Biology of the Uterus 9781468422733, 9781468422719, 1468422731, 1468422715, 9781468455892, 1468455893

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BIOLOGY OF THE

UTERUS

BIOLOGY OF THE

UTERUS Edited by

RALPH M. WYNN University of Illinois at the Medical Center Chicago, Illinois

PLENUM PRESS· NEW YORK AND LONDON

Library of Congress Cataloging in Publication Data Wynn, Ralph M Biology of the uterus. First ed. published in 1967 under title: Cellular biology of the uterus. Includes bibliographies. 1. Uterus. I. Title [DNLM: 1. Uterus - Anatomy & histology. 2. Uterus - Physiology. WP400 B615] QP265.W9 1976 599'.01'6 76-46382 ISBN-13: 978-1-4684-2273-3 e-ISBN-13: 978-1-4684-2271-9 DOl: 10.1007/978-1-4684-2271-9

Second edition of Cellular Biology of the Uterus

© 1967,1977 Plenum Press, New York Softcover reprint of the hardcover 1st edition 1977 A Division of Plenum Publishing Corporation 227 West 17th Street, New York, N.Y. 10011 All rights reserved No part of this book may be reproduced, stored in a retrieval system, or transmitted in any form or by any means, electronic, mechanical, photocopying, microfilming, recording, or otherwise, without written permission from the Publisher

Contributors

L. L. Anderson

University of London London, England

Department of Animal Science Iowa State University Ames, Iowa

J.

Randall L. Given Department of Human Anatomy University of California Davis, California

R. G. Challis Department of Obstetrics and Gynaecology University of Oxford, John Radcliffe Hospital, and Nuffield Institute for Medical Research Oxford, England

E. S. E. Hafez Reproductive Physiology Laboratories C. S. Mott Center for Human Growth and Development Wayne State University School of Medicine Detroit, Michigan

Allen C. Enders Department of Human Anatomy University of California Davis, California

Gabriel Hamoir Muscle Research Laboratory Institut Van Beneden University of Liege Liege, Belgium

Alex Ferenczy Department of Pathology McGill University and Gynecological Pathology and Cytology Laboratories of the Jewish General Hospital Montreal, Quebec, Canada

C. Y. Kao

Department of Pharmacology State University of New York Downstate Medical Center Brooklyn, New York

Colin A. Finn Department of Physiology Royal Veterinary College v

VI CONTRIBUTORS

Samuel S. Koide Bio-Medical Division The Population Council Rockefeller University New York, New York Hans Ludwig Department of ObstetricsGynecology University of Essen School of Medicine Essen, West Germany Edgar L. Makowski Department of Obstetrics and Gynecology University of Colorado Medical Center Denver, Colorado Kenneth W. McKern~ Department of Obstetrics and Gynecology University of Florida College of Medicine Gainesville, Florida Harland W. Mossman Department of Anatomy University of Wisconsin Madison, Wisconsin Ronan O'Rahilly Carnegie Laboratories of Embryology Department of Human Anatomy University of California Davis, California Elizabeth M. Ramsey Department of Obstetrics and Gynecology

University of Virginia School of Medicine Charlottesville, Virginia

J.

S. Robinson Department of Obstetrics and Gynaecology University of Oxford, John Radcliffe Hospital, and Nuffield Institute for Medical Research Oxford, England

William Scher Department of Cell Biology Mount Sinai School of Medicine New York, New York Sheldon J. Segal Bio-Medical Division The Population Council Rockefeller University New York, New York C. F. Shoen berg

Department of Anatomy University of Cambridge Cambridge, England C. D. Thorburn Department of Obstetrics and Gynaecology University of Oxford, John Radcliffe Hospital, and Nuffield Institute for Medical Research Oxford, England Ralph M. Wynn Department of Obstetrics and Gynecology The Abraham Lincoln School of Medicine University of Illinois Chicago, Illinois

Preface

In the decade following the publication of the first edition of Cellular Biology of the Uterus, advances in this field have been so rapid as to require not merely a revision of the earlier text but an essentially new volume. Even the title of the book has been changed, to Biology of the Uterus, to reflect the incorporation of more material based on classical anatomy and physiology. This histological and embryological information provides a necessary, though often lacking, background for the protein chemist and molecular biologist, and a bridge between biochemistry and biophysics, on the one hand, and clinical medicine, on the other. Thus, major practical problems in human reproduction, such as the mode of action of contraceptive agents and the cause of the initiation of labor, may be approached on a firm scientific footing. This text deals primarily with the biology of the uterus itself (comparative and human) rather than with placentation or pregnancy, and as such is a synthesis of data derived from many techniques, conventional and modern. Inasmuch as it is clearly beyond the competence of anyone scientist to prepare such a text on the basis of personal knowledge and experience, the aid of distinguished biologists from this country and abroad was enlisted. All of these authors, acknowledged experts in their respective fields, agreed to extensive revision of their chapters or preparation of entirely new contributions. A scholarly history of uterine biology has been added to illustrate the evolution of studies from superstitution to speculation to science. New material on comparative anatomy and an entirely new chapter on embryology of the human genitourinary tract, together with extensively revised chapters on vascular anatomy and physiology, precede the discussions of cell biology, which are emphasized in the subsequent chapters of the text. Comparative embryology of the miillerian derivatives provides the basis for predicting and analyzing the cellular response of the uterus to hormones. The chapters on vascular anatomy and physiology illustrate the dominant role of blood vessels in critical uterine functions, such as menstruation and pregnancy, and the effects on uterine blood flow of hormones, hypoxia, hyperoxia, and myometrial contractions. VII

Vlll

PREFACE

The endocrine regulation of uterine function is discussed in two chapters that describe the genetic control of synthesis of nucleic acids and proteins. The mode of action of steroidal hormones is related to the synthesis of enzymes and the associated production of the major types of ribonucleic acids and proteins. The manifold effects of estrogens on cellular structure and function are discussed in terms of molecular interaction at multiple sites of hormone receptors, and new information about the function of cyclic adenosine monophosphate and the role of lysosomes in uterine activity is added. The chapter on delayed implantation has been extensively revised and expanded to include a discussion of the uterus during early implantation, with detailed data derived from studies of marsupials and six orders of eutherian mammals. The chapter on decidualization has been replaced by a somewhat broader discussion of the implantation reaction, including experimental techniques and hormonal sensitization of the endometrium for implantation. Contributions of electron microscopy to the understanding of several fundamental problems in reproductive biology are well documented. The deciduoma is a virtually unique example of rapid histogenesis in a normal adult mammalian organ, and delayed implantation strikingly illustrates the mediation of the developmental rate of one organism (the unimplanted blastocyst) by the internal environment (the uterus) of another. The next two, entirely new, chapters present information based on techniques that were insufficiently developed in this field to justify separate chapters ten years ago. Scanning electron microscopy depicts details of the endometrial surface associated with ciliary function and glandular secretion, amplifying morphological data derived from other techniques. The chapter on transmission electron microscopy of the human endometrium deals with both basic problems, such as the origin and significance of the nucleolar channel system and the function of the decidua, and clinical applications, such as the effects of contraceptives and the cause of menstruation. The chapters on biochemistry, ultrastructure, and physiology of the myometrium have undergone extensive revision. The need for collaboration between the electron microscopist and the biochemist in elucidating the contractile mechanism of the myometrium is amply demonstrated. Although much more is known about skeletal than smooth muscle, the major data concerning the provision of energy and the proteins of the contractile mechanisms of mammalian myometrium are presented. The organelles of smooth muscle are discussed in detail and correlated with structural changes in the myometrium during immaturity, adulthood, and senility. The chapter on electrophysiology of the myometrium focuses on patterns of ionic distribution and resting potential and their modification by hormonal and gestational inf1uences. This chapter demonstrates shortcomings of a popular clinical explanation for the change in myometrial activity at the onset of labor. A new chapter on ultrastructural pathology provides criteria for the identification and classification of endometrial hyperplasia and neoplasia. The last two chapters, also new, discuss the uterine regulation of ovarian function and the endocrine control of parturition. Effects of hysterectomy and intrauterine devices on ovarian function, and luteolytic action of the uterus are analyzed. The effects of ovarian and adrenal steroids, prostaglandins, and oxytocin on uterine activity in six species including man are discussed. Although the emphasis of the volume as a

whole is not on pregnancy, this final chapter is obviously relevant to experimental biologists, endocrinologists, and obstetricians. Although each of the chapters is a self-contained unit, which provides a critical review and a comprehensive list of references for the graduate student, the intended interdisciplinary orientation is best achieved by reading the chapters in the order in which they appear in the book. The volume is designed to serve as an introduction of the academic clinician to the scientific foundations of reproduction and as a suggestion to the basic scientist of significant applications, which may be particularly pertinent in an era of "targeted" research. If new or continued productive collaborations between the laboratory scientist and the clinician are stimulated by this book, the efforts of the authors and the editor will be justified.

Chicago, Illinois

Ralph M. Wynn

IX PREFACE

Contents

1 History

1

ELIZABETH M. RAMSEY l. 2. 3. 4. 5. 6. 7. 8.

Greece Alexandria Rome. The "Dark Ages" Renaissance Sixteenth to Eighteenth Centuries Embryology and Microscopic Anatomy References

2 Comparative Anatomy

1

2 4 5 6 lO

14 18

19

HARLAND W. MOSSMAN l. 2. 3. 4.

5. 6.

Types of Uteri Distribution and Probable Evolution of Uterine Types Correlations of Uterine Types with Other Biological Features Miscellaneous Aspects of the Comparative Morphology of the Uterus Summary and Conclusions References

3 Prenatal Human Development

22 23 27 30 32 33

35

RONAN O'RAHILLY 1.

Urinary Preliminaries

36 Xl

XlI CONTENTS

2. 3. 4.

The Paramesonephric Ducts Fetal Development References

4 Vascular Anatomy

37 45 55

59

ELIZABETH M. RAMSEY 1.

2.

3. 4.

Menstrual Cycle 1.1 Distribution of Vessels 1.2 Histology Pregnancy 2.1 Anatomy 2.2 Histology Conclusion References

5 Vascular Physiology

60 60 64 66 66 68 74 75

77

EDGAR L. MAKOWSKI 1.

2.

3. 4.

Measurement of Uterine Blood Flow 1.1. Steady-State Diffusion . 1.2. Microsphere Technique 1.3. Electromagnetic Flowmeter Physiological Observations 2.1. Pressure-Flow Relationship 2.2. Distribution of Uterine Blood Flow 2.3. Reactivity of Uterine Vascular Beds 2.4. Effect of Estrogens on the Uterine Vascular Bed 2.5. Effects of Pregnancy on Uterine Blood Flow . 2.6. Effects of Acute Hypoxia and Hyperoxia on Blood Flow to the Pregnant Uterus 2.7. Effect of Uterine Contractions on Uterine Blood Flow Summary. References

77

77 79 80 82 82 83 84 86 92 96 96 97 98

6 Genetic, Biochemical, and Hormonal Mechanisms 101 in the Regulation of Uterine Metabolism KENNETH W. Mc KERNS 1.

Genetic Control of Metabolism 1.1. Pyrimidines, Purines, Nucleosides, and Nucleotides

102 103

2.

3.

1.2. Replication of DNA 1.3. Structure, Function, and Synthesis of RNA 1.4. Protein Biosynthesis and Enzyme Activity Biochemical Control of Metabolism . 2.1. Glucose Metabolism in Endocrine Glands and HormoneResponsive Tissues 2.2. Sources and Biosynthesis of Hormones Affecting the Uterus 2.3. Regulation of Uterus by Estradiol References

7 Estrogens, Nucleic Acids, and Protein Synthesis in Uterine Metabolism

106 107 113 118 118 125 133 137

139

SHELDON J. SEGAL, WILLIAM SCHER, AND S. S. KOIDE 1. 2.

3. 4.

Review of the Biosynthesis of Ribonucleic Acid and Protein Estrogen 2.1. Transport 2.2. Energy Supply. 2.3. Estrogen Receptor Sites 2.4. Ribonucleic Acid Biosynthesis 2.5. Protein Biosynthesis 2.6. Deoxyribonucleic Acid Biosynthesis 2.7. Adenosine 3' ,5' -Cyclic Monophosphate 2.8. Estrogen and Lysosomes 2.9. Estradiol-Sensitive Uterine Cell Cultures Conclusion References

141 144 144 147 149 160 174 178 180 181 181 182 183

8 The Endometrium of Delayed and Early

Implantation

203

ALLEN C. ENDERS AND RANDALL L. GIVEN 1.

2. 3. 4. 5. 6. 7. 8.

Marsupials Roe Deer. Armadillos Insectivores and Chiroptera Carnivores Rodents Discussion References

204 208 211 215 215 229 235 238

Xlll CONTENTS

XIV

9 The Implantation Reaction

245

COLIN A. FINN

CONTENTS

1.

2. 3.

4.

5. 6. 7. 8.

Preparation of the Endometrium 1.1. Cell Proliferation 1.2. Cell Differentiation Control of Endometrial Preparation 2.1. Hormonal Control of Cell Proliferation 2.2. Hormonal Control of Differentiation of Endometrium Sensitization of the Endometrium for Implantation 3.1. Experimental Techniques. 3.2. Role of Progesterone 3.3. Role of Luteal-Phase Estrogen 3.4. Role of Estrogen Secreted before Ovulation 3.5. Role of the Pituitary and Hypothalamus. 3.6. Mode of Action of Luteal-Phase Estrogen in Inducing Endometrial Sensitivity The Implantation Process . 4.l. Positioning of Blastocysts in the Uterus 4.2. The Attachment Reaction. 4.3. Activation of the Blastocyst 4.4. The Nidatory Stimulus 4.5. Formation of the Implantation Chamber Regression of the Decidua Significance of the Decidua . Concluding Remarks References

10 Scanning Electron Microscopy of the Endometrium

246 246 248 253 254 258 263 264 265 265 269 269 271 271 272 274 276 278 281 292 293 294 295

309

E. S. E. HAFEZ AND HANS LUDWIG 1. 2. 3. 4. 5. 6. 7. 8. 9.

Ciliated Cells . 1.1. Kinocilia 1.2. Solitary Cilia Secretory Cells Endometrial Secretions Endometrial Glands Species Differences Cyclical Variations Effect of Intrauterine Devices Changes during Implantation of Blastocyst Effect of Aging

310 310 310 312 315 317 317 326 329 329 333

10.

1l.

11

Concluding References

Remarks

Histology and Ultrastructure of the Human Endometrium

336 336

341

RALPH M. WYNN

l. 2.

Histology . Ultrastructure 2.l. The Normal Menstrual Cycle 2.2. The Nucleolar Channel System 2.3. The Decidua 2.4. The Arias-Stella Reaction . 2.5. Scanning Electron Microscopy 2.6. Ultrastructural Localization of Enzymes Clinical Correlations . 3.l. Effects of Contraceptive Agents 3.2. Mens truatio n References

341 347 347 356 357 364 365 368 371 371 373 374

12 Biochemistry of the Myometrium

377

3. 4.

GABRIEL HAMOIR

l. 2.

3.

The Biological Unit of Vertebrate Smooth Muscle Energy Provision . 2.l. Hormonal Influence on Glucose Metabolism and Respiration in the Uterus 2.2. Energy Sources 2.3. Lipids, Glycogen, A TP, and PC 2.4. Glycolytic and Respiratory Enzymes 2.5. Metabolism of Smooth Muscle Contraction Proteins of the Contractile Mechanism . 3.l. Nature and Organization of the Contractile Proteins of Striated Muscle 3.2. Extractibility of the Contractile Proteins of Vertebrate Smooth Muscle 3.3. Actomyosin 3.4. Myosin 3.5. Myosin Subunits 3.6. Actin 3.7. Tropomyosin 3.8. The Regulatory Proteins

381 383 383 384 385 388 392 394 394 396 399 400 403 406 407 408

xv CONTENTS

XVI

4.

CONTENTS

5. 6.

Special Characteristics of the Contractile Mechanism of Mammalian Smooth Muscle. 4.l. ATPase Activity 4.2. Solubility of Smooth Muscle Actomyosin at Low Ionic Strength 4.3. Calcium Regulation of Contraction Concluding Remarks References

13 Electrophysiological Properties of the Uterine Smooth Muscle c. l.

2.

3.

4. 5.

14 The Contractile Mechanism and Ultrastructure of the Myometrium 1.

412 412 413 414

423

Y. KAO

Review of Methodology l.l. Comparison of Uterine, Cardiac, and Skeletal Muscles l.2. A Brief Statement of the Ionic Theory of Excitation l.3. Methods of Recording Electrical Activities of the Myometrium Ionic Distribution Patterns and Resting Potential in Myometrium . 2.l. Problems in Analysis of Ionic Contents of Myometrium 2.2. Ionic Contents and Distribution 2.3. Resting Potential and Its Relation to Ionic Distribution . 2.4. Hormonal and Gestational Influences on Ionic Distribution and Resting Potential 2.5. Active Ion Transport Excitation in Myometrium 3.l. Cellular Phenomena 3.2. Ionic Basis of Spike Activity 3.3. Tissue Phenomena 3.4. Contractile Consequences of Electrical Activities 3.5. Actions of Drugs on the Myometrium Summary and Concluding Remarks References

c.

409 409

424 424 426 429 437 437 438 442 450 456 461 461 466 481 483 485 489 491

497

F. SHOEN BERG

The Contractile Mechanism l.l. Skeletal Muscles l.2. Vertebrate Smooth Muscles

498 498 501

2.

3.

4. 5.

l.3. Mechanism of Contraction The Organelles of Smooth Muscle 2.l. Surface Vesicles 2.2. Sarcoplasmic Reticulum 2.3. Mitochondria 2.4. Golgi Apparatus 2.5. Centrioles 2.6. Microtubules 2.7. Microsomes and Glycogen Granules Structural Changes of the Myometrium 3.l. Immature Myometrium 3.2. Adult Myometrium 3.3. Aging Discussion References

15 Ultrastructural Pathology of the Uterus

524 526 526 527 528 529 529 529 529 529 530 530 537 537 539

545

ALEX FEREN CZY l.

2. 3. .4. 5.

Endometrial Morphological Response to Hyperestrogenic Environment . l.l. Persistent (Anovulatory) Proliferative Endometrium and Endometrial Polyp l.2. Cystic Glandular Hyperplasia . l.3. Adenomatous Hyperplasia 1.4. Atypical Adenomatous Hyperplasia Endometrial Neoplasia Endometrial Morphological Response to Hormonal Therapy: Progestin Effect on Hyperplasia and Neoplasia Conclusions References

16 Uterine Control of Ovarian Function

547 548 550 552 564 566 572 582 583

587

L. L. ANDERSON

1.

2.

Ovarian Function 1.1. Corpus Luteum in Rat l.2. Corpus Luteum in Rabbit l.3. Corpus Luteum in Ewe and Sow Uterine Function 2.l. Development and Regression of Endometrium and Myometrium 2.2. Role of the Uterus in Cyclic Periodicity . 2.3. Uterine-Ovarian Function during Pregnancy

587 587 590 591 592 592 595 607

XVll CONTENTS

XVlll CONTENTS

3. 4.

5. 6. 7.

8.

9.

Effects of Hysterectomy on Ovarian Function . Luteolytic Action of the Uterus . 4.1. Amount of Uterus Required for Luteolysis 4.2. Local Luteolytic Action 4.3. Luteolytic Effects of Estrogens in the Ewe Ovarian Autotransplantation Uterine Transplantation . Intrauterine Devices and Ovarian Function 7.1. IUD in Rat and Rabbit 7.2. IUD In Ewe 7.3. IUD in Monkey and Woman Other Hormones Affecting Uterine-Ovarian Function 8.1. Prostaglandins 8.2. Relaxin. 8.3. Oxytocin References

17 Endocrine Control of Parturition

615 619 619 619 620 621 622 622 623 624 625 626 626 631 632 633

653

G. D. THORBURN, J. R. G. CHALLIS, AND J. S. ROBINSON 1. 2.

3.

4.

5.

Observations Implicating the Fetus in the Control of Parturition Parturition in the Sheep . 2.1. Early Experimental Evidence for the Role of the Fetus in the Initiation of Parturition 2.2. The Fetal Adrenal, Cortisol, and Parturition 2.3. The Trigger Mechanism 2.4. Hormonal Changes during Pregnancy Parturition in the Goat 3.1. Progesterone 3.2. Prostaglandins 3.3. Estrogens 3.4. Induction of Premature Parturition by PGF2a Infused into the Uterine Vein 3.5. Estradiol-Induced Premature Parturition Parturition in the Rabbit 4.1. Progesterone 4.2. Estrogens 4.3. Prostaglandins 4.4. Cortisol 4.5. Effects of Exogenous Glucocorticoids 4.6. Uterine Activity Parturition in the Guinea Pig 5.1. Progesterone 5.2. Estrogens 5.3. Cortisol

654 655 655 655 656 659 672 673 675 676 678 680 681 682 684 684 686 687 687 688 688 690 690

6.

7.

8.

9.

5.4. 5.5. 5.6. 5.7. 5.S.

Oxytocin Prostaglandins Uterine Activity and Relaxin Surgical Manipulations Comment Parturition in the Rhesus Monkey 6.l. Hormone Levels in Normal Pregnancy 6.2. Uterine Activity 6.3. Surgical Manipulations 6.4. Effects of Exogenous Compounds 6.5. Prostaglandins . 6.6. Dexamethasone 6.7. Comment Parturition in the Human 7.l. The Fetal Adrenal: Spontaneous Adrenal Hyperplasia and Hypoplasia . 7.2. Fetal Adrenal and Cortisol 7.3. Progesterone 7.4. Prostaglandins 7.5. Oxytocin and Vasopressin Concluding Remarks References

Index

691 692 692 693 694 694 695 700 701 703 703 704 704 705 705 706 709 710 712 713 715

733

XIX CONTENTS

1

History ELIZABETH M. RAMSEY

Our present knowledge of uterine structure and function has been achieved, slowly and laboriously, over the course of many centuries. Only bit by bit have facts emerged through the obscuring mists of superstition, tradition, and speculation. Since the human uterus, at least in its gross anatomy, is not a particularly complicated organ, one may wonder why anyone who had once held a uterus in his hand and perhaps made a simple sagittal section through it would have failed to grasp its pattern. That of course is the crux of the matter. The early physicians did not hold the uterus in their hands; many never even set eyes upon one. Religion and law both forbade dissection of human bodies until surprisingly recent times and all concepts of reproductive tract anatomy were based on findings in animals. Since most of the animals observed had duplex or bicornuate uteri, extrapolation to the human produced many erroneous and bizarre theories.

1.

Greece

In tracing the development of medical concepts, it is customary to regard the Golden Age of Greece as the beginning of "modern" medicine. This is an appropriate starting place for consideration of the history of the uterus, too. What then is to be found on this subject in the Hippocratic Corpus, which embodies the thought of the great Father of Medicine and his contemporaries in the fourth century B.C.? At that time, the uterus was believed to consist of a number of cavities exhibiting angulations and horns, its lining studded with "tentacles" or ELIZABETH M. RAMSEY . Department of Obstetrics and Gynecology, University of Virginia School of Medicine, Charlottesville, Virginia.

1

2 CHAPTER 1

"suckers." Tubes and ovaries were not identified or even mentioned. On the other hand, external portions of the reproductive tract which were accessible to inspection, the perineum, the vagina, the cervical os, and the labia, were carefully noted and described in terms still recognizable. The extensive studies of Aristotle fell within the same period. We tend to think of Aristotle as a philosopher rather than a scientist, but in classical times learned men were versed in all fields of knowledge, and Aristotle was the greatest biologist of his era. His studies dealt exclusively with animals and he frankly stated that he knew nothing of the reproductive tract of man. His concept of the uterus as bicornuate was arrived at by analogy with the animals he had studied and so too was his concept of "cotyledons" within the uterus, similar to those of cattle (cf. the Hippocratic "tentacles"). Unfortunately, Aristotle's drawings of the reproductive organs have been lost, for we may be sure that they would tell us a great deal about his ideas. An example of his careful and perceptive work survives in a sketch of a dogfish embryo attached to the maternal brood pouch. In it, incidentally, we can see a striking similarity to the condition of the mammalian embryo in utero. Aristotle's research in an indirectly related field, that of the development of the chick embryo, should be mentioned because it laid the basis for embryological investigation for centuries to come. Subsequently, as other types of embryos became available for study, investigators used the classic chick terminology to describe them, often causing confusion, some of which persists to our day.

2.

Alexandria

When the torch of intellectual eminence passed from Greece to Alexandria in Egypt, around the beginning of the Christian era, many Greek physicians journeyed to that great center. Here, for a time, dissection of human bodies was permitted, specifically those of executed criminals. The trio consisting of HerophiIus of Chalcedon and Rufus and Soranus both of Ephesus characterizes this era (Table 1). The first two continued the tradition of a bicornuate uterus but identified uterine tubes as separate entities. Herophilus thought that the tubes entered the urinary bladder but Rufus corrected this misconception. Both identified the ovary but were unaware of its function. Rufus modified earlier opinions of the shape of the uterus by describing it as similar to a "cupping vessel" and he differentiated a fundus with two cornua from the cervix, and both from the vagina. Soranus, who was especially cognizant and appreciative of his indebtedness to earlier workers and in his own turn profoundly influenced his successors until well into the sixteenth century, reminds us how knowledge and understanding were slowly growing as each successive investigator "stood upon the shoulders" of his predecessors. Soranus' Gynecology was one of the great works of the early Christian centuries (available to us in a fine translation by Owsei Temkin of the Johns Hopkins Department of Medical History). Although it deals chiefly with the clinical matters of Soranus' primary interest, it does incorporate the findings of his anatomical studies. The work is not illustrated, but a drawing in a ninth-century manuscript depicts the uterus as Soranus conceived it (Fig. 1). Shaped like a

3

Table 1. Chronology

Century

Practitioner

Birth and death (active)

Place of birth; training, activity

B.C.

Hippocrates Aristotle Herophilus of Chalcedon

460-377 384-322 (300)

Greece Greece Greece; Alexandria; Rome

First

Rufus of Ephesus

(98--117)

Second

Soranus of Ephesus

Greece; Alexandria; Rome Greece; Alexandria; Rome Pergamon, Asia Minor; Alexandria; Rome

Fourth

A.D.

Galen Ninth to twelfth Fourteenth Fifteenth

(110) 130-?200

Sixteenth

School of Salerno Mondino dei Luzzi Leonardo da Vinci Berengario da Carpi Vesalius

Seventeenth

Colombo da Cremona Eustachio Fallopio da Modena William Harvey

1516-1559 1520-1574 1523-1562 1578-1657

Marcello Malpighi Regnier de Graaf Caspar Friedrich Wolff John and William Hunter Johannes Peter Muller Hitschmann and Adler Robert Schroeder

1628-1694 1641-1673 1733-1794 (1774) 1801-1858 (1908) (1930)

Eighteenth Nineteenth Twentieth

1375-? 1452-1519 ?1480-1550 1514-1564

Italy Italy Italy Brussels; Louvain and Paris; Padua Italy Italy Italy England; Cambridge and Padua; London Italy Holland Germany England Germany Germany Germany

cupping vessel, as Rufus said, it has recognizable parts with modern names. Internally, Soranus described "two folds" in the fundus of nulliparae which disappear when the uterine cavity becomes "rounded and stretched" in pregnancy. Despite many progressive opinions, Soranus entertained some curious misconceptions. Thus he considered the cervix of girls before puberty to have a spongy consistency similar to the lungs and agreed with Herophilus that the tubes enter the urinary bladder (perhaps a confusion with the round ligaments?). He supported Aristotle's idea of cotyledons, regarding the nipplelike projections as provisions for intrauterine suckling to accustom the fetus to a function in which he would need to be proficient at birth. Soranus advanced the understanding of the function of the ovary to some degree but called the organ a female testis. His successors copied him in this idea down to the seventeenth century. The Gynecology is particularly distinguished by its tone of dispassionate objectivity. Soranus fought against superstition and espoused rationalism; he assailed many inherited and contemporary opinions but without rancor; his clinical histories were refreshingly

HISTORY

4 CHAPTER 1

clear in an age of verbosity and circumlocution. Indeed, they often sound more like the nineteenth than the second century A.D.

3.

Rome

Following the decline of Alexandria as an intellectual center in the first century of the Christian era, Rome became the lodestone for physicians as well as for other men of learning. The life and work of Galen may be regarded as spanning the period of this shift and as, in many ways, Rome's finest and most typical product. Galen did not have Soranus' special interest in gynecology, but his treatises included observations on all aspects of the human body and its normal and pathological function, so the reproductive tract received a share of his attention. As far as the uterus is concerned, he adopted the opinions of two of his predecessors: Rufus' belief that the tubes are connected with the uterus rather than the urinary bladder, and Aristotle's belief that the endometrial surface is

R

ORl

Figure 1. Earliest known representation of the anatomy of the uterus. It embodies Soranus' conception of the organ and appears a Muscio text of the ninth century. From Weindler (1908). Courtesy of the National Library of Medicine, Bethesda, Md.

cotyledonary. But to each he made additions of his own: to Rufus' that the tubes have large, patent lumina through which the ova are conducted to the uterine cavity, and to Aristotle's that the vessels supplying menstrual blood open into the crypts of the cotyledons. The animal background of his work led to the opinion that the uterus is multilocular-this although the monkey was one of the animal species he investigated. The great name of Galen was perhaps too great, for slavish adherence to his opinions stultified progress for more than 1000 years after his death. "According to Galen" or "as Galen said" provided the final word on all aspects of medical science and clinical practice throughout the Middle Ages. During the period of the Roman Empire, gynecology was an important facet of medical practice, but the invention of surgical instruments and the elaboration of operative techniques were of greater interest than the investigation of morphology and physiology. This preoccupation with practicalities was characteristic of the mode of Roman thought and was paralleled by the fact that organization of the hospital system was Rome's greatest contribution to medicine in general. When Rome in its turn declined, there developed an active and considerable body of Byzantine medicine in Constantinople. However, it was neither original nor creative, but was grounded in the study of ancient documents. In gynecology the major name we remember is that of Oribasius, who dealt almost exclusively with clinical practice, as did his colleagues.

4.

The "Dark Ages"

Throughout Europe the millennium following Galen's death, properly designated the Middle Ages, fully merits its more usual title, the "Dark Ages." The eager enthusiasm, the creative curiosity, the humanism of Greece and Alexandria were gone, replaced by dusty scholasticism and blind dependence on "the authorities." These attitudes were not confined to scientists, of course. In the broad field of philosophy, which covered a multitude of disciplines, "according to Aristotle" was as decisive an answer to all inquiries as "according to Galen" was in medicine. The writings of earlier scientists were copied, revised, interpreted, quoted (and often misquoted) in thousands of volumes. The practice of medicine, guided by inherited dicta, was of greater interest than investigation of its anatomical and physiological bases. The influence of Arabic medicine, reaching Europe at this time via Spain, gave added impetus to scholasticism and traditionalism. Scientific medicine was little advanced by the infusion, and gynecological knowledge hardly at all. In one spot the old spark was kept alive during the dark centuries. At Salerno in Sicily, inquiring spirits congregated and some advances were made-few, however, in the field of human reproduction. For the most part, even here, the era witnessed elaboration of inherited theories and opinions, particularly the theory of the multicompartmented uterus. The old magic number seven was the most favored one and particularly approved was the fanciful idea that male embryos develop in the three right-hand cells, females in the left three, and hermaphrodites in the middle one (Fig. 2). The reams of recorded debates upon

5 HISTORY

6 CHAPTER 1

this doctrine are remlmscent, in their heat and involved reasoning, of the discussions that engrossed theologians at the time as to how many angels could dance on the head of a pin! Another popular anatomical belief was that the female reproductive tract is the mirror image of the male: the vagina, a penis turned inside out: the uterus, analogous to the scrotum; and so forth. None of these theories, of course, was ancestral to present-day knowledge. They are cited only as illustrating the numerous blind alleys along the tortuous path by which that knowledge has been achieved.

5.

Renaissance

Although the fall of Constantinople in 1453 is usually accepted as the beginning of the Renaissance in Europe, with entry of fresh intellectual air and scientific curiosity, there was, of course, no sharp cutoff data for the Middle Ages. There had been numerous evidences of the "Medieval Awakening" long before that specific date. The first founding of universities in the thirteenth century and their subsequent growth and spread throughout Europe were in no small measure responsible. Most of the universities established medical faculties, and teachers and students proliferated. Slowly, dependence on the ancient authorities waned, particularly as study of the human body commenced. For the latter, the year 1315

tgura mattinG tIara ((ffJinalia . .'

.cOUSUI

.comua

Figure 2. A drawing illustrating the doctrine of the seven-chambered uterus. From Hundt, (ISO 1). Courtesy of the National Library of Medicine, Bethesda, Md.

may be considered a landmark date, for in that year at the University of Bologna, Mondino dei Luzzi performed the first authorized public dissection of a human body for scientific purposes in modern times. The burgeoning spirit of the Renaissance in the graphic arts was secondarily reflected in anatomical investigation, an effect both helpful and delightful to us in our present consideration. As never before, medical texts were now illustrated, often by skilled and famous artists. A full series of plates might be assembled describing, without a single word of text, the development of knowledge of the reproductive tract in these centuries. Typical examples in Figs. 3-9 show the feeling for artistic representation as well as for the scientific concepts that the writers wished to show. Thus in Mondino's drawing (Fig. 3) we see a uterus with cervix and vagina much as we know it. Tubes and ovaries are not shown, but we do see the lower ends of the vessels thought to convey menstrual blood to the mammary glands, there to be converted into milk in pregnancy. Mondino still believed that the interior of the uterus was divided into compartments, but he introduced the radically new idea that the organ is fixed and does not migrate in the abdominal cavity, as was previously maintained. Chronologically, Leonardo da Vinci comes next, and in his famous sketches are reflected the current ideas of his day as well as his own. In the drawing in Fig. 4, ovaries, tubes, and ligaments are depicted. In Fig. 5, the full extent of the uterus-to-breast vessels is clearly shown. The uterus appears to be lobular, though not actually divided, and from the beautiful drawing in Fig. 6 it can be confidently deduced that Leonardo considered the cavity of the pregnant uterus to be single. The very modern appearance of the uterus in this drawing may be compared with that of Rymsdyk's drawing for Hunter (Fig. 11) made nearly 300 years later. Almost contemporary with Leonardo was Berengario da Carpi, one of whose drawings is reproduced in Fig. 7. Here and in his text Berengario is unequivocal in statement of his belief that the uterus has a single chamber. "Es purum mendacium dicere ... "-it is a pure lie-to say that the uterus has seven cavities. This statement is reiterated by his equally dogmatic contemporary, Nicola Massa, who expressed it "Decepti sunt etiam ... "-deceived are they also-who believe that the uterus contains several cells, indeed there is only one. Long years of argument about this matter would thus seem to be definitively terminated, but some years later Dryander published a charmingly naive drawing (Fig. 8) in which a dimple at the fundus of the uterus denotes the presence of a septum dividing the cavity. Old ideas die hard! With Vesalius we reach one of the high points of Renaissance anatomy. He was born in Brussels, educated at Louvain and Paris, and eventually settled in Padua, and his career demonstrates both the commanding position of Italy at this time and the internationalism of science. The latter feature is reinforced by the record of students flocking to Vesalius' lectures and demonstrations from every country in Europe, including Britain, Poland, and Russia. In 4 years at Padua, on the basis of personally conducted anatomical studies, Vesalius prepared his epochmaking work, De humani corporis fabrica. Nothing in anatomical science has been the same since that publication, any more than cosmology has been the same since publication of Copernicus' De revolutionibus orbium coelestium. Interestingly enough, both treatises appeared in the same fateful year, 1543.

7 HISTORY

8 CHAPTER 1

Figure 3. Female genital tract according to Mondino as illustrated in a 1541 German translation of

his work. Note inde ntation at fundus of uteru s, indicating compartmentalization. From Mondino dei Lu zzi, per J. Dryandrum (1 54 1). Courtesy of the National Librar y of Medicine, Bethesda, Md.

We know that Vesalius dissected the female cadaver; the frontispiece of the Fabrica shows him presiding over such a dissection. He was clear about the size and shape of the uterus and about its single cavity, though he agreed with Galen that a septum partly divides it. He corrected opinion as to the shape and course of the adnexa and identified the muscular and decidual layers of the uterine wall. The terms "uterus" and "pelvis" were first used in his treatise, though he still called the ovaries the female testicles (Fig. 9). Three distinguished pupils of Vesalius' carried on the progress that he initiated. Colombo da Cremona described and named the labia and vagina and was the first to record a case of congenital absence of both uterus and vagina. Eustachio studied the vasculature of the whole body and one illustration in his Atlas includes an admirable drawing of the blood vessels of the pelvis (Fig. 10). Fallopio da Modena made the definitive description of the uterine tubes which we know by his name, though he disclaimed priority, remembering Herophilus' and Rufus' description some 1800 years earlier. Fallopio gave the "aqueous humor of the ovary" its modern name "corpus luteum" and described the hymen and clitoris correctly and noted that it is the integrity of the uterine ligaments which prevents uterine prolapse.

I

,;;"l(tI' -M.~ j;"~

1/""-'

.

..

~".

.~

Figure 4. Uterus and adnexa in a drawing by Leonardo da Vinci. From the Quademi d'Anatomia (1913). Courtesy of the National Library of Medicine, Bethesda, Md.

9 HISTORY

10

6.

Sixteenth to Eighteenth Centuries

CHAPTER 1

By the end of the sixteenth century the conditions outlined at the beginning of this chapter had been fulfilled in all essentials. Men had held the human uterus in their hands, had made sagittal and many other sorts of sections through it, and were clear about its gross morphology. Major attention now turned to other

,1Cf~· "

1N~

.... ~

,

:'.1

tltlllJ Figure 5. Organs of the female genital tract as drawn by Leonardo da Vinci. The uterus is lobular though not subdivided. The "milk vein" is clearly shown. From the Quaderni d'Anatomia (1913). Courtesy of the National Library of Medicine, Bethesda, Md.

11

-~.

HISTORY

.....~.htrCl'r

\): .;".I;!,.

"." , •• ,1, .. -.

.

\ j

._.... /- ) '

V

rq't;1 ,,!1~,m"~ "'~r'

.",', ... " tf~.J, I}

'-I'IT

I.: A

, t '"

" "

'.

Itrt .. ".....,. "'I'

.

-'t . ", '

Figure 6. Drawing by Leonardo da Vinci of an opened uterus with fetus in situ. The rim of the placenta and a coil of the umbilical cord are seen. From the Quaderni d'Anatornia (1913). Courtesy of the National Library of Medicine. Bethesda, Md.

components of the reproductive tract. To consider such important contributions as de Graafs well-known work on the ovary or Harvey's revolutionary concept, omne ex ova (all life comes from the egg), would carry us too far afield. The placenta, however, is closely enough allied to the uterus to justify a word about the work of John and William Hunter, who settled old problems about its nature and blazed new trails in the understanding of the mechanism whereby it acts as a nutritive and excretory pathway for maternal-fetal exchange. In particular, they scotched the old idea that maternal and fetal vessels are anastomosed end to end in the placenta, rendering the two bloodstreams continuous. They showed conclusively that there is maternal blood in the intervillous space and demonstrated the "curling arteries" of the endometrium (Figs. 11 and 12).

12

~-

CHAPTER 1

iL!~>:~~p~~~.~~:~~~~.~. :~;~~;:.w:

~.~~;,;;.f7r,~t:>~~:;~;;;;;;;;~;;;;;;;;;;~~~~~~~~~~~.

.. ::.;..

Figure 7. Female genital tract as illustrated by Bere ngario da Carpi. Note clear representation of the single cavity in the uterus. From Berengario (1521). Courtesy of the National Library of Medicine, Bethesda, Md.

13 HISTORY

Figure 8. Female genital tract shown by Dryander. The "dimple" at the fundus of the uterus

denotes the presence of a septum dividing the cavity. From Dryander (1547). Courtesy of the National Library of Medicine, Be th es da, Md .

14 CHAPTER 1

7.

Emlnyology and Microscopic Anatomy

Meanwhile, study of the embryology and histology of the reproductive tract was gaining momentum, made possible by development of a practical microscope and techniques of tissue fixation and staining. The prolonged influence of Aristotle's studies of avian embryology has been mentioned. By the middle of the sixteenth century, direct observation of mammalian embryos was under way, using relatively crude, simple lenses. The perfected compound microscope came two centuries later. The primary goal of the early embryological studies was to resolve the conflict between the theories of preformation ("a fully formed embryo exists in

V fG E·

Figure 9. Vesalius' illustration of the female genital tract. From Vesalius (1543). Courtesy of the

National Library of Medicine, Bethesda, Md.

15 HISTORY

.

1 's

TAlI, X

[I.

Figure 10. Drawing of the renal and reproductive tract circulations by Pini for Eustachio. Published 1761 by Albini. Courtesy of the National Library of Medicine, Bethesda, Md.

16 CHAPTER 1

the ovary") and epigenesis ("there is a gradual building up of parts"). Our present knowledge that the latter is in fact the case recognizes the work, among a great many others, of such men as Malpighi, a successor to Mondino in the chair of medicine at Bologna; Wolff, whose name is familiar as the eponym of the mesonephros and mesonephric ducts; and Miiller, whose name stands in similar relation to the paramesonephric ducts and therefore in closest possible relation to the uterus itself. The story of uterine microscopic anatomy has been built up very rapidly in comparison with the long history of gross anatomy from the time of Hippocrates to, say, Vesalius. The nineteenth and twentieth centuries in particular have seen an almost explosive growth of knowledge in this field. No development has been more dramatic and far-reaching than Hitschmann and Adler's demonstration of the hormone-controlled cyclic activity of the endometrium, which Robert. Schroe-

Figure 11. William Hunter's preparation of a uterus in the fifth month of pregnancy. Drawing by Rymsdyk. From Hunter (1774). Table XXII, Fig. I. Courtesy of the National Library of Medicine,

Bethesda, Md.

17

A.

HISTORY

:Fig. 11. .

G

;.. .

.-

u

11

Il

Il

t.. r

, '.

'"

.......... II

... ,.......

";

.""". .l1\

Figure 12. William Hunter's demonstra tion of curling arte ries of the endometrium in an injected specimen. CA , curling arte ries. Drawing by Rymsdyk. From Hunter (1774) . Table XXIlI, Fig. II. Courtesy of the Natio nal Libra ry of Medicine, Be th esda, Md.

der subsequently systematized into diagnostic stages (Chapter 11). It is hard to realize that well under 100 years ago all the histological appearances characteristic of those stages were lumped together as pathological conditions under the term "chronic endometritis." The electron microscope (Chapters 8--11, 14, and 15) is now opening new horizons fully as dramatic as those revealed to the first investigators who used the compound microscope. It is profitable to remember, however, that history is a continuum and that it is upon the foundations laid by the anatomical, embryological, and histological greats of past centuries that our present-and our futureunderstanding of the structure and function of the uterus is built. Indeed, everything presented in the following chapters of this book is part of the continuing history of the uterus.

18 CHAPTER I

8. I.

References Standard texts on the history of medicine. These works supply background and basic information about individuals.

Castiglioni, A., 1958, A History of Medicine, tr. and ed. by E. B. Krumbhaar, 2nd ed., Knopf, New York. Garrison, F. H., 1961, An Introduction to the History of Medicine, 4th ed., Saunders, Philadelphia. Mettler, C. C., and F. A. Mettler, 1947, History of Medicine: A Correlative Text Arranged according to Subjects, Blakiston, Toronto. Singer, C., and E. A. Underwood, 1962, A Short History of Medicine, 2nd ed., Clarendon Press, Oxford. II.

Histories of the reproductive tract.

Barbour, A. H. F., 1887-1888, Early contributions of anatomy to obstetrics, Trans. Edinburgh Obstet. Soc. 13: 127-154. Peillon, G., 1891, Etude Historique sur les Organes Genitaux de la Femme, O. Berthier, Paris. Ricci, J. V., 1943, The Genealogy of Gynecology, 1st ed., Blakiston Co., Philadelphia. III.

Works dealing in greater depth with specific individuals, discoveries, and theories.

Albini, B. S., 1791, Explicatio Tabularum Bartholomaei Eustachii, Joannes and Hermannus Verbeek, Leyden. Berengario, J., 1521, Carpi Commentaria cum amplissimis additionibus super anatomia Mundini, Impressum per Hieronymum de Benedictis, Bononiae. Corner, G. W., 1963, Exploring the placental maze: The development of our knowledge of the relationship between the bloodstream of mother and infant in utero, Am.}. Obstet. Gynecol. 86:408-418. da Vinci, L., 1913, Quademi d'Anatomia. III. Organi della Generazione-Embrione, Dodici Fogli della Royal Library di Windsor, Casa Editrice Jacob Dyburad, Christiana. Dryander, J., 1547, Arzenei Spiegel gemeyner Inhalt derselbigen, wes bede einem Leib unnd Wundtartzt, in der Theorie, Practic unnd Chirurgei zusteht, Christian Egenolph, Franckfurt am Meyn. Hitschmann, F., and L. Adler, 1908, Der Bau der Uterusschleimhaut des geschlechtsreifen Weibes mit besonderer Beriicksichtigung der Menstruation, Monatsschr. Geburtshilfe Gynaekol. 27: 1-82. Hundt, M., 1501, Antropologium de hominis dignitate, Impressum et finitum per Baccalarium Wolfgangum Monacenem, Liptzick. Hunter, W., 1774, The Gravid Uterus, Birmingham. Hunter, W., 1794, An Anatomical Description of the Human Gravid Uterus, and Its Contents, J. Johnson, London. Kudlien, F., 1965, The seven cells of the uterus: The doctrine and its roots, Bull. Hist. Med. 39:415423. Massa, N., 1559, Anatomiae liber introductoris, Venet. Mondino dei Luzzi, 1541, Anatomia Mundini, per Joannem Dryandrum, In officina Christiani Egenolph, Marburg. Ramsey, E. M., 1971, Maternal and foetal circulation of the placenta,lr.}. Med. Sci. 140:151-168. Schroeder, R., 1930, Weibliche Genitalorgane, Handb. Mikrosk. Anat. 7:329-566. Soranus, 1956, Gynecology, tr. by O. Temkin, Johns Hopkins Press, Baltimore. Vesalius, A., 1543, De Humani Corpor~, Fabrica, Basilae (facsimile, Brussels, 1964). Weindler, F., 1908, Geschichte der Gyniikologisch-anatomischen Abbildung, Zahn und J aensch, Dresden.

Comparative Anatomy

2

HARLAND W. MOSSMAN

In this brief consideration of some of the comparative aspects of the mammalian uterus, it is important to keep in mind that this organ develops from a pair of completely mesodermal tubes called, variously, miillerian, paramesonephric, or female ducts (see Nomina Embryologica, August 1974 revision, International Anatomical Nomenclature Committee). The whole female internal genital system of eutherian ("placental") mammals, excepting the vagina, is basically double-two ovaries, two oviducts, and two uteri. The vagina is completely paired in monotremes and marsupials, and has been reported to be partially divided by a longitudinal septum in the plains viscacha (Lagostomus) (Weir, 1971) and in the immature of some baleen whales, Mysticeti (Ohsumi, 1969); otherwise, it is single in Eutheria. In most mammals, the vagina is joined by the urethra. Together they open into a common tube, the definitive urogenital sinus, or vaginal vestibule, which connects both to the surface. Often this vestibule is nearly as long as the vagina proper. However, in woman it is represented by only the shallow space between the two labia minora. The female ducts arise as grooves in the mesothelial lining of the peritoneal cavity, lateral to, but closely alongside of, the mesonephric ducts about midway on the ventral surface of the mesonephroi, hence just lateral to the developing gonads. The caudal ends of the paired grooves differentiate into a pair of tubes that grow caudally in a retroperitoneal position until beyond the caudal poles of the mesonephroi (Fig. 1). Here they curve medialward until they meet at the midline, where they again turn and grow caudalward. In the human embryo they reach the urogenital sinus at about the ninth week, that is, in the early fetal stage. Examination of embryos around this period reveals that the gubernacula, the HARLAND W. MOSSMAN Wisconsin.

.

Department of Anatomy, University of Wisconsin, Madison,

19

20 CHAPTER 2

fibromuscular cords extending from the caudal poles of the gonads and mesonephroi to the internal inguinal rings, cross the female ducts somewhere along their medially directed portions (Fig. 1). Regardless of the mammalian species, this point of crossing marks the uterotubal junction. That portion of the female duct lateral and cephalic to the crossing becomes the oviduct, and the medial and caudal portions become the uterus plus whatever contribution the female ducts may give to the vagina (Cunha, 1975). The gubernacular fold cephalic to the crossing becomes the proper ligament of the ovary, and that caudal to the crossing becomes the round ligament of the uterus. These two ligaments may be difficult to distinguish in adults of some species. However, the proper ligament of the ovary can almost always be seen, and it always attaches at the uterotubal junction. The

Mesonephros _-+~ Ovary -----!-+f!!!f-' Mesonephric acid Female duct --+---,,!~Ii

A

B

Oviduct - - - - - + - + 1 Proper ligament of ovary ---+-~."....,"'. Round ligament of uterus - - - -....----Cl Uterus-----____~--_4~~~\ Vagina -------------_~,.._-+

c

o

Figure 1. Ventral view diagrams to show the relations of the gubernaculum to the female duct, and the relations of the derivatives of each to one another. Most other urogenital organs are omitted. A: Early fetal stage. B; Later fetal stage. C: Definitive stage in the case of a long bicornuate uterus with minimal caudal migration of the internal genital organs. D: Definitive stage in the case of a simplex uterus with maximal caudal migration.

round ligament of the uterus usually can be found attaching at the same area but on the opposite side of the uterotubal junction. Yet in some species, especially the ruminants, the round ligament of the uterus often can be traced from the internal inguinal ring only to about the center of the broad ligament, where it fans out and cannot be followed to its original connection with the proper ovarian ligament. The fact that these ligaments always attach, at least during development, to the uterotubal junction, regardless of the type of adult uterus (duplex, bicornuate, or simplex), is evidence that the simplex uterus is developed by fusion of the entire paired uterine portions of the female ducts, and is not derived just from their originally fused portion as seen in an early fetus. Also, the complexity of the arrangement of the musculature of the human uterus, and presumably that of other simplex uteri, is explainable on this basis (Goerttler, 1930). Clinicians know that partial or complete duplication of the uterus is common among anomalies of the tract in women, and that this is probably the result of failure of the female ducts to fuse in the normal manner. Another well-known abnormality is ectopic endometrium (endometriosis). This is sometimes explained as implantation of endometrium derived from menstrual detritus refluxed through the oviducts. When one realizes that, unlike the intestine, the whole uterus-mucosa, musculature, and serosa-is of mesodermal origin, one wonders whether ectopic endometrium may not possibly develop in situ as the result of the abnormal presence of factors comparable to those that induce the differentiation of endometrium in its normal location. Certainly from what is known of differentiation potential and induction mechanisms during embryonic development, this is perhaps the most logical explanation of ectopic endometrium. Discussion of the comparative anatomy of the uterus is hindered by lack of data on many mammalian genera and the unreliability of many of the data in the literature. Authors have frequently described uteri as bicornuate without mentioning whether or not they have examined the lumen carefully to be sure that there is a common endometrium-lined corpus and a single cervical canal connecting to the vagina. Then, too, in many eutherians, especially the smaller ones, it is necessary to study microscopic sections of the cervical area to determine where true endometrium of the gestational portion of the uterus meets the cervical mucosa. Even with microscopic examination, identification of the true cervical region and canal can be difficult because, as the literature shows, in many mammals, especially the smaller ones, the epithelium of the canal is essentially like that of the vagina; and its glands, if any are present, are often similar to those of the gestational endometrium. Colburn et at. (1967) found such histological conditions in the squirrel monkey (Saimiri), where the cervical region is, however, easily recognized by its gross appearance. In fact, most of the commonly studied mammals have cervices easily identified grossly and microscopically because they are in general similar to that of woman. However, the limited literature on less well-known and wild eutherians indicates that the more familiar histological and gross features are frequently absent (Graham, 1973), so that one must judge the extent of the true cervical region by a combination of features, including the nature and amount of muscle and connective tissue as compared with the vagina and gestational uterus, as well as by sometimes relatively minor variations of the mucosa from that of the rest of the uterus and the vagina. No wonder then that one finds statements that

21 COMPARATfVE ANATOMY

22 CHAPTER 2

the cervix is absent or that the vagina is absent in certain species, even when some microscopic study has been done. Perhaps these statements are true, but it seems best not to accept such allegations until thorough developmental and histological studies have been made on the species under consideration. Fortunately, the junction between the oviduct and uterus is usually quite abrupt and therefore obvious. Yet in thick-walled simplex uteri such as the human, an appreciable part of the oviduct is actually intramural, but can be easily identified by its typical oviductal mucosa. In one species of bat having a simplex uterus, implantation of the blastocyst takes place normally in what at first appears to be the intramural portion of the oviduct. However, the mucosa of the area proves to be much like that of the uterine corpus; hence the region no doubt represents a rudimentary cornu enclosed in the thick wall of the well-developed corpus (Fig. 2K) (Rasweiler, 1974). We have then a basic concept of the uterus of Eutheria with which we shall proceed. Simply stated, it is that the uterus consists of two different portions, either of which may be double or partially or completely united. They are (1) the gestational portion, lined by the specialized serous-type mucosa (endometrium), which is highly embryonic (i.e., capable of further differentiation during the estrous cycle and especially during pregnancy), and (2) the cervix, lined by a mucosa often but by no means always containing either compound mucous glands or a mucous epithelium, and so designed with connective tissue and smooth muscle that it acts as a sphincter for the gestational portion. With only a few dubious exceptions, all eutherian uteri are composed of these two portions. I shall also consider the following interesting questions and hope to throw some light on them. Why have various groups of mammals evolved so many modifications of the simple primitive paired tubular uteri? Why has the trend been toward a completely fused simplex type? Are uterine types correlated with such things as litter size, degree of maturity at birth, placental structure, body build, and size of the adult female? Do these comparative data give us any significant insight into uterine structure and function in women?

1.

Types

if Uteri

Three classic types of mammalian uteri are usually described. The duplex type has two separate tubes often joined externally at their cervical ends but always opening independently into two cervical canals. These canals usually open separately into the vagina, but in some species may join within the cervical region (Fig. 2C,D,G) and then communicate with the vagina by a single ostium. The bicornuate type has two tubes (cornua) joined externally beginning at their cervical ends for from about 5% to about 50% of their length, and always joined internally at their cervical ends to form the body (corpus), which opens by a single cervical canal into the vagina. The simplex type has a single unpaired corpus externally, usually with very small rudiments of the lumina of the cornua internally. Communication with the vagina is by a single cervical canal. An additional term, "bipartite," has been used, but unfortunately sometimes

for the long bicornuate types and sometimes for the medium to short bicornuate ones. Because of this confusion, the term should be dropped. Duplex uteri have often been called bicornuate simply because their cervical ends were joined externally and perhaps were assumed to be joined internally as well. Figure 2 is designed to illustrate, name, and give examples of the three basic types and some of their intermediates.

2.

Distribution and Probahle Evolution

if' Uterine Types

Table 1 lists the types of uteri found in the major groups of Eutheria. Two, sometimes three, distinctly different types are found in four of the groups--Megachiroptera, Microchiroptera, Rodentia, and Artiodactyla. These are cateD. chipmunk (tamias)

A. rabbit

B. rat

C. guinea pig

E. dog

F. pig

G. gnu

H. caw

I. horse

J. anthropoids

K. long - tongued bat

Figure 2. Diagrammatic frontal sections of the types of uteri found among Eutheria. One known possessor of each is given. Oviducts are indicated cut near the uterotubal junction and the vaginae just caudal to the cervices. Heaviest line represents cervical mucosa; lightest line represents endometrial surface epithelium; dashed line represents oviductal epithelium. A-D: Long duplex (A. most primitive; B, caudal ends fused enternally, simulating a corpus uteri; C,D, V-shaped and Yshaped cervical canals, respectively). E,F: Long bicornuate (E, very short corpus, rarely permitting a conceptus to extend from one cornu to the other; F, corpus large enough to permit a conceptus with its placenta in one cornu to extend into the other cornu). G: Medium-length duplex; V-shaped cervical canal. H: Medium-length bicornuate; corpus permits fetal membranes, but usually not the fetus, to extend into opposite cornu. I: Short bicornuate; very short septum, which is essentially obliterated during pregnancy, permitting fetal membranes and sometimes the fetus to extend into opposite cornu. j,K: Simplex G, cornua represented only by the lateral angles of the lumen and their surrounding tissues; K, cornua represented by small tubular pits, with surrounding tissues, connecting with the intramural portions of the oviducts). The blastocyst implants in one of these pits. Modified from Rasweiler (1974).

23 COMPARATIVE ANATOMY

24

gories with numerous genera, hence more likely to show a wide range of anatomical characters. Insectivora and Carnivora are also multigeneric, but at present are known to have only one uterine type each; however, this may change when more genera have been examined. The finding of duplex uteri in two artiodactyl genera, Hippotragus (sable antelope) (Fig. 3) and Connochaetes (blue wildebeest), was a surprise to the author, since all the domestic and the few wild genera of this group that had been studied up to this time had had bicornuate uteri. All of the other orders have only one distinct type each, but this is to be expected since most of them contain only a few living genera. Gestationally functional uteri probably evolved from a portion of each eggtransporting tube of primitive egg-laying mammals and were hence of the long,

CHAPTER 2

Table 1. Types of Uteri in the Major Groups of Eutheria a

Long cornua

Basic type: Examples:

Corpus absent Duplex Rabbit

Small corpus Bicornuate Pig

Medium cornua Corpus absent Duplex Gnu

Small corpus Bicornuate Sheep

Short cornua Large corpus Bicornuate Horse

Corpus only

Simplex Human

Litter size (range)

1-25

Insectivora (hedgehogs, tenrecs, shrews, moles) Dermoptera (flying "lemurs")

II

Megachiroptera (fruit bats)

II

Number of living genera (Simpson,

1945) 68

21

1-3

94

Prosimii (lemurs, lorises, galagos, tarsiers)

1-3

22

Anthropoidea (monkeys, apes, man)

1-2

36

1(4-12)P

14

II

Microchiroptera (all other bats)

Edentata (American anteaters, sloths, armadillos) Pholidota (pangolins) Lagomorpha (rabbits, pikas)

II

1-6

10

25

tubular, completely separate duplex type well known in marsupials. With the evolution of the unpaired vagina typical of eutherians it became possible for the two uteri to fuse, first externally and fmally internally, to have a single corpus and a single cervical canal. Once fusion occurred, it could logically continue from the long bicornuate condition through the medium to the short bicornuate and finally to the simplex type. Like most concepts of the pattern of evolution of soft parts, this is conjectural, but it has much support from both developmental and comparative anatomical evidence. Biologists long ago discarded the so-called law of recapitulation, that ontogeny obligatorily repeats phylogeny; yet the fact of the matter is that, although there are numerous exceptions, the developmental history of an organ or system usually

COMPARATIVE ANATOMY

Table 1. (Contd.)

Long cornua

Basic type: Examples: Rodentia (rodents)

Corpus absent Duplex Rabbit

Small corpus Bicornuate Pig

Medium cornua Corpus absent Duplex Gnu

IIY

Small corpus Bicornuate Sheep

Short cornua Large corpus Bicornuate Horse

Corpus only

Simplex Human

Number of living Litter genera size (Simpson, (range) 1945) 1-10

Cetacea (whales, porpoises)

35

Carnivora (dogs, weasels, seals) Tubulidentata (aardvarks)

337

1-8

113

II

Proboscidea (elephants)

2 1-4

Hydracoidea (hyraxes, dassies)

3

Sirenia (dugongs, manatees)

2

Perissodactyla (horses, tapirs, rhinos)

6

Artiodactyla (cloven-hoofed mammals)

Y

1-2

85

• The primary categories in this table are based on length or absence of uterine cornua, because general uterine function and litter size seem to be correlated more with this than with type of cervix. This table is a summary of observations by the author and data from a survey of the literature, the authors of which are too numerous to cite here. The range of litter size for each group is a combination of that of the genera within the group. For instance, some insectivores bear only one young, while others may average 25; hence the range is 1-25. Asdell (1965) was the source of most of the data on litter size. I, Single cervical canal; II, paired cervical canals; Y, V- or Y-shaped cervical canal; (4-12t, species having polyembryony.

26 CHAPTER 2

does in a broad general way repeat its phylogeny. The development of a simplex uterus from two female ducts certainly repeats the essential steps proposed for its evolution from the primitive paired-tube condition. The dual nature of the female genital tract in monotremes and marsupials and the occurrence of the duplex and long bicornuate types in some of the more primitive eutherians, together with the incidence of the short bicornuate and simplex types chiefly in the most specialized eutherians, point to an evolutionary trend from duplex to simplex uterus. The presence of two or more distinct uterine types in the same order or suborder, and of the duplex type in several widely unrelated orders, suggests that the more primitive duplex and long bicornuate uterine types probably persisted until several mammalian orders became well differentiated from one another. If this is true, then further evolution to the

- --

Cornu

Ovary in bursa

Level of t he two internal cerv ical ostia

Junction o f cervica l canals

Externa l cervical ostium

Figure 3 . Ute ru s of a sable an telope . The dorsal half of the base of each cornu a nd of the cervix has been cut away to show the V-sha ped cervical canal.

medium and short bicornuate and simplex conditions probably occurred more recently and also independently in several orders. This would account for the simplex uteri of such widely unrelated groups as the anthropoids, armadillos, and bats. Asymmetry in size of uterine cornua occurs in a few genera of each of three widely unrelated orders-Chiroptera (bats), Rodentia, and Artiodactyla (clovenhoofed mammals) (Wimsatt, 1975). In many of these, the right cornu is noticeably the larger, even in fetal and juvenile females. In one bat and five genera of ruminants, the right cornu is almost always significantly larger, although the right and left ovaries appear to be equally active in ovulation. In four other genera of bats and in one rodent, only the right ovary ovulates. In one other bat genus and another ruminant, the left ovary is more active, but the embryos almost always implant in the right cornu. These examples could represent a trend toward a type of simplex uterus. However, it is an asymmetrical pattern, and all known simplex uteri are clearly symmetrical; hence it is unlikely that they evolved in this manner.

3.

Correlations

if Uterine Types with Other Biological Features

Any attempt to correlate uterine type with reproductive or other characteristics of a species is somewhat risky because of the scattered nature of the data. Of the approximately 850 genera of eutherians (Simpson, 1945), some data as to uterine type are available on about 160, but at least a third of the data are more or less unreliable. Authors have called uteri bicornuate without making clear whether they have checked the actual nature of the cervices, either grossly or microscopically. However, a few correlations seem undeniable. For instance, transuterine migration of blastocysts is common in species with bicornuate uteri, but obviously cannot occur in those with duplex uteri: an egg or blastocyst certainly never passes through the cervix from one uterine tube into the vagina, and then up through the other cervix into the other uterus; nor is there any likelihood that it would pass down one arm of a V-shaped cervical canal and then back up the other arm into the opposite uterus. There is simply no hint of any physiological mechanism that could accomplish such a feat. The position of the implanted blastocyst, right, left, or otherwise in a simplex uterus, is apparently unrelated to which oviduct delivered it. The bat, Glossophaga, described by Rasweiler (1974), is apparently an exception, but here the blastocyst implants in the lumen of a rudimentary cornu from which it soon expands to occupy the corpus. Certainly long tubular uteri are ideally adapted to gestate large litters of relatively small fetuses, whereas medium and short bicornuate and simplex types appear best for only one or two relatively large fetuses. By and large, the data substantiate this, even though there are many species with long tubular uteri that bear only one or two large young. Examples of the latter range over several orders: certain elephant shrews of the Insectivora; the African rock hares (Pronolagus) of the Lagomorpha; several rodents, including the spring haas (Pedetes) and the North American porcupine (Erethizon); and several carnivores, including the panda (Ailurus), sea otter (Enhydra), and apparently all seals, sea lions,

27 COMPARATIVE ANATOMY

28 CHAPTER 2

and so forth (Pinnipedia). These cases seem best interpreted as trends toward one or two young per pregnancy that have occurred independently in several different orders, and that may well have been the factor resulting in the parallel evolution in several groups toward a short bicornuate or simplex uterus. In fact, the uteri of the porcupine and sea otter have relatively shorter cornua than are common in their respective orders. However, the lumen of the porcupine nonpregnant uterus is coiled, thus making it much longer than the externally relatively short cornu containing it. One of the most consistent correlations is that of the medium and short bicornuate and simplex uteri with species bearing only one or two relatively large and precocious newborn. This is especially characteristic of the hoofed mammals (Perissodactyla and Artiodactyla), primates, and edentates. The lone exception among the hoofed mammals is a breed of sheep, the Finnish Landrace, which normally bears an average of 3.4 young at each parturition (Bradford et at., 1971). The other exceptions are among the armadillos, in which a few species have apparently solved the problem of accommodating large litters to simplex uteri by "inventing" polyembryony, by which one fertilized ovum results in four to eight identical siblings. It is probable that simplex uteri were characteristic of this group long before polyembryony evolved in it. Development of four or more symmetrically arranged embryos from a single blastocyst seems an ideal way to assure equally shared space in an essentially ovoid uterine lumen. If this is valid, then one would expect human monozygous multiple pregnancies to result in a lower ratio of nongenetic anomalies and defects, such as those caused by crowding and inadequate blood supply, than do dizygous and polyzygous multiple pregnancies. However, this may be hard to prove because of the many difficulties in obtaining unbiased data on human pregnancies, especially on twinning. Multiple litter fetuses in long tubular uteri are delivered in order from the most caudal to the most cephalic. It would be almost impossible for a more cephalic fetus to be forced past a more caudal one without dislodging it. Also fetuses from such uteri are normally born enclosed in intact or nearly intact fetal membranes. Since the whole conceptus, membranes with placenta and fetus, is extruded at the same time, the fetuses must be quickly released from their membranes to prevent suffocation. This is accomplished either by the clawing action of the newborn or by the mother or both. The umbilical cords of such young are much too short to allow them to be born through the ruptured membranes and still to maintain a functional cord attachment to an undetached placenta as is normal in most double and single births of mammals such as anthropoids, man, and ruminants. The disposal of conceptuses that die before term is also somewhat different in tubular uteri from that in the shorter horned or simplex types. In the latter, spontaneous abortion is the rule, although occasionally the fetus is retained and either "mummified" or "skeletonized." In longer tubular uteri, retention and resorption in situ are almost universal, except in very late pregnancy. Even if the whole litter dies, mass abortion is not common. This is true even when there is only one conceptus, as in the American porcupine. Abortion late in pregnancy sometimes occurred in our porcupine colony, but much more often resorbing conceptuses were found of exactly the same type as those seen in multiple pregnancies in other rodents and in rabbits. Although I know of no literature on

the subject, it is my experience that resorption is much less frequent in insectivores and carnivores than it is in lagomorphs and rodents. Ipsilateral uterine horn influence on the ovary, especially relating to persistence of the corpora lutea, is currently of considerable interest to reproductive physiologists (Ginther, 1974). There is no indication that such an effect occurs in species with simplex uteri, but it has been demonstrated in those with duplex and bicornuate uteri (Fischer, 1967). There is a remote possibility that placentation limited to one side of a simplex uterus might produce such an effect, but so far there seems to be no evidence for this. Different uterine types could have evolved in correlation with differences in body build, which in turn is certainly correlated with habitat and behavioral patterns, but there is little to indicate that this happened. Simplex uteri occur in some of the most active groups, the anthropoids and bats, as well as in some of the most sh;ggish, the anteaters and sloths. Medium-length bicornuate uteri are found in species given to violent activity such as the ruminants, but also in cetaceans, in which the buoyancy of their aquatic environment and the smoothness of their movements would seem to minimize traumatic effects to which the ruminants are subject. Indirectly the form of the uterus may be related to some extent with numbers of litters per year and length of gestation. With one rare exception, Finnish Landrace sheep (Bradford et at., 1971), all eutherians with gestational periods of several months, excluding those with delayed implantation, bear only one or two relatively precocious young at a time, whereas those with very short gestational periods invariably have large litters of small altricial newborn. One of the more enigmatic situations is found in bats. Here the young at term are unusually heavy compared with the weight of the mother, and in many cases are even carried about for an appreciable period after birth. The uteri of bats tend to be short-horned or simplex as one would expect with precocious young, but why have these flying mammals not evolved low-weight young that could be left in shelters and fed by mothers unencumbered during their foraging flights? About all one can say is that the situation attests to the remarkably efficient flying ability of bats. The strictly anthropoid type of fetal membranes-i.e., hemochorial villous placenta, rudimentary yolk sac, and rudimentary or absent allantoic vesicleoccurs only in species having simplex uteri. Every other type of fetal membrane system and placenta is found at least occasionally in species having either the long or medium-long tubular uteri. Since both the simplex uterus and the anthropoid type of fetal membrane system probably represent the most recent evolutionary step of each, it is not surprising that they should be found together. However, it is unwarranted to assume that their evolution has been in any degree causally linked. Some simplex uteri bear labyrinthine endotheliochorial placentae (sloths, anteaters, and some bats). None has diffuse or cotyledonary placentae or the large allantoic vesicles that accompany these. Therefore, one can conclude only that among recent mammals the anthropoid type of fetal membrane system is limited to species having simplex uteri, whereas some occurrences of all other types of fetal membrane systems are known in either the long or medium-long tubular uteri, both duplex and bicornuate. In other words, there is no clear evidence of any evolutionary adaptive relationships between gross uterine type and the nature of the fetal membranes.

29 COMPARATIVE ANATOMY

of the Comparative Morphology of the

30

4.

CHAPTER 2

Comparative aspects of a number of uterine features should be mentioned, but cannot be discussed in detail in this chapter. There is considerable histological information about the endometrium of many mammals scattered among most of the orders, but it is found chiefly in literature pertaining to estrous cycles or to implantation and placentation. In every case, the cyclic and gestational changes are more conspicuous in the endometrium proper than in the cervical mucosa. Also, in most cases the endometrium is characterized by tubular glands of a serous type. In very small species, such as mice and shrews, glands are usually scarce, and the surface epithelium seems to be an adequate secretory substitute for them. A few, such as the elephant shrews (Macroscelididae), have definite "implantation sites," which differ from the remainder of the endometrium in various ways (Horst and Gillman, 1942; Horst, 1950). Also, the majority of ruminants have specialized aglandular endometrial caruncles to which the chorioallantoic cotyledons attach to form the placentomes. In fact, endometrial histology is so closely correlated with implantation and placentation that it scarcely makes sense to discuss it except in relation to these processes, and, again, this is beyond the purpose of this chapter. A start on the comparative histology of the cervix has been made by Hafez and his colleagues. Those interested should consult Hafez (1973a,b), Hafez and Jaszczak (1972), and Graham (1973). In my experience, all mammals have functional anastomoses between branches of the ovarian artery and branches of the uterine artery of the same side. These are so large that ligation of the uterine or the ovarian artery does not deprive either organ of an adequate blood supply. In fact, the human simplex uterus is reputed to have enough cross-anastomoses on its wall and between its muscle layers to allow ligation of both the ovarian and the uterine arteries on one side without serious consequences, at least to the uterus. Excellent illustrations and descriptions of the blood supply of the female internal genitals of several domestic and laboratory mammals are given in the following: Del Campo and Ginther (1972, 1973), Ginther (1974), and Ginther et at. (1974). They studied vascular injections primarily to find a pathway that might explain the well-known direct unilateral influence of a uterine horn on the ovary of the same side. Moffat (1959) demonstrated sphincteric structures at the origin of each segmental branch of the rat uterine artery. It is unknown how these may function and whether similar mechanisms occur in other mammals. However, Wragg (1955), during perfusion of rats' uteri with their own blood under near-normal pressure and temperature, noted brief periods of reverse flow in the segmentals from the uterus toward the main uterine artery. Presumably this was caused by intrauterine contraction of muscle in such a pattern and sequence that arterial blood was forced backward out of the capillaries and small arteries. This phenomenon occurred rather commonly, but no physiological explanation could be found for it. Such a mechanism could force blood that had been widely distributed in the uterus into arterial anastomoses with the ovarian arterial supply, and might be the pathway for the local unilateral effect of the uterus on the ovary. Obviously this is merely a possibility founded on very little evidence, and much more investigation must be done to indicate its validity or nonvalidity as an explanation for the "local effect" enigma.

