Reproductive Tissue Banking: Scientific Principles

I II REPRODUCTIVE TiSq~ BANKING Scientific Principles I II REPRODUCTIVE TiSq~ BANKING Scientific Principles Th...

Author: Armand M. Karow | John K. Critser

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REPRODUCTIVE TiSq~ BANKING Scientific

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REPRODUCTIVE Scientific Principles

Edited by

Armand M. Karow Department of Pharmacology and Toxicology Medical College of Georgia Augusta, Georgia and Xytex Corporation Augusta, Georgia

John K. Critser Cryobiology Research Institute Methodist Hospital of Indiana, Inc. Indianapolis, Indiana

ACADEMIC PRESS San Diego London Boston New York Sydney Tokyo Toronto

Photo credit: Reacting spermatozoon, exhibiting vesicles formed by the fusion of the plasma membrane with the underlying outer acrosomol membrane (see Chapter 6).

This book is printed on acid-free paper. ( ~

Copyright © 1997 by ACADEMIC PRESS All Rights Reserved. No part of this publication may be reproduced or transmitted in any form or by any means, electronic or mechanical, including photocopy, recording, or any information storage and retrieval system, without permission in writing from the publisher. Academic Press a division of Harcourt Brace & Company 525 B Street, Suite 1900, San Diego, California 92101-4495, USA http://www.apnet.com Academic Press Limited 24-28 Oval Road, London NW 1 7DX, UK http://www.hbuk.co.uk/ap/ Library of Congress Cataloging-in-Publication Data Reproductive tissue banking : scientific principles / edited by Armand M. Karow, John K. Critser p. cm. Includes bibliographical references and index. ISBN 0-12-399770-4 (alk. paper) 1. Human reproductive technology. 2. Sperm banks. 3. Cryopreservation of organs, tissues, etc. 4. Embryo transplantation. I. Karow, Armand M. II. Critser, John Kenneth. [DNLM: 1. Semen Preservation. 2. Oocytes--transplantation. 3. Embryo Transfer. 4. Ovary--transplantation. 5. Cryopreservation. WJ 834 R425 1997] RG133.5.R473 1997 612.6--DC20 DNLM/DLC for Library of Congress 96-43738 CIP

PRINTED IN THE UNITED STATES OF AMERICA 97 98 99 00 01 02 EB 9 8 7 6 5

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Contents

Contributors xv Preface xvii

Utility of Viable Tissue Ex Vivo

Banking of Reproductive Cells and Tissues Karen T. Gunasena and John K. Critser I. Semen Banking 2 A. Agriculture 2 B. Human Clinical Applications C. Genome Resource Banking II. Embryo Banking 8 A. Agriculture 9 B. Human Clinical Application C. Genome Resource Banking III. Oocyte Banking 13 IV. Ovarian Tissue Cryopreservation

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V. What Does the Future Hold for Reproductive Tissue Banking? 16 VI. Concluding Remarks 17 References 17

Tissue Maturation in Vivo and in Vitro Gamete and Early Emby o Ontogeny M. Lorraine Ieibfried-Rutkdge, Tanja DomMto, Elizabeth S. Critser, and John K. Critser

I. Introduction 23 11. Oocyte Maturation 25 A. Sources of Primary Oocytes 32 1. Laboratory Species 32 2. Domestic Animals 34 3. Primates 39 4. Exotics and Endangered Species 40 B. In Vitro Oocyte Maturation 40 1. Choice of Media 42 2. Gaseous Atmosphere 45 3. Macromolecular Supplements 47 4. Energy Substrates 49 5. Hormonal Supplements 49 6. Culture Methods 52 C. Follicular and Ovarian Factors Affecting Oocyte Competency 54 1. Oocyte Competency 54 2. Reproductive Status of the Donor 56 3. Age-Dependent Processes in Oocytes 57 D. Culture Systems for Oocytes from Earlier Stages of Oogenesis 60 1. Culture of Whole Antral Follicles 60 2. Culture of Primary Oocytes in Preantral Follicles 61 3. Culture of Stages Prior to the Primary Oocyte 62 111. Maturation of Spermatozoa 64 A. Sources of Mature Spermatozoa 72 1. Laboratory Species 74 2. Domestic Animals 75 3. Primates 76 4. Exotics and Endangered Species 77

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B. Culture Systems for Mature Spermatozoa 78 1. Sperm-Oocyte Interactions 78 2. Systems for Capacitation and Fertilization 81 3. Factors Affecting Sperm Fertility 85 C. Alternative Uses of Spermatogenic Stages in Vitro 87 1. Sperm Injection 88 2. Use of Cells at Earlier Stages of Spermatogenesis 90 IV. Preimplantation Embryonic Development 91 A. In Vivo Sources of Embryos 98 1. Laboratory Species 98 2. Domestic Animals 101 3, Primates 103 4. Exotics and Endangered Species 104 B. Culture of Mammalian Embryos 105 1. Biological Incubators 106 2. Coculture Systems 107 3. Defined Culture Systems 109 V. Concluding Remarks 111 References 111

Metabolic Support of Normothermia Roy H. Hammerstedt and Jane C. Andrews

I. Why Care about Metabolism at Normothermic Conditions? 139 A. A View of Integrated Cell Function 139 B. Effect of Cell Storage on Metabolic Balance 141 C. Integration of These Concepts into This Chapter 142 11. Overview of Metabolic Needs of Cells of Reproductive Interest 142 A. Heterogeneity in Metabolic Requirements 142 B. Selection of Cell Types for Discussion 144 111. Scope of This Review 144 A. General Bioenergetic Principles 144 B. Critical Questions to Be Developed in This Presentation 148

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IV. Overview of Integrated ATP Metabolism 149 A. The ATP Cycle 149 B. Modes of ATP Generation Used by Sperm 151 C. Modes of ATP Consumption Used by Sperm 152 1. Value of Information to Cell Storage 152 2. Allocation of ATP to Various ATP Consuming Pathways 153 3. Differences in ATP Turnover between Cauda Epididymal and Ejaculated Sperm 153 V. Examples of Metabolic Balance under Normothermic Conditions 153 A. Comparison of ATP Tunover in Cauda Epididymal and Ejaculated Bull Sperm 153 B. ATP Turnover in Bull, Ram and Ejaculated Rabbit Sperm 154 C. ATP Demands during Epididymal Storage 155 VI. Effect of Modest Changes in Temperature on ATP Turnover 155 A. Effect of Increased Temperature on Metabolism of Rooster Sperm 155 B. Effect of Decreased Temperature on Metabolism of Bull Sperm 156 VII. Literature Survey of Metabolic Needs of Other Cells of Reproduction Interest 159 VIII. A Primer for Construction of an ATP Balance Sheet 161 IX.Summary and Dedication 164 References 165

Pharmacological Interventions in Vitro Armand M. Karow

I. Introduction 167 11. General Characteristics of Drug Action 167 111. Receptor-Mediated Drug Action 169 A. Receptor Dynamics 170 B. Membrane-Bound Receptors 173 C. Nuclear Receptors 175 IV. Pharmacokinetics: Drug Access 176

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V. Gonadotropin Mediation of Folliculogenesis 182 A. Use of Gonadotropins in Tissue Preservation 182 B. Chemistry of Gonadotropins 183 C. Gonadotropin Receptors 187 IV. Reactive Oxygen Species (ROS) 187 A. Source and Biochemistry of ROS 188 B. ROS and Mammalian Spermatozoa in Vitro 192 C. Free Radical Scavengers 195 VII. Nonspecific Drug Action: Cryoprotectants (CPAs) 198 A. Sources of Cryoinjury 200 B. CPAs Limiting Freezing 201 C. CPAs Enhancing Freezing 207 VIII. Conclusions 208 References 209

Hypothermia and Mammalran Gametes John E. Parks

I. Introduction 229 11. Hypothermia and Mammalian Sperm 231 A. Overview of Mammalian Sperm Structure 232 B. Effects of Cold Shock on Mammalian Sperm 233 111. Membrane Organization and Thermotropic Phase Behavior 234 A. Organization and Structural Properties of Membrane Lipids 234 B. Thermotropic Phase Behavior of Membrane Lipids 235 IV. Sperm Membrane Lipid Composition 238 A. Sperm Phospholipid Composition 238 B. Sperm Glycolipid Composition 239 C. Sperm Sterol Composition 240 V. Relationship of Sperm Lipid Composition to Cold Shock 240 VI. Development Changes in Cold Shock Sensitivity 242 VII. Thermotropic Phase Behavior of Sperm Membrane Lipids 242

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VIII. Sperm Membrane Composition Relative to Phase Behavior of Mixed Lipid Systems 243 IX. Protection of Sperm from Hypothermic Effects 245 A. Protective Action of Egg Yolk and Its Components 245 B. Protective Action of Milk 246 C. Protective Action of Butylated Hydroxytoluene (BHT) and Its Analogs 247 D. Acquisition of Cold Shock Resistance in Boar Sperm 247 X. Conclusions 247 XI. Hypothermia and Mammalian Oocytes 248 XII. Overview of Mammalian Oocyte Structure 249 XIII. Effects of Cooling on Oocyte Structure 251 A. Effects of Cooling on the Oolemma 251 B. Effects of Temperature on the Spindle Apparatus on Mature Oocytes 253 C. Effects of Temperature on the Oocyte Cytoskeleton 254 D. Effects on Temperature on Cortical Granule Exocytosis 254 XIV. Interaction of Cooling and Cryoprotective Agents 255 XV. Effects of Hypothermia on Fertilization and Development 256 XVI. Preventing Hypothermic Damage to Mammalian Oocytes during Cryopreservation 257 XVII. Conclusions 257 References 258

Fundamental Cryobiology of M a x n f m h n Spermatozoa Dayong Gao, Peter Mazur, and John K. Critser

I. The Importance of and Need for Cryopreservation of Spermatozoa 263 11. Functional Aspects of Spermatozoa 265 A. SpermFunction 265 B. Assays of Sperm Function 266 1. Insemination and Pregnancy Initiation 266 2. Motility 268

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3. Plasma Membrane Integrity 272 4. Acrosomal Status 275 5. In Vitro Sperm-Egg Interactions 275 6. Temporal Aspects of Sperm Function 278 111. Fundamental Cryobiology of Mammalian Spermatozoa 278 A. Current Theory on Cell Cryoinjury 278 1. Cryoinjury during Cooling and Warming Processes 279 2. Preventing Injury During Slow Freezing 284 B. Spermatozoa as a Model Cell Type for Fundamental Cryobiology Research 285 C. Cryobiology of Mammalian Spermatozoa 286 1. Effect of Cryoprotective Agents (CPAs) 286 2. Effect of Cooling Rate: Cooling to the Freezing Point 288 3. Effect of Cooling Rate: Cooling below the Freezing Point 288 4. Warmingand Thawing 290 5. Fundamental Cryobiological Characteristics of Mammalian Spermatozoa 290 a. Osmotic Behavior of Spermatozoa 294 b. Sperm Water Permeability CoefJicient (L,) and Its Activation Energy (E,) 294 c. Permeability CoefJicient of Sperm to CPA (PcpA)and Its Activation Energy (E,) 297 d. Intracellular Ice Formation Temperatures 299 e. Sperm Tolerance Limits for Volume Excursion 300 j How to Use Determined Cryobiology Characteristicsto Optimize Cryopresewation Procedures 302 D. Future Research Areas 312 References 313

The Cryobiology of M a a n m aO hOn o c y t e s John K. Critser, Yuksel Agca, and Karen T. G u n a ~ e n a

I. Introduction 329 11. The History of Oocyte Cryopreservation 332

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111. The Current Status of Mammalian Oocyte Cryobiology 333 IV. The Cryobiology of Various Mammalian Species Oocytes 338 A. Mouse Oocytes 338 B. Bovine Oocytes 339 C. Rat Oocytes 341 D. Human Oocytes 343 V. Vitrification 345 VI. Summary 350 References 351

Cryopreservation of Mdticellular Embryos and Reproductive Tissues Sharon Paynter, Angela Cooper, Non Thomas, and Barry Fuller

I. Introduction 359 11. Cryopreservation of Reproductive Tissue 360 A. Structure and Physiology of the Ovary 361 B. Historical Review of Cryopreservation of Ovarian Tissue 361 111. Fundamental Aspects of Ovarian Tissue Cryopreservation 367 A. Physical Parameters of Ice Formation in Tissues 367 B. Permeation of Ovarian Tissue by Cryoprotectants 373 IV. Cryopreservation of Preimplantation Embryos 376 A. Development of the Pre-embryo 378 1. Stages of Pre-embryo Development 378 2. Embryo Culture 379 B. Fundamental Aspects of Cryobiology in Pre-embryos 382 V. Approaches to Embryo Cryopreservation 384 A. Techniques Using Slow Cooling 384 B. Techniques Using Rapid Cooling 385 C. Results of Embryo Cryopreservation 386 1. Slow Cooling Techniques 386 2. Rapid Cooling Techniques 389 3. Cryopreservation of Micromanipulated Embryos 391

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VI. Summary 392 References 393

Genome Resource Banking Impact on Biotic Conservation and Society David E. Wildt

I. Introduction 399 11. An Introduction to Biodiversity 400 111. Why Conserve Bio- and Genetic Diversity? 401 IV. How Complex Is the Task of Conserving Biological and Genetic Diversity? 405 V. A Role for Our Science in Conservation Biology 407 VI. General Types of Conservation Need 408 VII. Conservation of Crops and Livestock 409 VIII. Conservation of Laboratory Animals, Invertebrates, and Microorganisms 413 IX. GRBs for Wildlife Conservation-Advantages for the Endangered “Otboe” 414 A. Advantage 1: Easier and Cheaper Movement of Genetic Material 416 B. Advantage 2: Increased Efficiency in Captive Breeding; More Animals Become Successful Breeders 417 C. Advantage 3: Reduced Genetic Problems 417 D. Advantage 4: Fewer Space Problems 417 E. Advantage 5: Preserved Extant Genetic Diversity 418 F. Advantage 6: A Resource for Other Biomaterials (i.e., Blood Products, Tissue, andDNA) 418 G. Advantage 7: Economics 419 X. Organizational Planning for Effective Wildlife GRBs 421 XI. Science and Societal Needs to Achieve Biotic Cryoconservation 429 A. Knowledge and Support 429 B. Cooperation and Sharing 431 C. Birthing GRBs 432

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D. Specific Resources 433 E. Databases 434 XII. Summary 435 References 436

Implications of Tissue Banking for Human Reproductive Medicine Armand M. Karow

I. Introduction 441 11. Reproductive Technology Serving Medicine 443 111. Economic Impact of Reproductive Technology in America 444 IV. Social Issues in Reproductive Technology 447 A. Family Values 447 B. Moral Value of Being Human 449 C. Property Rights in Personal Tissue 451 D. Access to Health Services 452 E. Pursuit of Knowledge 452 V. American Regulation of Reproductive Technologies 453 VI. Social Interaction with Genetic Technology Working through Reproductive Medicine 455 VII. Conclusion 458 References 460 Index 465

Contributors

Numbers in parentheses indicate the pages on which the authors' contributions begin.

Yuksel Agca (329) Cryobiology Research Institute, Methodist Hospital of

Indiana, Inc., Indianapolis, Indiana 46202 Jane C. Andrews (139) Department of Biochemistry and Molecular Biology, The Pennsylvania State University, University Park, Pennsylvania 16802 Angela Cooper (359) Department of Obstetrics and Gynaecology, University of Wales College of Medicine, Heath Park, Cardiff CF4 4XN, Wales Elizabeth S. Critser (23) Advanced Fertility Institute, Methodist Hospital of Indiana, Inc., Indianapolis, Indiana 46202 John K. Critser (1, 23, 263, 329) Cryobiology Research Institute, Methodist Hospital of Indiana, Inc., Indianapolis, Indiana 46202 Tanja Dominko (23) Department of Meat and Animal Science, University of Wisconsin--Madison, Madison, Wisconsin 53706 Barry Fuller (359) Department of Obstetrics and Gynaecology, University of Wales College of Medicine, Heath Park, Cardiff CF4 4XN, Wales

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Contributors

Dayong Gao (263) Cryobiology Research Institute, Methodist Hospital of

Indiana, Inc., Indianapolis, Indiana 46202; and Department of Mechanical Engineering, Indiana University-Purdue University, Indianapolis, Indiana 46206 Karen T. Gunasena (1,329) Cryobiology Research Institute, Methodist Hospital of Indiana, Inc., Indianapolis, Indiana 46202 Roy H. Hammerstedt (139) Department of Biochemistry and Molecular Biology, The Pennsylvania State University, University Park, Pennsylvania 16802 Armand M. Karow (167, 441) Department of Pharmacology and Toxicology, Medical College of Georgia, Augusta, Georgia 30912 and Xytex Corporation, Augusta, Georgia 30904 M. Lorraine Leibfried-Rutledge (23) Department of Meat and Animal Science, University of Wisconsin~Madison, Madison, Wisconsin 53706 Peter Mazur (263) Fundamental and Applied Cryobiology Group, Biology Division, Oak Ridge National Laboratory, Oak Ridge, Tennessee 37831 John E. Parks (229) Department of Animal Science, Comell University, Ithaca, New York 14853 Sharon Paynter (359) Department of Obstetrics and Gynaecology, University of Wales College of Medicine, Heath Park, Cardiff CF4 4XN, Wales Non Thomas (359) Department of Obstetrics and Gynaecology, University of Wales College of Medicine, Heath Park, Cardiff CF4 4XN, Wales David E. Wildt (399) Conservation and Research Center, National Zoological Park, Smithsonian Institute, Fort Royal, Virginia 22630

Preface

The chief aim of the editors and authors of this book is to present the scientific basis of reproductive tissue banking to those able to enhance the circumstance of tissue banking. Reproductive tissues serve as a model for the technology of banking other living tissues. Reproductive tissues discussed here include gonads (primarily ovary), gametes, and preimplantation embryos. Technology for reproductive tissue banking is derived from principles basic to physics, chemistry, and biology. Current application and advancement of the technology are enhanced by knowledge of these principles. Our emphasis is on principles and theory, supported by examples drawn from mammalian reproductive biology. Readers seeking laboratory techniques for tissue banking, especially as applied to reproductive tissues, are directed to current literature. Scientific principles of tissue banking are presented in a manner accessible to readers who have a collegiate background in science. Presentations are intended to enable these readers to delve confidently into current research reports. Topics selected for presentation are representative rather than comprehensive. Chapters are self-contained presentations focused on a theme and closely related scientific principles. Chapter authors have developed con-

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Preface

cepts as required by the chapter's central theme, and therefore some concepts are developed more than once. Such presentations have been crossreferenced to other material in the book. The authors are scientists with laboratory experience in the topics presented. Presentations are made with the desire to involve others who will share in the scientific and technical advancement of tissue banking. Financial resources to support necessary research must come from persons recognizing the social benefits of such advancements. To this end, some social issues, risks, and opportunities are discussed herein. Reproductive tissue banking serves human medicine, the commercial livestock industry, specialty breeding such as laboratory animals, and conservation of global genomic resources of vertebrates. The editors are grateful for the valiant, sustained, and cheerful secretarial assistance of Tonya Montgomery and Katherine Vernon. Armand M. Karow John K. Critser

Utility of Viable Tissues ex Vivo Banking of Reproductive Cells and Tissues

K a r e n T. G u n a s e n a a n d J o h n K. Critser Cryobiology Research Institute Methodist Hospital of Indiana, Inc. Indianapolis, Indiana 46202

The term "banking" is defined literally as safekeeping or storage of utilities for emergency use. In the context of reproductive biology, gametes, embryos, and tissues are accumulated for use at a future time. However, these cells require manipulation with media and/or lowered temperatures to retain their functional and developmental capacity after a period of banking. The method employed depends primarily on the duration of storage required, which can be from a few days (extenders, usually semen and sperm) to several years (cryopreservation). This book concentrates primarily on long-term, subzero storage which has proven to be essential in maintaining viability of reproductive cells and tissues (semen, embryos, oocytes, ovarian tissue). These methods are applied in reproductive tissue banking for agriculture, human clinical treatment, and research programs and in the preservation of endangered species. This chapter is intended to give the reader an overview of the current technologies and approaches utilized in banking reproductive cells and tissues ex vivo, which will be described more fully in the following chapters. Reproductive Tissue Banking Copyright 9 1997 by Academic Press. All rights of reproduction in any form reserved.

