The Mammalian Pre implantation Embryo

Regulation of Growth and Differentiation in Vitro

The Mall1ll1alian Preill1 plan ta tion Ell1 bryo

Regulation of Growth and Differentiation in Vitro

Edited by

Barry D. Ba vister Department of Veterinary Science

and Wisconsin Regional Primate Research Center University of Wisconsin

Madison, Wisconsin

Plenum Press • New York and London

Library of Congress Cataloging in Publication Data

The Mammalian preimplantation embryo.

Includes bibliographies and index. 1. Embryology-Mammals. 2. Fertilization in vitro. 3. Cell culture. I. Bavister, Barry D.

QL959.M264 1987 599'.033 87-13986

ISBN-13: 978-1-4684-5334-8 001: 10.1007/978-1-4684-5332-4

© 1987 Plenum Press, New York

e-ISBN-13: 978-1-4684-5332-4

Softcover reprint of the hardcover 1 st edition 1987

A Division of Plenum Publishing Corporation 233 Spring Street, New York, N.Y. 10013

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

BARRY D. BA VISTER Department of Veterinary Science, University of Wisconsin, 1655 Linden Drive, Madison, Wisconsin 53706, USA, and the Wisconsin Regional Primate Research Center, 1223 Capitol Court, Madison, Wisconsin 53715, USA

JOHN D. BIGGERS Department of Physiology and Biophysics, Laboratory of Human Reproduction and Reproductive Biology, Harvard Medical School, Boston, Massachusetts 02115, USA

DOROTHY E. BOATMAN Wisconsin Regional Primate Research Center, University of Wisconsin, 1223 Capitol Court, Madison, Wisconsin -53715, USA

FOLK MAR ELSAESSER Institut fUr Tierzucht und Tierverhalten (F AL), Mariensee, 3057 Neustadt 1, Federal Republic of Germany

CHARLES J. EPSTEIN Departments of Pediatrics and of Biochemistry and Biophysics, University of California, San Francisco, Califomia 94143, USA

YVES HEYMAN I.N.R.A., Station de Physiologie Animale, 78350, Jouy-en­Josas,_France

SUSAN HEYNER Department of Obstetrics and Gynecology, Albert Einstein Medical Center, Northern Division, York and Tabor Roads, Philadelphia, Pennsylvania 19141, USA

MICHAEL T. KANE Physiology Department, University College, Galway, Ireland

GERALD M. KIDDER Department of Zoology, University of Western Ontario, London, Ontario, N6A 5B7, Canada

v

vi CONTRIBUTORS

TERRY MAGNUSON Department of Developmental Genetics and Anatomy, School of Medicine, Case Western Reserve University, Cleveland, Ohio 44106, USA

YVES MENEZO I.N.S.A., Laboratoire de Biologie, 69621, Villeurbanne CMex, France

HEINER NmMANN Institut fUr Tierzucht und Tierverhalten (F AL), Mariensee, 3057 Neustadt 1, Federal Republic of Germany

JAMES V. O'FALLON Department of Animal Sciences, Washington State University, Pullman, Washington 99164-6332, USA

ERIC W. OVERSTROM Department of Anatomy and Cellular Biology, Tufts University, Schools of Medicine and Veterinary Medicine, 136 Harrison Avenue, Boston, Massachusetts 02111, USA

ANGIE RIZZINO Eppley Institute for Research in Cancer and Related Diseases, University of Nebraska Medical Center, 42nd and Dewey Avenue, Omaha, Nebraska 68105, USA

HORST SpmLMANN Max v. Pettenkofer-Institut, Bundesgesundheitsamt (BGA), P.O. Box 33 00 13, 1 Berlin 33, West Germany

LYNN M. WILEY Division of Reproductive Biology and Medicine, Department of Obstetrics and Gynecology, University of California, Davis, California 95616, USA

RA YMOND W. WRIGHT, JR. Department of Animal Sciences, Washington State University, Pullman, Washington 99164-6332, USA

CAROL A. ZIOMEK Worcester Foundation for Experimental Biology, 222 Maple Avenue, Shrewsbury, Massachusetts 01545, USA