Miscellaneous Aspects

Uterus

In man and the few other mammals that have been studied, the lymph vessels draining a visceral organ almost parallel the arteries supplying the organ. Although too few species have been studied in this respect to make a comparison significant, it is reasonable to expect that the lymphatics of the viscera of all mammals conform to this basic pattern. The innervation of the human uterus is described in all the major anatomical textbooks, and the few studies on the gross anatomy of the innervation in common domestic and laboratory mammals show no essential differences. However, for a complete understanding of the innervation of such organs electron microscopic, histochemical, cytochemical, and physiological studies are necessary. With the exception of the vagina, the female internal genital organs of mammals are suspended by mesenteries. The early fetal ovary is attached to the ventromedial surface of the mesonephros by the mesovarium. An embryonic broad ligament attaches the cephalic portion of the female (miillerian) duct of the early fetus to the ventral or ventrolateral surface of the mesonephros as long as the latter persists, and the remainder of the duct to the dorsal or dorsolateral abdominal and pelvic walls. The gubernaculum crosses the female duct and traverses the broad ligament, dividing it into a more cephalic portion, the mesosalpinx, and a more caudal portion, the mesometrium. During the gradual caudal migration of the fetal ovary, the mesonephros degenerates, increasing the length and looseness of both the mesosalpinx and the mesovarium and thus allowing them to unite at their bases and to have a common attachment to the dorsal body wall. Because of this union, the mesovarium of the adult is sometimes considered part of the broad ligament, although it was originally a completely separate structure (Nomina Anatomica, 1966). Obviously the type of uterus and the degree of caudal migration of the ovary and uterus toward or into the pelvis have much to do with the nature of the broad ligament of the adult. For instance, the ovary, oviduct, and uterus of rabbits remain near their embryonic location, hence the rabbit's mesosalpinx and mesometrium are extensive in the cephalocaudal direction. Rats, on the contrary, have marked migration of the ovary and oviduct toward the uterus, the ovary being almost against the tubal end of the uterus and the oviduct tightly convoluted beside the ovary; thus the extent of the mesosalpinx, particularly at its tubal edge, is apparently much reduced. The relation of the uterine musculature to the broad ligament seems to vary greatly between mammalian groups, but apparently no comprehensive comparative study of this has ever been made. Frequently, as in the porcupine, Erethizon, the longitudinal layer from each side of the uterus extends out onto the mesometrium to form a very conspicuous band about as wide as the diameter of the nonpregnant cornu. In other species, the mesometrial musculature is an irregular network, heaviest along the major blood vessels and the round ligament. Hibernating mammals, such as the ground squirrels, marmots, hedgehogs, and tenrecs, use the broad ligament (and incidentally the mesovarium and mesorchium, also) as m;:uor fat storage depots. However, a narrow but sharply limited band adjacent to the cornu and oviduct remains fat free. It is a striking sight in a well-fattened hibernator to see the fat-laden portion of the broad ligament end abruptly in a thick, almost squared edge along the center of which is attached the delicate transparent uterine portion suspending a very narrow almost

31 COMPARATIVE ANATOMY

32 CHAPTER 2

threadlike anestrous uterus. Certainly in such mammals there are definitely delimited fields of potential steatoblasts, fields in which these cells are inconspicuous during most of the gestation and lactation periods.

5.

Summary and Conclusions

The eutherian internal genital system is basically paired, except for the vagina. Three primary types of uteri are recognized, duplex, bicornuate, and simplex; but there are significant intermediate types. The more specialized medium and short bicornuate and simplex types apparently have evolved independently in several mammalian orders. Uterine type is in some cases clearly correlated with other features of reproductive biology of the species: transuterine migration of blastocysts almost certainly cannot occur in duplex uteri; except for cases of polyembryony, large litters occur only in the longer tubular duplex or bicornuate uteri, not in the medium and short bicornuate or simplex types. Polyembryony is probably a recently evolved mechanism to adapt a species with a simplex uterus to simultaneous gestation of multiple young. Fetuses from long tubular uteri have short umbilical cords and are normally born with their membranes relatively intact; those from medium and short bicornuate and simplex uteri are typically large and precocious and are born through ruptured membranes, leaving the placenta with its umbilical cord still attached and functional until the newborn begins to breath air. Dead fetuses in long tubular uteri are usually resorbed in situ; only those in late gestation are aborted. Those of medium- and short-horned and simplex types are more commonly aborted. Anything comparable to the important ipsilateral influence of the uterine horn and conceptus on the ovary is unknown in species with simplex uteri. There is little evidence of a correlation between uterine type and such things as body build or activity of the female. As far as is known, all eutherians with several months of active gestation have large precocious newborn and medium to short bicornuate or simplex uteri, whereas those with very short gestational periods usually have large litters of small altricial newborn and long tubular uteri. Microscopic study of the cervical region is necessary, especially in smaller species, to ascertain whether a given uterus is duplex or bicornuate. Differences between mammalian groups in uterine mucosal histology are often great, especially with respect to changes during estrous and pregnancy cycles. Free anastomoses of uterine blood vessels with those of the ovaries appear to be universal among mammals. Man and the few other mammals investigated have closely similar uterine lymph drainage and innervation. The human uterus, although no doubt evolved from a long duplex type, is in its present form ill-adapted to the gestation of more than one fetus. The comparative anatomy and embryology of an organ or organ system can give a useful background and understanding of the biology of these structures.

ACKNOWLEDGMENTS

I am indebted to Dr. Oliver J. Ginther and his colleagues in our Department of Veterinary Science for their cooperation, to Dr. Archie S. Mossman for collection and preservation of the African mammal material used, and to Miss Lucy Taylor for preparation of the figures. Also, much of the information presented was gathered during preparation of a monograph on vertebrate fetal membranes, which is being supported by National Science Foundation Grant GB19732.

6.

References

Asdel!, S. A., 1965, Patterns of Mammalian Reproduction, 2nd ed., Constable, London. Bradford, J. E., Quirk, J. F., and Hart, R., 1971, Natural and induced ovulation rate of Finnish Landrace and other breeds of sheep, Anim. Prod. 13:627-635. Colburn, G. L., Walker, J. B., and Lang, C. M., 1967, Observations on the cervix uteri of the squirrel monkey,]. Morphol. 122:81-88. Cunha, G. R., 1975, The dual origin of the vaginal epithelium, Am. I Anat. 143:387-392. Del Campo, C. H., and Ginther, O. J., 1972, Vascular anatomy of the uterus and ovaries and the unilateral luteolytic effect of the uterus: Guinea pigs, rats, hamsters, and rabbits, Am. I Vet. Res. 33:2561-2578. Del Campo, C. H., and Ginther, O. J., 1973, Vascular anatomy of the uterus and ovaries and the unilateralluteolytic effect of the uterus: Horses, sheep, and swine, Am. I Vet. Res. 34:305-316. Fischer, T. V., 1967, Local uterine regulation of the corpus luteum, Am. I Anat. 121:425-442. Ginther, O. J., 1974, Internal regulation of physiological processes through local venoarterial pathways: A review, I Anim. Sci. 39:550-564. Ginther, O. J., Dierschke, D. J., Walsh, S. W., and Del Campo, C. H., 1974, Anatomy of arteries and veins of uterus and ovaries in rhesus monkeys, Bioi. Reprod. 11:205-219. Goerttler, K., 1930, Die Architektur der Muskelwand des menschlichen Uterus und ihre funktionel!e Bedeutung, Morphol. Jahrbuch 65:45-128. Graham, C. E., 1973, Functional microanatomy of the primate uterine cervix, in: Handbook of Physiology, Sect. 7: Endocrinology, Vol. II (R. O. Greep and E. B. Astwood, eds.), Female Reproductive System, Part 2, pp. 1-24, Williams and Wilkins, Baltimore. Hafez, E. S. E., 1973a, The comparative anatomy of the mammalian cervix, in: The Biology of the Cervix (R. J. Blandau and K. Moghissi, eds.), pp. 23-56, University of Chicago Press, Chicago. Hafez, E. S. E., 1973b, Anatomy and physiology of the mammalian uterotubal junction, in: Handbook of Physiology, Sect. 7: Endocrinology, Vol. II (R. O. Greep and E. B. Astwood, eds.), Female Reproductive System, Part 2, pp. 87-95, Williams and Wilkins, Baltimore. Hafez, E. S. E., and Jaszczak, S., 1972, Comparative anatomy and histology of the cervix uteri in non-human primates, Primates 13:297-314. Horst, C. J. van der, 1950, The placentation of Elephantulus, Trans. R. Soc. S. Afr. 32:435-629. Horst, C. J. van der, and Gillman, J., 1942, Pre-implantation phenomena in the uterus of Elephantulus, S. Afr. I Med. Sci. 7:47-71. Moffat, D. B., 1959, An intra-arterial regulating mechanism in the uterine artery of the rat, Anal. Rec. 134:107-124. Nomina Analomica, 1966, 3rd. ed., Excerpta Medica, New York. Ohsumi, S., 1969, Occurrence and rupture of vaginal band in the fin, sei, and blue whales, Sci. Rep. Whale Res. Inst. (Tokyo) 21:85-94. Rasweiler, J. J., IV, 1974, Reproduction in the long-tongued bat, Glossophaga soricina. II. Implantation and early embryonic development, Am. I Anat. 139: 1-35. Simpson, G. G., 1945, The principles of classification and a classification of mammals, Bull. Am. Mus. Nat. Hist. Vol. 85.

33 COMPARATIVE ANATOMY

34 CHAPTER 2

Weir, B. J., 1971, The reproductive organs of the female plains viscacha, Lagostomus maximus, J. Reprod. Fertil. 25:365-373. Wimsatt, W. A., 1975, Some comparative aspects of implantation, BioI. Reprod. 12: 1-40. Wragg, L. E., 1955, Adaptation in the uterine artery of the rat, Ph.D. thesis, Department of Anatomy, University of Wisconsin (Univ. Wise. Library, AW.W9213).

3

Prenatal Human Development RONAN O'RAHILLY

The prenatal development of the human uterus will be presented here primarily from the viewpoint of morphology. It should be needless to stress, however, that other considerations-from the fields of biochemistry and endocrinology, for example-are also necessary for a proper understanding of uterine development. Moreover, a developmental account of the uterus necessitates that attention be given to the urinary system, because "the urinary and reproductive organs ... form an inseparable whole in the adult organism" (Felix, 1912) as they do also in the embryo and fetus. The determination of the precise sequence and timing of developmental events necessitates the use of a staging system. The first S postovulatory weeks of human development (i.e., those following the ovulation and fertilization that resulted in pregnancy) have been subdivided into 23 Carnegie stages (O'Rahilly, 1973b), formerly termed "horizons" by Streeter. The stages, which are mostly 2 days apart, are based on morphological criteria such as the appearance of the eyes and the limb buds. The detailed use of the staging system has been described in a number of different regions, such as the nervous system (O'Rahilly and Gardner, 1971). It should be emphasized that such expressions as "at the IS-mm stage" should be replaced by "at IS mm C-R" because the single and variable criterion of embryonic length is not in itself sufficient to establish a stage. Unfortunately, at the present time a staging system for the fetal period (i.e., S weeks to birth) is not available.

RONAN O'RAHILLY California.

Carnegie Laboratories of Embryology, University of California, Davis,

35

36 CHAPTER 3

1.

Urinary Preliminaries

Although "the concept of the pronephros does not apply to the human embryo" (Torrey, 1954), the mesonephros is closely associated with the development of the reproductive pathway, particularly in the male. Hence a consideration of the urinary system is necessary. The hindgut appears at 20 days (stage 9). As described in detail elsewhere (O'Rahilly and Muecke, 1972), the intermediate mesoderm becomes visible at about 22 days (stage 10) and provides the nephrogenic cord. The ridge occupied by the developing mesonephros was described in about 1765 by the eminent embryologist of Berlin, Kaspar Friedrich Wolff, whose name has frequently been associated with mesonephric structures. At 24 days (stage 11), the mesonephric duct develops as a solid rod in situ from the nephrogenic cord, or from ectodermal buds lateral to the somites. At the same time, the nephrogenic tissue develops into nephric vesicles, which are connected by tubules to the mesonephric duct (Fig. 1). By 26 days (stage 12), the duct acquires a lumen. Although the mesonephric duct at first ends blindly, it soon becomes attached to the terminal part of the hindgut, which is henceforth known as the cloaca. (The cloaca maxima in Rome was the main sewer that led into the Tiber.) At about 28 days (stage 13), as the ureteric bud is about to form or has even appeared, the mesonephric ducts may already open into the cloaca.

Mesonephric glomerulus and tubule

Postanal gut Mesonephric duct

Urorectal septum Ureteric bud _ _ _.::;.../.,,"' Metanephros _ _ _....

LJ 0.1 mm Figure 1. Caudal end of a human embryo of approximately 32 days (stage 14). The embryo (inset drawing) is 5.7 mm in length and possesses 38 pairs of somites. In this left lateral view, the mesonehpric duct can be seen to enter the cloaca. Near the junction, the ureteric bud extends into the metanephros. The urorectal septum will shortly appear to "descend" and divide the cloaca into the primary urogenital sinus and the rectum. The paramesonephric duct will appear in less than a week. Based on Shikinami (1926).

Within a few days, the urorectal septum appears to "descend" and divide the cloaca into the primary urogenital sinus and the rectum. By the seventh week, urinary pressure is believed to cause rupture of the cloacal membrane, thereby allowing the urogenital sinus to communicate with the exterior, i.e., with the amniotic cavity. In summary, two features directly relevant to the establishment of the female reproductive pathway, namely the urogenital sinus and the two mesonephric ducts, have appeared by 6 weeks. The mesonephric ducts persist to a variable degree in the female. Koff (1933) found them intact at 56 mm but interrupted at 63 mm. Their openings were occluded by 75 mm, and, in a 77-mm fetus, only the rostral portions in the broad ligaments remained. The caudal parts are said usually to "lose their connections with the urogenital sinus and either disappear completely or migrate cranially .... and disappear at a later stage" (Koff, 1933). Others have denied the existence of a rostral migration, however, and it has been claimed that the caudal ends of the mesonephric ducts are quite durable structures (Witschi, 1970). Various mesonephric remains may be found in postnatal life. For example, the caudal portions of the mesonephric ducts may persist lateral to the uterus and vagina, where the unnecessary and historically unjustified name "Gartner's ducts" has been employed. When a mesonephric duct persists in the fetal cervix, it may present an ampulla, which sends branches into the substance of the cervix (Meyer, 1909). In addition, cranial to the ampulla, the mesonephric duct possesses a muscular coat that may persist even after degeneration of the epithelium (Huffman, 1948). Mesonephric remnants may appear in the adult cervix as either tubules or cysts, and the latter may give rise to adenomatous proliferations or to adenocarcinoma. Mesonephric structures may be seen also in the broad ligament, and have given rise to a ridiculous system of eponyms (Gardner et aI., 1948). The epoophoron, which includes mesonephric tubules and a portion of the mesonephric duct, was found to be present constantly (Duthie, 1925), although no evidence of secretory activity was observed on electron microscopy (Beltermann, 1965). As seen with the light microscope, the mesonephric duct is situated closely external to the musculature of the uterine tube. It is usually moderately convoluted, possesses its own muscular investment, and is lined by a nonciliated, low cuboidal epithelium. The mesonephric tubules are generally more highly convoluted, may display their own musculature, and are lined by ciliated and nonciliated, low columnar or cuboidal epithelium. Additional structures that may be found in the broad ligament include the paroophoron and rete tubules. Not all the structures in this area, however, are necessarily mesonephric in origin.

2.

The Paramesonephric Ducts

The paramesonephric duct was first noted in 1825 by the eminent physiologist of Koblenz, Johannes Muller, and hence was formerly termed the mullerian

37 PRENATAL HUMAN DEVELOPMENT

38 CHAPTER 3

duct. In accordance with one of the principles of the Nomina Anatomica, however, eponymous terms should be avoided whenever possible. The para mesonephric ducts arise as variable invaginations in the mesonephros during the sixth week (stage 16, Faulconer, 1951). The invagination (Figs. 2 and 3) involves a precise area of the celomic epithelium at the level of the third thoracic somite (Felix, 1912) and it occurs in both sexes. The site of the infolding later becomes the abdominal ostium of the uterine tube, and scattered irregulari-

Figure 2. Photomicrograph of coronal section through an embryo of approximately 41 days and 11 mm in C-R length (stage 17). The mesonephros is cut longitudinally and the celom, containing a portion of the liver, appears to its right. Mesonephric glomeruli and tubules are visible, as is a small portion of the mesonephric duct. Adjacent to the last, a funnel-shaped depression, the paramesonephric duct (P), can be seen to communicate with the celom. Section selected by Koff (1933).

39 PRENATAL HUMAN DEVELOPMENT

Figure 3. Photomicrograph showing transverse section of the mesonephric (M) and paramesonephric ducts at approximately 41 days (stage 17) in a 14.2-mm human embryo. The site of the paramesonephric invagination can be seen on the surface of the mesonephros. From Faulconer (1951).

ties of the margin form the beginnings of the future fimbriae. Accessory tubes, which end blindly, may be found in female embryos, and one of them may persist as the appendix vesiculosa (Monroe and Spector, 1962). The site of origin of the paramesonephric duct differs from the rest of the mesonephric ridge by virtue of the taller cells of its epithelium. The mesonephric duct lies close to this portion of the epithelium (hence the appropriate name "paramesonephric duct"), but it is clearly separated from it throughout (Gruenwald, 1941). It has been shown that, in the chick embryo, the mesonephric duct induces the "paramesonephric plaque," as the epithelial area may be termed (Didier, 1973b). As each paramesonephric duct grows caudally through the mesenchyme, the adjacent mesonephric duct acts as a guide for it, as has been shown experimentally in the chick embryo (Gruenwald, 1941, 1942). The caudal growth of the paramesonephric duct takes place probably by multiplication of its own cells, at least in the chick embryo (Didier, 1968). Three main segments (Fig. 4) may be distinguished rostrocaudally (Gruenwald, 1941): (a) a portion separated from the mesonephric duct by mesenchyme, (b) a portion separated by basement mem-

40 CHAPTER 3

bilsement membrilne

Figure 4. Diagram of longitudinal and transverse sections through the developing mesonehpric and paramesonephric ducts. Rostrally, at level (a), the two ducts are separated by mesenchyme and each has its own basement membrane. At level (b), the two ducts are no longer separated by mesenchyme but by basement membrane only. Caudally, at level (c), the two ducts are enclosed in a common basement membrane. The small arrows indicate cellular contributions from the celomic epithelium to the paramesonephric duct, and from the paramesonephric duct to the surrounding mesenchyme. Based on Gruenwald (1959).

brane only, and (c) a portion fused with the mesonephric duct without the intervention of a basement membrane. Thus the growing caudal tip of the paramesonephric duct lies within the basement membrane of the mesonephric duct, and it is possible that the mesonephric duct contributes cells to the paramesonephric canal (Frutiger, 1969). The sheath of connective tissue surrounding the paramesonephric duct develops in situ from a strip of differentiated mesothelium (Didier, 1973a,b). It has been claimed that a portion of the mesen-

chyme is derived from the cells of the paramesonephric duct itself (Gruenwald, 1959). It has recently been claimed that the mesenchyme surrounding the paramesonephric ducts shows differentiation from its earliest appearance in female (18.5 mm) but not in male (16 mm) embryos (Candreviotis, 1967). Abnormally, arrested growth of a mesonephric duct is accompanied by absence of the accompanying paramesonephric duct at corresponding levels. Furthermore, because of the absence of the mesonephric duct, the ureteric bud fails to develop, and hence unilateral renal agenesis is a frequent accompaniment of uterus unicornis and an associated defective uterine tube. The rate of elongation of the paramesonephric duct is so precisely regulated that "it provides us with an additional definable character for determining the level of development of any given specimen falling within this general period" (Streeter, 1948). The length of the paramesonephric duct can be correlated closely with the level of development of other parts of the embryo, such as the number of semicircular ducts present in the ear (Fig. 5). Thus, from the status of paramesonephric development, "one can arrive at the degree of development of an organ as remote and comparatively unrelated as the inner ear" (Streeter, 1948). Asymmetrical development of the urogenital system is characteristic of certain

Number of semicircular ducts

2

1.0

E E

...... j

"0

.!::!

~ Q)

c

~

E

3

-

'"~ 0.5r-

Figure 5. Graph to show the close relationship between the length of the paramesonephric duct and the number of semicircular ducts in the internal ear at approximately 44 days (stage 18). The paramesonephric duct attains a length of 0.5 mm only in those embryos that possess two semicircular ducts, and a length of 1 mm only in those that display all three semicircular ducts.

41 PRENATAL HUMAN DEVELOPMENT

42 CHAPTER 3

vertebrates. In most birds, for example, the right ovary is smaller and shows a tendency toward testicular differentiation, and the right oviduct ceases to develop. Indeed, from its first appearance, the left paramesonephric duct of the chick embryo is longer than that on the right. In the human, some isolated references to asymmetrical urogenital development may be found, such as a smaller right mesonephros. The length of the left uterine tube during fetal life is generally from 1 to 3 mm less than that of the right (Hunter, 1930). A systematic study of asymmetry in human urogenital development, however, does not seem to have been published. The mesonephric duct, as seen from "in front," develops two gentle curves that enable vertical, horizontal, and vertical portions to be distinguished successively in its course (Fig. 6). As the paramesonephric duct extends caudally, it adopts a similar arrangement, so that, during the eighth week, three parts can be detected: rostral vertical, middle transverse, and caudal vertical. Both the mesonephric and the paramesonephric ducts are enclosed in urogenital folds of peritoneum that later give rise to the broad ligaments. At 7 weeks, the para mesonephric ducts have generally not yet reached the urogenital sinus (Fig. 6), although a ventral projection of the dorsal sinusal wall is found between the openings of the mesonephric ducts (Vilas, 1934). This has been given the unsatisfactory name "Mullerian tubercle" but it will be referred to here as the "sinusal tubercle." The usual description that the solid tips of the fused paramesonephric ducts "push forward the epithelium of the posterior wall of the Stage 22

48mm

23

7%

9% Weeks

8

,1\ I

J

~rogenital SinUS

Stage 20 7 Weeks

Sinusal tubercle

,

\

Solid tip

I

I I

I

I \

,

I

~

I

Figure 6. Scheme to show the fusion of the paramesonephric ducts. At 7 weeks (stage 20), the ducts are separated widely from each other. Their paramesonephric position, i.e., the way in which they accompany the mesonephric ducts, is well shown. The rostral vertical and middle transverse portions of each duct have formed, and the caudal vertical part develops within a few days (stage 22). The ducts then become apposed (stage 22) and fused (stage 23). In addition, the sinusal tubercle has appeared and has become related to the solid tip of the fused para mesonephric ducts. Early in the fetal period, a remnant of the median septum can still be seen rostrally. The external contour of the developing uterus is shown by interrupted lines. Based on Hunter (1930) and Koff (1933).

urogenital sinus" to form the tubercle (Koff, 1933) does not appear to be correct. The projection first formed (the primary tubercle) is believed to subside and soon be replaced by the definitive tubercle, that is, a connective tissue proliferation that represents the future urethrovaginal septum (Frutiger, 1969). The primary and secondary tubercles are not formed by the pressure of the paramesonephric ducts, because those canals have not yet reached the urogenital sinus. The sinusal tubercle is perhaps best regarded as "a site at which three types of epithelium meet and very likely mingle" (Glenister, 1962): sinusal, mesonephric, and paramesonephric. As soon as the paramesonephric ducts "come into close contact with each other they begin to fuse even before their tips reach the urogenital sinus" (Koff, 1933). The fusion (Fig. 6) takes place initially by means of the external walls; then the cavities come together, being separated by merely a median septum. Finally, the septum becomes resorbed and the cavities become single. Remnants of the septum between the right and left ducts may be found either rostrally or caudally. By the end of the embryonic period proper (8 weeks), ductal fusion has resulted in the formation of the "genital canal" (Matejka, 1959) or so-called uterovaginal canal. The luminal fusion (Fig. 6) has been verified in a stage 23 female embryo and in a stage 23 male embryo by Nancy Kolzak in my laboratory. In the event that the paramesonephric ducts do not progress sufficiently caudally, a uterus may not be formed (uterine aplasia). In other instances, uterine nodules may be found (Rokitansky-Kiister-Hauser syndrome). Unilateral aplasia results when one paramesonephric duct fails to develop adequately (unicornvate uterus). In some cases, a uterine nodule may appear on the defective side (pseudounicornuate uterus). The paramesonephric ducts may retain their duality externally as well as internally, resulting in two hemiuteri, which may be bicornuate and bicervical or bicornuate and unicervical. All of these anomalies arise during the embryonic period proper, i.e., the first 8 postovulatory weeks. Unfortunately most of the classifications proposed for uterine and vaginal anomalies are either exceedingly complicated or oversimplified (Monie and Sigurdson, 1950). In addition to the mesonephric remains already described in the broad ligament, tubules believed to be of paramesonephric origin have been reported, and accessory uterine tubes have been recorded. Cysts have been noted, and the appendix vesiculosa may perhaps be considered as a hydropic accessory uterine tube. Primary paramesonephric epithelium consists of the cellular lining of the unfused and fused portions of the paramesonephric ducts (Lauchlan, 1972). Hence the term includes tubal, endometrial, probably endocervical, and possibly ectocervical epithelium, the prostatic utricle (at least in part), and the appendix testis. Secondary paramesonephric epithelium consists of cells similar to those lining the derivatives of the paramesonephric ducts but located external to the original ductal epithelium. The so-called germinal epithelium of the ovary is the chief source of the secondary cells but a far wider potential distribution has been recorded. That the celomic epithelium is a very unusual tissue has long been appreciated (Gruenwald, 1942). Its abnormal differentiation (peritoneal metaplasia) has frequently been invoked to account for at least some cases of endometriosis. Other

43 PRENATAL HUMAN DEVELOPMENT

44 CHAPTER 3

theories, however, are not lacking, such as the detachment of islands from the paramesonephric ducts. It would appear that "no uniform pathogenesis of dystopic endometriosis can be given" (Sanfilippo and Niedobitek, 1965). The development of the paramesonephric ducts in the male fetus has been described in detail by Glenister (1962). The caudal, fused portion of the ducts becomes joined to the urogenital sinus by a solid utricular cord. The cord then lengthens and becomes separated from the sinus by bilateral outgrowths of sinusal epithelium, which give rise to a single sinu-utricular cord. The caudal paramesonephric remnant becomes confined within the developing prostate and merges with the sinu-utricular cord. The composite rudiment then acquires a lumen and becomes greatly dilated. Postnatally the utricle is highly variable but, in some instances, is markedly glandular. In brief, "the prostatic utricle has a composite origin, its cranial portion being formed from the para mesonephric ducts and the caudal end from the mixed epithelium covering the colliculus seminalis" (i.e., the sinusal tubercle). It would seem, therefore, that the prostatic utricle corresponds developmentally to the uterus and perhaps to the vagina; hence the old term "utriculus masculinus," which was given to it by Weber in 1836, although the structure was probably first recognized by Morgagni in 1762. Variations in the position of the male vagina, or vagina-like diverticulum, have been recorded (Bowdler et aI., 1971). The appendix testis, present in 92% of adult males, is believed to be formed by the cranial end of the paramesonephric duct (Rolnick et aI., 1968). Frequently it

Table 1. Early Development of Reproductive Pathway

Stage

9 10 II

12 13 14 15 16 17 18 19 20 21 22 23

Embryonic length (mm)

Age (days)

1-3 4-12 13-20 21-29

1.5-2.5 2-3.5 2.5-4.5 3-5

20 22 24 26

30-?

4-6 5-7 7-9 8-11 11-14 13-17 16-18 18-22 22-24 23-28

28 32 33 37 } 41 44 48 51 52 54

27-31

56

Pairs of somites

Features Hindgut appears Intermediate mesoderm is recognizable Mesonephric ducts develop Mesonephric ducts become attached cloaca Ducts may open into cloaca Gonadal ridges appear Urogenital sinus is distinguishable

to

Paramesonephric ducts develop

Paramesonephric ducts are separated widely Sinusal tubercle has appeared; paramesonephric ducts are apposed Para mesonephric ducts have fused together and their solid tip is attached to urogenital sinus

45 PRENATAL H U MAN DEVELOPMENT

Figure 7. Photomicrograph of a sagittal section through th e caudal end of a male embryo o f the eighth week (stage 22) , 27.5 mm in length. The section is almost in the median plane. Fro m before backward , the genita l tubercle, urogenital sinus, urorectal septum, rectum, and cartilaginous verte bral centra can be ide ntified. Th e point wh e re th e fusi ng pa ramesonephric ducts reach the urogenital sinus is marked X.

contains tubular remnants of that duct, and is covered by an abundant and folded layer of paramesonephric epithelium that resembles closely that of the fimbriated end of the uterine tube (Sundarasivarao, 1953). The appendix epididymidis is generally, although not universally, held to be mesonephric in origin. The relevant events of the embryonic period proper are summarized in Table 1. Bilaterally situated paramesonephric ducts h ave grown caudally and fused together in both male (Fig. 7) and female embryos. In the latter, the future uterus is thereby established.

3.

Feml Development

The fetal period has not been staged, and the most widely employed single criterion of developmental progress is the crown-rump (C-R) length of the fetus,

46 CHAPTER 3

which corresponds to the sitting height postnatally. Body weight and foot length are also useful (O'Rahilly, 1975). Apart from possible differences in the differentiation of paramesonephric mesenchyme during the embryonic period (Candreviotis, 1967), no noticeable difference in the form and degree of development of the urogenital duct system in the male and the female is found until the ninth week, 35 mm C-R (Glenister, 1962). Sexual differentiation of the reproductive pathway begins early in the fetal period and is attributed to the influence of gonadal hormones. The fetal testis is believed to produce not only a masculinizing hormone but also probably a paramesonephric inhibitor (lost, 1972). Consequently, the mesonephric ducts become dominant in the male fetus whereas the paramesonephric ducts are allowed to pursue their development in the female. The heterologous ducts in each sex regress for the most part. Presumably once the paramesonephric ducts come into apposition and begin to undergo fusion, one may speak of a uterus in the female fetus. Hunter (1930) used the term "uterus" at 36 mm and Koff (1933) at 37 mm. The rostral limit of the organ is at first V-shaped, being formed by the approximation of the free portions of the paramesonephric ducts, which may now be termed uterine tubes (Fig. 6). Hunter (1930) found a distinct constriction between the cervix and body as early as at 36 mm. At 43 mm, he noted that the body of the organ appears to be slightly twisted, "a constant feature" that "persists throughout fetal life." Furthermore, the cervix is characterized by a fusiform thickening of the surrounding mesenchyme, which, although present at 48 mm (Fig. 6), becomes marked by 75 mm. In some instances, the internal median septum between the fusing paramesonephric ducts may persist in whole (uterus septus) or in part (uterus subseptus). Such anomalies would be expected to arise between 8 and 10 postovulatory weeks (30-50 mm C-R). In some cases (communicating uteri), an opening between the demicavities may be found at the level of the isthmus. Early in the fetal period (48 mm), the sinusal tubercle in the female attains its maximal development, and then declines and disappears (Koff, 1933). The fusion of the paramesonephric ducts to form the genital canal is complete at 56 mm (Koff, 1933) and, by that time, the septum between the fused ducts has generally disappeared entirely. The canal grows in length by further fusion of the paired paramesonephric ducts rostrally, by interstitial growth and cellular multiplication, and by continued caudal growth of the solid tip of the canal. Most writers have described some type of bilateral proliferation that unites with the solid paramesonephric tip and takes part in the formation of the vagina. Although some authors had considered these to be of mesonephric or paramesonephric origin, Koff (1933) maintained that the sinuvaginal bulbs were bilateral epithelial evaginations from the dorsal wall of the urogenital sinus. They were said to appear in the region of the sinusal tubercle, which thereby became obliterated at 63 mm. The bulbs became largely solidified by 77 mm through proliferation of their lining epithelium, and then fused with the solid paramesonephric tip. Bulmer (1957) referred to them as dorsolateral projections of the sinusal cells at 65 mm, and also described a third and median proliferation of the dorsal wall of the sinus, as noted previously by Vilas (1934). These three initial elements were said to

47 PRENATAL HUMAN DEVELOPMENT

Figure 8. Photomicrograph of a cross-section through the developing rectum, uterus, and bladder at 11 postovulatory weeks (69 mm C-R). The thick wall of the uterus is evident. The mesonephric duct is present immediately lateral (arrow) to the uterine lumen (cf. Koff, 1933, Plate I, Fig. G). The rectouterine pouch is visible behind the uterus.

fuse at 94 mm to form a single sinusal upgrowth. Wells (1959) concluded that the sinuvaginal bulbs "are not discrete evaginations of the urogenital sinus" but "merely regions of junction of two kinds of epithelium," namely, that of the paramesonephric ducts and that of the urogenital sinus. Witschi (1970) emphasized that the "old misconceptions" of previous authors were based on the assumption that the vagina grows rostrally whereas actually, he maintained, it grows caudally. The vaginal canal becomes occluded by a cellular mass termed the "vaginal plate" (Fig. 9), which is generally said to be, at least initially, of paramesonephric and sinusal (or sinuvaginal) origin (Koff, 1933; Bulmer, 1957). Histochemical studies seem to be more in accord with a mesonephric origin (Forsberg, 1963,

48 CHAPTER 3

1965). Witschi (1970) described a vaginal bud formed from the solid paramesonephric tip together with lateral wings of mesonephric duct blastema (Fig. 9). The vaginal plate is first seen distinctly at about 60-75 mm, and its formation is complete at about 140 mm. Finally, when the cells of the plate desquamate, the vaginal lumen is formed (Fig. 10). The formation of the vaginal plate is followed immediately by extensive growth caudally, so that at 105 mm the vaginal rudiment approaches the vestibule (Witschi, 1970). It has been assumed in the past that the tissue added, either to line or to replace the vaginal segment, was pushed rostrally from the caudal end of the vaginal rudiment. Witschi (1970), however, stressed that "the lower end of the vagina is sliding down along the urethra to its separate opening" into the vestibule. The uterovaginal canal at 75 mm presents a rostral dilatation, which represents the cavity of the body, and also a dilatation in the cervix, which "marks the region where the lateral fornices of the vagina develop" (Koff, 1933). The transition between pseudostratified columnar and stratified squamous epithelium at "17 weeks" has been assumed to represent the cervicovaginal junction (Davies and Kusama, 1962).

Figure 9. Photomicrograph of a cross-section through the developing cervix uteri and bladder at 13 postovulatory weeks (100.5 mm CoR). According to Koff (1933; see his Plate 2, Fig. C) , the section shows the original lumen and the vaginal plate. According to Witschi (1970), however, the fins on each side of the lumen consist of "irregularly arranged cells of mesonephric duct blastema."

49 PRENATAL HUMAN DEVELOPMENT

Figure 10. Sagittal section through the uterus and vagina at 17 postovulatory weeks (lSI mm C-R). The corpus is small and its lumen is relatively small, whereas glands are evident in the cervix. The posterior fornix appears higher in position than the anterior. The vaginal plate is still solid in its upper part. Degeneration of the central cells of the lower part of the plate indicates early formation of the vaginal lumen. A portion of the urethra can be seen in longitudinal section in front of the lower part of the vagina. From Hunter (1930, Plate 3, Fig. 15), courtesy of the Carnegie Institution of Washington. Compare Koff (1933, Plate 3, Fig. G).

50 CHAPTER 3

The cervix is generally believed to be of paramesonephric origin (Koff, 1933; Forsberg, 1965; Witschi, 1970) but it has been claimed that its mucous membrane is derived from the urogenital sinus (Fluhmann, 1960), so that the precise limits of the paramesonephric and sinusal contributions to the cervix remain uncertain (Davies and Kusama, 1962). The cervical glands appear at 100--120 mm (Koff, 1933; Eida, 1961). The position of the future ostium uteri is indicated at 112 mm by differentiation of the mesoderm surrounding the stratified polygonal epithelium of the caudal part of the genital canal (Bulmer, 1957). The vaginal rudiment reaches the level of the greater vestibular glands at 130 mm and makes contact with the vestibule, at which time the vaginal down growth equals the uterine rudiment in length (Witschi, 1970). At about this time the maternal and fetal organisms become flooded with increasingly high concentrations of estrogenic steroids, and organ responsiveness begins in the fetal vagina (Witschi, 1970). No indication of hormonal stimulation, however, is noted in the body of the organ (Witschi, 1970). At 130 mm, "the cervix is not sharply separated from the corpus, but at least two-thirds of the entire uterus are set off by a narrow isthmus against the short, bulbous corpus" (Witschi, 1970). The isthmus and the museau de tanche have been identified during the "5th month" of fetal life (Bouton and Maillet, 1971). A constriction between the body and cervix, however, has been recorded as early as at 36 mm (Hunter, 1930). Some stratification at 130 mm presages the differentiation of the mucosa, muscularis, and serosa (Witschi, 1970). By 139 mm, the uterine body is a single, rounded swelling, although a shallow notch remains at the point of junction of the right and left paramesonephric ducts. More of the free portion of the ducts continues to be taken into the body of the organ, so that the round ligaments, which were originally attached laterally, become anchored to the ventrolateral angles of the body (Hunter, 1930). By 139 mm (Hunter, 1930), and perhaps even as early as 37 mm (according to an illustration in Koffs article), the body, cervix, and vagina are concave ventrally (Fig. 11) in relation to the abdominal viscera, especially the well-developed urinary bladder. The glands of the corpus begin as outpouchings of the simple columnar epithelium at 151 mm (Koff, 1933). The solid epithelial primordia of the anterior and posterior fornices of the vagina can be seen (Fig. 11), and they may perhaps be detected even as early as at 130 mm (Matejka, 1959). Furthermore, "it is noteworthy that the anterior fornix is lower than the posterior fornix developmentally and not due to the cervicovaginal angulation as is commonly taught" (Koff, 1933). The anlagen of the palmate folds of the cervix are present at 160 mm (Hunter, 1930). Although estrogen sensitivity has spread over the entire length of the vagina by 162 mm, "the epithelium of the endocervix is only slightly stimulated. It has changed from cuboidal to cylindrical cells and mucoid development starts in the grooves of the many narrow folds" (Witschi, 1970). Cavitation in the vaginal plate can be observed at 151 mm (Fig. 10), and by 162 mm (Fig. 11) the vaginal lumen is complete except at its cranial end, where the fornices are still solid (Koff, 1933). The fornices become hollow at approximately 170 mm. By about 180 mm, the genital canal has access to the exterior (Bulmer, 1957).

51 PRENATAL HUMAN DEVELOPMENT

Figure 11. Median section through the uterus and vagina at about 171 postovulatory weeks (162 mm C-R). The corpus is small, and glands are more evident in the cervix. The fornices are still solid, but elsewhere the vaginal plate has become completely hollowed . Th e vaginal lumen communicates with the urogenital sinus through the hymeneal opening. The hymeneal membrane and the fossa (navicularis) vestibuli vaginae can be detected posteriorly. The posterior part of the hymen is lined internally by vaginal epithelium and externally by sinusal epithelium. The urethra can be seen in longitudinal section in front of the vagina. Compare Koff (1933, Plate 3, Fig. H).

52 CHAPTER 3

Although it is agreed that at least the body of the uterus is derived from the fused paramesonephric ducts, the development of the vagina has long been and still remains controversial. In brief, it is admitted that the epithelium of the vagina is derived from one or more of the following sources: the mesonephric ducts, the paramesonephric ducts, or the urogenital sinus. Thus the origin of the vaginal epithelium has been claimed to be (1) mesonephric ("wholly a derivative from sinus or Wolffian epithelium; it is difficult to establish which," Forsberg, 1973); (2) mesonephric and paramesonephric (the epithelium of the vagina "is contributed in almost equal measures by oviduct and mesonephric duct proliferations," Witschi, 1970); (3) paramesonephric (Walz, 1958); (4) paramesonephric and sinusal ("l'origine ... est double, miillerienne et sinusale," Agogue, 1965; Forsberg, 1973; and Cunha, 1975, in the mouse); or (5) sinusal (Politzer, 1955; "the sinus upgrowth ... forms the whole of its epithelial lining," Bulmer, 1957; Matejka, 1959; Fluhmann, 1960). Smooth muscle cells are found in the wall of the uterus immediately before the middle of prenatal life (Hunter, 1930; Valdes-Dapena, 1957; Song, 1964; Witschi, 1970). According to Hunter, the differentiation begins in the periphery, and the site of its greatest activity is at first in the area of the cervix. In another investigation, however, smooth muscle was observed to be well differentiated in the corpus at 180 mm, although it was absent from the wall of the cervix (Davies and Kusama, 1962). It has been estimated that muscular tissue in the uterus during fetal life is approximately 35--47% of the organ (Clarke, 1911). It has been claimed that, at 185 mm, not only the vagina and its fornices but also the cervical canal is lined by stratified squamous epithelium, so that the squamocolumnar junction is situated very high; i.e., entropion is present (Eida, 1961). In another study, however, the squamocolumnar junction has been found some distance external to the ostium, "so that the so-called 'congenital ectropion' already existed" (Davies and Kusama, 1962). Moreover, the ectropion was present "from 22 weeks to term when estrogenic effects were maximal and again at 22 months after birth when estrogenic effects were absent" (Davies and Kusama, 1962). These differing views may perhaps be attributed at least in part to individual variation. A well-marked fundus is visible by 227 mm, and "the change in the form of the upper limit of the uterus from a V-shaped notch to a convex curve ... is due to the general thickening of its walls, brought about by the growth and development of muscle tissue" (Hunter, 1930). The fundus projects well above the uterotubal junction and is distinctly bent ventrally. The corpus becomes anteverted upon the cervix (341 mm), and the cervix is somewhat, although not definitively, anteflexed upon the vagina (Hunter, 1930). The body of the uterus now presents its adult form, although not its adult position, and the peritoneal relations resemble closely those found in adult life. The endometrial alterations in the fetal uterus are said to resemble successively the cyclic changes in the adult mucosa (Song, 1964) (see Chap. 11). In the newborn, the endometrium generally resembles, but does not duplicate, either the proliferative or the secretory mucosa of the adult. In rare instances it resembles progestational endometrium, and decidual transformation as well as appearances analogous to menstrual changes (and attributed to estrogen withdrawal) may be encountered.

Table 2. The Development of the Uterus during the Fetal Period u C-R length Approximate age (mm) (postovulatory weeks)

36 36--43 48-75 56 60-75

10

62-130 63 100-120 112 130

15

135 151 162-180 170 180 190 215-295 227 240 305 341

a

9

lOt

11

17 19 20 21 22 24 25 26 34 40

Features Uterus is distinguishable as an organ Constriction occurs between body and cervix Thickened mesenchyme indicates cervix Median septum has usually disappeared Vaginal plate becomes distinct and fuses with solid paramesonephric tip Vaginal rudiment grows caudally Mesonephric ducts begin to disappear in female Cervical glands appear Position of future ostium is visible Isthmus is readily distinguishable; layers of uterus begin to be defined Cervix is about 5 mm in length Glands of body appear; solid fornices are evident Smooth muscle cells are distinguishable Fornices become hollow Canalization of vagina is complete Cervix is about 10 mm in length Body is about 10 mm in length Fundus is well marked Cervix is about 20 mm in length Cervix is about 25 mm in length Anteversion is noted, and an indication of anteflexIon

Modified slightly from O'Rahilly (1973a).

The endometrium at birth presents a low columnar or cuboidal epithelium (Fluhmann, 1960). The cervical epithelium during the last trimester reacts by proliferation and conversion to a mucinous epithelium, attributed either to estrogen alone or to a combination of estrogen and progesterone (Davies and Kusama, 1962). In the newborn, the cervical epithelium "appears typical of a stratified or pseudostratified columnar epithelium" and contains migrating leukocytes (Davies and Kusama, 1962). The basal cells are clear and the superficial, columnar cells are mucinous and periodic acid-Schiff positive. The vaginal epithelium is stratified squamous and shows intense estrogenic stimulation. Histochemical studies indicate that the development of the cervix precedes that of the corpus during fetal life (Szamborski and Laskowska, 1968). The junction between the cervical and vaginal epithelia is very variable at term. In some areas the vaginal epithelium extends up to the edge of the ostium, whereas in others "congenital ectropion" is observed. No evidence is found for an actual migration of the vaginal epithelium with displacement of the cervical epithelium or vice versa (Davies and Kusama, 1962). The site and pattern of the squamocolumnar junction vary considerably in postnatal life. The predominant pattern in childhood is one of entropion (Hamper! and Kaufmann, 1959).

53 PRENATAL HUMAN DEVELOPMENT

54 CHAPTER 3

Birth ~

60

E

50

E

1

Midpoint of prenatal life

'" 40 ~::l ::l

'0 30

Body

. .;:

....

Q)

spiral basol

~ ........

E

o

"0

;:

+-

Q)

E

o >E

Figure 1. Schematic representation of uterine arteries. Afte r Okkels and Engle (1938).

61

oO ,' !

.

'

..

...' (j~' ..

.

VASCULAR ANATOMY

o

Figure 2 . Diagram indicating correlated changes in ova ry and endometrium during a menstrual cycle. Based on obse rvations in the rh esus monkey , which are equa lly applicable to ma n. From Bartelmez (l957b).

menstrual cycle. The spiral arteries supply the stratum functionale and are extremely sensitive to the endocrine environment. The schema in Fig. 2 indicates the fluctuations they undergo during the cycle, correlated with the changes in the endometrium and with the hormonal stimuli emanating from the ovary (Bartelmez, 1957b). At the beginning of the growth phase of the menstrual cycle (Markee, 1940), the spiral arteries extend into the endometrium only a little farther than the stratum basale. They are connected with the subepithelial capillary plexus by long, straight precapillaries (Fig. 3). As the follicular phase continues, the arteries progress farther into the endometrium. Their absolute growth is greater than that statement implies at first glance, for the thickness of the endometrium is also increasing at this time. Convolution of the arteries becomes intensified (Fig. 4), particularly at the time of the brief endometrial regression coincident with ovulation. During the florid growth of the endometrium in the subsequent luteal phase of the cycle, arterial growth continues until the tips of the vessels are close to the base of the subepithelial capillary plexus. Two special features become apparent at this time: (1) the development of a few small branches given off at irregular intervals during the arteries' vertical course through the endometrium, and (2) the appearance, around the stems and the branches, of a cuff of dense connective tissue that forms a column conspicuously different from the looser endometrial stroma (Fig. 5). Through these "columns of Streeter" the vessels pursue their convoluted course. Branching of spiral arteries is apparently more common in man than in the monkey. It may be noted, however, that the branches do not intercommunicate but travel on a direct course to the subepithelial capillary bed; thus the familiar concept of these vessels as "end arteries," which has importance in the mechanism of menstruation, is not destroyed (Fanger and Barker, 1961). Columns of Streeter are quite similar in the two species. At the onset of premenstrual degeneration, the walls of the arteries resist

62 CHAPTER 4

Figure 3. Projection drawing of two endometrial spiral arteries in a monkey uterus, showing their connection with the subepithelial capillary network. Coils are limited to the stratum bas ale. Dilated veins drain the capillary net. From a uterus injected with India ink and removed on the twelfth day of the menstrual cycle. Mature graafian follicle. B-33S. 33.5x. From Bartelmez (l957a).

disintegration somewhat longer than the glands and stroma, and may project above the denuded endometrial surface for some hours (Markee, 1940). They eventually succumb, however, and participate in the progressive menstrual slough until viable vessel walls are once more to be found only at the inner margin of the stratum basale. Bartelmez (l957a), in describing the distribution of spiral arteries, says there is an average of 1.4 mm of stroma intervening between any two stems. The drainage of the uterus consists of a network of veins in which long, vertical vessels, roughly paralleling the course of the glands, are joined by short horizontal or oblique channels (Markee, 1940; Harris and Ramsey, 1966). At the

points of intersection of the connecting segments, large dilations may form, particularly in the stratum functionale. These "venous lakes" undergo frequent rapid changes in size and appear to serve as reservoirs to cushion change in blood volume and rate of flow through the endometrium. The familiar "blush and blanch" phenomenon (Markee, 1932) observed in the endometrium of rodents is occasioned by this mechanism. The existence of a comparable response in primates was established by Markee (1940) in his study of intraocular transplants of endometrium in monkeys. Drainage of venous blood from the uterus to the systemic circulation is accomplished via the uterine and ovarian veins. In monkeys, the latter play the major role, especially in pregnancy (Ramsey et at., 1966). In the human, the uterine veins are usually predominant, although in pregnancy, as Bieniarz's (1958) studies indicate, the region of the uterus in which placental insertion occurs is the determining factor. In general, placentas inserted low drain through the uterine veins, and those inserted closer to the fundus drain through ovarian veins. If there is serious exaggeration of the level of insertion in either direction, pathological clinical conditions may result. Bieniarz has suggested that placenta previa is associated with the abnormally low placenta draining principally through the uterine veins, and that preeclampsia is associated with abnormally high placental insertion and ovarian vein drainage. It must be noted that some statistical evidence to the contrary has been presented in the obstetrical literature.

Figure 4. Projection drawing of a n endometrial spiral artery in a monkey uterus. Coils extend into the midendometrium. Apical rami of th e vessel connect with the subepithelial capillary network but do not intercommunicate. From a uterus injected on the seventeenth day of th e menstrual cycle. Corpus luteum 6-7 days old. B-309. 33.5x. From Bartelmez (l957a).

63 VASCULAR ANATOMY

64 CHAPTER 4

The disputed problem of arteriovenous anastomoses is still unresolved, though the preponderance of opinion is that they do not occur either in monkey or in man (Harris and Ramsey, 1966; Bartelmez, 1957a; Danesino, 1950; Schlegel, 1945-1946). Certainly there is no evidence of them in radioangiographic visualizations of uteroplacental circulation (Donner et at., 1963; Borell et at., 1965).

1.2

Histology

The marked differences between the basal and the endometrial spiral arteries with respect to course and reaction to hormonal stimuli have their counterpart in differences in histological structure. The radial artery has the standard wall composition of distributing arteries. It is characterized by stout elastic membranes separating the endothelium and the media, scattered elastic fibrils intermingled with the muscle cells of the media, and irregular elastic membranes in the adventitia. The endometrial continuation of the radial, the endometrial spiral artery, loses the membrane formation when it crosses the myoendometrial border. A loose fibrillar network persists as far as the subepithelial region, according to Okkels and Engle (1938). However, study of specimens from early pregnancy suggests that the elastic tissue persists only as far as the midline of the endometrium in monkeys (Ramsey, 1949) and the stratum bas ale in man (Harris and

Figure 5. Endometrium ot a pregnant monkey on the tenth day after ovulation. showing the characteristic cuff of dense connective tissue surrounding a spiral artery in its course through the endometrium (column of Streeter). Carnegie monkey C-658, section 60b. lOx.

65 VASCULAR ANATOMY

Figure 6. Myoendometrial junction (arrow) in a monkey uterus during the eleventh day of the menstrual cycle, showing the basal portion of a spiral artery and the constriction of the associated radial artery. The segment of the radial artery that is out of focus has been strengthened. B-323 . 60x. From Bartelmez (l957a).

Ramsey, 1966). The basal arteries almost or entirely lack elastic tissue but have abundant muscle, predominantly of a type in which individual cells are especially large and have more cytoplasm and bigger nuclei than the muscle cells in other uterine vessels. As mentioned previously, endocrine response is absent in the basal arteries, though Markee (1940) noted their occasional transformation into spiral arteries. The spiral arteries respond to endocrine stimulation not only by growth but also by histological alterations. Thus spaying induces widespread fibroelastoid degener-

66 CHAPTER 4

ation in them (as in the myometrial arteries) but administration of estrogen restores them to normal. Some amplification of the description of spiral arterial "growth into the endometrium" is necessary, for this convenient expression is somewhat misleading. Even while reepithelialization of the endometrial surface is in progress following the menstrual slough, reconstitution of the capillary bed is commenced. The long precapillary channels described previously grow from the remaining segments of spiral arterial coils deep in the stratum bas ale (Fig. 3). The forward growth of the artery really consists in the formation of a media and adventitia around these precapillaries, thus converting them into arteries along an advancing front. A highly important property of the endometrial spiral arteries is their capacity to contract. As observed by Markee (1940) in intraocular endometrial transplants, and as described by Daron (1936), Bartelmez (l957a), Czekanowski (1975), and others, the contractions occur within the myometrium just before the artery crosses the myoendometrial border (Fig. 6). Individual arteries contract independently and at unrelated time intervals. In the discussion of the behavior of the uteroplacental arteries to follow, note will again be taken of this contractile property. In the menstrual cycle, arterial contractions, which are most frequent and intense in the luteal phase, appear to be the effective cause of the ischemia that occasions endometrial destruction and hemorrhage. The isolated, independent character of arterial contractions is reflected in the localized character of the necrosis and hemorrhage, the summation of which results in overt menstrual slough and bleeding (Markee, 1940; Bartelmez, 1957a). Bartelmez and Markee are of the opinion that fluctuations in hormone concentration occasion the arterial contractions. Attention must also be given to the role of myometrial contractions, both general and local (Csapo, 1959; Martin et at., 1964). This aspect is considered in greater detail in the discussion of intermittent functioning of arteries during pregnancy.

2. 2.1.

Pregrwncy Anatomy

The remarkable lability and adaptability of the endometrial spiral arteries are demonstrated by their behavior in the menstrual cycle, but the story is only half told until their metamorphosis into the uteroplacental arteries and their behavior as such are recounted. When the human or monkey blastocyst implants (interstitially in the human; superficially in the monkey), it finds the endometrium at the peak of development of the luteal phase. The tips of the spiral arteries lie immediately below the superficial capillary plexus, but still far enough away that several days elapse before the penetrating trophoblast taps them and effects free communication between the maternal arterial system and the developing intervillous space of the placenta. In the interval, connection is made first with segments of the capillary bed and then with superficial venules. While the trophoblast is penetrating the endometrium, the arteries continue

to elongate. They do so even after communication with the placenta has been established and they have come to merit the designation uteroplacental arteries. Indeed, they continue to grow until the placenta has attained its definitive form late in the first trimester. As a result of this progressive growth and of the necessity to accommodate themselves to a constantly shrinking endometrial diameter, the arteries become more and more coiled. Back-and-forth and lateral looping are added to the coiling until such time as stretching of the uterine wall causes the coils to be paid out (Fig. 7) (Ramsey, 1949; Ramsey and Harris, 1966). This straighten-

A Human

Figure 7. Diagrammatic representations of the course and configuration of the utero placental arteries in the rhesus monkey and man at comparable stages of gestation. From Harris and Ramsey (1966).

67 VASCULAR ANATOMY

68 CHAPTER 4

ing of the arteries is more marked in the monkey than in man. A concomitant feature is the progressive dilatation that the arteries undergo. Commencing in the monkey with the formation of a terminal sac just at the point of entry into the intervillous space, the dilatation progresses proximally until the entire radial artery is involved. The dilatation is somewhat more pronounced in man than in the monkey. Branches of the uteroplacental stems may be similarly involved, but only if they also communicate with the placenta. It is noteworthy that the paraplacental arteries exhibit none of the changes of pregnancy demonstrated by the uteroplacental vessels. They remain small and serve only to nourish the mural tissues. The veins undergo no such dramatic alterations as the arteries. With growth of the trophoblastic shell and pressure of the overlying conceptus, many of the channels are passively obliterated and the number of orifices of exit from the intervillous space is markedly curtailed. The remaining veins become distended by the increasing quantity of blood from the intervillous space that they must handle (Fig. 8). Orifices of exit from the placenta exist in all regions of the placental base (Ramsey, 1956; Harris and Ramsey, 1966). There is no predilection for the margin as drainage site and no separate and discrete "sinus" in the area, in the sense of Spanner (1935). Large veins often drain the margin and there is always a major wreath of veins within the endometrium at the epiplacental angle, but central drainage is equally important.

2.2.

Histology

The biology of the primate placenta is related to the present discussion only insofar as the mechanism by which it opens up the endometrial spiral arteries casts light upon properties of the vessels themselves and upon the histological changes that they undergo. Thus there is a suggestion that in the earliest contact between trophoblast and capillaries the delicate endothelial channels resist erosion or penetration and the cytotrophoblast flows around them and engulfs them (Harris and Ramsey, 1966). The observations supporting this concept in the human are (1) the presence of cavities containing maternal blood cells within the trophoblastic shell, (2) the early intercommunication of the chambers in the trophoblastic labyrinth, and (3) the lack of disintegration of tissue in the zone of maternofetal contact at that early stage. From another source comes a further indication that engulfment may occur, namely, observations made by Baving (1963) in the course of his experimental studies of implantation in the rabbit. When he applied sodium bicarbonate to the endometrial surface just before blastocystic attachment took place, there was dissociation of all of the fixed tissues except the endothelial lining of the vessels. This structure remained intact and no hemorrhage occurred. Later in the course of formation of the hemochorial placenta, of monkey and man, disruption does occur at the junction between fetal and maternal tissues, and the walls of arteries are involved in the process. Extravasation of blood results, with accumulations forming in the stroma and in the lumina of glands. In the human the trophoblast penetrates more deeply than in the monkey (Harris and Ramsey, 1966) and the walls of the arteries may be destroyed not only at their tips but also at deeper levels. The branches of main stems may be similarly undermined with resultant formation of nonfunctional branches and segments of stems. Another

~

PER ITONEAL SURFACE

....__ ... _._...

BA ~E

I [ID ~ th WEEK

ARCUATE VEIN

OF" ENDOMETRI UM

WEEK

OO19+h WEEK

.... ""

;;::~

--lC Or

~~

~< z~

t.O

en

Figure 8. Diagrammatic representations of the course and configuration of the uteroplacental veins of the rhesus monkey at various stages of pregnancy. The veins protrayed are, for the most part, those appearing in the blocks of tissue used for the models of utero placental arteries (Fig. 7). From Ramsey (1949).

3 .d WEEK

...-

70 CHAPTER 4

consequence of the multiple taps is the formation of several openings from a single stem into the intervillous space. These concomitants of deep trophoblastic penetration are uncommon in the monkey. Not all trophoblastic cells produce destruction of arterial walls. Simultaneously with the "tapping" of the distal tips of the spiral arteries, cytotrophoblastic cells begin to enter the lumina of the vessels (Ramsey et aI., 1976). They migrate along the inner walls, producing an appearance which has been likened to wax dripping down the side of a candle (Fig. 9). The cells may accumulate to the point of almost occluding the lumen. In man, the migration proceeds into the deeper portions of the arteries even involving myometrial segments (Fig. 10). The penetration is not so deep in monkey or baboon. At first, the vascular endothelium is simply stripped away (Fig. 11); subsequently, the wall itself is invaded (Fig. 10 and 12). The tunica media becomes disorganized and cytotrophoblast becomes incorporated into the vessel wall, eventually forming the primary element of its structure. Still later the cytotrophoblastic cells disappear from the lumina and the endothelium of the

Figure 9. Photomicrograph showing trophoblast in a human uteroplacental artery: the "candle wax dripping" stage. University of Virginia No. U32-2. 85x. From Ramsey et al. (1976).

71 VASCULAR ANATOMY

Figure 10. Photomicrographs illustrating trophoblast within the lumina of monkey and man uteroplacental arteries. A: Carnegie monk ey C-477, twenty-ninth day of pregnancy. A small branch of the artery is seen at the upper right with a little trophoblast in its lumen. A portion of uteroplacental vein just above the branch is enrjrely devoid of trophoblast. Intact endothelium is best seen in the branch. Section A2. 150x. B: Carnegie human specimen 9051, fourteenth week of pregnancy. In addition to the trophoblast within the arterial lumen, note the trophoblastic invasion of the stroma (arrows). Compare with Fig. I I. Section 27. 100 x .

72 CHAPTER 4

Figure 11. Another human uteroplacental artery showing, at a higher magnification than that of

Fig. lOB, the accumulation of trophoblastic cells within the lumen of the vessel as well as the cells within the stroma (arrows). Most of the cells lie within the lumen, touching the endothelium but not impairing it. Carnegie human specimen 2328, third month of pregnancy. 200x.

vessel is regenerated. The wall continues to consist preponderantly of cytotrophoblast associated with abundant fibrinoid. In man the cytotrophoblast also infiltrates the connective tissue stroma of the endometrium (Figs. lOB and II) and there is evidence that these cells may invade the arterial walls and participate in their replacement. In monkey and baboon, whose trophoblast appears to be "less invasive," there is no penetration of the stroma by trophoblast and therefore no suggestion of invasion of vascular walls from without (Fig. 12). The dilatation of uteroplacental arteries illustrated in Fig. 7 may well result from the weakening of the vessel wall brought about by this replacement of muscle and elastic tissue by trophoblast. This postulate gains support from comparison of monkey and man, since a somewhat lesser degree of dilatation in the monkey is associated with a lesser degree of trophoblastic action on the arterial walls and with persistence of elastic tissue to the level of the midendometrium. The intermittent constriction of individual arteries in the innermost layer of

the myometrium which was noted in considering the menstrual cycle is also prominent during pregnancy. The constrictions have been demonstrated by histological techniques in the monkey (Ramsey, 1959) (Fig. 13) and by radioangiography both in monkeys (Martin et at., 1964) (Fig. 14) and in man (Borell et at., 1965). During pregnancy as in the menstrual cycle the constrictions are not only intermittent but also independent from one arterial stem to another. In pregnancy an additional feature enters the picture, namely, the recurring myometrial contractions that culminate in labor. The pattern of these contractions has been extensively investigated (in man by Hendricks, 1958, 1962; in the monkey by Corner et at., 1963), as well as the effect they· have on blood flow through the placenta (Ramsey et at., 1963, 1966; Borell et at., 1965; Martin, 1972). These studies show that the arteries cease to deliver blood to the intervillous space during strong contractions and that delivery is curtailed during weaker contractions; i.e., fewer and slower "spurts" of arterial blood into the space occur (Fig. 14). Decrease or cessation of venous outflow is concomitant. But, although uterine contractions tend to "milk" blood from the mural veins, they do not "squeeze the placenta like a sponge" as the old concept suggested. Radioangiography shows that the placental pool is not materially diminished during contractions. The important implication of this is that the fetus is assured of a continued oxygen supply during contractions, though at a reduced level.

Figure 12. Monkey uterplacental artery at its point of entry into the intervillous space. India ink is present in the lumen. The trophoblast replaces the wall of the vessel. Note that there is no invasion of the endometrial stroma as in the human (see Fig. lOB). Carnegie monkey C-658, sixth week of pregnancy. Section 35-1. 60x.

73 VASCULAR ANATOMY

74 CHAPTER 4

. ..-.--- .

Figure 13. A: Cross-section of the wall of a monkey uterus on the 123rd day of pregnancy. Placenta in situ. Note central anemic area in the placenta. A patent cross-section of the spiral artery supplying this area is shown by the arrow. Carnegie monkey C-750. Section 123. 1.5x. B: The same specimen, 5 mm deeper in the block. The artery supplying the anemic area is here seen as it crosses the myoendometrialjunction. Its complete occlusion, apparently by vasospasm, explains the bloodlessness of the area of the placenta that it supplies. Carnegie monkey C-750. Section 113. 45X. From Ramsey (1959).

3.

Conclusion

Despite the remammg areas of ignorance, it becomes increasingly clear that the endometrial spiral arteries are responsible for a large part of the functional activity of the endometrium. They maintain it, through successive menstrual cycles, in a recurringly receptive state and, after implantation of a fertilized ovum,

75 VASCULAR ANATOMY

Figure 14. Three angiograms made after arterial injection of a radiopaque medium to demonstrate the pattern of uteroplacental arteries. Carnegie monkey 980, eighty-seventh day of pregnancy. A: Five and one-half seconds after injection during uterine relaxation. The medium is just beginning to enter the intervillous space in characteristic "spurts" (arrows) from the tips of the spiral arteries (SA). Also marked are HA (hypogastric artery) and UA (uterine artery). B: Ten seconds after injection , during relaxation. The size of the spurts increases as more medium is delivered to the intervillous space. C: Ten seconds after injection during a strong contraction. The absence of spurts shows that no medium is entering the placenta.

enable it to provide a suitable circulatory mechanism for embryonic development to term. It may be anticipated that further study of these remarkably sensitive and adaptable vessels will not only elucidate their specific functions and modes of operation but will also shed light on various facets of vascular physiology in general.

4,

References

Bartelmez, G. w. , 1957a, The form and the functions of the uterine blood vessels in the rhesus monkey, Contrib. Embryol. Carnegie Inst. Wash. 36:153-182. Bartelmez, G. W., 1957b, The phases of the menstrual cycle and their interpretation, Am. J. Obstet. Gynecol. 74:931-955. Bieniarz, j., 1958, The patho-mechanism of late pregnancy toxemia and obstetrical hemorrhages. 1. Contradiction in the clinical pictures of eclampsia and placenta previa depending upon the placental site, Am. J. Obstet. GynecoL. 75:444--453. Borell, U., Fernstrom, 1., Olson, L., and Wiqvist, N., 1965, Influence of uterine contractions on the uteroplacental blood flow at term, Am. J. Obstet. Gynecol. 93:44--57. Boving, B. G., 1963, Implantation mechanisms, in: Conference on Physiological Mechanisms Concerned with Conception, pp. 321-396, Macmillan (Pergamon), New York. Boyd, j. D., and Hamilton, W. j., 1970, The Human Placenta. Heffer and Sons, Cambridge, England. Corner, G. W., Jr., Ramsey, E. M., and Stran, H. M., 1963, Patterns of myometrial activity in the rhesus monkey in pregnancy, Am. J. Obstet. Gynecol. 85: 179-185.

76 CHAPTER 4

Csapo, A" 1959, Function and regulation of the mymometrium, Ann, N Y. Acad. Sci. 75:790-808. Czekanowski, R., 1975, Investigations into the spontaneous contractile activity of isolated human uterine arteries in vitro, Am. I Obstet. Cynecol. 121:718-722. Danesino, V., 1950, Dispositivi di blocco ed anastomosi arterovenosi nei vasi fetali della placenta umana, Arch. Ostet. Cinecal. 55:251-272. Daron, G. H., 1936, The arterial pattern of the tunica mucosa of the uterus in Macacus rhesus, Am. I Anat. 58:349-420. Donner, M. W., Ramsey, E. M., and Corner, G. W.,Jr., 1963, Maternal circulation in the placenta of the rhesus monkey: A radioangiographic study, Am. I Roentgenol. 90:638-649. Fanger, H., and Barker, B. E., 1961, Capillaries and arterioles in normal endometrium, Obstet. Cynecal. 17:543-550. Harris, J. W. S., and Ramsey, E. M., 1966, The morphology of human uteroplacental vasculature, Contrib. Embryol. Carnegie Inst. Wash. 38:43-58. Hendricks, C. H., 1958, The hemodynamics of a uterine contraction, Am. I Obstet. Cynecol. 76:969982. Hendricks, C. H., Eskes, T. K. A. B., and Saameli, K., 1962, Uterine contractility at delivery and in the puerperium, Am. I Obstet. Cynecol. 83:890-906. Markee, J. E., 1932, Rhythmic vascular uterine changes, Am. I Physiol. 100:32-39. Markes, J. E., 1940, Menstruation in intraocular endometrial transplants in the rhesus monkey, Contrib. Embryol. Carnegie Inst. Walh. 28:219-308. Martin, C. B., Jr., 1972, Uterine blood flow and uterine contractions in monkeys, Med. Primatol., Part I, pp. 298-307. Martin, C. B., Jr., McGaughey, H. S., Jr., Kaiser, 1. H., Donner, M. W., and Ramsey, E. M., 1964, Intermittent functioning of the uteroplacental arteries, Am. I Obstet. Cynecol. 90:819-823. Nelson, S., 1964, The arterial supply of the uterus and adnexa of the rhesus monkey, Master's Essays, Johns Hopkins University, Baltimore. Okkels, H., and Engle, E. T., 1938, Studies on the finer structure of the uterine blood vessels of the macacus monkey, Acta Pathol. Microbiol. Scand. 15: 150-168. Ramsey, E. M., 1949, The vascular pattern of the endometrium of the pregnant rhesus monkey (Macaca mulatta), Contrib. Embryol. Carnegie Inst. Wash. 35: 151-173. Ramsey, E. M., 1956, Circulation in the maternal placenta of the rhesus monkey and man, with observations on the marginal lakes, Am. I Anat. 98: 159-190. Ramsey, E. M., 1959, in: Oxygen Supply to the Human Foetus, O. Walker and A. C. Turnbull, eds.), pp. 67-79, Blackwell Scientific Publications, Oxford, England. Ramsey, E. M., 1962, Circulation in the intervillous space of the primate placenta, Am. I Obstet. Cynecol. 84:1649-1663. Ramsey, E. M., and Harris, J. W. S., 1966, Comparison of uteroplacental vasculature and circulation in the rhesus monkey and man, Contrib. Embryol. Carnegie Inst. Wash. 38:59--70. Ramsey, E. M., Corner, G. W., Jr., and Donner, M. W., 1963, Serial and cineradiographic visualization of maternal circulation in the primate (hemochorial) placenta, Am. I Obstet. Cynecol. 86:213-225. Ramsey, E. M., Martin, C. B., Jr., McGaughey, H. S., Jr., Kaiser, 1. H., and Donner, M. W., 1966, Venous drainage of the placenta in rhesus monkeys: Radiographic studies, Am. I Obstet. Cynecol. 95:948-955. Ramsey, E. M., Houston, M. L., and Harris, J. W. S., 1976, Interactions of the trophoblast and maternal tissues in three closely related primate species, Am. I Obstet. Cynecol. 124:647-652. Schlegel, J. U., 194511946, Arteriovenous anastomoses in the endometrium in man, Acta Anat. (Basel) 1:284-325. Spanner, R., 1935, Miitterlicher and kindlicher Kreislauf der menschlichen Placenta und seine Strombahnen, Z. Anat. Entwicklungsgesch. 105: 163-242. Wislocki, G. B., and Streeter, G. L., 1938, On the placentation of the macaque (Macaca mulatta), from the time of implantation until the formation of the definitive placenta, Contrib. Embryol. Carnegie Inst. Wash. 27: 1-66.

Vascular Physiology EDGAR L. MAKOWSKI

During pregnancy, considerable demands are placed upon the uterine vascular bed in order to meet the needs of a rapidly developing and growing fetoplacental unit. Although the literature contains numerous studies of the adaptation by the maternal cardiovascular system to pregnancy, data from different species are difficult to compare because of the techniques employed and dissimilar physiological conditions. As a result, this chapter will dwell on the circulatory dynamics of the nonpregnant and pregnant uterus of a single species, the sheep, because the best quantitative data available are in this species. Such data are most valuable In understanding certain physiological principles about the uterine vascular bed.