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Karen T. Gunasena a n d J o h n K. Critser

I. SEMEN B A N K I N G

A. A g r i c u l t u r e The effects of freezing temperatures on human semen were described as early as 1776 by Lazzaro Spallanzani. In the late 1930s and early 1940s many observers reported limited survival of sperm at temperatures of -269~ in the absence of a cryoprotectant. When cryoprotectants became known, investigations on cryopreservation of semen developed rapidly in the field of animal and veterinary science with an increasing demand for semen in artificial insemination (AI) breeding programs. Cryopreservation of bovine semen to -79~ yielded sufficient viable sperm post-thaw to result in pregnancies and calves (Polge and Lovelock, 1952; Polge and Rowson, 1952). Conception rates from the thawed sperm then averaged 65% in 208 cows (Polge and Rowson, 1952), a rate that is about as good as that generally achieved nearly 30 years later (Iritani, 1980; Pace, 1980). Developments in the ensuing 18 years were reviewed by Watson (1979, 1990) and Polge (1980). The major change was that Polge's original slow freezing technique gave way in the middle 1960s to rapid cooling techniques, yielding cooling rates of 100 to 200~ and to straw freezing, which is in current use today. Improvements in the cryopreservation of bovine sperm have been small and are due almost entirely to developments in techniques, rather than to advances in our understanding of cryoinjury and its prevention (Watson, 1979). Breeding of dairy and beef cattle by AI increased sharply in the late 1950s, due to the availability of cryopreserved semen with improved postthaw viability (Figure 1). Artificial insemination in beef cattle has historically been much lower than in dairy cattle. Cryopreserved semen is now the major source for AI of cattle bred for the meat and dairy industry (Herman, 1988). Following the successful application of the protective action of glycerol in the cryopreservation of bovine sperm, Polge (1956) attempted to apply the same approach to the low-temperature preservation of porcine sperm. However, it was immediately recognized that porcine sperm responded to cryobiological factors quite differently than bovine or human sperm. Either glycerol addition or cooling to temperatures below 15~ markedly reduced porcine sperm survival (Polge, 1956). Subsequently, it was found that shortterm storage of porcine semen (so-called preservation in the liquid state) could be performed fairly readily by diluting the semen with a variety of extenders combined with maintenance at 15 to 18~ (Reed, 1969; Watson, 1979, 1990; Pursel, 1979). During this period of time, commercial AI with liquid porcine semen became a viable industry in its own right, although markedly smaller than the bovine AI industry. Because of the success with liquid semen and because porcine sperm represented a rather difficult cell

Utility o f Viable Tissues ex Vivo: Banking of Reproductive Cells and Tissues

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The number of dairy (top, 1938 to 1978) and beef (bottom, 1963 to 1978) cows

bred artificially to bulls. Source: Dairy Herd Improvement Letter A R S (1963-1978), U S D A ; and NAAB reports (1947-1979). Reproduced with permission from Herman (1988).

type to cryopreserve, little work regarding the freezing of boar semen was conducted during the 1960s. Then in the 1970s interest in low-temperature storage of porcine sperm was rekindled with the increased use of the "pelleting" method developed by Nagase (1972). The rapid cooling rates of this method could be incorporated with low concentrations of glycerol (3-4%) necessitated by the boar sperm's sensitivity to this cryoprotectant. Polge et al. (1970) found that the pelleting method with low glycerol concentrations, enabled boar sperm to survive freezing and thawing. However, low fertility has been a persistent problem with the use of frozen-thawed boar semen and its commercial use remains relatively limited, representing only about 270,000 of a total of approximately 5.4 million or 0.5% of all commercial inseminations (Iritani, 1980; Reed 1985; Watson, 1990) (Table

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Karen T. Gunasena and John K. Critser

T A B L E 1 N u m b e r of Artificial Inseminations (AIs) P e r f o r m e d Worldwide Using Fresh and Frozen B o a r S e m e n a

Country

No. of AIs with fresh semen

No. of AIs with frozen semen

Percentage of Als with frozen semen ~

A ustri a Belgium Canada Denmark Democratic Republic of Germany Federal Republic of Germany Finland France Great Britain Hungary Italy Japan Netherlands Norway Peoples' Republic of China Poland Republic of China (Taiwan) Spain Sweden Switzerland USA

196,591 120,000 16,000 450,000 1,690,000 640,306 84,000 80,000 75,000 400,000 50,000 60,000 1,000,000 90,000 Widely used 100,236 103,542 350,000 36,000 50,000 100,000

n/a < 100 300 < 100 n/a 100 193 200 <100 300 15 1200 16 100 250 n/a 3000 200 10 66 7500

n/a <1 2 <1 n/a <1 <1 <1 <1 <1 <1 2 <1 <1 n/a n/a 3 <1 <1 <1 7.5

Note. n/a, not available. a Modified from Reed (1985). b Percentage of AIs with frozen semen is approximated from the published data.

1). Variability, high costs, and complexities in thawing and processing were among other factors contributing to the limited use of cryopreserved semen in AI programs.

B. Human Clinical Applications Artificial insemination using husband's semen was first reported by John Hunter in 1785 in England (Kleegman, 1967). Following the successful use of cryopreserved semen for AI in agricultural breeding programs, interest turned to a human clinical application. Therapeutic use of cryopreserved human semen was slow, beginning in 1953 with only 25 children born in the following 10 years (Sherman, 1979). Clinical use of frozen-thawed semen for AI continued at a slow rate until about 1985 when the increased incidence of human immunodeficiency virus (HIV) infection resulting in acquired immune deficiency syndrome (AIDS) raised concerns over the possibility of infecting patients during AI with flesh semen. Interest turned to cryopreservation as a method of storing semen until the donor could be

Utility of Viable Tissues ex Vivo: Banking of Reproductive Cells and Tissues

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verified as negative for HIV and a host of other sexually transmitted diseases. Around this time several U.S.-based professional societies issued guidelines (American Society of Reproductive Medicine, ASRM), standards (American Association for Tissue Banks, AATB), and regulations (state governments, New York and Indiana) requiring the exclusive use of cryopreserved sperm for human treatment. The ability to store frozen semen would allow sufficient time for seroconversion of donors so that possible carriers of the HIV could be detected (Masco!a and Guinan, 1986). The 1993 interim rule of the Food and Drug Administration (FDA), an agency of the United States federal government, enforced regulation of human tissue banking with the exception of reproductive cells and tissues. In 1995, the FDA attempted to redress this exclusion by compiling a discussion document regarding screening of donors of reproductive cells and tissues for transplantation (FDA, 1995). The proposed regulations specifically describe measures for controlling the transmission of communicable diseases (e.g., HIV-1 and -2, hepatitis, and other sexually transmitted diseases). The proposed regulations require medical, genetic, and social history of gamete donors (i.e., intravenous drug use, sexual behavior). When these regulations are enforced, semen banking in the United States should move from being an unregulated, voluntary, peer review system to a federally regulated one, with the establishment of a federal register of semen banks. Governments of other nations including Australia and many in Europe already regulate sperm banks. Sperm banks using anonymous donors are widespread throughout the United States, Canada, and Europe. A survey of major semen banks in the United States conducted by the AATB showed there to be 14 states without any anonymous semen banks and 2 with more than 10 in the state (Figure 2; Critser, 1995). However, there were perplexing fluctuations in the number of reported sperm banks over the late 1980s, varying from 493 in 1987 to 61 in 1988 to 135 in 1989 and only 42 in 1990 (Olson, 1995). This figure then apparently increased 10-fold by 1994 with 451 units recorded. These fluctuations are probably compounded by the different categories of sperm banks in the United States. Some centers, for-profit corporations, collect and bank samples for sale to assisted reproduction centers, which perform the inseminations. Alternately, centers providing inseminations may also have their own anonymous donors and bank samples for use in that program. These differences have contributed to the apparent inaccuracies in describing the number of centers that offer sperm banking. The demand for donor semen samples for clinical use continued to increase from 83,309 in 1992 to 85,446 in 1993 and 90,312 in 1994 (Figure 3; Critser, 1995). These samples are utilized for AI or in vitro fertilization. Concerns have been raised that conception rates with frozen-thawed sperm are lower than those with fresh (i.e., nonfrozen) sperm (Sherman, 1979; Corson et al., 1983). However, these concerns are somewhat academic,

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Karen T. Gunasena and John K. Critser

FIGURE 2

Numberof sperm banks, by state, in the United States (from Critser, 1995).

considering the imperative to use only cryopreserved quarantined semen samples to prevent transmission of several diseases. Semen banking is also utilized for the donors' own use. I n vitro fertilization treatments for infertile couples involves many days of ultrasound monitoring and physician visits. When oocytes are retrieved there is only a 4- to 6-hr window of time for insemination. If a sperm sample is not obtained at this time the treatment cycle has to be abandoned. As a preventative measure, some programs recommend sperm cryopreservation prior to treatment in the event that the partner is unavailable or unable to produce a semen sample on the day of oocyte retrieval. Male oncology patients who are at risk of iatrogenic infertility following chemo- and/or radiotherapy can also benefit from se-

FIGURE 3

Annual demand for semen samples 1992 to 1994 (from Critser, 1995).

Utility of Viable Tissues ex Vivo: Banking of Reproductive Cells and Tissues

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men cryopreservation, if semen is collected prior to treatment. Ideally, several semen samples should be collected at least 48 hr apart. Semen banking for oncology patients is an option that is often underutilized, due to urgency in moving from diagnosis to treatment, patients feeling too ill, inability to obtain a specimen, azoospermia, and no post-thaw motility. Low sperm numbers and poor motility are no longer exclusion criteria to cryopreserve semen samples from oncology patients, as intracytoplasmic sperm injection (ICSI) can be used to inseminate oocytes and only one sperm (per oocyte) is required. C. G e n o m e R e s o u r c e B a n k i n g A major effort is underway in many laboratories to characterize genes associated with embryonic development and disease in humans and other species. A powerful approach involves molecular analysis of mutations because the change in gene expression associated with a mutation often permits one to establish normal function of that gene. The mouse, because of its well-characterized genetic makeup and its accessibility to experimental manipulation, is an excellent system for studying the specific functions of genes in mammals. An ever expanding number of transgenic mouse lines is being produced, and the preservation of these strains is becoming a major issue. Normally once a transgenic mutant line is established, it is maintained like classical mutant lines by maintaining breeding colonies. However, that has disadvantages, namely, (a) it is costly, labor-intensive, and space demanding, and (b) strains may be lost by impaired reproductive efficiency, disease, or death. An alternative procedure for maintaining breeding colonies of transgenic or other mutant lines of mice is to cryopreserve embryos or even sperm. Cryopreservation of sperm is especially appealing, assuming that effective cryopreservation is feasible. Donor males would not have to be mated or hormonally treated, and collection of sperm and their subsequent manipulation may be somewhat faster and less demanding than the collection of embryos. Quite possibly, considerably fewer donor males would be required to produce an adequate sperm bank than the number of female donors required to produce an adequate embryo bank. Furthermore, preservation via sperm would permit cryopreservation of traits that become lethal in transgenic embryos. To rederive a desired strain from frozen sperm, ova from a wild-type line would be inseminated in vitro but possibly in vivo (Tanphaichitr et al., 1992) with the thawed sperm. In the former case the resulting embryos would be cultured in vitro to the two- to eight-cell stage and then transferred to recipient females for development to pups. In vitro fertilization techniques for mouse sperm have been well worked out during the past decade. In the simple case of a single copy of a gene being inserted, the desired

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Karen T. Gunasena a n d John K. Critser

trait would exist in heterozygous form in half the offspring. The trait could be obtained in homozygous form by appropriate backcrossing. Critical to the feasibility and cost-effectiveness of this approach is dependent upon whether mouse sperm can be cryopreserved in the sense of reasonable viability after freezing and thawing. Probably no mammalian cell type exhibits a greater range in cryobiological sensitivity than sperm. Human and bovine sperm can be frozen with reasonably high functional survival (i.e., ability to fertilize), although even in these cases, average survivals are mediocre and individual-to-individual variation is great. Until about 2 years ago the cryopreservation of mouse sperm had not been achieved. This may have been due in part to a lack of interest in attempting to do so but it probably also reflects inherent problems of unknown nature with the sperm of this species. Recently, two Japanese laboratories (Tada et al., 1990; Yokoyama et al., 1990) reported successful cryopreservation of mouse sperm using somewhat similar procedures, successful in the sense that thawed sperm produced embryos and mouse pups. However, three other groups (M. Wood and R. Rail, personal communications; Penfold et al., 1991) have been unable to obtain any viable sperm following these procedures. Mobraaten et al. (1991) reported modest success using very different procedures. The procedures so far examined have been derived purely empirically and more information on the cryobiology of mouse sperm is needed before semen banking to maintain transgenic animal models becomes routine. Cryopreservation of semen from endangered species is also becoming a valuable tool in conservation and maintenance of biodiversity (Dresser, 1988; Wildt et al., 1993). Semen samples can be stored for several decades and used for AI or to fertilize oocytes in vitro. Offspring from several species have been achieved in zoological parks across the world, although post-thaw survival is low and needs more detailed evaluation.

II. EMBRYO B A N K I N G

Applications of embryo cryopreservation are manifold, benefiting livestock producers, research laboratories, human clinical programs, and genome resource banks. The earliest attempt to store embryos at subphysiological temperatures was by Chang in 1947, who reported continued development of rabbit embryos at 10~ for 144 hr. The first successful postthaw survival of mouse embryos after cryopreservation was reported by Whittingham in 1971. Two years later the first birth of a calf after surgical transfer of frozen-thawed embryos was reported (Wilmut and Rowson, 1973). Since then, pregnancies and live births in many species have been reported (Leibo, 1986). Conventional cryopreservation techniques involve costly and sophisticated equipment for controlling cooling and warming

Utility o f Viable Tissues ex Vivo: Banking o f Reproductive Cells a n d Tissues

9

rates. An alternative to conventional equilibrium methods is vitrification, where embryos are exposed to high concentrations of cryoprotectants (i.e., 3-4 M dimethylsulfoxide or glycerol) and nonpermeating solutes (e.g., sucrose), followed by direct plunging in liquid nitrogen. When thawing is rapid, survival of mouse and rat embryos cryopreserved by this simple technique is reported to be comparable to other cryopreservation techniques (Takeda et al., 1984; Trounson et al., 1987). A. A g r i c u l t u r e During the late 1970s and early 1980s there was a sharp increase in the United States regarding importation of many beef cattle breeds commonly used in European countries (e.g., Chianina, Limousin, Maine Anjou, Simmental). Collectively these breeds were described as "exotics" and these animals became quite valuable over a short period of time. One reason for their perceived value was as a source of increased genetic vigor, although much of the value was a result of short-term marketing and speculation. During this time, because of the high market value for these animals and their offspring, many embryo transfer operations were initiated and thrived on this "exotics market." As more embryo transfers were performed, the importance of developing improved methods to cryopreserve, store, and transport bovine embryos increased dramatically. As more resources were committed to developing improved cryopreservation methods for the exotic driven market, these methods became more widely applied to other breeds and breeding scenarios, primarily in the dairy cattle industry. Today, bovine embryo transfer and embryo cryopreservation are routinely performed on thousands of animals, resulting in hundreds of thousands of offspring each year (Table 2). Cryopreserved embryos can be stored for long periods, maintaining genetic stocks at a small cost compared to that involved in housing and feeding the animals. In addition, cryopreservation of embryos from the control herd in a livestock population allows long-term maintenance of a "constant" genetic stock. Clearly, the costs and expertise required to establish successful cryopreservation procedures and achieve high post-thaw viability of embryos are not insignificant. Thus, the high proportion of cryopreserved embryo transfers in the cattle industry is indicative of the acceptable cost-benefit ratio in delivery rates from thawed embryos.