PREFACE

With a few notable exceptions, mammalian preimplantation embryos grown in vitro are likely to exhibit sub-optimal or retarded development. This may be manifested in different ways, depending on the species and on the stage(s) of embryonic development that are being examined. For example, bovine embryos often experience difficulty in cleaving under in vitro conditions, and usually cease development at about the 8-cell stage (Wright and Bondioli, 1981). The block to development is stage-dependent; embryos cultured for 24 hr from the I-cell stage are much more capable of developing into viable blastocysts after transfer to oviducts than embryos cultured for 24 hr from the 4-cell stage prior to transfer (Eyestone et oZ., 1985). Similar problems with in vitro embryo development are encountered in other species. Pig embryos can be grown up to the 4-cell stage in vitro but usually no further (Davis and Day, 1978). In the golden hamster, in the rat and in many outbred strains of mice, development of zygotes in vitro is blocked at the 2-cell stage (Yanagimachi and Chang, 1964; Whittingham, 1975). Even with some inbred mouse strains, embryo development is reduced if very early cleavage stages are used as the starting point for in vitro culture (Spielmann et oZ., 1980). A common finding is that embryos grown in vitro have reduced cell counts (Harlow and Quinn, 1982; Kane, 1985) and their viability is reduced (Bowman and McLaren, 1970; Papaioannou and Ebert, 1986) compared to equivalent developmental stages recovered from mated animals.

All of these difficulties show that we have a great deal to learn about culture conditions suitable for sustaining normal growth of preimplantation embryos. Resolution of these technical problems should be a high priority for investigators who are interested in analyzing mechanisms of embryo development in different species. Knowledge of culture requirements for embryos of various species will tell us much about their metabolism and the regulation of development, just as in the pioneering work of Biggers et oZ. (1971), Brinster (1971), Whitten (1957, 1971) and others using mouse embryos. Because of these pioneering efforts, mouse embryos have become firmly established as the models for the study of early (preimplantation) devel­opment. The notable advances in our understanding of the regulation of development that have derived from studies on mouse embryos, some of which

vii

viii PREFACE

are presented in this book, stand as elegant testimony to the validity of this approach. An enormous amount of information has been obtained on the cellular, molecular and genetic aspects of early embryonic development in the mouse. Yet the heavy emphasis placed on the study of the inbred mouse has fostered neglect of other important species. Cleavage stage embryos from some of these species, such as the domesticated animals, may be of consi­derable commercial interest, while others, such as non-human primate embryos, have great theoretical and practical interest because of their similarity to human embryos. Thus, there is an urgent need for comparative data on preimplantation embryogenesis using a wide variety of animal species.

It is rather ironic that human embryos, derived from in vitro fertilized (IVF) eggs, appear to be very easy to grow in vitro, even to the blastocyst stage. This would seem to undermine one major justification for studies with animal embryos, namely that information gained from such studies may help to increase understanding of human embryogenesis. However, the situation concerning experimental embryology in humans is quite complex. In many parts of the world, invasive experimentation on human preimplantation embryos, or even culture of embryos to the blastocyst stage, is proscribed for a variety of ethical and medico-legal reasons. Since the primary objective of human IVF is to transfer embryos, usually at the 4- to 8-cell stage, back to the infertile patient, "hard" information concerning (e.g.) optimal culture conditions for human embryos is difficult to obtain; factorially-designed experiments can hardly be done under these conditions. Moreover, about 90% of all IVF human embryos fail to develop to term following transfer, and we do not know what proportion of these failures is due to abnormalities of early embryo development in vitro. It is quite common for IVF primate embryos to undergo apparently normal cleavage in vitro, only to cease development before the morula stage (Bavister et aI., 1983a; Boatman et al., 1987). Early cessation of embryo development also seems to be common in vivo (Enders et aI., 1982). In view of all these considerations, a strong case can be made for the necessity of stUdies using animal embryos in order to obtain information that is potentially useful for understanding human reproductive problems.