1.

Measurement

if Uterine Blood Flow

Organ blood flow can be estimated by either direct or indirect methods. Each has its own advantages and limitations depending on the experimental design. Currently three reliable techniques are available to measure uterine blood flow (Table 1). Of these, the electromagnetic flow probe is the only direct method of measuring uterine blood flow.

1.1.

Steady-State Diffusion

Meschia et al. (1967) described the steady-state diffusion method for the simultaneous estimation of uterine and umbilical blood flows. A test substance, EDGAR L. MAKOWSKI . Department of Obstetrics and Gynecology, University of Colorado Medical Center, Denver, Colorado.

77

5

78

Table 1. Techniques to Estimate Uterine Blood Flow

CHAPTER 5

1. Steady-state diffusion 2. Microsphere 3. Electromagnetic flow probe

antipyrine, which is neither produced nor appreciably metabolized by the experimental animal, is infused at a constant rate in the fetal circulation with the fetus in utero. Approximately 70 min after starting the infusion, a steady state is achieved in which the rate of transplacental diffusion of the test substance is almost equal to the rate of infusion. A typical pattern of antipyrine concentrations in fetal and maternal blood in the steady state is shown in Fig. 1. When blood samples are obtained during the steady state from uterine and umbilical arteries and veins and analyzed for antipyrine, the flows to each can be estimated by applying Fick's principle. The validity of the calculated flow depends primarily on an accurate estimate of the transplacental diffusion rate and on the assumption that the uterine vein sample is representative of total uterine outflow. In addition to uterine and umbilical flows, this method can estimate the functional capacity of the placenta by measuring its ability to clear a test substance through diffusion (placental diffusion clearance). The placental diffusion clearance is defined as the milliliters of blood that in 1 min change a test substance concentration from the level in the maternal artery. This can be expressed in the following equation: Rf

C = [A1 - [A1

30 0 0 0

UMB. ART.

-I

aJ U.

0

E

20 UMB. VEIN

~

0 0

0; E w

z

a:

UTER. VEIN

10

>a..

~

i=

z

« 0 90

110

130

TIME (MINUTES)

150

Figure 1. Antipyrine concentrations In fetal and maternal blood during the steady stale.

The clearance (C) in milliliters of blood per minute is equal to the rate of loss of a test substance (Rf , mg/min) by the umbilical circulation divided by the concentration difference of the test substance in the fetal ([A]a, mg/ml) and maternal ([A]A, mg/ml) arteries. The major disadvantage of the steady-state method is its inability to measure acute changes in organ blood flow.

1.2.

Microsphere Technique

If microspheres large enough to be cleared completely by the target organ (uterus) are carried by the arterial blood from time zero to time x, the blood flow through the target organ may be calculated by the following equation:

' m I bl00 d/ mIn

=

number of microspheres in target organ t~x

Lo

Ct dt

where Ct represents the number of microspheres per milliliter of arterial blood at time t. The integration can be done mechanically by withdrawing arterial blood at a constant rate. Radioactive microspheres with a specific activity of 10 mCi/g and a mean diameter of 25 }Lm are employed. Either 141Ce_ or 51Cr-labeled microspheres are infused via a catheter in the left ventricle over 30-40 sec as described by Rosenfeld et al. (1973). A few seconds before the microsphere infusion, reference samples are withdrawn directly into counting vials by a hydraulic system, as described by Makowski et al., (196&), from both femoral arteries at a constant rate (3.99 mIl min) for 3 min. At the conclusion of the experiment, the animal is sacrificed and the uterus is removed. The various tissue layers are separated from the uterus, separately weighed, and homogenized. Aliquots from each homogenate are collected in preweighed vials. The micro spheres are collected at the bottom of the vial by centrifugation and their radioactivity is ascertained by counting the samples in a gamma counter. The total counts per minute for each homogenate is calculated from the mean counts per minute (cpm) per gram in the aliquots and the total weight of the homogenate. The fraction of uterine blood flow to each tissue layer is calculated as the ratio of the total radioactivity in one homogenate to the total radioactivity in the entire uterus. Uterine blood flow is then calculated according to the following equation: uterine blood flow (mllmin)

total cpm in uterus mean cpm in arterial samples

---------'~--,--------=-- X

rate of withdrawal

It is evident that the application of this method to the study of uterine circulation is based on the assumptions that microspheres with a diameter larger than blood capillaries i~ected in the arterial circulation will be distributed within

79 VASCULAR PHYSIOLOGY

80 CHAPTER 5

the uterus in proportion to the regional blood flows and that microspheres will not alter organ blood flow. Delaney and Grim (1964) measured the distribution of microspheres in the canine gastric mucosa, submucosa, and muscularis and found close agreement with the partition of 42K. Moreover, Rudolph and Heymann (1967) confirmed the validity of this assumption in an artificial system and showed satisfactory agreement in the ratios of flows in the two umbilical veins of fetal lambs measured by electromagnetic flowmeters and by the distribution of microspheres. Comparative studies have shown that microspheres in the amounts needed for one flow measurement do not alter organ flow, and there is excellent agreement of the microsphere method with both the antipyrine technique and electromagnetic flowmeter in measuring organ blood flow. The use of microspheres for measurement of regional blood flows requires certain precautionary measures such as the following: (1) the mixing of microspheres in the arterial blood must be homogeneous; (2) small microspheres should be used to allow for a better resolution of regional blood flows; (3) only a negligible number should escape the target organ; (4) radioactive isotopes incorporated in the microspheres should not react with biological fluids and become free; (5) a sufficiently large number of microspheres must be present in the structures to be analyzed; and (6) the tissue samples must be representative of the whole homogenate. The major drawback of this technique is that the organ under study must be removed at the end of the experiment in order to ascertain its flow.

1.3.

Electromagnetic Flowmeter

The electromagnetic flowmeter method is based on the principle that when an electrical conductor such as blood moves through a magnetic field, an electromotive force is produced at right angles to both the direction of motion of the conductor and the lines of magnetic force. Thus the flow probe can continuously measure the instantaneous pulsatile and mean blood flow of the arterial supply to an organ. Flow probes can be chronically implanted according to the technique described by Killam et at. (1973). This preparation, with the addition of catheters into the most lateral branch of each uterine artery distal to its bifurcation (Fig. 2), lends itself to study of the response of uterine blood flow to various drugs. The agent can be infused directly into one uterine artery without developing a systemic effect. Since the sheep has a bicornuate uterus, the contralateral horn can serve as a control. During an experiment, the arterial pressure is measured with a pressure transducer positioned at the level of the maternal heart. The uterine arterial blood flow is measured by a square-wave electromagnetic flowmeter and the fluctuations of the pressure and flow signals during the cardiac cycle can be electronically integrated and displayed on a pen recorder (Fig. 3). The accuracy of the flow measurement is dependent on both in vitro and in

vivo calibration characteristics of the flow probe. In vitro, saline is infused through uterine artery segments and the direct measurement of saline flow is compared with the flowmeter readings. The response should be linear; at flows of 200 mV min or higher, the flowmeter readings, based on the manufacturer's calibration factor, should be within 10% of the actual flow. In vivo, the combined flows through both uterine arteries are compared with measurements of total uterine blood flow by another technique. The correlation between blood flow to the uterus as measured by the microsphere method and the sum of the flows in the right and left uterine arteries as measured with the flow meters is shown in Fig. 4. The correlation coefficient is 0.94, and the slope of 0.86 suggests that the two major uterine arteries carry approximately 86% of the total uterine flow . It should be noted that in any of the animal preparations the effects of

FLOW PROBE UTERINE ARTERY

Figttre 2. Experimental model.

81 VASCULAR PHYSIOLOGY

82 CHAPTER 5

anesthesia and surgical stress may blunt the response of the uterine circulation to various stimuli. On the average, recovery from such stress takes from 3 to 5 days.

2.

Physiological Observations

2.1.

Pressure-Flow Relationship

Autoregulation has been described in such organs as the kidney, brain, skeletal muscle, intestine, myocardium, and liver. However, uterine perfusion pressure-flow studies by Greiss (1966) during the last 30 days of pregnancy in the sheep showed a linear relation (Fig. 5). Analyses of pooled data over the observed pressure range from 0 to 110 mm Hg demonstrated a linear regression with a correlation coefficient of 0.992. These results suggest that the uterine vascular bed is widely dilated and that the uterus is unable to maintain its blood flow during changes in perfusion pressure. Actually, such a linear uterine pressure-flow relationship is a composite of the reactivities of the myoendometrial and maternal placental vascular beds. According to the more recent studies of Greiss and Anderson (1974), it appears that the myoendometrial bed is capable of autoregulation whereas the maternal placental vascular bed shows a passive pressure-flow relationship during the last 30-40 days of gestation in the sheep. Thus the two vascular beds can respond differently from

SHEEP 573-056-00

~

CONTROL

RIGHT UTERINE ARTERY

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LEFT UTERINE ARTERY

100

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20

40

60

80

100 MINUTES

120

140

160

180

200

Figure 3. Recording of mean blood flows through the right and left uterine arteries and mean arterial blood pressure. Estrone was injected into the lumen of the left uterine artery and the right uterine artery served as a control.

•

2,000

83 VASCULAR PHYSIOLOGY

•

•

1,500

•

1,000

•

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Blood flow, ml/min

Figure 4. Comparison of uterine blood flows measured simultaneously by electromagnetic flow probe and microsphere techniques. Results from the electromagnetic flow probe method on the ordinate and from the microsphere technique on the abscissa.

and independently of each other to various stimuli. In both nonpregnant and early pregnant sheep, their studies show a curvilinear uterine pressure-flow relationship with the convexity toward the flow axis. This type of pressure-flow relationship indicates that the uterine vascular bed in nonpregnant and early pregnant sheep is capable of autoregulation.

2.2.

Distribution of Uterine Blood Flow

A distinct advantage of the microsphere technique is its ability to quantitate the distribution of organ blood flow to the various tissue layers. Rosenfeld et al. (1973) measured the distribution of uterine blood flow by the microsphere technique to the myometrium, endometrium, and caruncles in nonpregnant, oophorectomized ewes. The mean uterine flow was 20 ± 3 mllmin. The percentage distribution of uterine flow and the regional flows are summarized in Table 2. In the last month of pregnancy, about 84% of uterine blood flow is distributed to the placenta (cotyledons) and only 3% to the myometrium. The remaining 13% is distributed to the endometrium. When the regional blood flows are plotted against fetal weight (Fig. 6), it is evident that as the fetus grows the flow to the respiratory portion of the placenta (i.e., cotyledonary flow) increases markedly, while the flows to the myometrium and endometrium show no significant changes.

Reactivity of Uterine Vascular Beds

84

2.3.

CHAPTER 5

Greiss (1972) has demonstrated in term pregnant ewes and castrated ewes the presence of a-adrenergic and ,a-adrenergic receptors. In the castrated ewe, the myoendometrial vascular bed is exquisitely sensitive to a-adrenergic stimulation and resistant to a-receptor blockade. Furthermore, it responds to ,a-adrenergic stimulation with vasodilatation and shows epinephrine reversal. Both of these latter responses can be abolished by propranolol, which confirms the presence of ,a-adrenergic receptors. For precise quantitative analysis of the reaction of the uterine vascular bed to pharmacological agents, Barton et al. (1974) used the chronic animal preparation shown in Fig. 2. They were able to demonstrate in the castrated ewe, without estrogen stimulation, postvasoconstriction vasodilatation following epinephrine infusion directly into the lumen of an uterine artery. This response occurred 120

T

110 100

:t 2.0 S.D.

b = 1.035 r " 0 .992

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50 40 30

20 10

• = Origina l values

Experiment 13-1

10~~20o--:3~0~~4tcO~~50~--~6~0--~7~0--~8~0--~9~0----1~0-0---1~1-0----~ 6 Uter ine Blood Pressure (arterial.venous)(mm Hg I Figure 5. Pressure-flow relationship in the uterus. F. C. Greiss, courtesy of Am. J. Obstet. Gynecol.

85

Table 2. Distribution of Uterine Blood Flow in Nonpregnant, Oophorectomized Ewes a

Tissue layer Myometrium Endometrium Caruncles a

Percent of uterine flow

Tissue blood flow (ml/min)

39.2 ± 2.7 32.3 ± 1.9 28.5 ± 2.3

9.4 ± 1.8 8.2 ± 2.4 7.1 ± 2.1

VASCULAR PHYSIOLOGY

Results are expressed as the mean ± SEM.

without an a-blockade and could not be demonstrated after norepinephrine infusion. Since the response could be blocked with propranolol, these observations confirmed the presence of ,B-adrenergic receptors. Greiss (1972) suggested that in term pregnant ewes the myoendometrial circulation is more sensitive to catecholamines than is the maternal placental vascular bed. This observation was confirmed by Rosenfeld et at. (1976a). In pregnant sheep at 85-140 days of gestation, a constant systemic infusion of epinephrine at a mean rate of 0.29 ± 0.03 p,g/kg/min produced a 38.5% decrease in total uterine blood flow without any significant change in systemic pressure. The reduction of regional flows was greatest in the endometrium. The endometrial blood flow decreased by 58.7%, whereas the myometrial and placental blood flows decreased by 36.9% and 34.5%, respectively.

• cotyledonary flow

Blood Flow,ml/min

1,500 1,400 1,300

•

endometrial flow

o

myometrial flow

1,200

•

1,100 1,000

•

900 800 700

•

600 500 400

•

300 200 100

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0

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Fetal Weight in Grams

Figure 6. Relationship of uterine regional flows to fetal weight.

• 0

4,000

86 CHAPTER 5

Thus far, sympathetic cholinergic innervation of the uterine vascular bed has not been demonstrated. We have confirmed the observation by Greiss (1966) that, unlike the vascular beds supplied by the coronary arteries, skeletal muscle, and superior mesenteric artery, the vascular bed of the pregnant uterus does not show reactive hyperemia even after prolonged periods of arterial occlusion that are associated with clear signs of fetal hypoxia.

2.4.

Effect of Estrogens on the Uterine Vascular Bed

The role of estrogen in the physiological control of uterine circulation has long been of interest in reproductive physiology. That estrogens cause a marked dilatation of the endometrial vessels was first observed by Markee (1932) in intraocular transplants of uterine mucosa. More recently, it has been demonstrated that following the administration of estrogens a significant increase of uterine blood flow occurs in oophorectomized, nonpregnant ewes. Huckabee et at. (1970) observed a maximal uterine blood flow of 90 ml!minl 100 g after the intravenous infusion of large doses of estrone to ewes under acute surgical stress. Greiss and Anderson (1970) noted a maximum of 24 ml!min average blood flow through one of the two uterine arteries after the intramuscular injection of 2 p,g/kg of estradiol in oil. When estradiol-17{3 was infused either intravenously or directly into one uterine artery of chronic animal preparations, Killam et at. (1973) consistently noted a 30-min delay between injection and response (Fig. 7). The peak response occurred at about 90 min and was maintained for 1-3 hr, after which the flow progressively declined to the baseline level at approximately 8 hr following the infusion of estrogen. No significant changes in arterial blood pressure occurred during the observation. Furthermore, it was evident that the injection of one-tenth of the systemic dose of estrogen into one uterine artery evoked a flow response limited primarily to the site of injection (Fig. 7). The mean uterine blood flow prior to estrogen stimulation was 45 ± 5 ml!min SEM. Two hours following the intravenous administration of estradiol-17{3, 1 p,gl kg body weight, the mean uterine blood flow rose to 375 ± 24 ml!min SEM. This increase in uterine blood flow is much greater than that previously reported by Huckabee et at. (1970) and by Greiss and Anderson (1970). The discrepancy in results may be due, in part, to the route of estrogen administration and the type of animal preparation. When oophorectomized, nonpregnant sheep were divided into stressed (acute surgical manipulation) and unstressed (chronic animal preparation) groups, Rosenfeld et at. (1973) noted an obvious difference in the maximal response of the uterine blood flow to estradiol (Fig. 8). The increase in uterine blood flow in the stressed ewes following the systemic infusion of 1 p,g/kg body weight of estradiol17{3 went from 14 ± 4 to 161.1 ± 20 ml!min SEM, whereas that for the unstressed ewes rose from 24 ± 3 to 347 ± 20 ml!min SEM. This difference was significant at P = 0.05. In addition, the arterial pressures in the two groups were

significantly different (129.9 ± 7.0 mm Hg SD in the stressed group and 101.1 ± 3.5 mm Hg SD in the unstressed group). The effect of estradiol-17f3 on the blood flow to the various uterine tissues is shown in Table 3. The increase of blood flow relative to the control value is about tenfold in all the uterine tissues and there is no significant change of distribution of blood flow within the uterus.

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30 45 MINUTES • INFUSION TIME

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Figure 7. Upper tracing: Response of uterine blood flow to intraarterial injection of 0.1

~g/kg

estradiol-l 7 ,8. Lower tracing: Response of uterine blood f10w to systemic infusion of 1.0 j.tg/kg estradiol-17,8.

87 VASCULAR PHYSIOLOGY

88

STRESSED MEAN

CHAPTER 5 400

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cr

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100

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t

The relative vasodilatory effects of estriol, estrone, diethylstilbestrol, estradiol170:, and ent-estradiol-17{3 were then compared with that of estradiol-17{3 in oophorectomized, nonpregnant ewes by Resnik et at. (1974). The potency of the other estrogens relative to estradiol-17{3 is shown in Figs. 9 and 10. The order of uterine vasodilatory potency is as follows: estradiol-17{3 > estriol > diethylstilbesTable 3. Effect of Estradiol-17f3 on the Blood Flow to Various Uterine Tissues a Before estradiol Myometrium ml/min mllmin/g Uterine flow % Endometrium mllmin mllmin/gm Uterine flow % Caruncles mllmin ml/min/gm Uterine flow % a b

After estradiol

9.4 ± 0.8 0.16 ± 0.03 39.2 ± 2.7

101.9 ± 16.2b 1.92 ± 0.39 b 38.3 ± 4.5

8.2 ± 2.4 0.69 ± 0.15 32.3± 1.9

95.2 ± 19.6b 8.24 ± 0.62b 34.4 ± 3.2

7.1 ± 2.1 0.97 ± 0.23 28.5 ± 2.3

70.4 ± 9.4 b 10.4 ± 1.1 b 27.2 ± 2.6

Results reported as the mean ± SEM. Significantly higher than the control value (P < 0.005).

-+- ESTRADIOL

89

17 f3

···C···ESTRIOL

VASCULAR PHYSIOLOGY

S 100 -t.-ESTRONE 0 -l

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Figure 9. Comparison of the vasodilatory effects of estriol and estrone with that of estradiol-17{3.

--0-- DIETHYLSTILBESTROL

·····IIt···· ESTRADIOL 17a S 100

. -G - ENT-ESTRADIOL

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Figure 10. Comparison of the vasodilatory effects of diethylstilbestrol, estradiol-17a, and entestradiol-17{3 with that of estradiol-17{3.

90

Table 4. Comparative Estrogenic Effects on Uterine Blood Flow (UBF)

CHAPTER 5

Dose eliciting 50% UBF response (fLg)

Type of estrogen Estradiol-17 {3 Estriol Diethylstilbestrol Estradiol-17 a Estrone Ent-estradiol-17 {3 a

UBF

looa

70 160 320 2400 3400 >8000

44 22 3 2 estrone (Table 4). It should be noted that estradiol-17{3 and estriol have approximately the same potency in stimulating uterine blood flow (Fig. 9). It appears from these data that estriol has the specific role of stimulating uterine vasodilatation. Experiments involving the intraarterial infusion of estrogens have demonstrated two outstanding features of their vasodilatory effect. The first feature is that the presence of estrogens at high concentration in the arterial blood for only a few seconds evokes a maximal blood flow response, which lasts several hours. The second feature is the consistent delay of approximately 30 min between the time the uterus is exposed to estrogens in the blood and the onset of uterine vasodilatation. It appears that a fraction of the estradiol-l 7(3 injected into the uterine circulation rapidly diffuses into the cells of the uterine tissues and becomes

c

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co

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o

TIME (Hours)

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3

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Figure 11. Cycloheximide infused at a constant rate into the right uterine artery from time 0 to time 2 hr 25 min. Estradiol-17{3, 1 fLg/kg, infused systemically 30 min after time o. Injection of bradykinin into the right uterine artery produced a normal vasodilatory response after no response to the systemic administration of estrogen could be demonstrated. Twenty-five minutes after the discontinuance of cycloheximide infusion, an estrogen response appeared.

strongly bound to estrogen receptors present in the cytoplasm. The cytosol receptor-estrogen complex rather than the free estrogen may possibly be responsible for evoking the uterine blood flow response. Subsequently, the uterus responds to events triggered by the estrogen in the tissues. Some of these events invoke the increased rate of protein synthesis and the de novo appearance of specific proteins. The production of these proteins may be the rate-limiting factor that accounts for the 30-min delay between stimulus and response. In favor of such a hypothesis is the ability of an inhibitor of protein synthesis (e.g., cycloheximide), when injected intraarterially into one uterine artery, to block the blood flow in the ipsilateral horn while the contralateral horn shows an unequivocal response (Fig. 11).

6,000 5,000 II

4,000

•

,

3,000 2,000 1,000

a

o • 2,000 Figure 12. Uterine (a) total weight, (b) blood now, and (c) blood flow per gram of total weight plotted against gestational age.

1,500 iii E

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Gestational age, days

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Figure 14. Endometrial (a) weight. (b) blood flow, and (c) blood flow per gram of endometrium plotted against gestational age.

VASCULAR PHYSIOLOGY

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40 80 120 Gestational age, days

13a) and the blood flow to the myometrium increases approximately fivefold (Fig. 13b). The blood flow per gram of myometrium is greatest early in gestation (Fig. 13c). The endometrial weight increases rapidly from day 40 to day 80 of pregnancy, then levels off, and increases again in the last month of gestation (Fig. 14a). Endometrial blood flow increases rapidly and then follows a course similar to the increase in weight (Fig. 14b). As a result, the blood flow per gram of endometrium is high in the early stages of pregnancy (Fig. 14c). Prior to day 40 of gestation, the placental cotyledons are in the process of being formed and the fetal membranes are loosely attached to the caruncles, or the area of implantation in the uterine mucosa. After day 40, the placental cotyledons grow rapidly and attain their maximal weight at approximately day 90. Thereafter, the placental weight

94 CHAPTER 5

decreases to term (Fig. 15a). Throughout this period, there is a continuous rise in placental blood flow (Fig. 15b). Thus the placental blood flow per gram of placenta increases from a minimum of about 0.4 mVminig at 90 days to a maximum of approximately 3 mVminig near term (Fig. 15c). Of the total uterine blood flow, about 27% is distributed to the sites of implantation between 38 and 47 days of gestation, and then 64% is distributed to the placenta between 57 and 85 days of pregnancy. Makowski et aZ. (l968b) studied the percent of radioactivity shunted across the uterus during the last month of pregnancy and the percent of distribution of uterine blood flow to the myometrium, endometrium, and cotyledons by the microsphere method. Table 5 shows that no appreciable number of microspheres 0

800

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--100--+-200--+--300--400--500---200E 100 - - - - - - - - - - - - -_ _ _ _ _ _ _ __

o

Press. mmHg

1000E 500

o

Flow ml/min

Figure 16. Example of arterial blood pressure and uterine blood flow during inhalation of air (upper tracings) and during inhalation of a 15% O 2 plus 85% N2 mixture (lower tracings).

Figure 17. Example of arterial

blood pressure and uterine blood flow during inhalation of air (upper tracings) and during inhalation of 100% O 2 (lower tracings).

200E 100 - - - - - - - - - - -_ _ _ __ o Press. mmHg 1000E 500

Air

o Flow ml/min Sec. >-100--+--200--+--300--+-400---+---500200 100 E _ _ _ _ _ _ _ _ _ _ _ _ _ _- _ o Press. mm Hg 1000 500 E o Flow mi/min

decrease in uterine blood flow was correlated with the intensity of uterine contractions. These observations were confirmed by Greiss (1965). The decrease in uterine blood flow was related not only to the intensity of uterine contractions but also to the frequency. The recovery of uterine blood flow was dependent on the duration of uterine relaxation. Tetanic contractions resulted in uterine ischemia with ultimate fetal demise. In the first hour following fetal death in utero, Raye et ai. (1971) were unable to demonstrate any significant change in uterine blood flow. However, uterine blood flow did fall gradually in the following 26 hr to approximately 70% of the control value at 24 hr. During this period, there were no significant changes in intraamniotic pressure or in maternal arterial pressure. Such a gradual decrease in uterine blood flow could represent the release of vasomotor substances from degenerating tissues within the uterus or a gradual decline of estrogen production by the fetoplacental unit.

3.

Summary

In the past decade, considerable improvements have been made in the mensuration of uterine flood flow. Flow measurements can now be made in chronic animal preparations free from the stresses of anesthesia and surgical manipulations. Previously, uterine and umbilical blood flows had not been measured in the same animals, but had been measured separately, often by different techniques and under various experimental conditions. With the recent advances in methodology and technology, uterine and umbilical blood flows can be estimated simultaneously by the steady-state diffusion method. Several methods of total uterine blood flow measurement are now available. Provided that adequate precautionary measures are taken, total uterine blood flow and its distribution to the various tissue layers can be accurately measured by the microsphere technique. The recent addition of a biological preparation in which both ovine uterine arteries are fitted with electromagnetic flow probes and catheters are inserted into a branch of each uterine artery lends itself to the study of pharmacological effects of various agents on uterine vascular function. One horn of the bicornuate uterus serves as an experimental side and the other as a control. Such an experimental model obviates the objection of a systemic effect from pharmacological agents.

97 VASCULAR PHYSIOLOGY

98

ACKNOWLEDGMENT

CHAPTER 5

This work was supported Health Service.

4.

III

part by Grant HD 00781 from the U.S. Public

Riferences

Assali, N. S., Dasgupta, K., Kolin, A., and Holms, L., 1958, Measurement of uterine blood flow and uterine metabolism. V. Changes during spontaneous and induced labor in unanesthetized pregnant sheep and dogs, Am. J. Physiol. 195:614-620. Barton, M. D., Killam, A. P., and Meschia, G., 1974, Response of ovine uterine blood flow to epinephrine and norepinephrine, Proc. Soc. Exp. Bioi. Med. 145:99-1003. Delaney, J. P., and Grim, E., 1964, Canine gastric blood flow and its distribution, Am. J. Physiol. 207:1195-1202. Greiss, F. C., 1965, Effect of labor on uterine blood flow: Observations on gravid ewes, Am. J. Obstet. Gynecol. 93: 91 7-923. Greiss, F. C., 1966, Pressure-flow relationship in the gravid uterine vascular bed, Am. J. Obstet. Gynecol. 96:41-47. Greiss, F. C., 1972, Differential reactivity of the myoendometrial and placental vasculatures: Adrenergic responses, Am. J. Obstet. Gynecol. 112:20-30. Greiss, F. C., and Anderson, S. G., 1970, Effect of ovarian hormones on the uterine vascular bed, Am. J. Obstet. Gynecol. 107:829-836. Greiss, F. c., and Anderson, S. G., 1974, Pressure-flow relationship in the nonpregnant uterine vascular bed, Am. J. Obstet. Gynecol. 118:763-772. Huckabee, W. E., Crenshaw, C., Curet, L. B., Mann, L., and Barron, D. H., 1970, The effect of exogenous estrogen on the blood flow and oxygen consumption of the uterus of the nonpregnant ewe, Q. J. Exp. Physiol. 55: 16-24. Killam, A. P., Rosenfield, C. R., Battaglia, F. C., Makowski, E. L., and Meschia, G., 1973, Effect of estrogens on the uterine blood flow of oophorectomized ewes, Am. J. Obstet. Gynecol. 115: 10451052. Makowski, E. L., Meschia, G., Droegemueller, W., and Battaglia, F. C., 1968a, Measurement of umbilical arterial blood flow to the sheep placenta and fetus in utero: Distribution to cotyledons and the intercotyledonary chorion, eire. Res. 23:623-631. Makowski, E. L., Meschia, G., Droegemueller, W., and Battaglia, F. C., 1968b, Distribution of uterine blood now in the pregnant sheep, Am. J. Obstet. Gynecol. 101:409-412. Makowski, E. L., Hertz, R. H., and Meschia, G., 1973, Effects of acute maternal hypoxia and hyperoxia on the blood flow to the pregnant uterus, Am. J. Obstet. Gynecol. 115:624-631. Markee, J. E., 1932, Rhythmic vascular uterine changes, Am. J. Physiol. 100:32-39. Meschia, G., Cotter, J. R., Makowski, E. L., and Barron, D. H., 1967, Simultaneous measurement of uterine and umbilical blood flows and oxygen uptakes, Q. J. Exp. Physiol. 52: 1-18. Raye, J. R., Killam, A. P., Battaglia, F. C., Makowski, E. L., and Meschia, G., 1971, Uterine blood flow and oxygen consumption following fetal death in sheep, Am. J. Obstet. Gynecol. 111:917924. Resnik, R., Killam, A. P., Battaglia, F. C., Makowski, E. L., and Meschia, G., 1974, The stimulation of uterine blood flow by various estrogens, Endocrinology 94: 1192-1196. Rosenfeld, C. R., Killam, A. P., Battaglia, F. C., Makowski, E. L., and Meschia, G., 1973, Effect of estradiol-17 {3 on the magnitude and distribution of uterine blood flow in nonpregnant, oophorectomized ewes, Pediatr. Res. 7:139-148. Rosenfeld, C. R., Morriss, F. H., Jr., Makowski, E. L., Meschia, G., and Battaglia, F. C., 1974, Circulatory changes in the reproductive tissues of ewes during pregnancy, Gynecol. Invest. 5:252-268. Rosenfeld, C. R., Barton, M. D., and Meschia, G., 19700, Effects of epinephrine on distribution of blood now in the pregnant ewe, Am. J. Obstet. Gynecol. 124: 156-163.

Rosenfeld, C. R., Morris, F. H., Jr., Battaglia, F. C., Makowski, E. L., and Meschia, G., 1976b, Effect of estradiol-17{3 on blood flows to reproductive and nonreproductive tissues in pregnant ewes, Am. J. Obstet. Gynecol. 124:618-629. Rudolph, A. M., and Heymann, M. A., 1967, The circulation of the fetus in ulero: Methods of studying distribution of blood flow, cardiac output and organ blood flow, Cire. Res. 21: 163184.

99 VASCULAR PHYSIOLOGY

Genetic, Biochemical, and Hormonal Mechanisms in the Regulation of Uterine Metabolism

6

KENNETH W. McKERNS

The uterus is an organ markedly and dramatically influenced by hormones. The most obvious hormonally induced changes are those caused by estrogens and progesterones. Among the most striking are the changes that occur in the uterus at the time of puberty as a result of the increasing effect of the steroid hormones of the ovary. In addition, every normal adult woman experiences cyclic changes, especially in the stimulation of the growth of the endometrium and, to some extent, of the myometrium, followed by shedding of the endometrium down to the basal layer if pregnancy has not occurred. Additional uterine growth occurs during pregnancy, partly because of an increasing production of estrogen, progesterone, and other hormones. The final stages in the life of the uterus are the regressive changes that occur after the menopause with the decline of ovarian function. These physiological effects have been known for years to be induced by the steroid hormones of the ovary. This chapter concerns the newer concepts of how these changes are regulated by estradiol at the cellular level. By what possible mechanisms could this relatively simple steroid hormone so markedly influence growth and function of the uterus? The immature or ovariectomized rat has been a useful experimental tool in KENNETH W. Me KERNS . Department of Obstetrics and Gynecology, University of Florida College of Medicine, Gainesville, Florida.

101

102 CHAPTER 6

answering these questions. Administration of estradiol to the rat induces a rapid increase in the uptake of water, glucose, and amino acids in the uterus; increased glucose metabolism and an increased formation of glycogen; increased lipid synthesis; and an accumulation of lactate. There is an increased rate of synthesis of ribonucleic acid (RNA) and protein, increased cell division, and an eventual increase in uterine weight. Estrogen induces a sequence of metabolic events leading to an increase in the size of the uterus. However, a clear relationship of the sequence of these events is not immediately apparent. It may be that estrogen stimulates some rate-limiting metabolic step, such as the metabolism of glucose, either by increasing the rate of transport of glucose into the cell or by stimulating key enzymes concerned in the metabolism of glucose or one of its intermediates. On the other hand, it is currently fashionable to think that estradiol regulates the biological response by activating a few specific genes. In the cytoplasm of the uterine cell, estradiol binds to the specific "receptor protein." This complex migrates to the nucleus, and undergoes a conversion to a complex of higher molecular weight that binds to DNA or DNA protein and regulates RNA synthesis. The early effects are thought to be a specific increase in the limited number of RNA protein species, before the general increase in synthesis of RNA. However, this concept is not universally held and many details need to be filled in. The fundamental concept, how the modified estradiol protein complex actually stimulates RNA synthesis, remains to be determined. Various experimental approaches to these and other possible mechanisms that may explain the action of estradiol on the uterus will be examined and discussed. First, however, some consideration must be given to the genetic control of metabolism and to major metabolic pathways related to energy metabolism and to synthetic processes concerned in the formation of lipids, RNA, and protein. The sources and the pathways of synthesis of the hormones that affect the uterus during the endometrial cycle and in pregnancy will also be examined.

1.

Genetic Control

~f Metabolism

The function of a tissue cell is basically determined by the genetic material in the sense that the genetic material regulates not only the synthesis of structural protein but also the enzyme proteins that determine metabolic function. A cell will have a spectrum of enzymes, genetically determined, limiting its ability to metabolize substrates or synthesize products. However, in the case of certain organs, such as the uterus, function may not be completely realized in the absence of certain hormones. Thus hormones may modify the genetic expression of cell function and replication. The main genetic material of higher organisms is nucleic acid in the form of a double helical deoxyribonucleic acid (DNA), which is capable of replication during cell division. Each "unit of function," or gene, of the DNA molecule directs the synthesis of a protein molecule through a ribonucleic acid intermediate. A complementary copy of the gene is made in the form of messenger ribonucleic acid (mRNA). The amino acid sequence of the polypeptide chain of the protein is assembled in a special way corresponding to the purine and pyrimidine base

sequences of the mRNA, as will be shown later in this chapter. DNA is found chiefly in the nucleus and appears to be localized in the chromosomes. DNA has also been found in mitochondria. RNA is found associated with organelles of the cell as well as free in the cytoplasm. DNA increases during cell division but otherwise is constant in the somatic cells irrespective of the function or metabolic activity of the cell. RNA, on the other hand, increases as metabolic activity increases. These ideas are shown in a simplified fashion in Fig. 1. Before proceeding with this discussion of DNA-directed RNA synthesis of protein, it is desirable to consider briefly the components and synthesis of polynucleic acids such as DNA and RNA.

1.1.

Pyrimidines, Purines, Nucleosides, and Nucleotides

DNA and RNA consist of numerous nucleotides linked together by phosphoric acid. Nucleotides are made up of purine or pyrimidine bases linked through a C-N bond to ribose in the case of RNA, or deoxyribose sugars in the

DNA CHROMOSOME

NUC LEOLUS

( ,C""!;'"' )

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J

~

AMINO ACID

ATP

~

/000

RIBOSOMAL STRUCTURAL RNA

POLYSOME AMINOACYl~

tR NAs

POLYPEPTIDE (Translation)

Figure 1. Chromosomes are made up of a great number of genes in the cell nucleus. A gene is a sequence of genetic material consisting of numerous purine and pyrimidine nucleotides, which eventually is expressed as a single polypeptide. The genes serve as templates for the synthesis of various types of RNA such as messenger RNA and transfer RNA. For a messenger RNA (mRNA), a gene serves as a template for the synthesis (transcription) of a complementary structure of triplets of nucleotides (codons), which can bind specific transfer RNAs (tRNAs). Each tRNA carries a specific amino acid as an aminoacyl-tRNA. The amino acids are incorporated into polypeptide chains in a sequence (translation) according to the sequence of codons in the mRNA. Ribosomal structural RNA is synthesized in the nucleolus and, along with protein, forms the ribonucleoprotein particles and ribosomes in the cytoplasm. The ribosomes attach to each strand of mRNA, forming a polyribosome, where protein synthesis takes place. Each polysome synthesizes a polypeptide chain as the mRNA moves over the ribosomes. Growth of the polypeptide chain occurs sequentially from the amino end to the final carboxyl end. Each of 20 possible amino acids is carried to the site of polypeptide synthesis by a specific tRNA having a recognition site for an activating enzyme specific for a particular amino acid. The tRNA, as an aminoacyl-tRNA, also has a coding site of three nucleotides that is complementary to the codon on the mRNA, which represents the amino acid. Thus the base sequence of DNA is transcribed to a complementary mRNA containing codons that determine the amino acid sequence of the polypeptide. These concepts will be developed in a stepwise fashion in the text.

103 GENETIC, BIOCHEMICAL, AND HORMONAL METABOLIC MECHANISMS

104 CHAPTER 6

0

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Figure 7. Formation of the parent pyrimidine nucleotide uridine 5'-phosphate from orotate and 5-phosphoribosyl pyrophosphate. The triphosphate (UTP) can be formed from this. Cytidine 5'-triphosphate is formed from UTP. UTP and CTP are the two principal pyrimidine compounds used in the synthesis of RNA in higher animals, while DNA utilizes 2'-deoxycytidine 5'-triphosphate and 2'·deoxythymidine 5'triphosphate. The thymine base for this latter compound is illustrated. Also indicated are the two principal purine triphosphates of RNA. Again, DNA uses 2-deoxy derivatives.

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110

Trophic Hormone IACTHI

CHAPTER 6

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7 estrone> estradiol-17a (Brecher and Wotiz, 1968; Toft et al., 1967; Korenman et al., 1970; Kimball and Hansel, 1974; Somjen et al., 1974a). The binding potency of diethylstilbestrol is variable and may be due to the use of different assay methods or to different forms of the binding component or tissue of origin of receptor. In addition to estrogens, several synthetic steroids interact with the uterine receptor (Jensen et al., 1973a,b; Geynet et al., 1972; Jensen and DeSombre, 1972). Concentration of the estrogen-binding protein increases in the endometrium between day 15 and day 21 of the estrus cycle, which correlates· significantly with the plasma level of estradiol-17{3 and estrone. Hence the endometrial content of binding protein fluctuates with the plasma levels of estrogens (Ciaccio and Lisk, 1970; Clark et al., 1972; Hughes et al., 1969; Kimball and Hansel, 1974; Henderson and Schalch, 1972; Feherty et al., 1970; Lee and Jacobson, 1971; McGuire and Lisk, 1968; Somjen et al., 1973a). The hormone, on interacting with the cytosol-binding protein, forms a complex, which is rapidly transported into the nucleus. This translocation of the hormone-receptor complex is reflected by a decrease in the total estradiol receptor 8 S protein complex content of the rat uterine cytosol after administration of a large dose of the hormone and is gradually replenished 4 hr after the hormone (Jensen and DeSombre, 1972, 1973). The replenishment process is blocked by cycloheximide, suggesting that the replacement is dependent on protein synthesis. The depletion of the receptor from the cytosol in the uterus was also observed after incubation in vitro of the steroid added to uterine explants (Musliner et al., 1970; Gorski et al., 1968; Giannopoulos and Gorski, 1971a,b). Other factors such as age and hormonal state of the animals influence the concentration of estrogenbinding protein in the uterus of the immature rat, calf, and ovariectomized rats (Eisenfeld and Axelrod, 1966; Feherty et al., 1970; Clark and Gorski, 1970; Alberga and Baulieu, 1968; Ciaccio and Lisk, 1970; Gorski et al., 1971; Mester et al., 1974; Somjen et al., 1973c, 1974a). Progestational agents administered orally to women reduced significantly the estrogen receptor levels in the endometrium (Tseng and Gurpide, 1975). 2.3.3. Properties of Cytosol Receptor The uterine cytosol estrogen-binding receptor sediments at 8 S, which was originally reported to be 9.5 S (Toft and Gorski, 1966; Erdos, 1968; Siiteri et al., 1973; Pollow et at., 1975; DeSombre et al., 1969, 1971; Jensen et al., 1968, 1974a,b;

153 ESTROGENS, NUCLEIC ACIDS, AND PROTEIN SYNTHESIS

154 CHAPTER 7

Hunter and Jordan, 1975). The 8 S complex is dissociated into 4--4.5 S complexes by incubation in 0.3 M or higher concentrations of NaCI or KCI (Erdos, 1968; Korenman, 1968; Korenman and Rao, 1968; Sica et ai., 1973a; Pollow et at., 1975). The sedimentation coefficients of the cytosol and nuclear complex determined in salt-containing sucrose gradient are in the neighborhood of 3.8 Sand 5.2 S, respectively (Jensen et ai., 1973a; Chamness and McGuire, 1972). The complexes seem to be composed mainly of protein, for they are sensitive to proteases and not to nucleases (Jensen et at., 1969; Puca and Bresciani, 1968; Toft and Gorski, 1966). The molecular weight of the 8 S uterine cytosol complex is about 236,000 and its isoelectric point ranges from 6.6 to 6.8. The molecular weight of the 4 S or 4.5 S complex is 61,000 and the isoelectric point is 6.2 (DeSombre et ai., 1969; Puca et at., 1970, 1971, 1972). The estimates of the physicochemical properties of the uterine cytosol receptor vary somewhat depending on the experimental conditions in the presence of 0.4 M KCI at pH 7.4. The sedimentation coefficient of the rat uterine cytosol receptor is about 4.2 ± 0.04 S, the molecular Stokes radius is 44.0 ± 0.4 A, and the molecular weight is about 76,200. The uterine cytosol 4 S or 4.5 S binding unit is stabilized by the presence of calcium in the medium (DeSombre et at., 1969; Puca et ai., 1970, 1971). The interaction of the estradiol with the uterine receptor protein takes place by a strong noncovalent, physicochemical bonding. The approximate association constant for estradiol is in the range of 109_10 12 M- 1 at 37°C (Erdos et ai., 1969; Truong and Baulieu, 1971; Erdos, 1968; Puca and Bresciani, 1969; Baulieu et at., 1971, Baulieu and Raynaud, 1970; Erdos et at., 1970, 1971; Best-Belpomme et ai., 1970; Sanborn et at., 1971; Jensen and DeSombre, 1972; Puca et at., 1970, 1971; Ellis and Ringold, 1971; Truong et at., 1973). The approximate rate constant of association for estradiol at O°C is 6.4 X 10-5 M seC 1 at 20°C based on the first slope and 1.6 X 2.5 X 10- 1 sec- 1 at 20°C based on the first slope (Baulieu, 1973a). Evidence has been presented showing that an intermediate molecule with a sedimentation coefficient of 5.3 S and a molecular weight of about 110,000 is formed from the 8.6 S native receptor. The 5.3 S receptor protein is subsequently converted to a 4.5 S form by a Ca2+-dependent factor (Notides et at., 1973; Puca et at., 1972). However, the more generally accepted version of the transformation steps are that a 8 S cytoplasmic receptor is converted to a 4 S form and finally to a 5 S form as exist in the nucleus. It has been proposed that the cytosol receptor may function as a ribonuclease inhibitor (Zan-Kowalczewska and Roth, 1975). The evidence presented in support of this hypothesis is that the receptor protein (4 S) and the ribonuclease inhibitor have similar physicochemical properties. Moreover, ribonuclease inhibitor is present in immature rat uterus and absent in normal adult rat uterus. After ovariectomy, a decline in the uterine ribonuclease activity occurs with a parallel rise in the concentration of the inhibitor. These results suggest that the 4 S form of the uterine cytosol receptor and the ribonuclease inhibitor might be the same protein. Before this function is assigned to the receptor, the receptor should be purified to homogeneity and checked for its capacity to inhibit ribonuclease activity. 2.3 .4 . Receptor-Transforming Factor The conversion of the 8 S form of the receptor to the 4 S form on incubation in 0.3 M KCI (Erdos, 1968, 1971; Korenman and Rao, 1968; Jensen et at., 1973a;

Notides et al., 1973, 1975; Notides and Nielsen, 1974) can be carried out by treatment with various proteolytic enzymes. Mild treatment of immature calf uterus estradiol receptor (8 S) with trypsin, chymotrypsin, or pronase results in the formation of a molecule of 60,000 daltons with a sedimentation constant of 3.9 S, Stokes radius of 36 A and fifo of 1.36 (Erdos et al., 1969, 1971; Rat et al., 1974). This conversion of the 8 S receptor to a 4.5 S form is also mediated by a trypsinlike Ca2+ -dependent protease discovered in the human uterine cytosol. The protease, designated as the "receptor-transforming factor" or "human uterine protease," is capable of transforming the rat uterine estrogen-receptor complex from 8 S to a 4.5 S form (Notides et at., 1973; Puca et at., 1972). The transformation of the receptor from 8 S to the 4 S by the protease does not alter the estrogen-binding capacity. The transformation, however, is irreversible, and the transformed molecules are similar in size to those formed by the treatment with other proteolytic enzymes except that their ability to bind to chromatin is retained while their ability to bind to DNA is lost (Erdos et al., 1971). The properties and physiological role of this specific transforming factor need to be further investigated.

2.3.5.

Cytosol-Nuclear Receptor Transformation

The receptor undergoes a second transformation (4 S to a 5 S form) prior to, during, or after translocation into the nucleus, and the transformed receptor is bound to the nuclear acceptor site. Conversion of the cytosol 4 S receptor into the 5 S form appears not to require the presence of the nuclei, although in vivo the transformation may take place within the nucleus (Jensen et al., 1973a,b, 1974a; Gschwendt and Hamilton, 1972; Huber et al., 1975; Notides and Nielsen, 1974; Notides et at., 1975; Nielsen and Notides, 1975). This transformation or activation of the 4 S form into the 5 S form is temperature dependent and requires the presence of estradiol. The process of transformation occurs slowly at 4°C and rapidly at 25 and 37°C. The temperature-activated transformation of 4 S to 5 S form in vitro occurs without loss of estradiol-binding activity. The Arrhenius energy of activation is 2l.3 and 19.1 kcal mol- 1 in buffer without KCl and with 0.4 M KCI, respectively (Notides et at., 1975). The transformation appears to be the result of an association of the 4 S receptor (sedimentation coefficient 4.2 ± 0.04 S) of molecular weight about 76,000 with a second component or subunit of about 50,000 to form the 5 S estrogenbinding protein (about 130,000) (Notides and Nielsen, 1974; Nielsen and Notides, 1975). The transformed receptor is characterized by a sedimentation coefficient of about 5.5 ± 0.02 S, a molecular Stokes radius of 58.5 ± 0.5 A, and a molecular weight of 132,700 (Notides and Nielsen, 1974). More recent estimated molecular weights of the 4 Sand 5 S receptors are 60,000 and 105,000, respectively (Yamamoto, 1974). The transformation requires a second subunit that does not bind estradiol. The subunit is capable of binding to DNA independently of the receptor and is present in various rat tissues. The hormone that regulates the transformation in vivo is probably estradiol, for estrone does not induce the formation of the 5 S complex in the presence or absence of nuclei, although it may do so at high concentrations (Jensen et al., 1974a). It should be pointed out that the in vitro transformed 5 S receptor and the receptor extracted from uterine

155 ESTROGENS. NUCLEIC ACIDS, AND PROTEIN SYNTHESIS

156 CHAPTER 7

nuclei possess similar properties. The reported sedimentation coefficient of the receptor extracted from nuclei and of the transformed receptor differs somewhat with the method of preparation. The variation in results might be due to the process of transformation or to the fact that real differences in the hormonereceptor complex exist. 2.3.6.

Translocation

of Receptor to

the Nucleus

On administration of radioactive estradiol to immature or ovariectomized animals, the hormone is localized in the nucleus and found in association with chromatin (King et ai., 1965, 1969; Maurer and Chalkley, 1967; Teng and Hamilton, 1969; Somjen et ai., 1973c). The amount of estrogen receptor in uterine nuclei increases after estradiol administration while the concentration of highaffinity estrogen-binding receptor in the uterine cytosol decreases, suggesting a translocation of the uterine receptor from the cytoplasm into the nucleus (Gorski et ai., 1969; Chamness et al., 1974; Anderson et ai., 1975; So~en et ai., 1974a). Thirty minutes after hormone injection, the concentration of the nuclear binding sites of immature rat uterus increases to approximately 0.4 fmoI/1Lg DNA in the extractable fraction and about 1.2 fmoI/1Lg DNA in the residual fraction, while the cytosol estrogen receptor concentration decreases to approximately 10% of the initial value (Mester and Baulieu, 1975). Later the concentration of estrogen receptor in the nucleus decreases. The half-life values for the 0.5 M KCl extractable and residual forms of the nuclear receptor are approximately 140 and 120 min, respectively (Mester and Baulieu, 1975). Progesterone may influence the interaction of the receptor with the nuclei, for progesterone treatment of ovariectomized rats results in an increase in the in vitro binding of uterine estrogen-binding protein to uterine chromatin (Chatkoff and Julian, 1973). However, progesterone appears not to participate in the receptor---chromatin interaction. The hormone-receptor complex of the nucleus can be extracted with 0.3 M NaCI or 0.4 M KCI at pH 8.5 (jensen et al., 1967a; Puca and Bresciani, 1968). The nuclear estradiol-receptor complex sediments at about 4.5-5 S in physiological and in high-salt medium and can be separated from the cytosol receptor by chromatography on a DEAE-cellulose column (Chamness and McGuire, 1972; Siiteri et al., 1973). The sedimentation coefficient is variable and it can be altered by the concentration of polyanions such as heparin in the suspending medium. Upon acid dialysis, the complex aggregates to 8 Sand 9 S forms. Only the transformed receptor (5 S) is bound to acceptor sites of isolated chromatin or nuclei (McGuire et ai., 1973; Jackson and Chalkley, 1974). The transformed receptor interacts with isolated nuclei from several rat tissues under a variety of conditions of incubation (jensen et al., 1972a; Musliner et ai., 1970; Chamness et at., 1973). In spite of the earlier reports, there is no apparent qualitative or quantitative difference in the capacity of isolated nuclei from target and nontarget tissue to bind estrogen receptor (Chamness et ai., 1973; King and Thompson, 1974). On the other hand, evidence has been reported to show that chromatins from different tissues vary widely in their capacity to bind estradiolreceptor complex. The binding sites of isolated nuclei for estrogen receptor are of the unsaturable type, for isolated nuclei are capable of binding several times more receptors even after maximal in vivo stimulation of the uterus with the hormone

(Chamness et al., 1974; Shepherd et aI., 1974; Andre and Rochefort, 1975). These findings suggest that the acceptor sites in nuclei for estrogen receptors are present in large numbers and bind the receptor with relatively low affinity (Chamness et aI., 1974). The nuclear receptors are firmly associated with specific nuclear components and are partially extractable with 0.3 M NaCI (DeSombre et aI., 1969; Jensen and DeSombre, 1973). On fractionation of isolated nuclei, the receptors are found associated with the chromatin complex (King et al., 1965, 1969; Shyamala and Gorski, 1969), suggesting that they are bound to certain specific sites in the chromatin and that this interaction might activate and initiate the transcription of associated clusters of genes (Fig. 4). The translocation of estradiol receptor into the nuclei appears to follow different steps in immature and adult human uterine tissues. In the immature uterus, the hormone receptor is probably in the 8 S form, which is transformed to the 4 S form, whereas in the adult tissue it exists principally in the 4 S form (Huber et al., 1975). The estradiol-receptor complex of the cytosol of adult human uterine tissue is present primarily in the 4 S form and can be translocated into isolated calf endometrial nuclei by a temperature-independent step (Huber et aI., 1975). These results suggest that the site of transformation to the 5 S is in the nucleus and that hormone is bound initially with cytosol to the 4 S form of the receptor rather than to the 8 S form. On the other hand, the immature cytoplasm of 5-day-old rats contains preponderantly a 4 S estradiol-binding component (Somjen et at., 1974a). By age of 20 days, the uterine cytoplasm contains mainly an 8 S estradiol-binding protein. This apparent difference in the form of estradiolbinding receptor between human and rat uterine cytosol is not clear. The nuclear receptor is located within the nonhistone fraction of calf endometrium chromatin (Alberga et al., 1971). It possesses high affinity for estradiol (K ~ 10 14 M- 1 at 4°C) and is present in a small number of sites « 10) per cell (Alberga et aI., 1971). The nuclear acceptor site for the estradiol-receptor complex is probably present in the DNA and specific nonhistone proteins (King and Gordon, 1972; King and Thompson, 1974; Toft, 1972, 1973). The acceptor sites are saturated at about 10 fmol of nuclear-labeled estradiol receptor per 5 fLg of DNA, which is equivalent to two acceptor sites per 10 7 nucleotides (King and Gordon, 1972). This concentration of acceptor sites is equivalent to about 500 estradiol molecules per nucleus. Similar values are obtained with the use of estradiol-labeled cytoplasmic receptor. The concentration of nuclear receptor to half saturate the acceptor is about 2-4 x 10- 10 M, which is an approximate value of the dissociation constant at 4°C (King and Gordon, 1972). The 5 S receptor binds to DNA fifteen fold more tightly than does the 4 S form. The 5 S receptor binds equally well to all types of double-stranded natural and synthetic DNA (K = 300-400 fLg/ml) (Yamamoto and Alberts, 1974). The possibility that DNA may act as the acceptor is supported by the finding that the estrogen receptor binds to DNA and forms a stable complex. This complex is not dissociated by centrifugation in a sucrose gradient and can withstand extraction with 0.1 M KCI (Toft, 1972, 1973; Yamamoto, 1974; Yamamoto and Alberts, 1974). In addition, steroids can interact directly with DNA (Ts'o and Lu, 1964). The physicochemical studies on the interactions of estradiol-17{3 with DNA and polynucleotides indicate that the hormone interacts along the internal region of the polynucleotide chains and not at the end of the chains (Cohen et at., 1969; Kidson et aI., 1970).

157 ESTROGENS, NUCLEIC ACIDS, AND PROTEIN SYNTHESIS

158

2.3.7.

CHAPTER 7

The translocation of the estradiol-receptor complex into the nucleus may serve several functions. One role is that the receptor acts simply as a transport mechanism, whereby the hormone is delivered to its site of action in chromatin. Another function is that the steroid-protein complex or the receptor protein may interact with a nuclear acceptor site and in some manner influence nuclear functions and modify gene expression. Since the activation of a gene involves multiple factors and steps, measurements of the rate of mRNA formation or RNA polymerase activity would merely reflect one facet of the mechanism. Nevertheless, measurements of these factors may contribute information and shed some light on the molecular basis of gene activation induced by the receptor upon interacting with the nuclear acceptor site. Since one of the early events of estradiol action on the uterus is to stimulate mRNA synthesis and perhaps increase total RNA synthesis (Hamilton, 1964; Hamilton et at., 1968a,b; Hamilton and Luck, 1972), the estradiol-receptor complex was incubated with isolated uterine nuclei and the rate of RNA synthesis was ascertained. Although the direct addition of the hormone to isolated nuclei does not influence RNA synthesis, incubation of isolated nuclei with endometrial cytosol that contained the receptor protein results in a significant stimulation of RNA synthesis (Seshadri and Warren, 1969; Raynaud-Jammet and Baulieu, 1969, 1970; Raynaud-Jammet et at., 1969, 1971; Arnaud et al., 1971; Mohla et al., 1972; Jensen et at., 1973a). One possible mode of action is that the estradiol-receptor complex stimulates directly the Mg2+ -dependent RNA polymerase activity (Andress et al., 1974). This thesis is supported by the finding that estradiol-receptor complex (5 S) added in vitro augments nucleolar RNA polymerase activity, while the nucleoplasmic RNA polymerase activity is not affected. On the other hand, studies on the incorporation of RNA precursors into RNA by isolated uterine nuclei from estrogen-treated rats suggest that the stimulation is related to the synthesis of lesser chains of RNA rather than to an an increase in the number of chains. This suggests that the hormone accelerates the elongation of RNA rather than influences the initiation step. Another possibility is that the hormone stimulates the template activity for RNA synthesis (Barker and Warren, 1966; Barker, 1971). To affect the template, the hormone receptor should influence the nuclear protein-DNA association and cause some unwinding of the DNA (Wang, 1974). Another possible mode of action is that estrogens activate a specific protease that would partially hydrolyze the his tones or nucleoproteins, thereby increasing the accessibility of the DNA in the chromatin structure to RNA polymerase activity (Katz et al., 1972; Levitz et al., 1974). Consistent with this hypothesis is the probability that the receptor-acceptor interaction may activate or induce a protein or enzyme in the nucleus capable of activating RNA polymerase or the chromatin template. This thesis is supported by the finding that the migration of the cytoplasmic receptor into the nucleus is correlated with the formation of specific induced protein in the cytoplasm followed by an increase in ribosomal and other RN A production (Gorski et al., 1968; Teng and Hamilton, 1969; Katzenellenbogen and Gorski, 1972; Somjen et al., 1973c; Mayol, 1975). A cascade mechanism for the action of estrogen on RNA synthesis by uterine cells has been proposed (Baulieu, 1973b; Baulieu et at., 1971, 1972a,b). The proposition is that the hormone enters

Function of the Nuclear Receptor

the target cells and is transferred by the receptor to a specific nonhistone chromatin protein. Subsequent to this event, one or a few of the genes are transcribed with the formation of mRNA with a short half life. The mRNA is translated to form a key intermediate protein, which in turn activates the synthesis of essential components of the protein machinery, particularly ribosomal RNA. Further understanding of the mechanism of derepression of genes initiated by hormones awaits new data on the molecular basis of gene activation. 2.3.8.

Uterine Oxytocin Receptor and Action

One of the effects of estrogen on the uterus is to increase myometrial contractility, possibly by influencing oxytocin action. There is a relation between estrogen and oxytocin action, for estrogen administered to near-term pregnant women induces oxytocin-like effects on the uterus. Furthermore, estrogen potentiates the contraction of myometrial strips in vitro incited by oxytocin or epinephrine (Kumar, 1967; Stander and Barden, 1970). This potentiating effect of estrogen on oxytocin action might be caused by influencing the binding of oxytocin by uterine receptors. The oxytocin receptors are localized in the uterine particulate fraction (Soloff, 1975). Six hours after diethylstilbestrol administration to ovariectomized rats, the affinity of the receptors for oxytocin is increased (Ka of 1 X 10-8 M- 1 to 1.9 X 10-8 M-l), whereas 12 hr must elapse before the number of oxytocin-binding sites per uterus is elevated (Soloff, 1975). It can be concluded that estrogen enhances the affinity of an oxytocin receptor initially and increases the number of receptors as a later event. 2.3.9.

Uterine Progesterone-Binding Protein

Progesterone-binding receptor is present III the uterine cytosol of various mammals (Milgrom and Baulieu, 1970; Reel et at., 1971; Feil et at., 1972; Leavitt and Blaha, 1972; Faber et al., 1972a,b; McGuire and Bariso, 1972; Leavitt et at., 1974; Sar and Stumpf, 1974; Smith et at., 1974). Estradiol administered to castrated guinea pigs and rabbits results in an elevation of the concentration of specific progesterone-binding receptor in the uterus (Milgrom and Baulieu, 1970; Milgrom et al., 1970, 1972, 1973; Corvol et al., 1972; Toft and O'Malley, 1972; Rao et al., 1973) as early as 6 hr after the hormone is given. The peak concentration occurs at approximately 24 hr. The progesterone receptor is induced by estrogen since inhibitors of RNA and protein synthesis administered 15 min before the hormone prevent this effect (Milgrom et at., 1973). Stimulation of the uterine progesterone-binding activities by estrogen may account for the synergistic action of estradiol and progesterone on the uterus (Corvol et al., 1972). It is noteworthy that progesterone administered 20 hr after estrogen provokes a rapid fall in the progesterone receptor concentration so that less than 20% remains 1 day after progesterone administration. In contrast to the estradiol effect, the fall induced by progesterone is not prevented by inhibitors of protein synthesis. The results suggest that the increase in the concentration of progesterone receptor induced by estradiol is dependent on the synthesis of RNA and protein. On the other hand, progesterone in some unknown manner accelerates the inactivation of its own receptor (Milgrom et al., 1973).

159 ESTROGENS, NUCLEIC ACIDS, AND PROTEIN SYNTHESIS

160

2.4.

CHAPTER 7

2.4.1.

Ribonucleic Acid Biosynthesis Rate of Ribonucleic Acid Biosynthesis

. The rate of RNA biosynthesis, the kinetics of the effect of estrogen on the process, and the relation of these two processes to protein biosynthesis are of considerable importance in elucidating the action of estrogen. A chronological account of the study of the effect of estradiol on RNA biosynthesis is given in Table 3. As early as 1948, Jeener observed an increase in uterine RNA without a concomitant rise in DNA concentration 24 hr following administration of estradiol to mice. This finding was soon confirmed in the rat (Grauer et al., 1950), and since then most workers have used the rat in such studies. Grauer et al. (1950) were able to demonstrate an even more rapid effect of estrogen by using the incorporation of a radioactive RNA precursor as a measure of RNA synthesis. The uptake of 32P04 into the uterus was stimulated within 3 hr after estradiol treatment, and its incorporation into the acid-insoluble uterine subcellular fraction at 6 hr was enhanced. Meanwhile, using cytological techniques in combination with chemical measurements of DNA and RNA phosphorus, Drasher (1952, 1953) observed that chemical changes and variations in volume occur in nucleoli, nuclei, and cytoplasm of epithelial and stromal cells in the mouse during the course of the normal estrus cycle. Using the volume of the subcellular components in the diestrus state as a baseline, the volumes of nucleoli, nuclei, and cytoplasm increased in order during

Table 3. Time Required for Estradiol to Stimulate RNA Synthesis in the Uterus Time of measurement after treatment (hr)

24

Preparation

Estradiol-l 7f3 dose (,.Lg); route a

24

Ovariectomized mouse 10.0, in oil; s.c. Ovariectomized rat 1.0, in oil; S.c.

6

Immature rat

Estrus vs. diestrus 6 21

Mature rat

6-8 b

6-8 b

0.4, in 20% ethanol; s.c.

Radioactive precursor

32PO,

Ovariectomized rat Ovariectomized rat Uterine segments, ovariectomized rat

5.0, in oil; s.c. 5.0, in oil; S.c. 10.0; i.v.

[ 14

C]_ Formate

Uterine segments, ovariectomized rat

10.0; i.v.

[ 14

C] Glycine

Moiety measured

Approximate stimulation (%)

RN Ase-released 95 material Acid-insoluble purine 12.6 N Acid-insoluble 58 radioactivity P from mild alkaline hydrolysis Acid-insoluble ribose Acid-insoluble ribose Adenine and guanine of mixed nucleic acids Adenine and guanine of mixed nucleic acids

104

°

95 382; 734

150; 263

References Jeener (1948) Cole (1950) Grauer et al. (1950)

Drasher (1952, 1953) Telfer (1953) Telfer (1953) Mueller and Herranen (1956) Mueller and Herranen (1956)

Table 3. (Cont.)

Time of measurement after treatment (hr)

Preparation

Estradiol-17/3 dose (/Lg); route a

Radioactive precursor

6_8 b

Uterine segments. ovariectomized rat

24

Immature and 0.1, in oil for 32P04 ovariectomized rat 3 days; S.c. Ovariectomized rabbit 10.0 per day

120 6

10.0; i.v.

[3-'·C]Serine

2-4b

Uterine segments, ovariectomized rat Uterine segments, ovariectomized rat Uterine segments, ovariectomized rat Uterine segments, ovariectomized rat Uterine segments, ovariectomized rat Uterine segments, ovariectomized rat Uterine segments, ovariectomized rat Immature and ovariectomized rat Uterine slices, ovariectomized rat Uterine slices, ovariectomized rat Ovariectomized rat

1-2b

Immature rat

5.0; i.p.

1_2b

Immature rat

5.0; i.p.

Immature rat

5.0; i.p.

[8- 14C]Adenine [8-'·C]Adenine [3H]_ Uridine [3H]_ Cytidine [3H]_ Cytidine 32P04

Ovariectomized rat

10.0; i.p.

32P04

Immature rat, isolated whole uterine horn Immature rat, isolated whole uterine horn Immature rat, isolated whole uterine horn Immature rat

5.0; i.p.

12 6_7 b 12-13 b 6-7 b

4 4 4 4\12 24Y2

2-3 b 2_3 b 2-3 b 3-4 b

22-29b

Uterine segments, ovariectomized rat

10.0; i.v. 10.0; i.v. 10.0; i.v.

NaH'4C03

10.0; i.v.

NaH 14C0 3

10.0; i.v.

10.0; i.v.

[8- 14C]Adenine [2_14C]Glycine 32PO.

0.03; i.v.

32PO.

10.0; i.v.

10.0; i.v.

10.0; i.v. 10.0; i.v.

5.0; i.p. 5.0; i.p. 5.0; i.p.

0.1; i.!.

Approximate stimulation Moiety measured Adenine and guanine of mixed nucleic acids P from mild alkaline hydrolysis Acid-insoluble ribose

(%)

References

342; 734

Herranen and Mueller (1956)

134

Davis et at. (1956)

5.7 c 50.0 d Acid-insoluble isolated 0 U Acid-insoluble isolated 31 U Acid-insoluble adenine 387 and guanine 0.4 Acid-insoluble U

Telfer and Hisaw (1957) Jervell et at. (1958) Jervell et at. (1958) J ervell et at. (1958) Jervell et at. (1958)

Acid insoluble

109; 244

Mueller et at. (1958)

Acid insoluble

210

Acid insoluble

260

Acid-insoluble adenine and guanine Phenol, extractable (OD 260 nm) Phenol, extractable (OD 260 nm) RNAse-released radioactivity RNAse-released radioactivity RNAse-released radioactivity Acid-insoluble AMP

190

Mueller et at. (1961b) Mueller et al. (1961b) Hamilton (1963)

Small

Wilson (1963)

Large

Wilson (1963)

125

Ui and Mueller (1963) Noteboom and Gorski (1963a) Noteboom and Gorski (1963b) Gorski and Nicolette (1963) Gorski and Nicolette (1963) Gorski and Nicolette (1963) Gorski and Nicolette (1963) Gorski and Nicolette (1963) Gorski and Axman (1964)

Acid-insoluble nuclear AMP 32PO. Acid-insoluble nuclear AMP Acid-insoluble GMP [8- 14C]radioactivity Guanosine 32PO. Acid-insoluble GMP radioactivity [3H]_ Acid-insoluble RNAsereleased Cytidine radioactivity Acid-insoluble; not [U- 14C]Glycine acid soluble adenine and guanine

22-46 80 62-135 17-28 25 25 e 187f 16e 1061 90.5

130

Szego and Lawson (1964)

(continued)

Table 3. (Cant.) Time of measurement after treatment (hr)

Preparation

Estradiol-17 f3 dose (/Lg); route a

Radioactive precursor

12.4

Hamilton (1964)

290

Acid-insoluble AMP

224-270

[U- 14C]Glucose [3H]_ Cytidine [3H]_ Uridine [3H]_ Uridine [3H]_ Guanosine [3H]_ Uridine [3H]_ Uridine [3H]CTP [3H]UTP [3H]_ Uridine [3H]_ Guanosine [3H]_ Uridine [14C]Uridine [3H]_ Cytidine [3H]_ Uridine [3H]_ Guanosine

Acid-insoluble AMP

43

Phenol-extractable RNA Acid-insoluble radioactivity Acid-insoluble radioactivity Various types of DNAlike RNA

88 e 124f 520

Nicolette and Gorski (1964a) Nicolette and Gorski (1964a) Nicolette and Gorski (1964b) Gorski and Nelson (1965) Hamilton et al. (1965) Hamilton et al. (I 968b ) Billing et al. (1969)

Ovariectomized rat

10.0; i.v.

1

Ovariectomized rat

10.0; i.v.

5-6b

Immature rat, isolated whole uterine horn Immature rat, isolated whole uterine horn Immature rat, isolated whole uterine horn Immature rat

5.0; i.p.

5.0; i.p.

1 Ib

61

Ovariectomized rat

10.0; i.p.

12

Ovariectomized rat

10.0; i.p.

0.5-6

Immature rat

1.0; i.p.

2

Immature rat

5.0; i.p.

2

Immature rat

5.0; S.c.

1, 2--4

Immature rat

1.0; i.p.

2

Ovariectomized mouse 0.05; S.c.

4

Ovariectomized mouse 0.05; S.c.

1-2

Immature rat

1.0; i.p.

2

Nonpregnant oophorectomized sheep

1.0 /Lg/kg; i.v. [3H]_ Uridine

!-w i-l b

10.0; i.v. 5.0; i.p.