B. H u m a n Clinical Application A major breakthrough in the treatment of human infertility came in 1978 with the delivery of Louise Brown, conceived after retrieval of a single oocyte, fertilized in vitro, and the resulting embryo returned to the uterus. Recovery of oocytes from the ovary during a natural menstrual cycle often fails because

10

Karen T. Gunasena and John K. Critser

T A B t E 2 Number of Transfers of Fresh and Frozen-Thawed Bovine Embryos Performed Worldwide in 1992 to 1994 Continents

Embryos transferred

Fresh

% Fresh

Frozen

Africa North America South America Asia Europe Oceania Total

9,707 126,587 20,245 28,829 96,300 986 282,654

1992a 5,614 21,775b 12,941 20,221c 48,100 422 109,073

58 46b 64 74.5c 50 43 54

Africa North America South America Asia Europe Oceania Total

8,542 124,941 23,743 35,516 96,470 n/a 289,212

1993d 5,868 75,321 17,349 10,327 47,270 n/a 156,135

69 60 73 29 49 n/a 54

2,674 49,620 6,394 25,189 49,200 n/a 133,077

31 40 27 71 51 n/a 46

Africa North America South America Asia Europe Oceania Total

9,901 169,771 39,001 52,486 102,887 9,794 383,840

1994e 6,743 93,414 25,556 13,420 48,402 6,078 193,613

68 55 66 26 47 62 50

3,158 76,357 13,445 39,066 54,485 3,716 190,227

32 45 34 74 53 38 50

4,093 25,562b 7,204 6,921c 48,200 564 92,644

% Frozen

42 54b 36 25.5c 50 57 46

Note. n/a, no data available. Data from Embryo Transfer Newsletter, Vol. 11(4), 1993, and Thibier, personal communication. b Data (47,337 embryos) available only from Canada. c Data (27,142 embryos) not available from all Asian countries. d Data from Embryo Transfer Newsletter, Vol. 12(4), 1994. e Data from Embryo Transfer Newsletter, Vol. 13(~,), 1995.

a

o v u l a t i o n occurs b e f o r e the s c h e d u l e d retrieval. S t i m u l a t i o n r e g i m e n s with g o n a d o t r o p i n s t h a t e n a b l e d g r e a t e r c o n t r o l o v e r t h e o v a r i a n r e s p o n s e , prev e n t i n g o v u l a t i o n p r i o r to r e t r i e v a l a n d inducing the r e c r u i t m e n t a n d develo p m e n t of m o r e t h a n o n e o o c y t e ( s u p e r o v u l a t i o n ) , w e r e d e v e l o p e d . R e c o v ery of s e v e r a l o o c y t e s during an in vitro fertilization t r e a t m e n t cycle is c o m m o n p l a c e after s u p e r o v u l a t i o n a n d all r e c o v e r e d o o c y t e s are usually ins e m i n a t e d . S o m e couples m a y c h o o s e to h a v e only a few o o c y t e s i n s e m i n a t e d , t h e r e b y restricting the n u m b e r of e m b r y o s c r e a t e d , which t h e y see as t h e b e g i n n i n g of h u m a n life. H o w e v e r , m o s t couples wish to m a x i m i z e their

Utility of Viable Tissues ex Vivo: Banking of Reproductive Cells and Tissues

11

chances of a successful outcome and have all oocytes inseminated, often resuiting in supernumerary embryos, i.e., those that are not replaced in the uterus. Cryopreservation has become widely used as a means of rescuing these embryos that would otherwise ultimately perish in culture. Embryo cryopreservation was originally developed for agriculture and adapted for human clinical use in the early 1980s with the first pregnancy after transfer of a frozen-thawed human embryo reported in 1983 (Trounson and Mohr). Concerns were raised over the unknown effects of cryoprotectants and exposure to low temperatures on the health and development of the child. Babies born from frozen-thawed embryos indicate no significant differences in birth weight, development, and congenital abnormalities between children from fresh (i.e., nonfrozen) or frozen embryos (Wada et al., 1994; Sutcliffe et aL, 1995). Human embryo cryopreservation is now a routine clinical procedure enabling storage of supernumerary embryos for transfer during a future treatment cycle. Some couples may decide to donate their frozen embryos to research or for the treatment of another couple, usually if they have been successful and do not wish to have more children. The Society for Assisted Reproductive Technology (SART) maintains a database from American and Canadian centers performing in vitro fertilization, recording the number of procedures performed, pregnancy and delivery rates (Table 3). Since 1991 the number of programs, procedures, and pregnancy rates have increased gradually, due to a combination of increased practice and efficiency in centers performing these procedures. Pregnancy rates with cryopreserved embryos are slightly lower than those with fresh embryos (Table 3). Some reports suggest that pregnancy rates after replacement of frozen-thawed bovine embryos derived from in vitro fertilization are lower than those recovered after in vivo development (Touati et aL, 1992; Massip et al., 1995). Culture conditions to which embryos are subjected during in vitro fertilization must also be considered as a factor in post-thaw viability, not only the damage incurred during freezing and thawing. Human embryo cryopreservation and storage is accompanied by many ethical and legal considerations. Once fertilized the oocyte takes on a multitude of ethical questions. Emotions can often run high as some patients visualize the cryopreserved cells as a human in suspended animation. What are the "rights" of the frozen embryo? What happens to the embryo if one or both of the parents die or become incapacitated? Who becomes responsible for the embryo if the parents separate or divorce and then remarry? Several incidents have already occurred in which "custody" of the embryos has been disputed in the courts (Shuster, 1990; Robertson, 1996). Patients complete many consent forms to allow cryopreservation and storage of their embryos, but some couples lose contact with the program after their treatment is completed. The programs are then left to consider the disposition of the cryopreserved embryos. Sometimes embryos are lost through damage or failure of storage containers, resulting in great

12

K a r e n T. G u n a s e n a a n d J o h n K. Critser

N u m b e r of in V i t r o Fertilization ( I V F ) Cycles, Frozen E m b r y o Transfers (FETs), Clinical Pregnancies, Deliveries, and Birth Defects R e p o r t e d by Centers in the United States and Canada for 1990 to 1993

TABLE 3

1990 a

1991 b

1992 c

1993 d

No. of IVF cycles initiated

19,079

24,671

29,404

31,900

No. of oocyte retrievals performed

16,405

21,083

24,996

27,443

Deliveries per retrieval (%) Birth defects per neonate delivered after IVF (%) No. of programs performing FETs (%)e No. of FET procedures performed e

14 n/a

129 (72)

3,290

15.2

16.8

18.6

1.5

1.9

2.3

153 (82)

4,225

208 (83.5)

5,354

234 (87.6)

6,194

No. of clinical pregnancies (% per FET) e

383 (12)

559 (13.2)

820 (15.3)

984 (15.8)

No. of deliveries (% per FET) e

291 (9)

431 (10.2)

619 (11.6)

791 (12.8)

Birth defects per neonate delivered after FET e (%)

n/a

0.8

1.3

1.8

Note. n/a, no data available.

Data from Med. Res. Int./SART, 1992, Fertil. Steril. 57, 15-24. b Data from SART/AFS, 1993, Fertil. SteriL 59, 956-962. c Data from AFS/SART, 1994, Fertil. Steril. 62, 1121-1128. d Data from SART/ASRM, 1995, Fertil. Steril. 64, 13-21. e Cryopreserved embryos derived from the intended recipient, i.e., not donated embyros.

a

distress to couples. Although extreme and often misguided views can be detrimental to the assisted reproductive technology programs and patients, it is essential that debate and discussion about these issues continue.

C. G e n o m e Resource Banking In research laboratories, embryos are cryopreserved in an effort to develop and maintain stocks of a variety of strains and important genotypes, i.e., transgenics (Mobraaten, 1986). The ability to do so was first reported in the early 1970s by Whittingham et al. (1972). Some 75% or more of eight-cell mouse embryos survive such cryopreservation and the percentage of viable cryopreserved embryos that can develop to pups after embryo

Utility o f Viable Tissues ex Vivo: B a n k i n g o f Reproductive Cells a n d Tissues

13

transfer is about the same as the percentage that is capable of doing so in the absence of cryopreservation. Mouse genetic laboratories are currently making use of such cryopreservation for normal mutant lines. Although embryo cryopreservation is now, in a sense, "routine," it is not simple or inexpensive. Besides cryopreservation per se, it involves purchase or breeding of a sufficient number of donor females, their injection with superovulatory hormones, mating with males which have to be caged individually, and rather difficult surgical techniques to obtain embryos. After cryopreservation, it requires in vitro culture of thawed embryos, a colony of recipient females, a group of vasectomized males to prime the females, and difficult and precise surgical techniques to effect embryo transfer. Commonly, banks consist of 500-1000 frozen embryos per line. The number of donor animals required to produce the desired number of embryos, and hence the cost, is sensitive to the average litter size of breeders that produce donors, the average number of embryos that a superovulated donor female yields, and the variation in yield, which often is wide. Cryopreservation of embryos from inbred lines can incur costs of $20-$25 per embryo, excluding the cost of rederiving the line by embryo transfer. Most of the cost is in breeding and maintaining donor animals.

m.

BANKING

Ethical issues surrounding embryo cryopreservation would be somewhat less emotive if unfertilized oocytes were cryopreserved. The concerns would essentially be those already addressed for sperm held in semen banks. However, the ability to cryopreserve oocytes is limited by poor viability and chilling-induced chromosomal anomalies. Many women experience conditions that result in diminished or lost fertility (e.g., oncology treatment, premature menopause) when they are not ready to have children, due to age or circumstance. If feasible, oocytes could be retrieved from these patients, cryopreserved, and stored for their future use. In addition, there is a subset of infertile women who are unable to produce oocytes and whose only treatment alternative is to receive donated oocytes. Fertile women volunteer to donate oocytes and undergo superovulation and oocyte retrieval with notable discomfort and some surgical risks. Recovered oocytes are then inseminated with the recipient's partner's sperm. The limited viability of oocytes necessitates close coordination between donor and recipient's cycles, and often more than one recipient is prepared to ensure best use of the oocytes. In addition, donors must be screened for several diseases (as for semen donors) prior to and at the time of donation and development of diseases after donation is still a risk. The ability to cryopreserve oocytes would allow sufficient time for seroconversion of donors so that possible carriers of the HIV could be detected before oocytes are

14

K a r e n T. G u n a s e n a a n d J o h n K. Critser

utilized, as is currently done for sperm donors (FDA, 1995; van den Eede, 1995). Cryopreservation of nonhuman oocytes would also have potential applications for maintenance of biodiversity. Production of transgenic mice from frozen-thawed oocytes after microinjection of DNA has proven to be a viable method of maintaining such genetically desirable strains (Leibo et al., 1991). The stage at which an oocyte is cryopreserved is critical to the postthaw viability. In the ovary, follicle enclosed oocytes are maintained in a state of meiotic arrest. Throughout the mammalian female's reproductive life, follicles will grow and develop around the oocyte, which secretes the zona pellucida. Follicle-enclosed oocytes are at the germinal vesicle (GV) stage, where the nuclear material is surrounded by a membrane. Shortly before ovulation, meiosis resumes and the oocyte progresses to metaphase II, which is accompanied by the development of a microtubule arrangement of the nuclear material. The microtubules of metaphase II oocytes are fragile and sensitive to chilling. During cooling, the microtubule spindle formation is disrupted and some chromosomal dispersion may occur (Kola et aL, 1988; Carroll et al., 1989; Pickering et al., 1990; George and Johnson, 1993). An alternative to the problem of disrupting the microtubules would be to cryopreserve oocytes before metaphase II, i.e., at prophase I or GV stage. However, additional culture in vitro would be required to mature the GV oocytes to metaphase II prior to fertilization (Candy et al., 1994). Human prophase I oocytes demonstrated similar rates of maturation and fertilization after cryopreservation, although blastocyst development was 1/30 compared to 6/52 in the control, metaphase II, group (Toth et al., 1994). A thorough review on the cryopreservation of mammalian oocytes by Van Blerkom (1991) discusses the techniques and post-thaw outcomes of mature and immature oocytes. Although immediate post-thaw recovery may be in an acceptable range, long-term developmental potential may be reduced (Schroeder et al., 1990). These concerns about the competence of in vitro matured oocytes and potential detrimental embryonic and fetal development following cryopreservation and in vitro maturation must be resolved before oocyte cryopreservation becomes routine. As an alternative to oocyte cryopreservation some studies have investigated the potential to store oocytes in a concentrated salt solution at low temperatures, but not frozen, for periods of time over their normal viability in culture (Yanagimachi et al., 1979; Hammitt et al., 1993). Although some oocytes were fertilizable, generally this method yielded poor outcomes. Little effort has been placed in pursuing this methodology as reliable oocyte survival is essential. Previous attempts to vitrify mammalian oocytes have met with mixed results (Rail and Fahy, 1985; Critser et al. 1986; Kono et al., 1989).

Utility of Viable Tissues ex Vivo: Banking of Reproductive Cells and Tissues

15

A few reports of human pregnancies after transfer of embryos derived from frozen-thawed oocytes have been published, the first was by Chen in 1986. Two term pregnancies resulted from transfer of these embryos. Another report of clinical pregnancy after transfer of embryos derived from frozen-thawed oocytes was reported by A1-Hasani et al. (1986). In the following year van Uem et al. (1987) reported pregnancy and delivery of a baby girl derived from frozen-thawed oocytes. Siebzehnrubl et al. (1987) cryopreserved 38 unfertilized oocytes; only 14 were fertilized, seven of which reached syngamy prior to transfer to 10 patients, resulting in a single live birth. Subsequent investigations have found continued low clinical success and concern about potential sublethal damage during cryopreservation.

IV. OVARIAN TISSUE CRYOPRESERVATION Pioneering reports on the successful transplantation of cryopreserved ovarian tissue in mice and golden hamsters were published in the late 1950s and early 1960s by Parrott (1959, 1960) with restoration of ovarian tissue function and fertility to some animals. Investigations on ovarian tissue cryopreservation lulled for several decades after Parrott's work until the last few years. Ovarian tissue contains an abundance of primordial follicles, an oocyte surrounded by a layer of granulosa cells. Primordial follicles can, albeit with some degree of difficulty, be grown, matured, and ovulated in vitro. Investigators have successfully cryopreserved, thawed, and transplanted primordial follicles to recipient mice with pregnancies and live births (Carroll et al., 1990; Carroll and Gosden, 1993). Ovarian tissue sections have also been shown to maintain post-thaw viability after autologous (Gosden et al., 1994; Harp et al., 1994) or xenotransplantation to immune deficient mice (Candy et al., 1995). The encouraging results from animal studies have propelled intensive evaluation of the potential to cryopreserve human ovarian tissue. Clinical applications are extensive and could benefit oncology patients at risk of developing iatrogenic infertility after chemo- and/or radiotherapy. Ovarian tissue could be cryopreserved prior to treatment, banked, and replaced upon recovery. Banking and replacement of ovarian tissue would result in restoration of steroidogenic activity, which has well recognized general health benefits. Women who have not yet had children and wish to do so in the future could maintain their own gametes in storage. Once recovered from the disease, and ready to have children, the ovarian tissue could be thawed and either transplanted or oocytes could be recovered and fertilized in vitro. Young girls, teenagers, and women without a defined partner with which they want to have a child are another group of oncology patients for whom tissue cryopreservation is an opportunity to rescue steroidogenic

16

K a r e n T. G u n a s e n a a n d J o h n K. Critser

and gametogenic function of the ovary for their future use. Some centers are already offering ovarian tissue cryopreservation and banking to oncology patients, although human clinical trials have not yet been performed.

V. WHAT DOES THE ~ TISSUE BANKING?

HOLD FOR REPRODUCTIVE

Cryopreservation of reproductive cells and tissues has already benefited agriculture, human clinical applications, and in conservation of biodiversity via genome banking. Yet, there are still many unanswered questions and perplexing issues. How can post-thaw survival and viability be improved? At least 50% of the original motile population of sperm in a human semen sample becomes immotile after cryopreservation. Since sperm numbers are usually high, little effort is placed in assessing these sperm. Are they immotile and viable or are they nonviable? Sperm microinjection has opened up possibilities of utilizing sperm that are immotile, yet viable, bypassing the need for energy and motility in reaching and penetrating the oocyte. Fishel et al., 1995 have reported the delivery of a healthy baby girl after injection of a spermatid from the father, who did not produce mature sperm. This opens up a realm of possibilities in future ethical debates of reproductive technologies. Is only the paternal DNA necessary for fertilization, and does it have to be enclosed in a sperm? These advances have also opened up the possibility of utilizing testicular tissue for infertility treatment. If mature sperm or paternal DNA needed for oocyte microinjection could be successfully obtained from in vitro cultured testicular tissue, this tissue could also be cryopreserved and banked for a patient's own use or as part of a donated pool of gametes. Obviously, techniques to enable successful recovery of gametes from testicular tissue are needed before these options are available clinically. Embryo cryopreservation is a fairly routine procedure in all areas of reproductive tissue banking. However, improvements in post-thaw viability and developmental potential may be achieved by a better understanding of the underlying cryobiological properties of embryos. For example, in in vitro fertilization laboratories pronuclear and cleavage stage embryos are cryopreserved using the same protocols. However, do these two very different "embryos" have the same membrane permeability characteristics (mediating water loss and limiting ice crystal damage) or are they different enough as to drastically affect cryosurvival? Compounding these evaluations is the heterogeneity (in meiotic stage and developmental potential) seen in cohorts of oocytes, and hence embryos, in animals and humans. One of the current areas of intensive research in reproductive biology is that of ovarian follicle physiology, growth, and development, recently reviewed by Gosden et al. (1993). Interest is focused on primordial follicles,

Utility of Viable Tissues ex Vivo: Banking of Reproductive Cells and Tissues

17

as they are an abundant population of oocytes that are as yet inaccessible in terms of infertility treatment. Eppig and O'Brien (1996) reported the first successful culture and development of primordial follicles in vitro, with the birth of live mouse pups. Although the pregnancy and deliveries were low, this is an exciting advance that could benefit researchers and scientists in the human clinical and conservation field. If follicle cryopreservation is established, follicles from patients or endangered species could be banked and cultured in vitro or transplanted in a host animal. The quiescent nature of the oocyte in the primordial follicle is suggestive of successful cryosurvival and post-thaw viability, which remains to be seen.

VI. CONCLUDING REMARKS Tremendous advances in reproductive technologies occurred during the 20th century. Banking of reproductive cells and tissues for use ex vivo benefits agricultural and animal husbandry programs and human infertility treatments. These techniques are applied to animal model species for medical research (mouse, rat) and for conservation of endangered species. Cryopreservation is proving to be a safe approach to banking gametes and tissue, although post-thaw recovery can sometimes be erratic and inefficient. Many researchers are evaluating the cryobiological factors that may affect postthaw recovery and developmental potential of cells and tissues. Improvements in cryopreservation techniques and technology will benefit the whole field. The scope and applications for conservation and biodiversity will be manifold, and protection of endangered species will become an attainable goal. Advances in the ability to bank and use reproductive tissues ex vivo must be accompanied by thoughtful discussion and debate, to ensure safe and ethical application of these exciting technologies.

ACKNOWI~DGMENTS Dr. Michel Thibier of the Centre National d'Etudes Veterinares et Alimentaires, France, kindly compiled data for the comparative table of worldwide embryo transfers for 1992 to 1994. The authors thank Ms. Cara Willoughby for reviewing the manuscript.

REFERENCES A1-Hasani, S., Van der Ven, H., Diedrich, K., and Krebs, D. (1986). Successful in vitro fertilization of frozen/thawed human oocytes. In Workshop on Embryos and Oocytes Freezing (Reports), pp. 25-39. Collection Foundation Marcel Merieux, Annenc. The American Fertility Society and Society for Assisted Reproductive Technology (1994). Assisted reproductive technology in the United States and Canada: 1992 Results gener-

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Karen T. Gunasena and John K. Critser

ated from the American Fertility Society/Society for Assisted Reproductive Technology Registry. Fertil. Steril. 62, 1121-1128. Candy, C. J., Wood, M. J., Whittinghamn, D. G., Merrimen, J. A., and Choudhry, N. (1994). Cryopreservation of immature mouse oocytes. Hum. Reprod. 9, 1738-1742. Candy, C. J., Wood, M. J., and Whittingham, D. G. (1995). Follicular development in cryopreserved marmoset ovarian tissue after transplantation. Hum. Reprod. 10, 2334-2338. Carroll, J., and Gosden, R. G. (1993). Transplantation of frozen-thawed mouse primordial follicles. Hum. Reprod. 8, 1163-1167. Carroll, J., Warnes, G. M., and Matthews, C. D. (1989). Increase in digyny explains polyploidy after in-vitro fertilization of frozen-thawed mouse oocytes. J. Reprod. Fertil. 85, 489-494. Carroll, J., Whittingham, D. G., Wood, M. J., Teller, E., and Gosden, R. G. (1990). Extraovarian production of mature viable mouse oocytes from frozen primary follicles. J. Reprod. Fertil. 90, 321-327. Chang, M. C. (1947). Normal development of fertilized rabbit ova stored at low temperatures for several days. Nature (London) 159, 602-603. Chen, C. (1986). Pregnancy after human oocyte cryopreservation. Lancet 1, 884-886. Corson, S. L., Baxter, F. R., and Baylson, M. M. (1983). Donor insemination. Obstet. Gynecol. Ann. 12, 283. Crister, J. K. (1995). The Current Status of Semen Banking in the United States. Presented at the European Society of Human Embryology and Reproduction Workshop, Gamete Donation: Ethical Aspects. Corsendonck, Belgium, April 1995. Critser, J. K., Arneson, B. W., Aaker, D. V., and Ball, G. D. (1986). Cryopreservation of hamster oocytes: Effects of vitrification or freezing on human sperm penetration of zonafree hamster oocytes. Fertil. Steril. 46, 277-284. Dresser, B.L. (1988). In Biodiversity (E.O. Wilson, Ed.), 1st ed., pp. 296-308. National Academy Press, Washington, DC. Eppig, J.J., and O'Brien, M.J. (1996). Development in vitro of mouse oocytes from primordial follicles. Biol. Reprod. 54, 197-207. Fishel, S., Green, S., Bishop, M., Thornton, S., and Hunter, A. (1995). Pregnancy after intracytoplasmic injection of spermatid. Lancet 345, 1641-1642. [Letter] Food and Drug Administration (1993). Human tissue intended for transplantation. Fed. Regist. 58 (238), 65514-65521 (12/14/93). Food and Drug Administration (1995). Draft Discussion Points for Screening and Testing Donors of Human Tissue Intended for Transplantation and Human Reproductive Tissue, and for Establishment Registration. Intended for Discussion at the FDA Workshop on Human Tissue Intended for Transplantation and Human Reproductive Tissue: Donor Screening and Infectious Disease Testing, March 24, 1995, Washington, DC. George, M. A., and Johnson, M. H. (1993). Cytoskeletal organization and zona sensitivity to digestion by chymotrypsin of frozen-thawed mouse oocytes. Hum. Reprod. 8, 612-620. Gosden, R. G., Boland, N. I., Spears, N., Murray, A. A., Chapman, M., Wade, J. C., Zhody, N. I., and Brown, N. (1993). The biology and technology of follicular oocyte development in vitro. Reprod. Med. Rev. 2, 129-152. Gosden, R. G., Baird, D. T. M., Wade, J. C., and Webb, R. (1994). Restoration of fertility to oophorectomized sheep by ovarian autografts stored at -196~ Hum. Reprod. 9, 597-603. Hammitt, D. G., Syrop, C. H., Walker, D. L., and Bennett, M. R. (1993). Conditions of oocyte storage and use of noninseminated as compared with inseminated, nonfertilized oocytes for the hemizona assay. Fertil. Steril. 60, 131-136. Harp, R., Leibach, J., Black, J., Keldahl, C., and Karow, A. (1994). Cryopreservation of murine ovarian tissue. Cryobiology 31, 336-343. Herman, H. A. (1988). In Improving Cattle by the Millions. N A A B and the Development and Worldwide Application of Artificial Insemination, 2nd ed., pp. 36-49. Univ. of Missouri Press, Columbia and London.

