins, 146 Transmembrane pores, 150 Transmission electron microscope (TEM), 1, 20–22 Transmitter-gated channels, 400–401 Transmitter-gated Cl − channels, 400 Transmitter-gated Na + channels, 358, 358 , 400 Transmucosal delivery, 590–591 Transport active, 38, anterograde, 396–397, 56, axonal, 396–397 dendritic, 397 epithelial, 124, 144–145,

f

f

933

f

t

p

134 55

p

p

f

56

144

p

p

796

p

222 886

55

38

t

p

138

intraflagellar, 137–139, membrane, 37–44, neuronal, 392, 396–397 nucleocytoplasmic, 96 passive, , 38 posttranslational, of protein, 52, proteins, 38, 49, 144 retrograde, 396–397, 56, , vesicles, 29, , 58, 58–59, , 61 vesicular, 37–44, Transthyretin, 817, 834 Transurethral resection of the prostate (TURP), 887 –888 Transvaginal ultrasound, 917 Transverse colon, 660 Transverse rectal folds, 664, Transverse tubular system in cardiac muscle, 363–365, , in skeletal muscle, 355–356, , 359 Traumatic degeneration, in nerve injury, 429 Traumatic neuroma, 429 Treadmilling, in actin polymerization, 70, 128, , 16 Triads, skeletal muscle, , 356 Tricellular contact, 142, Tricellular zonula occludens, 142, 145 Tricellulin, 142, , 143 , 144, 145 Trichohyalin, 541 Trichromats, 1000 Tricuspid valve, , , 446–447, Trigeminal nerve (CN V), 735 mandibular division of, 596 Triglycerides, 280, 285, 296 , 648 Trigone, of urinary bladder, 798 Triiodothyronine (T 3 ), 695, 831–832, 832 Trilaminar embryo, 116 Triple helix, of collagen, 185 Triplet microtubules, 75–79, , 132, Trisomy of chromosome 21, 917 Tritanopia, 1000 Trophoblast, 930–931, Trophoblastic cells, 931 Trophoblastic diseases, 938 Trophoblastic lacunae, 933 Trophoblastic shell, 933–934, Tropic hormones, 823 Tropocollagen (collagen molecule), 180, Tropomodulin, 71, , 349–350, ,

37

37

56

56 60 60

38

55

f

665 363 364 355

Treponema pallidum

142

f 443 444

131

355 142 t

446

p

f

78

f

t

133

930

934

72

349 352

181

f

72

349 352 349

Tropomyosin, , 127, 349–350, , , 367 Troponin (troponin complex), , 350, 366 Troponin, Troponin-C (TnC), , 350 Troponin-I (TnI), , 350, 366 Troponin-T (TnT), , 350, 366 Trypsin, 648 , 713 Trypsin-like protease, 874 Trypsinogen, 648 , 713 Tryptase, 204 Tubal tonsils, 589, 1023 Tuberculosis, 241 , 750–752 Tubotympanic recess, 1018, Tubular gland, 164, 165 Tubular system, transverse in cardiac muscle, 363–365, , in skeletal muscle, 355–356, , 359 Tubules, renal, 770–771, , , 784–786, 806 –809 .

72

f

349 349 349

f

f

1020

t

363 364 355 773 774 p p See also specific tubules Tubules, testicular, 865, 866 , 875, 880, 881 , 896 p –899 p . See also Seminiferous tubules Tubuli recti (straight tubules of testes), 866, 866 , 875, 880, 881 , 896 p –899 p Tubulin dimers, 66–67, 67 , 68 , 395–396 Tubulin protofilaments, 132, 133 Tubuloacinar glands, 165 t Tubuloalveolar gland, 164 Tubuloalveolar glands, 904 p , 946–947, 946 Tubulointerstitial nephritis, 791 Tubulovesicular membrane system, of parietal cells, 642 Tuftelins, 604 Tumor necrosis factor (TNF), 105, 250, 281 Tumor necrosis factor receptors (TNFRs), 109 Tumor necrosis factor α (TNF-α), 204, 285, 286 , 309, 488 Tumor necrosis factor β (TNF-β), 286 Tumor suppressor(s), 97, 106, 667 Tumor-suppressor protein p53, 97 Tunica (lamina) propria, of testes, 866 Tunica adventitia, 206, , 451, 451 , 474 –475 of aorta, 476 –477 of large (elastic) arteries, , , 460, 476 –477 of medium (muscular) artery, , , 478 –479 of small arteries, 462 of veins, 465, 467, , 478 –479 Tunica albuginea female, 910, 957 –958 male, , 866, , , 890, 896 –899 Tunica intima, 450, , 451 , 474 –475