References

(%)

Acid-insoluble RNAse released Acid-insoluble RNAse released Acid-insoluble AMP

4

5-6 b

Moiety measured

[2_14C]Uridine [2_ 14C]_ Uridine [2- 3H]Glycine 32P04

1

2

Approximate stimulation

Hamilton (1964)

500-600 50-500

32 Sand 45 S RNA

None

Acid-insoluble radioactivity 45 S rRNA precursor

80 400 (1 hr)

4 S, 18 S, 28 S RNA

Increased

Miller and Baggett (I 972a,c)

acid-insoluble nuclear RNA 18 S, 28 S, 32 S, 45 S RNA

75

Miller and Baggett (l972b) Knowler and Smellie (1973)

Acid-insoluble radioactivity

400-100 (1 hr heterogeneous mRNA) 200-300

Joel and Hagerman (1969) Raynaud-Jammet et al. (1971) Knowler and Smellie (1971)

Resnik et al. (1975)

Abbreviations: s.c., sulxutaneous; i.v., intravenous; i.p., intraperitoneal; i.I., intraluminal. Radioactive precursors were provided during range indicated and measurement was made at completion of time period. Endometrium. d Myometrium. e Nucleus. f Cytoplasm. a

b

C

the active stages of the cycle. The RNA-phosphorus:DNA-phosphorus ratio was found to be maximal at estrus. Injected estradiol increases formate and glycine incorporation into nucleic acid moieties of surviving rat uterine segments within 6--8 hr (Mueller and Herranen, 1956). Davis et ai. (1956) demonstrated an increased incorporation of radioactive phosphorus into rat uterine RNA 24 hr after injection of estradiol. An increase in the specific activity of the uterine DNA was also noted. An increase in total RNA of incubated rat uterine segments, measured as acid-insoluble uridine and adenine, occurs 12 hr after an injection of estradiol Oervell et at., 1958; Mueller, 1957). There is no concomitant increase in the thymine of DNA. In addition, incorporation of radioactivity from NaH 14C03 into acid-insoluble uridine increases 12-13 hr after estradiol treatment. Using the same in vitro system, it can be shown that estradiol promotes the incorporation of [814C]adenine into the adenine and guanine residues of RNA within 7 hr (Mueller et ai., 1958). Synthesis of rat uterine protein in vivo (under estradiol stimulation) is inhibited nearly 90% after the intraperitoneal injection of 15 mg of puromycin (see Fig. 1). The estrogen stimulation of nucleic acid synthesis is also inhibited by puromycin (Mueller et at., 1961a,b; Gorski et ai., 1961). Hamilton (1963) confirmed these findings with a much lower estrogen dose, which is presumably closer to a physiological concentration. As little as 0.03 ILg estradiol or estriol or 0.6 ILg estrone injected intravenously into an ovariectomized immature rat 4 hr prior to sacrifice was sufficient to stimulate incorporation of [214C]glycine into uterine protein and incorporation of [32P]phosphate into nucleotides of the uterine mixed nucleic acid fraction. These doses are in the range that Hisaw (1959) found to be needed for accumulation of uterine water. Puromycin was shown to inhibit [2- 14C]glycine incorporation into protein and to inhibit almost all of the estrogen stimulation of this reaction. Ui and Mueller (1963) studied the effect of intraperitoneally administered actinomycin D, an inhibitor of DNA-dependent RNA synthesis (Kirk, 1960; Reich et ai., 1961; Hurwitz et at., 1962), on in vivo RNA synthesis, protein synthesis, and increase in wet uterine weight in ovariectomized rats. Actinomycin D blocked RNA synthesis about 90% when the radioactive tracers were administered during the interval 2.5-4.5 hr following the administration of inhibitor. The administration of estradiol intravenously 4 hr prior to sacrifice (30 min after the actinomycin D) had no effect. Late (2-4 hr) protein synthesis that was responsive to estradiol administration (see Section 2.4.2) was prevented by actinomycin D, but the baseline protein synthesis was not prevented. The acceleration of mixed lipid synthesis in the 2-4-hr period following administration of estradiol was 270% of the control value. By a mechanism similar to the inhibition by puromycin of protein synthesis, actinomycin D did not change the baseline of synthesis of lipid, but did reduce the estradiol-stimulated portion of mixed lipid synthesis (by approximately 90%). Again, in the consideration of the change in wet weight, which in these relatively short periods of time was due to accumulation of water, actinomycin D caused no alteration. The drug, however, did diminish the usual estradiol stimulation of protein synthesis by 70%. By administering actinomycin D at different times in the 4-hr test period, Ui and Mueller (1963) demonstrated that (1) the actinomycin D inhibition of the estradiol-stimulated increase in wet weight of the uterus occurred

163 ESTROGENS, NUCLEIC ACIDS, AND PROTEIN SYNTHESIS

164 CHAPTER 7

during the first 2 hr of the period and (2) the estradiol stimulation of synthesis of protein and phospholipid depended to some extent on continued synthesis of RNA. Gorski and Nicolette (1963) were able to demonstrate, in ovariectomized adult and immature female rats, greater stimulation by estradiol of RNA synthesis (incorporation of injected [32 P]orthophosphate into AMP or the GMP-UMP area of chromatograms obtained from fractions solubilized by hydrolyzing the acidinsoluble residue) at an earlier time period, 0-1 hr, than the incorporation of [32P]orthophosphate into uterine lipids. A more detailed kinetic study of the rate of estrogen stimulation of RNA synthesis and its comparison with the rate of estrogen stimulation of protein synthesis was completed by Hamilton (1964). RNA and protein synthesis were found to be enhanced 30 min after intravenous administration of estradiol. The increase in protein synthesis was maintained at 1 hr. It reached a plateau until another and larger increase was noted at 3 hr, and still another increase was observed at 4 hr. This result was in contrast to the stimulation of RNA synthesis, which continued almost linearly for 3 hr and then reached a plateau. Gorski and Nelson (1965) reported the stimulation of incorporation of [3H]cytidine into immature rat uteri in vivo in the 40-60 min period following intraperitoneal injection of estradiol. More recently, maximal stimulation (520%) of [3H]uridine incorporation into uterine RNA within the 10-20 min period following an estradiol injection was demonstrated by Hamilton et al. (1965). The evidence is abundant, therefore, that after an increase in the level of estradiol in the uterus (1) there is very little immediate change in DNA synthesis, (2) RNA synthesis increases linearly up to 40 hr, (3) the increase in RNA synthesis may begin as early as 10-20 min, (4) actinomycin D is capable of preventing the estrogen stimulation of bulk protein synthesis as well as a great majority of the RNA synthesis, and (5) some early protein synthesis may be necessary to evoke the estrogen stimulation of RNA synthesis. An increase in uterine RNA polymerase activity appears approximately 30 min after estradiol administration (Hamilton et al., 1965; Gorski, 1964) and is therefore one of the earliest known responses to estrogen. The RNA polymerase activity was assayed in crude uterine nuclear fractions from estradiol-treated and nontreated immature rats. The polymerase activity was noted to possess the characteristics reported for other mammalian tissue RNA polymerase activities. Actinomycin D administered in vivo or in vitro inhibits rat uterine RNA polymerase activity, thus indicating that this activity is DNA dependent (Gorski, 1964). Furthermore, the addition of deoxyribonuclease to the in vitro assay destroys the activity (Hamilton et al., 1965). 2.4.2.

Types of Ribonucleic Acid Produced

The first attempt to characterize the type of RNA synthesized under the influence of estrogen was made by Jervell et al. (1958). The uridine from the acidinsoluble material in subcellular fractions separated by centrifugation was measured. The majority of the uterine RNA uridine was found in the sediment after low-speed centrifugation. An estradiol injection 24 hr prior to sacrifice increased the RNA uridine in both the sediment and the supernatant fluid fractions.

Subsequently, incorporation of [8- 14C]adenine into specific fractions of rat uterine RNA was investigated using the sucrose density gradient technique (Britten and Roberts, 1960). Since it was known that DNA synthesis did not proceed at an appreciable rate during the first 24 hr following estrogen treatment in an ovariectomized animal, the [8- 14C]adenine incorporation that was measured soon after hormone treatment was presumably incorporation into RNA. During 30-min incubations of uterine slices from ovariectomized rats, [8- 14C]adenine was incorporated primarily into a 4 S-6 S fraction that was believed to contain a substantial amount of tRNA. The total RNA and the [8- 14C]adenine incorporation into the 4 S-6 S RNA fraction were both found to increase 4 hr after injection of estradiol, and to increase even further after 24 hr. Very little increase in radioactivity in the heavy, presumably structural, ribosomal RNA was noted until 24 hr after estradiol treatment. In other experiments, the 30-min incubation with [8- 14C]adenine was followed by a 3-hr incubation in the presence of an excess of nonradioactive adenine. Essentially the same results were obtained, except that in the uterine slices from rats pretreated for 4 hr with estradiol some increase in radioactivity was noted in the rapidly sedimenting (heavy) fraction, and this increase was further exaggerated in the slices from rats pretreated for 24 hr. It appeared, therefore, that in the bulk portions of uterine cellular RNA the lighter moieties (tRNA and incomplete ribosomal RNA) were synthesized first, followed by the heavier RNA molecules. Ribosomal RNA (structural RNA) is produced in the nucleolus in L strain fibroblasts (Perry, 1962) and in the nucleus (which does not exclude the nucleolus) in HeLa cells (Tamaoki and Mueller, 1962). In order to investigate the site of production of ribosomal RNA in uterine mucosa, histological studies were done at 0, 4, and 24 hr following intravenous administration of 10 Il-g of estradiol to ovariectomized rats. At 0 and at 4 hr, the nucleoli were small and dense, the epithelial cells were cuboidal, and the cytoplasm contained only small amounts of basophilic material. There was no significant change at 4 hr, but after 24 hr the nucleoli were large and pale, the epithelium was columnar, and the cytoplasm contained dense basophilic material (RNA). It seems, therefore, that ribosomal RNA appears histologically at some time between 4 and 24 hr after estradiol treatment, whereas the bulk of the incorporation of radioactive labeled precursors of RNA into RNA is found at 2 hr and in slowly sedimenting (light) RNA (Wilson, 1963). Protein synthesis can be increased in a uterine cell-free system consisting of a supernatant fluid fraction obtained from nontreated rats by the addition of a fraction from rats treated with estradiol for 4-5 hr (Greenman and Kenney, 1964). The increased protein synthesis can be correlated with an increase in the activity of the ribosomal fraction. These findings are consistent with the interpretation that estradiol increases the number of ribosomes as well as the level of messenger RNA that is probably associated with the ribosomes. The nature of the uterine RNA that is rapidly synthesized following estrogen treatment was also investigated by Noteboom and Gorski (1963a). Immature rats were given estradiol intraperitoneally, followed in 1 hr by an injection of [3H]cytidine, and then sacrificed at the end of the next hour. The uteri were removed and homogenized, and subcellular fractions were separated by differential centrifugation. The fractions were termed "nuclear," "mitochondrial," "ribosomal," "postribosomal," and "cytoplasmic," according to their sedimentation properties: 800g for 10 min, 15,000g for 15 min,

165 ESTROGENS, NUCLEIC ACIDS, AND PROTEIN SYNTHESIS

166 CHAPTER 7

10S,000g for 1 hr, lOS,OOOg for 3 hr longer, and the remaining supernatant fluid fraction, respectively. The radioactivity incorporated into cytosine S'-phosphate (CMP) of a residue prepared from each fraction, by acid precipitation, lipid solvent precipitation, and ribonuclease release, was measured. During the 1-2 hr period after estradiol treatment, the incorporation of [3H]cytidine was increased in both of the two fractions observed in this experiment: the nuclear fraction by 17S% and the cytoplasmic fraction by 2S0%. During this period, there was a 2246% increase in the incorporation of radioactivity from injected [2- 14C]glycine or eH]leucine into the ribosomal fraction protein, but no significant increase in "protein synthesis" in the other subcellular fractions examined, namely, the nuclear, mitochondrial, postribosomal, and cytoplasmic fractions (Noteboom and Gorski, 1963a). Gorski and Nicolette (1963) were able to demonstrate, in vivo, stimulation of incorporation of [32P]orthophosphate into rat uterine RNA during the I-hr interval after estradiol administration. The percentage increase in RNA synthesis was greater than the percentage increase in the incorporation of [32P]orthophosphate into lipids. No increase in phospholipid synthesis was noted until the 1-2 hr period after treatment with estradiol, except in the microsomal fraction. At 3-4 hr the estradiol-mediated increase in protein synthesis was even higher: 47-239% of the control values. During the 1-2 hr period following estradiol or saline injection, prior puromycin administration was shown to reduce protein synthesis to 20% of control values. In contrast, puromycin did not depress the level of RN A synthesis in control animals, but it did depress RNA synthesis in estradiol-treated animals. The net effect of puromycin was to reduce the high level of RNA synthesis in estradiol-treated rats to approximately the level noted in control animals. The RNA polymerase activity (Fig. 1) in the crude nuclear fraction obtained from rats pretreated with estradiol for 1-2 hr was then measured. It was found to be 118% greater than that measured in similar preparations from nontreated rats. Puromycin blocked the activity stimulated by estradiol in this RNA polymerase system without diminishing the baseline level of activity. This blocking action of puromycin on the estradiol-stimulated activity of the RNA polymerase is similar to the action of puromycin on the estradiolstimulated uterine RNA synthesis. It was postulated, therefore, that estrogen first caused an increase in RNA polymerase activity, and perhaps small amounts of other critical proteins, followed by increases in synthesis of phospholipid, RNA, and bulk protein. Further characterization of the RNA product of estrogen action at an earlier period (1 hr) was carried out by Gorski and Nicolette (1963). An increase in uterine RNA synthesis was found 1 hr estradiol treatment to the nuclear, mitochondrial, microsomal, and soluble subcellular fractions. The RNA from the nuclear fraction was studied further. Its sensitivity to ribonuclease was examined. The specific activity of the radioactive nuclear RNA that was ribonuclease-sensitive was 3-S times higher than that of the ribonuclease-resistant fraction. One to two hours after estradiol treatment, the synthesis of the ribonuclease-sensitive nuclear RNA was stimulated approximately fivefold, and that of the ribonuclease-resistant nuclear RNA was stimulated approximately threefold. An in vitro method was also used to study intracellular differences in RNA synthesis. Estradiol was injected into rats intraperitoneally, and 2 hr later the uteri were excised. These uteri were studied for their ability to incorporate [32 P]orthophosphate or [8- 14C]guanosine

into the guanosine 5' -phosphate (GMP) residue of various RNA subcellular fractions during 1 hr of in vitro incubation. The estradiol stimulation was nearly abolished in the nuclear RNA fraction and was greatly diminished in the cytoplasmic RNA fraction by this incubation. Stimulation of incorporation of [32P]orthophosphate into lipid in several fractions was not diminished. The evaluation of data on the use of uterine preparations in vitro is difficult because of the state of the tissue. An intact uterus, incubated in vitro, is too thick to allow adequate oxygenation at its center. A uterine slice may not be suitable because slicing a muscular tissue generally results in a great percentage of damaged cells (Kipnis and Cori, 1957). Using another inhibitor of protein synthesis, cycloheximide, Gorski and Axman (1964) reinvestigated the relationships between protein synthesis and RNA synthesis. During the period 3-4 hr after injection of estradiol into an immature rat, a 93% inhibition of [2- 14C]glycine incorporation into uterine protein was noted in the presence of cycloheximide. This inhibition included nearly all of the protein synthesis occurring in the tissue at that time, irrespective of the presence of estradiol. Cycloheximide alone did not change uterine wet weight significantly, nor did it inhibit [3H]cytidine incorporation into RNA. Cycloheximide did, however, inhibit estradiol-stimulated increases in wet weight and in RNA synthesis (Gorski and Axman, 1964). The same results were obtained when the tissue was examined at the end of the 1-2 hr period following estradiol administration when there was little or no detectable estradiol stimulation of the protein synthesis in the uterus (Noteboom and Gorski, 1963a). The results of the studies that made use of two inhibitors of protein synthesis, cycloheximide and puromycin, in uterine systems indicated a similar site of action of estradiol (Noteboom and Gorski, 1963b; Gorski and Axman, 1964). This fact takes on added importance when the sites of action of the two inhibitors are compared (see Fig. 1). Puromycin apparently stops protein synthesis by speeding the release of the partially formed protein (peptide) from the microsome-bound messenger RNA complex resulting in some manner from its covalent attachment to the growing peptide (Nathans, 1964), whereas cycloheximide stops the process in a different manner by inhibiting the step in which the amino acid is transferred from charged aminoacyl-tRNA to the growing polypeptide (Ennis and Lubin, 1964), presumably by damaging the ribosome in some way (Siegel and Sisler, 1965). A complete comparison of the effects of actinomycin D and puromycin administered in vivo on macromolecular syntheses in rat uterus with and without estradiol was made by Hamilton (1964). Actinomycin D inhibited synthesis of both RNA and protein in the uteri of control and estradiol-treated rats. Although actinomycin D inhibited RNA synthesis, a partial stimulation of protein synthesis by estradiol in the presence of actinomycin D did occur when compared with the actinomycin D-treated control. This finding indicated again that estradiol could enhance some protein synthesis that cannot be inhibited by actinomycin D (i.e., that does not depend on prior DNA-dependent RNA synthesis). Puromycin, however, inhibited protein synthesis severely in the presence or absence of estradiol as well as in the presence of actinomycin D. Gorski and Nelson (1965) used in vivo studies in further efforts to characterize the uterine RNA synthesized in response to estradiol activity. Incorporation of

167 ESTROGENS, NUCLEIC ACIDS, AND PROTEIN SYNTHESIS

168 CHAPTER 7

intraperitoneally irUected [3H]cytidine into uterine RNA subcellular fractions (prepared by differential centrifugation and by sucrose density gradient centrifugation) was employed. During the 40-60 min period following estradiol injection into immature rats, [3H]cytidine incorporation into uterine RNA was increased approximately 88%. The radioactive label was found at that time almost entirely in the nuclearmyofibrillar subcellular fraction. Very little labeling of the RNA of the cytoplasmic fraction occurred. The estrogen-stimulated RNA synthesis that occurred during the 40-60 min and the 45-105 min periods after estradiol administration was not localized to any single RNA fraction (tRNA, mRNA, or ribosomal RNA). By irUection of [3H]cytidine directly into the uterine lumen 15 min prior to sacrifice, rapidly sedimenting RNA was labeled. Estradiol did not, however, stimulate this labeling when injected 45 min prior to sacrifice. In order to investigate the possibility that some RNA produced in response to estradiol might be high in messenger RNA characteristics, hybridization experiments were carried out with uterine DNA and [3H]cytidine-Iabeled RNA from estrogen-treated and nontreated rats. Less than 5% of these RNA preparations hybridized with the DNA, using the Nygaard and Hall (1963) technique. This method did not show an estrogen stimulation of messenger RNA, and sucrose density gradient centrifugation did not show an estrogen-mediated rise of any specific RNA fraction. Earlier studies suggested that there was a prompt and significant increase in the rate of RNA synthesis in the uterus commencing immediately after estradiol administration (Hamilton, 1964; Moore and Hamilton, 1964; Means and Hamilton, 1966; Barker and Warren, 1966). This increase in RNA synthesis was accompanied by a stimulation of the in vitro template activity of uterine chromatin for RNA synthesis and an increase in RNA polymerase activity (Hamilton, 1964; Hamilton et al., 1965, 1968b; Barry and Gorski, 1971). Although estradiol induces an increase in all types of RNA, the rate-limiting event is the synthesis and intracellular accumulation of specific mRN A (Hamilton et al., 1968b; Luck and Hamilton, 1972; Baulieu et al., 1972b; Miller and Baggett, 1972a,b,c; O'Malley et al., 1972). Estrogen administered to ovariectomized adult rat induced an increase in the rate of synthesis of ribosomal RNA in the uterus and the rate or efficiency of processing of precursor RNA species of high molecular weight (Moore and Hamilton, 1964; Billing et al., 1969; Raynaud-Jammet et al., 1971; Luck and Hamilton, 1972; Miller and Baggett, 1972a; Knowler and Smellie, 1971, 1973). The rate of incorporation of radioactive uridine into rapidly labeled RNA in the ovariectomized rat uterus increases 20 min after estradiol administered intraperitoneally (Hamilton et al., 1965, 1968b; Billing et al., 1969; Knowler and Smellie, 1971, 1973). On the other hand, estradiol administered to immature rats did not affect the pattern of distribution of high-molecular-weight, rapidly labeled RNA (Joel and Hagerman, 1969). The variation in results may be due to the ages of the animals or to the method of extraction of the RNA. Moreover, the RNA being synthesized in the estradiol-stimulated uterus has a very short half-life, presumably composed of species having DNA-like composition and not stable species such as ribosomal precursor RNA (Miller and Baggett, 1972c). It appears that the hormone induces an increase in the total RNA activity of chick oviduct polysomes, resulting in an increase in the ribosomes' translation capacity and stimulation of the activity of peptide chain initiation (Comstock et al., 1972). It has been suggested

that the increase in RNA synthesis after administration of estradiol to ovariectomized mice is due primarily to an increase in the incorporation of administered nucleotides into nucleotide pools (Miller and Baggett, 1972b). Estradiol and progesterone induce different effects on the turnover and synthesis of ribosomal RNA, tRNA, and mRNA in the uterus (Miller and Baggett, 1972a,c; Moriyama, 1974). The half-life of uterine ribosomal RNA in ovariectomized mice is about 7 days, decreasing to about 4 days in mice that received 25 /-Lg of estradiol daily. The half-life of uterine tRNA of estradiol-treated and untreated mice is about 3 days. Hence estrogen administration results in a shortened half-life of ribosomal RNA, whereas that of tRNA remains unchanged. Progesterone administration results in a stimulation of all classes of RNA synthesis in the uterus of ovariectomized mice (Miller, 1973c). The rate of total RNA synthesis increased from l.46 to 2.56 nmol of UMP incorporated into RNA per minute per uterus of control mice 4 hr after estradiol-17 ~ (Miller and Baggett, 1972c). With progesterone the rate increased to 3.10 nmol of UMP incorporated into RNA per minute per uterus (Miller, 1973b). The increase in ribosomal RNA and tRNA, however, is only about 0.6-l.3%, suggesting a greater increase in mRNA synthesis. Likewise, the rate of RNA synthesis in decidualizing mouse uterus induced by intraluminal administration of sesame oil is increased by 64% (Miller, 1973a). In addition to estrogen and progesterone, uterine RNA synthesis is increased by other hormones. The administration of insulin to alloxan-diabetic rats, growth hormone to hypophysectomized rats, and thyroxine to thyroidectomized rats results in a marked increase in the rate of [3H]uridine incorporation into uterine RNA (Miura and Koide, 1970, 1971). The effect of estrogen on protein and RNA synthesis is complicated by the fact that they are interrelated. For example, stimulation of ribosomal RNA synthesis by estrogen would increase the rate of peptide synthesis. Furthermore, estrogen may elevate the capacity of ribosomes for peptide synthesis in several ways (Moore and Hamilton, 1964; Teng and Hamilton, 1967; Suvatte and Hagerman, 1970; Whelly and Barker, 1974): by (1) increasing the number of ribosomes in uterine cells, (2) activating the ribosomes in some indeterminate manner, (3) stimulating the rate of peptide chain initiation and elongation in the ribosomes, and (4) facilitating translocase activity of the ribosomes. Elevation in any of these activities associated with ribosomes will stimulate protein synthesis. On the other hand, estradiol may induce the synthesis of a specific factor required for RNA synthesis (Teng and Hamilton, 1969). Such a possibility is suggested by the finding that estradiol induces an early synthesis of a specific nuclear factor that can activate nucleolar RNA polymerase activity or template activity (Raynaud-Jammet et at., 1969, 1972). However, the production of this specific factor may be secondary to an initial induction of the mRN A for the specific factor, whereas generalized protein synthesis takes place as a later event. 2.4.3.

Biological Activity of Uterine Ribonucleic Acid

The concept that estrogens may exert their stimulatory actions on the uterus by activation of RNA synthesis has been developed chiefly through biochemical studies, as described in the preceding section. The evidence includes the observations that estrogen stimulation leads quickly to an increase in the rate of incorporation of precursors into RNA (Mueller, 1965; Gorski et at., 1965), the

169 ESTROGENS, NUCLEIC ACIDS, AND PROTEIN SYNTHESIS

170 CHAPTER 7

initial increase being noted in the nuclear fraction of RNA (Talwar, 1964; Gorski and Nelson, 1965), and that specific inhibitors of DNA-dependent RNA synthesis prevent the stimulatory action of estrogen (Talwar and Segal, 1963; Hamilton, 1964). It has been noted, also, that RNA polymerase activity in uterine cells is enhanced by estrogen stimulation (Gorski, 1964). Although there is no evidence that the mixed RNA moieties of estrogenstimulated uterine cells differ qualitatively from those produced by the estrogendeprived cell, the quantitative difference in RNA production is evident. The biological activity of RNA extracted from estrogen-stimulated rat uteri has been studied (Segal, 1964; Segal et aI., 1965a; Mansour and Niu, 1965), and the results reveal that several estrogen-like phenomena can be induced by the material. In the reported studies, mixed uterine RNA was extracted by methods that do not preferentially separate species of RNA. It is therefore not possible to associate the reported activities with a particular nucleic acid component of estrogen-stimulated cells. These procedures do, however, involve several ethanol precipitation steps that tend to minimize the likelihood that biological activity of RN A extracts can be accounted for on the basis of contaminating estrogens in the excised uteri. The first reported RNA-induced estrogen-like effect was the stimulation of the endometrium of ovariectomized rats. The in vivo system used was the ovariectomized rat with indwelling catheters in each uterine horn. A total of ISO I-tg of uterine RNA was applied locally in divided doses over a 48-hr period and the histological pattern of the endometrium was compared with that of the contralateral horn, which received saline by means of the same tubal irrigation. Uterine RNA obtained from estrogenized rats caused endometrial stimulation, including all criteria usually associated with hormonal influence, namely, transition of the lining epithelium to high columnar cells with basal nuclei and prominent nucleoli, uterine glandular proliferation, and separation of stromal cells (Fig. SA,B). That the activity is due to RNase-sensitive material is evidenced by the almost complete loss of activity when the extract is preincubated with RNAse (Fig. SC,D). The activity is destroyed, also, by preincubation with phosphodiesterase but not by DNAse or trypsin. Other tissues from estrogenized animals have been used as controls, and the RNA extracted from liver, vagina, and kidney does not cause endometrial stimulation (Fig. SE,F). Corresponding results have been obtained using the mouse as the experimental animal, and uterine alkaline phosphatase activity as the biological end point (Mansour and Niu, 1965). In the mouse, ovariectomy results in a regression of activity of uterine alkaline phosphatase, and estrogen treatment restores the activity to above the normal level (Leathem, 19S9). Uterine RNA from normal mice administered intraluminally induced a SO% increase in uterine alkaline phosphatase activity of ovariectomized animals. Liver RNA failed to restore the enzymatic activity. Boiling the uterine RNA extract or pretreating with RNAse at room temperature for 2 hr destroyed the enzymestimulating potency (Table 4) (Mansour and Niu, 1965). Blastocystic nidation in the rat, a uniquely estrogen-dependent physiological process, has proved also to be responsive to uterine RNA (Segal et at., 1965b). Experimentally induced delay of nidation in the rat has been used by several investigators to study the hormonal requirements for nidation of the blastocyst (Nutting and Meyer, 1964). Postcoital females are ovariectomized on day 4 of pregnancy, which is

maintained subsequently on progesterone. The blastocysts remain viable and unattached in the uterine lumen and will not implant until some estrogen is provided. A dose of 0.01 f-tg of estradiol injected into the parametrial fat will induce nidation (Wada et at., 1965). Equally effective in causing nidation is the parametrial injection of 0.75 f-tg of RNA extracted from the uteri of estrogen-

Figure 5. (A) Cross section of endometrium of ovariectomized rat that received intrauterine application of estradiol-17 f3. A total dose of 6 x 10-4 P,g was administered in divided doses every 4 hours for 48 hours and the animal was sacrificed 4 hours after the twelfth intraluminal application. (B) Opposite uterine horn of animal shown in (A). This horn was treated with saline on same schedule. (C) Cross section of endometrium of ovariectomized rat that received intrauterine application of uterine RNA e. A dose of 12.5 p,g was administered every 4 hours for 48 hours and the animal was sacrificed 4 hours after the twelfth intraluminal application. (D) Opposite uterine horn of animal shown in (C). This horn was treated with uterine RNA e incubated with 5 p,g/ml RNase (pancreatic) for 60 minutes at 37°C. (E) Cross section of endometrium of ovariectomized rat that received intrauterine application of uterine RNAe (as described for animal shown in (C). (F) Opposite uterine horn of animal shown in (E). This horn treated with liver RNA e on identical schedule and concentration used for contralateral horn. Magnification: x81O.

171 ESTROGENS, NUCLEIC ACIDS, AND PROTEIN SYNTHESIS

172

Table 4. Effect of Different RNAs on Alkaline Phosphatale Activity in Ovariectomized Mouse Uterus Homogenate a

CHAPTER 7

Treatment Control Liver RNA Uterine RNA (U-RNA) RNAse-treated U-RNA Boiled U-RNA a b

Number of Body Uterus animals weight (g) weight (g) Enzyme activity" 55 37 61 5 6

31.52 2S.70 29.01 29.S0 2S.40

241 267 454 269 ISO

23.15 21.67 22.46 25.30 IS.00

± ± ± ± ±

(T

units/O.l ml

15.7 15.5 15.5 39.1 33.1

3.5 4.0 6.7 3.7 3.2

After Mansour and Niu (1965). The enzyme activity is expressed by changes in optical density E (1 cm x 400 nm) x 10"' Numbers indicate the mean ± standard error of the mean.

Table 5. RNA-Induced Blastocyst Implantation in Postcoitally Ovariectomized Ratl,a Material (0.01 ml) Sham operated Saline Estradiol-17 f3 Estradiol-17 f3 U-RNAe c U-RNA' c a b C

Doseb (/Lg)

Uteri treated

Uteri with nidation 0

0.001 0.01 0.075 0.75

100 100 20 20 20 20

Table based on unpublished data of E. Schuchner. K. Wada. and S. Parametrially injected. U-RNA' extracted from uterus of estrogenized rats.

J.

15 1

16

Segal.

treated rats (Table 5). RNA extracts from other organs are not active (Table 6) and pretreatment of the extract with RNAse or phosphodiesterase eliminates the activity (Table 7). The studies of enzymatic degradation are particularly significant in the interpretation of these results. Although the active preparation is termed "RNA extract," the possibility that the active constituent may be a component or contaminant other than RNA cannot be rejected. Activity by a contaminating macromolecular fraction such as protein or DNA is not likely since neither DNAse nor proteolytic enzymes influence the activity. The enzymes that do inactivate the preparation share the property of degrading RNA. An explanation of activity based on slight contamination by estrogen does not take into account the results of enzymatic degradation. The observation that RNA extracted from the uteri of estrogen-treated rats when instilled into the uteri of immature mice induces changes characteristic of estradiol administration was confirmed by other investigators (Mansour and Niu, 1965; Mansour, 1967, 1968; Unhjem et aI., 1968; Fencl and Villee, 1971; Galand et al., 1971; Galand and Dupont-Mairesse, 1972, 1973; Hayashi et at., 1973). Estradiol contamination of the RNA preparations was considered in these studies and found not to account for the hormone-inductive properties of the RNA (Fencl and Villee, 1971; Dupont-Mairesse, 1972; Tuohimaa et aI., 1972). Moreover,

instillation of uterine RNA from estrogen-treated rats induces an increase in the activities of uterine glucose 6-phosphate dehydrogenase and ornithine decarboxylase, simulating estradiol effects in vivo (Villee and Loring, 1975; Villee, 1974). The estrogen-mimetic activity resides in the poly(A)-rich fraction, which corresponds to mRNA-rich fraction (Villee, 1974; Villee and Loring, 1975). It is noteworthy that the RNA fraction is active in vitro. The addition of poly(A)-rich RNA to the incubation medium containing explants of immature uteri maintained in organ culture (Villee and Loring, 1975) stimulates the incorporation of amino acids into protein. These results suggest that mRNA can induce the changes attributed to estrogen and is biologically active when instilled into the lumen of the untreated uterus. Similarly, RNA extracted from oviducts of estrogen-progesterone-treated chicks induces avidin synthesis when instilled into estrogen-treated chick oviduct and when incubated in vitro with oviduct explant culture (Tuohimaa et at., 1972). Intraoviductal instillation of poly(A)-rich RNA purified by nitrocellulose chromatography is capable of inducing avidin synthesis (Segal et al., 1973, 1974), suggesting that mRNA is probably the responsible agent. In addition, the chick RNA preparation is capable of inducing avidin synthesis when instilled into pigeon oviduct, suggesting that the uterine mRNA fraction may be active in inducing hormonal effects in the uteri of other closely related species. In addition to mRNA, tRNA may influence hormone action. When tRNA isolated from target organs of animals after hormone treated is added to target organ explants, the effect~ of the hormone are potentiated. For example, tRNA

Tahle 6. RNA-Induced Blastocyst Implantation in Postr:oitally Ovariectomized Ratl· a Material" (0.01 ml) Uterine RN Ae Vaginal RNA" Liver RNAe Kidney RNA" a b C

Dose c (JLg)

Uteri treated

Uteri with nidation

7.5 7.5 7.5 7.5

20 10 10 10

17 0 0

From unpublished data of E. Schuchner, K. Wada, and S. J SegaL RNAe extracted from organ of estrogenized rats in each case. Parametrially injected.

Table 7. RNA-Induced Bif/stocyst Irnplfmtation in Postcoitally Ovariectomized Rat,a Enzyme" DNAse RNAse DPEase RNAse a b

Substance" 7.5 JLg U-RNAe 7.5 JLg U-RNA" 7.5 JLg U-RNA" 0.0 I JLg Estradiol-I 7{3

From unpublished data of E. Schuchner. K. Wada. and S Substance injected parametrially after enzyme incubation.

J

SegaL

Uteri treated

Uteri with nidation

10 12 10 10

8 I 0 7

173 ESTROGENS, NUCLEIC ACIDS, AND PROTEIN SYNTHESIS

174 CHAPTER 7

extracted from the oviducts of laying hens potentiates ovalbumin synthesis when added to oviduct magnum explants of immature chicks treated with estrogen, whereas rooster liver tRN A or rat liver tRN A has no effect (Sharma et aI., 1973; Sharma and Borek, 1974). The results suggest that the hormone stimulates the formation of specific tRNA, which facilitates the translocation process of mRNA induced by the hormone.

2.5. 2.5.1.

Protein Biosynthesis Increase in Ti5sue MaliS

Many reactions are stimulated in the uterus by estrogens. These reactions have been demonstrated by measuring actual increases in size of uterine structures, increase in the weight of uterus or some of its components, increase in cellular or enzymatic activities, and an increase in the rate of turnover of a cellular component. Some of these reactions have already been discussed. A more complete list of these increases, with selected references, follows: (1) There are changes in uterine cellular structure: the cells become swollen, the mitochondria vesiculated, and the endoplasmic reticulum spread apart during the first 6 hr after estrogen administration. Up to 24 hr after these changes, the endoplasmic reticulum becomes more abundant and the nucleolar size increases (Mueller et aI., 1958); there are changes in the epithelial basal lamina (Nilsson and Wirsen, 1963) and changes in the cell membrane (Nilsson and Norberg, 1963). (2) Nuclear volume (Alfert and Bern, 1951). (3) Mitotic rate (Allen et aI., 1937). (4) Hyperemia (Szego and Roberts, 1953). (5) Water accumulation (Astwood, 1939). (6) Dry weight (Velardo, 1959). (7) Oxygen utilization (David, 1931). (8) Small-molecule accumulation in addition to water, see Section 2 of Fig. 1. (9) Ribosome formation (Moore and Hamilton, 1964). (10) Glycolysis (Kerly, 1937). (11) Lipid biosynthesis (Emmelot and Bosch, 1954). (12) Phospholipid biosynthesis (Borell, 1952). (13) Glycogen synthesis (Bitman et aI., 1965). (14) Polysaccharide synthesis (Zachariae, 1959). (15) Cholesterol synthesis (Aizawa and Mueller, 1961). (16) Phosphorus utilization and incorporation into macromolecules (Borell, 1951a,b). (17) DNA synthesis (not seen until at least 25 hr after estrogen administration, reaching a maximum after 40--72 hr) (leener, 1948). (18) RNA synthesis. (19) Protein synthesis, see following. (20) Increases in enzyme activities: (a) acid and alkaline phosphatases (Atkinson and Elftman, 1947; Leathem, 1959); (b) serine aldolase (Herranen and Mueller, 1956); (c) aldolase and proteolytic activity (Goodall, 1965); (d) glutamat,eoxalic acid transaminase and glutamate-pyruvate transaminase (Puchol and Carballide, 1959); (e) peptidase (Albers et aI., 1961); (f) ,B-glucuronidase (Fishman and Farmelant, 1953); (g) carbonic anhydrase (Lutwak-Mann, 1955); (h) phosphorylase (Bo, 1961; Leonard and Crandall, 1963; Rinard, 1972); (i) glycogen synthetase (UDPC-glucosyltransferase) (Bo and Smith, 1963; Rubulis et ai., 1965); G) peroxidase and reduced diphosphopyridine nucleotide oxidase (Hollander and Stephens, 1959; Rubulis et aI., 1965; Hilf et aI., 1965); (k) dehydrogenases and transhydrogenase (Scott and Lisi, 1960; Williams-Ashman and Liao, 1964); (1) fibrinolysin

(Page et at., 1951); (m) indoxyl esterase (Fuxe and Nilsson, 1963); (n) glycerylphosphorycholine diesterase (Wallace et at., 1964); (0) succinoxidase and cytochrome oxidase (Telfer and Hisaw, 1957); (p) eight amino acid-activating enzymes (see Section 2.2.2; McCorquodale and Mueller, 1958); (q) aspartate carbamoyl transferase (Trembley, 1965; Mayol and Thayer, 1966); (r) deoxyribonuclease (Brody and Wiqvist, 1961); (s) ribonuclease (Goodall, 1965); (t) RNA polymerase (Gorski, 1964); (u) histone acetyltransferase (Libby, 1968, 1972; Harvey and Libby, 1976); (v) glucose 6-phosphate oxidoreductase (Barker, 1967; Barker et at., 1966; Smith and Barker, 1974; Moulton and Barker, 1971, 1973); (w) tRNA-methyltransferase (Baliga and Borek, 1974; Sharma and Borek, 1974); (x) peroxidase (Churg and Anderson, 1974; Anderson et at., 1975; Kang et at., 1975). The data from the investigations just listed show that estrogen increases the tissue mass of the uterus, and delineate particular cellular components that contribute to the increment. Very little is known about tissue degradative reactions or tissue mass-reducing activities that occur rapidly in the uterus following estrogen administration (Goodall, 1965). Astwood (1938) observed a rapid increase in water accumulation following estradiol administration to an estrogen-deprived animal and noted a fairly linear increase in uterine dry weight beginning at approximately 4 hr and reaching its maximum 31 hr after a single estradiol injection. Approximately 80% of this increase is protein (see Table 1). Talwar (1964) demonstrated a maximal increase (70%) in protein nitrogen 48 hr after a single dose of 5 ILg estradiol injected subcutaneously into ovariectomized rats. 2.5.2.

Incorporation of Radioactive Precursors

With the advent of a more sensitive technique involving the use of radioactive isotopic protein precursor for measuring newly formed proteins, it was shown that an increase in protein synthesis did indeed occur in the rodent uterus shortly after administration of a single dose of estradiol (Frieden et at., 1952; Mueller and Yanagi, 1952; Mueller, 1953). An experimental design was needed to study synthesis of macromolecules in vitro under conditions in which the isotope pool would not be diluted throughout the organism. This method was attempted by Szego and Lawson (1964) and Mueller (1965). A single injection of estradiol was given to immature or ovariectomized rats. At specified times, the uterus was isolated and divided into segments. The segments were incubated in vitro in the presence of a radioactive protein precursor. A crude protein fraction was isolated from the incubated tissue and weighed, and the radioactivity of the fraction was ascertained. These techniques also permit quantitative assays of the effect of estrogens and of other compounds on biosynthesis of RNA, DNA, and phospholipid within uterine tissue. Wilson (1963) made use of rat uterine slices incubated in vitro to allow optimal perfusion of oxygen throughout the tissue preparation. In general, no increase in protein or in any macromolecular synthesis has been demonstrated in any of these in vitro systems when the estradiol is administered to the tissue in vitro. The only completely in vitro stimulations of protein synthesis mediated by steroids were demonstrated by Mueller (1955) using the 2-hydroxy and the 4-hydroxy derivatives of estradiol. These estradiol derivatives were shown to stimulate incorporation

175 ESTROGENS, NUCLEIC ACIDS, AND PROTEIN SYNTHESIS

176 CHAPTER 7

of [14C]formate into protein of uterine segments incubated in vitro. Estradiol stimulated incorporation of 32P04 into ethanolamine phospholipid in a similar system (Aizawa and Mueller, 1961). To the frustration of many investigators, however, no organelle-free, cell-free biological system is yet known in which increased synthesis of macromolecules had been demonstrated in response to a physiologically produced hormone added in vitro. The synthesis of a great variety of proteins is enhanced by estrogen stimulation, particularly after the hormone has gained access to the tissue for relatively long periods of time (see item 20 in the list at the beginning of Section 2.5.1). The increased reactions noted in the list are probably the result of increased de novo protein synthesis. Changes in conformation of proteins are not likely to account for the vast range of increased reactions. This contention is supported by studies using inhibitors of protein biosynthesis, puromycin (Mueller et al., 1961a) and cycloheximide (Gorski and Axman, 1964; Nicolette and Gorski, 1964a). The findings that emerge from studies with these substances are that (1) very little protein is produced in uterine preparations in the presence of appropriate concentrations of these inhibitors, (2) these inhibitors do not inhibit the baseline control values of RNA synthesis, phospholipid synthesis, or accumulation of water in the uterus, and (3) in the presence of both an inhibitor and estradiol either no stimulation or only very little (and possibly insignificant) stimulation of protein, RNA, and phospholipid synthesis or of water accumulation occurs. These results suggested the requirement of some protein production (and not of enzymatic activation) in order for the increase in the anabolic reactions to occur in the uterus after an increase in the supply of estrogen. Some reservation remains with regard to interpretation of experiments involving the in vivo administration of metabolic inhibitors (Lippe and Szego, 1965). In support of the significance of these studies is the observation that the local application of actinomycin D prevents the stimulatory effect of estrogen on one target organ (vaginal mucosa) whereas another target organ (uterus) in the same animal responds normally to the hormone (Talwar and Segal, 1963). Characterization of the specific proteins, the formation of which is enhanced in the uterus after estrogenic stimulation, has been attempted. Szego and Roberts (1953) utilized an electrophoretic technique to fractionate the soluble proteins from a single uterus obtained from a rat treated for 20 hr with estradiol. Most of the radioactivity incorporated into protein from [2- 14C]alanine was found in a single electrophoretically separated fraction obtained from estradiol-treated uteri, but not from the untreated controls. This fraction contained neither the most acidic nor the most basic of the soluble uterine proteins. The alanine used as tracer did not, of course, contribute to any deviation of pH from neutrality. Some specificity of protein synthesized in response to estrogen treatment was therefore indicated. Frieden (1956), amplifying earlier results of Telfer (1953) and using differential solubility for characterization of protein, found that the water-soluble guinea pig uterine fraction reaches a maximal incorporation of [l-14C]glycine into protein 12 hr after an injection of estradiol and then steadily declines. The alkalisoluble fraction maintains a constant specific activity from the eleventh to the thirty-sixth hour. The specific activity of the alkali-insoluble collagen fraction, however, reaches a maximum at 24 hr and then diminishes at 36 hr. Synthesis of

proteins in the soluble fractions is stimulated earlier than synthesis of protein in the collagen fraction. Noteboom and Gorski (1963a) separated uterine extracts, prepared from estradiol-treated and nontreated rats, into five fractions by differential centrifugation (see Section 2.4.2). No stimulation of protein biosynthesis occurred at the end of 2 hr of estrogen treatment. During the interval of 3-4 hr after estradiol injection, however, some increase in protein synthesis was measured in all of the subcellular fractions investigated. This method did not detect any increase in a specific protein or group of proteins consequent upon an increase in estrogen level. Anyone of several reasons might have accounted for this result: (1) the method was not sensitive enough to detect the subcellular localization of small amounts of newly synthesized specific proteins after estrogen stimulation; (2) there was no subcellular localization of these proteins; (3) the specific proteins synthesized in response to estrogen did localize in subcellular fractions, but there were roughly equivalent amounts of several different proteins in the various fractions; or (4) there was no preponderance of a specific group of proteins produced following estrogen stimulation, but rather a general increase in protein synthesis. 2.5.3.

A Specific Uterine Protein Induced by Estradiol

The administration of estrogen to ovariectomized rats results in the synthesis of a specific uterine protein designated as "specific induced protein." This protein is formed within 40 min following estradiol administration and after 1 hr of incubation of the uteri in vitro (Barnea and Gorski, 1970; Katzenellenbogen and Gorski, 1972, 1975; Somjen et al., 1973b,c; Katzenellenbogen and Williams, 1974; Katzenellenbogen and Greger, 1974; Katzenellenbogen, 1975). The induced protein was identified by measuring the incorporation of labeled amino acid into proteins and separating the protein by polyacrylamide gel electrophoresis. Estradiol-17{3, estrone, estriol, and diethylstilbestrol induce formation of the specific protein, while 17a-estradiol has a slight inductive ability and progesterone is ineffective (Ruh et al., 1973). The specific protein is found in the endometrial stroma and myometrium (Katzenellenbogen and Leake, 1974; Dupont-Mairesse and Galand, 1975a,b). The amount increases during the proestrus phase of the normal rat estrus cycle (Iacobelli, 1973; Dupont-Mairesse and Galand, 1975a,b; Katzenellenbogen, 1975; Katzenellenbogen and Leake, 1974). The supernatant and sediment fractions of uterine homogenate contain the induced protein, indicating that the localization of the specific protein is not restricted to a particular cellular compartment. This induced protein can be synthesized in isolated explants of uteri under in vitro conditions by incubating with physiological concentration of estrogens (Katzenellenbogen and Gorski, 1972; Wira and Baulieu, 1972; Mayol, 1975). A cell-free extract system obtained from estradiol-treated rat uterus has been also developed that is capable of producing the specific protein (Somjen et al., 1974a). The formation of the specific protein does not require the participation of cAMP, because cAMP at 10-3-10- 5 M in vitro is unable to induce the synthesis of the specific protein (Katzenellenbogen and Gorski, 1972). The specific protein is formed by de novo synthesis and not by activating or altering the character of a

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precursor protein. The decline in the concentration of induced protein in the uterus 3-4 hr after estrogen administration appears to be due to a block in the synthesis of the protein rather than to an increase in the rate of degradation (DeAngelo and Fujimoto, 1973). The protein is composed of a single subunit with a molecular weight of 39,000-45,000 daltons and an isoelectric point at 4.5-4.7 at 4°C as ascertained by isoelectric focusing in polyacrylamide gels (Iacobelli, 1973; Iacobelli et at., 1973; Somjen et aZ., 1973a; Katzenellenbogen and Williams, 1974; King et aZ., 1974). However, others reported that the specific induced protein is composed of multiple components with isoelectric points ranging from 3.5 to 4.0 (Mayol and Thayer, 1970; King et at., 1974). The uterine estrogen-induced protein was reported to possess protein phosphatase activity with phosphorylated protamine and his tones as substrates (Vokaer et at., 1974). The phosphatase activity is optimal at neutral pH, inhibited by Zn2+, and independent of cAMP or cGMP. However, this claim is negated by the finding that the phosphoprotein phosphatase activity can be separated completely from the induced protein by ammonium sulfate fractionation and by cellulose acetate electrophoresis (Kaye et at., 1975). 2.5.4.

Other Estrogen-Induced Proteins

A nonhistone protein is also induced in the uteri of ovariectomized rats within 15 min after estradiol administration. This induced protein is found to be associated with the arginine-rich F3 histone fraction (Barker, 1971; Kaye et aZ., 1974). The isoelectric point of this protein is in the range of 4-5. Intrauterine application of actinomycin D did not inhibit the induction of the protein, indicating that the synthesis of the induced protein was not dependent on prior RNA synthesis. A nuclear acidic protein is also induced on estradiol administration (Teng and Hamilton, 1970). This protein was detected by allowing [3H]tryptophan to be incorporated into the nuclear acidic protein fraction of the uterus upon estradiol administration. The induced radioactive protein was analyzed and identified on acrylamide gel electrophoresis. Estradiol administered to guinea pigs induces on electrophoresis an additional protein band that is associated with the 60 S subunits of the hormone-treated uterine ribosomes (Shapiro et aZ., 1975a). The properties, function, and relation of this protein to the other induced proteins need to be established. The molecular basis for the induction of the synthesis of specific induced proteins by estradiol is an interesting problem for study. The basis for the induction of specific proteins might be stimulation by the hormone of a common pathway of protein synthesis (Figs. 1 and 2) or acceleration of the synthesis of a common precursor that may be transformed into the various proteins. Whether each induced protein plays a role in mediating the action of estradiol is not yet known.

2.6.

Deoxyribonucleic Acid Biosynthesis

Estradiol and progesterone influence uterine DNA synthesis (Bronson and Hamilton, 1972; Somjen et aZ., 1973a,b; Lee, 1974). During diestrus, plasma

estrogen concentration rises in association with maximal cell renewal of uterine tissues (Epifanova, 1966; Finn and Martin, 1973). At late diestrus the duration of DNA synthesis (S phase) of the uterine epithelia is shortened markedly, and at proestrus cell proliferation is high. During late diestrus, cell division in the uterine epithelia declines to the lowest level, associated with a decrease in the secretion of sex hormones. On the other hand, the endometrial stroma becomes sensitive to decidual stimulation, and cell division is accelerated (Leroy and Bogaert, 1973). The inhibition of epithelial cell division and stimulation of stromal mitotic activity are related to a high progesterone and a low estrogen concentration. Estradiol and progesterone have divergent effects on DNA synthesis of the endometrium and myometrium. The administration of estradiol-17{3 to the ovariectomized mouse results in many mitoses in the luminal and glandular epithelia but not in the connective tissue stroma (Martin and Finn, 1968; Beato and Dienstbach, 1968; Beato et at., 1968; Smith et al., 1970; Lee, 1972; Kaye et at., 1972; Finn and Martin, 1973; Martin et at., 1973a,b,c; Clark, 1973; Lee et at., 1974). Progesterone reverses the pattern (so that many mitoses are produced in the stroma and few in the epithelia) and suppresses epithelial cell division whereas large doses of estradiol tend to overcome the inhibitory effects of progesterone and induce epithelial mitoses (Martin et at., 1970; Finn and Martin, 1973; Martin et at., 1973a; Das and Martin, 1973). These findings suggest that progesterone stimulates stromal cells in the resting phase to enter the cell cycle, and estrogen subsequently accelerates division by shortening the period between mitosis and DNA synthesis (Galand et at., 1971; Martin et at., 1973b). Estrogen administered to ovariectomized rats results in a marked hypertrophy of the myometrium and connective tissue stroma with minimal proliferation, whereas the luminal epithelium undergoes marked hyperplasia, which is rapid, highly synchronized, and brief (Lee, 1972, 1974; Martin et al., 1973b,c; Lee et at., 1974). The mean duration of the S phase is 10.5 hr in the uterine glandular and luminal epithelium of ovariectomized mice, shortening to 6 hr after estrogen treatment (Das, 1972), and is associated with a shortened G1 phase. Hence the increase in the rate of DNA synthesis is correlated with a shortening of the Sand G 1 phases. The stimulation of uterine DNA synthesis by estradiol might be related to the induction of enzymes associated with chromatin. The administration of estradiol17{3 to the immature mouse results in a rise in the uterine poly(adenosine diphosphoribose)synthetase activity, which has been implicated in DNA synthesis (Miura et at., 1972). This increase in the synthetase activity may be related to the elevation of uterine pyridine nucleotide levels that is observed after the administration of estradiol-17{3 to ovariectomized rats (Barker, 1967; O'Dorisio and Barker, 1970). The NAD+ level rises during the initial 2 hr, whereas NADP levels are constant for 2 hr and increase to 160% by the sixth hour. It was observed that the increase in [3H]thymidine incorporation into uterine DNA induced by estradiol is inhibited by nicotinamide (Miura et at., 1972). The basis for the block in DNA synthesis effected by nicotinamide is not known; however, it is conceivable that nicotinamide might act by increasing the levels of N AD+ or by inhibiting poly(adenosine diphosphoribose)synthetase activity. The incorporation of [3H]thymidine into uterine DNA of immature mouse is blocked during the early phase (30 min-5 hr) after estradiol-17{3 administration (Tsong and Koide, 1975), whereas a stimulation of the incorporation occurs 12 hr

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after the hormone. The effect of the hormone might be related to histone synthesis or its modification (Smith et al., 1970; Libby, 1972; Barker, 1971; Anderson and Gorski, 1971; Kaye et al., 1974), for the two processes are closely coupled. Within 4 hr after estradiol is administered to ovariectomized mouse, a marked stimulation in the incorporation of [14C]lysine into his tones and acidic nuclear proteins of the uterine epithelium takes place (Smith et al., 1970). The rate of incorporation of labeled leucine and arginine into various histone fractions is increased gradually within a few hours after estrogen treatment of immature rat (Anderson and Gorski, 1971). The stimulation of the rates of synthesis of various his tones is significantly elevated 24 hr after the hormone. Histones can be modified by acetylation, methylation, and phosphorylation (Koide, 1969). Acetylation of uterine his tones is stimulated within 5 min after estradiol treatment of immature rat (Libby, 1968, 1972). Moreover, the addition of estradiol to a cell-free uterine system causes a stimulation of acetylation of his tones, suggesting that the hormone is capable of activating a kinase or a histone acetyltransferase system. The acetylation process is somewhat selective in that the Fz and F3 histones are acetylated primarily under estradiol influence. The relationship of histone acetylation to RNA and DNA synthesis has not been clearly established.

2.7.

Adenosine J',5'-Cyclic Monophosphate

Recent reports indicate that estrogens administered to ovariectomized rats incite an increase in the level of adenosine 3' ,5'-monophosphate (cAMP) in uterine cells (Hechter et al., 1967; Szego and Davis, 1967, 1969) and stimulate uterine adenyl cyclase activity (Rosenfeld and O'Malley, 1970). The mechanism involved in cAMP formation induced by the hormone has not been established (Andress et al., 1974) (Fig. 2). Furthermore, no evidence has been presented to show that the estradiol-17f3 interacts directly with the cell membrane or with a membrane receptor. In addition, the role of cAMP as a second messenger in mediating estrogen action has not been clearly defined. The notion that cAMP may be the mediator of estrogen action is supported by the findings that cAMP is capable of stimulating the rat uterus (Szego, 1965; Hechter et al., 1967; Griffin and Szego, 1968; Mohla and Prasad, 1970) and that theophylline, an inhibitor of cAMP phosphodiesterase, potentiates the effect of estrogen (Lafreniere and Singhal, 1971). Although evidence has been reported to show that estrogen induces an early increase in cAMP concentration in rat uterus (Szego and Davis, 1967, 1969; Szego, 1971), later studies indicate that estrogen did not elevate cAMP levels in castrated rat uteri as an early event (Sanborn et al., 1973; Zor et al., 1973; Andress et al., 1974). In addition, propranolol, a f3-adrenergic blocking agent, suppresses the early increase in cAMP concentration and inhibits adenyl cyclase activity (Szego and Davis, 1969; Szego, 1971; Dupont-Mairesse et al., 1974; Dupont-Mairesse and Galand, 197 5a), suggesting that catecholamines might participate in the induction of cAMP formation (Korenman et al., 1974a). Moreover, several known responses of the rat uterus to estradiol administration remain unaffected by propranolol treatment: (1) hypertrophy of luminal epithelium, (2) stimulation of RNA, protein, and glycogen synthesis, (3) increase in the activities of a certain glycolytic enzyme, and (4) augmentation of the in vitro

incorporation of [14C]phenylalanine into proteins are not affected by propranolol administration to estrogen-treated immature or spayed adult rats (Singhal et at., 1972; Dupont-Mairesse et al., 1974; Dupont-Mairesse and Galand, 1975). These findings suggest that many estrogen-induced events are independent of cAMP participation. Ascertaining the role of cAMP is further complicated by the finding that the uterine content and metabolism of substances that affect cAMP formation, such as epinephrine, histamine, and serotonin, are influenced by estrogen administration. Furthermore, epinephrine and the {3-adrenergic agonist isoproterenol cause uterine cAMP accumulation (Triner et al., 1970; Korenman et a!., 1974b) and increase cAMP-independent protein kinase activity in the uterus of ovariectomized rats (Korenman et at., 1974a). Hence one should be cautious in concluding that a cause-and-effect relation exists between estrogen action and uterine cAMP levels. It is reasonable to conclude that estrogen may provoke an elevation of uterine cAMP levels, probably by an indirect or secondary mechanism, and that cAMP mediates only one facet, if any, of the total metabolic effects triggered by estradiol.

2.8.

Estrogen and Lysosomes

It has been proposed that steroid hormones may act by influencing the metabolism or the release rate of hydrolases from lysosomes. Hormonally active steroids, namely testosterone, estradiol-17{3, and progesterone, accelerate markedly the release rate of hydrolases from lysosomes of rat liver in vitro, whereas hydrocortisone has a retarding effect (deDuve et at., 1963). The in vivo influence of estradiol on lysosomes was studied by observing the changes in the lysosomal structure of uterine and preputial gland cells of ovariectomized rats induced by estradiol administration. Estrogen initiates a rapid migration of lysosomes to a perinuclear position (Szego, 1974). When nuclei are isolated from these hormonetreated rats, the preparations contain a high concentration of lysosomes, suggesting translocation of lysosomes in proximity to the nucleus or adhering to the nucleus (Szego and Seeler, 1973). In addition, the lysosomes of hormone-treated rats are more fragile to chemical agents and are more susceptible to fragmentation and dissolution on incubation. Furthermore, nuclei isolated from preputial gland 2-15 min after hormone treatment possess substantial lysosomal hydrolase activity. (Szego et at., 1974). Moreover, the lysosome-enriched fraction of preputial gland contains macromolecules with estradiol-binding property of low capacity, which is characteristic of the uterine cytosol estrogen receptor protein. The results of these studies suggest that lysosomes may participate in the translocation of hormone from the cell membrane to the nucleus and that lysosomal enzymes traverse the nuclear membrane and may serve to initiate gene expression.

2.9.

Estradiol-Sensitive Uterine Cell Cultures

Studies on the in vitro response of various cell cultures to estrogen have become increasingly numerous with the advent of improved culture techniques

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and media. The addition of estradiol at 10-9 M to cultures of isolated rat endometrial cells provokes a significant rise in sodium and water contents and increases the yield of 14C02 production from [U- 14C]glucose within 2 hr (Pietras and Szego, 1975a). A permanent line of cells derived from a human endometrial carcinoma in which estrogen does not influence the rate of protein synthesis by these cells has been cultured. These cells contain a protein with a low-affinity, nonspecific capacity to bind estradiol and lack a specific estrogen receptor (Shapiro et al., 1975b), indicating that the ability of cells to respond to a hormone is dependent on the content of specific hormone receptors. Cultured human endometrium treated with estrogen and progesterone shows higher concentrations of glycogen and an increased rate of leucine incorporation, as compared with treatment with estrogen alone (Shapiro and Hagerman, 1974). Uridine incorporation remains the same on treatment with a single hormone or with both hormones. In contrast, it was reported that marked variation in RNA and DNA synthesis is observed in endometrium obtained at different phases of the menstrual cycle (Nordquist, 1970). The addition of progesterone to the culture medium depresses synthesis of both RNA and DNA. These results support the finding that progesterone suppresses endometrial cell division (Martin et al., 1970, 1973a; Finn and Martin, 1973).

3.

Conclusion

Intensive investigations have been undertaken during the past decade to unravel the molecular basis for the effects of estrogens on uterine metabolism. To this end, investigators in the field of endocrinology, biochemistry, and molecular biology have contributed extensively to our understanding of hormone receptors and their possible modes of action. The action of estrogen on uterine cell metabolism is diverse and complex. A current hypothesis is that the hormone interacts with cytosol receptor (8 S or 4 S), which is transformed and translocated into a 5 S form in the nucleus. The receptor interacts with chromatin, resulting in a stimulation of transcription by activating clusters of genes. Other early events associated with estrogen action are the formation of cyclic AMP, Ca2+ influx, alteration of lysosomal function, eosinophil infiltration, inhibition of ribonuclease, and induction of specific proteins. The sequence of events of RNA vs. protein synthesis has not been established definitively. The information derived from the studies on hormone receptors and on the molecular mechanisms of induction of protein and nucleic acid biosynthesis aids us in our understanding of the complex internal structure, organization, transport, and communication within eukaryotic cells. In addition, intercellular communication and interaction are being more intensively studied. From these studies, the cause and mechanisms leading to teratological changes, endocrine dysfunction, genetic aberrations, and carcinogenesis will be elucidated. One of the challenges to investigators conducting research in reproductive physiology is to modulate reproductive processes with the aim of regulating

fertility. For this purpose, an important area for future research is the elucidation of the action of hormones on biomembranes and on microcirculation. Information from these studies may uncover greatly needed facts to give proper direction and impetus for the development of more effective antifertility agents and methods.

ACKNOWLEDGMENTS

The authors are grateful to Dr. Martin Sonenberg, Memorial Sloan-Kettering Cancer Center, New York, for helpful suggestions, discussion, and editorial comments, and to Dr. Luis Burzio, Population Council, New York, for Figs. 2-4.

4.

Rrferences

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201 ESTROGENS, NUCLEIC ACIDS, AND PROTEIN SYNTHESIS

The Endometrium of Delayed and Early Implantation ALLEN C. ENDERS and RANDALL L. GIVEN

The length of gestation in a given species is generally proportional to the size of the young at birth and the relative maturity of the neonates. However, there are several variations in reproductive patterns that can extend the length of time from copulation to parturition. Delayed fertilization is a rare phenomenon found in a few species of bats (Wimsatt et at., 1966). Characteristically, sperm from a fall mating remain viable at the tubouterine junction until ovulation in the spring (Racey and Potts, 1970), and there is no retardation in the development of the zygote once fertilization has occurred. Conversely, in retarded development, now reported in at least two genera of bats, implantation occurs shortly after the blastocyst appears in the uterus, but formation of the placenta is slow, expecially the establishment of appropriate vascularization, and the early developmental stages of the embryo are retarded (Fleming, 1971; Bleier, 1975; Burns and Wallace, 1975; Wimsatt, 1975). Delayed implantation is a more common phenomenon. It is particularly interesting because it appears to be a condition under which the development of the blastocyst is controlled by the uterus. Although the term "delayed implantation" suggests that only the attachment of the blastocyst to the uterus per se is prevented, the prolongation can occur at any time during the preimplantation ALLEN C. ENDERS and RANDALL L. GIVEN . Department of Anatomy and Neurobiology, Washington University School of Medicine, St. Louis, Missouri. Present address for both authors: Department of Human Anatomy, University of California, Davis, School of Medicine, Davis, California.

203

8

204 CHAPTER 8

period from entry into the uterus to adhesion of blastocyst and uterine luminal epithelium. In carnivores, marsupials, and the roe deer, the delay occurs early in the preimplantation period, in that there is a period of quiescence in growth of the blastocyst followed by an appreciable increase in size (activation) before implantation is initiated. In the mink, repeated matings can occur during the delay period, resulting in blastocysts of different ages but in a similar developmental stage (Enders, 1952). In the rat and mouse, lactational delay is relatively short, the shedding of the zona pellucida is retarded, and after an initial increase in size of the blastocyst there is no further growth until after implantation (Surani, 1975b). In the roe deer, there may be slow growth of the blastocyst during the delay period (Short and Hay, 1966). The zona pellucida may be present during the entire delay period (carnivores, Wright, 1963), or may be absent for all of delay (armadillo, Enders, 1966). In the rat and mouse, delayed implantation can be readily induced by a variety of experimental procedures. Ordinarily, delayed implantation cannot be induced experimentally in animals lacking a normal delay (e.g., hamster, Orsini and Meyer, 1962). Delayed implantation has proved to be a useful phenomenon in studies of reproduction because it permits separation of a variety of events preceding implantation from those of implantation per se. In particular, it is a period in which the blastocyst is dependent on the intrauterine environment, and consequently on the condition of the endometrium.

1.

Marsupials

In the two decades since Sharman (l955a,b) documented the presence of delayed implantation in marsupials, a great deal has been learned concerning reproduction in this intriguing group. To date, delay of implantation is confined to the macropod marsupials, but the patterns of reproduction within this group are extremely diverse, not only because of species variations but also because of the responsiveness of the animals to local seasonal conditions (Tyndale-Biscoe et at., 1974). When a delay of implantation is interposed in the reproductive cycle, it is lactation-induced, and is apparently produced by an interference with full luteal development. The basic pattern of delay of implantation in macropod marsupials is as follows. An ovulation occurs around the time of parturition. Following fertilization of this ovum, the cleaving zygote receives an albumen coat and egg shell membrane in the oviduct as it moves down to enter the ipsilateral uterus. It reaches the stage of a small unilaminar blastocyst and subsequently undergoes a period of quiescence. At the termination of the period of quiescence, there is a period of several days during which the activated blastocyst increases in size and number of cells prior to loss of the shell membrane and attachment to the endometrium. Because of the period of blastocystic enlargement prior to implantation, the Australian workers frequently refer to the interposed period as "embryonic diapause" or blastocyst "quiescence." It should be noted, however, that a similar period of blastocystic enlargement at the end of a period of delay of

implantation is found in many carnivores and in the roe deer, and is missing only in those species that do not show a marked increase in blastocyst size prior to "normal" implantation. However, in macropods the conditions for activation of the blastocyst are not the same as those for subsequent maintenance of the embryo (the latter requiring a fully luteal endometrium, Tyndale-Biscoe, 1970), and the postimplantation period is shorter than the "activation" period. The two uteri of the marsupial reproductive tract are short, saccular, and fusiform, in both monotocous and in polytocous species. They are surprisingly similar in widely diverse marsupials, including the New World forms such as the philander opossum and the Virginia opossum as well as in macro pod and nonmacropod Australian species, and do not show the diversity of gross or histological structure found in Eutheria. In non delaying species such as the ringtail opossum (Hughes et al., 1965) and the philander opossum (personal observation), the endometrium is richly glandular, composed in early pregnancy of a luminal epithelium of columnar epithelial cells covering a series of folds into which the simple tubular and branched tubular glands enter (Fig. 1). The glands have tall columnar epithelial cells with nuclei in a distinctly basal position. Both glandular and luminal epithelia have some ciliated cells, especially in the neck region of the glands. A particularly prominent feature of pregnancy is the pronounced edema of the lamina propria of the endometrium (Padykula and Taylor, 1971). In most species, the tendency toward pseudostratification of the luminal epithelium decreases as pregnancy progresses, whereas stromal edema increases. During delayed implantation in marsupials, the endometrium is more highly developed than during anestrus, but less highly developed than after the initiation of blastocystic activation and subsequent gestation. Tyndale-Biscoe (1963) listed the range of diameters of the anestrous uterus of the Rottnest Island wallaby, Setonix, as 3-5 mm, and that during delay as 5-7 mm. He also reported that the endometrial glands were longer and more coiled in delay than in anestrus, and that, as well as having the typical columnar cells with basally situated nuclei, the lumina of the glands were large and patent. There was apparently no difference in structure of the uterus between lactating animals in delay and nonpregnant lactating animals. In early pregnancy in species both with and without a quiescence, the glandular epithelium is composed of slender elongated epithelial cells, while the luminal epithelial cells are broader with a more centrally placed nucleus and less extensive apical cytoplasm (Figs. 1 and 2). When intrauterine fluids were collected from the tammar wallaby, Macropus eugenii, and the proteins analyzed by electrophoretic mobility, it was found not only that the quantities of these proteins differed from other body fluids but also that there were uterine-specific prealbumins present (Renfree, 1973). Consequently, the uterus is seen to have a role in controlling its luminal contents. During delayed implantation in this species, the uterus is smaller than after the initiation of activation of the blastocyst, and both the amount of uterine secretion and the protein concentration of this secretion are less in the quiescent period (TyndaleBiscoeetal., 1974). In non delaying marsupials such as the Virginia opossum and the phalangerid Trichosurus, the increase in uterine weight and secretions following ovulation is similar in pregnant and nonpregnant females (Tyndale-Biscoe et at., 1974; Renfree, 1975). Although this relationship apparently holds true in the tam mar

205 ENDOMETRIUM OF DELAYED AND EARLY IMPLANTATION

206 CHAPTER 8

';,

'.. .,

..:

Figure 1. Micrograph of a section through a uterus from a philander opossum in early pregnancy. The blastocyst is separated from the uterine luminal epithelium by the shell membrane (SM). The zona pellucida is the thin light band between the protoderm and the albuminous coat (AC). Note the well-developed glands surrounded by edematous stroma. X 150. Figure 2. Micrograph of a uterine gland from the red kangaroo during delayed implantation.

wallaby during the activation phase, the further increase in uterine weight and secretions seen after attachment of the blastocyst does not occur in the contralateral uterus or in nonpregnant animals under similar conditions, and is consequently considered to be a response to the conceptus. An additional difference between the delaying and nondelaying species of marsupials is the greater similarity of the uterine fluids in the latter to the composition of maternal blood serum. Glutaraldehyde-fixed portions of uteri from six red kangaroos killed during the delayed implantation period were available for this study.* One of the kangaroos was in the "activation" stage of blastocyst enlargement. In general, the development of the uterine glands of the red kangaroo during the delay period is greater than that of rodents during the delay period. The endometrium is moderately vascular and mildly edematous, and macrophages are common in the connective tissue (Fig. 2). A comparison of the glandular epithelium of four animals containing quiescent blastocyst with that of one containing an activated blastocyst showed that although both were composed of tall columnar cells, the gland cells in the latter were approximately twice as tall as in the quiescent stage (Fig. 3), and were considerably larger than the gland cells of late pregnancy in a nondelaying species (Fig. 5). Lipid droplets are more abundant in gland cells during the quiescent period, and vacuoles are present in these cells in both stages. However, large granules are prominent in the gland cells from the activation period. By electron microscopy, both types of gland cells have appreciable granular endoplasmic reticulum, but extensive dilation of the cisternae is seen during the activation period (Fig. 4). Gland cells at this stage also have well-developed Golgi complexes and numerous large granules containing flocculent material of varying density. From the evidence concerning both uterine fluid and uterine histology, it appears that the kangaroo uterus is neither atrophied nor devoid of secretory activity during the quiescent period, but that the gland cells are appreciably more active in their secretory capacity during the activation stage. It is interesting that a dilated granular endoplasmic reticulum has been reported during the blastocystswelling stages not only of the red kangaroo but also of the roe deer and the mink.

* This

material was kindly supplied by Dr. C. H. Tyndale-Biscoe.

There is a mixed population of tall columnar cells surrounding a lumen containing dense-staining extracellular material. X 630. Figure 3. Micrograph of a uterine gland from the red kangaroo during the stage in which the blastocyst is enlarged and activated. Note the extreme hypertrophy of the gland cells and the numerous small granules and vacuoles situated between the nuclei and the lumen. x630. Figure 4. Electron micrograph of a gland cell from the red kangaroo during the activation stage of delayed implantation. The cisternae of endoplasmic reticulum (ER) are highly dilated and contain flocculent material. x 16,800. Figure 5. Uterine glands from a philander opossum in late pregnancy. The stroma is highly edematous and the apical ends of the gland cells are filled with small vacuoles. x48S.

207 ENDOMETRIUM OF DELAYED AND EARLY IMPLANTATION

208

2.

Roe Deer

CHAPTER 8

The roe deer (Capreolus capreolus) is the only artiodactyl in which the existence of a delayed implantation period has been established. Short and Hay (1965, 1966) reviewed the early literature on delayed implantation in the European roe deer (Ziegler, 1843; Bischoff, 1854), and have reported their own observations on this species. Aitken et al. (1973) and Aitken (1974a,b, 1975) confirmed the earlier findings that fertilization occurs in late July or early August, and that soon afterward the zona pellucida is lost and the blastocyst begins a period of delayed implantation lasting until late December or early January. During the delay period, the blastocyst grows slowly and shows evidence of change in the trophoblast and differentiation of the endoderm. After the period of relative quiescence, but prior to implantation, rapid elongation of the blastocyst takes place. Actual attachment of the trophoblast to the caruncles does not occur until after this increase in length. Contrary to the observations of Prell (1938) and of Stieve (1950), Short and Hay (1966) did not find any evidence of ovulation during second rut in the fall. Some preliminary evidence reported by Lincoln and Guinness (1972) indicates that the time of implantation is not under photoperiodic control, although the annual molt was advanced with altered photoperiod in the two animals observed. The uterus of the roe deer, like that of other artiodactyls, is bicornuate. There is a relatively large region of communication between the two horns, from which the cornua pass cranially and then laterally in two broadly curved arcs. The endometrium surrounding the large lumina of the cornua is composed of irregular folds that are interrupted mesometrially by a variable number of aglandular caruncles (Fig. 6). (See Harrison and Hyett, 1954, and Harrison and Hamilton, 1952, for a description of the structure of the uterus of several deer species and the distribution of caruncles.) The histological description of the roe deer uterus that follows is based on three blocks of endometrium, two from roe deer in the delay period and one from a roe deer with a 22-mm (early postimplantation) embryo, kindly sent to us by Roger Short, and on the descriptions of Aitken and Short. In delay uteri, the surface epithelium covering the protruding caruncles is tall columnar, the basal regions of the cells are vacuolated, and the nuclei are occasionally flattened on their basal end. The cells are not ciliated. A central vascular stalk is present in the pedicel forming the base of the caruncle. The vessels spread fanlike into the richly cellular connective tissue that underlies the surface epithelium. Neither glands nor crypts were present in caruncles of the two delay specimens, although the latter are well developed in the endometrium of the postimplantation animal (Figs. 6 and 8). The surface epithelium of the intercaruncular endometrium is similar to that covering the caruncles, except that the columnar cells are a little shorter and are interrupted periodically by the openings of the numerous glands. Aitken (1974a) reports that at the onset of embryonic elongation the number and diameter of the gland openings increase. The glands are simple branched tubular structures. The necks of the individual glands are broadly dilated and contain scattered ciliated cells. The neck region is slightly coiled. It branches shortly to give rise to the

209 ENDOMETRIUM OF DELAYED AND EARLY IMPLANTATION

Figure 6. Section through the uterus of a roe deer during delayed implantation. The caruncle is aglandular and has relatively dense stroma. The rest of the endometrium contains numerous branched tubular glands. x60. Figure 7. Roe deer uterus during delayed implantation. At this magnification the differential dilation of some of the basal endometrial glands is seen. Note the arteriole in the center of the micrograph and the edematous connective tissue. x 540. Figure 8. Crypt formation on the caruncular surface is seen in this section of a roe deer uterus at the stage of embryonic elongation. x 160.