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Iritani, A. (1980). Problems Freezing Spermatozoa of Different Species. Proceedings, 9th International Congress on Animal Reproduction Artificial Insemination, Madrid, Vol. 1, pp. 115-132. Kleegman, S. J. (1967). Therapeutic donor insemination. Conn. Med. 31, 705-713. Kola, I., Kirby, C., Shaw, J., Dave, A., and Trounson, A. (1988). Vitrification of mouse oocytes. Results in anueploid zygotes and malformed fetuses. Teratology 38, 467-474. Kono, T., Kwon, O. Y., and Nakahara, T. (1989). Development of vitrified mouse oocytes after in vitro fertilization. Cryobiology 28, 50-54. Leibo, S. P. (1986). Cryobiology: Preservation of mammalian embryos. Basic Life Sci. 37, 251-272. Leibo, S. P., DeMayo, F.J., and O'Malley, B. (1991). Production of transgenic mice from cryopreserved fertilized ova. Mol. Reprod. Dev. 30, 313-319. Mascola, L., and Guinan, M. E. (1986). Screening to reduce transmission of sexually transmitted diseases in semen used for artificial insemination. N. Engl. J. Med. 314, 1354-1359. Massip, A., Mermillod, P., and Dinnyes, A. (1995). Morphology and biochemistry of invitro produced bovine embryos: Implications for their cryopreservation. Hum. Reprod. 10, 3004-3011. Medical Research International, The Society for Assisted Reproductive Technology (1992). In vitro fertilization-embryo transfer (IVF-ET) in the United States: 1990 results from the IVF-ET Registry. Fertil. Steril. 57, 15-24. Mobraaten, L. E. (1986). Mouse embryo cryobanking. J. In Vitro Fertil. 3, 28-32. Mobraaten, L. E., Champlin, A. K., Johnston, D.S., Schroeder, A. C., and Gordon, J. W. (1991). Cryopreservation and in vitro fertilization with mouse sperm. Cryobiology 29, 527-528. Nagase, H., Tomizuka, T., Hanada, A., Hosoda, T., and Morimoto, H. (1972). Cryoprotection of some amide solutes to spermatozoa of domestic animals. I. Effects of formamide, acetamide and lactamide on the motility of bovine spermatozoa in pellet freezing. Jpn. J. Anita. Reprod. 18, 15-21. Olson, J. H. (1995). History of Sperm Banking and New Frontiers. Presented at the American Association of Tissue Banks Reproductive Cryotechnology Specialist Review Course, March 29, 1995, Raleigh, NC. Pace, M. M. (1980). Fundamentals of assay of spermatozoa. Proceedings, 9th International Congress on Animal Reproduction and Artificial Insemination, Madrid, Vol. 1, pp. 133-146. Parrott, D. M. V. (1959). Orthoptic ovarian grafts in the golden hamster. J. Endocrinol. 19, 126-138. Parrott, D. M. V. (1960). The fertility of mice with orthoptic ovarian grafts derived from frozen tissue. J. Reprod. Fertil. 1, 230-241. Penfold, L. M., Moore, H. D. M., and Holt, W. V. (1991). In vitro fertilization and embryo development in mice using frozen/thawed spermatozoa. Cryobiology 29, 573. Pickering, S. J., Cant, A., Braude, P. R., Currie, J., and Johnson, M. H. (1990). Transient cooling to room temperature can cause irreversible disruption of the meiotic spindle in the human oocyte. Fertil. Steril. 54, 102-108. Polge, C., and Lovelock, J. E. (1952). Preservation of bull semen at -79~ Vet. Rec. 64, 396-397. Polge, C., and Rowson, L. E. A. (1952). Fertilizing capacity of bull spermatozoa after freezing at -79~ Nature (London) 169, 626-627. Polge, C. (1956). Artificial insemination in pigs. Vet. Rec. 68, 62-76. Polge, C., Salamon, S., and Wilmut, I. (1970). Fertilizing capacity of frozen boar semen following surgical insemination. Vet. Rec. 87, 424-428. Polge, C. (1980). Freezing of spermatozoa. In Low Temperature Preservation in Medicine and Biology (M.J. Ashwood-Smith and J. Farrant, Eds.), pp. 45-64. Pitman, London. Pursel, V. G. (1979). Advances in the preservation of swine spermatozoa. In Animal Reproduction (H. W. Hawk, Ed.), Beltsville Symp. Agric. Res. 3, pp. 145-157. Allanheld, Osmun & Co., Montclair.

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Rail, W. F., and Fahy, G. M. (1985). Ice-flee cryopreservation of mouse embryos at -196~ by vitrification. Nature (London) 313, 573-575. Reed, H. C. B. (1969). Artificial insemination and fertility of the boar. Br. Vet. J. 125, 272-280. Reed, H. C. B. (1985). Current Use of Frozen Boar Semen-Future Need of Frozen Boar Semen. Proceedings 1st International Conference on Deep Freezing of Boar Semen, pp. 225-237. Swedish Univ. of Agricultural Sciences, Uppsala, Sweden. Robertson, J. A. (1996). Legal troublespots in assisted reproduction. Fertil. Steril. 65, 11-12. Schroeder, A. C., Champlin, A. K., Mobraaten, L. E., and Eppig, J. J. (1990). Developmental capacity of mouse oocytes cryopreserved before and after maturation in vitro. J. Reprod. Fertil. 89, 43-50. Sherman, J. K. (1979). Synopsis of the use of frozen human semen since 1964: State of the art of human semen banking. Fertil. Steril. 24, 397-416. Shuster, E. (1990). Seven embryos in search of legitimacy. Fertil. Steril. 53, 975-977. Siebzehnrubl, E. R., Todrow, S., van Uem, J., Kock, R., Wildt, L., and Lang, N. (1987). Cryopreservation of human and rabbit oocytes and one-cell embryos: A comparison of DMSO and propanediol. Hum. Reprod. 4, 312-317. Society for Assisted Reproductive Technology, The American Fertility Society (1993). Assisted reproductive technology in the United States and Canada: 1991 Results from the Society for Assisted Reproductive Technology generated from The American Fertility Society Registry. Fertil. Steril. 59, 956-962. Society for Assisted Reproductive Technology and American Society for Reproductive Medicine (1995). Assisted reproductive technology in the United States and Canada: 1993 Results generated from the American Society for Reproductive Medicine/Society for Assisted Reproductive Technology Registry. Fertil. Steril. 64, 13-21. Spallazani, L. (1776). Opuscoli di Fisca animale e Vegetabile, Opuscolo I and II. Presso La Societfi Tipographica, Modena. Sutcliffe, A. G., D'Souza, S. W., Cadman, J., Richards, B., McKinlay, I. A., and Lieberman, B. (1995). Minor congenital anomalies, major congenital malformations and development in children conceived from cryopreserved embryos. Hum. Reprod. 10, 3332-3337. Tada, N., Sato, M., Yamonoi, J., Mizorgi, T., Kasai, K., and Ogawa, O. (1990). Cryopreservation of mouse spermatozoa in the presence of raffinose and glycerol. J. Reprod. Fertil. 89, 511-516. Takeda, T., Eldsen, R. P., and Seidel, G. E. (1984). Cryopreservation of mouse embryos by direct plunging into liquid nitrogen. Theriogenology 21, 266. Tanphaichitr, N. A., Tayabali, Gradil, C., Juneja, S., L6veill6, M. C., and Lingwood, C. A. (1992). Role of germ cell-specific sulfolipid-immobilizing protein (SLIP1) in mouse in vivo fertilization. Mol. Reprod. Dev. 32, 17-22. Thibier, M. (1993). Statistics describing the international embryo transfer industry. IETS Newsl. 11, 10-13. Thibier, M. (1994). Statistics of the embryo transfer industry around the world, lETS Newsl. 12, 13-16. Thibier, M. (1995). Statistics of the worldwide embryo transfer industry, lETS Newsl. 13, 18-121. Toth, T. L., Jones, H. W., Baka, S. G., Muasher, S., Veeck, L. L., and Lanzendorf, S. E. (1994). Fertilization and in vitro development of cryopreserved human prophase I oocytes. Fertil. Steril. 61, 891-894. Touati, K., Delcroix, P., Boccart, C., and Ectors, F. (1992). Proceedings, 8th Scientific Meeting of the European Embryo Association, pp. 220. Lyon, France. Trounson, A., and Mohr, L. (1983). Human pregnancy following cryopreservation thawing and transfer of an eight-cell embryo. Nature 305, 707-709. Trounson, A., Peura, A., and Kirby, C. (1987). Ultrarapid freezing: A new low cost and effective method of embryo cryopreservation. Fertil. Steril. 48, 843-850.

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van Blerkom, J. (1991). Cryopreservation of the mammalian oocyte. In Current Communications in Cell and Molecular Biology, Animal Applications of Research in Mammalian Development (R. A. Pedersen, A. McLaren, and N. L. First, Eds.), pp. 83-119. Cold Spring Harbor Laboratory Press, Cold Spring Harbor, NY. van den Eede, B. (1995) Investigation and treatment of infertile couples: ESHRE guidelines for good clinical and laboratory practice. Hum. Reprod. 10, 1246-1271. van Uem, J. F. H. M., Siebzehnrubl, E. R., Schuh, B., Koch, R., Trotnov, S., and Lang, N. (1987). Birth after cryopreservation of unfertilized oocytes. Lancet 1, 752-753. Wada, I., Macnamee, M. C., Wick, K., Bradfield, J. M., and Brinsden, P. R. (1994). Birth characteristics and perinatal outcome of babies conceived from cryopreserved embryos. Hum. Reprod. 9, 543-546. Watson, P. F. (1979). The preservation of semen in mammals. In Oxford Reviews of Reproductive Biology (C. A. Finn, Ed.), pp. 283-350. Oxford Univ. Press, London. Watson, P. F. (1990). Artificial insemination and the preservation of semen. In Marshall's Physiology of Reproduction (G. E. Lamming, Ed.), 4th ed., Vol II, pp. 747-896. Churchill Livingstone, London. Whittingham, D. G. (1971). Survival of mouse embryos after freezing and thawing. Nature 233, 125-126. Whittingham, D. G., Leibo, S. P., and Mazur, P. (1972). Survival of mouse embryos frozen to -196~ and -269~ Science 178, 411-414. Wildt, D. E., Byers, A. P., Howard, J. G., Weise, R., Willis, K., O'Brien, S., Block, J., Tilson, R. L., and Rall, W. F. (1993). Genome Resource Banking (GRB) action plan: Global need and a plan for the North American region. Presented at AAZPA Annual Meeting, Omaha, Nebraska, August, 1993. Wilmut, I., and Rowson, L.E.A. (1973). Experiments on the low temperature preservation of cow embryos. Vet. Rec. 92, 686-690. Yanagimachi, R., Lopata, A., Odom, C. B., Bronson, R. A., Mahi, C. A., and Nicholson, G. L. (1979). Retention of biologic characteristics of zona pellucida in highly concentrated salt solution: The use of salt-stored eggs for assessing the fertilizing capacity of spermatozoa. Fertil. Steril. 31, 562-574. Yokoyama, M., Akiba, H., Katsuki, M., and Nomura, T. (1990). Production of normal young following transfer of mouse embryos obtained by in vitro fertilization using cryopreserved spermatozoa. Exp. Anim. 39, 125-128.

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van Blerkom, J. (1991). Cryopreservation of the mammalian oocyte. In Current Communications in Cell and Molecular Biology, Animal Applications of Research in Mammalian Development (R. A. Pedersen, A. McLaren, and N. L. First, Eds.), pp. 83-119. Cold Spring Harbor Laboratory Press, Cold Spring Harbor, NY. van den Eede, B. (1995) Investigation and treatment of infertile couples: ESHRE guidelines for good clinical and laboratory practice. Hum. Reprod. 10, 1246-1271. van Uem, J. F. H. M., Siebzehnrubl, E. R., Schuh, B., Koch, R., Trotnov, S., and Lang, N. (1987). Birth after cryopreservation of unfertilized oocytes. Lancet 1, 752-753. Wada, I., Macnamee, M. C., Wick, K., Bradfield, J. M., and Brinsden, P. R. (1994). Birth characteristics and perinatal outcome of babies conceived from cryopreserved embryos. Hum. Reprod. 9, 543-546. Watson, P. F. (1979). The preservation of semen in mammals. In Oxford Reviews of Reproductive Biology (C. A. Finn, Ed.), pp. 283-350. Oxford Univ. Press, London. Watson, P. F. (1990). Artificial insemination and the preservation of semen. In Marshall's Physiology of Reproduction (G. E. Lamming, Ed.), 4th ed., Vol II, pp. 747-896. Churchill Livingstone, London. Whittingham, D. G. (1971). Survival of mouse embryos after freezing and thawing. Nature 233, 125-126. Whittingham, D. G., Leibo, S. P., and Mazur, P. (1972). Survival of mouse embryos frozen to -196~ and -269~ Science 178, 411-414. Wildt, D. E., Byers, A. P., Howard, J. G., Weise, R., Willis, K., O'Brien, S., Block, J., Tilson, R. L., and Rall, W. F. (1993). Genome Resource Banking (GRB) action plan: Global need and a plan for the North American region. Presented at AAZPA Annual Meeting, Omaha, Nebraska, August, 1993. Wilmut, I., and Rowson, L.E.A. (1973). Experiments on the low temperature preservation of cow embryos. Vet. Rec. 92, 686-690. Yanagimachi, R., Lopata, A., Odom, C. B., Bronson, R. A., Mahi, C. A., and Nicholson, G. L. (1979). Retention of biologic characteristics of zona pellucida in highly concentrated salt solution: The use of salt-stored eggs for assessing the fertilizing capacity of spermatozoa. Fertil. Steril. 31, 562-574. Yokoyama, M., Akiba, H., Katsuki, M., and Nomura, T. (1990). Production of normal young following transfer of mouse embryos obtained by in vitro fertilization using cryopreserved spermatozoa. Exp. Anim. 39, 125-128.

Tissue Maturation in V i v o a n d in Vitro: Gamete and Early Embryo Ontogeny

M. L o r r a i n e L e i b f r i e d - R u t l e d g e and Tanja Dominko Department of Meat and Animal Science, University of WisconsinmMadison, Madison, Wisconsin 53706 E l i z a b e t h S. Critser Advanced Fertility Institute, Methodist Hospital of Indiana, Inc., Indianapolis, Indiana 46202 J o h n K. C r i t s e r Cryobiology Institute, Methodist Hospital of Indiana, Inc., Indianapolis, Indiana 46202

I. INTRODUCTION As advances in basic biology continue to provide a wider selection of assisted reproductive technologies for use in clinical (Beier and Lindner, 1983; Betteridge and Rieger, 1993; Trounson et al., 1994a) and agricultural applications (Brackett et al., 1981; First, 1991; Gordon, 1994; Trounson et al., 1994a) and for preserving biodiversity (Loskutoff and Betteridge, 1992; Wildt et al., 1992a,b,c; Loskutoff et al., 1995), tissue banking of gametes and early embryos at different stages of their life history becomes critical. Ability to cryopreserve embryonic and gametogenic cells (Niemann, 1991; Rall, 1992; Pollard and Leibo, 1994) at any stage of development and differentiation will allow flexibility in choice of genetics, make better use of scarce or hard to obtain material, and increase the scenarios available for application. In order to provide the raw material for cryopreservation or ensure appropriate utilization after thawing, the reproductive cell types that are the subject of this chapter may have to be cultured or matured in Reproductive Tissue Banking Copyright 9 1997 by Academic Press. All rights of reproduction in any form reserved.