The present situation is that we know a great deal about regulation of embryo development in the mouse and very little in other species. This difference could soon be eliminated if techniques (e.g., culture media) devised for mouse embryos were applicable to other mammalian species, and if information obtained on (e.g.) the nutritional requirements for mouse embryos were found to be representative of mammals in general. Unfortunately, neither of these situations appears to be true. From the limited amount of data available, it appears that pre implantation embryos of several mammals are much more demanding in their requirements for growth in vitro than are mouse embryos, and perhaps are also more sensitive to the trauma associated with collection and culture. For example, mouse embryos have been grown from the 2-cell stage to blastocysts in the absence of protein (Cholewa and Whitten, 1970) and do not require any amino acid source for development in vitro up to the late zonal blastocyst stage. In contrast, rhesus monkey embryos (at least from the 8-cell stage) need a complex culture medium with protein supplement (Morgan et al., 1984), while growth of rabbit and hamster embryos is very dependent on exogenous amino acids (Kane and Foote, 1970; Bavister et al., 1983b).

PREFACE Ix

Information is urgently needed that will allow us to bridge the gulf of knowledge between embryos of the mouse and those of other mammals, not only in terms of our ability to culture these embryos successfully, but also in reaching a consensus about the similarities and differences between species. Then advances made using mouse embryos could rapidly be tested in other species, and embryos of the latter group could also be investigated for their own particular developmental characteristics. The production of this book represents one effort to build such a bridge. Each contributing author is in the forefront of his or her particular area of embryogenesis research. Subjects of the chapters were chosen to represent a range of topics, in terms of species and of analytical approach. It will be obvious, for reasons already mentioned, that research described using mouse embryos is, in general, at a more advanced stage than studies using other species. Authors were requested to provide technical details of their research as well as the embryological data obtained using these methods; this dual emphasis should help other investigators to confirm or extend the work and to facilitate start-up for those who are beginning to study embryo development in vitro.

Mechanisms involved in the fertilization process have not received attention in this book, partly because this would have excessively broadened the scope of the work, and partly because several books dealing specifically with fertilization have been published in the last few years. For similar reasons, I have largely avoided inclusion of IVF techniques, although there is presently considerable interest in IVF and the consequences for embryo­genesis. Readers interested in the topics of fertilization and IVF are referred to works by Mastroianni and Biggers (1981), Beier and Lindner (1983), Hartmann (1983), Trounson and Wood (1984) and Seppala and Edwards (1985). By focusing on developmental events following fertilization and up to peri­implantation stages, the contributing authors have brought a wide variety of interests and techniques to bear on a single topic: the mechanisms regulating growth of pre implantation embryos in vitro. The contributors have generally described their own most recent work in specialized areas of interest, while presenting this information against a background review of work from other laboratories. The bibliographic lists compiled by each contributor should by themselves be valuable aids to research, since the literature on preimplant­ation embryogenesis tends to be scattered widely among many different journals, books and symposium proceedings.

At the end of the book are two Appendices. In Appendix I, I have attempted to synergize some of the ideas of the contributing authors in order to point out some problem areas and lines of enquiry that should help to accelerate the pace of comparative research using pre implantation embryos. In addition, I have provided some technical notes on embryo culture, focusing on areas cited by the authors as being critically importan4 and also drawing from experiences in my own laboratory. Appendix II lists the names, and the addresses as far as possible, of suppliers of materials and equipment used in embryo culture research. The sole criterion for inclusion of names and items in this list was that the supplier or manufacturer was cited by one or more of the contributing authors. Not only is it convenient to group this information in one place, but it also avoids unnecessary duplication and (I hope) makes the text more readable.

x PREFACE

A major reason for undertaking this work was to stimulate wider interest in the study of pre implantation embryogenesis. Time will tell if we have succeeded in this goal; however, the authors have all risen to the occasion splendidly in their attempts to meet it, and I thank each of them for their unstinting efforts.