210 CHAPTER 8

fundic portions of the glands, which in turn rebranch before extending down to the myometrial surface. The fundic portions of the glands are slightly coiled and are smaller in diameter than the neck segments. The cells are low columnar, with basally situated nuclei, and few cilia are present (Fig. 7). Aitken et al. (1973) noted a sharp decrease in cell height in glandular fundi at the time of embryonic elongation. Although most of the fundic portions are only slightly dilated, some individual glands are more broadly dilated. Small amounts of periodic acid-Schiff positive material can be found throughout the glands and overlying the surface epithelium. Apical granules are also seen in many of the cells of the neck segment. The endometrial stroma in the intercaruncular area is more edematous and less cellular than in the caruncular pedicel (Fig. 6). Aitken et al. (1973) further noted that the endometrium is most edematous in early August, subsides gradually, and then increases again at the time of placental attachment. The endometrium is quite vascular, and the vessels in the subsurface region are moderately dilated. The connective tissue in this region is slightly more cellular. Not only is the postimplantation uterus hypervascular and edematous, but also the glands, although exhibiting the same regions, are highly dilated and have areas in which colloidal contents distend the lumen. The caruncular region, as well as showing well-formed caruncular crypts, is more vascular. Aitken et al. (1973) and Aitken (1974a, 1975) have described the endometrium of the row deer at the ultrastructural level from the time of fertilization until after implantation. They have found that the most striking changes in the endometrium occur at the onset of embryonic elongation when the fundic cells suddenly become divested of the apical vesicles that had accumulated during the delay period. These clear vesicles are apparently derived from the Golgi apparatus and their release results in a marked decline in the height of these cells. The Golgi apparatus, which proliferates during the delay period, shows a decrease by the end of the period of embryonic elongation. The endoplasmic reticulum is never well developed in this cell type and remains poorly developed through the implantation period. The fundic cells remain free of apical vesicles throughout the implantation period. Aitken (1975) has also reported that the nonciliated cells of the necks of the endometrial glands contain clear supranuclear vesicles and more basal lipid than is seen in the cells of the gland fundi. The apical vesicles are lost in these cells also at the beginning of embryonic elongation. However, these cells do not appear as inactive as the glandular cells. The lipid material is retained and electron-dense lysosome-like granules appear in the cells. During the early stages of implantation, the granular endoplasmic reticulum and the continuous nuclear membrane contain moderately electron-dense material in their cisternae that could possibly be secreted. At this time granular inclusions near the apical cell membrane also seem to be releasing material into the lumen. Lipid deposits disappear from the cells but the lysosome-like granules are still seen. Aitken (1975) reported that at the beginning of embryonic elongation and at the time of release of the clear vesicles from the glandular epithelium the lumina of the necks and fundic portions of the glands are filled with clear vesicles, much cellular debris, and electron-dense cellular material. At implantation, large amounts of the material are still present. Another phenomenon that was noted by

Aitken (1974a, 1975) was the presence of large apical protrusions from the surface of the luminal and neck epithelium at the time of embryonic elongation and implantation. These protrusions are seen in the caruncular and intercaruncular luminal epithelium and were suspected of being a type of apocrine secretion. The number of these protrusions seemed to decrease later in the implantation period. The sudden decrease in clear apical vesicles in the glandular fundi and neck epithelia at about the time of rapid embryonic elongation led Aitken et al. (1973) and Aitken (1974a,b, 1975) to suggest that the released material may stimulate embryonic elongation and subsequent development. Aitken (1975) also suggested that the secretion of material from the granular endoplasmic reticulum in the neck region of the glands may not effect embryonic elongation, but could possibly be a result of embryonic growth. Analysis of uterine flushings revealed that there was a significant rise in calcium, protein, carbohydrate, and a-amino nitrogen content during embryonic elongation (Aitken, 1974a,b). Studies also indicated that there was an elevated estrogen level at this time that may act to stimulate secretory activity in the endometrium (Aitken, 1974a). However, since no change was noted in the ovaries of the roe deer during the delay period (they remain activeappearing), this estrogen rise may be a result rather than a cause of increased embryonic growth (Aitken, 1974a).

3.

A rrtUldillos

Delayed implantation was first reported in the nine-banded armadillo (Dasypus novemcinctus) by Patterson (1913). It was later studied by Hamlett (1932a), who also pointed out that Dasypus hybridis, too, probably has a delay of implantation (Hamlett, 1935). A series of studies established that in Texas the delay of implantation in the nine-banded armadillo lasts from the time of breeding in midsummer until implantation in November and December (Enders, 1966). During the delay period, the corpus luteum is apparently active (Talmage et ai., 1954; Labhsetwar and Enders, 1968) and is well developed (Enders, 1966). The blastocyst remains in the fundic portion of the uterus simplex (Patterson, 1913) in a chamber bordered laterally by the openings of the oviducts, and ventrally and dorsally by endometrial folds formed where the thicker endometrium of the body of the uterus overlaps the thinner endometrium of the fundus (Enders and Buchanan, 1959a). The length of the delay period can be experimentally shortened by bilateral ovariectomy (Buchanan et al., 1956; Enders and Buchanan, 1959b). Implantation occurs approximately 18 days following bilateral ovariectomy in the absence of exogenous hormones (Enders, 1966). The endometrium of the armadillo during the delay period has been described by Enders et al. (1958) and by Enders (1961). The endometrium is richly glandular. The luminal epithelium is pseudostratified columnar and is interrupted periodically by straight, simple tubular glands, which pass with little branching directly toward the myometrium (Fig. 9). In the body of the uterus, the glands are slightly dilated and consist of a straight neck region of columnar epithelial cells

211 ENDOMETRIUM OF DELAYED AND EARLY IMPLANTATION

212 CHAPTER 8

with basally situated nuclei, a more sinuous portion, and a short coiled basal portion lying beneath the venous sinusoids. During delay, lipid droplets are numerous in the glandular and in the luminal epithelial cells but are most conspicuous in the deeper portions of the glands. Glycogen can be seen in only occasional scattered cells. Alkaline phosphatase activity is limited largely to the stroma, but acid phosphatase and esterase activity can be detected in the epithelial cells. Succinic dehydrogenase activity is readily demonstrable throughout the epithelium. The stroma consists of loose fibroelastic connective tissue, containing blood vessels with an unusual pattern. C~iled arterioles penetrate the basal stroma from the myometrium, with relatively straight capillaries extending toward the luminal epithelium. These capillaries ramify throughout the endometrium and pass back toward the myometrial surface of the endometrium. The capillaries enter large venous sinuses that form an anastomotic network over the body and fundus of the uterus. The sinuses in turn are drained by numerous channels passing through the basal endometrium into the myometrium. The stroma is richly cellular rather than edematous. In the fundic portion of the uterus, the sinuses are well developed, but the endometrium is thinner. The glands are more highly coiled and lack a distinctive neck portion. Ciliated cells are found scattered in the epithelium throughout the uterus, and are common in both the glands and luminal epithelium of the fundic region (Fig. 9). The ciliated cells are large and pale, and have numerous rod-shaped mitochondria and abundant uniform long microvilli in additon to cilia (Fig. 11). Although they contain some lysosomes and a well-developed Golgi complex, there are relatively few granules in this cell type. The nonciliated "secretory" cells have numerous apical granules, and larger mitochondria than those in the ciliated cells (Figs. 11 and 12). Some of the mitochondria are unusually large, being several times the diameter of those of the ciliated cells. More polyribosomes are found in the secretory than ciliated cells, and granular endoplasmic reticulum is sparse. Moreover, the endoplasmic reticulum is neither dilated nor abundant. Both ciliated and secretory cells have some lipid droplets, especially basally. The surface of the secretory cells is irregular, with caveolae between microvilli and occasional larger cavities with flocculent material suggesting release of granules. The impression given by the cytological appearance of these cells is that they are secretory cells producing a granular content at a relatively low rate. They do not appear inactive. Appreciable change in the endometrium occurs at implantation. The epithelial cells store glycogen and lipid, they become broader, and the sinuses enlarge (Fig. 10). Ciliated cells diminish in number. Microvilli on the gland cells are longer, and although there are appreciable agranular endoplasmic reticulum and some strands of granular endoplasmic reticulum there is no dilation of the latter and the apical secretion granules disappear. It should be noted that the armadillo blastocyst loses its abembryonic trophoblast shortly after attaching to the endometrium, d~.us inverting the yolk sac at a stage when the endoderm extends only slightly beyond the embryonic cell mass. The subsequent growth of the conceptus pushes the endoderm out of the fundic recess into the body of the uterus, where it is directly exposed to the patent uterine glands. There is some evidence that the

213 ENDOMETRIUM OF DELAYED AND EARLY IMPLANTATION

Figure 9. Glandular epithelium of an armadillo during delayed implantation. The epithelium is composed of light ciliated cells and interposed darker secretory cells. Note the dense stroma. X4S5. Figure 10. Glandular epithelium of an armadillo just after implantation. The light areas in the cells are lipid droplets and the darker patches are glycogen. x4S5. Figure 11. Electron micrograph of two gland cells from an armadillo uterus during delayed implantation. The ciliated cell (right) has significantly smaller mitochondria than the secretory cell (left). The secretory cell contains numerous dense apical granules. X40,500.

214 CHAPTER 8

Figure 12. Electron micrograph of the apical region of gland cells from an armadillo in delayed implantation. Note irregular microvilli and numerous large secretory granules. X24,OOO.

Figure 13. Electron micrograph of the apical region of a gland cell from an armadillo just after implantation . The extensive areas of glycogen granules (Gly) are partially extracted and the microvilli are longer and more regular than before implantation. x23,OOO.

fundic area and the body of the armadillo uterus are physiologically different (Buchanan, 1967). The accumulation of glycogen and lipid in gland cells after implantation, coupled with their short stature and lack of granular endoplasmic reticulum, indicates a reduced secretory activity, although it is difficult to correlate this with the concurrent events of implantation. In the absence of data on uterine secretions or autoradiography, it is difficult to ascertain from cytological evidence alone the relative activity of the cells. In comparison with other delaying species, it can be seen that the armadillo also appears to release granules that were present during delay, but in contrast to the case of the mink, roe deer, or kangaroo there is no extensive dilated endoplasmic reticulum after implantation, and the gland cells are not hypertrophied. Consequently, the ways in which the endometrium nurtures the postimplantation conceptus in the armadillo remain enigmatic.

4.

Imectivores and Chiroptera

Brambell (1935) described the presence of a delay period in the European common shrew, Sorex araneus, and Tarkowski (1957) obtained similar data. In this species, a postpartum mating occurs and the resulting pregnancy is thought to be prolonged by a delay of implantation. Although Brambell does not describe the histology of the endometrium of delay of implantation per se, he does describe the endometrium of the second pregnancy. As in the initial pregnancy, the endometrium undergoes a number of changes prior to the swelling of the blastocyst that accompanies implantation. The changes occurring before implantation include displacement of the nucleus of the luminal epithelial cells from a single row to multiple levels and an enhanced development of the uterine glands, which are confined to the antimesometrial portion of the endometrium. From this evidence it seems that the delay in the common shrew occurs while the endometrium is in a preimplantation condition. Brambell has also suggested that a delay may exist in the lesser shrew (Brambell, 1937; Brambell and Hall, 1936). A delay of implantation has not been reported in any of the other insectivores. Delayed implantation has not been extensively studied in the Chiroptera. It has been reported in the fruit bat, Eidolon (Mutere, 1967; Fayenuwo and Halstead, 1974), and in Miniopterus (Peyre and Herlant, 1967). The interesting climatic factors associated with the delay in these species have been considered (Wimsatt, 1975), but the condition of the endometrium in relation to delay and implantation has yet to be studied.

5.

Carnivores

Delayed implantation occurs in both the pinniped and fissiped carnivores. Among the pinniped carnivores, it is a common feature in seals, having been

215 ENDOMETRIUM OF DELAYED AND EARLY IMPLANTATION

216 CHAPTER 8

reported in the common seal (?hoca vitulina) (Fisher, 1954; Harrison, 1960), grey seal (Halichoerus grypus) (Backhouse and Hewer, 1964; Hewer and Backhouse, 1968), cape fur seal (Arctocephalus pusillus) (Rand, 1954), northern fur seal (Callorhinus ursinus) (Pearson and Enders, 1951; Craig, 1964), and elephant seal (Mirounga leonina) (Laws, 1956). Among the fissiped carnivores, a period of delay of implantation is probably the general rule in the bears (Hamlett, 1935), and has been confirmed in the black bear (Wimsatt, 1963) and grizzly bear (Craighead et at., 1969). It is also extremely common in the mustelids, but has not been reported in any of the other fissiped families. The uterus of carnivores is bicornuate, with the lumina of the two horns becoming confluent at the cervical region. The endometrium is generally richly glandular in adult animals, and is composed of a series of longitudinal folds, usually five or more in number, that give the lumen a pentaradiate form when seen in cross-section. The glands of young animals are simple, straight, tubular sacculations arising periodically from the surface of the folds and passing parallel to one another toward the myometrium. Because of this arrangement, the glands arising near the apices of the folds are longer than those at the margins and have a somewhat straighter course. Generally there is a short communicating portion of the gland, which is the isthmic portion; a relatively straight continuation, which may be dilated during the breeding season; and an extensive body of the tube, which is often convoluted and frequently terminates in a basal coiled portion. In general, the luminal epithelium is tall columnar, and the epithelium of the glands, although exhibiting regional variation, is lower. Cilia are not commonly found in carnivore uteri. In seals, mating takes place within a week after parturition. The uterine horns bear a pup alternately. Thus, while one horn is in the state of postpartum reorganization, a blastocyst may well be present in the contralateral horn. Whether the length of lactation has any bearing on the time of implantation is not known at present, but current evidence suggests that it does not. Despite the widespread interest in reproduction in seals, there are relatively few observations on the endometrium. Hewer and Backhouse (1968) in a study of the embryology and fetal growth of the grey seal have observed an increasing hypertrophy of uterine glands during the period of embryonic enlargement prior to implantation. Harrison et at. (1952) have described the histology of the uterus of late fetuses and nursing pups. They also illustrated the endometrium from "a recently ovulated lactating fur seal"; in this animal, the endometrium is only moderately glandular, but the glandular epithelium is columnar. Pearson and Enders (1951) state that the uterine glands of fur seals in which unattached blastocysts were found were well developed and "secreting actively." They found that both luminal and glandular epithelium was tall columnar, and that the lumen usually contained both secretory material and cellular debris. In a study of the histology of the reproductive tract of the fur seal, Craig (1964) has further noted that the glandular lumina contain acidophilic secretory material. With the onset of implantation, the glandular cells become more active in appearance, with coiling of the glands and dilation of the lumina with acidophilic secretion. The necks of the gland also become more open in the uterine lumen and the stroma becomes more edematous. However, more information is needed

to characterize fully the pinniped endometrium during the delay and implantation periods. The only nonmustelid fissiped carnivore in which delayed implantation has been studied is the black bear. Wimsatt (1963) has made detailed histological observations of the endometria from the preovulatory to the postimplantation period from bears in the upper New York State area. In this species, breeding takes place in the beginning of the summer, probably by mid-June. Implantation of the blastocysts occurs in the fall; specimens from animals killed early in December are implanted. The luminal epithelium of the endometrium is composed of columnar cells with round bulging apices. Lipid droplets are present both basally and supranuclearly. The glands take origin from the entire endometrial surface. They have a short, constricted isthmus leading into a relatively narrow straight neck segment, which joins the wider convoluted tubular secretory crypts. The secretory crypts of the glands occupy the greater width of the endometrium, extending down to the deep layers, where relatively coarse bundles of collagen and the intervascular plexus separate the stroma of the endometrium from the circular muscle layer. The epithelial cells of the secretory crypts are tall columnar with a rounded nucleus close to the base of the cell. Lipid droplets are present both beneath and above the nucleus, but there is a clear apical region devoid of lipid droplets. Some diastase-resistant periodic acid-Schiff positive material is sometimes present within the crypts, as well as in the neck segments, which histologically resemble crypt cells without the apical end. Wimsatt (1974), in a further study of the morphogenesis of fetal membranes and placenta of the black bear, states that implantation is central and superficial. In the uterus of a recently implanted specimen, Wimsatt (1963) noted that the lipid was still plentiful in the epithelium of the neck and upper crypt cells of the gland but that it had completely disappeared from the lower two-thirds of the crypts and the surface epithelium. During the implantation period, the endometrial glands show futher growth and hypertrophy. The mustelids exhibit a wide variety of gestation periods. The ferret (Mustela fura), for example, does not delay at all. The mink (Mustela vison) has a short delay period of variable length depending on how early in the breeding season the individual animal mates. The longest delay periods are exhibited by the fisher (Martes pennanti) (Wright, 1963; Wright and Coulter, 1967), otter (Lutra canadensis) (Hamilton and Eadie, 1964), and individual European badgers (Meles meles) (Canivenc and Laffargue, 1963), in all of which breeding occurs in the postpartum estrus with the result that the animals are pregnant most of the year. In some species, such as the short-tailed weasel (stoat, Mustela erminea) , breeding does not ordinarily occur until some time after parturition, but takes place while the animal is still lactating. In the western spotted skunk (Spilogale putorius latifrans) (Mead, 1968b), marten (Martes americana), American badger (Taxidea taxus) , long-tailed weasel (Mustelafrenata), and wolverine (Culo gulo) (Wright, 1963), breeding occurs at the end of the summer after the termination of lactation, and the delay period lasts until the next spring. Consequently, most of these mustelids have a relatively long delay period. The sea otter (Enhydra lutris), however, has no distinct breeding season but breeds throughout the year (Sinha et al., 1966) and has an estimated gestation

217 ENDOMETRIUM OF DELAYED AND EARLY IMPLANTATION

218 CHAPTER 8

Figure 14. Uterus from a badger (Taxidea taxus) during delayed implantation. The large cells with the centrally situated nuclei extend from the luminal surface into the necks of the glands (arrows). These cells contain masses of glycogen. x485. Figure 15. Uterus from a short-tailed weasel (Musteta erminea) during delayed implantation. As in the badger, the luminal epithelial cells are large and contain abundant glycogen. X485.

period of S-9 months (Barabash-Nikiforov, 1935). In a brief histological description of the sea otter uterus during the delay period, Sinha et al. (1966) noted that the endometrium is filled with coiled glands that show secretory activity. The endometrium of implanted animals is edematous and congested with coiled glands. In the area of the placental attachment, the glands become progressively dilated and hypertrophied, and appear secretory as development proceeds. The four wild mustelids most thoroughly studied are the badger, long-tailed and short-tailed weasels, and spotted skunk. In the European badger, bilateral ovariectomy does not result in death of the blastocysts, although histological evidence of regression of the endometrium has been reported (Harrison and Neal, 1959). Harrison (1963) states that the endometrium of delayed implantation in the badger does not vary throughout the delay period, although the vaginal epithelium shows structural modifications during delay. He describes the mucosa as having a luminal epithelium of tall columnar cells with palely staining cytoplasm. The glands are relatively straight and exhibit litde secretory activity. Although Hamlett (1 932b ) and Wright (1966) thoroughly discussed the reproductive cycle of the American badger, there is litde information concerning the endometrium during the delay period. We have had an opportunity to study the uteri from two animals killed during the delay period. The endometrium is glandular and highly vascular (Fig. 14). The luminal epithelial cells have extensive deposits of glycogen in both subnuclear and supranuclear positions. The luminal epithelium extends well down into the neck of the gland, where a sharp transition to the neck epithelial cell type occurs ( Fig. 14 and IS). The neck cells have abundant glycogen, but far less than that seen in the luminal epithelium. A few sparsely filled granules are seen, and some dilated rough endoplasmic reticulum is present. The straight neck portion ends as the gland becomes more coiled, and there is a gradual transition to glandular epithelial cells, which are large with spherical nuclei situated in the lower third of the columnar cells. The apices of these gland cells are filled with a sparse flocculent material and dilated rough endoplasmic reticulum (Fig. 19). The glandular lumen contains PAS-positive material and debris. The endometrium is among the more active seen in delayed implantation. The spotted skunk (Spilogale putorius) is the only mustelid species that shows both obligate delay and no delay of implantation. Eastern forms (S. putorius interrupta, S. p. ambarualis, and probably S. p. putoriils) breed in April and have a gestation period of 50-65 days (Mead, 1968a), while the western forms (S. p. gracilis, S. p. leucoparia, S. p. latifrons, and S. p. phenax) breed in September, with parturition occuring in May (Mead, 1965b). The gestation period has been further delineated in S. p. latifrons into a total period of 230-250 days in which the delay period lasted from 200 to 220 days (Foresman and Mead, 1973). Mead (196Sb) suggests that the eastern and western forms are actually distinct species.

Figure 16. Uterus of a mink (Mustela vison) during delayed implantation. The luminal epithelium is

tall columnar and glands are abundant. However, there is no accumulation of glycogen in the luminal cells and the amount of glycogen in the gland cells varies during this stage. x4S5. Figure 17. Uterus of a mink at an early postimplantation stage. Note the general hypertrophy of

both luminal and glandular epithelia and the subepithelial capillary plexus. x4S5.

219 ENDOMETRIUM OF DELAYED AND EARLY IMPLANTATION

220 CHAPTER 8

Figure 18. Electron micrograph of a badger uterus during delayed implantation. Note transition between large luminal epithelial cells fille d with glycogen (Gly) and the smaller more typical glandular epithelial cells at the right. XII, 700. Figure 19. Electron micrograph of a badger uterine gland during delayed implantation . The presence of short segments of dilated endoplasmic reticulum (ER) and especially the numerous vacuoles with flocculent content suggests some secretory activity. x 16,200.

The appearance of the uteri of both the eastern and western forms of the spotted skunk is similar except during the period of delay in the western form. The uteri have a smaller diameter than in estrus, with deep straight glands that show signs of increased activity and slighdy dilated necks, and a tall columnar luminal epithelium (Mead, 1968b). With the approach of implantation, the uterine cornua begin to enlarge, preimplantation blastocystic swelling occurs, and the cornua become coiled and tortuous beginning about 6 days before implantation (Foresman and Mead, 1973). After implantation the endometrium is thick, well-developed, and wellvascularized, and contains active-appearing glands that are straight to moderately coiled. At the implantation sites, the endometrium is thicker and the glands are deeper, coiled, and more dilated (Mead, 196&). Wright (1963) has made some preliminary observations on the histochemistry of the endometrium of the long-tailed weasel. He noted that the endometrium in the typical delay state has glycogen in the luminal epithelium. He also reported that when weasels were ovariectomized during the delay period, the endometrium underwent regression. In this sense, he believes that the uterus is not in an anestrous condition but is being maintained (possibly by the interstitial cells of the ovary). Wright (1963) also reports that, in one instance, ovariectomy resulted in death of the blastocysts. It is interesting that Deanesly (1935), in describing the uterus of the pseudopregnant stoat (before she ascertained that these animals had a delay period, 1943), described the endometrium as being in a luteal phase, exhibiting the same features as did uteri in early implantation. Portions of uteri were obtained by unilateral hysterectomy from two shorttailed weasels at four times during the delay period. This endometrium showed a remarkable concentration of glycogen in the tall columnar luminal epithelial cells. The nuclei are displaced to the apical ends of the luminal epithelial cells, with the cells becoming virtual sacs of glycogen. The glands have relatively low columnar epithelial cells with litde glycogen except in the neck regions, where a transition to the surface epithelium occurs. Some secretory material is present in some of the cells of the basal portions of the glands (Fig. 15). In electron micrographs, the luminal epithelial cells are truly remarkable and appear very similar to the luminal cells of the American badger. Glycogen fills the cytoplasm except at the very apex and a thin zone at the margin of the cells (Fig. 20). The microvilli are well developed and have a glycocalyx similar to that seen in the intestine. There are numerous agranular tubules in the cytoplasm adjacent to the microvilli. Mitochondria lie just below this cytoplasm and interdigitate with the glycogen-rich lower portions of the cell. A small Golgi zone is located between the apical tips and the nuclei. The few strands of granular endoplasmic reticulum that are present are situated largely along the margins of the cells. Ribosomes are associated with the peripheral and perinuclear regions of the cytoplasm, which are largely devoid of glycogen. In contrast to the luminal epithelial cells, the glandular epithelial cells during the delay period are largely devoid of glycogen (Fig. 21). The microvilli of the cells are shorter than those of the luminal epithelial cells. Caveolae can frequently be found at the bases of these microvilli, as well as at the lateral margins of the cells. A small subapical vacuolated region is apparent in many of the gland cells. The Golgi

221 ENDOMETRIUM OF DELAYED AND EARLY IMPLANTATION

222 CHAPTER 8

Figure 20. In this section through the uterine luminal epithelium of a weasel in delayed implantation, the cells are seen to be largely filled with fine granules of glycogen (Gly). The rest of the cytoplasmic components are largely confined to the apical, peripheral, and perinuclear regions. XS,OOO.

223 ENDOMETRIUM OF DELAYED AND EARLY IMPLANTATION

Figure 21. Weasel gland cells from the period of delayed implantation. The lumen of the gland contains granular material. A number of large vesicles are seen above the Golgi complex at the apex of the glandular cells. More basally there is granular endoplasmic reticulum (ER) with a flocculent content. The dilated intercellular space (ICS) is characteristic. x 22 ,700.

224 CHAPTER 8

zone is more highly developed, and some of the cisternae are dilated and often show fusion with associated Golgi vesicles. The endoplasmic reticulum shows numerous small, slightly dilated cisternae. Ribosomes are numerous not only in association with the endoplasmic reticulum but also free within the cytoplasm. Mitochondria are large, but no extraordinarily large mitochondria are seen. An intracellular space between cells near the basal border is a common feature of the glandular epithelium. Some glandular epithelial cells have a few lipid droplets, and most of the epithelial cells of the basal glands have large supranuclear granules that appear to be lipid pigment. Sheldon (1972) reported that after ovariectomy during the delay period, the luminal epithelium declined sharply in height, apparently because of loss of stored material. The glandular epithelium, however, was not so sensitive. Unlike those of the European badger, the blastocysts in this group of weasels largely deteriorated within 3 weeks and all were fragmented by 9 weeks after ovariectomy, even though the immediate reaction was a proliferation of embryonic cell mass elements. Sheldon concluded that mitotic activity in the blastocyst was stimulated even though the uterine environment could not support further embryonic differentiation. More information is available concerning the uterus of the mink than concerning that of any of the other mustelids. The mink delay period is dependent on the time of breeding, since implantation occurs at approximately the same time in all individuals (Enders, 1952). In standard breeding practice, a single female will be bred more than once. Both superfetation and superfecundation result from multiple breeding and ovulation during the delay period (Hansson, 1947; Enders, 1955). The mink thus exhibits an unusual type of delay in that it occurs during the breeding season, the length of which depends on how early in the season the individual female is bred. Histological descriptions of the mink endometrium have been made by Hansson (1947) and Enders (1952). Histochemical studies were reported by Enders (1961), Enders and Enders (1963), and Murphy and James (1974). In general, the endometrium during delay in the mink exhibits appreciable variation, especially as viewed histochemically. The luminal epithelium is columnar (Fig. 16). The surface of the luminal and the glandular epithelium is covered by a P ASpositive coat, and alkaline phosphatase activity is exhibited in the apices of these cells. However, the thickness of the PAS-positive coat, the extent of the alkaline phosphatase activity, and the amount of glycogen in the cells all vary during the delay period. It has been suggested (Enders and Enders, 1963) that this variation is due to the fluctuations in follicular development superimposed on the partial luteinization of the corpora lutea of delay, and that the fluctuations consequently represent relative follicular or luteal ascendency. Murphy and James (1974) agreed with Enders and Enders (1963) regarding the variability in the presence of glycogen. However, they reported no variability in the PAS-positive material but rather saw a uniform distribution of mucosubstances. The mink endometrium exhibits relatively little lipid or phospholipid during the delay period (Enders, 1961). Murphy and James (1974) reported hyaluronidase- and sialidase-susceptible material in cells of luminal epithelium and particularly in gland necks and gland bases during the delay period. During the delay

period, sulfated mucopolysaccharides were also found in luminal epithelial cells and the gland necks. The onset of implantation is reflected in the uterus by a hypertrophy of the luminal epithelial cells (Fig. 17), an increase in the alkaline phosphatase activity and decrease in acid phosphatase activity, and a broadening of the opening of the isthmus of the glands onto the luminal epithelium, resulting in a dentate appearance similar to that described in the cat in early implantation (Dawson and Kosters, 1944). Murphy and James (1974) also noted a general increase in PASpositive diastase-resistant material but a depletion of glycogen in the uterine epithelium during the postimplantation period. Also, sulfated mucopolysaccharides appear in the glands during this period while acidic mucopolysaccharides show no change from the delay appearance. Electron microscopy of the mink endometrium during the delay period reveals a number of significant features. The glandular epithelial cells from the bodies of the glands have moderate amounts of saccular granular endoplasmic reticulum. In the apical ends of these cells are numerous membranebound granules (Fig. 23). Many of these granules exhibit a localized region of increased density. The Colgi complex is extensive, with numerous vesicles, but the cisternae are not dilated, nor can any dense material be seen within the Colgi cisternae or vesicles. Although mitochondria are not especially abundant within the glandular cells, many of the mitochondria are unusually large and spherical, with peculiar spiral cristae. Numerous capillaries indent the glands during this period. The luminal epithelial cells of the delay period are more elongate than those of the glands and have longer microvilli projecting from their conical apical borders (Fig. 22). The mitochondria are more normal in appearance, being rodshaped with lamelliform cristae. There is a concentration of mitochondria in the apical region of the cell. No membrane-bound granules of the type described in gland cells are present, but a few dense pigment granules are present in the supranuclear cytoplasm. Occasional lipid droplets are present basally. The granular endoplasmic reticulum is composed of a few elongate undilated cisternae. Scattered portions of agranular endoplasmic reticulum are common along the margins of the cell, both laterally and basally. A few subapical vacuoles are usually seen in these cells. The nuclei of the luminal epithelial cells are farther from the base than in glandular cells, and the Colgi zone is more compact. The cytology of the gland suggests that these cells are secreting small amounts of protein. The luminal epithelial cells, although taller, show no evidence of secretory activity, but have features that are compatible with the function of absorption of luminal fluid. The glandular epithelia during the late delay and postimplantation periods show notable changes from that seen earlier (Enders et at., 1963). The endoplasmic reticulum of the subnuclear region is extensively dilated, leaving only thin strands of cytoplasm between the cisternae (Fig. 24). The giant mitochondria disappear and are replaced by smaller rodlike forms as well as the large secretion granules. Smaller granules are seen, especially near the Colgi zone, where the cisternae are more dilated than seen previously. During this period, the luminal epithelium shows little change from that seen in delay. In the mustelids, the endometrium contains two to three distinct cell types

225 ENDOMETRIUM OF DELAYED AND EARLY IMPLANTATION

226 CHAPTER 8

Figure 22. The tall columnar luminal epithelial cells of the endometrium of a mink in delayed implantation are shown in this micrograph. Note the rounded apical surfaces with protruding microvilli. The Golgi zones (G) are compact and the endoplasmic reticulum (ER) is in the form of elongated undilated cisternae. A few lipid granules (L) are found in some of these cells, but no secretion granules are present. X 11,000.

227 ENDOMETRIUM OF DELAYED AND EARLY IMPLANTATION

Figure 23. Gland cell from mink endometrium during delayed implantation. Note the numerous membrane-bound granules (Gr) in the apical cytoplasm and the large mitochondrion (M) above the nucleus. The endoplasmic reticulum is dilated at this stage. X 12,900.

228 CHAPTER 8

Figure 24. Mink uterine gland cells just after implantation. Polyribosomes are abundant in the cells and the endoplasmic reticulum is extensively dilated. Golgi cisternae are well developed and there are numerous small vesicl es associated with this region. Despite the absence of large secretory granules, these cells give the impression of intense activity. x 14,700.

during the delay period. The appearance of these cells varies somewhat among species but all appear active during, in some cases, a long delay period. Marked changes occur at the onset of blastocystic expansion and implantation, including loss and apparent release of materials found within the cells and appearance of the characteristics of more highly secretory cells. The general appearance of the endometrium also changes, with increasing dilation of glands and general hypertrophy of the epithelium. In several species undergoing obligate delay, the concentration of uterine fluid protein has been shown to be low during the delay period, but it increases strikingly in two species (mink and northern fur seal) at about the time of implantation (Daniel and Krishnan, 1969; Daniel, 1971). The changes in uterine histology and composition of uterine fluid are coincidental with the sharp rise in plasma progesterone levels noted at about the time of implantation in several delay species (mink, M\1lller, 1973; stoat, Gulamhusein and Thawley, 1974; spotted skunk, Mead and Eik-Nes, 1969). Exogenous steroids and gonadotropins, however, fail to induce implantation in several species (reviewed by Foresman and Mead, 1974). At this time, our understanding of the interrelationships involved in the delay period and implantation is not complete.

6.

Rodents

The subject of delayed implantation in rodents was first investigated by Lataste (1891), who noticed that when female rats were allowed to breed at the postpartum estrus those that were suckling young had a longer gestation period than did mated nonlactating females. He correctly attributed this prolongation of pregnancy to a delay in the time of implantation. Lataste also must be considered the first to study delayed implantation experimentally. He altered the implantation time, as judged by gestation length, both by removal of young from lactating females and by cauterization of nonlactating females. Since Lataste's time, extensive studies have been made of delayed implantation in the rat and in the laboratory mouse, and the gerbil has been added to the group of laboratory animals that have a lactational delay (Marston and Chang, 1965; Norris and Adams, 1971). Whereas little work has been done concerning the distribution of delayed implantation in wild rodents (exceptions, in addition to Lataste's work, are the report in the vole, Clethrionomys glareolus, by Brambell and Rowlands, 1936, and in the deer mouse, Peromyscus sp., by Svihla, 1932) a great deal of information has been added to our understanding of the endocrine control of delay in the rat and mouse, and to studies of the blastocyst under these conditions. Although it is clearly impossible to summarize all of the data accumulated concerning the various ways the delay of implantation may be experimentally induced in the rat and mouse in one sentence, the overwhelming mass of the data suggests that, from an endocrine view, delay of implantation in these species is a matter of progesterone dominance of the uterus in the absence of a sufficiency of estrogen. Rat blastocysts during the delay period grow to a size as great as or greater than those that can be flushed from early postimplantation (day 6) normal pregnant uteri (Schlafke and Enders, 1963; Enders and Schlafke, 1965; Surani, 1975a). As such, the blastocysts can be considered "postdeveloped," as opposed to

229 ENDOMETRIUM OF DELAYED AND EARLY IMPLANTATION

230 CHAPTER 8

Figure 25. Light micrograph of the endometrium of a mouse on day 8 of delayed implantation. Note the tall columnar luminal epithelium containing supranuclear and subnuclear lipid. Lipid is also seen in the glandular epithelium. Note numerous capillaries closely apposed to the glandular coils. x520. Figure 26. Transmission electron micrograph of the uterine luminal epithelium of a rat on day 11 of delayed implantation. The edge of a large pinopod is seen on the apical surface of the cell.

the "preswelling" condition for the blastocysts in animals exhibiting central implantation. The uterus of the myomorph rodents, compared with that of other groups exhibiting delay, is relatively rich in stroma and poor in glandular epithelium. The lumen is a nearly closed trench bordered by columnar epithelial cells with sparsely distributed simple coiled glands entering it. During delay, there is some interdigitation of the adjacent microvilli of luminal epithelial cells of the two sides of the uterus (Hedlund and Nilsson, 1971). A number of low longitudinal folds of endometrium are present, lending a serpentine appearance to a cross-section of the lumen. An additional fold is often found in a mesometrial position, resulting in a T or inverted L shape to the luminal cross-section. When implantation follows delay, the lumen withdraws from the antimesometrial portion of the endometrium and the "trench" becomes shorter. The epithelial "attachment reaction" becomes more pronounced, possibly after an intermediate period of transient fluid accumulation (Nilsson, 1974). In both rat and mouse, the luminal epithelium is clearly the most abundant epithelial constituent of the endometrium. The tall columnar cells of this epithelium have abundant basal lipid droplets (Fig. 25). The glands tend to have fewer lipid droplets, although some may be present in the neck regions and, in the mouse, a few droplets extend into the basal coils of the glands. The glandular epithelium is composed of more cuboidal cells than the luminal epithelium, and the nuclei are more basally situated. The lamina propria is highly vascular, with subluminal and periglandular plexuses as well as radially situated vessels throughout the highly cellular stroma (Fig. ~5). A number of studies have been concerned with the histochemistry of the mouse (Hall, 1973, 1975) and rat (Enders, 1961) uterus in delay and preimplantation. The lipid, in particular, which is pronounced in luminal epithelial cells, appears to be restudied every few years (Alden, 1947; Elftman, 1958; Enders, 1961; Christie, 1967; Boshier and Holloway, 1973; Hall, 1975). Although there is some variation in results concerning the amount of free fatty acids and lipid, most of the authors are agreed that lipid is abundant when progesterone predominates, and that estrogen can produce a mobilization of the lipid (Elftman, 1963). There is little or no glycogen present in the epithelial cells during delay, and very little alkaline phosphatase activity in the epithelium, although the vascular bed is well demonstrated by this enzyme. On the other hand, acid phosphatase and {3glucuronidase can be demonstrated in the apical ends of both luminal and glandular epithelial cells, especially in the area where the Colgi complex and most of the lysosomes are situated (Enders, 1961). The general cytology of the epithelium of the rat uterus during delay has been described (Warren and Enders, 1964; Enders, 1967). There is little cytological evidence of secretory activity in this uterus. However, a puzzling feature of the luminal epithelial cells is the presence of numerous small vacuoles that are

Peroxidase is present within vesicles in the pinopod. A few irregular strands of granular endoplasmic reticulum are present, as well as large secondary Iysosomes (Ly). x20,300. Figure 27. Scanning electron micrograph of the luminal surface of a rat on day 11 of delay. Several

pinopods are seen projecting from the microvillous surface.

X

5900.

231 ENDOMETRIUM OF DELAYED AND EARLY IMPLANTATION

232 CHAPTER 8

apparently not derived from pinocytosIS m the apical cytoplasm (Fig. 26). Both multivesicular bodies and more typical lysosomal structures can be found in both luminal and glandular epithelial cells. In the latter, a few small granules can be found in the apical cytoplasm that have an appearance similar to some of the material found in the lumina of the glands. Recent evidence indicates that the luminal epithelial cells of the rat uterus are active in pinocytosis during delayed implantation (Fig. 27). Large ectoplasmic projections seen both in preimplantation stages of normal pregnancy and during delay of implantation (Psychoyos and Mandon, 1971) have been found to be processes involved in pinocytosis (Enders and Nelson, 1973; Parr and Parr, 1974). It has been shown that under experimental conditions these pinopods are capable of removing tracers introduced into the lumen and transferring at least some of the contained material to the lateral intercellular borders (Fig. 26). Micropinocytotic activity also takes place in the uterus, but at a low rate as judged by the markers used to date. The luminal epithelium of the mouse uterus during experimentally delayed implantation has been described as having fungus-like apical protrusions similar to pinopods. The protrusions make crater-like imprints in the blastocysts of delay when viewed by scanning electron microscopy (Bergstrom, 1972; Bergstrom and Nilsson, 1972). These protrusions are present throughout the experimental delay period, while the apical microvilli show some modification with time (Nilsson, 1974). Both the microvilli and the pinopods have a negatively charged glycoprotein cell surface coat (Enders and Schlafke, 1974). The glandular epithelium during delay in the mouse has not yet been described at the ultrastructural level. Our observations indicate that the glandular lumina contain a dense material that may contain small vesicular structures. Prominent apical microvilli protrude into the lumen (Fig. 28). Near the apical border of gland cells, clear vesicles are commonly seen, along with some elements of smooth endoplasmic reticulum. Multivesicular bodies are frequently seen and scattered strands of rough endoplasmic reticulum are found throughout the cell. The Golgi apparatus is prominent and usually lies somewhat lateral to the nucleus. Although many recent studies have been concerned with the mouse uterus and blastocyst during normal implantation (Finn and Hinchliffe, 1965; Potts, 1966, 1968; Finn and McLaren, 1967; Potts and Wilson, 1967; Reinius, 1967; Wong and Dickson, 1969; M.S.R. Smith and Wilson, 1971; Calarco and Epstein, 1973; A. F. Smith and Wilson, 1974), the appearance of the endometrium during the implantation that follows the delay period has not been reported extensively. McLaren (1968) stated that after estrogen administration or removal of the litter during lactational delay, several changes could be seen in the mouse endometrium including disappearance of shed zonas, appearance of pontamine blue reactivity, stromal edema, and primary decidual formation. The same sequence of changes was seen by Finn and McLaren (1967) during normal implantation. Nilsson (1974) has also noted some of the progressive changes seen after estrogen induction of implantation during experimental delay. The luminal epithelium lacks the large apical protrusions seen during delay, and the blastocysts are separated from the luminal epithelium by material that he states is a uterine secretion and is similar to a material seen by Potts (1968). Nilsson (1974) also noted a loss of apical vesicles, which he interpreted as a discharge into the lumen.

233 ENDOMETRIUM OF DELAYED AND EARLY IMPLANTATION

Figure 28. Electron micrograph of an apical portion of mouse gland cells on day 6 of delayed

implantation. Note several large multivesicular bodies (MVB). Portions of the Golgi complexes are lateral to the nuclei. x 17,300. Figure 29. Electron micrograph of a basal portion of a gland cell at the time of a postdelay

implantation (day 11). Note the two large Golgi complexes, the ordered arrangement of the extensive granular endoplasmic reticulum, and numerous Iysosomes. x 17,300.

234 CHAPTER 8

Subsequently, the luminal cell surface becomes progressively less microvillous and eventually closely apposed to trophoblast surface. We have preliminary observations that indicate that the glandular epithelium in the mouse endometrium undergoes changes during this time as well. The Golgi complex becomes more prominent, while the amount of rough endoplasmic reticulum increases and is more regularly arranged (Fig. 29). The number of smooth profiles of endoplasmic reticulum increases in the apical region of the cell. Numerous lysosome-like inclusions are also present. Jenkinson (1913) described the uterine glands of the mouse during the implantation period as "open glands with long necks. These secrete a coagulable, presumably proteoid, material. These secretions are absorbed by the free blastocyst." More recently, Gwatkin (1969) has further characterized the protein and amino acid composition of mouse uterine fluid during normal pregnancy and during the delayed implantation period. He found that there was a drop in amino acid and protein concetration until about the third day of pregnancy and then a rise until day 6. Amino acid concentration during the delay period was only slightly higher than that found on the day of implantation (day 5), and no difference in kinds of amino acids was found during the normal and delayed implantation periods. In other studies, Mintz (1970) proposed the term "implantation-inducing factor" (IIF) for an agent found in the uterine fluid of mice. The presence of this agent in the uterine fluid was indicated by in vivo removal of the zonae pellucidae of dead morulae just prior to the time that blastocysts begin the implantation process (day 4). This agent appeared to be present after estrogen administration but absent during the early delayed implantation period, for zonae of dead morulae were present during the delay period while blastocysts hatched normally. Proteinase activity, as detected by a casein-substrate assay of mouse uterine fluid, coincided with the time period that IFF was thought to be present (Pinsker et aI., 1974). Sacco and Mintz (1975) have reported in immunological studies of mouse uterus the presence of two antigens shared only with duodenum, one of which is most abundant on days 4 and 5 of pregnancy. They believe that this antigen may be the proteolytic agent found previously, and hypothesize that this agent may act on blastocystic cell surface receptors, causing attachment to the uterus, as well as lysis of the zona. The uterine fluid of the rat has been analyzed for several components. Recently, Joshi and Murray (1974) have reported an endopeptidase isolated from estrous rats. After an antibody to the endopeptidase was absorbed with rat serum, they demonstrated using immunofluorescent methods the presence of the enzyme in the apex of glandular epithelial cells of rats on days 5 and 6 of pregnancy and in the luminal and glandular epithelia of proestrous, estrous, and immature rats. They speculated that this endopeptidase could possibly be a zona lytic factor. Analysis of electrolytes found in uterine fluid and endometrium has led to the suggestion that shifts in potassium concentration after estrogen administration may cause adhesion of the trophoblast and uterus (Clemetson et at., 1972). However, Setty et al. (1973) reported no significant changes in electrolyte concentration after estrogen stimulation during experimental delay. In addition, Levin (1973) has pointed out that most studies have confused transluminal potential with

cell surface charge. As a consequence, the electrolyte changes seen may not be so critical to implantation and delay as once thought. In recent studies, Surani (1975b) has analyzed uterine secretions using polyacrylamide gel electrophoresis of bilaterally ovariectomized rats that received progesterone for 10 days followed by progesterone and estrogen. He found that 1 hr after estrogen treatment a transient 70,000 molecular weight protein band appeared. At 13 hr and 20 hr after estrogen, there was a shift to higher molecular weight proteins not seen under progesterone support alone. These proteins were similar to those seen on day 5 of normal pregnancy. It was proposed that presence of these proteins may be related to the process of implantation. As indicated in the preceding paragraphs, analysis of the intraluminal contents of the uterus is now well underway. The precise role of the constituents and the cellular basis of their appearance remain to be ascertained.

7.

Discussion

It is not surprising that there is variation in the endometria from delayed implantation, since the phenomenon is found in such a wide variety of species. In addition, the term "delayed implantation" is used to describe any condition in which the period of time when there is an unimplanted blastocyst in the uterus is prolonged and in which there is evidence of a temporary reduction or cessation of blastocystic development for part of that time. However, even if we restrict our comparison of endometria to those from the quiescent portion of delay, there is considerable variety. In the tammar wallaby, in which part of the delay period is in the anestrus season, the glandular development of the uteri is greater than in nonmated animals. The weasel and badger are extraordinary in appearing to have a tremendous energy reserve in the form of glycogen in the more superficial epithelial cells of the endometrium during the period of delayed implantation. Several other species such as the armadillo have a highly vascular endometrium that has typical secretory glandular cells, such as might be anticipated in a preimplantation condition in nondelaying species. The presence or absence of ciliated cells is also variable. It is not possible to generalize concerning classes of compounds found in endometrial epithelial cells during delay. Lipid is abundant in the rat and mouse in delay, but is seen in abundance in the armadillo and mink only after implantation. Glycogen, which is very prominent in the weasel, is almost absent in several other species, and appears only after implantation in the armadillo. In many species, the cytological differences between luminal epithelial cells and glandular epithelial cells are rather remarkable during the delay period. Clearly, information concerning the time sequence of formation of glandular secretion products by the endometrium is needed to increase our understanding of the role of the endometrial secretions in delay of implantation. It has been shown that, in the rat, a product with peroxidase activity is synthesized in response to high levels of estrogen (Brokelmann and Fawcett, 1969; Churg and Anderson, 1974). However, at

235 ENDOMETRIUM OF DELAYED AND EARLY IMPLANTATION

236 CHAPTER 8

implantation, the only evidence of endogenous peroxidase actIvIty is a peculiar granule in the cisternae of the Golgi complex (Enders and Nelson, 1973). Although it seems obvious that the uterus controls blastocystic development during delay, it is encouraging to see evidence from wild species such as the roe deer that endometrial changes coincide with blastocystic elongation (Aitkin, 1974a). In vitro cultivation of blastocysts from delay in mice shows that the rate of protein synthesis by the blastocyst is depressed by the intrauterine environment (Weitlauf, 1974). Another encouraging trend has been the analysis of intrauterine fluid during delay of implantation and at implantation. Although some aspects of the nature of these fluids and the quantitative data can be influenced by the method of collection, data of this type and data on oxygen tension and on uterine motility will help us understand the environment of the delayed blastocyst. The constituents of the uterine lumen are the net result of influx and efflux (Fig. 30). Potentially, materials can enter the uterus from the oviduct (peritoneal fluid, oviductal secretions, and transudates) and from the endometrium (secretion

\./

c::::l':

INTERSTITIAL FLUID

(:;t ~

".

..

../'"

EFFLUENT

- - -- - ---~ SPECIFIC REABSORPTION

)

Figure 30. A diagrammatic representation of the possible factors responsible for maintaining the intrauterine environment during delayed implantation.

by glandular or luminal epithelium, transudation from the vessels and stroma, direct diffusion of small molecules, dehiscence of luminal cells, and leukocyte migration), and it is possible that the blastocyst may contribute substances either acting locally (proteases?) or having a more widespread effect (hormones). Although there are almost always alternative explantations, the literature concerning effects of blastocysts seems to be increasing, e.g., the increase in size of corpora lutea in progesterone-delayed implantation when the blastocysts are permitted to enter the uterus rather than remain restricted by ligature at the oviducts (Chatterton et al., 1975). On the efflux side, there is not only the possibility of direct loss from the cervix but also transudation from the lumen into stroma, pinocytosis such as that reported in the rat, and micropinocytosis, which could include specific reabsorption of selected components of uterine fluid. It is at least theoretically possible that the removal of a specific component by the blastocyst could be significant. Different aspects of intrauterine exchange have been emphasized by individual studies in particular species. Vascular exchanges are important for both oxygen and pH changes and are potentially quite variable, as seen in the rat (Yochim, 1975). The finding by Surani (1975b) that apparently specific proteins are found after the injection of estrogen to induce implantation in a delay situation in rats implicates secretion as an important part of the normal implantation environment, as does the work on uteroglobin in nondelay (Beier, 1974). In mink, in which the blastocysts survive in a highly variable climate during delay, including occasional augmentation by seminal fluid, specific secretion to either inhibit or stimulate blastocystic development seems probable. It is not clear whether blastocysts are inhibited from further development during delay or implantation or are simply in a deficient, especially proteindeficient, environment (McLaren, 1973). The "recovery" of blastocysts in culture (Weitlauf, 1974) and the inhibition of blastocysts by uterine extracts (Psychoyos, 1973) have been interpreted as indications of the presence of a blastocyst inhibitor. On the other hand, the increase in secretory activity in the roe deer, which undergoes a preimplantation swelling, has been interpreted as a release of accumulated secretion rather than a sudden change in nature of material synthesized (Aitken, 1974b). The delayed removal of the zona pellucida and the abundance of shed zonas in the early stages of delayed implantation have been taken as an indication of a reduction in uterine lytic factors during the delay period (Surani, 1975a). Only recently has the role of absorption by the uterus received much attention. In particular, the role that pinocytosis may play both in limiting the uterine environment and in permitting close apposition of uterus to blastocyst has been brought out in studies using tracers in the rat. These studies also indicate that the apical junctional complexes of the luminal epithelial cells are an effective barrier to larger molecules. Time sequence and quantitative studies of pinocytosis could add further to our understanding of fluid movements. Delay of implantation remains an excellent phenomenon with which to study uterine-blastocyst interrelationships. During this period, the general uterine environment can clearly be considered to be the environment of the blastocyst. After the initiation of implantation, the local response of the uterus to the blastocyst results in altered local conditions, so that the general luminal environ-

237 ENDOMETRIUM OF DELAYED AND EARLY IMPLANTATION

238

ment of the uterus can no longer be monitored with any assurance that it reflects the conditions actually encountered by the implanting blastocysts.

CHAPTER 8

ACKNOWLEDGMENTS

We are happy to acknowledge the aid of the following investigators: Dr. R. K. Enders, who provided seal, mink, and marten material; Dr. P. L. Wright, who not only provided badger material but also made several live weasels available; Dr. R. V. Short, who kindly provided paraffin blocks of roe deer uterus; Dr. Hugh Tyndale-Biscoe, who provided glutaraldehyde-fixed red kangaroo material; and Dr. John Biggers, who provided some of the philander opossum material. Part of this work was supported by Grant HD 04962 and Training Grant GM 02025 from the National Institutes of Health.

8.

References

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239 ENDOMETRIUM OF DELAYED AND EARLY IMPLANTATION

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241 ENDOMETRIUM OF DELAYED AND EARLY IMPLANTATION

242 CHAPTER 8

Prell, H., 1938, Die Tragzeit des Rehes, Zuchtungskunde 13:325-345. Psychoyos, A., 1973, Hormonal control of ovoimplantation, Vitam. Horm. 31:201-256. Psychoyos, A., and Mandon, P., 1971, Scanning electron microscopy of the surface of the rat uterine epithelium during delayed implantation,]. Reprod. Fertil. 26:137-138. Racey, P. A., and Potts, D. M., 1970, Relationship between stored spermatozoa and the uterine epithelium in the pipistrelle bat (Pipistrellus pipistrellus)J. Reprod. Fertil. 22:57-63. Rand, R. W., 1954, Reproductin in the femle cape fur seal Arctocephalus pusillus, Proc. Zool. Soc. (London) 124:717-740. Reinius, S., 1067, Ultrastructure of blastocyst attachment in the mouse, Z. Zellforsch. 77:257-266. Renfree, M. B., 1973, Proteins in the uterine secretions of the marsupial Macropus eugenii, Dev. Bioi. 32:41-49. Renfree, M. B., 1975, Uterine proteins in the marsupial, Didelphis marsupialis virginiana, during gestation,]. Reprod. Fertil. 42:163-166. Sacco, A. G., and Mintz, B., 1975, Mouse uterine antigens in the implantation period of pregnancy, Bioi. Reprod. 12:498-503. Schlafke, S. j., and Enders, A. C., 1963, Observations on the fine structure of the rat blastocyst,]. Anal. 97:353-360. Setty, B. S., Singh, M. M., Chowdhury, S. R., and Kar, A. B., 1973, The role of electrolytes of the endometrium and uterine fluid during delayed implantation in rats,]. Endocrinol. 59:461-464. Sharman, G. B., 1955a, Studies on marsupial reproduction. II. The oestrous cycle of Setonix brachyurus, Anst.]' Zool. 3:44-55. Sharman, G. B., 1955b, Studies on marsupial reproduction. III. Normal and delayed pregnancy in Setonix brachyurus, Aust.]' Zool. 3:56-70. Shelden, R. M., 1972, The fate of short-tailed weasel, Muslela erminea, blastocysts following ovariectomy during diapause,]. Reprod. Fertil. 31:347-352. Short, R. V., and Hay, M. F., 1965, Delayed implantation in the roe deer, Capreolus capreolus, ]. Reprod. Fertil. 9:372-373. Short, R. V., and Hay, M. F., 1966, Delayed implantation in the roe deer Capreolus capreolus, in: Comparative Biology of Reproduction in Mammals (I. W. Rolands, ed.), pp. 173-194, Academic Press, London. Sinha, A. A., Conaway, C. H., and Kenyon, K. W., 1966, Reproduction in the female sea otter,]. Wildl. Manage. 30:121-130. Smith, A. F., and Wilson, 1. B., 1974, Cell interaction at the maternal-embryonic interface during implantation in the mouse, Cell Tiss. Res. 152:525-542. Smith, M. S. R., and Wison, 1. B., 1971, Histochemical observations on early implantation in the mouse,]. Embryol. Exp. Morphol. 25: 165-174. Stieve, H., 1950, Anatomische-biologische Untersuchungen liber die FortpflanzungsUitigkeit des europaischen Rehes (Capreolus capreolus capreolus L.), Z. mikrosk. Anat. Forsch. (Leipz) 55:427530. Surani, M., 1975a, Zona pellucida denudation, blastocyst proliferation and attachment in the rat,]. Embryol. Exp. Morphol. 33:343-353. Surani, M., 1975b, Hormonal regulation of proteins on the uterine secretion of ovariectomized rats and the implications for implantation and embryonic diapause,]. Reprod. Fertil. 43:411-417. Svihla, A., 1932, A comparative life history study of the mice of the genus Peromyscns, Univ. Mich. Mus. Zool. Misc. Publ., No. 24. Talmage, R. V., Buchanan, G. D., Kraintz, F. W., Lazo-Wasem, E. A., and Zarrow, M. X., 1954, The presence of a functional corpus luteum during delay implantation in the armadillo,]. Endocrinol. 11:44-49. Tarkowski, A. K., 1957, Studies on reproduction and prenatal morality of the common shrew (Sorex araneus L). II. Reproduction under natural conditions, Ann. Univ. Mariae CurieSklodowska 10: 177-244. Tyndale-Biscoe, C. H., 1963, The role of the corpus luteum in the delayed implantation of marsupials, in: Delayed Implantation (A. C. Enders, ed.), pp. 15-32, University of Chicago Press, Chicago. Tyndale-Biscoe, C. H., 1970, Resumption of development by quiescent blastocysts transfered to primed, ovariectomized recipients in the marsupial, Macropus eugenii,]. Reprod. Fertil. 23:2532.

Tyndale-Biscoe, C. H., Hearn, J. P., and Renfree, M. B., 1974, Control of reproduction in macropodid marsupials,]. Endocrinol. 63:589-614. Warren, R. H., and Enders, A. C., 1964, An electron microscope study of the rrat endometrium during delayed implantation, Anat. Rec. 148: 177-195. Weitlauf, H. M., 1974, Metabolic changes in the blastocysts of mice and rats during delayed implantation,]. Reprad. Fertil 39:213-224. Wimsatt, W. A., 1963, Delayed implantation in the Ursidae, with particular reference to the black bear (Ursus americanus Pallus), in: Delayed Implantation (A. C. Enders, ed.), pp. 49-76, University of Chicago Press, Chicago. Wimsatt, W. A., 1974, Morphogenesis of the fetal membranes and placenta of the black bear, Ursus americanus (Pallus), Am.]. Anat. 140:471-496. Wimsatt, W. A., 1975, Some comparative aspects of implantation, Bioi. Reprod. 12: 1-40. Wimsatt, W. A., Krutzsch, P. H., and Napolitano, L., 1966, Studies on sperm survival mechanism in the female reproductive tract of hibernating bats. I Cytology and ultrastructure of intrauterine spermatozoa in Myotis lucifugus, Am.]. Anat. 119:25-60. Wong, Y. C., and Dickson, A. D., 1969, A histochemical study of ovoimplantation in the mouse,]. Anat. 105:547-555. Wright, P. L., 1963, Variations in reproduction cycles in North American mustelids, in: Delayed Implantation (A. C. Enders, ed.), pp. 77-95, University of Chicago, Press, Chicago. Wright, P. L. 1966, Observation on the reproductive cycle of the American badger (Taxidea taxus), in: Comparative Biology of Reproduction in Mammals (I. W. Rowlands, ed.), pp. 27-45, Academic Press, New York. Wright, P. L., and Coulter, M. W., 1967, Reproduction and growth in Maine fishers,]. Wildl. Manage. 31:70-87. Yocbim, J. M., 1975, Development of the progestational uterus: Metabolic aspect, Bioi. Reprad. 12:106-133. Ziegler, L., 1843, Beobachtungen uber die Brunst und den Embryo der Rehe, Hannover.

243 ENDOMETRIUM OF DELAYED AND EARLY IMPLANTATION

The Implantation Reaction

9

COLIN A. FINN

Implantation involves the attachment of the blastocyst to the wall of the uterus with, in some cases, the embedding of the entire ovum in the uterine wall. Associated with it are very profound and characteristic changes in the endometrium both during preparation for implantation and following attachment of the blastocyst. In this chapter, the uterine changes that take place during preparation will be considered first and then the changes during the process of implantation. The former occur regularly and frequently throughout the life of most female mammals, whereas the latter are rarer events, especially in women. This division of the subject is also convenient for the consideration of the control mechanism involved. Whereas the preparative changes are largely directed by the varying levels of the hormones secreted by the ovary, once the implantation process has been initiated hormones playa largely permissive role. There is considerable species variation in the reaction of the uterus in preparation for and during implantation. De Feo (1967) reviewed this subject very thoroughly in the first edition of this book. The great majority of research investigations have been carried out on laboratory rodents, with very little on primates or the larger domestic animals. It follows therefore that any general account of implantation will lean heavily on information acquired from laboratory animals. Although one would like to have a much more general coverage, one cannot escape the reality that information acquired from many experiments on large numbers of laboratory animals provides a sound foundation on which to build. Information from primates or wild mammals will always tend to be fragmentary because of the difficulty of obtaining large numbers of specimens. Anyone who has worked with rats or mice knows how easy it is to be misled by the results from two or three animals. The only sensible course therefore is to get as COLIN A. FINN London, England.

Department of Physiology, Royal Veterinary College, University of London,

245

246 CHAPTER 9

much basic information as possible from laboratory animals to provide a springboard for the much more difficult task of discovering what takes place in other speCIes.

1.

Preparation

of the Endometrium

In the majority of mammals, the endometrium is prepared for implantation following every ovulation. All the three main tissues of the endometrium-luminal epithelium, glandular epithelium, and stroma-undergo characteristic and welldefined changes under the influence of the ovarian hormones. An exception to this general picture occurs in the case of some of the myomorph rodents, which, in the absence of copulation, have an abbreviated ovarian cycle without the formation of a functioning corpus luteum. A full-length cycle occurs, however, if copulation takes place, even if the mating is infertile. The animal is then said to be pseudopregnant, although it is probably more correct to consider the luteal phase that is induced by the male to be the normal cycle and the abbreviated cycle to be an evolutionary adaptation peculiar to the mode of existence of these species. Under experimental conditions, it is possible to induce the extended luteal phase by stimulating the cervix of the rat electrically (Shelesnyak, 1931) or mechanically during estrus, although this is not possible in the mouse. To obtain a luteal phase without pregnancy in the latter, it is necessary to mate the females with a vasectomized male. The changes taking place in the tissues result in proliferation of the cells followed by their differentiation, with at the same time profound changes in the blood supply. They reach a climax at the time the blastocyst is ready to attach and the uterus is then sensitive to the presence of the blastocyst and can respond to it. The period of full sensitivity is of very limited duration and in the absence of an implanting blastocyst the endometrium regresses with considerable cell death. This is particularly pronounced in women, in whom overt shedding of the endometrial lining occurs at menstruation, but in most mammals there is probably a cycle of proliferation-differentiation-regression. Again, there is a slight deviation from this general rule in that some animals are able to hold the uterus in a state of partial preparation in which the blastocyst is maintained in a dormant state for an extended period before attachment to the wall of the uterus. This condition of delayed implantation will be discussed further in this chapter and in Chapter 8. Coincidental with the cellular changes are vascular changes in the stroma (Bacsich and Wyburn, 1940; Williams, 1948; Prill and Goetz, 1961; Greiss and Anderson, 1969; Bindon, 1969a) and in the extracellular components (Fain stat, 1960, 1963). These are related to the increased metabolic needs of the cells and are an important part of the hormonal response of the uterus.

1.1.

Cell Proliferation

It has been known for many years that cell division occurs frequently in the endometrium and that the extent varies with the state of the ovarian cycle (Schmidt, 1943; Perrota, 1962).

Two main methods are used to demonstrate and quantify cell proliferation in the uterus. In the first, a drug such as colchicine or vinblastine, which arrests mitosis in metaphase, is administered a few hours before autopsy (Allen et al., 1937), and the number of cells undergoing mitosis counted in histological sections. This method collects the cells going into mitosis over the period of drug activity and allows an estimate of proliferation in the tissue at that time. The other method involves labeling of the cells in the S phase of the cell cycle with tritiated thymidine and subsequently counting the cells that have taken up the isotope after autoradiography on histological sections (Beato et at., 1968; Leroy et at., 1969). By a combination of these methods, it is also possible to estimate the duration of the various phases of the cell cycle. The pattern of cell division in preparation for implantation has been investigated in the mouse (Finn and Martin, 1967; Zhinkin and Samoshkina, 1967), rat (Leroy et al., 1969; Chaudhury and Sethi, 1970; Marcus, 1974b), guinea pig (Mehrotra and Finn, 1974; Marcus, 1974a), and hamster (Krueger and CraigMaibenco, 1972). A distinct pattern of mitosis in the mouse is illustrated in Fig. 1. Just before ovulation the number of mitoses is maximum in the luminal epithelium, with few in the glandular epithelium and none in the stroma. At the time of ovulation and mating there is little proliferation in any tissue. On the third day after ovulation a sudden burst of mitosis takes place in the glands and there is a second small peak of activity in the luminal epithelium. On the next day there is a characteristic switch in the pattern of cell division; no cells undergoing mitosis are found in the epithelial tissues but many are found in the stroma. This is continued on the fifth day, when implantation takes place. In the absence of implantation, proliferation then remains low until the next proestrus. There is considerable variation between species in the extent of glandular proliferation. The guinea pig (Mehrotra and Finn, 1974; Marcus, 1974a) and

50 40 L u me n ~

! hr, Gulyas and Daniel, 1969). Whatever the nature of the stimulus, neither it nor the mechanism for its reception is blocked by the administration of actinomycin D (Finn and Martin, 1972b; Finn and Bredl, 1973). However, since the drug was administered systemically to the mother, it probably did not reach very high concentrations in the blastocyst, and the experiments do not therefore provide evidence for or against any mechanism for the production of the decidual stimulus by the blastocyst, although they suggest that the uterine reception and transduction of the message are not dependent on transcription of DNA. From the blocking of the later epithelial response, it is apparent that the drug reaches the uterine epithelial cells. The early stages of implantation thus involve a complex interplay among the hormones of the ovary, the endometrium, and the blastocyst. Removal of the zona and activation of the blastocyst, triggering of the uterus, and attachment of the trophoblast to the uterine epithelium take place over a very short period. It is clear that the ovarian hormones start the process going, but the sequence of events and their control after that are very difficult to untangle and there are many problems still awaiting answers.

4.5.

Formation

if the Implantation Chamber

For species in which implantation progresses beyond simple attachment of the trophoblast to the uterine epithelium, the next stage involves passage of the blastocyst into the stroma, which is modified to receive it. Even in species with epitheliochorial placentation, some changes occur in the stroma, but they are much less extensive than in species having eccentric or interstitial implantation.

4.5.1.

Vascular Changes

The first observable change, which seems to be common to many species, affects the blood vessels supplying the endometrium. This was shown very clearly by Psychoyos (1960), who injected a dye of high molecular weight, pontamine sky blue, intravenously into rats at the time of expected implantation and demonstrated a greatly increased permeability of the blood vessels in areas of the uterus containing an implanting blastocyst. In these areas, the dye, probably mostly bound to albumin, leaves the blood vessels and remains in the tissues after most of the dye has been removed from the circulation. Animals killed 15 min after the dye injection therefore show blue bands across the uterus, indicating areas of implantation (Fig. 13). This response is one of the earliest discernible signs of implantation. It also occurs in mice, first appearing during the night between the

Figure 13. Uterus of a mouse killed early on the fifth day of pregnancy 15 min after the injection of a solution of pontamine blue. One ovary had been removed before the animal mated. This picture illustrates the dark bands caused by the implanting blastocysts and the increase in length of the fertile horn. From Finn and Porter (1975), by permission of Elek Science, London.

281 THE IMPLANTATION REACTION

282 CHAPTER 9

fourth and fifth days of pregnancy (Finn and McLaren, 1967), or approximately 16 hr after the injection of a nidatory dose of estradiol into mice undergoing delayed implantation. It has also been demonstrated in the guinea pig (Orsini and Donovan, 1971), sheep (Boshier, 1970), and hamster. Although there is little quantitative information about the blood flow to the implantation site, visual observation suggests the development of new blood vessels in response to implantation. Bouda (1969) describes such a development in the human uterus, and Hall (1969) has shown mitoses in the endothelial cells around the blastocyst in the mouse uterus. Senger et al. (1967) suggest that the level of blood supply may be an important factor in embryonic survival. Boving (1954) considers that the rabbit trophoblast penetrates toward a blood vessel, although it is equally possible that the blood vessels grow toward the invading trophoblast. The increased vascular permeability is followed rapidly by the development of edema in the stroma underlying the blastocyst. Presumably the increased osmotic pressure in the intercellular spaces, due to the passage of plasma proteins through the permeable blood vessels, attracts water into the tissue spaces. The functional significance of the edematous tissue is unclear, although since the edema occurs immediatf'ly preceding decidualization one assumes that it is related in some way to the increased metabolic needs of the tissue at that time. The permeability changes in the blood vessels and consequent edema of the stroma appear very soon after the triggering of the implantation reaction by the blastocyst. Whether they occur before or after the attachment of the trophoblast is difficult to decide. In investigations using the electron microscope, the pontamine blue reaction is normally employed to locate the blastocysts during early implantation, and it is extremely difficult to find blastocysts before this stage. In my experience, most blastocysts located by pontamine blue have undergone the attachment reaction. However, occasionally one finds blastocysts that have not attached (Fig. 14), suggesting that the blueing reaction and attachment occur at about the same time. Obviously the blood vessels must receive some message, via the epithelial cells, indicating impending attachment. This message is not blocked by actinomycin D; in fact, after the administration of the drug, the uterus is maintained in the edema stage and fluid may continue to accumulate so that an excessively edematous stroma results (Fig. 15). Another early change in the stroma, which rapidly follows the above changes, is the induction of the enzyme alkaline phosphatase (Finn and Hinchliffe, 1964). Until the start of the implantation reaction, very little of this enzyme can be found in the stroma, the little there is being associated with the glands and blood vessels. A few hours after the first appearance of the pontamine sky blue reaction, a crescent-shaped area in which the enzyme is present can be detected in the stroma near the implanting blastocyst (Fig. 16) (Finn and McLaren, 1967). In the human uterus, alkaline phosphatase is found associated with the predecidual cells that form in the late luteal stage of the cycle (Wilson, 1969). Again, unfortunately, the functional significance of the sudden appearance of the enzyme at this time is not known. It has been detected in other tissues just before differentitation (Moog, 1944), and it is reasonable to assume that it is related in some way to decidual differentiation.

4.5.2.

Epithelial Changes

It is in the reaction of the trophoblast with the epithelium that the differences between the various methods of implantation are most obviously seen. In intrusive, or interstitial, implantation, as occurs in the guinea pig (von Spee, 1893; Samson and Hill, 1931), chimpanzee (Heuser, 1940), and man (Heuser and Streeter, 1941), the epithelial cells appear to separate, thus making a passage for the blastocyst (Blandau, 1949; Enders and Schlafke, 1972). The factor responsible for the breakdown of the junctional complexes at the apices of the epithelial cells is not known, and there is some doubt about the extent to which cell death is involved in allowing passage of the blastocyst. In the guinea pig, it is rare, in material prepared for electron microscopy, to find blastocysts on the point of passing through the epithelium (Schlafke and Enders, 1975) and so far as I am aware this stage has not been seen in the human uterus. Consequently, very little is known of the forces initiating the passage beyond the suggestion that lysosomes from the blastocyst may be involved (Owers, 1971). To attribute it to "invasiveness of the trophoblast" says little about the physiological mechanisms involved beyond

Figure 14. Section through the junction hetween trophoblast and uterine epithelium be±ore attachment of blastocyst (Courtesy of Mrs. Jennifer Downie) .