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m. Lorraine Leibfried-Rutledge et al.

vitro to a particular stage in their life history appropriate for their intended use. For the purpose of this chapter, the assumption is made that the culmination of tissue maturation and banking for gametogenic or embryonic cells will be to produce live offspring. Since we are not yet at the point of being able to mature or culture reproductive cells at all stages of development, this exposition will focus on stages where success is currently possible while also providing an insight into new directions in tissue maturation that may be brought to fruition in the near future (Seidel, 1991). The guiding principle for this discussion will be to impart an understanding of factors that need to be considered during tissue maturation or culture that will result in the greatest potential to produce live, healthy offspring in mammals. The earliest progenitor cells for gametes are found in the extraembryonic tissue of the early fetus, yolk sac, and allantoic membrane (Chiquoine, 1954; Mintz, 1960, 1961; Peters, 1970; Baker, 1972a). These primordial germ cells migrate into the embryo to colonize the genital ridge (Haffen, 1977; Zuckerman and Baker, 1977; McLaren, 1988). After proliferation of the population has occurred, the early gametogenic tissue of the male and female diverge in their life history. In association with nongerminal tissue of the developing gonad, the female's oogonia begin meiosis and become entrapped in primordial follicles as primary oocytes (Baker, 1972b; Mauleon and Mariana, 1977; Byskov, 1986), while the male's germ cells become residents of the seminiferous cords as spermatogonia and will not begin meiosis until after birth when the animal becomes sexually mature (Bishop and Walton, 1960; Roosen-Runge, 1962; Johnson et al., 1970; Ortavant et al., 1977; Hecht, 1986; Bedford, 1991). In mammals then, the pool of gametogenic material available to the female during her lifetime is set prior to or just after birth in some species, and these oocytes will not complete meiosis unless the exigencies of ovulation are forced on selected cells after puberty. In the male, spermatogonia will continue to serve both proliferative and differentiative functions by continually undergoing meiosis and spermiogenesis to yield spermatozoa throughout the animals adult existence. In vitro culture of the primary oocyte to yield a gamete that is ready for fertilization has been the primary focus of tissue maturation in the female mammal since its ovarian location makes it fairly accessible. A large volume of literature has shown that this cell stage is quite capable of yielding live progeny after in vitro manipulations of many types and will therefore be central to further discussion. Mature spermatozoa have been the focus of cryopreservation and hence tissue banking and utilization in vitro in male mammals. These cells are both abundant and easily accessible and come prepared to undergo fertilization under appropriate conditions after collection by routine methods. Uniting the matured oocyte and sperm during fertilization yields a zygote which resumes mitotic cell cycles, proliferating from a one-celled entity into the multicelled blastocyst (McLaren, 1974; Anderson, 1977;

Tissue M a t u r a t i o n in Vivo a n d in Vitro

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Leibfried-Rutledge, 1996). During this preimplantation development, the embryo has managed to transition from maternal regulation of development to utilization of the new embryonic genome for gene expression and also exhibits the first morphologically recognizable signs of embryonic differentiation (Telford et al., 1990; Schultz, 1993; Schultz et al., 1995). The zygote in some species can be persuaded to undergo tissue maturation under in vitro conditions, yielding a blastocyst capable of pregnancy initiation and maintenance through to production of a unique member of the species. The conditions necessary for tissue maturation during this period of preimplantation development which normally occurs during passage from the oviduct to the uterus has spawned a large volume of literature. This period of development has also been central to developing new assisted reproductive technologies and embryo banking strategies and therefore will be the emphasis of discussion on early embryo ontogeny. As pointed out in previous discussion, the primary oocyte, mature spermatozoon, and preimplantation embryo are reasonably abundant and accessible reproductive cell types for collection in vivo or can be successfully produced by in vitro methods. Most importantly, there is much precedent in the literature for in vitro tissue maturation and cryopreservation of these cell types. Although each species and reproductive cell type at a particular stage of development or differentiation may have specialized requirements for successful in vitro tissue maturation, there are some common considerations when contemplating culture systems for cells when readying them for cryopreservation or utilizing them after thawing. The following sections will consider both common requirements and unique aspects of in vitro tissue maturation for the primary oocyte, using the mature sperm for fertilization and culturing the.preimplantation embryo. Since three authors have chosen the oocyte as the focus of their research, some slight preference is given to this part of the succeeding textual material. Due to the breadth of material encompassed in this chapter, we have relied on reviews and basic reference material in order to provide a starting point for those beginning to consider tissue preservation for a particular species. To all the investigators whose previous and/or continuing work in basic or applied biology have made possible the many assisted reproductive techniques available, we offer our most sincere gratitude and apologies for not always using original citations.

II. OOCYTE M A ~ T I O N The primary oocyte stage of gamete differentiation in most female mammals is reached during fetal life after proliferation of primordial germ cells and oogonia with subsequent entry into meiosis and transformation of the gamete into the primary oocyte (Peters, 1970; Baker, 1972a; Gondos,

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m. Lorraine Leibfried-Rutledge et al.

1978; Tsafriri, 1979; Baker, 1982, 1987; Byskov, 1986; Racowsky, 1991). The life history of the female gamete is diagrammatically shown in Figure 1. The oocyte, arrested in prophase of meiosis I, is entrapped by follicle cells that form a squamous, single-layered epithelium around individua; oocytes; and the total population of such units found in the ovarian cortex, termed the primordial follicle, form the resting pool of gametogenic material that must last throughout an animals reproductive life history (Biggers and Schuetz, 1972; Zuckerman and Weir, 1977; Zuckerman and Baker, 1977; Gondos, 1978; Hardisty, 1978; Peters, 1978; Gosden and Teller, 1987; Hirshfield, 1992). At various times throughout the female animal's life, cohorts of primordial follicles begin a developmental and differentiative period of DEVEIDPMENTAL EVENTS

STATE OF GERM

(D.. CELLS

Multiplication by mitosis

|

Migration to genitalridge

~,RTH-rat)t~, ferret,mink: ~:)ie, Fi~ i n t ~

DNA synthesis Meiotic prophese begins

~IRTH- Mo~'mamrnaLs

~-| ->

~ >

Growth of oocyte and follicle [PuBER:rY.,

_..~) GERMCELLS ~(~)"(~)

>

(~)l"(~)

O0~N~

|

Q 1

PRIMARY OOCYTE

1

Follicular maturation

1 First meiotic division begins

Sperm penetration-dog, fox First polar body emitted (may divide)

~!~)

[OVU'LATION-Most mammals

s.co.DA.v

OOCYTE

,Sperm peneb'atJon- Most mammals

,Second meiotic division, fertilization,and emission of second IX:Carbody

~,1~)

E-GG(OOfK))

Generalized diagram of oogenesis in mammals. The life cycle of female gametes from embryonic stages through to reproductive maturity is represented. (Baker, 1972b. In: Reproduction in Mammals: C. R. Austin and R. V. Short [eds.], Cambridge University Press, Cambridge.)

FIGURE 1

Tissue M a t u r a t i o n in Vivo a n d in Vitro

27

growth, which will result in production of the mature, preovulatory follicle under the appropriate hormonal conditions in vivo (Mauleon and Mariana, 1977; Baird and McNeilly, 1981; Foxcraft and Hunter, 1985; Spicer and Echternkamp, 1986; Tonetta and diZarega, 1986, 1990; Hirshfield and Schmidt, 1987; Sakai and Hodgen, 1987; Franchimont et al., 1988; Hirshfield, 1991). Entry of primordial follicles into the growth phase has been observed in fetuses and prepuberal animals, throughout menstrual and estrus cycles, and even during pregnancy (Casida et al., 1935; Williams et al., 1956; Rajakoski, 1960; Erickson, 1966; Perry and Rowlands, 1962; Lobel and Levy, 1968; Greenwald, 1978), but conditions appropriate for producing the mature follicle in vivo with an oocyte ready for fertilization are found only in reproductively mature animals during specific stages of the reproductive cycle and during the breeding season in those species responsive to seasonal influences (Mossman and Duke, 1973; Weir and Rowlands, 1977; Greenwald, 1978; Donoghue et al., 1993). Growing follicles not attaining dominance, in other words not selected for ovulation, will eventually become atretic and be lost from the ovarian population (Byskov, 1978; Jones, 1978; Richards, 1978; Baker, 1982, 1987). It is these follicles and the enclosed oocyte on which much effort has been directed toward perfecting protocols for in vitro tissue maturation, since they form a large pool of genetic material that can potentially be salvaged for use in assisted reproductive protocols, research, or tissue banking. This material is more abundant than the few follicles that do attain dominance, and the timing for recovery of this material is much less critical than that for the limited number of in vivo matured oocytes in the preovulatory follicles. During growth and differentiation of the follicle unit that has left the resting pool of primordial follicles, the somatic component begins to replicate and forms a single layer of columnar or cuboidal cells around the primary oocyte which has also begun to increase in size (follicular development reviewed in Zuckerman and Weir, 1977; Bjersing, 1978; Jones, 1978; Tsafriri, 1978; Staigmiller and England, 1982; Guraya, 1985; Schuetz, 1985; Spicer and Echternkamp, 1986; Hirshfield, 1991; Fortune, 1994). This morphological stage is identified as the primary follicle. A schematic of oocyte growth and folliculogenesis is shown in Figure 2. After the follicle unit has begun growth and differentiation the oocyte's nonliving extracellular coat, the zona pellucida, is first observed to form (Baker, 1971). The zona pellucida is composed of several glycoproteins (Wassarman, 1988b, 1991a, 1994) that modulate later sperm-egg interactions at fertilization such as species specificity, sperm binding, the acrosome reaction, and the zona mediated block to polyspermy (Bleil, 1991; Wassarman, 1993). In at least one species, mice, secretion of the zona can be attributed to the oocyte (Bleil and Wassarman, 1980). Proliferation of the follicle cells continues until a multilayered epithelium is formed, intercellular associations are established, creating a unique environment around the oocyte, and an

28

m. Lorraine Leibfried-Rutledge et al.

Primordial

Terti~

Primary

0

Preovulatory maturation

Secondary

Secondary oocyte Generalized diagram of folliculogenesis in mammals. Growth and differentiation of the follicle unit starting from the nongrowing pool of primordial follicles, entering the growth phase and ending with the oocyte-cumulus complex undergoing meiosis I in the tertiary follicle prior to ovulation.

FIGURE 2

outer layer of squamous cells forming the thecal layer is acquired. In this secondary follicle the primary oocyte continues to grow. After continued proliferation of both types of follicle cells and division of theca into interna and externa, the follicle now begins to form a fluid-filled lumen in which the oocyte remains attached to the cells, now termed granulosa, at one side of the follicle wall. Fully grown follicles of a few species of mammals such as the Madagascar hedgehog (Setifer setosus), tenrec (Hemicentetes semispinosus), and moon rats (Echinisorex gymnurus) never acquire an antrum (Mossman and Duke, 1973). In the tertiary or antral follicle of most mammals, the oocyte remains surrounded by a hillock of cells called the cumulus oophorus, which maintains cellular associations with the granulosa, cumulus cells themselves, and with the oocyte. Those cells immediately surrounding the oocyte that have villi interdigitating with those of the oocyte plasmalemma are the corona radiata cells (Eppig, 1985; Dekel, 1988; Larsen and Wert, 1988; Buccione et al., 1990; Eppig, 1991b; Canipari, 1994). In some mammals, the oocyte has now attained its maximum diameter and thus has completed growth. This has mostly been observed in short-cycle species such as rodents (Szybek, 1972; Iwamatsu and Yanagimachi, 1975;

Tissue Maturation in Vivo a n d in Vitro

29

Sorenson and Wassarman, 1976), while in long-cycle species such as domestic animals and primates, growth of the oocyte continues to occur (Motlik and Fulka, 1986; Schramm et aL, 1993; reviewed by First et al., 1988). Proliferation of the follicle cells continues in the tertiary follicle, as does fluid secretion into the lumen, resulting in an increasingly larger follicle, almost up to the time of ovulation. Many reviews exist which can provide details of the hormonal requirements for follicular growth, components and mechanism for antral fluid formation, and functions of the follicle cells. See for example: Jones, 1978; McNatty, 1978; Guraya, 1985; Glasier et al., 1989; Hirshfield, 1991; Findlay, 1993; Lunenfeld and Insler, 1993; GoreLangton and Armstrong, 1994; Greenwald and Roy, 1994; Hillier, 1994; Cain et al., 1995; Campbell et al., 1995; Monget and Monniaux, 1995; Erickson and Danforth, 1995). During follicular growth and formation, the primary oocyte which is still in meiotic arrest, also undergoes a large increase in volume, begins a very active synthetic phase, stockpiles large stores of both protein and mRNA which is very stable, and also acquires what has been termed meiotic competence (Tsafriri, 1978; Wassarman, 1983; Bachvarova, 1985; Schultz, 1986b; Bachvarova, 1988; First et al., 1988; Wassarman, 1988a; LeibfriedRutledge et aL, 1989b; Eppig, 1991a; Racowsky, 1991; Albertini et al., 1993). Part of a histological section taken from the ovary of an adult hamster illustrates in Figure 3 the dramatic growth of the oocyte once it has left the

Histological section of an adult hamster ovary. Examples of primordial, primary, secondary, and an early tertiary follicle are shown. (Leibfried-Rutledge et aL, 1989. In: The Molecular Biology o f Fertilization. H. Schatten and G. Schatten [eds.], Academic Press, New York.)

FIGURE 3

30

m. Lorraine Leibfried-Rutledge et al.

nongrowing pool of primary oocytes. Unlike oocytes in lower vertebrates or invertebrates where a hormonal signal for induction of meiotic maturation (i.e., resumption and completion of meiosis I) is necessary, mammalian oocytes removed from antral follicles and placed into culture spontaneously undergo meiotic maturation (Pincus and Enzmann, 1935; Edwards, 1965). Acquisition of meiotic competence coincides with attainment of maximum diameter and antrum formation in the follicle in rodents studied (Szybek, 1972; Erickson and Sorensen, 1974; Iwamatsu and Yanagimachi, 1975; Sorensen and Wassarman, 1976; Kuar and Guraya, 1983). In long-cycle species, meiotic competence is not acquired until later during growth of the antral follicle (McGaughey et al., 1979; Motlik and Fulka, 1986; Schramm et aL, 1993). Conditions necessary for acquisition of competence in vivo have been studied in rats and involve the presence of FSH and estrogen (BarAmi and Tsafriri, 1981, 1986). At the time that meiotic competence is attained, the oocytes decrease synthetic processes and dismantle the interphase array of microtubules, the nucleolus morphology becomes indicative of very low levels of mRNA production, and chromatin begins condensing in the oocyte nucleus or germinal vesicle (summarized by Albertini, 1992; Albertini et al., 1993). The growth phase of the oocyte through to meiotic competence is highly reminiscent of the transversal of G2 phase in somatic cells. At the point at which the oocyte becomes able to resume and complete meiosis I, the oocyte has stored a genetic program that will later be expressed during fertilization and early preimplantation embryonic development (Bachvarova, 1985). There are many excellent reviews concerning mammalian oocyte development and meiotic regulation in the literature (Baker, 1972a; Crosby and Moor, 1984, 1985; Schultz, 1986b; Moor and Gandolfi, 1987; First et al., 1988; Moor, 1988; Leibfried-Rutledge et al., 1989b; Eppig, 1991a; Schultz, 1991; Moor et aL, 1992; Parrish et aL, 1992; Downs, 1993; Gosden et al., 1993; Wassarman and Albertini, 1994). Meiotically competent, primary oocytes from antral follicles are currently being utilized in a number of scenarios in both commercial and research settings to provide material for in vitro fertilization (IVF) and the in vitro production (IVP) of embryos (Wildt et al., 1992b; Lasely et al., 1994; Gordon, 1994; Looney et al., 1994; Hasler et al., 1995; Lohuis, 1995; Loskutoff et al., 1995). Since meiotically competent primary oocytes of mammals spontaneously resume and complete the first meiotic division in culture and are convenient to obtain compared to earlier stages of the female gamete, the process of in vitro oocyte maturation (IVM) has received much attention with the focus on producing a metaphase II-arrested, secondary oocyte capable of yielding live offspring after IVF. This was slightly more difficult to achieve than first thought, due to the fact that although meiotic maturation took place spontaneously and appeared normal, other processes now referred to as cytoplasmic maturation appeared to be more demanding as to the preferred in vitro environment for complete maturation

Tissue M a t u r a t i o n in Vivo a n d in Vitro

31

of the oocyte (discussed by First et aL, 1988; Leibfried-Rutledge et al., 1989a,b; Schultz, 1991). The actual processes involved in cytoplasmic maturation are as yet not defined and the only good assay for this endpoint is to fertilize the IVM oocyte and evaluate development. We do know that there are competencies other than meiotic that the oocyte must acquire to be fully competent, and these appear to be attained in a stepwise fashion (with meiotic ability apparent first) and are associated with the oocyte's stored genetic program (First et al., 1988; Eppig and Schroeder, 1989; Eppig et al., 1994). Previous work has shown that oocytes become progressively able to undergo normal fertilization, begin cleavage which must involve ability to transition to mitotic cell cycles, form blastocysts, and initiate and maintain a pregnancy. Part of the tissue maturation of the competent, primary oocyte should involve the ability to release its stored genetic program that allows complete expression of the various oocyte competencies. Rearrangement of cytoplasmic organelles also occur during meiotic maturation of the oocyte (Van Blerkom et al., 1990). Despite the fact that in vitro maturation yielding a fertilizable oocyte that results in a new individual is still a rather black-box process, success has been achieved in many species. These range from laboratory species, domestic animals, and primates (Chang, 1955; Moor and Trounson, 1977; Newcomb et al., 1978; Shalgi et al., 1979; Staigmiller and Moor, 1984; Schroeder and Eppig, 1984; Critser et al., 1986; Hanada et al., 1986; Sirard et aL, 1988; Mattioli et aL, 1989; Cha et al., 1991; Trounson et aL, 1994b) to exotics (summarized by Loskutoff and Betteridge, 1992; Loskutoff et aL, 1995). With the obvious reliance on IVM as central to many assisted reproductive technologies and research endeavors, further discussion of oocyte maturation will focus on tissue maturation of the meiotically competent, primary oocyte taken from tertiary follicles of mammals. This will be followed by sections concerning its utilization in IVF and embryo culture. For each species of interest one must keep in mind that there are many variations on this main goal that can be utilized to overcome the road blocks presented when a specific part of the process has not yet been brought to maturity. An example is the horse where after completion of maturation in vitro, IVF and embryo culture are not yet reliable. Here the matured oocyte can be placed back into the oviduct for fertilization and initial development after which the embryos are recovered from the uterus and transferred to recipient animals (Carnevale and Ginther, 1995). In cattle prior to development of a wide choice of in vitro embryo culture systems, biological incubators such as rabbits and sheep were used to take the zygote resulting from IVF after in vitro maturation of oocytes to the blastocyst stage which was compatible with transfer to a recipient or cryopreservation (Lawson et al., 1972; Boland, 1984; Eyestone et al., 1987). Thus many in vivo and in vitro techniques are available which can be joined in a wide variety of combinations to maximize use of the female gamete.

32

m. Lorraine Leibfried-Rutledge et al.