I am indebted to Mary Born, Kirk Jensen, John Matzka and their staff at Plenum Press for their advice and practical help with the editorial process; to Amy Magulski for her tireless efforts in helping me to prepare the book for publication; and to Jean Lasecki for her heroic work on the index. I am grateful to the Department of Veterinary Science and to the Regional Primate Research Center, University of Wisconsin-Madison, for support during the preparation of this book.

Finally, I would like to dedicate this book to my Ph.D. supervisor, Dr. C. R. "Bunny" Austin, who is emeritus Charles Darwin Professor of Animal Embryology at Cambridge University. I am immensely indebted to him for his help in getting my research career started, and for his advice and generous encouragement of my work.

References

Barry D. Bavister Madison, Wisconsin

Bavister, B.D., Boatman, D.E., Leibfried, M.L., Loose, M., and Vernon, M. W., 1983a, Fertilization and cleavage of rhesus monkey oocytes in vitro, BioI. Reprod. 28: 983-999.

Bavister, B.D., Leibfried, M.L., and Leiberman, G., 1983b, Development of preimplantation embryos of the golden hamster in a defined culture medium, BioI. Reprod. 28: 235-247.

Beier, H.M., and Lindner, H.R. (eds.), 1983, Fertilization of the Human Egg In Vitro, Springer-Verlag, Berlin.

Biggers, J.D., Whitten, W.K., and Whittingham, D.G., 1971, The culture of mouse embryos in vitro, in: Methods in Mammalian Embryology (J.C. Daniel, ed.), Freeman & Co., San Francisco, pp. 86-116.

Boatman, D.E., Morgan, P.M., and Bavister, B.D., 1987, Culture of in vitro fertilized rhesus monkey oocytes to peri-implantation stages of embryo development, BioI. Reprod. (submitted).

Bowman, P., and McLaren, A., 1970, Viability and growth of mouse embryos after in vitro culture and fusion, J. Embryol. Exp. Morph. 23: 693-704.

Brinster, R.L., 1971, In vitro culture of the embryo, in: Pathways to Con­ception: the Role of the Cervix and the Oviduct in Reproduction (A. I. Sherman, ed.), Charles C. Thomas, Springfield, pp. 245-277.

Cholewa, J.A., and Whitten, W.K., 1970, Development of 2-cell mouse embryos in the absence of a fixed nitrogen source, J. Reprod. Fertil. 22: 553-555.

Davis, D.L., and Day, B.N., 1978, Cleavage and blastocyst formation by pig eggs in vitro, J. Anim. Sci. 46: 1043-1053.

Enders, A.C., Hendrickx, A.G., and Binkerd, P.E., 1982, Abnormal develop­ment of blastocysts and blastomeres in the rhesus monkey, BioI. Reprod. 26: 353-366.

PREFACE xi

Eyestone, W.H., Northey, D.L., and Leibfried-Rutledge, M.L., 1985, Culture of I-cell bovine embryos in the sheep oviduct, Biol. Reprod. 32 (Suppl. 1): 100a.

Harlow, G.M., and Quinn, P., 1982, Development of preimplantation mouse embryos in vivo and in vitro, Aust. J. Biol. Sci. 35: 187-193.

Hartmann, J.F. (ed.), 1983, Mechanism and Control of Animal Fertilization, Academic Press, New York.

Kane, M.T., 1985, A low molecular weight extract of bovine serum albumin stimulates rabbit blastocyst cell division and expansion in vitro, J. Reprod. Fertil. 73: 147-150.

Kane, M.T., and Foote, R.H., 1970, Culture of two- and four-cell rabbit embryos to the expanding blastocyst stage in synthetic media, Proc. Soc. Exp. Biol. Med. 133: 921-925.

Mastroianni, L., Jr., and Biggers, J.D. (eds.), 1981, Fertilization and Embry­onic Development In Vitro, Plenum Press, New York.

Morgan, P.M., Boatman, D.E., Collins, K., and Bavister, B.D., 1984, Complete preimplantation development in culture of in vitro fertilized rhesus monkey oocytes, Biol. Reprod. (Suppl. 1): 96a.

Papaioannou, V.E., and Ebert, K.M., 1986, Development of fertilized embryos transferred to oviducts of immature mice, J. Reprod. Fertil. 76: 603-608.