f

p

864

p

450

468 p p 866 867 450 t

t

t

t p p 457 459 p 459 460 p p p p p

p

p

p

p

p

p

of aorta, 476 –477 of large (elastic) arteries, 456–460, , 476 –477 of medium (muscular) artery, , , 461, 478 –479 of small arteries, 461 of veins, 465, 466–467, , , 478 –479 Tunica media, , 451, 451 of aorta, 476 –477 of large (elastic) arteries, , 459, , 476 –477 of medium (muscular) arteries, , 460–461, , 478 –479 of small arteries, 462 of veins, 465, 467, , , 478 –479 Tunica vaginalis, of testis, , 866, Tunica vasculosa, 866 Turbinates (conchae), 732, , 733 Turbulent precipitation, 733 Tylenol, 700 Tympanic cavity, 117, 1019, , Tympanic membrane, 1018–1020, , , Tympanic membrane perforation, 1021 Tyrosinase, 546 Tyrosine kinase, 161, Tyrosine kinase system, 817

457 p 459 460 p 467 468 p p t p 457 459 p 459 460 467 468 p p 863 866 732

450 p

f

p p p

p

p

1019 1022 1019 1021 1022

161

U

54

Ubiquitin, 53, Ubiquitin-activating enzymes, 53 Ubiquitination, in posttranslational modifications, 36, 53–54, Ulcer, peptic, 641 Ulcerative colitis, 664 Ultimobranchial bodies, 830 Ultrafiltrate, glomerular, 768, 771, , 784 Ultrasound biomicroscopy (UBM), 987 Ultrasound, transvaginal, 917 Ultraviolet (UV) microscope, 18 Ultraviolet (UV) radiation, 561 , 985, 1007 Umami taste, 594–595, Umbilical arteries, 936–937, Umbilical vein, , 937 Umbo, of tympanic membrane, 1020 Umbrella cells, of urothelium, 766, Unbent confirmation, of myosin head, 354–355 Uncoupling protein (UCP-1), 288, 290 Undescended testes, 864 Undigested food, 588 Unicellular glands, 164, Unilocular adipose tissue. White adipose tissue

f

936

f

775

595

f 936

796

164 See

54

f

Union, in wound healing, 572 Unipolar neurons, 390, Universal acceptors, of blood, 303 Universal blood donors, 303 Universal system, for dentition, 597 –598 Unmyelinated axons, 407, , 414 Unmyelinated fibers, 403 Unreplicated-DNA checkpoint, 98, Upper esophageal sphincter, 635 Upper pole, of kidney, 769 Uranyl acetate, 21 Uranyl nitrate, 21 Urate oxidase (uricase), 66 Urea, 695 Urea frost, 566 Urea transporters, 787, 788 Uremia, 566 Ureter(s), 768, , , 797–798, 810 –811 epithelial tissue of, 795–798, epithelial tissues of, 795–798, 810 –811 urine flow to, 795 Ureteric orifices, 798 Urethra, 768, 799 compression by prostate gland, 886 epithelial tissue of, 117, 795–797, 799 female, 799, 908 male, 799, Urethral glands, 799 Urethral orifices, 798, 908 Urethral sphincter, 798 3H-uridine, 10 Urinalysis, 784 Urinary bladder, 768, 798–799, 812 –813 epithelial tissue of, 117, 124, 174 –175 , 795–799, , 812 – 813 innervation of, 799 openings of, 798 trigone of, 798 urine flow to, 795 Urinary casts, 784 , 787 Urinary pole, of renal corpuscle, 744, , , 806 –807 Urinary space, , , 780, 806 –807 Urinary system, 768–799. Kidney(s) components of, 768 epithelium of, 124, 125 , 174 –175 , 795–799, , 810 –813 Urinary tract infections, 796 Urine, 769