Literature concerning culture of earlier stages of primary oocytes will be summarized since this would salvage even more genetic material for other uses (Skoblina, 1988; Staigmiller, 1988; Jinno et al., 1990). A. S o u r c e s o f P r i m a r y

Oocytes

Primary oocytes from tertiary follicles are most frequently recovered postmortem, surgically, with the aid of a laparoscope or most recently via ultrasound-guided follicular aspiration. Each in vitro protocol has its own rate of success (usually less than unity) and the outcome of the total process will be a multiplicative factor comprising all the steps (Rutledge and Seidel, 1983). Reducing the number of intervening steps when possible will result in the greatest success for a particular type of reproductive cell. Yet this approach must be balanced by trying to rescue the greatest amount of genetic material possible for any specific species or in any specific instance, so often prevailing circumstances will dictate the stage at which donor material will be collected. Circumstances, species, value of the donor animal, and relative abundance or scarcity of the genetic material, and in some situations policy and ethical considerations, are some of the factors that will determine which source of cell will be used in tissue culture to reach the point at which the genetic material can best be stored or banked. The most frequent means of obtaining primary oocytes from various species will be discussed below. 1. Laboratory Species These species have classically been the paradigm for developing new cryopreservation protocols due to the ease and affordability of obtaining research material. In recent years, preservations of gametogenic and embryonic cells in rodents has become increasingly important on their own merit. Many unique strains of mice and rats have been produced by classical breeding methods or new transgenic technologies. Increasing restraints on resources does not allow maintaining large colonies of every strain available nor would this be advisable in case of unforeseen accidents. Recovery of oocytes from short-cycle species, exemplified by laboratory rodents, is usually done after death of the donor following excision of the ovaries and either pricking the antral follicles or mincing the ovarian tissue with a sharp blade while ovaries are submerged in an appropriate medium or sometimes under oil (Hafez, 1970; Moore, 1977; Oliphant and Eng, 1981). Caution must be taken in methods used for animal sacrifice since some types of anesthesia may cause changes in the gametes or embryos sought as will too long a lag between death and gamete collection (Keefer and Schuetz, 1982; Farrell and Bavister, 1984). The stage of the estrous cycle at which antral follicles are found is critical in short-cycle species. The day prior to that of expected estrus is probably the best time to observe antral follicles

Tissue M a t u r a t i o n in Vivo a n d in Vitro

33

containing oocytes in GV arrest (Greenwald, 1978). Vaginal smears or observation of discharges or a copulation plug allows easy definition of stage of the cycle in many laboratory species (Ward, 1946; Orsini, 1961; Bennett and Vickery, 1970). As estrus approaches, the luteinizing hormone (LH) surge will commence with concomitant induction of oocyte maturation and ovulation. Since most of the laboratory species are typically polytocous, a fair yield of oocytes enclosed in cumulus cells is realized from a single donor. Induction of superovulation prior to oocyte collection will obviously increase the yield and hence utilization of the animals sacrificed for gametogenic material. Superovulation protocols for use with specific species are well documented in the literature (Edwards and Fowler, 1960; Greenwald, 1962; Kennelly and Foote, 1965; Hogan et al., 1986). Only the guinea pig seems unresponsive to superovulation. Some studies have shown that an increased frequency of abnormal karyotypes are found in embryos collected after superovulation protocols in a wide range of species. Since the literature seems to include information that is conflicting on this point, it is recommended that a search be done for individual species to assess the potential for genetic damage after superovulation. Follicular matured or freshly ovulated secondary oocytes are very often used as donor material for IVF (Oliphant and Eng, 1981) and embryo culture purposes in smaller laboratory animals with good success. Since recovery of gametogenic material occurs postmortem whether obtaining immature or mature primary oocytes, secondary oocytes, zygotes, or various stages of preimplantation embryos, it is recommended that in the smaller laboratory animal species the stage of gamete or embryo desired for tissue banking be collected postmortem at the appropriate stage of the animal's reproductive history. This prevents the addition of intervening steps to reach the stage of tissue maturity most compatible with successful cryopreservation. Addition of unnecessary steps will only reduce the overall success rate of producing progeny. Much literature exists to document the timing necessary to obtain specific stages of oocytes or embryos for various species whether off of natural cycles or hormonally regulated cycles (see Hogan et al., 1986, for mice and Hafez, 1970, for other species of laboratory animals; also Orsini, 1961; Moore, 1977; Swanson et al., 1994). Domestic carnivores are popular models for developing assisted reproductive technologies for their wild relatives. Initial success in transfer of this technology from a domesticated lab animal to a wild animal particularly within the felids has been encouraging (Wildt et al., 1992b). A good number of reference books in the fields of veterinary medicine and lab animal care have extensive material on the reproductive management and life history of cats and dogs. Techniques for collecting oocytes are well documented in the literature (Mahi and Yanagimachi, 1976; Mahi-Brown, 1991; Goodrowe et al., 1988; Johnston et al., 1989).

34

m. Lorraine Leibfried-Rutledge et al.

2. D o m e s t i c A n i m a l s

Meiotically competent oocytes from antral follicles are commonly obtained postmortem in domestic animal species quite easily by collecting ovaries at an abattoir. In cattle, transport of ovaries in a simple, physiological saline is compatible with production of live offspring after recovery and maturation of the oocytes from antral follicles (Sirard et al., 1988). A few reports exist concerning the most appropriate temperature at which to transport the ovaries prior to recovery of the oocytes (Sato et aL, 1977; Kruip and Vernooy, 1982; Shioya et al., 1988b; Risopatron et al., 1991; Sekine et aL, 1992; Solano et al., 1994), but on the whole, little attention has been paid to the thermodynamics of tissue cooling, rewarming, etc., in obtaining the most successful results afterward. In the experience of one of the authors and also from literature citations, it is fair to say that exposure of excised ovaries containing primary oocytes or the oocytes themselves to cooler temperatures impairs the gamete's ability to undergo meiotic maturation in vitro and hence negates success of any further chances for producing live offspring (Moor and Crosby, 1985; Pickering et al., 1990; Yang et al., 1990; Leibfried-Rutledge, unpublished). Length of time that ovaries can be held prior to recovery of oocytes has also been little explored beyond those references dealing with temperature for transport. Ovaries of elite cattle obtained postmortem can be transported via a commercial courier and the oocytes used to produce calves after oocyte maturation, fertilization, and embryo culture in vitro. The oocyte enclosed in the tertiary follicle is in a rather "deprived" environment with its closest blood supply being outside the basal lamina and the blood barrier maintained by tight junctions of the granulosa (Mossman and Duke, 1973; Bjersing, 1978). This may be the saving factor in the success currently encountered using postmortem tissue as source of female gamete. Changes in the follicular environment after excision of the ovary are certainly dramatic as demonstrated by the difference in glucose values in bovine ovaries freshly recovered compared to after transport from an abattoir (Susko-Parrish et al., 1992). Yet this model may offer a means for banking of reproductive tissue from exotics and endangered species as these in vitro technologies become available for the individual species. As an example, gaur ovaries were flown from the Henry Doorly Zoo in Omaha, Nebraska, to a laboratory at the University of Wisconsin, Madison, Wisconsin. After an 8-h delay in oocyte recovery following removal of the ovaries, a live gaur calf was obtained after submitting the primary oocytes recovered to in vitro technologies (Johnston et al., 1994). The embryo recipient was a Wisconsin Holstein as shown in Figure 4. Live offspring have been produced from primary oocytes recovered postmortem in cattle (Hanada et al., 1986; Critser et al., 1986; Sirard et al., 1988), pigs (Mattioli et al., 1989), sheep (Cheng et al., 1986; Crozet et al., 1987), goats (Crozet et al., 1993), and buffalo (Suzuki et al., 1992; Madan et al., 1994).

Tissue M a t u r a t i o n in Vivo a n d in Vitro

35

Gaur calf born to a Holstein recipient at the Henry Doorly Zoo (Omaha NE). Surface visible antral follicles on ovaries taken from adult animals and shipped to the University of Wisconsin (MadisonWI) were aspirated to obtain primary oocytes. After in vitro maturation, the oocytes were fertilized with frozen-thawed gaur semen and cultured to the blastocyst stage. Interspecies transfer of two blastocysts resulted in one successful outcome. (Courtesy of J. J. Rutledge and L. A. Johnston.)

FIGURE 4

36

m. Lorraine Leibfried-Rutledge et aL

A variety of methods have been used to recover oocytes from postmortem ovarian tissue in domestic species (Gordon, 1994). Aspiration of antral follicles with hypodermic needles and a syringe or vacuum pump is common. Figure 5 illustrates the variety in morphological appearances seen in cumulus-oocyte complexes aspirated from antral follicles 2-6 mm in diameter visible on the surface of ovaries taken from adult cows at slaughter. A fair number of attempts have been made to develop rating criteria associated with selecting the complexes with the highest likelihood of maturing in vitro and developing into a transferable blastocyst after IVF (Leibfried and First, 1979; Fukui and Sakuma, 1980; Shioya et al., 1988a; de Loos et al., 1989; Madison et al., 1992; Hawk and Wall, i994). These selection systems rely on morphology of the oocyte and surrounding cumulus investment. Many show significant skewing of developmental success within classifications depending on criteria used for ranking. Some success in attaining the blastocyst stage of development is demonstrated across classes in most of the schemes, so if producing a blastocyst is the primary objective then it might be argued that all oocytes obtained should be cultured for reasons of expediency. This is true if the female gamete is limiting or has desirable genetics. Classification schemes have not progressed beyond evaluating ability to form a blastocyst. Ability to initiate and maintain a pregnancy

Variety of morphological appearances of bovine cumulus-oocyte complexes. Complexes were aspirated from antral follicles (2-6 mm diameter) visible on the surface of abattoir-derived ovaries. Complexes with many layers of cells still adhering to the underlying granulosa (a) are found along with oocytes lacking cellular investment (b).

FIGURE 5

Tissue M a t u r a t i o n in Vivo a n d in Vitro

37

or produce normal offspring has not been correlated with any of the systems. Slicing of the ovarian cortex with sharp blades or dissection of individual follicles with rupture of the follicle wall to release the enclosed oocyte have also been used to obtain oocytes in domestic animals. The latter method appears the most successful in horses (Del Campo et al., 1994) because of the structure of the ovary. Gamete collection in horses has been recently reviewed (Fayrer-Hosken et al., 1993). Gordon (1994) has summarized some of the reported efficiencies and expectations for different methods of obtaining oocytes from follicles of domestic animals. Slicing of the ovarian tissue or dissection of all follicles including those buried in the ovarian cortex will release more material than just aspiration or dissection of follicles visible on the ovarian surface (Katska, 1984; Sato et al., 1990; Kay and Frylinck, 1992; Mogas et al., 1992; Carolan et al., 1994; Arlotto et al., 1996) and hence is always recommended for scarce ovarian material to release every possible oocyte. Differences in maturational competence and hence potential for further development has been observed for oocytes taken from peripheral antral follicles vs those deeper in the ovarian cortex in cattle, implying that location in the ovary may indicate different populations of follicles containing oocytes with different maturational competencies (Iwasaki et al., 1987; Sato et al., 1990; Martino et al., 1992; Takagi et al., 1992; Arlotto et al., 1996). Researchers in the laboratory of one of the authors have observed that primary oocytes in antral follicles located deeper in the ovarian cortex of adult cows tend to have smaller diameters on the average and a decreased potential to complete the first reduction division (Arlotto et al., 1996). This may indicate that growing follicles and oocytes are located below the surface and rise toward the germinal epithelium at later stages of development and differentiation of the follicular unit (Harrison, 1962; Greenwald and Peppler, 1968; Spicer et al., 1987). Other reports indicate that oocytes from the two follicle populations do not differ in the various endpoints observed after in vitro protocols (Hamano and Kuwayama, 1993). Although gametogenic material obtained postmortem is useful in many situations, it does not allow for choice of genetics that may often be desired nor is it the best way to utilize scarce living resources. Surgical means of recovering oocytes have been utilized in specific situations via laparotomy or standing flank incisions depending on the species and facilities available. Large antral follicles may be aspirated or the ovary excised. Ovaries in larger domestic species may also be removed via salpingectomy using a vaginal approach, but this again negates further reproductive usefulness of the animal. Secondary oocytes matured in the follicle, ovulated secondary oocytes, and even early embryos can be recovered in this manner. Since surgical intervention is cumbersome and expensive, most frequently secondary oocytes taken from follicles or oviducts are the intended cell stage for recovery. Laparoscopy can also be used successfully in some domestic

38

m. Lorraine Leibfried-Rutledge et al.

animals to recover oocytes from antral follicles (Lambert et al., 1983) and again has resulted in birth of live offspring after IVF or transfer back to the oviducts for fertilization and development (cattle: Brackett et al., 1982a; Sirard and Lambert, 1986; Lambert et al., 1986; Armstrong et al., 1992; swine: Cheng et al., 1986; sheep: Crozet et al., 1987). Again, secondary oocytes or those that have begun maturation in the follicles are the most frequent stage recovered using laparoscopy in domestic animals. In small ruminants such as sheep, laparoscopic folliculocentesis can be used to recover oocytes for use in IVM (Baldassarre et al., 1994). If this is done after a hormonal regime to induce increased follicular development and obtain control of the estrous cycle, a single animal can increase its total contribution to the genetic pool dramatically. A technique being applied very recently to domestic animals is that of ultrasound-guided, follicular aspiration (Pieterse et al., 1988, 1991; Kruip et al., 1991; Cook et al., 1992). Positioning of the ovary in relation to the ultrasound transducer and puncture needle during transvaginal ultrasoundguided follicular aspiration in cattle is shown in Figure 6. This is currently being used on a commercial basis in cattle to recover primary oocytes from antral follicles that will be matured, fertilized, and cultured to the blastocyst stage using in vitro procedures (Looney et al., 1994; Hasler et al., 1995). It can be done on an individual animal repeatedly over an extended period of time without damage to the ovary or subsequent reproductive impairment. Unlike conventional methods for increasing fecundity in cattle such as nonsurgical embryo recovery normally used in conjunction with superovula-

4

J:

~.~m $ I

~i o

Ib

~

i~ #

Im

II

Diagram of transvaginal ultrasound-guided follicular aspiration in cattle. Ovary (1) is positioned against the transducer by rectal manipulation (2) while the ultrasound transducer containing the needle guide and aspiration needle (4) is placed against the anterior portion of the vagina beside the cervix (3). (Kruip et al., 1991. Vet. Rec. 128, 208-210.)

FIGURE 6

Tissue M a t u r a t i o n in Vivo a n d in Vitro

39

tion, no gonadotropin is required so response does not decrease. Older animals, pregnant animals, prepuberal animals, or animals with clinical infertility may also be utilized as donor animals (Ryan et al., 1993; Kruip et al., 1994; Meintjes et aL, 1994, 1995b; Duby et aL, 1996) using this method for follicular retrieval of immature oocytes with ensuing production of live offspring after IVM-IVF-IVC and transfer to recipients. This procedure has been used successfully in cattle and buffalo to produce calves (Ryan et al., 1990; Looney et al., 1994; Hasler et al., 1995; Madan et al., 1994) and has been adapted for a smaller ruminant, the goat (Dorn et aL, 1989; Han et al., 1996). A transvaginal follicular aspiration method has also been developed for field situations that does not require ultrasound visualization of the ovary (Hill, 1995), both reducing the initial outlay for equipment and allowing application to smaller animals such as young or prepuberal heifers. This technique may be well suited for many ungulates. 3. P r i m a t e s

Use of postmortem or surgically excised material (Alak and Wolf, 1994) in nonhuman primates to obtain primary oocytes does make best use of an animal if clinical or other research treatments have not impaired the currently growing population of follicles. As with domestic species, slicing of the ovarian cortex with sharp blades will liberate most of the genetic material available in antral follicles. Evaluating quality of oocytes recovered from many nonhuman primates has been somewhat difficult (Wolf et al., 1990) and often relies on morphology of associated cumulus cells. Use of genetic material from human ovaries removed for various clinical reasons could also be made available for tissue banking after application of in vitro technologies. However, many investigators have observed limited recovery of quality oocytes (Racowsky and Kaufman, 1992; Racowsky et al., 1992). More importantly, work in humans will be guided by countries' regulations and ethical considerations for gamete and embryo work which should be consulted prior to any experimentation with human gametes and embryos. Sources of guidelines for several countries are included at the end of the references. Because of the obvious clinical demands, development of laparoscopic (Steptoe and Edwards, 1970; Feitchinger and Kemeter, 1984) and ultrasound-guided (Lenz et al., 1981; Wikland et al., 1983) technologies were available in primates before such use was considered in most other species. Most typically, the focus has concentrated on recovery of secondary oocytes most frequently after hormonal induction of a cohort of large antral follicles which makes best use of the mostly monotocous primate family (Wolf et al., 1990). Live offspring have been produced after IVF of secondary oocytes in a number of primate species (Balmaceda et al., 1984; Bavister et al., 1984; Boatman, 1987; Lopata et al., 1988; Cranfield et al., 1990). However, use of the primary oocyte has been limited due to the need for developing protocols for IVM of these oocytes. Births have very recently

40

m. Lorraine Leibfried-Rutledge et aL

been reported after IVM of human primary oocytes (Trounson et aL, 1994b) but success has been more limited in other primates. These initial reports have spawned a renewed interest in the development of in vitro tissue maturation for primates. 4. Exotics a n d Endangered Species

As conservation efforts are increasingly directed toward tissue banking of genetic materials to maintain species diversity, assisted reproductive technologies will reach maturity for the individual species (For an overview see Wildt et al., 1992a,b,c; also in this book). This effort is further advanced by the development of model species (Wildt et aL, 1986; Lasley et aL, 1994; Loskutoff et aL, 1995) exemplified by work with felids (Goodrowe et aL, 1991; Johnston et aL, 1989, 1991a,b; Donoghue et aL, 1990, 1993), bovids (Johnston et aL, 1994; Armstrong et aL, 1995; McHugh et aL, 1995), and other ungulates (Hearn and Summers, 1996; Loskutoff et aL, 1995). Historically, much of the available tissue used in research efforts to develop necessary technologies has recovered the female gametes postmortem, as tissue adventitiously became available. The introduction of nonsurgical methods of recovering oocytes (ultrasound-guided technologies and laparoscopy) have greatly facilitated efforts with endangered and exotic species (gaur: Armstrong et aL, 1995; camelids: Brogliatti et aL, 1996; Del Campo et al., 1995). The ability to collect primary oocytes from living animals to use for in vitro tissue maturation will greatly expand the material available and allow careful manipulation of genetics to maintain as large a gene pool as possible in rare species. Efforts have been initiated in a variety of species (Pavasuthipaisit et aL, 1992; Christensen et aL, 1993; Del Campo et aL, 1995; Meintjes et aL, 1995a) with variable success. Collection of oocytes from antral follicles will avoid the intensive work and the number of living animals needed to bring to maturity for individual species, assisted reproductive technologies such as superovulation, surgical or nonsurgical recovery of embryos, and embryo transfer. These require detailed knowledge of an individual species' reproductive characteristics (Barone et al., 1994; Donoghue et aL, 1993) and response to hormonal intervention (Loskutoff et aL, 1990) that may prolong obtaining the end result, tissue banking, when animals are scarce. Despite the vast number of technological hurdles to overcome, a number of live offspring have been produced with these technologies (Indian desert cat: Pope et aL, 1989; gorilla: Dresser et al., 1996; tiger: Donoghue et aL, 1990; gaur: Johnston et aL, 1994; Armstrong et aL, 1995; reviewed by Loskutoff and Betteridge, 1992). B. In V i t r o

Oocyte

Maturation

The goal of culturing primary oocytes that are meiotically competent through completion of the first reduction division to yield a secondary

Tissue M a t u r a t i o n in Vivo a n d in Vitro

41

oocyte capable of development after fertilization is the same for all species of interest. Oocytes that have been cultured for various periods of time then fixed (Gordon, 1994) are shown in Figure 7. This figure illustrates the various stages of meiotic maturation, or meiosis I, which must be completed successfully prior to contemplating IVF. The protocols utilized for specific species that support meiotic maturation may vary in details due to individual differences in reproductive characteristics. Culture systems found successful in one species of a family can be applied to other members, as in the case of domestic cattle. Here the entire system commonly used for the in vitro production of cattle embryos has been successfully applied to the gaur (Johnston et al., 1994; Armstrong et al., 1995) and buffalo (Totey et aL, 1992; Madan et al., 1994) to produce progeny while the IVM, IVF, and culture systems work well in banteng, bison, and yak (Barnes et aL, 1988; Dorn et al., 1990; Dorn, 1995; McHugh et al., 1995; J. J. Rutledge, personal communication) to produce blastocysts. Systems for producing bovine embryos in vitro have actually worked at some level of efficiency for a number of nondomestic ungulates (Lasley et al., 1994; Loskutoff et al., 1995). This approach of applying successful strategies developed from an abundant member of a family to members where material is more difficult to obtain is being used in a number of exotics and endangered species and should be borne in mind when attacking a new species of interest (Wildt et al.,

7 The stages of meiosis I in mammalian oocytes. Whole mounts of oocytes were fixed and cleared at various times after the start of culture for in vitro maturation and then photographed using Nomarski optics. (A) Swine primary oocyte before culture showing the germinal vesicle. (B) Bovine oocyte at metaphase I. (C) Bovine oocyte at anaphase I. (D) Bovine oocyte at metaphase II after expulsion of the first polar body.