SeppaHi, M., and Edwards, R.G. (eds.), 1985, In Vitro Fertilization and Embryo Transfer, Ann. N. Y. Acad. Sci., Volume 442, New York Academy of Sciences, New York.

Spielmann, H., Eibs, H.G., and Jacob-MUller, U., 1980, In vitro methods for the study of the effect of teratogens on preimplantation embryos, Acta Morphologica Acad. Sci. Hung. 28: 105-115.

Trounson, A., and Wood, C. (eds.), 1984, In Vitro Fertilization and Embryo Transfer, Churchill Livingstone, Inc., New York.

Whitten, W.K., 1957, Culture of tubal ova, Nature (London) 179: 1081-1082. Whitten, W.K., 1971, Nutrient requirements for the culture of preimplantation

embryos in vitro, in: Schering Symposium on Intrinsic and Extrinsic Factors in Early Mammalian Development, Advances in the Biosciences, Vol. 6 (G. Raspe, ed.), Pergamon Press, Oxford, pp. 129-141.

Whittingham, D.G., 1975, Fertilization, early development and storage of mammalian ova in vitro, in: The Early Development of Mammals (M. Balls, and A.E. Wild, eds.), Cambridge University Press, Cambridge, U.K., pp. 1-24.

Wright, R.J., Jr., and Bondioli, K.R., 1981, Aspects of in vitro fertilization and embryo culture in domestic animals, J. Anim. Sci. 53: 702-728.

Yanagimachi, R., and Chang, M.C., 1964, In vitro fertilization of golden hamster ova, J. Exp. Zool. 156: 361-376.

CONTENTS

Chapter 1

PIONEERING MAMMALIAN EMBRYO CULTURE

John D. Biggers

1. Introduction •••••••••••••• 2. Early Period Using Media Prepared From

Biological Fluids ••••••• 3. Successful Culture of a Rodent

Preimplantation Embryo •••••• 4. Culture of Rabbit Embryos Revisited • 5. Conclusion •••• 6. References •••••••••••••

Chapter 2

CELL POLARITY IN THE PREIMPLANTATION MOUSE EMBRYO

Carol A. Ziomek

1. Introduction ••• . • • • 2. The Unfertilized Egg •• 3. The Fertilized Egg ••• 4. 2-Cell and 4-Cell Stages 5. The 8-Cell Stage Embryo ••

xiii

1

2

6 11 15 17

23 23 27 28 31

xiv CONTENTS

6. Developmental Significance of Embryonic Polarities • • • • • • • • • • • • • •• 36

7. References ...••...............•.• 37

Chapter 3

INTERCELLULAR COMMUNICATION DURING MOUSE EMBRYOGENESIS

Gerald M. Kidder

1. Introduction · · · · · · · · · · · · · 2. Communication Via Cytoplasmic Bridges. · 3. Communication Via Intercellular Membrane Channels · · · · · · · · · · · · · · · · 4. Communication Via Cell Surface Interaction

5. On the Role of Intercellular Communication Pathways in Embryogenesis · · · · · · · 6. References · · · · · · · · · · · · · · · ·

Chapter 4

· · · · · · · · · · · · · · · ·

DEVELOPMENT OF THE BLASTOCYST: ROLE OF CELL POLARITY IN CAVITATION AND CELL DIFFERENTIATION

Lynn M. Wiley

1. 2.

Introduction •••••.••••••••••••

3.

4. 5.