283 THE IMPLANTATION REACTION

284 CHAPTER 9

. ... ~.: .r '. " • ' . 1" ' : ,.' , ..,

j

.~ .

.

..

' ~'

:,

i

Figure 15. Cross-section through the uterus of a mouse showing excessively edematous stroma resulting from blockage of the artificial oil-induced DCR by actinomycin D.

implying that the endometrium is passive. At the present state of knowledge, even this implication is unwarranted. The eccentric, or displacement, method of implantation occurs in rats and mice and has been more widely studied. The characteristic feature of this type of implantation is that the uterine epithelial cells surrounding the blastocyst die and are, at least partly, phagocytosed by the trophoblast (Fig. 17). There has been some

debate about whether the death of the epithelial cells is caused by the "invading" trophoblast or whether their destruction is controlled from within the cells by "programmed cell death" as an inherent part of the implantation reaction. Two lines of evidence favor the latter. As mentioned earlier, the injection of oil into the uterus initiates the implantation reaction. Histologically, the endometrial response to oil resembles very closely the implantation reaction, including breakdown of the epithelium on the antimesometrial side of the uterus (Finn and Hinchliffe, 1965). The fact that death occurs selectively in the same cells as would be involved were a blastocyst present and at approximately the same time after the application of the nidatory stimulus suggests that these cells are programmed to degenerate given the appropriate stimulus. It is unlikely that the oil itself destroys the cells because instillation into an insensitive uterus does not lead to cell degeneration. Further evidence favoring controlled cell death comes from experiments using actinomycin D (Finn and Bredl, 1973). Although the administration of this drug does not affect blastocyst activation or attachment or stromal edema, it stops the degeneration of the uterine epithelial cells. This has the rather interesting result that the blastocyst continues to grow but is surrounded by a mass of epithelial cells and the trophoblastic cells cannot therefore approach the maternal

Figure 16. Cross-section of mouse uterus soon after attachment of the blastocyst. showing the crescent of alkaline phosphatase in the stroma.

285 THE IMPLANTATION REACTION

286 CHAPTER 9

blood vessels. At points, however, the trophoblastic cells can sometimes be seen penetrating between the epithelial cells (Fig. 18). It is suggested (Finn and Bredl, 1973) that the evolutionary advantage of allowing the embryo to develop within the wall of the uterus, and thus closer to the maternal blood vessels, has led to the evolution of two mechanisms to achieve it; one causes the trophoblastic cells to penetrate through the epithelium, as seen, for example, in the guinea pig, and the other promotes the breakdown of the uterine epithelial cells. In normal implantation in mice, the latter is predominant, but after

Figure 17. Embryo of a mouse after nidation into the decidualized stroma. The uterine epithelium has disappeared and trophoblastic giant cells can be seen "invading" the stroma. Courtesy of Mr. John Bred!.

287 THE IMPLANTATION REACTION

Figure 18. Section through the uterus of a mouse in which the death of the uterine epithelial cells has been blocked by actinomycin D. From Finn and Bredl (1973) by permission of the editor, J. Reprod. F ertil.

actinomycin D, when programmed cell death is prevented, the trophoblast shows some ability to penetrate, albeit not very effectively. The breakdown of the epithelial cells has been studied in detail by Hinchliffe and El-Shershaby (1975), who have similarly concluded that it is a result of autolytic breakdown. The trophoblastic cells, especially after differentiation into giant cells, phagocytose the dead epithelial cells (Jollie, 1962; Mulnard, 1967; Finn and Lawn, 1968) and presumably utilize the ingested material (Galassi, 1967). These remarkable

288 CHAPTER 9

cells exceed even the decidual cells (see later) with a DNA content up to 2048 times that of the haploid (Nagl, 1972). The uptake of isolated dead epithelial cells can be seen very early in the implantation reaction, before the general breakdown of the epithelium (Fig. 19) (Finn and Lawn, 1968). With the light microscope the cells appear as darkly staining bodies. When originally seen, they were interpreted as cells passing from the blastocyst into the maternal tissue (Wilson, 1963b). They were subsequently designated "W bodies" and shown to appear soon after the permeability changes in the blood vessels, at about the same time as the appearance of stromal edema (Finn and McLaren, 1967). Recognition that they were dead epithelial cells in the process of phagocytosis by the trophoblast was not possible until they were visualized with the electron microscope, when tongues of trophoblast could be seen encircling a condensed dead cell (Finn and Lawn, 1968). Wholesale breakdown of the epithelium does not take place until considerably after the onset of the implantation reaction, by which time decidual transformation is well under way. Thus the trophoblast does not come into contact with the undifferentiated stromal fibroblasts. This is thought to be an important factor in restraining trophoblastic penetration. The uterine epithelial cells initially present a barrier to the trophoblast that is later replaced by the decidua (see later). The importance of the epithelium in restraining the blastocyst was demonstrated by Cowell (1969), who transferred blastocysts to the uteri of cycling mice and showed that the trophoblast would penetrate into the stroma if the epithelium was damaged. After the removal of the epithelium, progress of the trophoblast appears to be halted for a short time at the basement membrane (Schlafke and Enders, 1975) until it also disappears. Subsequently, the uterine epithelium remaining mesome-

Figure 19. Electron micrograph of a dead uterine epithelial cell in the process of being engulfed by the trophoblast. From Finn and Lawn (1968), by permission of the editor,]. Reprod. Fertil.

trially to the blastocyst rejoins, so that the embryo is completely within the wall of the uterus with the uterine lumen above it. A most unexpected method of epithelial penetration occurs in the rabbit (Larsen, 1961; Enders and Schlafke, 1971; Steer, 1970, 1971). This starts with fusion of the trophoblast to the uterine epithelium. The trophoblastic cells form a syncytium, and initially pegs of tissue pass through the blastocystic coverings and fuse with the epithelial cells to form a single cell. At first, the line of fusion can be recognized by the presence of vesicles; later, the maternal nuclei dis·appear and the cell is "taken over" by the fetal component. The problems in cell biology and immunology presented by this process are fascinating and completely unsolved. In some animals the uterine epithelial cells proliferate at the site of implantation to form a plaque, as in the rhesus monkey (Wislocki and Streeter, 1938), or syncytial masses, as in ferrets and other carnivores (see Amoroso, 1952). Both of these can be induced by traumatization (Rossman, 1940) or injection of oil into the uterus at the appropriate time (Marston et at., 1971; Beck, 1974). The cells later become necrotic, as in normal pregnancy, indicating that the epithelial response is an intrinsic part of the maternal pregnancy reaction and requires only the trophoblast, or an artificial stimulus, for its initiation. 4.5.3.

Changes in the Stroma

Stromal changes in preparation for nidation occur coincidentally with the epithelial changes, and the early vascular responses have already been described. The appearance of alkaline phosphatase is the first sign of decidual transformation, and this enzyme is a useful marker of the extent of decidualization (Finn and Hinchliffe, 1964). While the inner cells are undergoing decidual transformation, the stromal cells peripheral to them are dividing. This is best shown after colchicine treatment (Allen, 1942; Finn and Martin, 1967). This increased rate of mitosis is restricted to the implantation site and is part of the implantation reaction, distinct from the hormonally induced mitosis discussed earlier. Much has been written about decidual cells since they were first described in the last century. In his review of anatomy over a century ago, Turner (1873) states that "the epithelium is shed from areas to which the ova are attached, and a rich new formation of cells takes place, by which the ova are embraced." In the first edition of this book, 100 pages were devoted to decidualization (De Feo, 1967). Cellular transformation of this complexity must be rare in adult mammals. The only similar case I can bring to mind is the differentiation of the corpus luteum from granulosa cells in the ovary (a conversion, incidentally, on which decidual differentiation is very much dependent). For a detailed account of the decidual reaction, the reader should consult the classic paper by Krehbiel (1937). It is customary to divide decidual cells into three basic types-antimesometrial (or primary), mesometrial, and metrial gland cells. The last are not strictly decidual cells, for they are not shed, although they are part of the implantation reaction. Primary decidual cells form initially in the crescent-shaped area surrounding the blastocyst, shown first by the presence of alkaline phosphatase (Fig. 16). They differentiate from stromal fibroblasts (Galassi, 1968). When fully formed they are large cells, usually containing two or more nuclei, each with several nucleoli. The

289 THE IMPLANTATION REACTION

290 CHAPTER 9

nuclei are polyploid (Sachs and Shelesnyak, 1955; Dupont et aI., 1971), with DNA increasing up to 64 times the haploid content (Zybina and Grishenko, 1972; Ansell et aI., 1974). Studies of decidual cells in chimeric mice (Ansell et al., 1974) have shown that the increase in DNA does not result from cell fusion, but is probably a result of endoreduplication, and the multinucleate cells arise from mitoses without subsequent cell division. The uptake of tritiated thymidine is high in decidual cells in the first half of pregnancy in the rat (Bulmer and Peel, 1974), although mitoses are rarely seen even after colchicine treatment (Hall, 1969; Finn and Martin, 1967). Apart from their large nuclei the cells have abundant basophilic cytoplasm with well-developed inclusions. With the electron microscope Qollie and Bencosme, 1965; Wynn, 1965; Stegner et al., 1971), an abundance of smooth endoplasmic reticulum, well-developed Golgi complexes, and many mitochondria and lysosomes are seen. The endoplasmic reticulum is dilated and contains a flocculent material. A few profiles of rough endoplasmic reticulum are occasionally seen. Jollie and Bencosme (1965) make the interesting observation that the stromal fibroblasts before decidualization contain either smooth or rough endoplasmic reticulum, and the cells in the future primary decidual area are predominantly of the former type. In view of the finding that smooth endoplasmic reticulum predominates in the mature decidual cell, they suggest that the stromal cells are already partly differentiated into future decidual cells before the application of the decidual stimulus. With the light microscope, the decidual cells are seen to form a solid mass of cells around the implanting embryo, and they have been described as "epithelioid." This description is confirmed by electron microscopic studies, in which the individual cells are seen to be joined by gap junctions (Fig. 20) (Finn and Lawn, 1967). These are absent from undifferentiated stromal tissues but are present in the predecidual cells around the arterioles of the human uterus during the late luteal phase of the menstrual cycle (Lawn et aI., 1971). In mice the decidual cells are tightly packed together, and fingerlike processes from the cell frequently indent a neighboring cell with a cup-shaped gap junction linking the two cells (Finn and Lawn, 1967).

Figure 20. Electron micrograph of a gap junction between two decidual cells in a mouse uterus. Courtesy of Dr. Alan Lawn.

The first-formed decidual cells around the implantation chamber contain granules of glycogen. They are not so prominent in the antimesometrial cells farther away from the chamber but are a prominent feature of the mesometrial decidual cells. In cleared specimens of uteri the glycogen renders the decidual areas opaque (Foster et at., 1963). The mesometrial decidual cells appear 1 or 2 days after the antimesometrial ones and are considerably smaller, usually with only one nucleus. These cells form the decidua basalis of the placenta, while the antimesometrial cells give rise to the decidua capsularis. By the time the mesometrial cells have differentiated, the blood vessels of the decidua have grown considerably. When fully formed they are large sinusoids lined by prominent large endothelial cells in very close association with the decidual cells. A particularly interesting feature is the absence of a basement membrane in the endothelial cells (Hall, 1968, 1969). Associated with this is the disappearance of the enzyme adenosine triphosphatase (Hall, 1968). In the late decidua these sinuosids open into the lumen so that the maternal blood flows in channels formed by the trophoblastic giant cells. Around the outside of the decidua a fibrinoid capsule forms consisting of small condensed cells with a fibrinoid material (Bulmer and Dickson, 1961), and outside of this structure a few undifferentiated stromal cells remain. With the growth of the decidua the uterine glands retreat to the periphery and are eventually completely lost in the implantation site. The so-called metrial gland is not a discrete gland in the accepted sense of the word. It is a collection of cells with characteristic histological features situated mostly around the blood vessels of the mesometrial triangle. The triangle is bound on one side by the inner circular muscle layer and on the other two by the reflection of the longitudinal muscle layer into the mesometrium (Selye and McKeown, 1935). Although there have been suggestions that the cells secrete hormones, the evidence is not yet complete enough to be certain that the cells are glandular. The structure has been demonstrated in the rat (Selye and McKeown, 1935; Dixon and Bulmer, 1971), mouse (Smith, 1966), and guinea pig and rabbit (Asplund et at., 1940), and cells with a similar appearance are found in some primates including humans (Hamperl and Hellweg, 1958), although they are distributed throughout the stroma and not confined as in the metrial gland. In rodents the cells are found in pregnancy or after the induction of deciduomata in pseudopregnancy. They appear after decidual transformation is fairly well advanced (day 8 or 9 of pregnancy in the rat) and persist well into lactation (Baker, 1948). Their development follows three overlapping stages, so that three cell types can be found (Baker, 1948): (1) basophilic cells, (2) cells containing eosinophilic granules and glycogen, and (3) cells containing lipid. The second type probably represents the fully differentiated cell, the basophilia of the first type representing increased ribosomal activity preceding granule formation. The lipid-containing cells are prominent during lactation and may represent fatty degeneration of the cells (Baker, 1948). With the electron microscope the granules of the second type of cell appear as electron-dense spherical structures (Wislocki et at., 1957; Dixon and Bulmer, 1971), located peripherally to a large Colgi apparatus (Larkin and Cardell, 1971). These cells reach a peak of development on days 13-15 of pregnancy in the rat

291 THE IMPLANTATION REACTION

292 CHAPTER 9

and are found mostly as cuffs around the blood vessels of the mesometrial attachment. The granules react positively for esterase (Bulmer, 1965), exhibit metachromasia with toluidine blue, and stain intensely with the PAS technique, indicating the presence of glycoprotein (Peach and Bulmer, 1965). The similar granular cells that appear in the stroma of the primate uterus appear to develop, at least initially, in response to hormonal stimulation (estrogen and progesterone, Cardell et al., 1969) and are found during the luteal phase of the menstrual cycle (Dallenbach-Hellweg, 1967). In this respect they differ from those in rodents, in which an implantation stimulus is necessary. They have been given various names: "Kornchenzellen" or .oK cells" (Hamperl, 1955; Hamperl and Hellweg, 1958), "amphophils" (von Numers, 1953), "Schollen leukocytes" (Weill, 192i), and "granulocytes" (Dallenbach-Hellweg, 1967). There is a suggestion from experiments using immunohistological and other techniques that the metrial gland cells in rodents (Wislocki et al., 1957; DallenbachHellweg et al., 1965) and the granulated cells in primates (Dallenbach-Hellweg et al., 1966) secrete relaxin.

5.

Regression if the Decidua

In rodents, toward the middle of pregnancy, the antimesometrial decidua regresses as the implantation chamber is enlarged to accommodate the growing embryo. Eventually it forms the thin rim of decidua capsularis. The mesometrial decidua continues until later in pregnancy as the decidua basalis. This too does not last the full length of gestation. The life span of the artificially stimulated decidua in rodents is also of welldefined duration. It is, of course, dependent on a continuous supply of progesterone, and if this is withheld the decidua will quickly regress. However, even if the progesterone supply is maintained the life of the decidua is finite. Characteristic changes take place in the uterus during regression to bring about removal of the decidual cells and reestablishment of the normal endometrial structure (Warbrick, 1954; Lobeletal., 1965; Hall, 1969). Human decidual cells are shed with the placenta at the end of pregnancy. It will be recalled from an earlier section that, unlike rodent decidual cells, they are formed initially during the luteal phase of the cycle in response to hormonal stimulation. They are also very occasionally found in the endometrium of the newborn (Ober and Bernstein, 1955). If ovarian hormones or their synthetic equivalents are administered to women for long periods, decidualizion of the stroma continues until virtually all the stroma is decidualized. Regression of the decidual tissue does not occur while hormonal treatment continues. This hormone-induced decidualization thus differs from the blastocyst-induced decidualization in rodents. In normal human pregnancy, blastocyst-induced decidualization probably augments that initiated by the hormones of the cycle. Whether the cells then have a finite life span in the presence of hormones is not known.

6.

Significance

0/ the Decidua

293 THE IMPLANTATION REACTION

The encircling of the embryo by a mass of polyploid, binucleate glycogencontaining cells intimately joined by gap junctions presumably has some functional significance. Many biologists encountering the tissue must have pondered the evolutionary advantage to the animal of the decidua, and several hypotheses have been put forward (Mossman, 1937; Amoroso, 1952; Huggett and Hammond, 1952; McLaren, 1965; De Feo, 1967; Finn, 1971). The first fact that must be taken into account is the extent to which the cells are found among the various species of mammals. Decidualization does not occur in all animals (see Mossman, 1937), and the development of a functional placenta is not dependent on it. A clue to the significance of decidual transformation might emerge if its presence could be correlated with any other facet of the implantation response in those species in which it occurs. This is difficult because detailed information about implantation is available for only a few of the 6000 or more species of mammals. From the information available, it appears that decidualization is most common in those animals in which the blastocyst establishes itself within the stroma. In them, the uterine epithelium no longer intervenes between the trophoblast and the maternal blood vessels. It should be noted, however,that decidualization starts while the epithelium is still intact; indeed, in women it commences in the absence of a blastocyst. Thus, although decidual transformation may have evolved because it conferred some advantage after removal of the epithelium, it does not generally occur in response to the presence of the blastocyst within the stroma. The next step is to inquire whether there is any property acquired by the decidual cells that was previously presented by the uterine epithelium. One possibility is the development of junctional complexes (gap junctions, Fig. 20). Epithelial cells are usually joined by junctions, albeit of a complex nature, at their apices. Could this be an essential property of cells in contact with the trophoblast? An observation that has already been referred to is the invasiveness of the trophoblast. This appears to be due to the ability of the trophoblastic cells to pass between cells and encircle them. Within the uterus this is controlled so that the invasion is limited; however, if blastocysts are transferred to sites outside the uterus, invasion is extensive (under the kidney capsule, Fawcett, 1950; Kirby, 1960; Porter, 1967; in the spleen, Kirby, 1963b; in the testis, Kirby, 1963a; Porter, 1967, in the anterior chamber of the eye, Fawcett et al., 1947). The giant cells of the trophoblast rapidly infiltrate among the cells of the host tissue, causing widespread damage. Blastocysts transferred to uteri of mice during the estrus cycle, however, do not invade unless the epithelium is damaged (Cowell, 1969). This suggests that the intact uterine epithelium restrains trophoblast invasion, possibly because the junctional complexes at the cell apices prevent penetration. It is tempting to think that the joining of the newly formed decidual cells in preparation for the encroachment of the blastocyst into the stroma might be related to limiting and controlling the penetration of the trophoblast. As long

294 CHAPTER 9

ago as 1876, Turner suggested a protective function for the decidua, and it was later likened to scar tissue (Huggett and Hammond, 1952). This rather simple explanation is almost certainly not the whole story. From what is known of gap junctions in other tissues, they do not seem to have evolved primarily as a defense mechanism. Their main function appears to be as a means of direct communication between cells (Kanno and Loewenstein, 1966). They are involved in the rapid propagation of impulses in excitable tissue (Dewey and Barr, 1962; Revel and Karnovsky, 1967), and from their occurrence at certain stages of development in embryos it is likely that they are involved in integrating control of embryonic differentiation (Trelsad et al., 1966). Their formation in the uterine stroma presumably converts a collection of isolated motile cells, floating in tissue fluid, into a solid, sessile mass of intercommunicating cells in which integrated and controlled activity is possible. The presence of large quantities of glycogen and fat in decidual cells suggests that the decidua may also have a nutritive function. Following activation and attachment, the embryo grows rapidly and a readily available source of carbohydrate might be advantageous, especially in the period before the chorioallantoic placenta has formed. The blood vessels in the decidua are very well developed and their endothelial walls are in very close contact with the decidua. Substances synthesized in decidual cells might pass into the sinusoids and be taken directly to the embryo. From their histological and electron microscopic appearance, the decidual cells appear to be actively synthesizing something, and it would not be surprising if this were for the benefit of the embryo. One important characteristic of decidual cells in many species, for which it is difficult to surmise a functional basis, is the presence of two or more nuclei, which are usually polyploid. To speculate on the significance of this at present would be futile.

7.

Concluding Remarks

It will be apparent from this chapter that there are many aspects of the control of the implantation process about which very little is known. The endocrine involvement of the ovary in the preparation of the endometrium for implantation is reasonably well established. Once the uterine reaction has been triggered, however, hormones no longer seem to playa directive role. Progesterone must be present in sufficient quantity for the reaction to proceed, but this a permissive rather than a directive role. The mechanisms controlling the series of changes in the endometrium following the triggering of the implantation reaction must presumably be directed by intracellular control mechanisms (Finn and Porter, 1975). Investigation of these mechanisms is only just beginning, at present using relatively crude techniques, such as the administration of drugs affecting cell processes. In writing this chapter, I am particularly conscious of the number of areas in which information is extremely scanty. It is to be hoped that improvements in knowledge and techniques in developmental and cell biology will provide

better means of studying the cellular mechanisms involved reaction.

8.

III

the implantation

Riferences

Aitken, R. j., 1974, Delayed implantation in the roe deer (Capreolus capreolus), I Reprod. Fertil. 39:225-236. Alden, R. H., 1947, Implantation of the rat egg. 2. Alteration in osmophilic epithelial lipids of the rat uterus under normal and experimental conditions, Anal. Rec. 97:1-13. Allen, E., 1922, The oestrous cycle in the mouse, Am. I Anal. 30:297-348. Allen, E., 1942, The rates of growth in genital tissue and the hormonal regulation involved, Growth 6:73-82. Allen, E., Smith, G. M., and Gardner, W. U., 1937, Accentuation of the growth effect of theelin on genital tissues of the ovariectomized mouse by arrest of mitosis with colchicine, Am. I Anat. 61:321-341. Allen, W. M., 1931, Cyclical alterations of the endometrium of the rat during the normal cycle, pseudopregnancy and pregnancy, Anat. Rec. 48:65-84. Alloiteau, j. j., 1961, Hypophysectomy at the beginning of gestation and implantation of the ovum in the rat, C. R. Acad. Sci. Paris 253: 1348-1350. Amoroso, E. C., 1952, Placentation, in: Marshall's Physiology of Reproduction, Vol. 2 (A. S. Parkes, ed.), pp. 127-311, Longmans, Green, London. Andary, T. J., Dabich, D., and Van Winkle, L. j., 1972, Changes in proteinase activity in the early vs late mouse blastocysts,I Cell Bioi. 55:3a. Ansell, j. D., Barlow, P. W., and McLaren, A., 1974, Binucleate and polyploid cells in the decidua of the mouse,I Embryol. expo Morphol. 31:223-227. Asplund, j., Borell, V., and Holmgren, H., 1940, In der Uteruswand graviditatauftretende metachromatische granulierete Zellverbande und ihr SteHung zur "Glandula myometralis," A. Z. Mikrosk. Anat. Forsch. (Leipz.) 48:478-528. Astwood, E. B., 1941, The regulation of C. L. function by hypophysial luteotrophin, Endocrinology 28:309-320. Atkinson, W. B., and Engle, E. T., 1947, Studies on the endometrial alkaline phosphatase during human menstrual cycle and in hormone treated monkeys, Endocrinology 40:327-333. Bacsich, P., and Wyburn, G. M., 1940, Cyclic variations in the vascular architecture of the uterus of the guinea pig, Trans. R. Soc. Edinburgh 60:79-86. Baker, B. L., 1948, Histochemical variations in the metrial gland of the rat during pregnancy and lactation, Proc. Soc. expo Bioi. Med. 68:492-496. Banik, U. K., and Ketchel, M. M., 1964, Inability of histamine to induce deciduomata in pregnant and pseudopregnant rats,I Reprod. Fertil. 7:259-261. Baranczuk, R., and Greenwald, G. S., 1973, Peripheral levels of estrogen in the cyclic hamster, Endocrinology 92:805-812. Barraclough, C. A., and Sawyer, C. H., 1959, Induction of pseudopregnancy in the rat by reserpine and chlorpromazine, Endocrinology 65:563-571. Beato, M., and Dienstbach, F., 1968, Effects of estrogens and gestagens on the duration of DNA synthesis in the genital tract of ovariectomized mice, Virchows Arc. Abt. B: Zellpathol. 1: 197-200. Beato, M., Lederer, B., Boqui, E., and Sandritter, W., 1968, Effect of estrogens and gestagens on the initiation of DNA synthesis in the genital tract of ovariectomized mice, Exp. Cell Res. 52: 173-179. Beck, F., 1974, The development of a maternal pregnancy reaction in the ferret, I Reprod. Fertit. 40:61-69. Beier, H. M., 1968, Uteroglobin: A hormone sensitive endometrial protein involved in blastocyst development, Biochim. Biophys. Acta 160:289-291. Bell, W. Blair, and Hick, P., 1909, Observations on the physiology of the female genital organs. 3. The correlation of the uterus and ovaries, Br. Med. I, pp. 655-658.

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Bensley, C. M., 1951, Cyclic fluctuations in the rate of epithelial mitosis in the endometrium of the rhesus monkey, Contrib. Embryol. 34:87-98. Bergstrom,S., and Nilsson, 0., 1975, Embryo-endometrial relationship in the mouse during activation of the blastocyst by oestradiol, I Reprod. F ertil. 44: 117-120. Biggers, j. D., Finn, C. A., and McLaren, A. 1962, Long term reproductive performance of female mice. l. Effect of removing one ovary,I Reprod. Fertil. 3:303-312. Bindon, B. M., 1969a, Blood flow in the reproductive organs of the mouse after hypophysectomy, after gonadotrophin treatment, during the oestrous cycle and during early pregnancy, I Endocrinol. 44:523-536. Bindon, B. M., 1969b, The role of the pituitary gland in implantation: Inhibition of implantation by hypophysectomy and neurodepressive drugs,I Endocrinol. 4~:225-235. Bjorkman, N., 1972, An Atlas of Placental Fine Structure, Bailliere, Tindall, and Cassell, London. Blandau, R. j., 1949, Observations on implantation of the guinea pig ovum, Anat. Rec. 103: 19-47. Bloch,S., 1943, Uber die Wirkung der Milchdriisensekretion auf die Nidation bei der Maus, Schweiz. Med. Wochenschr. 73:245-262. Boshier, D. P., 1970, The pontamine sky blue reaction in pregnant sheep uteri, I Reprod. Fertii. 22:595-596. Bouda, j., 1969, Arterioles and capillaries of human decidua during the first trimester of pregnancy,Obstet. Gynecol. 33: 182-188. Bouin, P., and Ancel, P., 1910, Recherches sur les fonctions du corps jaune gestatif. l. Sur Ie determinisme de la preparation de l'uterus a la fixation de l'oeuf,I Physiol. Pathoi. Gen. 12: 116. Boving, B., 1954, Blastocyst-uterine relationships, Cold Spring Harbor Symp. Quant. Bioi. 19:9-25. Brambell, F. W. R., 1937, The influence of lactation on the implantation of the mammalian embryo, Am.]. Obstet. Gynecoi. 33:942-953. Brinsfield, T. H., and Hawk, H. W., 1974, Ultrastructure of sheep endometrial stromal cells after ovariectomy and hormone treatment, BioI. Reprad. 10:98-102. Brouha, L., 1928, Production of placentomata in rats injected with anterior hypophysial fluid, Proc. Soc. expo bioi. Med. 25:488-489. Buchanan, G. D., Enders, A. C., and Talmage, R. V., 1956, Implantation in armodillos ovariectomized during the period of delayed implantation,]. Endocrinoi. 14:121-128. Bullock, D. W., and Bhatt, B. M., 1973, Oestrogen binding to rabbit blastocysts and its possible role in implantations,I Reprad. Fertil. 35:614-615. Bulmer, D., 1965, Esterase and acid phosphatase activities in the rat placenta,]. Anat. 99:513-525. Bulmer, D., and Dickson, A. D., 1961, The fibrinoid capsule of the rat placenta and the disappearance of the decidua, I Anat. 95:300-310. Bulmer, D., and Peel,S., 1974, An autoradiographic study of cellular proliferation in the uterus and placenta of the pregnant rat, I Anat. 117:433-441. Burrows, H., 1949, Biological Actions of Sex Hormones, Cambridge University Press, Cambridge. Canivenc, R., and Bonnin-Laffargue, M., 1963, Inventory of problems raised by the delayed implantation in the European badger (Meles meles L.), in: Delayed Implantation (A. C. Enders, ed.), pp. 115-129, Chicago University Press, Chicago. Canivenc, R., and Laffargue, M., 1957, Survival of rat blastocysts in the absence of ovarian hormones, C. R. Acad. Sci. Paris 245: 1752-1754. Canivenc, R., Laffargue, M., and Mayer, G., 1956, Nidations retardees chez la ratte cas tree et injectee de progesterone: Influence du moment de la castration sur la chronologie de l'ovoimplantation, C. R. Soc. Bioi. 150:2208-2212. Cardell, R. R., Hisaw, F. L., and Dawson, A. B., 1969, The fine structure of granular cells in the uterine endometrium of the rhesus monkey (Macaca muiatta) with a discussion of the possible function of these cells in relaxin secretion, Am. I Anat. 124:307-340. Carter, j., and McLaren, A., 1975, The effect of oestrogen and progesterone on the incorporation of tritiated thymidine in mouse uteri in vitro,I Reprad. Fertii. 42:439-445. Challis, j. R. G., Heap, R. B., and Illingworth, D. V., 1971, Concentrations of oestrogen and progesterone in the plasma of non-pregnant, pregnant and lactating guinea pigs,]. Endocrinol. 51:333~345.

Chambon, Y., 1949, Besoins endocriniens qualitatifs et quantitatifs de l'ovoimplantation chez Ia lapine, C. R. Soc. Bioi. Paris 143:1172-1175.

Chambon, Y" 1950, Essais de realisation du deciduome par traumatisme apres la date de l'ovoimplantation chez la lapine, C. R. Soc. Bioi. Paris 144:258-260. Chambon, Y., 1957, Influence de la chlorpromazine sur la function luteotrophe de I'hypophyse dans la realization du deciduome et de I'ovo-implantation, Bull. Acad. Med. 141:535-539. Chambon, Y., 1960, Phenothiazines, ovoimplantation et decidualization, Bull. Soc. R. Beige Cynecol. Obstet. 30:573-584. Chaudhury, R. R., and Sethi, A., 1970, Effects of an intra-uterine contraceptive device on mitosis in the rat uterus on different days of pregnancy,]. Reprod. Fertil. 22:33-40. Clark, B. F., 1971, The effects of oestrogen and progesterone on uterine cell division and epithelial morphology in spayed, adrenalectomized rats,]. Endocrinol. 50:527-528. Clark, B. F., 1974, Effect of oestrogen priming on the uptake, metabolism and biological activity of progestins in the mouse uterus,]. Endocrinol. 63:343-349. Clark, M. J., 1968, Termination of embryonic diapause in the red kangaroo Megaleia rufa, by injection of progesterone or oestrogen,]. Reprod. Fertil. 15:347-355. Clauberg, 0., 1930, Zur Physiologie und Pathologie der Sexualhormone im besonderen des Hormons des Corpus-luteum, Zentralbl. Cynaekol. 54:2757-2770. Clemetson, C. A. B., Mallikarjuneswara, V. R., Moshfeghi, M. M., Carr, J. J., and Wilds, J. H., 1970, The effects of oestrogen and progesterone on the sodium and potassium concentration of rat uterine fluids,]. Endocrinol. 47:309-319. Cochrane, R. L., and Meyer, R. K., 1957, Delayed nidation in the rat induced by progesterone, Proc. Soc. Exp. BioI. 96: 155-159. Cochrane, R. L., and Shackleford, R. M., 1962, Effect of exogenous estrogen alone and in combination with progesterone in the intact mink,]. Endocrinol. 25:101-106. Corner, G. W., 1928, Physiology of the corpus luteum: The effect of very early ablation of the corpus luteum upon embryos and uterus, Am.]. Physiol. 86:74-81. Corner, G. W., and Warren, S. L., 1919, Influence of the ovaries upon the production of artificial deciduomata, confirmatory studies, Anat. Rec. 16: 168-169. Cowell, T. P., 1969, Implantation and development of the mouse eggs transferred to the uteri of non-progestational mice,]. Reprod. Fertil. 19:239-245. Cox, R. I., Mattner, P. E., and Thorburn, G. D., 1971, Changes in ovarian secretion of oestradiol17f3 around oestrus in sheep,]. Endocrinol. 49:345-346. Curtis, A. S. G., 1967, The Cell Surface: Its Molecular Role in Morphogenesis, Logos Press, London. Cutuly, E., 1941, Implantation following mating in hypophysectomized rats injected with lactogenic hormone, Proc. Soc. expo Bioi. 48:315-318. Dabich, D., and Andary, T. J., 1974, Prevention of blastocyst implantation in mice with protease inhibitors, Fertil. Steril. 25:954-957. Dallenbach-Hellweg, G., 1967, Endometrial granulocytes and implantation, Excerpta Med Int. Congr. Ser. 133:411-418. Dallenbach-Hellweg, G., Battista, J. V., and Dallenbach, F. D., 1965, Immunohistological and histochemical localization of relaxin in the gland of the pregnant rat, Am.]. Anat. 117:433-450. Dallenbach-Hellweg, G., Dawson, A. B., and Hisaw, F. L., 1966, The effect of relaxin on the endometrium of monkeys, Am.]. Anat. 199:61-78. Daniel, J. C., 1968, Comparison and electrophoretic patterns of uterine fluids from rabbits and animals having delayed implantation, Compo Biochem; Physiol. 24:297-300. Daniel, J. C., 1970, Coincidence of embryonic growth and uterine protein in the ferret,]. Embryol. expo Morphol. 24:305-312. Das, R. M., 1972, The effects of oestrogen on the cell cycle in epithelial and connective tissues of the mouse uterus,]. Endocrinol. 55:21-30. De Feo, V. J., 1963a, Temporal aspects of uterine sensitivity in the pseudopregnant or pregnant rat, Endocrinology 72:305-316. De Feo, V. J., 1963b, Determination of the sensitive period in the rat by different inducing procedures, Endocrinology 73:488-497. De Feo, V. J., 1967, Decidualization, in: Cellular Biology olthe Uterus (R. M. Wynn, ed.), pp. 191290, Appleton-Century-Crofts, New York. Denker, H. W., 1972, Blastocyst protease and implantation: Effect of ovariectomy and progesterone substitution in the rabbit, Acta Endocrinol. 70:591-602.

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12:5-13.

Nilsson, 0., 1972, Ultrastructure of the process of secretion in the rat uterine epithelium at implantation, I Ultrastruct. Res. 40:572-580. Nilsson, 0., 1974, Changes of the luminal surface of the rat uterus at blastocyst implantation: Scanning electron microscopy and ruthenium red staining, Z. Anat. Entwicklungsgesch. 144:337-

342. Nilsson, 0., Lindqvist, 1., and Ronquist, G., 1973, Decreased surface charge of mouse blastocyst at implantation, Exp. Cell Res. 83:421-423. Noyes, R. W., Hertig, A. T., and Rock, J., 1950, Dating the endometrial biopsy, Fertil. Steril. 1:3-25. Ober, W. B., and Bernstein, J., 1955, Observations on the endometrium and ovary of the newborn, Pediatrics 16:445-460. Ober, W. B., Clyman, M. J., Decker, A., and Roland, M., 1964, Endometrial effects of synthetic progestagens, Int. I Fertil. 9:597-608. Orsini, M. W., 1963a, Attempted decidualization in the hamster and rat with pyrathiazine, I Reprod. Ferti!. 5:323-330. Orsini, M. W., 1963b, Induction of deciduomata in hamsters and rats by injection of air, I Endocrinol. 28: 119-121. Orsini, M. W., and Donovan, B. T., 1971, Implantation and induced decidualization of the uterus in the guinea pig, as indicated by pontamine blue, Bioi. Reprod. 5:270-281. Orsini, M. W., and Meyer, R. K., 1959, Implantation of castrate hamsters in the absence of exogenous estrogen, Anat. Rec. 134:619-620 (abst.). Orsini, M. W., and Meyer, R. K., 1962, Effect of varying doses of progesterone on implantation in the ovariectomized hamster, Proc. Soc. Exp. BioI. Med. 110:713-715. Owers, N. 0., 1971, Ingestive properties of guinea pigs trophoblast grown in tissue culture: A possible lysosomal mechanism, in: The Biology of the Blastocyst (R. J. Blandau, ed.), pp. 225-241, University of Chicago Press, Chicago. Owers, N. 0., and Blandau, R. J., 1971, Proteolytic activity of the rat and guinea pig blastocyst in vitro, in: Biology of the Blastocyst (R. J. Blandau, ed.), pp. 207-224, University of Chicago Press, Chicago. Parkes, A. S., 1929, The functions of the corpus luteum. 2. The experimental production of placentomata in the mouse, Proc. R. Soc. London Ser. B 104: 183-188. Parkes, A. S., Dodds, E. C., and Noble, R. L., 1938, Interruption of early pregnancy by means of orally active oestrogens, Br. Med. I 2:557-559. Parr, M. B., and Parr, E. L., 1974, Uterine luminal epithelium: protrusions mediate endocytosis, not apocrine secretion, in the rat, Bioi. Reprod. 11:220-233. Peach, R., and Bulmer, D., 1965, Cytochemistry and electron microscopy of the granulated metrial gland cells of the rat placenta,I Anat. 99:415. Peckham, B. M., and Greene, R. R., 1950, Endocrine influences on implantation and deciduoma formation, Endocrinology 46:489-493.

Perrota, C. A., 1962, Initiation of cell proliferation in the vaginal and uterine epithelia of the mouse, Am. I Anat. 111: 195-204. Perry, j. S., Heap, R. B., and Amoroso, E. C., 1973, Steroid hormone production by pig blastocysts, Nature (London) 245:45-47. Pinsker, M. C., and Mintz, B., 1973, Changes in cell surface glycoproteins of mouse embryos before implantation, Proc. Natl. Acad. Sci. USA 70:1645-1648. Pinsker, M. C., Sacco, A. G., and Mintz, B., 1974, Implantation-associated proteinase in mouse uterine fluid, Dev. Bioi. 38:285-290. Pollard, R. M., 1973, A functional and morphological study of the response of mouse endometrium to ovarian hormones, Ph.D. thesis, London University. Pollard, R. M., and Finn, C. A., 1972, Ultrastructure of the uterine epithelium during the hormonal induction of sensitivity and insensitivity to a decidual stimulus in the mouse, I Endocrinol. 55:293-298. Pollard, R. M., and Finn, C. A., 1974, Influence of the trophoblast upon differentiation of the uterine epithelium during implantation in the mouse,I Endocrinol. 62:669-674. Pollard, R. M., Bredl, j. C. S., and Finn, C. A., 1973, The effect of actinomycin D on the attachment reaction of implantation in mice,I Reprod. Fertil. 33:343-345. Pollard, j. W., Finn, C. A., and Martin, L., 1976, Actinomycin D and uterine epithelial protein synthesis,I Endocrinol. 69:161-162. Porter, D. C., 1967, Observations on the development of mouse blastocysts transferred to the testis and kidney, Am. I Anat. 121:73-86. Potts, M., 1966, The attachment phase of ovoimplantations, Am. I Obstet. Gynecol. 96(8): 11221128. Potts, M., and Psychoyos, A., 1967, Evolution de l'ultrastructure des relations ovoendometriales sous l'influence chez la ratte en retard experimental de nidation, C. R. Acad. Sci. Paris 264:370-373. Prenant, L. A., 1898, De la valeur morphologique du corps jaune, son action physiologique et therapeutique, Rev. Gen. Sci. 9:646-650. Prill, H. j., and Goetz, F., 1961, Blood flow in the myometrium and endometrium of the uterus, Am. I Obstet. Gynecol. 82: 102-108. Psychoyos, A., 1960, La reaction deciduale est precedee de modifications precoces de la permeabilite capillaire de l'uterus, C. R. Seances Soc. Bioi. 154: 1384. Psychoyos, A., 1961, Nouvelles recherches sur I'ovoimplantation. C. R. Hebd. Seances Acad. Sci. Paris 252:2306-2307. Psychoyos, A., 1963a, Precisions sur l'etat de "non-receptivite" de l'uterus, C. R. Acad. Sci. Paris 257:1153-1156. Psychoyos, A., 1963b, A study on the hormonal requirements for ovum implantation in the rat by means of delayed-nidation inducing substances (chlorpromazine, trifluoperazine), I Endocrino I. 27:337-343. Psychoyos, A., 1966, Influence of oestrogen on the loss of the zona pellucida in the rat, Nature (London) 211:864. Psychoyos, A., 1974, Hormonal control of ovo-implantation, Vitam. Horm. 32:201-256. Psychoyos, A., and Bitton-Casimiri, V., 1969, Captation in vitro d'un precurseur d'acide ribonucleique (ARN) (uridine-5- 3 H) par Ie blastocyst du rat; differences entre blastocystes normaux et blastocystes en diapause, C. R. A cad. Sci. Paris 268:188-190. Psychoyos, A., and Mandon, P., 1971, Etude de la surface de l'epithelium uterin au microscope electronique a balayage. Observation chez la rate au 4 e et 5 e jour de la gestation, C. R. Acad. Sci. Paris 272:2723-2725. Rabii, j., and Kragt, G. L., 1972, Plasma levels of prolactin, FSH and LH in the pseudopregnant rat, PrOG. Soc. Exp. BioI. Med. 141:359-362. Raud, H. R., 1974, The regulation of ovum implantation in the rat by endogenous and exogenous FSH and prolactin; possible role of ovarian follicles, BioI. Reprod. 10:327-334. Reinius, S., 1967, Ultrastructure of blastocyst attachment in the mouse, Z. Zellforsch. 77:257-266. Revel, j. P., and Karnovsky, M. j., 1967, Hexagonal array of subunits in intercellular junctions of the mouse heart and liver,I Cell. Bioi. 33:c7-cl2. Reynolds, S. R. M., 1949, The Physiology of the Uterus. Hoeber, New York.

305 THE IMPLANTATION REACTION

306 CHAPTER 9

Rossman, 1., 1940, The deciduomal reaction in the rhesus monkey (Macaca mulatta), Am. I Anat. 66:277-365. Rothchild, 1., and Meyer, R. K., 1942, Studies of the pretrauma factors necessary for placentoma formation in the rat, Physiol. Zool. 15:216-223. Rothchild, 1., Meyer, R. K., and Spielman, M. A., 1940, A quantitative study of estrogenprogesterone interaction in the formation of placentomata in the castrate rat, Am. I Phsiol. 128:213-224. Sachs, 1., and Shelesnyak, M. C., 1955, The development and suppression of polyploidy in the developing and suppressed deciduoma in the rat,I Endocrinol. 12:146-151. Sadlier, R. M. F. S., 1972, Cycles and seasons, in: Germ Cells and Fertilization (C. R. Austin and R. V. Short, eds.), Cambridge University Press, Cambridge. Samson, G. S., and Hill, J. P., 1931, Observations on the structure and mode of implantation of blastocysts of cavia, Trans. Zoo I. Soc. 21:295-355. Schlafke, S., and Enders, A. C., 1975, Cellular basis of interaction between trophoblast and uterus at implantation, Bioi. Reprod. 12:41-65. Schmidt, 1. G., 1943, Proliferation in the genital tract of the normal mature guinea pig treated with colchicine, Am. I Anat. 73:59-80. Segal, S. J., Davidson, O. W., and Wada, K., 1965a, Role of RNA in the regulatory action of estrogen, Proc. Natl. A cad. Sci. USA 54:782-787. Segal, S. J., Wad a, K., and Schuchner, E., 1965b, Role of RNA in estrogen-induced nidation of blastocysts, in: Proceedings of the Forty-seventh Annual Meeting of the Endocrinological Society, p. 26. Selye, H., and McKeown, T., 1935, Studies on the physiology of the maternal placenta of the rat, Proc. R. Soc. London B 119:1-31. Senger, P. 1., Lose, E. D., and Ulberg, 1. C., 1967, Reduced blood supply to the uterus as a cause for early embryonic death in the mouse,I Exp. Zool. 165:337-343. Shaikh, A. A., 1971, Estrone and estradiol levels in the ovarian venous blood from rats during the estrous cycle and pregnancy, Bioi. Reprod. 5:297-307. Shaikh, A. A., 1972, Estrone, estradiol and 17a-hydroxyprogesterone in the ovarian venous plasma during the estrous cycle of the hamster, Endocrinology 91:1136-1140. Shaikh, A. A., and Abraham, G. E., 1969, Measurement of estrogen surge during pseudopregnancy in rats by radioimmunoassay, Bioi. Reprod. 1:378-380. Shaikh, A. A., and Harper, M. J. K., 1972, Ovarian steroid secretion in estrous, mated and HCG treated rabbits, determined by concurrent cannulation of both ovarian veins, BioI. Reprod. 7:387-397. Shelesnyak, M. C., 1931, The induction of pseudopregnancy in the rat by means of electrical stimulation, Anat. Rec. 49: 179-183. Shelesnyak, M. C., 1933a, The production of deciduomata in immature rats by pregnancy urine treatment, Am. I Physiol. 104:693-699. Shelesnyak, M. C., 1933b, The production of deciduomata in spayed immature rats after estrin and progestin treatment, Anat. Rec. 56:211-217. Shelesnyak, M. C., 1954, Ergotoxine inhibition of deciduoma formation and its reversal by progesterone, Am. J. Phsiol. 179:301-308. Shelesnyak, M. C., 1955, Disturbance of hormone balance in the female rat by a single injection of ergotoxine ethanesulfate, Am. I Physiol. 180:47-49. Shelesnyak, M. C., 1957a, Gonadotrophin content of pituitary of pregnant and pseudopregnant rats following single injection of ergotoxine, Endocrinology 60:802-805. Shelesnyak, M. C., 1957b, Some experimental studies on the mechanisms of ovoimplantation in the rat, Recent Prog. Hormone Res. 13:269-322. Shelesnyak, M. C., 1958, Maintenance of gestation in ergotoxine-treated pregnant rats by exogenous prolactin, Acta Endocrinol. 27:99-109. Shelesnyak, M. C., 1960, Nidation of the fertilized ovum, Endeavour 19:81-86. Shelesnyak, M. C., and Kraicer, P. F., 1959, La pyrathiazine est un antihistaminique capable de provoquer Ie deciduome uterin par liberation d'histamine dans l'organisme, C. R. A cad. Sci. Paris 248:2126-2128. Shelesnyak, M. C., Kraicer, P. F., and Zeilmaker, G. H., 1963, Studies on the mechanism of nidation. 1. The estrogen surge of pseudopregnancy and progravidity and its role in the process of decidualization, Acta Endocrinol. 42:225-·235.

Shirai, E., Iizuka, R., and Notake, Y., 1972, Analysis of human uterine fluid protein, Ferti!. Steril. 23:522-528. Smith, D. M., 1968, The effect of implantation of treating cultured mouse blastocysts with oestrogen in vitro and the uptake of (3H) oestradiol by blastocysts,]. Endocrinol. 41(1): 17-29. Smith, D. M., and Biggers, J. D., 1968, The oestrogen requirement for implantation and the effect of its dose on the implantation response in the mouse,]. Endocrinol. 41(1): 1-9. Smith, L. J., 1966, The changing pattern of basophilia in the mouse uterus from mating through implantation, Am.]. Anat. 119: 1-14. Smith, M. J., and Sharman, G. B., 1969, Development of dormant blastocysts induced by oestrogen in the ovariectomized marsupial, Ausl.]. Bioi. Sci. 22: 171-180. Somerville, B. W., 1971, Daily variation in plasma levels of progesterone and estradiol throughout the menstrual cycle, Am.]. Obstet. Gynecol. 111:419--426. Steer, H. W., 1970, The trophoblastic knobs of the preimplanted rabbit: A light and electron microscopic study,]. Anal. 107:315-325. Steer, H. W., 1971, Implantation of the rabbit blastocyst: The adhesive phase of implantation,]. Anat. 109:215-227. Stegner, H. E., Sachs, H., and Uthmoller, E., 1971, Electron microscopic study of experimental deciduoma in the rat, Z. Geburtshilfe Gynaekol. 174:241-253. Stone, G. N., and Emmens, C. W., 1964, The effect of oestrogens and antioestrogens on deciduoma formation in the rat,]. Endocrinol. 29:147-157. Surani, M. A. H., 1975, Hormonal regulation of proteins in the uterine secretion of ovariectomized rats and the implication for implantation and embryonic diapause,]. Reprod. Fertil. 43:411417. Szego, C. M., 1972, Lysosomal membrane stabilization and antiestrogen action in specific hormonal target cells, Gynecol. Invest. 3:63-95. Tachi, c., Tachi, S., and Lindner, H. R., 1970, Action of antihistaminics on the endometrium and the histamine theory of decidual induction,]. Reprod. Fertil. 23: 169-172. Tachi, C., Tachi, S., and Lindner, H. R., 1972, Modification by progesterone of oestradiol-induced cell proliferation, RNA synthesis and oestradiol distribution in the rat uterus,]. Reprod. Fertil. 31:59-76. Tachi, C., Tachi, S., and Lindner, H. R., 1974, Effect of ovarian hormones upon nucleolar ultrastructure in endometrial stromal cells of the rat, Bioi. Reprod. 10:404-413. Takewaki, K., 1970, Formation of deciduomata in response to uterine trauma in reserpinized immature rats, Proc. Jpn. Acad. 46:552-555. Takewaki, K., and Machida, T, 1970, Deciduomal response to uterine trauma following placement of hypothalamic lesions in immature rats with ovaries luteinized by injections of PMS and HCG, Annol. Zool. Japon. 43:23-27. Trelsad, R. L., Revel, J. P., and Hay, E. D., 1966, Tight junctions between cells in the early chick embryo as visualized with the electron microscope,]. Cell Bioi. 31:c6-clO. Turner, W. M., 1873, Report on the progress of anatomy,]. Anal. Physiol. 8: 159-178. Turner, W., 1876, Lectures on the comparative anatomy of the placenta, Edinburgh. Van Winkle, L. J., Dabich, D., and Andary, T J., 1973, Effect of proteinase inhibitors on amino acid incorporation in mouse blastocysts, Fed. Proc. 32:214a. Velardo, J. T, and Hisaw, F. L., 1951, Quantitative inhibition of progesterone by estrogens in development of deciduoma, Endocrinology 49:530-537. Vokaer, R., 1952, Recherches histophysiologiques sur l'endometre du rat, en particulier sur Ie conditionnement hormonal de ses proprietes athrocytaires, Arch. Bioi. 63:3-84. Vokaer, R., and Leroy, F., 1962, Experimental study on local factors in the process of ova implantation in rats, Am.]. Obstet. Gynecol. 83: 141-148. von Numers, C., 1953, On the specific granular cells (globular leukocytes) of the human endometrium: with special reference to their occurrence in different pathological conditions and to their staining reactions, Acta Pathol. Microbiol. Scand. 33:250-256. von Spee, G., 1893, Beitrag zur Entwicklungsgeschichte der friiheren Stadien des Meerschweinchens bis zur Vollendung der Keimblase, Arch. Anal. Physiol. 7:44-60. Warbrick, J. G., 1954, The regeneration of the uterine epithelium at the placental site in postpartum rats,]. Anat. 88:573. Watson, J., Anderson, F. B., Alam, M., O'Grady, J. E., and Heald, P . .J., 1975, Plasma hormones

307 THE IMPLANTATION REACTION

308 CHAPTER '9

and pituitary luteinizing hormone in the rat during the early stages of pregnancy and after postcoital treatment with tamoxifen (ICI 46,474),I Endocrinol, 65:7-17, Webb, F, T G" 1975, Implantation in ovariectomized mice treated with dibutyryl adenosine 3', 5'monophosphate (dibutyryl cyclic AMP),J Reprod, FertiL 42:511-517. Weichert, C. K., 1928, Production of placentomata in normal and ovariectomized guinea pigs and albino rats, Proc. Soc. Exp. BioL Med. 25:490-491. Weichert, C" K., 1942, The experimental control of prolonged pregnancy in the lactating rat by means of oestrogen, Anat. Rec. 83: 1-15. Weill, P., 1921, Les cellules, granule uses des muqueuses intestinale et uterine, Arch. Anal. Microsc. 17:77-82. Weitlauf, H. M., 1971, Influence of ovarian hormones on the incorporation of amino acids by blastocysts, in vitro, in: The Biology of the Blastocyst (R. j. Blandau, ed.), pp. 277-290, University of Chicago Press, Chicago. Weitlauf, H. M., 1973, In vitro uptake and incorporation of amino acids by blastocysts from intact and ovariectomized mice, I Exp. ZooL 183:303-308. Weitlauf, H. M., and Greenwald, G. S., 1968, Survival of blastocysts in the uteri of ovariectomized mice,J Reprod. Fertil. 17:515-520. Whitten, W. K., 1956, Endocrine studies on delayed implantation in lactating mice,I Endocrinol. 13: 1-6. Whitten, W. K., 1958, Endocrine studies on delayed implantation in lactating mice: Role of the pituitary in implantation,I Endocrinol. 16:435-440. Williams, M. F., 1948, Vascular architecture of rat uterus influenced by estrogen and progesterone (corpus luteum hormone), Am. I Anat. 83:247-307. Wilson, E. W., 1969, Alkaline phosphatase in pre-decidual cells of the human endometrium, I Reprod. FertiL 19:567-568. Wilson, I. B., 1963a, A tumour tissue analogue of the implantating mouse embryo, Proc. Zool. Soc. London 141:137-151. Wilson, I. B., J963b, A new factor associated with the implantation of the mouse egg, I Reprod. Fertil. 5:281-282. Wislocki, G. B., and Streeter, G. L., 1938, On the placentation of the macaque (Macacca mulatta) from the time of implantation until the formation of the definitive placenta, Contrib. Embryol. 27: 1-66. Wislocki, G. B., Weiss, L. P., Burgos, M. H., and Ellis, R. A., 1957, The cytology, histochemistry and electron microscopy of the granular cells of the metrial gland of the gravid rat, I Anat. 91:130-140. Wu, j. T, and Chang, M. C., 1973, Hormonal requirement for implantation and embryonic development in the ferret, Bioi. Reprod. 9:350-355. Wu, j. T, and Meyer, R. K., 1970, The effect of implantation of culturing delayed rat blastocysts in medium containing 17{3 estradiol, Bioi. Reprod. 3:201-204. Wynn, R. M., 1965, Electron microscopy of the developing decidua, Fertii. Steril. 16: 16-26. Yochim, j. M., and De Feo, V. j., 1962, Control of decidual growth in the rat by steroid hormones of the ovary, Endocrinology 71:134-142. Zhinkin, L. N., and Samoshkina, N. A., 1967, DNA synthesis and cell proliferation during formation of deciduomata in mice,I EmbryoL Exp. Morpho!. 17:593-603. Zybina, E. V., and Grishenko, T A., 1972, Spectrophotometrical estimation of ploidy level in decidual cells of the endometrium of the white rat, Dokl. Akad. Nauk SSSR 14:284-290 (in Russian).

Scanning Electron Microscopy of the Endometrium

10

E. S. E. HAFEZ and HANS LUDWIG

The uterine epithelium plays a major role in the secretion of endometrial fluids that are important for the survival and transport of gametes. Transmission electron microscopy has been used extensively to study the ultrastructure of the endometrium in a variety of animals (Stinson et al., 1962; McQueen, 1964; Kojima and Selander, 1970; Nilsson, 1962b) and man (cf. Wynn, 1967; Lawn, 1973). Functional aspects of the uterine epithelium seem, in some way, to be related to alterations in the fine surface structure of the secretory cells and these alterations can conveniently be studied by scanning electron microscopy. It has recently been applied to study surface ultrastructure of the endometrium of several mammalian species (Hafez, 1975), including man (Johannisson and Nilsson, 1972; Ferenczyet al., 1972; Ferenczy and Richart, 1973; Hafez et ai., 1975a,b; Ludwig and Metzger, 1976b). As observed by scanning electron microscopy, the endometrium is composed of ciliated cells covered with kinocilia or solitary cilia, and secretory cells covered with apical microvilli. The surface ultrastructure of the endometrial cells varies with the state of reproductive cycle, onset of implantation, species, aging of the female, administration of steroid contraceptives, and the presence of intrauterine devices. E. s. E. HAFEZ . Reproductive Physiology Laboratories, C. S. Mott Center for Human Growth and Development, Wayne State University School of Medicine, Detroit, Michigan. HANS LUDWIG . Department of Obstetrics-Gynecology, University of Essen School of Medicine, Essen, West Germany.

309

310

1.

CHAPTER 10

The endometrium has two types of ciliated cells: ciliated cells with kinocilia common to endometria of all mammalian species studied, and those with solitary cilia, which are found in endometria of certain rodents and under certain hormonal conditions in other species, including the postmenopausal woman.

1.1

Ciliated Cells

Kinocilia

Ciliated cells with typical kinocilia (motile) have nine peripheral and two central filaments. These ciliated cells, less abundant in the endometrium than in the oviductal epithelium, are found singly or in clusters. Although the basic ciliary structure and its variations throughout biological systems are well documented (Brenner, 1969; Fawcett, 1961; Satir, 1965), little is known about the mechanisms of ciliary motion. The directional beat of the cilia suggests that the action of kinocilia is to facilitate the release and distribution of the endometrial secretions and to propel fluid currents within the uterus. It was originally believed that ciliated cells are present only in pathological uteri (Novak and Rutledge, 1948). It is now known that ciliated cells are found in the endometria of several mammals (Hafez, 1973, 1975), including humans (Schueller, 1968, 1973; Armstrong et at., 1973; Hafez et at., 1975a,b). The dynamics of the population of ciliated cells during the menstrual cycle has been controversial. Furin and Tiche (1967) suggested that they are seen only in the proliferative human endometrium. Flemming et al. (1968) and Schueller (1968, 1973), on the other hand, described them in the secretory endometrium. Ciliated cells, not uniformly distributed in the endometrium, are found in greatest numbers around the isthmus and at the cornua (Schueller, 1968; Johannisson and Nilsson, 1972; Ferenczy et at., 1972). The frequency of ciliated cells varies with the stage of the reproductive cycle. In the human endometrium the percentage of ciliated cells increases during the proliferative phase to reach a maximum of around 20% (see Chapter 11). This percentage is maintained during the ovulatory phase, and then declines (Masterson et at., 1975). Ciliated cells (Fig. 1) may undergo deciliation, reciliation, or necrosis in situ. Ciliated cells may also develop some secretory capability, thus losing their histochemical and ultrastructural characteristics (Schueller, 1968). In cases of endometrial hyperplasia with high estrogen titers, there is a significant increase in the expected percentage of ciliated cells, followed by a decrease in numbers during the secretory phase of the menstrual cycle in response to progestrone levels (Flemming et al., 1968). The abundance of ciliated cells varies in most species in different parts of the reproductive organs (Fig. 2). Maximal numbers of ciliated cells are observed in the fimbriae and the cervix and minimal numbers in the corpus.

1.2

Solitary Cilia

Solitary cilia ("9 + 0" type), equipped with striated rootlets, are found in the connective tissue of the uterus of both estrous and pseudopregnant hamsters

311

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487 ELECTROPHYSIOLOGY OF UTERINE SMOOTH MUSCLE

488 CHAPTER 13

be a direct result. The initiation of spike activity and the increase in the number of burst and spike discharges can be explained by an increased probability of successful spikes arising out of pacemaker potentials, which, in the absence of oxytocin, would have been abortive. There is an interesting detail of oxytocin action that requires some comment. In conjunction with his progesterone block hypothesis, Csapo (1961) proposed that the action of oxytocin was voltage dependent. If the membrane potential were more negative to the spike threshold, oxytocin would have a depolarizing action, taking the membrane potential to the spike threshold, resulting in activity. Such a condition would exist in the myometrium of the term uterus. If the membrane potential were highly negative such as in midpregnancy, then oxytocin would be ineffective, because it could not depolarize the membrane enough to reach the spike threshold. If the membrane potential were slightly less negative to the spike threshold such as might be produced experimentally by lowering (Ca 2+)o, oxytocin would be effective, because it could reverse its depolarizing action by repolarizing the membrane potential to the spike threshold (Kuriyama and Csapo, 1961). However, if the membrane potential were considerably less negative than the spike threshold such as might exist in immature and spayed animals, then oxytocin would be ineffective, because it could not repolarize enough to reach the spike threshold. Out of these observations came the conclusion that a prerequisite for oxytocin to be effective is that the membrane potential must be close to the spike threshold (see Marshall, 1974). The idea that oxytocin can be depolarizing as well as repolarizing, both toward some assumed spike threshold, although provocative, is not supported by evidence obtained by other investigators. As stated before, the depolarizing actions of oxytocin were observed only with unphysiologically high doses of oxytocin, and not observed when microunit ranges of oxytocin were used (Kleinhaus and Kao, 1969; Osa and Taga, 1973). The alleged repolarizing action of oxytocin, produced in media containing lowered [Ca 2+]o, could not be reproduced by Kleinhaus and me (1969). Moreover, rabbit myometrium slightly depolarized either by slight elevation of [K+]o or by potassium-free media responded readily to oxytocin with increased frequency of spike discharges without any observable changes in existing resting potential (Kleinhaus and Kao, 1969). Similarly, in the rat myometrium depolarized with excess [K+]o oxytocin did not have any repolarizing action (Marshall, 1968). Considerably less information is available on the actions of other oxytocic drugs, but ergonovine, ergotamine, and sparteine all increase the frequency of spike discharges in a manner similar to that of oxytocin (Kleinhaus, 1968).

3.5.2.

Autonomic Drugs

Most uteri respond with some depolarization or increased spike discharges to acetylcholine and more stable choline esters such as methacholine (Kleinhaus, 1968) and carbaminylcholine (Osa and Taga, 1973). The responses to adrenergic agents are variable, and depend on both the species and the hormonal state. In some species such as the rabbit, epinephrine or norepinephrine caused spike discharges and contractions (Kleinhaus, 1968). In

other species such as the rat, these agents caused a hyperpolarization, cessation of spike discharges, and relaxation (Marshall, 1974). In still others such as the cat, epinephrine caused a contraction in the nongravid state and relaxation in the gravid state. A few comments will be made on the hyperpolarizing action of catecholamines on the rat myometrium, because the lesson learned from studies of this tissue is possibly of general interest to investigations of smooth muscles. In an orthodox approach to the hyperpolarization, the phenomenon can be explained by an increase in the potassium conductance of the myometrial membrane. Such a change would necessarily move the membrane potential toward the potassium equilibrium potential, which is at a more negative level. This explanation was indeed used by Ohashi (1971) for the rat myometrium. On investigating the catecholamine-induced hyperpolarization with the voltage-clamp technique, it became obvious that there was no change in the potassium conductance (Kao et al., 1971). The hyperpolarization was due to a combination of an increased electromotive force of the potassium cell and a change in cationic selectivity in favor of potassium. The change in electromotive force is manifested also in a real increase in [K+]i (Kao et al., 1976). The implication of the conclusion is that for smooth muscles with small individual cells, electrical changes can be produced by alternative routes to actions on the membrane, such as biochemical and metabolic changes resulting in changes in ionic concentrations.

4.

Summary and Concluding Remarks

As in all other mammalian tissues, the myometrial cell has a high concentration of potassium and a low concentration of sodium intracellularly. Compared with those of skeletal and cardiac muscles, the intracellular concentrations of sodium and chloride in the myometrium are higher. The distribution of chloride between the intracellular and extracellular phases does not appear to be passive, but may involve some active accumulation mechanism, concentrating chloride intracellularly. Maintenance of the distribution patterns of sodium and potassium involves active transport processes, which, under normal physiological conditions, appear to be coupled. The circumstantial evidence for this conclusion is that the net fluxes of sodium and potassium are reciprocal and stoichiometrically related, and that absence of potassium from the medium prevents all net efflux of sodium, and markedly reduces the efflux of 22Na from the intracellular compartment. Data available at present indicate that the primary source of energy for sodium extrusion is the oxidation of glucose, and that potassium accumulation, but not sodium extrusion, can derive some energy from glycolysis. The resting potential of the myometrial cell is about 50 m V inside negative. It is influenced by some permeability of the membrane to sodium and most probably also to chloride. The resting potential of myometrium deprived of estrogenic stimulation is low, and administration of estrogens causes an increase of resting potential to - 50 m V. There is some question of whether additional treatment with

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progesterone further increases the resting potential. There are enough fundamental differences, however, between the electrophysiological behavior of the progesterone-dominated nonpregnant myometrium and the pregnant myometrium to cast serious doubt on the assumption that the increase in resting potential of the pregnant myometrium can be attributed solely to the action of progesterone. The characteristic action potentials of the myometrium appear as irregular bursts of spikes. Two types of pacemaker activities set the pattern of excitation: one, of a periodicity on the order of tens of seconds, initiates the burst discharges; the other, of a periodicity in the range of 0.5-1 sec, controls the number of individual spikes in each burst. The forms of spikes are usually complex because of asynchronous discharges of neighboring cells; at parturition they become synchronous and simple. The basic simple spike lasts about 50 msec and usually has an overshoot. Removal of external sodium stops spike activity. The sodium influx during each spike is 7-8 pM!cm 2 , which is capable of producing a spike of 67-77 mV across a membrane capacity of 10 p.,F/cm 2 • Recent observations using a voltage-clamp technique show that the ionic currents in the myometrium are qualitatively similar to those in better-studied excitable membranes: upon threshold depolarization, an initial inward current was followed by a sustained outward current. Sodium ions are the principal charge carriers for the early current, and potassium ions are the principal charge carriers for the late current. Calcium ions influence the membrane conductance and thereby exert an effect on the excitatory phenomena. The sodium equilibrium potential is about 25 m V, and the potassium equilibrium potential is about 15-20 m V more negative than the natural resting potential. Contractions of the myometrium are activated by action potentials. The burst discharges set the frequency of contractions encountered in clinical experience, whereas the number of spikes in each burst controls the intensity of each contraction. The active state of the rabbit myometrium is long lasting, remaining measurable 35 sec after the stimulus. The onset of the active state appears to be gradual and there may not be a plateau to the active state. A high rate of stimulation by action potentials may lead to a decline in contractile strength. Oxytocin is capable of initiating spike discharges in quiescent myometrial preparations and of increasing the frequency of burst discharges as well as prolonging the duration of each burst. It can also increase the amplitude of spikes. These actions are unaccompanied by preceding changes in the resting potential. The maximum rate of rise of the action potential in the rabbit myometrium is rather low, but can be increased by the action of oxytocin. The relation between the maximum rate of rise and the resting potential is steepened by oxytocin as well as shifted toward more positive levels. This mechanism is believed to be capable of explaining the electrophysiological actions of oxytocin. Although notable progress has been made in the last 10 years in the study of electrophysiological and mechanical behavior of the myometrium, much remains to be learned. There persists a gap between the new information and the application of such information to the relief of human sufferings. It is to be hoped that the attainment of practically applicable information will be based on scientifically sound investigations, which, in the long run, are the most economic way toward progress.

ACKNOWLEDGMENT

All work from my laboratory, published and unpublished, was supported by a grant from the National Institute of Child Health and Human Development HD 378.

5.

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Specht, P. C., and Kao, C. Y., 1973, Phase-plane analysis of action potentials in uterine smooth muscle, Fed. Proc. 32:389 Abs. Sperelakis, N., and Shumaker, H. K., 1968, Phase-plane analysis of cardiac action potentials, j. Electrocardiol. 1:31-42. Stampfli, R., 1954, A new method for measuring membrane potentials with external electrodes, Experientia 10:508-509. Talbot, N. B., Lowry, O. H., and Astwood, E. B., 1940, Influence of estrogen on the electrolyte pattern of the immature rat uterus,j. Bioi. Chem. 132: 1-9. Taylor, G. S., Paton, D. M., and Daniel, E. E., 1970, Characteristics of electrogenic sodium pumping in rat myometrium,j. Gen. Physiol. 56:360--375. Thiersch, J. B., Landa, J. F., and West, T. C., 1959, Transmembrane potentials in the rat myometrium during pregnancy, Am. j. Physiol. 196:901-904. Thomas, R. C., 1969, Membrane current and intracellular sodium changes in a snail membrane during extrusion of injected sodium,j. Physiol. (London) 201:495-514. Thomas, R. C., 1972, Electrogenic sodium pump in nerve and muscle cells, Physiol. Rev. 52:563594. Tomita, T., 1970, Electrical properties of mammalian smooth muscle, in: Smooth Muscle (E. Biilbring, A. F. Brading, A. W. Jones, and T. Tomita, eds.), pp. 197-243, Williams and Wilkins, Baltimore. Ussing, H., 1960, The alkali metal ions in isolated systems and tissues, in: Handbuch der Experimentellen Pharmakologie, Vol. 13 (0. Eichler and A. Farah, eds.), pp. 1-195, Springer, Berlin. Vassort, G., 1975, Voltage-clamp analysis of transmembrane ionic currents in guinea-pig myometrium: Evidence for an initial potassium activation triggered by calcium influx, j. Physiol. (London) 252:713-734. Weidmann, S., 1952, The electrical constants of Purkinje fibers,]. Physiol. (London) 118:348-360. Wells, J. A., 1958, Historical background and general principles of drug action, in: Pharmacology in Medicine, 2nd ed. (V. A. Drill, ed.), pp. 11-12, McGraw-Hill, New York. Wolfs, G., and Rottinghuis, H., 1970, Electrical and mechanical activity of the human uterus during labor, Arch. Gynaekol. 208:375-385. Woodbury, J. W., and Brady, A. J., 1956, Intracellular recording from moving tissues with a flexibly mounted ultramicroelectrode, Science 123: 100-1 1. Woodbury, J. W., and McIntyre, J. M., 1954, Electrical activity of single cells of pregnant uteri recorded with intracellular ultramicroelectrode, Am. j. Physiol. 177:355-360.

°

14 The Contractile Mechanism and Ultrastructure of the Myometrium c. F. SHOENBERG This chapter is dedicated to Dr. Dorothy M. Needham on the occasion of her eightieth birthday.

It is usual to stress the resemblance between vertebrate smooth muscles and other muscles (striated and cardiac muscles of vertebrates, and invertebrate smooth muscles). An actomyosin can be extracted from vertebrate smooth muscles in solutions of high ionic strength (Csapo, 1948), which has properties closely resembling those of skeletal muscles (see review by Needham and Shoenberg, 1967). The muscles appear to contract by a sliding filament mechanism (Shoenberg, 1962, 1969b) and develop tensions comparable to those developed by skeletal muscles (Herlihy and Murphy, 1974; Murphy et at., 1974). There exist, however, very striking differences between vertebrate smooth muscles and skeletal muscles, and there are some aspects in which these muscles resemble much more closely nonmuscle cells, fibroblasts in particular. Particular emphasis will be placed in this chapter on the different ways in which smooth muscles resemble nonmuscle cells. Most of our knowledge on the structure and function of muscles is derived from studies on skeletal muscles; therefore, these are briefly described before approaching the subject of smooth muscles. C. F. SHOENBERG

Department of Anatomy, University of Cambridge, Cambridge, England.

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1. 1.1.