FIGURE

42

m. Lorraine Leibfried-Rutledge et al.

1986, 1992b; Loskutoff et aL, 1995). There are aspects of culture that may also be very species dependent. Choice of antibiotics or macromolecular supplements for example, are much more sensitive considerations in humans, where anaphylactic shock, disease transmission, or ethical considerations are involved, than in domestic animals. In domestic animals and exotics, source of animal material used as media supplements or coculture tissue needs consideration with respect to animal health regulations, especially if IVP embryos will be used for export or used for transfer to recipients. Disease transmission must be considered for all components of in vitro systems and not just the product (Stringfellow and Wrathall, 1995; Wrathall, 1995). In mice there are examples where different steps in the production of embryos in vitro may have to be modified for specific strains since they show strain differences for each step of the I V M - I V F - I V C sequence. These species and strain specific considerations should be taken into account before beginning work in the laboratory so that results will not be nullified later for practical usage. The following discussion of culture systems will avoid some basic technicalities such as quality and storage of reagents, water source, and source of disposables. There are a number of excellent books on basic tissue culture (Nuzzolo and Vellucci, 1983; Freshney, 1987; Morgan and Darling, 1993; Davis, 1994) and books and reviews on culture pertaining specifically to gametes and embryos (Bavister, 1981a; Rogers, 1981; Bavister, 1982, 1986; Hogan et al., 1986; Leibfried-Rutledge et al., 1989a; Wolf et al., 1988; Boone and Shapiro, 1990; Leibo, 1990; Gordon and Lu, 1990; Brackett and Zuelke, 1993; Gordon, 1994; Pinkert, 1994; Bavister, 1995; Thompson, 1996). These should be consulted even if the investigator already has some experience in these methods. The remainder of this section will be devoted to aspects of culture that will be common considerations for in vitro oocyte maturation, fertilization, and embryo culture or that pertain specifically to the female gamete and its in vitro maturation. 1. Choice o f M e d i a

There are two basic types of media to be considered. Simple balanced salt solutions that can be made up easily in the laboratory comprise one category, although they have become a little more complex than that first used to develop mouse one-cell embryos to the blastocyst stage outside the tubal environment (Whitten and Biggers, 1968). More complete media usually obtained from commercial sources make up the second category (categories reprised by Thompson, 1996). The simple salt solutions are typified by Tyrodes' modifications (Bavister and Yanagimachi, 1977; Bavister et al., 1983), Whitten's (1971), Brinster's (1969), CRlaa (Rosenkrans et al., 1993; Rosenkrans and First, 1994), Menezo's B2 (Menezo, 1976), SOF (Tervitt et al., 1972), CZB (Chatot et al., 1989), and HECM (Schini and Bavister, 1988, 1990; Seshagiri and Bavister, 1990). Formulations of

Tissue M a t u r a t i o n in Vivo a n d in Vitro

43

balanced salt solutions that are commonly used for in vitro technologies with various species are listed in Table 1 along with their major uses and species applications. TCM199, Ham's F10, Eagle's basal medium, etc., exemplify the class considered as more complete media (reviewed by Staigmiller, 1988; Thompson, 1996). Despite the basic type of media, both categories rely on either bicarbonate or phosphate buffering systems with some of the more complete media coming with either buffering system depending on whether the balanced salts that provide osmolarity and electrolytes are supplied by Earle's or Hank's salts. The vast majority of tissue culture in gametes and embryos has been done in media that rely on the bicarbonate buffering system (Mahadevan et al., 1986), which in addition to functioning

Physiological Saline Solutions C o m m o n l y Used for G a m e t e and E m b r y o Culture

TABLE 1

Medium

Reference

M o s t c o m m o n use

Species

BMOC BO

Brinster, 1965, 1 9 7 2 Brackett and Oliphant, 1975 Seidel et al., 1991 Rosenkrans et al., 1 9 9 3 Chatot et al., 1989 Schini and Bavister, 1988 Quinn et al., 1985 commercially available

Ova/embryo culture Sperm capacitation

Mouse Rabbit, cow, sheep Cow Cow Mouse, cow Hamster, cow Human, mouse Many species

CDM CRlaa CZB HECM a HTF KRB KSOM M16 M2 Menezo's B2 PBS SOF Sperm-TALP (BGM1)b TALP c

TL-Hepes r Whitten

Embryo culture Embryo culture Embryo culture Embryo culture Embryo culture Gamete/embryo culture, collection Lawitts and Biggers, 1992, Embryo culture 1993 Whittingham, 1971 Embryo culture Hogan et al., 1986 Gamete/embryo collection Menezo, 1976 Gamete/embryo culture Versions commercially available Tervit et aL, 1972 Parrish et al., 1986, 1 9 8 8 Bavister and Yanagimachi, 1977 Bavister et aL, 1983 Bavister et al., 1 9 8 3

Gamete/embryo collection Embryo culture Sperm processing and capacitation Fertilization

Oocyte/embryo handling, collection Whitten and Biggers, 1968 Embryo culture Whitten, 1971

Mouse, cow Mouse Mouse Human, cow, sheep Many species Sheep, cow Cow, other species Hamster, cow, other domestic Hamster, cow, other domestic Mouse

Series of modifications as improvements in ability to culture hamster embryos are made. b Series of modifications for processing and handling of bovine spermatozoa. c Glucose eliminated for work in cattle, other domestic animals, and some exotics. a

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as a buffering system is a metabolic requirement for many cell types including preimplantation embryos (Wales et al., 1969; Brinster, 1972). Maintenance of both extracellular and intracellular pH is important for the production of viable gametes and embryos (Blatz, 1993; Blatz et al., 1995). A limited amount of work has been done on the medium used for a specific species that yields matured oocytes with the greatest chance of becoming blastocysts (Eppig et al., 1990; van de Sandt et al., 1990; Bavister et al., 1992; Rose and Bavister, 1992; Hawk and Wall, 1993). Work in mice, cattle, hamsters, and nonhuman primates indicates that species does affect choice of medium (Bavister et al., 1992; Gordon, 1994) and that the mouse may have limited value as a model for other species (Winston et al., 1991). Even within a species, different laboratories may have greater success with one medium than other laboratories. Many small technicalities are probably associated with these differences so each laboratory must determine what medium and protocols work best for their uses. To provide some foreshadowing to the reader who has stayed with the chapter to this point, medium selected for maturation of the oocyte of a particular species may not be the same one selected for use in other phases of the reproductive process such as fertilization or embryo culture. Some effort has been made to determine if one medium will support maturation, fertilization, and embryo development for cattle (Waterman et al., 1991; Nagao et aL, 1995; T. Dominko, unpublished) as is done in mice (Hogan et al., 1986), but more work is necessary to optimize each process before this can be done for many species. Processing reproductive tissue is normally done at atmospheric conditions so both simple salt solutions and more complete media have been modified by reducing bicarbonate levels, addition of synthetic buffers such as Hepes, both of the afore-mentioned, or switching to a phosphate-buffered medium alone. This is to maintain a relatively constant pH, which would not be possible using a bicarbonate-buffered medium in air. Medium having a high phosphate concentration is detrimental to sperm motility and early development of murine embryos (Quinn and Wales, 1973; White, 1974) and one of the present authors believes that prolonged holding of gametes or embryos in similar formulations may not be compatible with maintaining optimum viability of these cells. As a reminder, media such as TCM199 with Hepes buffer with the normal level of sodium bicarbonate for Earle's salts (commercially available) or Tyrode's medium modified for use with bovine sperm (Parrish et al., 1986) will produce a drift in pH over time under atmospheric conditions, presenting uncontrolled changes in pH. These will be slower than in media using a bicarbonate-based buffering system only, but they do occur. Choice of media not only for final culture, but for processing material ahead of time under ambient atmospheric conditions should be selected with care.

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When choosing media for processing or culture of tissue, considerations must be given to the osmolarity of the medium selected. Although only a limited amount of work has been done on effects of osmolarity with regard to oocyte maturation or fertilization, a greater amount of work has been compiled concerning effects on early embryo development beginning almost from the inception of in vitro tissue maturation as applied to preimplantation mammalian embryos (Whitten, 1957) and continuing to the present (Brinster, 1965; Whitten and Biggers, 1968; Lawitts and Biggers, 1992, 1993; Biggers et al., 1993). Whitten (1971), during this innovative period, received the information from Waymouth, "that many cells in culture can tolerate very wide variations in osmolarity if the sodium to potassium ratio is kept constant." Keep in mind that effects of osmolarity are tightly linked to that of media constituents, particularly the ratios of various electrolytes, and also the gaseous atmosphere used for culture with a specific media. Introduction of macromolecules such as BSA can also shift osmolarity if products are not sufficiently purified. Monitoring osmolarity is one means of detecting possible drift in media quality. Vigorous monitoring of all media components is another critical quality control measure necessary for success during in vitro manipulations. Their storage is also critical. Some media components degrade spontaneously during storage of media (Schiewe et al., 1990). Diluted and aliquated proteins even stored at low temperatures will also loose their biological activity after a period of several months as cautioned by sources such as the National Institutes of Health gonadotropin program. Some deliberation must be given to each component in regard to how it should be stored (most chemical companies indicate this on the container) and whether it can be stored as a diluted stock or should be diluted and added to media just prior to use with tissue. Media once prepared for in vitro manipulations will also deteriorate during storage (Stewart-Savage and Bavister, 1988). Length of storage for the water used to make up media can also affect the success of in vitro embryo production (Nagao et al., 1995). Quality control is essential. There have been many media additives tested to determine if they improve tissue maturation of reproductive cells in culture. These range from anti-oxidants, chelators of heavy metals, vitamins, amino acids, cofactors, antibiotics, and on into countless other factors, most recently including growth factors and cytokines. It is suggested that the excellent references cited in the literature be consulted for the many possible modifications of culture media that have been tested and found useful in laboratories around the world to find the system that works most effectively for a particular laboratory, species, or cell type. 2. G a s e o u s A t m o s p h e r e

Media considerations have been discussed slightly in regard to selecting media for processing of tissue and gametes vs choice of media for final

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culture of the oocyte. Also the buffering system in the medium selected for maturation will dictate the CO2 concentration required for culture. Oxygen and N2 are the other two gases normally considered when choosing the atmosphere for culture. Nitrogen is fairly inert in most mammalian culture systems and is used to make up the balance of the incubator atmosphere when ambient air is not selected for use during culture. Oxygen at concentrations found in atmospheric conditions may be toxic to many cell types such as embryonic cells (summarized for domestic animals by Wright and Bondioli, 1981; Thompson, 1996) and so is frequently reduced to comprise 10% or less of the constituents of the gas atmosphere. 02 concentrations in oviduct and uterus is known for a species and is consistently less than 10% (Fischer and Bavister, 1993; reviewed by Bavister, 1988; Leese, 1991). Despite our early awareness of oxygen toxicity to the early embryo (Whitten, 1957, 1971; Brinster, 1965, 1970), a high proportion of the laboratories involved in the in vitro production of embryos continue to use a gas phase with atmospheric levels of oxygen. Live and seemingly normal offspring continue to be successfully produced in this manner. Interactions between media constituents, osmolarity, and atmospheric components are exceedingly complex. Studying changes in any one of these factors must be considered in relation to the others. This fact is often forgotten and may affect conclusions of experiments designed to study just one aspect of the total culture environment. Elevated 02 content of atmospheric air may lead to increased formation of radicals which damage intracellular processes. In fact the generation of free radicals in culture media in general is a topic receiving considerable attention in efforts to develop improved conditions for in vitro maturation of mammalian embryos (Noda et aL, 1991; Goto et al., 1993; Johnson and Nasr-Esfahini, 1994; Nagao et al., 1994). This focus is just beginning to be addressed for oocyte maturation and deserves more attention in the future. Perhaps, it has been held for too long that the oocyte's surrounding cumulus cells lend protection from the potential damage by free radicals. These cells are necessary in some fashion to the oocyte for final maturation as determined from in vitro studies concerning developmental competence of oocytes matured with or without surrounding cumulus (Staigmiller and Moor, 1984; Leibfried-Rutledge et al., 1989a; Canipari, 1994). Follicle cell derivatives are a major route for the entry of metabolites and precursor molecules via gap junctions between cumulus and oocyte (Schultz, 1986b). Along with proteins from body fluids, they also appear to prevent a phenomenon referred to as zona hardening with impairs subsequent attempts to fertilize the in vitro matured oocyte (discussed for several species by Gordon, 1994). Cumulus cells may even have a positive inductive role in mammalian oocyte maturation, and in mediating the gonadotropin effects on the oocyte (Dekel, 1988; Schultz, 1991; Aktas et aL, 1991). In vitro maturation of

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mammalian oocytes must actually be considered as an interaction of several types of tissue, both gametogenic and somatic.

3. Macromolecular Supplements Macromolecular supplements used in tissue culture perform two basic roles: one is to provide a fixed source of nitrogen; the other is to reduce stickiness of the cell surfaces to make handling and manipulation easier. As mentioned previously, blood serum used as macromolecular supplement during in vitro oocyte maturation prevents zona hardening, possibly by a blood component identified as fetuin (Schroeder et al., 1990). A minor amount of buffering capacity is also supplied by proteinaceous supplements such as found in body fluids. Two basic groups of supplements are utilized. Body fluids and their products are one type. This includes fluids such as blood serum or products isolated from serum such as bovine serum albumin (BSA). Culture systems utilizing these tissue-derived supplements are often termed undefined systems, since even purified components from them contain contaminants so amounts of specific substances and composition are not completely known. Different lots of blood serum from bovine fetuses and even lots of supposedly purified components such as BSA or human serum albumin (HSA; Ashwood-Smith et al., 1989) have differing abilities to support successful outcomes after tissue culture (Kane, 1978, 1987). Variability in frequency of development to the blastocyst stage, rate of hatching, and cell number of the resulting product after culture of rabbit and hamster embryos in different lots of BSA has been reported (Kane, 1983; McKiernan and Bavister, 1992). In rabbit embryo culture, the differences between lots appears to be a result of different levels of citrate (Kane, 1990; Gray et al., 1992) which is now known to enhance rabbit blastocyst development (Kane and Fahy, 1993). Variability in lots of blood serum or its components and whether this is due to differing amounts of actual stimulatory factors or toxic effects of contaminants is not known for most reproductive cell types being cultured. This fact must be kept in mind when performing studies to determine the most appropriate tissue-derived supplement to use with various cell types. Several different lots or sources of a particular supplement should be planned into the experimental design so that the investigator is actually testing or characterizing the effect of the component rather than one lot on the desired cell type. Fetal calf serum compared to BSA has been observed to improve viability of both hamster and bovine cumulus cells and their expansion (Leibfried-Rutledge et al., 1986) during maturation of the oocyte while supporting a higher frequency of oocytes that complete meiosis I (Cross and Brinster, 1970; Shea et al., 1976; Leibfried-Rutledge et al., 1986). Gonadotropin-induced expansion of cumulus cells requires a serum component that is not supplied by BSA (Eppig, 1979, 1980). Choice of supplement will depend on the final purpose of the tissue culture. Media conditioned by cell secretions also can be

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included in the group of tissue-derived supplements. This type of macromolecular supplementation is most often encountered in embryonic culture. The second group of macromolecular supplements is comprised of large synthetic polymers or defined serum replacements. Polyvinylpyrrolidone (Cholewa and Whitten, 1970) and polyvinyl alcohol (Bavister, 1981b) are examples of polymers that are often used in current culture systems when a completely defined environment is desired to study cell phenomena. Since these are both synthetic polymers they can be bought in a number of chain lengths or molecular weights. Not all give equal results for tissue culture purposes. These substances from some sources may also have to be cleaned up by dialysis or chromatography prior to use in culture. Serum replacements are typified by Plasmanate (TM; Cutter Biological, Elkhart IN; Adler et al., 1993), Plasmatene (TM; Alpha Therapeutics, Los Angeles; Pool and Martin, 1994), or Synthetic Serum Substitute (Irvine Scientific, Irvine CA). These are characterized by having defined amounts of highly purified known components, such as defined percentages of albumin and a- and/3-globulins. On the whole, serum replacements have not been frequently utilized for gamete or embryo culture, with the human system being the most notable exception. The defined culture system can be utilized successfully (Menezo et al., 1984; Caro and Trounson, 1986; Schini and Bavister, 1990) as demonstrated by production of normal offspring and is useful for several reasons. First, when transfer to recipients, or international transport is a feature of the use scenario, than considerations for control of disease vectors is simplified. Analysis of the parental genotypes is easier as is verification of an embryo's health status more readily achieved. The less defined culture systems are workable in this setting but take more documentation to provide supporting health certifications. The defined culture environment is also a useful model for studying specific cellular processes such as energy metabolism or amino acid metabolism where total control of these substances in the culture media is required for the most definitive results (Gardner, 1994; Bavister, 1995; Leese et al., 1995). Unfortunately, except for a very few species (references previously cited) demonstration of normal offspring resulting from completely defined systems, or rigorous testing of defined vs. those using blood serums or its less pure components has not been accomplished although they have been used to produce blastocysts in a fair number of species. The investigator must be sure that the totally defined environment is not distorting the outcomes and forcing cellular processes down alternative pathways, and this may often be difficult to determine. The objective for using in vitro technologies will dictate whether a defined or less defined culture system is chosen. With greater attention being paid to the potential for inducing perturbations to normal developmental mechanisms with in vitro manipulations (Willadsen et al., 1991; Walker et al., 1992a,b, 1996; Seamark and Robinson, 1995), we as researchers, clinicians, agriculturists

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or environmentalists are increasingly obliged to examine offspring produced both as the neonate and at stages of its future life history to verify normality of the product. 4. Energy Substrates Oocytes, as do all cell types, require sources of energy to maintain cell processes and hence viability. As in the mouse preimplantation embryo (Brinster, 1965, 1969), it was noticed early in developing successful culture systems that murine primary oocytes require a three-carbon energy metabolite (Biggers et al., 1967; Eppig, 1976; reviewed by Leese, 1991, Rieger, 1992) for resumption and completion of meiotic maturation in vitro, which implies that oxidative metabolism is the major source of energy in the oocyte (Rieger and Loskutoff, 1994). Oocytes of the cow and rhesus monkey show a similar preference (Brinster, 1971; Rushmer and Brinster, 1973). More recent work extends this idea to support the notion that metabolic substrates may be capable of regulating meiotic maturation in vivo (Downs, 1992). As stated previously, current oocyte maturation systems must actually be considered as the culture of several different cell types since we are, on the whole, unable to obtain a developmentally competent oocyte if it is matured without follicular companion cells. The cumulus cell appears able to convert a six-carbon sugar, such as glucose, into the three-carbon metabolite favored by the oocyte (Biggers et al., 1967). In cattle, addition of both glucose and pyruvate to the oocyte complex in culture results in the most successful oocyte maturation and development to the blastocyst stage after fertilization (Susko-Parrish et al., 1992). More blastocysts were obtained with glucose present during culture of the cumulus-oocyte complex during maturation, indicating a requirement for glucose by cumulus cells, while the oocyte showed a preference for pyruvate. Since glucose is thought to be inhibitory to development of early cleavage-stage embryos in some species (Schini and Bavister, 1988; Takahashi and First, 1993) and does hinder capacitation of sperm (Parrish et al., 1989), it was thought that a similar principle might apply to the oocyte. On the whole, we know much more about the metabolic pathways and substrate preferences of sperm and embryos compared to those of the oocyte complex. The area of nutrient requirements needs more attention in the future. Reviews concerning substrate utilization throughout the various stages of maturation, fertilization, and development should be consulted when selecting culture medium for specific purposes and species where available (Bavister, 1990b; Leese, 1991; Rieger, 1992; Conaghan et al., 1993; Bavister, 1995; Barnett and Bavister, 1996). 5. Hormonal Supplements Preovulatory oocyte maturation in situ in the Graafian follicle is induced at a specific stage of the estrous or menstrual cycle in response to a specific