391

f 409

f

f

f

f

101

f

770 773

p p

796

p p

863 f

p

p

f

774 775

p See also t p

p

p

p

774 777 p

p

796

p

p

796

p

p

p

Uriniferous tubule, 770 Urogenital diaphragm, 799 Urogenital ridges, 815, , 864 Uromodulin, 784 , 787 Uropathogenic , 796 Uroplakins, 796 Urothelial permeability barrier, 797 Urothelial plaques, 796–797 Urothelium, 124, 125 , 174 –175 , , 797, 810 –813 Uterine arteries, ovarian branches of, 923 Uterine contractions, 826 Uterine part, of uterine tube, 923 Uterine tubes, 908, , 923–925, , 960 –961 bidirectional transport in, 924–925 cyclic changes in, 908–909 ectopic pregnancy in, 917, 924–925 layers of, 923–924 segments of, 923 Uteroplacental circulatory system, 933, Uteroplacental malperfusion, 934 Uterus, 908, , 925–933, , 964 –965 anatomic regions of, 925 cyclic changes in, 908–909, 925, 926–928, 926 –927 oxytocin action in, 826 pregnancy changes in, 926 wall, layers of, 925 Utricle, macula of, , 1026, 1030, , Utriculosaccular duct, 1025 Uvea, 981, , 987–992, 1010 –1011

839

f E. coli t 926

p

p 796

909

909

981

V

924

925

1024

909

p

p

p

p

934 p p

p

p

f

f

1031 1032

938 p p p p 932 p p

Vagina, 908, , 938–939, , 974 –975 epithelial tissue of, 172 –173 Vaginal part, of cervix, 932, , 968 –969 Vaginal vestibule, 908, 939–941 Vaginal wall, 938 Vagus nerve (CN X), 592, 596, 637 Valve cusps, 447–448, Valves, 635 heart, 443, , 446–448, , , 457, 474 –475 lymphatic vessel, 471, 480 –481 spiral (of Heister), of cystic duct, , 708 venous, 465, 466 Valves of Kerckring (plicae circulares), 633, 651, 680 –683 Valvular heart disease, 448

443

447

446 447 p p

707

p

p

p

p

Valvular interstitial cells, 447–448 Valvulitis, 448 Vas deferens. Ductus deferens Vasa recta, in kidney, 770–771, 791–792, Vasa vasorum, 451, 460 Vascular coat, of eye, , 987–992, 9481 Vascular endothelial cadherin (VE-cadherin), 471 Vascular endothelial growth factor (VEGF), 256, 664 Vascular endothelial growth factor (VEGF) inhibitor, 992 Vascular endothelial growth factor C (VEGF-C), 471 Vascular endothelial growth factor receptor-3 (VEGFR-3), 471 Vascular endothelium, 124, 450, 452–456, Vascular pole, of renal corpuscle, 744, , 806 –807 Vascular resistance, 454, 456 , 462 Vascularis, 451, 4640 Vasectomy, 879 , 884 Vasectomy reversal, 874 Vasoactive agents, 459 Vasoactive intestinal peptide (VIP), 402, , 647 , 656, 717 , 718 , 719, 822 Vasoconstriction, 454, , 464–465 Vasoconstrictors, 454 Vasodilation, 454, , 464 Vasodilators, 454 Vasomotion, 464 Vasopressin. Antidiuretic hormone Vater, hepatopancreatic ampulla of, , 708, 711–712, VEGF. Vascular endothelial growth factor Vein(s), 442, 465–467 characteristics of, 451 general features of, 450–456 large, 451 , 465, 467, , layers of wall (tunics), 450–451, , 451 , 465, 466–467,

See

794

981

t

455

646

455

See

t

458 774 p

t

f

See

f

t

711

t 467 468

Venipuncture, 300 Venous sinuses dural, 1039, 423, 468 splenic, , 517–518, , , 532 –535 Ventral (anterior) horns, 422, 440 –441 Ventral motor neurons, 422 Ventricles of brain, 413 of heart, 443–444, , , ,

519 520 p

p

t

707

450 468 medium, 451 t , 465, 466–467, 478 p –479 p muscular, 468 small, 451 t , 465–466 Vellus hair, 566 f Vena cava, 443–444, 443 , 444 516