The Contractile Mechanism Skeletal Muscles

Striated muscles consist of numerous well-aligned myofibrils, which run the entire length of the muscle. In longitudinal sections one can see that each myofibril is composed of a number of identical units, each of which is in register with the units of the adjacent fibrils. These units, known as sarcomeres, are separated from each other by cross-striations. These cross-striations, or "Z bands," lie perpendicular to the length of the fibrils. Each sarcomere is almost entirely filled by thick and thin filaments running parallel to the length of the myofibrils. The thick filaments, consisting almost entirely of myosin, lie at the center of the sarcomere; this region is known as the "A band." The thin filaments, composed mainly of actin, are attached at one end to the Z bands and with their free ends interdigitate with the thick filaments. In the resting muscle only part of each thin filament lies between two thick filaments. The region separating the A band from the Z bands is known as the "I band" and the region of the A band in which the thin filaments do not penetrate is called the "H band." At the center of the H band (the M band) the thick filaments are connected with one another, which helps to hold them in register. In longitudinal sections, projections can be seen on both sides of the thick filaments, except in the central region (Huxley, 1957). During contraction the projections or bridges move toward the thin filaments (Huxley, 1969) and attach and detach themselves cyclically to and from the thin filaments and in this manner draw them toward the middle of the sarcomere. The interdigitation between thick and thin filaments is consequently much greater in a contracted muscle, and as shortening proceeds both I and H bands gradually disappear. This is the classical theory of contraction first described by Hanson and Huxley in 1955. In transverse sections of skeletal muscles the thick and thin filaments are seen to lie in hexagonal arrays with the thin filaments at the trigonal positions, and six thin filaments surround each thick filament. In 1963, Huxley showed that if a skeletal muscle is homogenized both thick and thin filaments are present in the preparation and can be visualized when coated with an electron-dense stain. These thick and thin filaments closely resemble the thick and thin filaments seen in tissue sections and also the myosin and actin filaments obtained by lowering the ionic strength of purified myosin and actin preparation~. The myosin filaments are formed by the ag~regation of myosin molecules (l520A long), which consist of a long thin rod (20 A wide) terminating in two globular heads. These molecules respond to tryptic digestion by separating into two halves called "light" and "heavy" meromyosin, respectively (LMM and HMM). The LMM rods are 20 A in diameter and vary in length from 600-900 A, depending on the degree of digestion; the rods are insoluble in solutions of low ionic strength and can form spindle-shaped paracrystals with a smooth surface. The HMM fraction consists of short rods terminatin& in two globular heads; it can be separated by papein digestion into short rods 20 A in diameter (subfragment 2 or S2) and globular heads 40 A in diameter and 120-150 A long (subfragment 1 or SI). Both SI and S2 are soluble in solutions of low ionic strength and cannot aggregate. Lowey and Cohen (1962) showed that SI has ATPase activity and that

this activity is equal to that of either HMM or the intact myosin molecule. With the electron microscope, Huxley (1963) examined LMM paracrystals, myosin molecules, and their subfragments and suggested that the LMM parts of the myosin molecules aggregate in a staggered fashion to form the myosin filament and that the projections on the surface of the filament consist of HMM. As the directionality of the molecules is reversed on both sides of the filament, this leaves a central bare portion consisting of LMM only (Fig. 1). From low-angle X-ray diffraction diagrams it was deduced that the protrusions, or bridges, are arranged in a 6/2 helix around the thick filaments, so that at any given level there are always two bridges opposite each other. It was also found that each pair of bridges is separated from the next pair by a distance of 143 A and an angle of rotation of 120°, and that since the rotation is continuous, two pairs of bridges will be in the same plane every 429 A (Fig. 2) (Huxley and Brown, 1967). When thin filaments and actin filaments were examined in detail with the electron microscope and by X-ray diffraction methods, it was found that the filaments consist of beadlike G-actin monomers arranged in two chains twisting around each other. The twistin&: chains cross over at intervals of 360-370 A and have a helical pitch of 720-740 A. Long, rod-shaped tropomyosin molecules lie in the grooves on both sides of the actin helix and give support to the filament (Hanson and Lowy, 1964b), whereas the small globular Ca2+-sensitive troponin molecule lying near the end of each tropomyosin molecule controls the stoichiometric changes that take place at the onset of contraction (Fig. 3) before the myosin bridges can attach themselves to the actin monomers (Moore et at., 1970). Huxley (1963) also found that thin filaments could take up HMM or Sl from a solution and form complexes between the G-actin monomers and the myosin fragments, giving a general appearance of arrowheads. When an isolated Z band to which thin filaments were still attached on both sides was treated with HMM, the direction of the arrowheads was reversed on both sides of the Z band, showing that the thin filaments have opposite directionality, as do the two halves of a thick filament. At the onset of contraction, the bridges move away from the body of the filament (Fig. 4), but they can attach themselves to the thin filaments only after the tropomyosin has moved in the groove. Both the outward movement of the bridges and the stoichiometric changes in the thin filaments are Ca2+ dependent and take place only when these ions are released after stimulation. In the resting muscle the level of free Ca2+ is very low (10- 7 M) and most of the ions are stored in the sarcoplasmic reticulum, which consists of smooth-walled vesicles, tubules, and cisternae. These organelles, which run both longitudinally and transversely in each sarcomere, lie in an ordered array, which repeats from sarcomere to sarcomere

Figure 1. Arrangement of myosin molecules in the thick filaments. The backbone of the filaments is formed from the assembly of the "tails" of the molecules, and the molecules from two anti parallel sets, one in each half of the filament, with a reversal of structural polarity at the center. By permission of Dr. H. E. Huxley from Proc. R. Soc. London Ser. B (1971).

499 CONTRACTILE MECHANISM AND ULTRASTRUCTURE OF MYOMETRIUM

500 CHAPTER 14

429.6.

Figure 2.

Diagram of crossbridge arrangement on thick myosin-containing filaments of frog sartorius muscle, which would account for the observed X-ray pattern. By permission of Dr. H. E. Huxley fromProc. R. Soc. London Ser. B (1971).

and is in register with the reticulum of the adjacent fibrils (Porter and Palade, 1957). A striking feature of the reticulum is the presence of three large vesicles occurring at regular intervals. The two outer vesicles are continuous with the longitudinal component of the reticulum, but the middle vesicle is in register and continuous with a transverse component known as the "T-tubules." Of the reticulum the T-tubules alone open out into the plasma membrane (FranziniArmstrong and Porter, 1964; Huxley, 1964). Using the freeze-etching technique, Bertaud et at. (1970) have shown that the T -tubules and the sarcoplasmic reticulum belong in reality to two different systems; the T-tubules have smooth-walled membranes, whereas the sarcoplasmic reticulum has a slightly rough surface. It is thought that in the resting muscle the calcium ions are stored in the terminal

Act in

Troponin ,

,

Tropomyos in

Figure 3. Double helix formed by the globular actin monomers. The long thin tropomyosin

molecules form a continuous strand, which lies alongside each groove of the actin chains. A globular troponin molecule is attached near one end of each tropomyosin molecule. There are two tropomyosin and troponin molecules for each twist of the actin chains, one on each side. At the onset of contraction, the troponin molecule takes up calcium and induces the tropomyosin molecule to shift its position to one nearer the center of the groove; the myosin bridges can now interact with the actin monomers. Reproduced by permission of Professor Ebashi (Ebashi et al., 1969).

501

Actin

Figure 4. Relative positIons of filaments and crossbridges in (a) a relaxed muscle and (b) a contracted muscle. In the relaxed muscle, the crossbridges do not project very far toward the actin filaments; in the contracted muscle, the crossbridges could attach to the actin filaments by bending at two flexible junctions. By permission of Dr. H. E. Huxley from Science (1969).

Q .J

I

Q .,

Myosin

Q

0,

a

?~~: :t

Myosin

:,

b

vesicles of the sarcoplasmic reticulum and that on stimulation the surface membranes of the muscle and the T -tubules are depolarized, inducing an electrical field that causes the terminal vesicles of the sarcoplasmic reticulum to release the Ca 2+; these ions rapidly diffuse to the center of the sarcomere and turn on both the thick and thin filaments, which are then able to interact with one another (Ebashi and Endo, 1968). The storage of the Ca2+ in the terminal vesicles in the resting muscle and its transfer to the A bands after stimulation was demonstrated by autoradiographic methods (Winegrad, 1965).

1.2. 1.2.1.

Vertebrate Smooth Muscles Background

Vertebrate smooth muscles contract much more extensively on stimulation than do skeletal muscles. Isolated pieces of muscle shorten readily to one-third of their excised length and these pieces can easily be stretched to at least twice their excised length and still respond to a stimulus. It is not so easy to stretch a skeletal muscle, and at twice its resting length there would be no overlap between the thick and thin filaments, the muscle would not respond to a stimulus, and the process would be irreversible. Incubated striated muscles are quiescent unless stimulated, but many smooth muscles incubated in an oxygenated physiological saline contract rhythmically for several hours. Structurally, smooth muscles differ even more strikingly from skeletal muscles. The muscles consist of cells, not fibrils, and are arranged in small bundles separated from one another by a thick meshwork of collagen. These bundles are well aligned only in a few muscles such as taenia coli and retractor penis. In both these muscles the bundles lie parallel to the longitudinal axis of the muscle. Each of the cells in a bundle is surrounded by a fine meshwork of collagen and has a long cylindrical shape, which tapers at both ends. The cells have a more or less centrally situated elongated nucleus and are almost entirely filled with three types of filaments: the thick, the thin, and the intermediate, or 100 A, filaments. In addition to the filaments there are some micro tubules, a litde sarcoplasmic reticulum, a more or less well-developed Golgi apparatus, some mitochondria, and more or less numerous ribosomes and

CONTRACTILE MECHANISM AND ULTRASTRUCTURE OF MYOMETRIUM

502 CHAPTER 14

glycogen granules. There are no well-defined Z bands; instead, their place appears to be taken over by dense regions lying close to and adhering to the cell membranes. There are also some irregularly distributed dense bodies to which various functions have been attributed. In addition, there are numerous vesicles opening out into the cell membranes (Fig. Sa). The 100 A filaments and microtubules are never seen in adult skeletal muscles. Vesicles opening out into the cell membranes, although absent from skeletal muscles, are seen in some regions of the heart. Both the 100 A filaments and microtubules are frequently found in nonmuscle cells. The various filaments and organelles in smooth muscle cells do not lie in an ordered array as they do in all other muscles. Whereas the thick and thin filaments lie in a perfect geometric array in skeletal muscles, their distribution in smooth muscles is much less nearly perfect. The thick filaments, when visualized, often appear to be irregularly distributed, and the number and distribution of the thin filaments around them vary also, although the thin filaments are themselves in hexagonal arrays. The sarcoplasmic reticulum and other organelles are irregularly

Figure 5. (a) Longitudinal section of taenia coli from an adult guinea pig. Note the clusters of vesicles opening out into the cell membrane. These clusters alternate with dense patches adhering to the cell membrane. Some mitochondria lie in close juxtaposition to the vesicles; others are irregularly distributed in the cell, as are the dense bodies. There is very little sarcoplasmic reticulum. From Shoenberg (unpublished).

503 CONTRACTILE MECHANISM AND ULTRASTRUCTURE OF MYOMETRIUM

Figure 5. (b) Longitudinal section of a mesenteric arteriole from a rat. Note the tubular elements of the sarcoplasmic reticulum irregularly distributed in the body of the cell. Some of the tubular elements lie very close to the surface vesicles. An unpublished micrograph reproduced by courtesy of Dr. C. E. Devine.

distributed in the cell and do not appear to be in any way in register with the reticulum and other organelles of the adjacent cells. The only feature that appears with any degree of regularity are the vesicles opening out into the plasma membranes (Fig. 5b).

1.2.2.

Thin Filaments

The thin filaments of vertebrate smooth muscles resemble closely all other Factin filaments (Hanson and Lowy, 1964b). They have a diameter of 60-95 A (average 80 A), but their length has not yet been ascertained (Lowy and Small, 1970). Although it is not clear whether troponin is associated with the tropomyosin in these filaments (see Chapter 12), there are changes in the X-ray diffraction pattern of the actin filaments when the muscles contract (Vibert et at., 1972). These changes show that in vertebrate smooth muscles, as in skeletal muscles, contraction is associated with a movement of the tropomyosin molecule toward the center of the groove in the actin filament. The regulation of the calcium ions in these muscles is discussed in Chapter 12. The ratio of thin to thick filaments is much greater in smooth muscles than in either skeletal or cardiac muscles. In a well-

504 CHAPTER 14

preserved specimen there appear to be 11-15 thin filaments for every thick filament or ribbon (Heumann, 1969; Devine and Somlyo, 1971; Small and Squire, 1972); this ratio is in agreement with that calculated by Tregear and Squire (1973) from their observations after polyacrylamide gel electrophoresis. The thin filaments of smooth muscles are, however, much more fragile than those from skeletal muscles (Sobieszek and Bremel, personal communication; Shoenberg, unpublished). The thin filaments lie parallel to the cell membranes in both resting muscles and muscles contracted under tension (Shoenberg, 1962, 1969b). It has been suggested that all the thin filaments are anchored at one end to the dense regions, or patches, adhering to the cell membranes (Rosenbluth, 1965). From the micrographs of Ashton et al. (1975) it is clear that some of the thin filaments are anchored in this way (Fig. 13c). Since the cells are usually 50 /-Lm long (and may be several hundred micrometers long at the end of pregnancy) and are 5-8 /-Lm wide, it seems unlikely that all the filaments could be attached to these dense patches and the filaments remain parallel to the cell membranes in both relaxed and contracted muscle cells, as they do when the muscles are kept under slight tension during equilibration and fixation (Shoenberg, 1969b). At present, it is only possible to surmise how the other filaments are attached. Filaments have been shown to pass through the dense bodies irregularly distributed in the cells (Shoenberg, 1958; Heumann, 1971; Ashton et ai., 1975). Heumann and Ashton and his collaborators suggest that the filaments passing through these bodies are the thin filaments, whereas Cooke and Fay (1 972a) and Small (1975) suggest that it is the 100 A filaments which are anchored in this way. Since the dense bodies appear to be granular (in fixed preparations), it is impossible to distinguish the filaments passing through them from the granules in a transverse section. In longitudinal sections the filaments passing through the bodies are clearly visible, but in these sections the thin filaments are virtually indistinguishable from the 100 A filaments, so that it is not possible to state categorically which type is seen in such a preparation. Examination of transverse sections shows that the thin filaments lie in clearly defined hexagonal arrays (Fig. 6a) in muscle preparations equilibrated and fixed under stretch (Lowy and Small, 1970; Heumann, 1970; A. P. Somlyo et ai., 1971b; Rice et al., 1970). The single 120 A equatorial X-ray reflection first observed by Elliott and Lowy (1968) confirms that this array is genuine and not an artifact. Striated muscles develop maximal tension when there is complete overlap between the thin filaments and the bridge-bearing parts of the thick filaments. At this stage all the thick and thin filaments of these muscles are involved. In smooth muscles it is impossible for the thick filaments to interact simultaneously with all the thin filaments whatever the degree of contraction. Heumann (1971) proposed models for relaxed and contracted muscles; it is not clear from these models how the thick filaments can interact with all the available thin filaments. Since it is impossible for the thick filaments to interact simultaneously with all the thin filaments, perhaps they interact with only some of them for partial contractions, but when there is considerable shortening of the muscles they interact successively with different sets of filaments. To resolve this question it will be necessary to have more information about the mode of attachment of all the thin filaments.

1.2.3.

Thick Filaments

1.2.3a. Background. The myosIn of skeletal muscle is in filament form in solutions of low ionic strength. The myosin of vertebrate smooth muscle is extractable in solutions of low ionic strength (Laszt and Hamoir, 1961; Huys, 1961), as is the myosin of many nonmuscle cells (Pollard and Weihing, 1974). It also resembles the myosin of certain nonmuscle cells in some other very significant aspects, such as the light chain structure (Burridge, 1974) and response to antibodies (Groschel-Stewart et at., 1975). Since myosin forms thick filaments in all other types of muscle and since they are essential for the classical sliding filament theory of contraction, it was expected that thick filaments must also be present in smooth muscles (particularly since these muscles develop tensions comparable to those developed by skeletal muscles). Nevertheless, there were no thick filaments in the first electron micrographs of smooth muscle cells, although the cells appeared to be well preserved and tightly packed with thin filaments. It was thought that with further improvement in the preparatory techniques, thick as well as thin filaments would be seen in smooth muscle preparations, and in due course thick filaments were observed. At first, the thick filaments, when seen, were small in size and numbers, but after a few years they were found in larger numbers and in more, although not by any means in all, preparations. The thick filaments were found in all types of smooth muscles, but they varied in number, size, and shape. These differences were not related to the type of smooth muscle examined, but depended on the method of preparation. Attempts were made to find out the factors controlling the structure and visualization of the thick filaments in fixed preparations. They showed that it is at present impossible to assess the in vivo structure of smooth muscle myosin from electron microscopic observations of fixed preparations. Thick filaments have also been obtained in homogenates and extracts of smooth muscles, but they have been seen only under special conditions and are no indication of the state of the myosin in vivo. X-ray diffraction experiments, which have the advantage that they can be carried out on living muscles, have also been inconclusive, but have shown clearly that some of the myosin must be in filamentous form and that the thick filaments may be labile even in the living muscle. 1.2.3b. Electron Microscopic and X-Ray Observations. For the early electron microscopic and X-ray work, see reviews by Needham and Shoenberg (1967,1968) and Needham (1971). Thick filaments were first seen in vertebrate smooth muscles by Choi (1962). They had narrow oblong shapes in cross-section, but were not so easily identified in longitudinal section. In the latter, the thick filaments were very short (about 0.5 /-tm long) and so thin that they might equally well have been an artifact caused by the superimposition of two thin filaments within one section. The thick filaments also were too few to account for all the myosin known to be present in these muscles from biochemical observations (Needham and Williams, 1959). It was subsequently found that their presence in a preparation depended not only on the method of fixation but also on the pretreatment of the living muscle (Needham and Shoenberg, 1964). During the 1960s thick filaments were seen in sections of smooth muscle cells

505 CONTRACTILE MECHANISM AND ULTRASTRUCTURE OF MYOMETRIUM

506 CHAPTER 14

Figure 6. (a) Transverse section from guinea pig taenia coli equilibrated in hypertonic saline in the cold before fixation. Note the regular arrangement of the thin filaments and the ribbon-shaped structure of the thick filaments . Each ribbon is surrounded by a clear zone of 70-200 A; myosin bridges are only occasionally seen. Fixation with acrolein dichromate followed by postfixation with osmium tetroxide. (b) Edge-on view of a ribbon in a longitudinal section. Myosin bridges are clearly visible on both sides of the ribbon; they occur at 140 A intervals. (c) The optical diffraction pattern of the ribbon in (b); the meridional spacing is 140 A. Parts (a-c) are reproduced by permission of Dr. ]. V. Small from Lowy et al. , (1972). (d) Transverse section from vertebrate smooth muscle equilibrated in a physiological saline before fixation in glutaraldehyde followed by postfixation in osmium tetroxide. In cross-section the thick filaments are circular, polygonal, or ribbon-shaped. (e) Longitudinal section from vertebrate smooth muscle equilibrated and fixed as above. A regular array of crossbridges can be seen on both sides of the filament. Parts (d) and (e) are reproduced by permission of Drs. A. P. and A. V. Somlyo from Somlyo et al. (1973) .

in a number of laboratories, but they were observed only in muscles that had received unphysiological pretreatment (see review by Shoenberg and Needham, 1976). By the late 1960s it became possible for most workers in the field to observe thick filaments in sections of muscle that had not undergone unphysiological pretreatment. The filaments were best visualized in muscles stretched and equilibrated in an oxygenated physiological solution before fixation (Devine and Somlyo, 1971). The thick filaments observed by different workers varied enormously in size, shape, and number. Their diameter might be as little as 130 A or as great as 1000 A (see Table 1); their length, when measured recently by Ashton et al. (1975) and by Small (in press), was found to be 3 fJ-m by the first group and 9 fJ-m by Small. Most workers found what they called "conventional filaments," whereas others found ribbon-shaped filaments or none at all. The conventional filaments had diameters varying from 130 to 328 A and appeared in cross-section as oblong, polygonal, or circular (Fig. 6d). The differences in the diameters of the thick filaments in different preparations did not result from different methods of measurement, for there was no comparable variation in the diameters of the thin filaments, which varied in size only from 65 to 95 A. It was assumed that the differences in the size and number of thick filaments seen in different preparations and the irregularity of their shape were fixation artifacts caused by their extreme lability. Panner and Honig (1967, 1970), who did not find any thick filaments in their preparations, suggested that the smooth muscle myosin must be in dimer form in vivo, whereas Lowy and his colleagues, who examined precooled muscles, found ribbon-shaped, thick filaments in their preparations (Fig. 6a). The shape that Lowy attributed to the thick filaments was based on both electron microscopic and X-ray diffraction observations (Lowy and Small, 1970;

Table 1. Thick Filament Diameters Found by Different Workers

Author Campbell and Charnley (1975) Cooke and Fay (1972b) Devine and Somlyo (1971) Garamvolgyi et al. (1971) Heumann (1973) Heumann (1973) Kelly and Arnold (1972) Keyserlingk (1970) Kris tensen et al. (1971) Nonomura (1968) Rice et al. (1970) Rosenbluth (1971) Small and Squire (1972) Uehara et al. (1971)

Diameter of thick filaments

Type of tissue

Animal

(A)

Vas deferens Taenia coli Portal vein Ileum Small intestine Small intestine Iris sphincter Small intestine Small intestine Taenia coli Taenia coli Small intestine Taenia coli Gizzard

Mouse Guinea pig Rabbit Guinea pig Mouse Mouse Rat Rat Rat Guinea pig Guinea pig Toad Guinea pig Chicken

120-180 170 160-328 130 160 170 220-230 150-200 150 120-170 100-200 250-300 200-1000 150-200

507 CONTRACTILE MECHANISM AND ULTRASTRUCTURE OF MYOMETRIUM

508 CHAPTER 14

Lowy et at., 1970). Since X-ray diffraction reflections were obtained from living muscles, it seemed that the structure of the thick filaments derived from these observations must represent the state of the myosin in vivo. It will be shown that artifacts may arise even in vivo. To obtain good X-ray diffraction reflections, it is essential to have a muscle in which the fibers or cells are all aligned in one direction. Taenia coli from guinea pig was therefore used for these experiments. Skeletal muscles give two good myosin reflections, the 143 A reflection, which is indicative of the distance between bridges (along the axis), and the 430 A reflection, which measures the axial repeat of the helix formed by the bridges. Both of these reflections are strongest in relaxed muscles. When taenia coli muscles are equilibrated in an oxygenated physiological saline at 37°C, they go into rhythmic contractions, which are maintained as long as the muscles are in good condition. At this temperature these muscles can relax for any length of time only under the influence of drugs or when Ca2+ ions are absent from the bathing solution, but if the temperature of the muscle bath falls below 15°C the muscles lose all tension and activity and remain relaxed for many hours, and this state is completely reversible. Lowy and his colleagues thought that cooling would be the most physiological method of obtaining relaxed taenia coli muscles. In their experiments the muscles were either equilibrated in physiological ,aline for 2-4 hr at 12-15°C and then X-rayed at this temperature or equilibrated in the same solution for the same time at 4°C and fixed at this temperature for electron microscopic observations. They obtained, as did Elliott (1964, 1967), layer lines from actin at 59 A and an equatorial reflection showing lateral organization of the thin filaments. They also observed the meridional reflections from collagen. In addition, they observed a 143 A reflection characteristic of myosin in filament form. (They did not observe the other important myosin reflection, the 430 A.) The 143 A reflection was much stronger in muscles equilibrated in hypertonic saline at 15°C or at 37°C (Lowy et at., 1970, 1973) and further work was carried out in such salines. From the width of the 143 A reflection they deduced that the myosin filaments must be broad and probably ribbon-shaped. Their electron microscopic observations (Lowy and Small, 1970) confirmed this supJ;0sition. In cross-sections the cells were filled with small dense dots 30--65 A in diameter (the thin filaments); slightly larger dots with a light central core, 100 A in diameter (the 100 A, or intermediate, filaments); and lines of different lengths 80 A by 200--1100 A, which they concluded must be the thick filaments, or ribbons (Fig. 6a). The ribbons were separated from the surrounding thin filaments by a clear halo of 70--200 A; they bore few projections and did not seem to be aligned in any way. Although the thin filaments appeared to be distributed in regular (sometimes) hexagonal arrays, the number surrounding each ribbon varied greatly and did not seem to depend on the size of the ribbon. Both ribbons and thin filaments were clearly seen in longitudinal sections. The ribbons and thin filaments were very distinct and the ribbons could be followed for a distance of up to 5 /.tm. The projections, which were irregular and not always clearly distinguishable in cross-section, were very clear in the longitudinal sections and were distributed at fairly regular intervals along the ribbons(Fig. 6b). The clear halo and scarcity of bridges surrounding cross-sections of thick filaments were also a feature of the more conventionally shaped filaments (Fig.

6d), as were crossbridges regularly distributed along the sides of the filaments in longitudinal sections (Fig. 6e) (Somlyo et at., 1973), but again they could not be followed along the entire filament. Combining electron microscopic and optical diffraction methods, Small and Squire (1972) concluded that the assembly of myosin molecules in ribbons is very different from that in filaments. An examination of the broad surface of the ribbons showed that they were traversed by fine lines along their entire length. Small and Squire found that these lines lie 140 A apart and that numerous dots can be distinguished on each one; they concluded that each dot corresponds to an individual crossbridge. In edge-on views it was also possible to see that the axial spacing between the bridges is 140 A; it was clear that they have opposite directionality on the two broad faces of the ribbon. By tilting the microscope stage it became clear that each projection on a ribbon corresponds to a row of bridges on the broad face of the ribbon. Since the polarity of the bridges is reversed on the two faces of the ribbon and not as in striated muscle at both ends of the filament, the myosin molecules in a ribbon must be assembled differently from those in a filament. Small and Squire suggested that the myosin molecules are attached to a non myosin core and that the reverse polarity on opposite broad surfaces of the ribbon is due to a reverse polarity of the nonmyosin core. Small (1976) no longer holds the view that the myosin molecules are attached to a nonmyosin core. Small and Squire also observed that optical diffraction patterns of edge-on views of the ribbons showed not only a meridional reflection of 140 A but also an off-meridional reflection, which indicated that the bridges could not be symmetrically arranged along the long axis of the ribbon. To find out the correct distribution of the bridges on the ribbons, they built a number of models, compared the optical diffraction patterns obtained from these models with that obtained from a ribbon, and concluded that the bridges curving in opposite direction on the two surfaces of the ribbon do not originate at the same level, but that there is a 40-50 A shift on the opposite faces (Fig. 7). It was very puzzling why some workers observed ribbons in their preparations, whereas others saw more conventionally shaped filaments, or why the diameter of the thick filaments should vary so much in different laboratories. A number of attempts were made to find out the factors controlling the visualization of thick filaments in a preparation and also those controlling their size and shape when present. Various factors were closely examined, in particular: 1. The effect of changes in osmolarity of the equilibration solution and the fixative on the shape of the filaments. 2. The effect of changes in temperature during equilibration and fixation on the visualization of thick filaments. 3. The effect of changes in ion content of the tissue during either equilibration or fixation on the shape of the thick filaments. 4. The effect of stretch during equilibration and fixation on the shape of the thick filaments. 5. The effect of different fixatives on the shape of the filaments. A. P. Somlyo and his colleagues studied the effect of stretch and hypertonicity. In their experiments, muscles were stretched to a length at which they no

509 CONTRACTILE MECHANISM AND ULTRASTRUCTURE OF MYOMETRIUM

510 CHAPTER 14

longer responded to stimulation with norepinephrine. The muscles were then incubated at these lengths in isotonic and hypertonic solutions (A. P. Somlyo et at., 1971c); others less stretched were also incubated in these solutions. All the muscles were then fixed in a hypertonic fixative and in addition some of the muscles incubated in isotonic physiological saline were fixed in an isotonic fixative. They found that when muscles were overstretched there was an increase in the number of ribbons in their preparations. The number of ribbons was even greater when overstretched muscles were both incubated and fixed in hypertonic solutions (Fig. Sa). Fixation alone in hypertonic solutions did not influence the shape of the filaments. They then had the same irregular shapes as the filaments in preparations from muscles equilibrated and fixed in isotonic solutions. (Both equilibration and fixation were carried out at room temperature.) The authors concluded that since equilibration and fixation under conditions of overstretching and hypertonicity caused the formation of ribbons, the ribbons in Small's preparations were artifacts. It is true that in Small's experiments the muscles were loaded with 10 g weights during equilibration and fixation and were, moreover, in many instances kept throughout in hypertonic solutions; but ribbons were also observed in preparations of muscles equilibrated and fixed in isotonic solutions. Furthermore, these muscles responded to stimulation by caffeine, although they developed only half the normal tension. Shoen berg and Goodford and their colleagues were concerned with the effect that changes in the ionic content of the muscles during either equilibration or fixation might have on visualization of thick filaments. Goodford had found that whereas the ionic content of taenia coli remained constant for several hours when these muscles were equilibrated at 37°e in an oxygenated phosphate-free physiological solution (Goodford's saline), the ionic content changed considerably if equilibration took place at 4°e. In muscles equilibrated at 4°e there were a slow loss of potassium ions and a gain in sodium ions (Freeman-Narrod and Goodford, 1962). It was also pointed out in this paper that even at room temperature the

Figure 7. Schematic diagram of a longitudinal edge-view through a ribbon. The projections on opposite faces have opposite polarity and tilt toward opposite ends of the ribbon. There is an axial shift between projections on opposite faces of about 40-50 A. The distance between projections on one face is 144 A. Reproduced by permission of Dr. J. V. Small from Small and Squire (1972).

content of potassium ion fluctuates. Goodford found that when muscles were equilibrated in the cold there was a rise in the tissue Ca2+ content just as in conditions of shock (Bauer et al., 1965); this observation was particularly disturbing because Shoenberg had observed that thick filaments are formed in vitro only in the presence of ATP, Mg2+, and traces of Ca2+ (see Section 1.2.3c). Hence the additional tissue calcium in muscles equilibrated in the cold might affect the aggregation of the myosin filaments in these preparations. The effect of the temperature of equilibration and fixation on visualization of thick filaments was therefore examined. Taenia coli of guinea pig was equilibrated in oxygenated Goodford saline at either 37 or 4°C. Specimens from both groups were then either warm-fixed or cold-fixed (Shoenberg, 1973). Cold fixation was carried out entirely at 4°C, but warm fixation was only initiated at 37°C (for a period of 10 min) and was then completed at 4°C. When muscles were equilibrated at 37°C and then subjected to warm fixation, thick filaments were seen only in very thin specimens such as taenia coli from very young guinea pigs or strips of longitudinal muscles from the ileum. Jones et al. (1973) looking at taenia coli muscles fixed at room temperature also found thick filaments only in sections obtained from thin muscles. After equilibration in the cold, thick filaments were found after either cold or warm fixation in sections from quite thick muscles, such as taenia coli from adult guinea pigs. Subsequently muscles were cold-fixed after different periods of equilibration in cold isotonic saline and it was found that the shape of the thick filaments appeared to be related to the time of equilibration of the muscles (Shoenberg and Haselgrove, 1974). When taenia coli muscles were equilibrated for 30 min, 2-4 hr, or 6 hr in the cold before fixation, the thick filaments in the preparations had the usual irregular appearance, varying in crosssection from rectangles to polygonal and circular shapes. When the period of equilibration was prolonged to 24 hr, the thick filaments were predominantly ribbon-shaped (Fig. 8b). Some quite large ribbons were observed in these preparations and occasionally some cell shrinkage. The size of the ribbons and the degree of cell shrinkage were much enhanced when equilibration and fixation took place in hypertonic solutions. It was noted also that when muscles equilibrated for 24 hr in the cold were transferred to a warm saline they developed tension and rhythmic activity only after 30-40 min. It was assumed that the length of the recovery process probably corresponded to the time required for the ionic content of the muscle to return to normal. Shoenberg and Goodford and their colleagues (1973) studied the changes in tissue ionic content that take place during fixation. They confirmed that the ionic content of taenia coli remained unaltered for several hours when the muscles were equilibrated in Goodford saline at 37°C. Furthermore, measurements of the extracellular space by the 4C]sorbitol method showed that (as previously observed by Goodford) there was an initial rapid uptake of the tracer, after which there was little further exchange. These muscles were then either warm-fixed or cold-fixed by the addition of 2i or 5% glutaraldehyde to the bathing solution. On addition of the fixative (warm fixation), there was a rapid uptake of the [14C]sorbitol, which continued until it corresponded to the volume of the tissue, indicating that immediately on addition of the glutaraldehyde the permeability of the cell membrane was altered. These changes in permeability must be the reason for the

e

511 CONTRACTILE MECHANISM AND ULTRASTRUCTURE OF MYOMETRIUM

Figure 8. (a) Rabbit portal anterior mesenteric vein. Transverse section of muscle stretched to 2.5 x its excised length and incubated at this length in physiological saline plus 12% sucrose (hypertonic) for 30 min before fixation in 0.1 M cacodylate-buffered 2% glutaraldehyde made hypertonic with 12% sucrose (postfixed in osmium tetroxide). Some of the thick filaments are ribbon-shaped. Reproduced by permission of Drs. A. P. and A. V. Somlyo from A. P. Somlyo et al. (l971b). (b) Guinea pig taenia coli. Transverse section of muscle equilibrated for 24 hr in physiological saline at 4°C before fixation in 0.1 M cacodylate-buffered 2~% glutaraldehyde. Postfixed in osmium tetroxide. Many

of the thick filaments are ribbon-shaped. From Shoenberg (unpublished). (c) Guinea pig taenia coli transverse section of muscle equilibrated 2 hr in physiological saline at 4°C before fixation as in (b). The thick filaments are heterogeneous in shape: circular, polygonal, and rectangular. From Shoenberg (unpublished). (d) Guinea pig taenia coli. Transverse section of muscle equilibrated for 2 hr in physiological saline at 4°C before fixation in acrolein followed by postfixation in osmium tetroxide. The muscles in (c) and (d) were treated in the same way before fixation, but after acrolein fixation many of the thick filaments are ribbon-shaped. From Shoenberg (unpublished).

enormous ionic fluxes that also took place. It was observed that the potassium ion content, which remained virtually unchanged during the period of equilibration, fell sharply on addition of 5% glutaraldehyde during warm fixation and was less than half after the first 10 min of fixation at 37°C. The loss of potassium ions from the tissue continued when the muscles were transferred to the cold fixative, and by 120 min of fixation 90% of the total tissue potassium was lost. With cold fixation there was a similar loss of potassium ions, which although initially slower nevertheless reached the same end point after 120 min (Fig. 9a). The sodium ion content, which like that of the potassium ions remained unchanged during equilibration, rose rapidly with warm fixation and was nearly double after the. first 10 min. It continued to rise so that the exchange of potassium and sodium was complete after 120 min. With cold fixation the gain of sodium was considerably slowed and the tissue sodium ion content had not even doubled after 120 min (Fig. 9b), so that the loss of potassium ions far exceeded the gain of sodium ions at this time. The magnesium and calcium ion contents also remained unchanged in the equilibrated living muscles, but whereas the magnesium ion content remained constant with either warm or cold fixation (Fig. 9c) the calcium ion content rose rapidly (particularly at first) with both warm and cold fixation. The increase in tissue calcium was more than threefold; it rose from an initial 3.5 to 12 mmollkg fresh wt after 240 min (Fig. 9d). Indeed, at the end of fixation the calcium ion concentration of the tissue was approximately 5 times higher than that of the saline buffering the fixative (glutaraldehyde) and much greater than that of the fixative. These experiments showed that considerable ion exchanges take place in smooth muscles during fixation and were in good agreement with those of Krames and Page (1968) on cardiac muscle whether fixed in osmic acid or formaldehyde. It was also observed that the osmolarity of the fixative did not affect the volume of the muscle cells after warm fixation, but (as was to be expected from the differences in ionic movements during cold fixation) an increase in osmolarity of the fixative used for cold fixation caused considerable cell shrinkage. To achieve good preservation of the muscles fixed at 4°C it was necessary to avoid hypertonicity as much as possible. Jones et al. (1973) observed similar osmotic effects when fixing at room temperature. Jones et al. (1973) found that if equilibration of the muscle takes place under conditions that cause swelling of the cells, the muscles may continue to respond to an outside stimulus, but both thick and thin filaments are destroyed during fixation. They demonstrated by extracellular space measurements that when muscles are incubated in a Krebs saline in which KCl is substituted for the NaCl, the cells are more permeable to the KCl and become swollen. This observation was subsequently confirmed by electron microscopic observations. The swelling of the cells (and consequent disruption of the filaments during fixation) was prevented by making the KCl Krebs hypertonic by the addition of 29--58 mM sucrose or by substituting K2 S04 for the KCl, because the cell membrane is impermeable to S04 ions. In these preparations both thick and thin filaments were well preserved. It was also observed that although calcium ions were necessary for the formation of thick filaments in vitro, removal of these ions from the bathing solution did not affect visualization of the thick filaments in fixed preparations, whether equilibration took place at 37°C (A. P. Somlyo et al., 1971a; Shoenberg, unpublished) or in the cold (Wolowyk, personal communication). Fay and Cooke

513 CONTRACTILE MECHANISM AND ULTRASTRUCTURE OF MYOMETRIUM

SODIUM

514 CHAPTER 14 POTASSIUM

40

0

I

a

I

b b

0

240

120

0

I

120

I

240

TIME AFTER PRE-EQUILIBRATION (min)

TIME AFTER PRE-EQUILIBRATION ( min

CALCIUM

12

MAGNESIUM

5·4

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~

)

1

~ ....... J15·O ~

8

!. 4

4·6 I

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c

I

120

I

240

TIME AFTER PRE-EQUILIBRATION (min)

0

d

I

0

I

120

2lo

TIME AFTER PRE - EQUILIBRATION (min

Figure 9. (a) Total potassium, (b) total sodium, (c) total magnesium, and (d) total calcium content of

guinea pig taenia coli measured after at least I hr pre equilibration in Goodford's physiological saline at 36°C. Symbols: • 0, control samples at 36°C in Goodford's saline throughout; ., 0, muscles transferred to Goodford's saline containing 5% glutaraldehyde at 36°C for 10 min and thereafter to the same solution at 4°C (the arrow marks the lO-min changeover point), ., 6, muscles transferred to Goodford's saline containing 5% glutaraldehyde at 4°C throughout. Each point represents the mean of six samples. Vertical bars show the standard error of the mean when it is greater than the size of the symbol. Note (I) the significant effect of cooling on the rate of sodium uptake, (2) the enormous effect of fixation on the calcium uptake, and (3) how only the magnesium content remains virtually unchanged during fixation. From Shoenberg et at. (1973).

(1973) incubated muscles in a Krebs solution to which they had added either EGTA or EDT A in excess of its calcium ion content. They observed the usual reversible loss of tension and activity, but found no thick or thin filaments in muscle preparations fixed during incubation in either EGT A or EDT A Krebs. Since the cells in these preparations were swollen, probably because of the use of the chelating agents, it seems likely that the disruption of the filaments took place during fixation and not as a consequence of calcium depletion. It has been shown how different conditions of equilibration affect the final visualization of thick filaments in a preparation; the type of fixative used may have an equally striking effect. Heumann (1973) found that when he fixed with glutaraldehyde the thick filaments had a greater diameter than when he used formol-osmic as a fixative. Shoenberg observed in muscle preparations equilibrated in the cold for 4 hr before fixation that the shape of the filaments varied with the type of fixative used. When muscles were fixed with glutaraldehyde, they had the mixed shapes of the so-called conventional filaments (Fig. 8c), but after acrolein fixation the filaments were ribbon-shaped (Fig. 8d) (Shoenberg and Haselgrove, 1974). If the mere use of a different fixative can alter the shape of the thick filaments finally visualized in a preparation, it becomes impossible to assess with any degree of certainty the shape of the myosin filaments in vivo from electron microscopic observations. It has not even been conclusively shown that there are any thick filaments in the living muscle; they might conceivably even be formed during fixation. The X-ray diffraction pictures of muscles, whether equilibrated in the cold in isotonic or hypertonic saline or at 37°C in hypertonic saline (Lowy et at., 1973), were controversial on the score of either temperature or hypertonicity; it was therefore necessary to repeat these experiments on muscles equilibrated and relaxed at 37°C in an isotonic saline. Since at this temperature the muscles go into rhythmic contraction, it was necessary to find a way of relaxing them without affecting the contractile proteins. Shoenberg and Haselgrove (1974) did not think that removing the Ca2+ from the bathing solution would be a satisfactory method, since these ions were known to affect myosin aggregation in vitro. A suitable relaxant drug was therefore sought, and the anesthetic xylocaine was found to be satisfactory in all respects. On addition of 1.5 mM of xylocaine to the bathing solution, the muscles lost all tension and activity and remained completely inert for periods of as long as 24 hr. On withdrawal of the xylocaine even after 24 hr, the muscles recovered both tension and activity after 5 min or so, so that it could be assumed that there was no significant change in ion content of the tissue or an appreciable change in cell volume. The latter was confirmed by extracellular space measurements (Table 2) (Good ford and Wootton, personal communication). It was concluded that the xylocaine must act solely on the cell membranes and was therefore unlikely to affect the contractile system. Using this method, taenia coli muscles were equilibrated and relaxed at 37°C, and X-ray diffraction pictures were then obtained. At this temperature Haselgrove and Shoenberg found no 143 A reflection, although both the actin and collagen reflections were good. When a muscle was X-rayed first at 37°C and subsequently cooled and X-rayed at 4°C, no 143 A reflection was obtained at the higher temperature, but a good reflection was observed at the lower one. These findings were related to electron microscopic observations of muscles equilibrated and relaxed in the same way and then fixed

515 CONTRACTILE MECHANISM AND ULTRASTRUCTURE OF MYOMETRIUM

516 CHAPTER 14

throughout the primary fixation (in glutaraldehyde) at 37°C. In transverse sections of the preparations, thick filaments were present in many of the cells. These filaments were irregular in shape and usually not sufficiently numerous to account for all the myosin in the muscle. Lowy (personal communication) obtained weak 143 A reflections from muscles equilibrated and relaxed under identical conditions at 37°C. The widths of these reflections differed considerably from those obtained at 4°C, and he concluded from his observations that at 37°C the thick filaments must be narrow. He could not tell whether under these conditions the 143 A reflection came from filaments or ribbons. These experiments showed that a strong 143 A reflection could be obtained only when the thick filaments are broad and ribbon-shaped, i.e., when the muscles are equilibrated in the cold or in a hypertonic solution. The experiments showed also that when muscles are equilibrated under more physiological conditions the 143 A reflection is at best weak, although it is too early to state whether this is because the thick filaments are very short or poorly aligned or whether perhaps only some of the myosin is aggregated into filaments at 37°C. Lowy's observations on the width of the 143 A reflections obtained at 37°C and at 4°C would seem to suggest that the myosin of vertebrate smooth muscle is very remarkable and can undergo structural changes in vivo. Campbell and Charnley (1975) have also observed structural changes of the myosin in vivo. They found that if smooth muscles are fixed after different periods of incubation in Locke's saline at 37°C the thick filaments become increasingly larger and denser the longer the period of incubation (Fig. lOa,b).

Table 2. Effect, of Xylocaine

Control rl

Xylocainee

a b C

d

14C ECS b

lCS e

Time

W/F a

(milk g)

(mllkg)

15 30 60 240 15 30 60 240

993.5 974.3 974.5 936.5 940.2 960.5 932.7 908.8

391 398 453 450 423 447 395 405

602.5 576.3 521.5 486.5 517.2 513.5 537.7 503.8

Wet weight/fresh weight ratio. Extracellular [14C]sorbitol space. Intracellular space. Muscles were incubated in Biilbring-Golenhofen saline, before measurements were taken.

'Muscles were equilibrated in Biilbring-Golenhofen saline, 1.5 mM xylocaine was then added, and measurements were taken. There was a gradual decrease in the cell volume of the controls, whereas the cell volume of the muscles incubated in saline with additional xylocaine remained almost unchanged.

The third method of approach, the study of homogenates and extracts, has also failed to reveal the state of the myosin in vivo. 1.2.3c. Thick Filament;

==

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RN A, > DN A, and protein synthesis in dormant mouse and rat blastocysts in vitro and in vivo (Weitlauf and Greenwald, 1965; Mohla and Prasad, 1971; Smith and Smith, 1971). Intraluminal uterine infusion of tritiated estradiol in the rat results in rapid concentration of radioactiv-

605 UTERINE CONTROL OF OVARIAN FUNCTION

606 CHAPTER 16

ity in uterine stromal, glandular, and muscle cells but no retention of the labeled steroid in the blastocysts (Prasad et at., 1974). Estrogenic activation of the blastocysts in diapause is evident within 5 min after subcutaneous administration of estradiol, as indicated by increased incorporation of [3H]uridine into RNA. The action of estradiol in activating blastocysts may be through an initial activation of RNA synthesis, which does not involve the continual binding of estradiol to the cells of the blastocysts. Estrogen-induced uterine RNA mimics the action of estrogen in stimulating morphological changes in the uterus and blastocysts in a manner similar to implantation evoked by estradiol (Segal et at., 1965a). RNA synthesis in rat blastocysts also may be evoked by cyclic AMP (Mohla et at., 1970). Histochemical evidence of .il5-3,8-hydroxysteroid dehydrogenase (3,8-0HSDH) activity, first appearing about 30 hr before implantation in rat embryos, suggests steroidogenic capability by the embryo (Dickmann and Dey, 1974). Preimplantation embryos incubated in a medium containing dehydroepiandrosterone (DHA) exhibit 3,8-0H-SDH activity. Steroidogenesis by morulas and preimplantation blastocysts may playa role in embryonic development and in preparation of the endometrium at the prospective sites for implantation. In ovariectomized rats, estrogen is required in addition to progesterone (Nutting and Meyer, 1963); steroidogenic capacity of the embryo alone is inadequate to accomplish implantation in this species. No .il5 -3,8-hydroxysteroid dehydrogenase (3,8-0H-SDH) activity is found in unfertilized rat ova or in embryos at days 1, 2, and 3 (Dey and Dickmann, 1974). 3,8-0H-SDH activity is first observed on day 4, peaks on day 5, declines on day 6, and is absent on day 7. The possible function of the embryos as a source of steroid hormones rapidly declines by day 6. In the rabbit, histochemical evidence for .il 5-3,8-hydroxysteroid dehydrogenase (3,8-0H-SDH) activity indicates that enzymatic activity begins in preimplantation embryos (morula stage) 48 hr after mating. Activity is sustained through the late blastocyst stage (144 hr), and absent during early postimplantation period (168 hr). Embryos at the blastocystic stage contain estradiol-17,8. The rabbit blastocyst seems capable of steroidogenesis, which may playa role in the implantation process. In the ewe, the embryo must be present within the uterus by day 12.5 after mating if the corpus luteum is to remain functional and sustain the pregnancy. Physical distention of the uterus during this time does not in itself influence the maintenance of the corpus luteum (Rowson and Moor, 1968). After fetectomy, with placental membranes remaining in situ, the interval from the fertile estrus to the immediate postoperative estrus was found to increase to 56, 91, and 118 days when the surgical intervention was performed at days 30, 50, and 70 after mating, respectively. The placenta in this species seems involved in maintaining luteal function during pregnancy. The pig blastocyst elongates rapidly between days 11 and 14 (Anderson, 1974), with attachment to the endometrium occurring about day 18 (Perry et at., 1973). During this brief period, maternal recognition of pregnancy is established, and the corpora lutea are then maintained for the duration of the gestation, whereas in unmated pigs the corpora fail and estrus recurs by about days 20-21. Incubation of pig blastocysts with radiolabeled androstenedione, dehydroepiandrosterone, or pregnenolone reveals biochemical evidence within the blastocyst of aromatase, 17-20-desmolase, and 3-sulfatase for production of estrogens, progesterone, and conjugated steroids (Perry et at., 1973).

2.3. 2.3.1.

Uterine-Ovarian Function during Pregnancy Rat

Astwood and Greep (1938) reported that extracts of rat placentas could sustain pseudopregnancy in the rat, presumably by continuing progestin secretion. Extracts of rat placentas were found to provide luteotropic activity and mammotropic function as indicated by their capability to induce mammary growth and early lactational changes (Ray et ai., 1955; Matthies, 1967). During the first half of gestation, the adenohypophysis is essential for maintaining pregnancy (Pencharz and Long, 1933; Greenwald and Johnson, 1968), whereas placental luteotropins seem to stimulate ovarian function during the later half of gestation (Ray et al., 1955; Lyons and Ahmad, 1973). Serum levels of prolactin, LH, and FSH remain below those that characterize the diestrous phase of the estrous cycle (see Fig. 4) (Linkie and Niswender, 1972). Extracts of placentas and sera from rats on days 11, 12, and 13 of pregnancy support deciduomata in hypophysectomized pseudopregnant rats, but extracts or sera obtained earlier (day 10) or later (days 14, 16) do not sustain deciduomata in the test animals (Linkie and Niswender, 1973). In hypophysectomized ovariectomized rats, the pregnancies were maintained during the critical period of days 10-13 by injecting extracts of day 12 placentas (1 placenta/day and estradiol 5 JLg/day). Evidence of participation of the uterus in luteotropic action during midpregnancy in the rat has been evaluated by measurement of ovarian venous blood levels of progesterone and 20a-hydroxypregn-4-en-3-one (20a-OH-P) (Sin et al., 1971). During midpregnancy there is a marked rise in progesterone and 20a-OHP levels during placentation (Hashimoto et al., 1968) and a corresponding increase in these two progestins occurs after hypophysectomy on day 12 (Sin et at., 1971). The luteotropic effect of the conceptuses was not of pituitary origin. When the hypohysectomized rats are, in addition, hysterectomized, a marked decline results in ovarian venous levels of both progesterone and 20a--OH-P. Fetectomy with the placentas remaining in situ can not produce a similar increase in these progestin levels as found during normal pregnancy, but the steroid levels are sustained to the same extent as found in pseudopregnant rats bearing deciduomata. Thus the fetuses are considered essential for the placentas to exert luteotropic action; production of the luteotropins during mid pregnancy may result as an interaction of placentas and fetuses. The development of a radio receptor assay for rat chorionic mammotropin allows measurement of serum levels in peripheral circulation (Shiu et al., 1973; Kelly et al., 1975). Rat chorionic mammotropin (placental lactogen) shows two major peaks in peripheral serum during pregnancy, the first at day 12 and the second between days 17 and 21 (see Fig. 4). The appearance of the first lactogenic peak corresponds to the period when both serum and placenta possess the greatest luteotropic activity, and thus a correlation exists between luteotropic and lactogenic activity in serum at days 11-13 of pregnancy in the rat (Kelly et al., 1975). The placental lactogen appearing at days 17-21 of pregnancy has a different molecular weight, shorter half-life (1.2 vs. 19.5 min), and different pattern on gel electrophoresis, as compared with that at day 12. During the later part of pregnancy, the secretion of progesterone declines, and 20a-OH-P and 20a-OH-SDH increase before parturition (see Fig. 4) (Fajer

607 UTERINE CONTROL OF OVARIAN FUNCTION

608 CHAPTER 16

and Barraclough 1967; Hashimoto et al., 1968; Wiest et at., 1968; Fuchs et at., 1974; Labhsetwar and Watson, 1974). The pattern of progesterone concentration in ovarian venous plasma is characterized by a small peak in early pregnancy and a larger peak in the second half of pregnancy (Ichikawa et at., 1974). The secretion rates of 5a-pregnane compounds are highest at mating, decline markedly to low values by day 3, and remain low through parturition. Only 20a-OH-P increases markedly just preceding parturition. Exogenous LH increases the levels of 5apregnane-3, 20-dione, and 3a-hydroxy-5a-pregnane-20-one throughout all stages of pregnancy. The levels of 20a-hydroxy-5a-pregnane-3-one, 5a-pregnane-3a20a-diol, and 20a-OH-P remain unresponsive to exogenous LH during the entire gestation. In pregnant rats, serum progesterone levels are consistently lower in the early morning and steadily increase to reach highest levels at 11 P.M., which indicates a daily rhythm of progesterone secretion (Dohler and Wuttke, 1974). A semicircadian increase in prolactin levels occurs during pregnancy, with highest values during the afternoon and early morning (Butcher et at., 1972; Dohler and Wuttke, 1974), which is out of phase with the rhythm of progesterone secretion. Blood levels of estrogen (Yoshinaga et al., 1969; Shaikh, 1971), LH (Linkie and Niswender, 1972; Bast and Melampy, 1972), and prolactin (Amenomori et at., 1970; Nagasawa and Yanai, 1972; Linkie and Niswender, 1972) also increase before parturition (Figs. 3 and 4). Uterine venous levels of PGF remain constant from day 18 to day 20, when progesterone declines and 20a-OH-P increases (Labhsetwar and Watson, 1974). Prostaglandin levels increase markedly on day 21, coinciding with the increase in estradiol in ovarian venous plasma. PGF levels in uterine venous blood remain on day 22, then drop within 24 hr after parturition and continue at a low level during the remainder of the postpartum period (Labhsetwar and Watson, 1974). Rat placentas are steroidogenic as indicated by their ability to metabolize [7 a3H]pregnenolone to [3H]progesterone in vitro (Chan and Leathem, 1975). The 5areduced metabolites of progesterone and other compounds including 17a-hydroxyprogesterone, androstenedione, and testosterone were isolated and characterized from placental tissue; however, there was no indication of estrogen formation. Administration of progesterone prolongs pregnancy in the rat (Boe, 1938; Barrow, 1970), and ovariectomy late in pregnancy prevents parturition (Csapo, 1969). An increased in vitro release of prostaglandins from the pregnant rat's uterus occurs on the day of expected delivery, and neither exogenous progesterone nor ovariectomy prevents this release (Harney et ai., 1974). Ovarian estrogen and progesterone do not seem to affect this release of prostaglandin. The role of fetal corticoids in inducing parturition is unclear in this species. Neither removal of fetal brain (Swaab and Honnebier, 1973) nor complete removal of the fetuses (Selye et at., 1935; Kirsch, 1938) alters duration of pregnancy. Parturition may depend on the maturity of the fetus (Yoshinaga, 1971) but may be unrelated to total uterine volume. During normal pregnancy, in vitro prostaglandin release increases from barely detectable levels on day 17 to maximal values by day 22, the expected day of delivery (Harney et at., 1974). Progesterone levels decline (Csapo and Wiest, 1969; Hashimoto et ai., 1968), estrogen secretion increases (Yoshinaga et al., 1969; Shaikh, 1971), the fetal adrenal glands increase in size (Milkovic et at.,

1973), and fetal plasma corticosterone secretion increases (Cohen, 1973). Removal of fetuses with placentas remaining in situ on day 16 or 17 reduces prostaglandin F release and spontaneous activity when measured in vitro on day 22, the expected day of delivery (Parnham et al., 1975). Phospholipase A makes available prostaglandin precursors from tissue lipids (Kunze and Vogt, 1971) and arachidonic acid serves as a precursor for prostaglandin synthesis (Anggard and Samuelson, 1965), and uterine activity increases in

6

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2400 ~ 2000

> c

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1600

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DAY OF PREGNANCY Figure 3. Ovarian function during normal pregnancy in the rat. Progesterone levels in ovarian venous blood are from Hashimoto et at. (1968) and in peripheral serum from Pepe and Rothchild

(1974). Data for 20a-hydroxysteroid dehydrogenase activity in ovarian tissue are from Bast and Melampy (1972), on 20a-hydroxypregn-4-en-3-one in peripheral serum from Wiest (1970), on estrogens in ovarian venous plasma from Shaikh (1971), and on relaxin in ovarian tissue from Anderson et at. (1973a).

609 UTERINE CONTROL OF OVARIAN FUNCTION

10000c-----------------:::I

610 CHAPTER 16

....C

Figure 4. Ovarian and placental function during normal pregnancy in the rat. Data on prolactin in peripheral serum are from Linkie and Niswender (1972). Data on rat chorionic mammotropic activity (_) as compared with prolactin levels (e) in peripheral serum throughout pregnancy are from Shiu et at. (1973) .

a:

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a: en

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... ...a: e......... a:

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c:

~ Z ~ u C

....

80

.

60

(

40 20

0

a:

Q.

o

4

8

12

16

20

24

DAY OF PREGNANCY

quiescent rat uteri by the addition of either precursor (Parnham et al., 1975). When the animals are made unilaterally pregnant after ligation of one uterine horn, there is a decrease in output of prostaglandin F from both horns, but the horn containing viable fetuses remains at a higher level of activity. Thus it seems necessary to have viable fetuses for increased activity of the prostaglandin synthetase system, and this can be demonstrated by rats having only one horn remaining pregnant. 2.3 .2.

Rabbit

In the estrous rabbit, mating induces ovulation 10-12 hr later. Progesterone, 20a-OH-P, and estradiol levels, expressed as units per ovary per hour, increase to peak values during the first 4 hr after mating, seemingly in response to endogenous release of LH (Fig. 5) (Hilliard and Eaton, 1971). By the time of ovulation, the levels of these steroids are low, and they remain low during the

period of 3-4 days required for tubal transport of embryos. When the blastocysts begin implantation between days 7 and 10, the progestin and estradiol concentrations gradually increase. Levels of the progestins continue to increase to peak values between days 12 and 24 and then decline to low values near the time of parturition on day 32 (Hilliard et ai., 1973); progesterone in peripheral plasma follows a similar pattern (Baldwin and Stabenfeldt, 1975). Estradiol levels gradually increase to day 28 and decline before parturition. A progesterone-binding protein (receptor) in the cytoplasmic fraction of the rabbit myometrium was found to indicate a decrease in receptor-site concentration after midpregnancy (Davies et ai., 1974). In nonpregnant rabbits, the concentration of progesterone receptor sites in myometrial cytosol was 4.5 pmol/mg protein; this level dropped to 1.3 pmol/mg within 3 days after mating. It remained low and declined (e.g., 0.6 pmol/mg) after mid pregnancy and was followed by an increase to 1.9 pmole/mg by day 30 of pregnancy. This pattern of change in myometrial progesterone receptor concentration in pregnant rabbits is qualitatively similar to that observed in the rat (Davies and Ryan, 1973). The concentration of progesterone in the myometrium is related to plasma levels of the steroid, which may

10 8 6

4

2 Figure 5. Ovarian function durIng normal pregnancy In the rabbit. Data on progesterone concentrations In peripheral plasma are from Baldwin and Stabenfeldt (1974). Data on progesterone, 20a-hydroxy-pregn4-en-3-one. and estradiol levels in units per ovary per hour are adapted from Hilliard and Eaton (1971) and Hilliard et ai. (1971. 1973). Relaxin activities In the blood during pregnancy are data adapted from Marder and Money (1944).

o

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8

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16 20 24 28 32

DAY

611 UTERINE CONTROL OF OVARIAN FUNCTION

612 CHAPTER 16

indicate equilibration by simple diffusion (Davies et aI., 1974). During the first twothirds of pregnancy, receptor concentration exceeds plasma progesterone, whereas during the last third of pregnancy receptor concentration is less than plasma progesterone. Myometrial concentrations of progesterone, estrone (E 1), and estradiol-17f3 (E2f3) during pregnancy in the rabbit are severalfold greater than found in peripheral plasma, and the levels are, in part, unrelated to the changes that occur in the plasma (Challis et at., 1974a). The myometrial concentrations of E1 and E2f3 remain severalfold greater than plasma levels throughout gestation, whereas progesterone concentrations in the myometrium closely parallel those found in the plasma. During the last half of pregnancy, myometrial concentrations of E2f3 increase, the Ezf3: E1 ratio increases, and the E1f3 myometrial: plasma ratio increases. None of these changes is attributed to changes in the peripheral plasma. The decline in peripheral plasma levels of progesterone is a prerequisite for onset of myometrial activity in the rabbit (Challis et aI., 1974a). A significant increase in plasma prostaglandin F concentration occurs between days 21 and 30 of gestation (Challis et at., 1973), which is coincident with the prepartum decrease in progesterone levels in plasma. Thus, during the normal pregnancy, the increasing levels of prostaglandin F may be luteolytic and induce the preparturient decline in plasma progesterone concentration.

2.3.3.

Guinea Pig

Progesterone in peripheral plasma of the guinea pig increases markedly between days 15 and 30 of pregnancy, with high levels maintained during the remainder of the gestation (Challis et at., 1971). Unconjugated estrogens in the plasma are undetectable before day 25 and increase gradually to maximal levels (e.g., 50 ng/ml) between days 55 and 60 after mating, with a slight decline occurring just preceding parturition. The increase in progesterone production rate between days 15 and 30 in intact pregnant guinea pigs results mainly from increased ovarian secretion of the steroid. The embryo seems to exert a luteotropic effect on corpora lutea. Placental production of progesterone becomes important later in pregnancy. Guinea pigs ovariectomized during the last half of pregnancy continue their pregnancies to term (Courrier et at., 1929). Plasma progesterone levels are highest at the end of gestation and are closely correlated with placental development (Heap and Deanesly, 1966). Ovariectomy, adrenalectomy, or both, do not affect plasma concentrations of total unconjugated estrogens throughout pregnancy (Illingworth and Challis, 1973). Thus both progesterone and estrogen are primarily of extraovarian sources, particularly during the second half of gestation.

2.3.4.

Cow

A preovulatory increase in the concentration of progesterone in peripheral plasma occurs about 16 hr before the onset of estrus (Ayalon and Shemesh, 1974). The surge is brief, lasting about 4 hr, with subsequent values steadily declining until the onset of estrus. This proestrous rise in plasma progesterone precedes by about 12 hr the peak in plasma estradiol, which occurs about 4 hr before the onset of estrus. In intact heifers, the preovulatory surge of increased plasma progeste-

rone levels seems to facilitate the manifestation of estrus and sexual receptivity (Ayalon and Shemesh, 1974). Peripheral blood levels of progesterone are similar between days 3 and 12 in pregnant and nonpregnant heifers, whereas by day 50 of pregnancy the progesterone levels are higher (e.g., 5.0 ng/ml) than those found in nonpregnant heifers (e.g., 0.7 ng/ml) (Hasler et at., 1975). The corpus luteum remains functional throughout pregnancy, with peripheral blood levels of progesterone declining markedly only during the last few days of gestation (Stabenfeldt et at., 1970). Peripheral plasma levels of estrone increase from about 250--400 pg/ml to about 600 pg/ml 10-13 days before parturition (Edqvist and Johansson, 1972). Estrone values increase to 900 pg/ml within a few days of parturition; they then drop to less than 300 pg/ml within 5 hr after parturition and continue to decline to less than 25 pg/ml the day after parturition. Estradiol-17/3 levels are consistently low « 100 pg/ml) during this period. Robertson (1973) found similar changes in peripheral plasma levels of El and E2/3 as well as a marked increase in E2o: a few days preceding parturition and a drop to low levels at parturition. Estrogens in bovine urine increase during late stages of pregnancy and consist of estrone, estradiol-17a, and small amounts of estradiol-17/3 (Mellin and Erb, 1965). Cotyledons extracted from bovine placenta recovered at term indicate prolactin-like activity as assessed by the biological response of alveolar distention of the mammary gland in the rat (Matthies, 1975). 2.3.5.

Ewe

Peripheral plasma levels of progesterone during pregnancy increase steadily from low values (e.g., 1-2 ng/ml) at day 1 to peak levels of 2: 9 ng/ml by day 125 and then decline until parturition (Bassett et at., 1969; Stabenfeldt et at., 1972). In ewes carrying twins, progesterone levels are significantly higher between days 60 and 130. Within 9 days before parturition, progesterone concentrations in ewes carrying single fetuses or twins show a coincident decline to 2 ng/ml by onset of parturition. Concentrations of unconjugated estrone and estradiol-17/3 begin to rise during the 48 hr preceding delivery and decline to low levels within 12 hr after parturition (Challis, 1971; Charnley et at., 1973). Pregnancy is maintained in ewes ovariectomized after day 50 (Denamur and Martinet, 1955), which implicates placental contribution to steroid production and may explain the steady decline in progesterone levels during the last 2 weeks of pregnancy. 2.3.6.

Sow

The corpus luteum is required for maintenance of pregnancy in the sow; ovariectomy as late as day 110 results in abortion within a few hours (Belt et at., 1971). Progesterone in peripheral plasma increases to peak values by days 12-20 and then declines to levels of 10-12 ng/ml until the last few days before parturition (Fig. 6). With onset of parturition, there is a rapid decline in progesterone to levels that remain low after delivery. Plasma levels of unconjugated estrone and estradiol-17/3 in peripheral blood are measurable by day 80 and rapidly increase to peak values just before parturition (Fig. 6). Two peaks of estrone sulfate occur at day 30 and day 112, respectively, and then drop at onset of parturition. Urinary estrogens show similar patterns of excretion (Bowerman et at., 1964; Rombauts,

613 UTERINE CONTROL OF OVARIAN FUNCTION

614

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Figure 6. Ovarian function during normal pregnancy in the pig. Data on progesterone levels in peripheral plasma are adapted from Baldwin and Stabenfeldt (1975), Killian et al. (1973), Robertson and King (1974), and Tillson et al. (1970). Relaxin concentrations in luteal tissue and corpus luteum weight during pregnancy are data from Anderson et al. (I 973b) and relaxin levels in peripheral plasma during the last days of pregnancy are data from Sherwood et al. (1975). Estrogens in peripheral plasma throughout pregnancy are data from Robertson and King (1974).

1962). The fetoplacental umt IS the major source of estrogen production as indicated by similar urinary excretory patterns in intact controls compared with those in sows after ovariectomy, hypophysectomy (Fevre et al., 1968), or adrenalectomy (Fevre et al., 1972). 2.3.7.

Mare

Peripheral serum levels of progesterone in the mare are low on the last days of estrus (e.g., 0.3 ng/ml) before ovulation, and then steadily increase to peak values by day 10 of diestrus and decline to less than 2 ng/ml by day 18 of diestrus (Squires et al., 1974a). In pregnant mares, progesterone levels decline between days 7 and 19, from 13 to 10 ng/ml, increase steadily to peak values of 15 ng/ml by days 90-150, and decline gradually to day 220. Implantation does not occur until approximately day 60, a time when endogenous blood levels of PMSG then begin a rapid increase for a rather brief part of the gestation (Allen, 1969). The fetus stimulates development of endometrial cups and production of PMSG at days 3740; removal of the fetus on or after this time results in normal production of PMSG (Allen and Moor, 1972).

).