Three Models for Cavitation: Cell Polarity and the Production of Nascent Blastocoele Fluid •

Electrical Polarity of Outer Blastomeres from Mouse Morulae ••••••

SUllITlary • • • • • • • • • • • • • • References •••••••••••••

Chapter 5

Eric W. OverstrOm

IN VITRO ASSESSMENT OF BLASTOCYST DIFFERENTIATION

1. Introduction - Overview of Blastogenesis · · · · 2. Oxidative Metabolism · · · · · · · · · · · · · · 3. Trans-Trophectodermal Sodium Transport · · · · · 4. Protein Synthesis During Blastogenesis •••• · 5. Characterization of Blastocyst Plasma Membranes · 6. Conc lusi ons: Future Perspectives · · · · · 7. References · · · · · · · · · · · · · · · · · ·

· · · · · · · · · · · · · · · · · ·

· · · ·

· · · · · ·

43 44

46 58

59 61

65

67

77 89 91

95 97

100 105 109 110 112

CONTENTS

Chapter 6

STEROID HORMONES IN EARLY PIG EMBRYO DEVELOPMENT

Heiner Niemann and Folkmar Elsaesser

1. Introduction ................... . 2. Methodo logy . . . . . • . . . . . . . . . . . . . • . 3. Progesterone: Embryonic Uptake and Its Specificity 4. In Vitro Culture of Morulae in the Presence of

Supplementary Steroids ••••••••••••• 5. Estradiol-17 Beta: Embryonic Uptake and

Its Specificity • • • • • • • • • • • • • • • • • 6. Estradiol Withdrawal and In Vitro Development of

Morula Stages • • • • • • • • • • 7. Further Considerations 8. References ••••••

Chapter 7

GENETIC EXPRESSION DURING EARLY MOUSE DEVELOPMENT

Terry Magnuson and Charles J. Epstein

1. Introduction •••••••••••••••••••• 2. Synthesis of Maternally-Derived Products •• 3. Activation of the Embryonic Genome ••••••• 4. Are Maternally-Derived Products Important for

Development Beyond the 2-Cell Stage? ••• 5. Both the Maternal and Paternal Genomes Are

Required for Normal Development •••••••••• 6. Is It Possible To Clone Mammals by Nuclear Transfer? 7. Mutations and Chromosomal Abnormalities That

Affect the Preimplantation Mouse Embryo •••• 8. Genetic Control of Early Mammalian Development:

9. 10.

Future Approaches • • • • • • • • • • SUDII1ary • • • • • • • • • • • • • • • • • • References • . • • • • • • • • •

Chapter 8

DEFINING THE ROLES OF GROWTH FACTORS DURING EARLY MAMMALIAN DEVELOPMENT

Angie Rizzino

1. Introduction ••••••••••••••• 2. Preimplantation Mouse Embryos Cultured In

Serum-Supplemented Media • • • • • • • •

117 117 122

123

124

126 129 130

133 133 134

135

135 137

137

142 143 144

151

152

xvi CONTENTS

3. Mouse Embryos Cultured in Serum-Free Media •• 4. Design of Serum-Free Media for

Mouse Blastocysts ••••••••••••• 5. Production of Growth Factors by Early Embryos • • 6. Possible Roles of Growth Factors During Early

Postimplantation Development ••••••••••••• 7. Possible Effects of Growth Factors on the

Process of Differentiation •••• 8. 9.

10.

Conclusions • • • • • • • • • • • • • • • • Appendix •• • • • • • • • • • • References • • • • • • • • • •

Chapter 9

INTERACTION OF TROPHOBLASTIC VESICLES WITH BOVINE EMBRYOS DEVELOPING IN VITRO

Yves Heyman and Yves Menezo

l. Introduction · · · · · · · · · · · · · · · · · · 2. Working With Trophoblastic Vesicles • · · · · · · 3. Effect of Trophoblastic Vesicles on Early Stage Embryo Development In Vitro · · · · · · · · · · 4. Evidence for Release by Trophoblastic Vesicles of Signals For Corpus Luteum Function · · 5. Conclusions • · · · · · · 6. References · · · · · · · · · · · · · · · · ·

Chapter 10

·

· · ·

IN VITRO GROWTH OF PREIMPLANTATION RABBIT EMBRYOS

Michael T. Kane

l. Introduction · · · · · · · · · · · · · · · · · · · 2. Relevance of Rabbit Embryo Culture to Studies of Embryonic Development · · · · · · · · · · 3. How Does Growth of Cultured Rabbit Embryos Compare With Growth In Vivo? · · · · · · · 4. In Vitro Growth Requirements of Rabbit Embryos