f

p p

443 444 445 446

t

p

467,

737 738 p 737 738 f

p p

of larynx, 736, 737–738, , , 760 –761 Ventricular folds, , 737–738, , , 760 –761 Ventricular hypertrophy, of heart, 458 Ventricular tachycardia, polymorphic, 365 Ventricularis, of heart valve, 446–447 Venulae rectae, 792, Venules, 465, 465–466, , 480 –481 . high endothelial, 124, 465–466, 501, , 509–510, , 528 – 531 muscular, 451 , 465, 466 postcapillary, 124, 307, , 372, 442, 451 , 465–466, 501 terminal hepatic, , 696–699, – , , 707, 722 –726 Vermiform appendix, 482, , 505, 660, 663, , 688 –689 Vernix caseosa, 566 Versican, 196 , 224 Vertebral ligaments, 189–190 Vertical growth phase, of melanoma, 552 Vertigo, 1034 , 1039 Very low-density lipoproteins (VLDLs), 454, 692–693, 694 Vesicle(s). Vesicle budding, 37 Vesicle-specific SNARE (v-SNARE), 43, Vesicular transport, 37–44, Vessels, lymphatic, 464 Vestibular (Reissner’s) membrane, 1032, , Vestibular (oval) window, 1019, 1020–1022, , 1025, 1032 Vestibular dysfunction, 1034 , 1039 Vestibular folds, 736 Vestibular ganglion (of Scarpa), 1036, Vestibular glands, 939–941 Vestibular labyrinth, of internal ear, , 1025, 1030 Vestibular nerve, , 1036, Vestibular system, 1018 Vestibular veins, 1039 Vestibule of ear, , , 1025, 1042 –1043 of nasal cavity, 731–732, of oral cavity, 589 of vagina, 908, 939–941 Vestibulocochlear nerve (CN VIII), , 1036 Vestigial organs, 505 Vibrissae, 731, 732 Villi, 633 chorionic, , 933–935, , 970 –973 intestinal, 633, 651, , 680 –685 Villin, 127, Villous chorion, 935 Villous maturity, 934

732

794 465

p

t

693

t

p

308 483

p See also specific types 502 509 p

t 696 698 699 665 f

f f See specific types

39

1021

1033 1035 1024

f

1038 1024

1038

1019 1024

732

p

p

1019

931 128

653

934

p

p

p

p p

p

f

43

f

p

p

p

t 75

Vimentin, 74, 74 , in adipocytes, 283 in microglia, 415 in Sertoli cells, 877 in smooth muscle, 367, 368 in valvular interstitial cells, 448 Vinblastine, 80 Vincristine, 80 Vinculin, 146, , 161, , 250, Viral infection, 483 viral particles, 35 Viral replication compartments, 421 Viral rhinitis, 742 Virtual microscopy, 1, 25, Virtual slide, 25 Viruses, and junctional complexes, 142–144, 148 Visceral epithelial cells, of Bowman capsule, 776 Visceral ganglia, 419 Visceral layer, of Bowman capsule, 776, 806 –807 Visceral layer of serous pericardium, 445, Visceral motor (efferent) neurons, 390, 418, Visceral pleura, 754, 764 –765 Visceral sensory (afferent) neurons, 390, 416, 418 Visceral striated muscle, 344–345 Visfatin, 281, 286 Visible mucus, 639 Vision accommodation in, 981, 982, 1003 adaptation in, 981 color sensitivity in, 995, 1000 light sensitivity in, 995 process of, 998–999, stereopsis in, 980 vitamin A and, 693, 702, 998 Visual fields, 980, 992 , 993 Visual pigment/purple (rhodopsin), 702, 998 Visual processing, 998–999, Vitamin A, 693, 702, 998 Vitamin B 12 , 644 Vitamin B 12 deficiency, 306 , 641, 641 , 644 Vitamin B 2 (riboflavin), 488 Vitamin C, 185, 265 Vitamin D, 693, 695, 769, 769 , 839 deficiency of (rickets), 265 , 693, 769 regulation of, 839 renal regulation of, 769, 769 Vitamin E, 693

f f 147

161

f

253

25

f

p

p p 445 418

p

t

f

999

f

999

f

f

f

f

f

f

f

Vitamin K, 693 Vitamin K–dependent proteins, bone-specific, 239 Vitamin(s), hepatic storage and metabolism of, 693, 702 Vitreous body, 982, , , 1004 Vitreous chamber, 982, , 1010 –1011 Vitreous humor, 982 Vocal folds, 189, , 736–737, , , 760 –761 Vocalis muscle, 736, Voice production, 736–738 Volkmann canals, 242, , 245, 270 –271 Voltage-dependent anion channels, 62, 63, Voltage-gated Ca 2+ channels, 399, 594, Voltage-gated ion channels, 38 Voltage-gated K + channels, 414 Voltage-gated Na + channels, 356, 359, 400, 414 Voltage-sensitive Ca 2+ channels, 596 Voltage-sensitive Na + channels, , 596 Voltage-sensor proteins, 356, 359, , 365 Volume receptors, 450 von Ebner (lingual salivary) glands, 592, 608, 624 –625 von Willebrand factor, 457 Vulva, 908, 939–941 Vulvovaginitis, 945