Effects

of Hysterectomy on Ovarian Function

The effects of hysterectomy on ovarian function, particularly luteal function, in several species (at least 17) have been reported by numerous investigators, and recent reviews include those of Anderson et al. (1969), Anderson (1973), Bland and Donovan (l966b), and Rothchild (1965). The duration of cyclic periodicity or pseudopregnancy as affected by hysterectomy is shown for several species in Table 1. In the hamster, mouse, and rat, hysterectomy seems not to alter the brief cyclic intervals; pseudopregnancy, however, is increased to a duration similar to that of pregnancy. In the estrous rabbit, uterine removal does not alter occurrence of ovulation but extends the duration of pseudopregnancy. Cyclic periodicity is interrupted after hysterectomy in the guinea pig, ewe, sow, and heifer, and the corpora lutea are maintained for a period equal to or exceeding that of pregnancy. When the corpora eventually fail, estrus and ovulation occur, and the newly formed corpora lutea in these animals are maintained again for an equally prolonged period. Likewise, subsequent prolonged pseudopregnancies can be induced after hysterectomy in animals that experience brief estrous cycles. If the animals are hysterectomized too late in the estrous cycle or pseudopregnancy, the duration of that cycle or pseudopregnancy is not extended, but subsequent cycles or pseudopregnancies are prolonged. In species such as the ground squirrel (Drips, 1919), ferret (Deanesly and Parkes, 1933), dog (Cheval, 1934), badger (Canivenc et al., 1962), opossum (Hartman, 1925), brush possum (Clark and Sharman, 1965), rhesus monkey, and human, ovarian function and estrous or menstrual cycles continue uninterrupted after hysterectomy. Ovulations are induced in the estrous ferret, and the duration of the pseudopregnancy is similar to that of pregnancy (about 42 days); the

615 UTERINE CONTROL OF OVARIAN FUNCTION

616 CHAPTER 16

functional actlVlty of the corpora lutea, as indicated by peripheral plasma progesterone levels, shows similar patterns of secretion during these two reproductive states (Heap and Hammond, 1974). In the 4-day cyclic rat, the first indications of conversion of a corpus luteum of the cycle into a corpus luteum of pseudopregnancy occur on the morning of day 2, as indicated by maintenance of progesterone secretion (Smith et at., 1975). Hysterectomizing the pseudopregnant rat on day 4 or 5 increases the duration of that pseudopregnancy to about 21 days, which is equivalent to the duration of pseudopregnancy in rats bearing decidual tissue or to the length of normal pregnancy. The peripheral serum levels of progesterone (Pepe and Rothchild, 1974) and of LH and prolactin (Bast and Melampy, 1972) and the ovarian tissue levels of 20a-OH-SDH show similar patterns during prolonged pseudopregnancy in hysterectomized rats and in rats bearing decidual tissue. Ovarian venous blood levels of 20a-OH-P are inversely related to progesterone during prolonged pseudopregnancy resulting from hysterectomy or induction of deciduomata (Hashimoto et al., 1968). As progesterone levels decline toward the later part of the psueodpregnancy, 20a-OH-P levels and 20a-OH-SDH activities increase abruptly. Luteolytic action of the uterus in the rat is eliminated by decidualization, presence of conceptuses, or uterine removal. Dependence of the corpus luteum on luteotropic support during pregnancy is suggested by the production of rat chorionic mammotropin (Shiu et al., 1973) and during prolonged pseudopregnancy by the possibility of luteotropic action by decidual tissue (Gibori et al., 1974); prolonged pseudopregnancy after hysterectomy is explainable in terms of the removal of luteolytic action by the uterus. Patterns of secretion of prolactin are similar during these three reproductive states. In hypophysectomized rats, highly purified preparations of prolactin maintain luteal function for periods of 20 days (Macdonald et al., 1970), which supports and confirms earlier findings of Astwood (1941). Both bovine and ovine LH decrease the duration of luteal function in prolactin-treated, hypophysectomized animals (Macdonald et at., 1970), indicating a direct luteolytic effect on the ovary in the continued presence of prolactin. When the uterus is removed in these hypophysectomized rats given prolactin, the duration of luteal function is extended; exogenous LH in addition to the prolactin in these animals does not reduce the duration of luteal function. Thus in hypophysectomized rats there is an interrelation of the uterus and the ovary mediated by LH when given in the presence of prolactin. Hysterectomy during the last half of pregnancy in the rat leads to a rapid onset of maternal behavior within 2-5 days as compared with 6-7 days required for maternal behavior after hysterectomized nonpregnant animals have been exposed to young pups (Siegel and Rosenblatt, 1975). When the ovaries of pregnant rats are removed at the time of hysterectomy, the acceleration of maternal behavior fails to appear; exogenous estradiol benzoate (5 /-Lg) restores the timing sequence of this behavior. Hysterectomy between days 10 and 22 of pregnancy stimulates resumption of estrous cycles (Waynforth, 1971; Klein, 1935; Morishige and Rothchild, 1974). Hysterectomy during pregnancy leads to a decline in progesterone levels in peripheral circulation, a decline in function of corpora lutea (Takayama and Greenwald, 1973; Rothchild et al., 1973), and increased estrogen secretion, as indicated by changes in vaginal epithelium (Klein, 1935). The rise in estrogen secretion may trigger the onset of early maternal

behavior after hysterectomy during pregnancy in the rat (Siegel and Rosenblatt, 1975). The functional life of the corpus luteum of the estrous cycle in the ewe can be prolonged a variety of ways: by pregnancy (Moor and Rowson, 196fu,b), hysterectomy (Denatnur et a!., 1966; Moor et a!., 1970), exogenous estrogen (Denamur and Mauleon, 1963; Piper and Foote, 1968), and exogenous LH (Karsch et at., 1969a,b). The effects of pregnancy and hysterectomy may be attributed to removal of a uterine luteolytic stimulus. The luteotropic action of exogenous estradiol benzoate (1 mg/day) beginning on day 3 of the estrous cycle in the ewe results in prolonging the life of the corpora lutea; this luteotropic effect persists after hypophyseal stalk transection but not after hypophysectomy (Denamur et at., 1970). After hypophyseal stalk transection, the corpora lutea develop normally to day 12, but progesterone secretion is not maintained beyond day 15. Thus, in the presence of the uterus, progesterone secretion from the ovine corpus luteum is not maintained (Denamur et at., 1966, 1970). Functional corpora lutea, as indicated by progesterone in ovarian venous blood, are maintained after hypophyseal stalk transection by daily injections of estradiol benzoate. Luteal function also is prolonged after hypophyseal stalk transection combined with hysterectomy, but progesterone secretion is slightly lower than that found in ewes given estrogen. The experimental results are interpreted to mean that estrogen, like hysterectomy, prolongs the life of cyclic corpora lutea in the ewe, but it can also extend luteal function when the pituitary gland is separated from the brain (Denamur et at., 1970). Implantation of small amounts of estrogen directly in the ovine corpus luteum fails to sustain a luteotropic effect. Estrogen, like hysterectomy, prolongs the life span of ovine corpora lutea even in animals after hypophyseal stalk transection. The luteotropic role of estrogen in this species may be to inhibit or override the production of uterine luteolysin. Prolonging the life of corpora lutea in ewes during pregnancy by the infusion of LH or by hysterectomy also may result through inhibition of a uterine luteolytic effect. Removal of the uterus, even as late as day 15 in the ewe, can arrest luteolysis (Moor et at., 1970), whereas pregnancy, estrogen treatment, or infusions of LH must be initiated several days earlier to produce luteotropic effects (Karsch et at., 1969b; Stormshak et a!., 1969; Kann and Denamur, 1973). After sexually mature sows have been hysterectomized, the corpora lutea remain in a functional state, as indicated by blood progesterone levels, similar to that found during pregnancy and for a period exceeding that of gestation (Table 1) (Masuda et a!., 1967). The induction of ovulation by PMSG and HCG in prepubertal sows results in maintenance of the induced corpora lutea when these animals are either hysterectomized (e.g., days 130-140 of age) or mated, as indicated by sustained plasma levels of peripheral progesterone (Rampacek et a!., 1975), but the response may not be consistent (Salisbury et a!., 1975). Luteolysis occurs in prepubertal pigs in which ovarian function was induced by exogenous gonadotropins with the uterus remaining in situ and nongravid. Luteotropic support by adenohypophyseal hormones is required for sustaining luteal function in hysterectomized pigs. Corpora lutea develop and produce progesterone when the pig is hypophysectomized during the first few days after

617 UTERINE CONTROL OF OVARIAN FUNCTION

618 CHAPTER 16

estrus, but they regress by the end of that cycle (du Mesnil du Buisson and Leglise, 1963). Hypophysectomy of previously hysterectomized pigs results in rapid luteal regression (du Mesnil du Buisson and Leglise, 1963). Hypophysectomy between days 4 and 90 results in immediate termination of pregnancy (du Mesnil du Buisson et at., 1964; du Mesnil du Buisson and Denamur, 1969; Kraeling and Davis, 1974). After hypophyseal stalk transection of hysterectomized pigs, the previously maintained corpora lutea regress (Anderson et at., 1967b); however, if the stalk transection is delayed until day 50, only partial luteal regression occurs, and it can be prevented by daily injections of estradiol benzoate; if the stalk transection is delayed until day 70, no further estrogen treatment is required for luteal maintenance (du Mesnil du Buisson and Denamur, 1969). Hypophyseal stalk transection of pregnant pigs at days 50 results in abortion within a few days, but pregnancy is maintained when the stalk transection is delayed until day 70 or 90 (du Mesnil du Bussion and Denamur, 1969). Injections of desiccated porcine or ovine anterior pituitary, LH, or HCG sustain corpora lutea in hysterectomized hypophysectomized pigs (Anderson et at., 1965a, 1967b). Neither the pituitary preparation nor gonadotropins prevent luteal regression in hypophysectomized animals in which the uterus remains in situ. However, administration of estradiol benzoate together with LH sustains luteal function in hypophysectomized pigs, suggesting that the estrogen can effectively neutralize the luteolytic effects of an intact uterus. These results indicate that LH provides luteotropic support in hysterectomized pigs, but the relative importance of prolactin and FSH remains unclear in this luteotropic process. In heifers, hysterectomy results in an extension of the life of the corpus luteum to a period equivalent to or exceeding that of pregnancy (Anderson et at., 1962, 1965b). Adenohypophyseal hormones required for luteotropic support after hysterectomy implicate LH and prolactin. When the hypophyseal stalk is transected in previously hysterectomized heifers, the adenohypophysis sustains the corpus luteum at least 48 days as indicated by maintenance of peripheral blood levels of progesterone (Anderson, 1968; Anderson et at., 1969; Hendricks et at., 1969). Other in vivo and in vitro experimental evidence is interpreted as suggesting luteotropic action by LH in the cow (Donaldson and Hansel, 1965; Snook et at., 1969). Probable luteotropic action of LH is based on partialluteolytic effects of LH antiserum in hysterectomized animals, whereas prolactin antiserum does not alter luteal function in hysterectomized cows (Hoffmann et at., 1974). These data support the contention that LH is likely a dominant luteotropic factor in cattle. Hysterectomized mares retain the primary corpus luteum to day 30 (Ginther and First, 1971) and in a pattern similar to that of pregnant mares to day 70 (Squires et at., 1974b,c). Ovulation with formation of secondary corpora lutea does not occur by day 70 in hysterectomized mares, whereas ovulations and secondary corpora occur in a majority of pregnant mares by this time. The development of large ovarian follicles increases markedly during early pregnancy and then decreases at about the time when secondary corpora lutea begin to form (days 3470). A similar pattern of follicular development occurs after hysterectomy, which may indicate dependence on hypophyseal or ovarian factors other than PMSG during this time. An acyclic secretory pattern of progestin levels in peripheral plasma after hysterectomy during the luteal phase of the estrous cycle in the mare is indicative of a prolonged life span of the corpus luteum (Ginther and First, 1971;

Stabenfddt et al., 1974). Cyclic regression of the corpus luteum in the nonpregnant mare usually is dependent on the presence of the uterus. Follicular growth and ovulations may continue after hysterectomy in the presence of a functional corpus luteum, but behavioral estrus is not observed. When the mares are hysterectomized, progesterone levels decline steadily from 10 ng/ml at day 7 to less than 1 ng/ml by day 140. Changes in progesterone levels are similar in pregnant and hysterectomized mares from day 7 to day 19, but progesterone in the hysterectomized animals continues to decline, whereas it increases after day 32 in pregnant mares. Continuation of ovarian cycles after hysterectomy in the rhesus monkey indicates no dependence on a uterine luteolytic stimulus for cyclic periodicity (Knobil, 1973; 1974; Neill et aI., 1969). Antiserum to HCG, which binds monkey LH, given during the luteal phase of the cycle results in premature onset of menstruation (Moudgal et at., 1971). Endogenous levels of LH remain low during the luteal phase of the menstrual cycle and are interpreted as providing a permissive rather than controlling influence on luteal function (Knobil, 1973, 1974). Exogenous estradiol benzoate induces premature luteal regression (Dierschke et at., 1973), whereas endogenous estrogens are produced within the corpus luteum and may lead to its own regression, and thus play a role in cyclic periodicity (Knobil, 1973). Luteal function soon after conception depends on chorionic gonadotropic stimulation, with the corpus luteum being sustained throughout the gestation.

4. 4.1.

Luteolytic Action Amount

of the

Uterus

of Uterus Required for Luteolysis

In those species in which hysterectomy prolongs the luteal phase, only a small nongravid uterine segment is required for maintaining cyclic periodicity. In the rat, the duration of pseudopregnancy is increased after unilateral as well as bilateral hysterectomy, but the initiation of luteal regression during pseudopregnancy occurs earlier, as the amount of nontraumatized endometrium increases (Melampy et at., 1964). When the endometrium in the remaining uterine segment is converted to decidual tissue, the pseudopregnancy is prolonged. Examples of species in which normal ovarian cycles are maintained after partial hysterectomy are guinea pigs with almost a whole of one uterine horn remaining (Butcher et at., 1962), sows with almost one quarter of one horn (Anderson et al., 1961), and heifers with only the cervix and a small posterior segment of the uterine body (Wiltbank and Casida, 1956; Anderson et al., 1962).

4.2.

Local Luteolytic Action

In guinea pigs, sows, cows, and ewes, the uterus influences luteal regression (Anderson et at., 1969), possibly through release by the uterus of a luteolytic

619 UTERINE CONTROL OF OVARIAN FUNCTION

620 CHAPTER 16

substance that reaches the adjacent ovary. When a small segment of uterine horn (e.g., < 26 cm) remains after partial hysterectomy in the sow, the corpora lutea in the ovary ipsilateral to that uterine segment regress earlier than those in the contralateral ovary (du Mesnil du Buisson, 1961). Local control of luteal function has been found in other species such as the hamster, guinea pig, ewe, and cow, as indicated by unilateral luteal regression after partial hysterectomy. The luteolytic effect of a nongravid segment of uterus also may be found during early pregnancy. For example, in the sow (du Mesnil du Buisson, 1961; Anderson et aI., 1961; Anderson, 1966; Niswender et aI., 1970) and ewe (Moor and Rowson, 1966b), the presence of conceptuses overrides uterine luteolytic action, but a small non gravid uterine segment near an ovary induces luteolysis in that ovary while luteal function continues in the contralateral ovary acUacent to the gravid horn. The comparative anatomy of uterine and ovarian vasculature in several laboratory and domestic species indicates that presence or absence of local pathways between a uterine horn and the ipsilateral ovary could be explained on the basis of anatomical relations (Ginther, 1974). Transport of a uterine luteolysin through venoarterial pathways may involve veins draining the uterus and the ovarian artery. Barrett et al. (1971) suggested that the luteolysin passes from the utero-ovarian vein to the ovarian artery. This is based on maintenance of corpora lutea in ewes after surgical separation of the vein and artery. By surgical anastomosis of the main uterine vein of one uterine horn to the corresponding vein in the opposite horn in bilaterally ovulating ewes, luteotropic effects can be ascertained when an embryo resides in one of the uterine horns (Mapletoft and Ginther, 1975; Mapletoft et at., 1975). In the sham-operated controls, the corpus luteum regresses in the ovary contralateral to the uterine horn containing the conceptus. Diversion of uterine venous blood to either the nongravid or the gravid horn results in maintenance of corpora lutea in both ovaries, but the corpus luteum is consistently heavier in the ovary ipsilateral to the horn containing the conceptus. The corpus regresses when the adjacent uterine vein contains blood from only the nongravid horn. Thus the uterine venous effluent from a gravid horn in the ewe either exhibits a luteotropic effect or prevents luteolytic action. In heifers, surgical isolation of the broad ligament along the ovarian pedicle, which effectively isolates the ovary from all uterine connections, extends luteal function to day 30, as indicated by ovarian structure and maintenance of progesterone levels (Hixon and Hansel, 1974). Intrauterine administration of a rather high dose (6 mg) of prostaglandin F2a in the uterine horn ipsilateral to the ovary containing the corpus luteum induces luteolysis, as indicated by a rapid decline in plasma levels of progesterone and reduced luteal weight. Peripheral plasma levels of estrone and estradiol-17f3 increase to peak values within 9 hr after prostaglandin treatment. The authors interpret these experimental results to indicate local countercurrent transfer of uterine luteolytic effects to the ovary. Futhermore, they suggest that the increased estrogen levels may play a role as possibly a second factor in the PGF2a-induced luteolysis in heifers.

4.3.

Luteolytic Effects of Estrogens in the Ewe

Exogenous estrogens have both luteotropic and luteolytic effects in the ewe (Stormshak et aI., 1969; Hawk and Bolt, 1970; Denamur et aI., 1970; Howland et

al., 1971; Bolt et al., 1971), whereas removal of endogenous estrogens by destruction of ovarian follicles results in luteal maintenance (Karsch et al., 1970; Ginther, 1971). Both luteinizing hormone and prolactin are luteotropic in the ewe (Schroff et al., 1971; Denamur et al., 1973), and exogenous prostaglandin (PGF 20J is luteolytic (McCracken et al., 1970). Luteotropic actions of estrogens are attributed to release of endogenous LH (Howland et al., 1971; Bolt et al., 1971) and luteolytic actions of estrogens by stimulation of uterine synthesis and release of a luteolytic substance that induces regression of the corpora lutea (Caldwell et al., 1972). Denamur et al., (1970) administered estrogen daily, beginning early in the cycle, and concluded that the estrogen probably acts on the uterus to prevent normal luteolysis. Removal of the uterus before beginning a series of injections of estradiol prevents luteolysis (Akbar et al., 1971). Thus the luteolytic effect of the estrogen requires the presence of the uterus (Bolt and Hawk, 1972). During the estrous cycle in the ewe, [3H]estradiol-17{3 is taken up and retained by the luteal cells at maximal rates between days 8 and 12, with little or no retention during luteinization (days 2-5) or luteal regression (days 13-15) (Sheridan et al., 1975). Uptake and retention of estradiol-17{3 are inhibited when progesterone synthesis by the ovine corpus luteum is stimulated by exogenous LH. Warren et al., (1973) administered estradiol alone or in combination with progesterone at different stages of the estrous cycle in the ewe to ascertain the onset of luteal regression. They found that when the estradiol (1 mg/day) is given on days 5 and 6, the corpora lutea maintained; however, when both progesterone (25 mg/day) and estradiol are administered, luteolysis occurs by day 9. Estradiol alone effectively induces luteal regression by day 14 when given on days 9 and 10, but maintains luteal function if administered from the beginning of the estrous cycle. When a small daily dosage (200 JLg) of the steroid is given before treatment with 1 mg estradiol on days 9 and 10, the corpora lutea remain functional to day 14. Luteolysis occurs when progesterone is given concurrently with the lower dosage of estradiol. Progesterone may facilitate luteolysis by acting at sites that are partly or entirely in the uterus and involved in the maintenance or regression of the corpus luteum.

5.

Ovarian Autotransplantation

Autotransplanting ovaries to a site distant from the uterus prolongs the life of corpora lutea in the rat (Anderson et al., 1969), guinea pig (Bland and Donovan, 1968), hamster (Duby et al., 1969), heifer (Hansel and Snook, 1970), and ewe (Goding et al., 1967b). In rats bearing autotransplanted ovaries in the renal capsule, pseudopregnancies are extended 3--4 days beyond those in sham-operated animals (13 days). When one or both uterine horns are removed in these animals with transplanted ovaries, the pseudopregnancies are prolonged to 20-24 days. Autografting ovaries to the uterus, however, reduces the duration of pseudopregnancy to less than that in sham-operated rats (Anderson et al., 1967a) and in guinea pigs reduces the duration of vaginal cycles (Bland and Donovan, 1968).

621 UTERINE CONTROL OF OVARIAN FUNCTION

622

6.

Uterine Transplantation

CHAPTER 16

Uterine transplantation (autotransplants, homotransplants, or heterotransplants) has been used to ascertain whether uterine innervation plays a role in the regulation of ovarian function. Experimental evidence from several species (e.g., rat, hamster, rabbit, guinea pig, dog, pig, and sheep) indicates that major nervous pathways to the uterus are not essential for uterine luteolytic action (Anderson et al., 1969; Anderson, 1973). Transplants with viable endometrial glands and epithelium are more capable of inducing luteolysis than those that contain few glands. In hysterectomized hamsters, transplants of endometrium to the cheek pouch reduce the duration of pseudopregnancy to 13.5 days, as compared with 18 days in hysterectomized controls; transplants of myometrium are ineffective (Caldwell et al., 1969). In sows, uterine autotransplants to abdominal muscles or to the peritoneum effectively induce luteolysis (Anderson et al., 1963), but similar transplants to the small intestine are ineffective because the liver inactivates the luteolytic effect of the autograft (du Mesnil du Buisson and Rombauts, 1963). Endometrial autotransplants to flank muscles reduce the life span of corpora lutea in hysterectomized ewes, but endometrial tissue grafted in the corpus luteum at hysterectomy induces only local degeneration of lutein cells and a prolonged diestrous phase (Caldwell et at., 1969). Local luteolytic effects are evident from autotransplantation of the uterus, ovaries, or both to the neck (Goding et ai., 1967a,b; Harrison et at., 1968). When the ovary is autotransplanted to the neck in unilaterally ovariectomized animals, the corpus luteum is maintained (Baird et at., 1968). A similar prolonged luteal phase occurs when a uterine horn is transplanted to the neck and an ovary remains in the abdomen. However, transplanting an ovary and a uterine horn to the neck of a ewe in which the other ovary and horn are removed results in estrous intervals of normal duration. These results favor a local, as opposed to a systemic, action of the uterus on the function of the corpus luteum. Thus experimental evidence provided by surgical techniques utilizing uterine transplantation in several species suggests that the uterine luteolytic effect is mediated by local and systemic pathways, and only endometrium is essential for this action.

7.

Intrauterine Devices and Ovarian Function

A foreign body inserted into the uterine lumen alters the length of ovarian cycles by affecting development and maintenance of the corpus luteum in several mammalian species. The presence of the intrauterine device results in an asynchronous maturation of the endometrium, which is not compatible with implantation. An intrauterine device (IUD) shortens estrous cycles in the cow, ewe, guinea pig, and goat by interfering with development and maintenance of the corpora lutea. The length of an estrous cycle in the rat, hamster, or pig or that of a menstrual cycle in the monkey and human is unaffected by the presence of an IUD, but implantation is prevented in these species as well as in the rabbit

(Anderson et at., 1969; Corfman and Segal, 1968; Duncan and Wheeler, 1975; Hawk, 1968). When an IUD is introduced into the uterine lumen in the early part of the estrous cycle in the cow, ewe, or guinea pig, the corpora lutea regress prematurely, whereas insertion in the later part of the cycle can prolong that estrous-cycle interval. In the ewe, the initial and subsequent cyclic intervals are reduced to ~13 days when an IUD is introduced on day 3, but the estrous intervals are extended beyond 20 days when the device is inserted on day 8 (Moore and Nalbandov, 1953). The cycle length is unaltered when the IUD is inserted as late as day 13 (Nalbandov et at., 1955). These ewes resume normal estrous cycle intervals (16 days) after the IUD is removed. The premature regression of the corpus luteum in the presence of an IUD is induced by a luteolytic effect from the stimulated uterus. This luteolytic action may be local, i.e., induction of luteolysis in the adjacent ovary whereas corpora lutea in the opposite ovary remain unaffected. Denervation of the uterine segment containing the IUD nullifies the effect, and normal estrous intervals then continue. A local luteolytic action is demonstrated in the guinea pig by the insertion of glass beads into only one of the uterine horns. Although corpora lutea develop in both ovaries, those in the ovary adjacent to the horn containing the beads regress earlier, whereas luteal regression occurs at the normal time in the opposite ovary (Bland and Donovan, 1966a). In those species in which only one ovulation occurs, an IUD exerts a greater luteolytic effect, as indicated by reduced estrous-cycle intervals, when the ovary containing the corpus luteum is adjacent to the horn containing the IUD. Several actions may be considered in the contraceptive effects of an IUD; these include increased mechanical traumatization of the endometrium, increased peristalsis of oviductal and uterine musculature, increased infiltration by leukocytes, or induced luteolysis. The IUD may act by disrupting the implanting embryo, inducing premature transport of eggs into a hostile uterine environment, altering luteal function to a degree incompatible with implantation, or producing leukocytic infiltration into the uterine lumen.

7.1.

IUD in Rat and Rabbit

An IUD inserted into the uterine lumen of only one horn of the rat does not interfere with early embryonic development in the contralateral uterine horn. Presence of an IUD is associated with increased levels of cyclic AMP and a slight increase in phosphodiesterase activity in the uterus during the estrous cycle and early pregnancy (Sim, 1974). The increase in uterine weight caused by an IUD in the uterine horn of the rat can be partly prevented by exogenous indomethacin and aspirin (Chaudhuri, 1975). The IUD-induced increased uterine weight may be mediated, in part, by endogenous release of prostaglandin. The leukocytic invasion of the endometrium renders the uterine horn containing the IUD sterile in the rat, while conceptuses develop in the opposite intact horn (Doyle and Margolis, 1963). An inflammatory response results from leukocytic infiltration, particularly near the site of the IUD. Leukocytic extracts recovered from the horn containing the IUD are embryotoxic (Parr, 1969). An IUD in the uterine horn of

623 UTERINE CONTROL OF OVARIAN FUNCTION

624 CHAPTER 16

the rabbit prevents implantation, but only at sites near the IUD; embryonic development proceeds in the other locations within that horn (Adams and Eckstein, 1965a,b). In both rats and rabbits, there is a quantitative relation between the amount of luminal leukocytic infiltrate and contraceptive action. The blastocyst is particularly vulnerable to toxic cellular substances migrating from the segment of the horn containing the IUD (EI Sahwi and Moyer, 1971; Joshi and Gunn, 1971). By using the technique of double transfer of embryos, which is well suited for distinguishing uterine from embryonic effects, the embryotoxic effects of the intrauterine environment can be ascertained in rats having an IUD (DeBoer and Anderson, 1971). The embryos are subjected to the environment of the IUDbearing uterus for only a few hours before being retransferred. It was found that exposure of embryos to an IUD-bearing uterus for 2~4 hr resulted in failure to recover 85% of them. Even the few embryos recovered during these periods of exposure to the IUD environment show greatly increased mortality when transplanted into a normal pseudopregnant recipient. Removing the IUD 6, 24, 48, or 72 hr before embryo transfer increases both the recovery and survival rates over those obtained with the IUD in situ. Thus embryo transfer experiments indicate that an IUD inhibits pregnancy in the rat by directly causing the death of embryos within 4 hr.

7.2.

IUD in Ewe

In the ewe, an IUD intensifies the inflammatory response of the uterus, resulting in bactericidal and spermatocidal activity (Hawk, 1970; Warren and Hawk, 1971). Furthermore, the IUD in this species interferes with transport of spermatozoa and the fertilization process, and reverses the direction of uterine contractions (Hawk, 1965, 1967, 1970). Insertion of a large IUD (8-mm-diameter polyethylene spiral of 50 mm length) in ewes on day 3 of the estrous cycle was found to reduce cycle intervals to 5-8 days in length, and peripheral progesterone levels remain consistently low from day 3, even in those ewes not expressing estrous behavior after insertion of the IUD (Hecker et aI., 1974). When the size of the IUD is reduced (2-mmdiameter, 50-mm-Iong polyethylene spiral), half the cycle intervals are shortened, but blood progesterone levels remain low. An IUD consisting of monofilament nylon (0.2 mm diameter) fails to interrupt normal cycle intervals, and peripheral progesterone levels remain similar to those found in intact controls. When a large IUD (8 mm diameter) is inserted into each uterine horn on each day of the estrous cycle, cyclic intervals are consistently reduced and peripheral progesterone levels remain low for ewes subjected to insertions from day 0 to day 3 (WodzickaTomaszewska et at., 1974). IUD insertions after day 3 result in normal or prolonged cycles, and progesterone concentrations up to day 14 are similar to those in control ewes at the same stage of the estrous cycle. In some ewes, there is an extended period between the end of luteal function and the exhibition of behavioral estrus. Ovarian hormones affect the responsiveness of ewes to the luteolytic effect of an IUD. Destruction of follicles at days 1 and 3 by electrocautery delays luteolytic action of an IUD inserted on day 1 (Ginther, 1971). Progesterone prevents IUD-

induced luteolysis (Bhalla and Casida, 1970; Warren and Hawk, 1972) and decreases levels of prostaglandin F2a in endometrium after ovariectomy (Wilson et at., 1972b).

The insertion of an IUD on day 2 of the estrous cycle in ewes initiates premature regression of corpora lutea and an increase in the concentration of prostaglandin F2c< in endometrium by day 5 (Wilson et at., 1972a). Endogenous concentrations of prostaglandins in the ovine endometrium are greatest near an IUD, as compared with non-IUD segments of the uterus (Spilman and Duby, 1972). An IUD in either one or both uterine horns of the ewe results in higher concentrations of prostaglandin F in uterine venous plasma from both horns as compared with levels found in controls (rexton et at., 1975a). The uterine tissue from ewes with an IUD contains higher concentrations of prostaglandin F in each horn as compared with intact controls. Furthermore, caruncular tissue in the uterine horn containing the IUD has a higher concentration of rGF than does the non-IUD horn, while intercaruncular tissue responds with rGF synthesis whether the IUD is in the same or the contralateral horn; myometrial levels of rGF remain low. Thus since levels of rGF remain similar in venous plasma collected ipsilateral or contralateral to the corpus luteum, the corpus does not seem to exhibit a local effect upon the release of rGF from uterine tissue. Anatomical evidence indicates that the main uterine vein serves as a proximal component of a direct pathway between the uterine horn and ipsilateral ovary in luteolysis induced by the uterus in the ewe (Baird and Land, 1973; Ginther et at., 1973). Ginther and Bisgard (1972) found that the luteolytic action of an IUD contralateral to an ovary containing the corpus luteum involved a direct pathway between uterine horns and that this pathway was not through the uterine luteum or uterine tissues. Numerous prominent veins anastomose the right and left uterine venous systems in nonpregnant ewes (Del Campo and Ginther, 1973) and are responsive to exogenous estrogen as indicated by increased uterine blood flow (Rosenfeld et at., 1972). Indomethacin, an anti-inflammatory drug, blocks the increase in prostaglandin caused by insertion of the IUD and prevents IUD-induced luteal regression. Uterine inflammation induced early in the estrous cycle of the ewe by intrauterine irUections of chemicals or bacteria induces premature luteal regression (Brinsfield and Hawk, 1968; Woody et at., 1969). Actinomycin D injected into the uterine lumen on day 11 of the estrous cycle delays cyclic regression of the corpora lutea in ewes (French and Casida, 1973). It has been suggested that synthesis of a luteolytic substance by the uterus at the termination of the cycle may depend on induction of RNA synthesis. Intrauterine administration of actinomycin D on day 2 blocks the luteolytic effect of an IUD on day 8, but actinomycin D alone also reduces the mean weight of the corpora lutea (French and Casida, 1974).

7.3.

IUD in Monkey and W011Uln

In women and monkeys, an IUD does not alter ovulatory cycles or corpus luteum development but causes localized response of the endometrium. Soon after IUD insertion, polymorphonuclear leukocytes, lymphocytes, monocytes, and

625 UTERINE CONTROL OF OVARIAN FUNCTION

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erythrocytes are found in the stroma and in the uterine glands, whereas macrophages, neutrophils, and lymphocytes increase in number later (Sagiroglu, 1971; Mastroianni and Rosseau, 1965). The large number of macrophages accumulating in the IUD-bearing uterus may phagocytize the blastocyst and thus account for the action of the IUD in women (Sagiroglu and Sagiroglu, 1970).

8.

Other Horrrwnes Affecting Uterine-Ovarian Function

8.1.

Prostaglandins

Prostaglandins are 20-carbon unsaturated hydroxy acids grouped in four main series as A, B, E, and F, according to differences in the 5-carbon ring structure. At least 15 prostaglandins have been isolated from mammalian tissues, and they induce a variety of physiological actions within the body (for reviews, see Bergstrom, 1967; Berhman and Anderson, 1974; Pharriss and Shaw, 1974; Pike, 1973; Walpole, 1975). They are synthesized from phospholipid and arachidonic acid precursors and those of the E and F series are quickly metabolized. Pharmacological doses of certain ones (e.g., from the E and F series) as well as synthetic analogues produce marked responses of reproductive organs in both sexes. Of particular interest is the capability of administered prostaglandins to induce premature luteolysis for regulation of sexual cycles (McCracken et al., 1972b; Mauleon and Ortavant, 1975), to affect ovulatory processes, and to terminate pregnancy. Evidence of possible roles of endogenous prostaglandins in regulation of cyclic ovarian function and interaction with uterine physiological events exists for certain animals (e.g., ewe; McCracken et al., 1972a,b, 1973; Goding, 1974) and is suggested for other animals such as the guinea pig, sow, cow, and mare. 8.1.1.

Rat and Rabbit

8.1.1 a. Physiological Effects. Levels of PGF2o: in blood and uterine tissue vary with the stage of the estrous cycle in the rat (Saksena and Harper, 1972). Estrogens induce uterine hyperemia and hypertrophy; similar uterine vasodilatation results when PGEl is administered to the rat (Clark et al., 1973). Indomethacin, a potent anti-inflammatory agent, also is a potent inhibitor of prostaglandin synthetase (Lerner et at., 1975). Estrogens (e.g., diethylstilbestrol) and nonsteroidal antiinflammatory agents are capable of inhibiting prostaglandin synthetase. Under in vitro conditions, there is a gradual increase in PGF output from low levels at day 17 of pregnancy in the rat to maximal levels by day 22, the day of expected delivery (Harney et al., 1974). When fetuses are surgically removed on day 16 or 17 from only one uterine horn, fetuses in the remaining uterine horn as well as placentas from the intact and fetectomized uterine horn are delivered normally (Parnham et al., 1975). Removal of fetuses from one or both horns reduces PGF output and spontaneous uterine activity in vitro, which is interpreted as indicating that the presence of viable fetuses exerts some control over prostaglandin synthesis. Quiescent uteri respond to exogenous PGF2a by restoration of rhythmic contrac-

tions. Furthermore, actIvity is increased in quiescent uteri by arachidonic acid (5ILg/ml) or phospholipase A (160 mlL/ml), but is inhibited by indomethacin (20 ILg/ml). 8.1.1 b. Pharmacological Effects. Infusion of prostaglandin F2a at selected stages after mating terminates pregnancy in the rat (Nutting and Cammarata, 1969; Fuchs and Mok, 1973; Fuchs et al., 1974). The PGF2a is particularly effective in terminating pregnancy when given at days 9-12 or days 18-20; pregnancy is interrupted at these stages at levels of 125 ILg or more. PGE 1 and PGE2 are much less effective than PGF2a in terminating pregnancy in this species. Treatment of pregnant rats with PGF2cx or PGE2 has no effect on il5-3f3-0H-SDH enzyme activity in the ovary, whereas histochemical evidence for 20a-OH-SDH enzyme activity appears in the corpora lutea after infusion of PGF2cx (Fuchs and Mok, 1974). PGF2cx given on day 9 or later is associated with a rapid increase in 20a-OH-SDH activity in the corpora lutea of pregnancy. PGF2a terminates pregnancy only at periods when intense 20a-OH-SDH activity appears in the ovaries after infusion of the fatty acid. During normal pregnancy, progesterone levels in blood are declining when 20a-OH-SDH levels increase (Wiest et al., 1968; Bast and Melampy, 1972). A negative correlation is found in all instances between prostaglandin-induced alterations in 20a-OH-SDH activity and plasma progesterone levels (Fuchs et at., 1974). The action of PGF2cx on 20a-OH-SDH activity is masked by exogenous LH, but not by exogenous prolactin, which would suggest an inhibition by PGF2cx of the action of LH on luteal function. Neutralization of endogenous LH by LH antiserum leads to similar results as does PGF2a treatment in pregnant rats with regard to ovarian 20a-OH-SDH activation and interruption of pregnancy (Loewit et aI., 1969). LH may control the activity of this enzyme in rat ovary, and thus the luteolytic action of PGF2a results from an inhibition of the suppressive effect of endogenous LH on 20a-OH-SDH activity (Fuchs and Mok, 1974). Intravenous infusion of prostaglandin F2a during a brief period in pseudopregnant rabbits causes no consistent changes in vascular resistance of the corpora lutea, whereas there is a pronounced vasodilatation in the interstitial tissue (Janson et at., 1975). Luteal blood represents a predominant part of the total ovarian flow, whereas the interstitial vasodilatation causes only negligible changes in blood flow to the whole ovary. The experimental results do not support the hypothesis of a prostaglandin-induced reduction in luteal blood flow preceding luteolysis. 8.1.2.

Sow, Cow, and Mare

8.1.2a. Physiological Effect). Phospholipid, free fatty acid, and microsomal fractions of bovine endometrium are luteolytic, as indicated by reduction of luteal weight and peripheral plasma levels of progesterone in pseudopregnant hysterectomized hamsters (Shemesh et at., 1974). The active luteolytic substance is further characterized as arachidonic acid by R f values in thin-layer chomatographic systems, by retention times in gas-liquid chromatography, and by mass spectra for comparison with arachidonic acid. When arachidonic acid, isolated from bovine endometrium, is infused into the ovarian bursas of hamsters at levels of 100 ILg, luteolysis occurs (Hansel et al., 1975). Intraperitoneal injections of arachidonic acid (12-25 mg) also are luteolytic in pseudopregnant rabbits (Hoffman, 1974). Injections of the fatty acid directly into the corpus luteum of heifers on day 12 or

627 UTERINE CONTROL OF OVARIAN FUNCTION

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13 of the estrous cycle results in partial regression of luteal tissue (Shemesh and Hansel, 1975b). Thus the bovine corpus luteum seems capable of converting arachidonic acid to prostaglandin. Prostaglandin F (PGF) levels in bovine endometrium and uterine venous blood rise at the time the corpus luteum begins to regress and plasma progesterone levels fall (Table 2) (Shemesh and Hansel, 1975a). By the time the signs of estrous behavior are detected, the PGF levels have already begun to decline. The rise and fall of PGF before onset of estrus correspond with the elevation and decline in peripheral plasma testosterone (She mesh and Hansel, 1974) and estradiol (Hansel et al., 1973), which occur 3 days preceding estrus. Increasing levels of testosterone may reflect a role of testosterone as a precursor of estrogen, as well as a role in behavioral estrus. Thus endogenous levels of prostaglandin in the bovine endometrium are correlated with events leading to luteolysis during the estrous cycle. 8.1.2b. Pharmacological EfJect~. In the pig, an intramuscular injection of either 10 or 20 mg PGF2" during the luteal phase of the estrous cycle induces luteal regression, but the animals do not return to estrus or ovulate before day 16 (Douglas and Ginther, 1975a); lower levels of PGF2" (2 or 5 mg) given during the micllllteal phase fail to cause luteolysis (Diehl and Day, 1974). In heifers, the introduction of prostaglandin F2" into the uterine horn ipsilateral to the corpus luteum induces premature luteolysis (Liehr et al., 1972; Louis et al., 1974) provided that it is given after day 4 of the estrous cycle (Rowson et al., 1972). Intrauterine infusion requires only 0.5 mg PGF2" as compared with 1530 mg of the fatty acid when injected intramuscularly (Anderson, 1975). A single intrauterine administration of 350 f.-tg of a PGF2" analogue (ICI 79939) effectively induces luteolysis; intramuscular injection of 800 f.-tg and 200 f.-tg on 2 consecutive days also results in precise synchronization of estrus in heifers (Tervit et al., 1973). In heifers, the increase in luteinizing hormone at 6 hr after injection of PGF2" seems related to effects of progesterone withdrawal rather than to a direct action of the PGF2" on the hypothalamic-hypophyseal axis (Louis et al., 1975). Evidence from several trials indicates that an intramuscular injection of PGF2" (e.g., 30 mg) beginning after day 4 of the estrous cycle effectively induces premature luteolysis, with estrous and ovulation occurring within 2-4 days after treatment (Inskeep, 1973; Lauderdale et al., 1974). Administration of lower doses (e.g., 8 and 4 mg on

Table 2. Endugenuus Lel!els of Prustaglandin F in BUliine Endometrium and Uterine Venuus BLood during the Estrous Cyde a Endometrium Days of estrous cycle

(ng/g)b

1-5 10-14 15-17 20-0

54 ± 15 42 ± 9 128±8 135 ± 10

a b

From Shemesh and Hansel (1975a). Mean ± SE.

Uterine venous blood (ng/ml)b

0.138 0.187 2.850 l.470

± ± ± ±

0.031 0.057 0.220 0.103

consecutive days) of the prostaglandin is equally effective in reducing cycle intervals (Anderson, 1975). Abortion is induced within 1 week as a result of PGF2", (e.g., 45 mg) injected before day 120 of the pregnancy (Lauderdale, 1975). In mares, PGF2", induces premature luteolysis when given subcutaneously, intramuscularly, or by intrauterine infusion (Noden et al., 1973; Allen and Rowson, 1973; Douglas and Ginther, 1975b). Estrus and ovulation are synchronized when PGF2", is administered as a single injection on day 7, 10, or 13 after the previous ovulation, but not when mares are given the PGFZty on day 1 or 4 post ovulation (Douglas and Ginther, 1975a). When the prostaglandin is injected on day 7 post ovulation there results a rapid decline in progesterone levels in jugular blood within 24 hr, regardless of route of administration (intramuscular, intralateral, or intrauterine). The mares return to estrus and ovulation occurs approximately 11 days later. Intraovarian arterial injection of small amounts (125 and 250 f.Lg) of PGF2", reduces estrous intervals and peripheral plasma levels of progesterone and results in increased luteinizing hormone levels in plasma in mares during the early luteal phase (Douglas et al., 1975). Intracarotid injections of the same dose of the prostaglandin do not induce premature luteolysis. The pharmacological effects of prostaglandin-induced luteolysis in mares may result as a direct action on the ovary rather than mediation via the hypothalamic-hypophyseal axis. 8.1.2c. Pharmacological Effects in Hysterectomized Animals. In sows hysterectomized during the early part of the estrous cycle, luteolysis is induced by exogenous PGF2", (5, 10, or 20 mg) (Muljono et aI., 1974; Kraeling et aI., 1975). Injections of estradiol benzoate partly protect the corpora lutea from luteolytic effects of prostaglandin in these animals. In heifers and cows hysterectomized during the estrous cycle, PGF2", (e.g., 10 or 30 mg) induces luteolysis consistently, whereas a smaller dose (5 mg) effectively induces luteolysis after hysterectomy but not before operation (Stellflug et aI., 1975; LaVoie et al., 1975). The period during luteolysis in these hysterectomized animals is indicated by reduced blood levels of progesterone and reduced weight of the corpus luteum, as compared with maintained luteal function in salineinjected controls. Thus the presence of the nongravid uterus is not essential for induction of luteolysis by PGF2m although the response is dose dependent. 8.1.3.

Ewe

8.1.3a. Physiological Effects. In the ewe, preliminary evidence indicates that PGF2m released from the uterus, may reach the ovary containing the corpus luteum by a countercurrent mechanism (McCracken et aI., 1970, 1971). Ligation of the uterine artery and vein, but not the artery alone, inhibits luteal regression (Kiracofe et aI., 1966), and separation of the structures between the ovary and the uterus also results in prolonged maintenance of luteal function (Inskeep and Butcher, 1966). When the uterine vein remains intact, ovarian cycles continue, which suggests that a luteolytic substance is transported from the uterus through the uterine vein (Baird and Land, 1973). Endogenous levels of PGF2", in ovine endometrium indicate a significant increase by day 14 of the estrous cycle, as compared with earlier stages (days 3-11) (Wilson et al., 1972b). PGF2", also increases significantly in uterine venous blood by late stages (day 15) of the estrous cycle (Bland et al., 1971; Thorburn et aI., 1972). Uterine venous plasma collected from

629 UTERINE CONTROL OF OVARIAN FUNCTION

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donor ewes around the time of luteal regression during the estrous cycle (day 15) depresses progesterone secretion when the plasma is infused into the ovarian arteries of ewes bearing an ovarian autotransplant (Baird et ai., 1973). The luteolytic activity of uterine venous plasma collected at days 14-16 is greater than that of plasma obtained at days 9-11. Also, the PGF2a concentrations are higher in plasma samples collected during the last days of the cycle than in those from day 9 to day 11. The mechanism by which the ovine embryo prevents luteolysis may involve uterine prostaglandin. In pregnant and nonpregnant ewes, plasma concentrations of prostaglandin F on day 15 are similar in the ovarian artery (1.1 vs. 0.6 ng/ml) and in the ovarian vein (0.8 vs. 0.6 ng/ml) (Pexton et at., 1975b). Blood flow from the ovarian arteries is approximately 4.5 times greater in the pregnant animals; the quantity of PGF transported via the ovarian arterial plasma is higher in pregnant ewes (2.1 vs. 0.5 ng/ml). Concentrations of PGF in ovarian venous plasma are not correlated with levels of PGF in ovarian arterial plasma or luteal tissue in either pregnant or nonpregnant ewes. Mattner and Thorburn (1969) found that ovarian venous blood flow declines progressively from day 13 to day 16 of the estrous cycle. Distribution of radiolabeled microspheres indicates a decrease in blood flow in the ovarian arteries from day 12 to day 16 of the estrous cycle, but not in pregnant ewes (Niswender et al., 1973). Pexton et al. (l97Sb) concluded that prevention of luteolysis in the ewe by pregnancy, as diagnosed by day 15, does not seem to be caused by changes in the levels of PGF in the ovarian artery or vein, or in ovarian or luteal tissue. Other evidence indicates that, during early pregnancy in the ewe, prostaglandin levels in uterine vein blood remain low at day 15, whereas in the cycle the prostaglandin increases markedly with approach of the next estrous period (Thorburn et at., 1973; Barcikowski et at., 1974). Furthermore, mechanical stimulation (e.g., massage) of the ovine uterus fails to stimulate PGFfu release at day 15 in pregnant ewes, but results in a marked release of the lipid as early as day 13 in nonpregnant animals (Barcikowski et al., 1974). A single injection of estradiol-17f3 given during the late luteal phase of the estrous cycle in ewes induces premature luteolysis (Stormshak et al., 1969; Hawk and Bolt, 1970); this effect is abolished by hysterectomy. Endogenous estrogens in the ewe may playa role in the synthesis or release of a uterine luteolysin. Infusion of physiological amounts of estradiol-17f3 directly into the arterial supply of the ovine uterus results in a marked release of PGF2", whereas systemic infusion of the same amount of the steroid does not stimulate PGF2a release (Barcikowski et at., 1974). Local infusion of estrogen into the uterus late in the estrous cycle induces PGF2a release. The mechanism by which the estrogen stimulates PGF2a release is unknown. The estradiol-17f3 could induce lipolytic enzymes, which in turn could provide substrate or precursor arachidonic acid for prostaglandin synthesis. Protein synthesis seems to be a requirement of the estrogen-induced release of PGF2a as indicated by indirect evidence of the effects of actinomycin D injected into the uterus of the ewe on day 11 of the estrous cycle (French and Casida, 1973). The luteolytic effect of the estradiol-17f3 is abolished, which is interpreted as an indication of the necessity of DNA-dependent RNA synthesis for the production of ovine luteolysin (French and Casida, 1973). 8.1.3b. Pharmacological Effects. The injection of 10-20 mg PGF2a systemically (McCracken et at., 1972b; Bolt et al., 1974) or into the uterine lumen (Goding,

1974) induces premature luteolysis during the estrous cycle in the ewe. Much smaller amounts (2-100 /Lg/hr) induce luteal regression when infused into the ovarian artery.

8.2.

Relaxin

The ovaries of pregnant rats contain a uterine relaxing factor (Bloom et at., 1958). Dallenbach-Hellweg et al., (1965) reported localization of relaxin activity in metrial glands during pregnancy by using a fluorescent antibody to relaxin. Relaxin levels in ovarian tissue of the rat increase during pregnancy to peak values just preceding parturition and then decline precipitously to extremely low levels during lactation (see Fig. 3) (Anderson et at., 1973a). The accumulation and disappearance of the smaller membrane-bound granules correlate with the rise and fall in relaxin during pregnancy. Ultrastructural features of the corpus luteum during pregnancy in the rat include electron-dense, membrane-bound granules (Long, 1973). By day 14 of pregnancy, membrane-limited granules (about 500 nm diameter) are found in increasing numbers in the cytoplasm of the lutein cell. These electron-dense granules increase in numbers by day 17 when a second group of smaller granules (about 270 nm diameter) becomes evident. The numbers of the smaller-sized granules increase to day 20, then decline after parturition, and are absent during lactation. The larger granules exhibit acid phosphatase and aryl sulfatase activities and are probably lysosome-like. The smaller granules show no reaction products of these two enzymes and probably are not lysosomes. It was suggested these smaller granules may be sites for the storage of relaxin. In the rabbit, relaxin levels increase in the blood serum to peak values during the last half of pregnancy, and then drop precipitously with onset of parturition (see Fig. 5). During this time, peripheral plasma levels of progesterone decline, reaching low levels at parturition (Hilliard et at., 1968; Challis et at., 1974b). This decline in progesterone coincides with an increase in myometrial sensitivity to oxytocin during the last days of the gestation and culminates in spontaneous myometrial activity with the onset of labor. By using cross-circulation techniques for exchange of blood of pregnant and nonpregnant rabbits, it was found that between days 20 and 29 of pregnancy the blood contains a myometrial inhibitory factor that is not attributable to the inhibitory effects of progesterone (Porter, 1974). It was suggested that this myometrial inhibitory factor may be relaxin or a relaxin-like substance. In the sow, the corpus luteum is a rich source of relaxin, particularly during the late stages of pregnancy and after hysterectomy (Hisaw and Zarrow, 1948; Anderson et aI., 1973b). Biochemical characterization and purification of porcine relaxin reveal three contiguous peaks on elution from a carboxymethyl cellulose column (Sherwood and O'Bryne, 1974). The three relaxin preparations are similar in biological activity, molecular weight (approximately 6500), isoelectric points, and amino acid composition. Investigations of the fine structure of porcine luteal cells in pregnant and hysterectomized animals reveal accumulation of membranelimited cytoplasmic granules that have no demonstrable acid phosphatase activity and, therefore, are not lysosomes (Belt et at., 1970). Development of a radioimmu-

631 UTERINE CONTROL OF OVARIAN FUNCTION

632 CHAPTER 16

noassay for a radioiodinated, biologically active polytyrosyl-relaxin allowed measurement of serum relaxin in peripheral circulation of the pig (Sherwood et at., 1975). Preliminary results comparing serum levels of relaxin by this radioimmunoassay technique confirm earlier findings by Belt et al., (1971) and Anderson et al., (l973b) of an increase to peak values of relaxin in extracts of porcine luteal tissue and ovarian venous plasma during late pregnancy in the sow, and a precipitous decline in luteal tissue and plasma levels of relaxin within 1-6 hr preceding parturition, with low levels continuing during lactation (Fig. 6). The appearance and accumulation of dense granules in the cytoplasm of the lutein cells in early and middle gestation and their disappearance in the last days of pregnancy parallel the rise and fall of relaxin in the corpora lutea (Fig. 6). After hysterectomy, the corpora lutea are maintained for an extended period (e.g., > 130 days), and these corpora accumulate considerable amounts of relaxin as they increase in age, with peak values occurring by day 110 (Anderson et al., 1973b). Although the corpora remain morphologically large, the levels of relaxin in them decrease by day 128. When the life span of corpora lutea is extended by injecting estrogen during the luteal phase of the estrous cycle, the maintained corpora contain considerable levels of relaxin by day 110, but levels decline by day 128, as in hysterectomized animals. Relaxin levels increase as the age of the corpus luteum increases during pregnancy, after hysterectomy, and in estrogen-treated pigs. Although porcine corpora contain abundant relaxin activity, particularly during late pregnancy in the pig, a role in physiological action of the hormone in preparation for parturition in this species is undefined. Extreme relaxation of the interpubic ligament occurs near the time of parturition in such species as the guinea pig and mouse, and this relaxation can be induced by injecting relaxin. However, extensive relaxation of this ligament near onset of parturition is not evident in the pig.

8.3.

Oxytocin

Endogenous oxytocin stimulates myometrial contractions at the onset of labor, and injected oxytocin stimulates myometrial activity in nonpregnant animals. Exogenous oxytocin also induces luteolysis in heifers, but has little effect in altering ovarian function in species such as the rat, rabbit, guinea pig, sheep, and pig. Oxytocin given during the early part of the estrous cycle causes premature luteolysis of the developing corpus luteum and results in reduced cycle intervals (Armstrong and Hansel, 1959; Harms et at., 1969). The cyclic periodicity is uninterrupted when the regimen of oxytocin injections begins during the luteal phase of the cycle. Futhermore, oxytocin fails to induce luteolysis in hysterectomized heifers (Anderson et al., 1965b), but causes luteal regression in partially hysterectomized animals (Ginther et at., 1967). A localluteolytic action of the uterus is initiated by the injected oxytocin, as indicated by induced luteolysis in unilaterally hysterectomized heifers in which the corpus luteum is ipsilateral to the remaining uterine horn, but luteal persistence results in those animals in which the remaining horn is contralateral to the corpus luteum. Epinephrine and atropine, which inhibit uterine contractions, can block luteolytic action of the oxytocin (Black and Duby, 1965). The luteolytic effect does not seem to be entirely dependent on oxytocin-

induced uterine contractions, however, for similar contractions produced by electrical stimulation fail to induce luteolysis in the heifer (Oxenreider, 1968).

9.

References

Abdul-Karim, R. W., and Bruce, N., 1973, Blood flow to the ovary and corpus luteum at different stages of gestation in the rabbit, Fertil. Steril. 24:44-47. Adams, C. E., and Eckstein, P., 1965a, Effect of intrauterine foreign bodies on pregnancy in the rabbit, Fertil. Steril. 16:508-521. Adams, C. E., and Eckstein, P., 1965b, Effect of intrauterine silk threads on location and survival of conceptuses in the rabbit,]. Reprod. Fertil. 9:351-354. Akbar, A. M., Rowe, K. W., and Stormshak, F., 1971, Estradiol induced luteal regression in unilaterally hysterectomized and luteinizing hormone-treated ewes,]. Anim. Sci. 33:426-429. Alberga, A., and Baulieu, E., 1968, Binding of estradiol in castrated rat endometrium in vivo and in

vitro, Mol. Pharmacal. 4:311-323. Allen, W. M., and Corner, G. W., 1929, Physiology of the corpus luteum. III. Normal growth and implantation of embryos after early ablation of the ovaries under the influence of extracts of the corpus luteum, Am. ]. Physiol. 88:340-346. Allen, W. R., 1969, Factors influencing pregnant mare serum gonadotropin production, Nature

(London) 223:64-66. Allen, W. R., and Moor, R. M., 1972, The origin of the equine endometrial cups,]. Reprod. Fertil. 29:313-316. Allen, W. R., and Rowson, L. E., 1973, Control of the mare's estrous cycle by prostaglandins,].

Reprod. Fertil. 33:539-543.

Alloiteau, J. J., and Psychoyos, A., 1966, Y a-t-il pour l"oeuf de ratte deux fa 9 o CI:

w

I-

I:J

en w

(!)

0

CI:

a..

o

0

o

12

24

36

HOURS AFTER INFUSION STARTED

Figure 4. Estradiol-induced premature parturition in the sheep. Changes in the concentration of estradiol-17{3 (e) and progesterone (0) in jugular venous plasma and prostaglandin F (0) in the utero-ovarian venous plasma of a sheep during intravenous infusion (top graph) of estradiol-17 {3. The time of premature parturition is indic. 184 days). Plasma progesterone did not fall until delivery of the placenta occurred or hysterectomy was performed (Bosu et at., 1974). These data clearly indicate the potential importance of both the ovary and the placenta as sources of progesterone, and serve to emphasize the interaction between these endocrine glands particularly with respect to an apparent compensatory feedback mechanism. These studies illustrate the requirement for extreme care in the interpretation of blood hormone levels after extirpation of an endocrine gland, and the general applicability of the principles that have been illustrated cannot be overemphasized.

6.4.

Effects

if Exogenous Compounds

The effects of a variety of compounds on the length of gestation in the rhesus monkey have been examined by the authors. The results were, in general, unspectacular. Progesterone, estradiol, and dexamethasone administered to either the mother or the fetus and ACTH administered to the fetus were all without effect on the length of gestation, using the protocols that were adopted. The only group of compound to emerge as being clearly implicated in primate parturition from these types of experiments were the prostaglandins.

6.5.

Prostaglandins

Exogenous PGF2a reliably precipitates premature delivery or abortion when administered to pregnant rhesus monkeys (Kirton et at., 1970a, 1971; Challis et at., 1974c). In early pregnancy, while the plasma progesterone is still produced exclusively by the corpora lutea, PGF2a appears to exert its effect through influencing corpus luteum function, either directly, or indirectly through impairment of the luteotropic support supplied by the conceptus to the corpus luteum. This effect could be resultant upon an increase in myometrial activity and changes in the blood flow to the conceptus (Kirton et at., 1970b, 1971). Evidence for a direct luteolytic action of exogenous PGF2G' on the primate corpus luteum is less than convincing, and it is necessary to achieve very high infusion rates in order to provoke luteolysis (Auletta et at., 1973). We have discussed previously the evidence suggesting that the myometrium of the pregnant rhesus monkey is highly responsive to exogenous PGF2G' or PGE2 , and that around day 100 of gestation PGE2 is approximately 10 times more effective on a dose basis than PGF2G' in stimulating myometrial activity (Kirton et at., 1971), although this relation may change at term (Oshima and Matsumoto, 1973). The abortifacient action of exogenous PGF2G' during the latter part of gestation appears to be independent of changes in the maternal peripheral plasma hormone concentrations, the levels of which are somewhat variable during the period of prostaglandin administration (Challis et at., 1974c). In some animals a decline in plasma progesterone was suggested, but the recurrent criticism that peripheral plasma hormone concentrations may not reflect changes in the levels of these substances in the myometrium is valid. In this series of animals, delivery was preceded by a decline in maternal

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704 CHAPTER 17

estrogen concentrations, which probably reflected an impairment of fetoplacental endocrine function, and all the fetuses were found dead in their cages on the morning following delivery. The involvement of endogenous prostaglandins in monkey parturition is suggested by studies showing that there was a significant prolongation of the length of gestation (> 180 days) after the administration to pregnant monkeys of indomethacin, an inhibitor of the prostaglandin synthetase system (Novy et at., 1974). Any change in prostaglandin production associated with term labor could be in response to the increase in fetal and maternal estrogen titers. In the nonpregnant ovariectomized rhesus monkey, estradiol (25 /Lg/day) provoked a striking increase in the uterine fluid PGF concentrations, while progesterone treatment had little effect (Demers et at., 1974).

6.6.

Dexamethasone

In sheep, dexamethasone induces premature delivery when administered to the fetus, or in the latter part of gestation to the mother. The endocrine changes associated with the induced delivery resemble those seen at spontaneous term. Attempts to reproduce these effects by the administration of large amounts of dexamethasone to pregnant rhesus monkeys have proved unsuccessful and delivery occurred at the normal time (Bosu et at., 1973b; Challis et at., 1974d). During the period of dexamethasone administration, maternal plasma progesterone was unaltered. However, maternal peripheral plasma estrone and estradiol gradually decreased during the period of dexamethasone administration, maternal plasma progesterone was unaltered. However, maternal peripheral plasma estrone and estradiol gradually decreased during the period of dexamethasone administration, and parturition occurred with maternal plasma estrogens at values only 10-20% of those seen before the administration of dexamethasone. The decline in maternal estrogens was due in part to suppression of the maternal pituitaryadrenal axis, and a decline in the concentration of androstenedione and testosterone in maternal plasma was noted (Challis et at., 1975a). In addition, dexamethasone crossed the placenta and suppressed the fetal pituitary-adrenal axis. The adrenal glands of newborn monkeys whose mothers had received dexamethasone were considerably atrophied, and premature regression of the fetal zones of these glands occurred in utero, implying a suppression of fetal pituitary ACTH (Challis et at., 1974d). The differences between the effects of dexamethasone in the rhesus monkey and the sheep are important but poorly understood. In part they may be due to greater amounts of dexamethasone crossing the hemochorial placenta of the primate compared with the epitheliochorial placenta of the sheep. In addition, there appear to be differences in the effects of exogenous glucocorticoids on placental enzymes in these species.

6.7.

Comment

It is apparent that whereas some of the results obtained in the rhesus monkey suggest that parturition in this species may be accomplished in a manner similar to that described in the sheep and in the human, there are important differences.

Observations of endogenous hormone concentrations have shown that in the rhesus monkey there is an increase in cortisol in the fetal compartment and estrogens in the maternal compartment, as in the sheep. Hypophysectomy of the macaque fet~s but not of the mother appears to predispose to a prolongation of gestation. The use of sensitive and specific immunoassays has allowed an understanding of the apparent anomaly of the fetectomy experiments. Prostaglandins are implicated in the control of monkey parturition, but other experiments with exogenous compounds have emphasized the differences between the sheep and the monkey. If prostaglandins represent a common denominator in the parturient mechanisms of different animal species, then future efforts directed toward understanding the control of prostaglandin production in the primate at term may result in a clearer appreciation of the fundamental endocrine mechanisms involved in parturition.

7.

Parturition in the Human

In previous sections of this chapter we have discussed the evidence implicating the fetus in the initiation of labor. We have seen that in the ruminants there is good evidence of a primary role for the fetal pituitary-adrenal axis in parturition, and an early clue was the association of prolonged pregnancy with fetal pituitaryadrenal hypofunction. Similarly, in the human certain malformations provide support for the concept of a fetal role in parturition, although the evidence is by no means conclusive.

7.1.

The Fetal Adrenal: Spontaneous Fetal Adrenal Hyperplasia and Hypoplasia

In 1898, Rea observed the association of prolonged pregnancy with anencephaly. Malpas (1933) extended this observation, suggesting that the failure of adrenal development pointed to the involvement of the fetal pituitary and adrenal glands in the onset of labor. Anencephaly is associated with apparently normal development of the fetal adrenal gland until about 20 weeks' gestation, at which time the fetal zone involutes prematurely and the definitive cortex fails to develop (Benirschke, 1956). This is similar to the time when it has been suggested that the hypothalamus and pituitary may become a functional unit (Fisher et at., 1970). Anderson et at. (1969) and Milic and Adamson (1969) have further explored the association of the size of the fetal adrenal gland and the length of gestation. Prolonged pregnancy was observed in anencephaly provided that there was no hydramnios and also in cases of fetal adrenal hypoplasia (O'Donohoe and Holland, 1968; Roberts and Cawdery, 1970; Fliegner et at., 1972), while pregnancies terminating in premature delivery may involve fetal adrenal hyperplasia (Anderson et at., 1971). O'Donohoe and Holland (1968) describe an interesting family in which three sibs with adrenal hypoplasia went past term whereas three normal sibs did not. However, these results without detailed endocrine investigation must be viewed with caution, because Pakravan et at. (1974) failed to find prolonged pregnancy in association with absence of the fetal adrenal gland. In their study,

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the cord cortisol concentration was 13 ng/ml, about 20-25% of normal, and presumably derived from the maternal circulation. Our knowledge of pituitary hormones in normal or malformed fetuses is scanty, and certainly there is a need for more data before attempts are made to correlate structure and function. However, Allen et al. (1973) and Winters et al. (1974) have reported low levels of ACTH in blood obtained from anencephalic fetuses. There was no relation between fetal and maternal ACTH concentrations even when maternal ACTH was very high (Nelson's syndrome), indicating that the ACTH in fetal plasma is of fetal origin. Honnebier et al. (1974) attempted to assess the role of the fetal adrenal in the mechanisms responsible for the onset of labor by injecting HCG or ACTH directly into anencephalic fetuses. HCG had no effect on the size of the fetal adrenal glands, steroid hormones in the cord blood at delivery, or the initiation of labor (Honnebier et al., 1974). Likewise, ACTH injected into the fetus failed to induce labor, although the adrenal glands were somewhat larger than those of control anencephalics and DHAS was then present in the cord blood. These observations, which should be contrasted with those of Johannisson (1968), suggest that perhaps prolonged treatment with ACTH with or without the addition of other pituitary hormones (e.g., prolactin, Winters et al., 1975) may be required for normal fetal adrenal function.

7.2.

Fetal Adrenal and Cortisol

The human fetal adrenal gland has the ability to synthesize a considerable range of steroids. This capacity and the interrelationship of the adrenal and the placenta (the fetoplacental unit) have been the subject of many excellent reviews (Diczfalusy and Mancuso, 1969; Ryan, 1969; Villee, 1969; Oakey, 1970; Davies and Ryan, 1972), and only a brief description will be given here. The fetal adrenal cortex has the ability to synthesize the steroid nucleus from acetate and to convert cholesterol to pregnenolone. However, there is a relative deficiency of 3{3hydroxysteroid dehydrogenase (3{3HSD); thus the conversion from pregnenolone to progesterone is not favored. The 17a-hydroxylase, C 17- 20 desmolase, and sulfokinase activities result in the secretion of large amounts of DHEA and DHAS by the fetal adrenal cortex, and Oakey (1970) has suggested that the secretion of DHAS instead of cortisol leads to a lower. feedback inhibition of ACTH, which in turn would have the effect of stimulating adrenal growth and steroidogenesis. Although there is the relative deficiency of 3{3HSD, cortisol may be formed from placental progesterone, possibly in discrete zones of the adrenal. Later in pregnancy an increase of 11{3-hydroxylase and 21-hydroxylase increases cortisol production (Villee, 1969). Cortisol produced by the fetus has a high clearance rate in the fetal liver and also in the placenta, where it is metabolized to cortisone or transferred to the maternal circulation (Beitins et al., 1973). The latter is favored by the greater cortisol-binding globulin activity in the maternal plasma. The cortisol concentrations in cord blood and in amniotic fluid have been measured throughout pregnancy. Cortisol in cord blood was low in early pregnancy (about 7 ng/ml), and only slightly higher levels were found in infants delivered by elective cesarean section or vaginal delivery following the induction of labor. Much higher values were found following the spontaneous onset of labor,

and vaginal delivery (72.6 ± 17.3 ng/ml). Murphy (1973) and Ohrlander et al. (1975b) obtained fetal scalp blood during the course of labor by the Saling technique and found similar levels in both spontaneous and induced labor. However, cortisol increased early in spontaneous labor and more slowly in induced labor (Ohrlander et al., 1975b), perhaps suggesting increased adrenal sensitivity in the former as is found in the sheep. In amniotic fluid, there is an increase of cortisol during pregnancy with a more rapid rise in the last few weeks (Murphy et al., 1974; Fencl and Tulchinsky, 1975). These observations may provide some evidence of a role for the fetal pituitary-adrenal axis in the mechanisms resulting in the onset of labor, but not all the cortisol present in the fetal circulation is of fetal origin and the increase may be an effect of labor, rather than the stimulus for it. Beitins et al. (1973) have reported that about 25% of cortisol in the fetal circulation is of maternal origin; indeed, in the complete absence of the fetal adrenal glands Pakravan et al. (1974) found 13 ng/ml of cortisol in the cord plasma. However, a protective mechanism exists in the placenta whereby cortisol is converted to cortisone, a less active glucocorticoid. Murphy et al. (1974) injected eH]cortisol into mothers 20-30 min before abortion between 13 and 18 weeks and recovered 85% of the radioactivity as cortisone. This is similar to the interconversion observed by Beitins et al. (1973) for their term pregnancies. Anderson and Turnbull (1973) reported different rates of conversion of isotopically labeled cortisol and cortisone by the placenta at 18 and 40 weeks' gestation. There was a 94.6% conversion of cortisol to cortisone at 18 weeks but only 21 % conversion at term. However, the numbers in each group were low. In contrast to the rise of cortisol in cord blood and amniotic fluid before and during normal labor, infants delivered following prolonged pregnancy have a relative adrenocortical insufficiency (Nwosu et al., 1975b). Compared with control infants, low peripheral cortisol levels were found in these infants between 12 and 36 hr following delivery. Indeed, this has been extended to include infants with fetal distress in labor (Nwosu et al., 1975a). However, it should be pointed out that the cord cortisols were similar in infants delivered at or past term in a distressed condition, suggesting that both groups have similar responses to asphyxia or hypoxia. Information is now available concerning the interrelationship between fetal cortisol and fetal ACTH. It is apparent in the intact human fetus that intrafetal injection of ACTH stimulates adrenal steroidogenesis, as judged by an increase in maternal estrogen excretion (Arai et al., 1972). More recently, Seron-Ferre et al. (1975) tested the responses of different layers of the fetal adrenal cortex to ACTH in an in vitro perfusion system. While the definitive adrenal cortex responded with an increase in cortisol secretion, the fetal zone did not, thus conflicting with earlier data that indicated that both ACTH and HCG stimulated the cells of the fetal zone Qohannisson, 1968). It will be extremely difficult to obtain resting levels of pituitary hormones in the human fetus because of the problem of sample collection. The levels of ACTH in the human fetus reported by Winters et al. (1974) have to be interpreted with caution. ACTH was measured in blood obtained from fetuses removed by termination hysterectomy and in the older ones from the umbilical cord following vaginal delivery. Higher ACTH levels were found in the former, when hypoxemia and asphyxia were more likely to have stimulated ACTH secretion. Finally, the

707 ENDOCRINE CONTROL OF PARTURITION

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human placenta has been found to contain large amounts of ACTH (Genazzani et al., 1975b; Rees et al., 1975a), which will make the interpretation of human pituitary function in utero more complicated, although it is conceivable that this ACTH may be secreted mainly into the maternal circulation as is placental lactogen. The DHAS from the fetal adrenal is converted by the fetal liver to 16a-OHDHAS and then aromatized to estriol (E3) by the placenta. The production of E3 in the human is greater than in other primates (Ryan and Hopper, 1974), although the function of the massive quantities of E3 that are produced remains obscure. In contrast to estriol, estrone (E 1) and estradiol-17 {3 (Ez{3) can be formed from both fetal and maternal C 19 precursors in roughly equal proportions (Siiteri and MacDonald, 1963; MacDonald and Siiteri, 1965). This observation was confirmed by the report that the E2{3 production rate was 50% of normal when the mother had been adrenalectomized (Siiteri and MacDonald, 1966). Similarly, the absence of fetal precursors in anencephaly reduces maternal Ez{3 to 25-50% of normal (Siiteri, 1974). These observations may be related to the suggested role of E2{3 in the initiation of labor. Dawood and Ratnam (1974) and Turnbull et al. (1974) found that maternal peripheral plasma Ez{3 in humans gradually increases during the period 8-30 weeks, and then rises more rapidly to 20-30 ng/ml between 30 and 40 weeks, followed by a decline after delivery. The latter group have also reported elevated levels in association with premature labor (TambyRaja et al., 1974), although this remains to be confirmed. If the human is analogous to the ruminant in the mechanisms responsible for the onset of labor, then it is necessary to look for a relation between ACTH and cortisol and estrogen production. Simmer et al. (1974) have examined the role of cortisol and ACTH in the regulation of estrogen production in term pregnancies ended by cesarean section. Cortisol, given to the mother during the 10 hr preceding delivery, lowered ACTH in both mother and the fetus. In addition, fetal DHEA and DHAS and maternal estrogens were reduced. ACTH given to the mother increased maternal cortisol but lowered E3 in the maternal blood and in the fetus. ACTH given to the fetus directly, however, increased estrogen excretion (Arai et al., 1972). These data indicate that the human fetal adrenal will respond to ACTH with an increase in C 19 steroid secretion. Moreover, it can be suppressed by elevated maternal cortisol levels crossing the placenta and exerting a feedback inhibition on the fetal pituitary. These data are consistent with the kinetic data discussed earlier, and point to important differences between the primate and the ruminant that may be related to the placental transfer and metabolism of glucocorticoids. In ruminants exogenous glucocorticoids will induce premature parturition, but in the human there is little evidence for this effect. Anderson and Turnbull (1973) failed to induce labor in 20 normal women at 39 weeks with betamethasone (20 mg). However, Mati et al. (1973) were able to show a reduced injection--delivery interval in women classified as past term who were given betamethasone into the amniotic fluid. Exogenous glucocorticoids given to women have long been known to reduce E3 excretion. Ohrlander et al. (1975b) demonstrated that a brief 3-day course of 6 mg betamethasone phosphate and 6 mg betamethasone acetate reduced maternal estriol excretion for 3 weeks. TambyRaja et al. (1974) have similarly observed a depression of maternal plasma E2{3 following

betamethasone; indeed, this has been proposed as a method for the prevention of premature parturition in humans.

7.3.

Progesterone

The evidence has been discussed that, in the rabbit, ovarian progesterone is necessary for pregnancy maintenance and a decline in the secretion of this steroid precedes parturition. In the human in early pregnancy progesterone is first formed by the corpus luteum under the trophic support of HCG. Although ovarian progesterone secretion continues throughout gestation (Le Maire et al., 1970), the placenta becomes the principal site of progesterone secretion between the sixth and ninth weeks of pregnancy. Before this time, excision of the corpus luteum will lead to abortion (Csapo et al., 1974b), but after this time it is more difficult to assess the precise role of progesterone in the onset of labor. Progesterone is synthesized by the placenta from maternal cholesterol, and the pathways involved have been discussed in detail (Diczfalusy and Mancuso, 1969; Siiteri, 1974; Ryan, 1974). Measurement of the major urinary metabolite of progesterone, pregnanediol, was one of the first hormonal tests of placental function to be used clinically. However, pregnanediol excretion can continue normally after fetal death (Cassmer, 1959) and its diagnostic use is limited. Plasma progesterone increases from 25 ng/ml in early pregnancy to about 180 ng/ml at term, when the secretion rate is about 300 mg/day. Higher plasma progesterone is found in association with twin pregnancy (Aitken et al., 1958) corresponding to a secretion rate of about 500 mg/day (Sitteri, 1974). Although a few authors (e.g., Csapo et al., 1971, 1974a; Turnbull et al., 1974) have suggested that there is a fall in progesterone preceding the onset of spontaneous labor or abortion, thus supporting the concept of progesterone withdrawal (Csapo, 1961), most independent investigators have failed to substantiate these results (e.g., Siiteri, 1974; Tulchinsky et al., 1972; Llaura et al., 1968). Because most of the progesterone in plasma is protein bound, Yanonne et al. (1969) measured both free and bound progesterone throughout pregnancy and serially before the onset of labor. The total progesterone concentration increased throughout pregnancy, whereas the proportion that was bound to high-affinity globulin remained constant at about 91 % both ante natally and in labor. Thus the free progesterone was highest in late pregnancy and there was no evidence of a fall in either free or total progesterone before labor. Short et al. (1965) were unable to find a reduction of progesterone in uterine venous blood following intraamniotic saline in midpregnancy, although Csapo et al. (1971) have shown a fall in progesterone after intraamniotic saline. Observations on the distribution of progesterone in different parts of the uterus have indicated that the highest concentrations are found beneath the placenta (Pulkkinen and Enkola, 1972), although this may be true only earlier in pregnancy (Runnebaum and Zander, 1971). In the human, myometrial sensitivity to oxytocin increases during late pregnancy (see Csapo, 1969). Johansson (1968) observed that this increased sensitivity was found when the progesterone concentration was low. However, Kumar et al. (1964), Llaura et al. (1968). and Ances et al. (1971) were unable to

709 ENDOCRINE CONTROL OF PARTURITION

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relate myometrial sensitivity to oxytocin with progesterone. In this context, Csapo et al. (1966) were unable to demonstrate convincingly that exogenous progesterone in large doses could delay delivery. Similarly, progesterone administration is unable to prevent induced abortion (M