5. Methodological Considerations • · · · · · 6. Evaluation of Embryos • · · · · · · · · · 7. Conclusions · · · · · · · · · 8. References · · · · · · · · · · · · · ·

· · ·

· · · · ·

· · ·

·

154

157 158

162

164 164 165 168

175 176

181

187 188 189

193

194

195 198 206 209 212 213

CONTENTS

Chapter 11

STUDIES ON THE DEVELOPMENTAL BLOCKS IN CULTURED HAMSTER EMBRYOS

Barry D. Bavister

1. Introduction · · · · · · · · · · · · · · · · · · 2. Technical Procedures · · · · · · · · · · · 3. The 2-Cell Block to Development · · · · · · 4. Growth of 8-Cell Embryos In Vitro · · · · · · · · 5. Conclusions · · · · · · · · · 6. References · · · · · · · · · · · · · · · · Chapter 12

GROWTH OF DOMESTICATED ANIMAL EMBRYOS IN VITRO

Raymond W. Wright, Jr. and James V. O'Fallon

· · · · · · · · ·

· · · · · ·

xvii

219 222 228 236 245 246

1. Introduction ••••••••••••••••• 251 2. Media, Supplements and Antibiotics •••••• 253 3. Glucose Metabolism During Embryo Development 258 4. Embryo Co-Culture • • • • • • • • • • • • • • • •• 262 5. References .................. 266

Chapter 13

IN VITRO GROWTH OF NON-HUMAN PRIMATE PRE- AND PERI- IMPLANTATION EMBRYOS

Dorothy E. Boatman

1. Introduction · · · · · · · · · · · · 2. Technical Considerations · · · · · · 3. Supply of In Vivo Fertilized Embryos 4. In Vitro Fertilized Embryos ••••• · · · 5. Embryo Growth Characteristics •••• · · · 6. Assessment of Embryonic Normalcy after

In Vitro Culture · · · 7. Conclusions · · · · · · · · · · · · · · · · 8. Appendix: Culture Media · · · · · · · · · 9. References · · · · · · · · · · · · · · · · · ·

273 275

· · · · 276

· · · · · 279

· 282

· · · · 294

· 299

· · · 299

· · · · · 303

xviii CONTENTS

Chapter 14

ANALYSIS OF EMBRYO'l'OXIC EFFECTS IN PREIMPLANTATION EMBRYOS

Horst Spielmann

1. Introduction · · · · · · · · · · · · · · · · · · · 2. Sensitive Toxicological Endpoints · · · · · · 3. Exposure of Embryos In Vitro · · · · · · · · 4. Exposure of Embryos In Vivo • · · · · 5. Conclusions · 6. References · · · · · · · · · · · · · · Chapter 15

· · · ·

APPLICATIONS OF ANIMAL EMBRYO CULTURE RESEARCH TO HUMAN IVF AND EMBRYO TRANSFER PROORAMS

Susan Heyner

1. Introduction •••••••••••••••• 2. Studies on Animal IVF. Embryo Culture and

Transfer • . • • • • . . • • • . . · · · · 3. Present Status and Prospects of Human IVF-ET · · · · 4. Conclusions. • ••• · · · · · · · · 5. References ••••••••••••• · · · · APPENDIX I

1. Introduction · · · · · · · · · · · · · · · · · 2. Conditions for Embryo Culture · · · · · 3. Equipment . · · · · · · · · · · · · · · · · 4. Experimental Design · · · · · · · · · · · · · · 5. Evaluation of Responses to Embryo Culture · · 6. Blocks to Development · · · · · · 7. References · · · · · · · · · · · · · · · · · · APPENDIxn

1. Supplies for Embryo Culture Experiments 2. Equipment ••••••••••••••.•• 3. Manufacturers and Distributors of Supplies

and Equipment • • • • • • • • • • • • • •

INDEX • • . . • • • . . • . . . . • • • • . .

· · · · · ·

· · · · · · · · · · · ·

309 310 320 322 326 327

333

334 337 338 338

341 342 350 351 351 351 352

357 359

360

363