982 983 982 732 738 242

p p 737 738 p

595

p

p

p 63 595

359

p

f

W

Waldeyer tonsillar ring, 589 Wallerian degeneration, in nerve injury, 426–429 Wandering cell population, of connective tissue, 197 Warts, genital, 950 Washing, of tissue specimen, 2 Water barrier, of epidermis, 541, 542, 543, Water channels (aquaporins), 785, 791 ADH action on, 791, 791 , 825 in gallbladder, 711 in glomerular capillaries, 775 in high endothelial venules, 509–510 in water homeostasis of brain, 425 Water homeostasis, 425, 542, 825 Water reabsorption, 660, 784–786 Wave of mineralization, 261, 606 “Wear and tear” pigment, 82 Weibel–Palade bodies, 457, 466 Weight regulation, 283–285 Wet age-related macular degeneration, 992 Wharton (submandibular) duct, 589 Wharton’s jelly, 177,

f

f

178

544

f

f

p

Wharton’s jelly mesenchymal stem cells, 177 White adipocytes, 281–285, , 292–293 White adipose tissue, 281–285, 296 –297 features of, 292 function of, 281 regulation of, 283–285 structure of, 283, transdifferentiation of, 292–293 White blood cells (WBCs). Leukocyte(s) White matter brain, 421, 436 –437 spinal cord, 422, , 440 –441 White muscle fibers, 346 White pulp, of spleen, , 532 –533 White-to-brown transdifferentiation, in adipocytes, 291 Wilms tumor 1 gene , 863 Wilms tumor, familial, 863 Wilson ATPase, 694 Wilson disease, 694 Wirsung, duct of, , 711–712, , 713 Wisdom teeth, 597 Wolffian duct, 880–882, , 885 Wolfring, glands of, , 1007 Wolman disease, 51 Wound repair (healing), 99, 197, 200, 202 , 206, 312, 572 Woven bone, 242, 254, 1 gene, 863

282

t

283

p

p

See

p 423 p p 516 p p (WT-1)

707 f

WT-

X

p

f

881 1005

711

f

254

f

X91 disease, 311 X chromosome, 90, 91, , Xanthomatosis, familial, 51 Xenobiotics, 694 XY female embryo with gonadal dysgenesis, (Swyer syndrome), 863

Y

91 865 f

865

Y chromosome, 90, Yellow bone marrow, 241, 333 Yolk-sac phase, of hemopoiesis, 321, Young’s syndrome, 139

f

Z

Z line (Z disc) cardiac muscle, 363,

364

323

347 1005

349 352 355

skeletal muscle, , 348–349, , , Z matrix, 349 Zeis glands, 566, , 1006 Zellweger syndrome, 66 ζ-Tubulin, in MTOC, 77 ZO-1 protein, 143 , 144, 149 ZO-2 protein, 143 , 144 ZO-3 protein, 143 , 144 Zollinger-Ellison syndrome, 642 , 716 Zona glomerulosa, 781, 843–844, , 858 –859 Zona pellucida, 911, , , 920–921, 957 –958 Zona reaction, 921 Zona reticularis, , 843, 846, 858 –859 Zone 1, of liver acinus, 697 Zone 2, of liver acinus, 697 Zone 3, of liver acinus, 697 Zone of calcified cartilage, 257, , 276 –277 Zone of hypertrophy, 257, , 276 –277 Zone of proliferation, 257, , 276 –277 Zone of reserve cartilage, 257, , 276 –277 Zone of resorption, 257, , 276 –277 Zonula adherens, , 145–149, , 152 composition of, 146–148 electron microscopy of, , 148 function of, 146 Zonula occludens, , 141–145, , 152 bicellular, 142 permeability of, 145 proteins of, 142–144, 143 strand formation, 142–144 transport across, 144–145, tricellular, 142, , 145 viruses exploiting, 142–144, 148 Zonular fibers, of eye, , , 990–991, 1003, Zonule of Zinn, 982 ZP (zona pellucida) glycoproteins, 911 Zygote, 102, , 920–921, 930 Zygotene, , 102 Zymogen(s), 648 , 659 Zymogen granules, 40, 610, 642, , , 713

t t t

811

912 913

140

140

100

917 f

844

p

p

258 258 p 258 p 258 258 p 147 147 143 t

142

f

144

987 988

p

p

p

p

p p p p p p p t

f

642 712

t

1003