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hormonal milieu. Initial attempts to obtain offspring using oocytes matured in vitro tried to mimic this situation by supplementing culture medium with gonadotropins and estrogen (Crosby et al., 1981; Staigmiller and Moor, 1984; Sirard et al., 1988). Since then, oocytes have been matured in vitro in media without hormonal supplements using blood serum or follicular fluid as protein supplement or in completely defined culture systems without hormonal supplementation. Many studies in a wide variety of species have been conducted to determine if gonadotropins and steroids are actually needed alone or in combination (as examples see Moor, 1978; Moor et al., 1980; Prins et al., 1987; Behrman, 1988; Fukui and Ono, 1989; Younis et al., 1989; Saeki et al., 1990; Gomez et al., 1993; Alak and Wolf, 1994; Schramm et al., 1994; Schramm and Bavister, 1994, 1995). From looking at the numerous variations in culture systems in use today, the literature at this point appears conflicting, with no definitive answer in sight. Very few studies have gone past blastocyst formation as an endpoint to assess the effect of hormones during oocyte maturation in culture. Complete developmental competence as affected by hormone treatment, in other words, has not been rigorously evaluated. In the majority of studies conducted to date, success rate for completing meiotic maturation does not appear to be affected by the presence of hormonal supplements although cumulus maturation is gonadotropin-dependent (Eppig, 1979, 1980; Hillensjo and Channing, 1980; Ball et al., 1982). The expansion of the cumulus oophorous during in vitro maturation of cattle oocytes is shown in Figure 8. This is not anywhere near as extensive as that observed after in vivo maturation. Duration of time required for resumption and completion of meiosis I is changed in response to supplementation with hormones (Suss et al., 1988; Dominko and First, 1992). Gonadotropin-induced cumulus maturation uses cAMP as a second messenger and the increased levels of cAMP may be transiently inhibitory to the oocyte (Downs et al., 1988) so meiotic resumption is delayed. Events involved in regulating the time required for resumption and completion of meiosis I may have an impact on success in blastocyst formation, since the earliest maturing cattle oocytes appear to have the greatest success in developing to blastocysts after fertilization (Dominko and First, 1992). In some studies, addition of hormonal supplements is associated with higher rates of fertilization or development to the blastocyst stage (Shalgi et al., 1979; Fukushima and Fukui, 1985; Downs et al., 1986; Brackett et al., 1989; Younis et al., 1989; Zuelke and Brackett, 1990; Galli and Moor, 1991; Saeki et al., 1990, 1991). In the rhesus monkey, the influence of exogenous gonadotropins appears to be dependent upon the stage of the menstrual cycle (Alak and Wolf, 1994), highlighting the complexity of potential interactions. Although no receptors for gonadotropin have been localized to the oocyte, the surrounding cumulus cells and granulosa cells adhering to the complex do possess receptors (reviewed by Dekel, 1988). In vitro tissue maturation of the oocyte complex involves several types of differentiated cells and complete success of the process may depend on

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8 In vitro maturation of the cumulus investment. During culture of the primary oocyte the cumulus oophorus undergoes expansion, changing from a many layered epithelial-

FIGURE

like conformation (A) to a loose arrangement of cells entrapped in a sticky matrix (B).

getting conditions "right" for all the cells involved. Cytoplasmic maturation of the oocyte has been thought to be at least partially responsive to steroids since the early days of extrafollicular culture of oocytes (credited to Soupart by Tsafriri, 1978; Moor and Trounson, 1977; Moor et al., 1980) and so may need careful consideration as a component of media used for oocyte culture. Although cytoplasmic maturation can be achieved without addition of supplementary gonadotropins or steroids as verified by production of

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blastocysts after IVF and in some species full-term offspring, consistency in achieving respectable rates of blastocyst formation after IVF is realized if hormonal supplements are added to the medium used for maturation. Since clearly defined increases in steroids and both follicle stimulating hormone (FSH) and LH are manifest prior to ovulation, it is not unreasonable to suppose that final oocyte maturation may be mediated at least indirectly by the changing hormonal content of the preovulatory follicle. In humans where the greatest number of studies have been done to correlate follicular fluid contents with success in producing pregnancies after IVF of mature secondary oocytes recovered in vivo, hormonal balance does appear to be correlated with a successful outcome (Schramm and Bavister, 1994, 1995). Using a whole follicle culture system, oocyte maturation can be induced in rat oocytes with either FSH or LH (Lindner et al., 1974; Daniel et al., 1989). Whether this is due to action of the hormones on their own receptors or cross-reaction with the individual receptors has not been clearly defined. Ovulation in vivo can be induced only by molecules with LHlike activity. A recent area of research has focused on a possible role for various growth factors in oocyte maturation (Downs, 1989; Das et al., 1991; Jewgenow and Pitra, 1993; Gomez et al., 1993; Smith et al., 1995). Both ligands and receptors have been identified in growing follicles of various species, levels analyzed in follicular fluid of antral follicles or cellular secretions (Adashi et al., 1985; Hammond et al., 1991; Webb et al., 1994). Current studies have examined the effects of growth factors added to culture media for various stages of in vitro embryo production starting with maturation of the oocyte. Although no consensus has been reached for oocyte maturation, this area may prove fruitful in the future, especially if culture results are extended to transferring embryos to recipient females. 6. C u l t u r e M e t h o d s

Once a medium and its constituents have been selected and a gas atmosphere chosen, the method of culture for the particular cell type must be picked. Petri dishes, tubes, multiwell plates and chamber slides have all been frequently used as culture vessels. If disposable plastics are chosen, they should be tested for toxicity since not all sources yield the same success rates (Boone and Shapiro, 1990; Schiewe et al., 1990). The ratio of number of cells being cultured per volume of medium has become a topic of interest for both oocytes and embryos and is best determined for an individual laboratory since many subtle factors affect this variable. In the case of the oocyte, the time required for maturation in vitro can actually be prolonged if not inhibited completely at too high a cell density (Leibfried-Rutledge et al., 1989a; Petr et al., 1989). Microdroplets of medium are often utilized as described by Bavister (1981a). This is very sparing of media and supplements. Depending on volume of culture media placed in containers, an oil overlay might be considered to prevent evaporation. The oil itself should

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be tested prior to use with critical material. Oil used for overlay is most often saturated with medium or saline. As described by Gordon (1994), the oil overlay reduces water evaporation when using small volumes of medium, provides some protection from microbial contamination, attenuates temperature and gas fluctuations and allows easier observation of cultures compared to other systems. The investigator should be aware that the oil overlay forms a two-phase separation system for hydrophobic substances. We observed this effect in some of our initial studies while supplementing steroids during oocyte maturation by adding a radiolabeled estrogen to our medium and counting the oil overlay after 24 hours. This has also been demonstrated by Miller and Pursel (1987). Since the oil is usually equilibrated with a water-based medium prior to use, the oil overlay will also allow diffusion of water-soluble compounds out of the medium. One can empirically demonstrate this for oneself by adding hyaluronidase to one of a pair of oil-overlaid microdrops containing oocytes having expanded cumulus. Culture treatments examining differences between concentrations of test substances should not be put in the same dishes. All medium used for culture, no matter what physical aspects are part of the culture system, should be equilibrated with the gas phase prior to adding the cells for culture. The first system used for in vitro maturation of oocytes from domestic species that resulted in the subsequent production of live born offspring utilized coculture of the oocyte-cumulus complex with granulosa cells in a system that was continually agitated (Staigmiller and Moor, 1984). Since there are benefits associated with the oocytes' intercellular associations (Fulka and Motlik, 1980; Staigmiller and Moor, 1984; Leibfried-Rutledge et al., 1989a; summarized by Gordon (1994) for domestic species), this is a system that might be contemplated for new species in lieu of much background knowledge concerning its tissue culture requirements for successful oocyte maturation. Again it must be cautioned that high numbers of granulosa cells can cause either a transient or complete maintenance of meiotic arrest during culture of the oocyte complex (Leibfried-Rutledge et al., 1989a; Sirard and Bilodeau, 1990; Hinrichs and Schmidt, 1993; Kalous et al., 1993; Grocholova et al., 1995). During maturation, during both growth and development of the oocyte and final maturation, cell-to-cell interactions play an important part in maintaining oocyte viability and competency (Moor, 1983; Schultz, 1986b; Larsen and Wert, 1988; Buccione et aL, 1990; Eppig, 1991b; Racowsky, 1991). Passage of metabolites and precursors are mediated by intercellular passage of substances from cumulus cell to oocytes, as are regulators of the meiotic process. Cytoplasmic maturation of the oocyte during final maturation also appears to involve cumulus cells in some fashion (Staigmiller and Moor, 1984; Leibfried-Rutledge et al., 1989a; Grocholova et al., 1995). It becomes apparent then that maturation of the oocyte cannot occur in isolation. Too high a density of complexes can

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change the kinetics of meiotic resumption, yet attempts to culture single complexes have also not been completely successful and gives lower frequencies of blastocyst production than pools of complexes. Addition of granulosa cells or additional cumulus cells may aid oocyte maturation when limited quantities of cells are available. Again, too many cells added as helper cells may impair the meiotic process so some prudence needs be exerted. Further research in the area of coculture may be one avenue capable of improving our ability to produce embryos in vitro. More recently, coculture systems have been developed using a variety of cell types which have been shown to have some potential beneficial effects on oocyte maturation and development (Danedaker et aL, 1991; Grocholova et al., 1995; Janssenswillen et al., 1995; Bongso, 1995). C. F o l l i o f l a r a n d O v a r i a n F a c t o r s A f f e c t i n g Oocyte Competency Primary oocytes found in antral follicles may not all be of equal competency to undergo maturation and normal fertilization and sustain development and pregnancy to result in a new individual of the species. Many factors such as growth stage of the follicular unit, age of animal used for donor and stage of the estrous or menstrual cycle may affect the status of the oocyte. One of the many potential uses of embryo production in vitro is to study the effect that these factors have on oocyte competency to learn more about the development of the oocyte and its enclosing follicle in respect to hormonal and environmental factors affecting fecundity. The following discussion will attempt to point out what is known about these factors at present to introduce some aspects that must be considered when trying to identify a source of material useful for tissue banking or embryo production. 1. O o c y t e C o m p e t e n c y

Primary oocytes in primordial follicles that have left the nongrowing pool are destined either for ovulation or in most cases, atresia (reviewed by Fortune, 1994). During their growth phase there is a stepwise acquisition of capabilities or competencies that eventually culminate in the ability to produce a full-term young of the species after the appropriate interaction with the paternal genotype (discussed by First et al., 1988). The first observable competency is the ability to resume meiosis I, evidenced by germinal vesicle breakdown, after release from the follicular environment. This has been shown to be a hormone-dependent event in rats (Bar-Ami and Tsafriri, 1981; Bar-Ami et al., 1983). Obviously, oocytes that are meiotically incompetent would not be useful for current systems dealing with maturation of oocytes to be used in producing embryos. Only in mice have culture systems been developed to support growth of oocytes in secondary follicles through

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to meiotic competence with birth of live offspring (Eppig and Schroeder, 1989). When we can do this for an increasing number of species, then more genetic material can be salvaged from female animals. In short-cycle species such as rodents, meiotic competence is acquired coincidentally with completion of growth and the beginning of antrum formation in the secondary follicle (Sybek, 1972; Erickson and Sorensen, 1974; Iwamatsu and Yanagimachi, 1975; Sorensen and Wassarman, 1976). In long-cycle domestic animals, acquisition of meiotic competence and completion of growth do not occur until after formation of the tertiary follicle and may not coincide (Tsafriri and Channing, 1975a; Moor and Trounson, 1977; Anderson and Hillensjo, 1982; Motlik et al., 1984; Motlik and Fulka, 1986). In humans (Tsuji et al., 1985; Carson et aL, 1995) and nonhuman primates (Lefevre et al., 1989; Schramm et aL, 1993), acquisition of meiotic competence and ability to complete meiosis I may not occur until preovulatory maturation, although ability to resume meiosis in the marmoset does appear at the time of antrum formation (Gilchrist et al., 1995). In only a few instances have live offspring been produced after in vitro maturation of primary oocytes from primates (Cha et al., 1991; Trounson et al., 1994b). Much still needs to be learned about oocyte development in primate species. Ability to resume meiosis is separate from the ability to complete meiosis I, that is to undergo the first reduction division with resulting formation of the metaphase II spindle and expulsion of the first polar body (Thibault, 1972; Tsafriri and Channing, 1975a; Sorensen and Wassarman, 1976; Bar-Ami and Tsafriri, 1981; Motlik et al., 1984), at which point the oocyte is considered a haploid despite the 2C chromatin amount. Abilities for normal fertilization, initial cleavage of the early embryo, development to blastocyst, and initiation and maintenance of pregnancy have all been shown to develop during growth and differentiation of the primary oocyte using various species as model systems (summarized by First et aL, 1988). Again, in only one species, the mouse, has a culture system been developed that allowed acquisition of competence by immature primary oocytes such that live offspring were produced after in vitro maturation and fertilization. In primates, competencies yet to be acquired after ability for meiotic resumption are probably not attained until relatively late in follicular development compared to other species (Westergaard et al., 1985; Schramm et aL, 1993). This probably accounts for current difficulties in using primary oocytes from antral follicles for embryo production (Schramm and Bavister, 1994). The stage of antral follicle development at which the enclosed oocytes are completely competent to undergo normal maturation, fertilization, and development is known for only a limited number of species. This aspect should be taken into account for each new model system utilized. If one attempts to go directly to assessing development after fertilizing cultured oocytes, it is difficult to assess where breakdown of the culture systems

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m. Lorraine Leibfried-Rutledge et al.

occurred if cleavage is not successful. Since in vitro production of embryos involves linking several different parts of the reproductive process in sequence and each is dependent of the previous part, assurance that each prior process has been completed successfully is essential before adding the next step. With maturation of oocytes often being the initial step in utilizing in vitro systems for embryo production, knowledge concerning the stage of development at which the oocyte is taken is crucial to further use of the donor genotype. 2. R e p r o d u c t i v e Status o f the Donor

Presence of antral follicles on the ovary, besides having unique species and strain or breed differences, is even within a species dependent on many factors including age of the animal, stage of the reproductive cycle, and reproductive status. This is ignoring the many exogenous factors affecting folliculogenesis and oogenesis such as climatic and nutritional effects. Depending on species, antral follicles may be found from the fetal stage through to the senescence of the female mammal (see previous reviews concerning oogenesis and folliculogenesis). Oocytes in antral follicles of fetal or prepuberal animals may not have acquired complete maturational competence, even if oocytes are taken from follicles equivalent in size to those used successfully as source of oocyte in adult animals (Eppig and Schroeder, 1989; Arlotto, 1994; Revel et al., 1995; Arlotto et al., 1996, Duby et al., 1996). Conditions endowing the oocyte with complete maturational and developmental competence may not be entirely in place even during the first few estrous cycles of postpuberal animals as seen in swine (Menino et al., 1989; Archibong et al., 1992; Koenig and Stormshak, 1993). If incompetent oocytes from more species could be cultured through to complete competence, then not only could more genetic material be salvaged, but it would broaden the material available from all ages of animals which would increase the numbers of donor animals available. This may be of value when concerned with populations with few individuals. In cattle that have commenced regular estrous cycles, oocytes taken from antral follicles have resulted in equivalent rates of embryo production after in vitro maturation regardless of stage of the cycle (Tan and Lu, 1990; Takagi et al., 1992; Arlotto et al., 1996). Oocytes possessing cumulus investment taken from antral follicles of all sizes and all stages of the estrous cycle are equally competent to undergo meiotic maturation and morphologically normal fertilization in vitro (Leibfried and First, 1979; Leibfried-Rutledge et al., 1985). In cattle, antral follicles are also found on ovaries of pregnant animals (Choudary et al., 1968) and these also appear quite capable of yielding oocytes that can be matured and used for embryo production. Follicular aspiration has been used to retrieve oocytes from antral follicles of aged cattle (Hasler et al., 1995) and horses (Carnevale and Ginther, 1995), and these oocytes yield pregnancies or offspring after

Tissue M a t u r a t i o n in Vivo a n d in Vitro

57

in vitro maturation although oocyte competence appears less than optimum

in aged females of long-lived species. At least in postpuberal cattle, antral follicles from all ages of donor and despite reproductive phase, appear able to enclose primary oocytes capable of undergoing meiotic maturation and yielding blastocysts after fertilization (see Gordon, 1994). Whether this will apply for many species has yet to be determined. Yet size of the antral follicle from which primary oocytes are taken does influence both the capacity and time to complete meiosis I in culture and also undergo early development (Bae and Foote, 1975; Tsafriri and Channing, 1975a; Moor and Trounson, 1977; Fukui and Sakuma, 1980; Gougeon and Testart, 1986; Motlik and Fulka, 1986; Sato et al., 1990; Tan and Lu, 1990; Morgan et aL, 1991; Anderson and Hillensjo, 1992; Eppig et al., 1992; Pavlok et al., 1992, 1993; Schramm et aL, 1993; Gordon, 1994; Lonergan et aL, 1994; Crozet et al., 1995; Arlotto et al., 1996). There remain many aspects determining follicular status of ovaries for which we have little information on how oocyte competence is affected. Factors such as nutritional state, season, stage of cycle, breed, reproductive status, age of animal, number of gestations, and genetics not only have the potential to affect numbers of follicles developing on an ovary but also potentially the competence of the enclosed oocytes. The in vitro technologies that will be discussed throughout this chapter will aid in increasing our knowledge concerning the influence of these many factors on growth, differentiation, and acquisition of the maternal developmental program by the mammalian oocyte. Until greater understanding of how these factors affect oocyte competency, the investigator should obtain as much information on the life history of the species and donors as possible prior to selecting the culture protocols to be used. 3. A g e - D e p e n d e n t P r o c e s s e s in Oocytes

Once the female germ cell has left the nongrowing pool and completed growth and development in the postpuberal animal, delay prior to ovulation or fertilization causes changes in the ability of the oocyte to undergo fertilization and/or development (reviewed by Austin, 1970; Thibault, 1971; Abramson, 1973; Mikamo and Hamaguchi, 1975; Szollosi, 1975; Edwards, 1986; Epel, 1990). Delays at these two points in oogenesis result in an aging oocyte with a compromised program for maternal development. Preovulatory aging is a problem in induced ovulators unless care is taken to avoid collecting such impaired material for further use. Postovulatory aging can occur in many species and also after in vitro maturation of competent oocytes from antral follicles. In fact, indications of aging after completion of meiosis I in vitro may occur quicker than in the in vivo situation (Longo, 1980; M. L. Leibfried-Rutledge, unpublished hamster data). Loss of ability to maintain pregnancy is one of the first manifestations of aging. Inability

58

M. Lorraine Leibfried-Rutledge et al.

to form blastocysts after fertilization (Figure 9A) or parthenogenetic activation (Figure 9B) is another lesion found early in the aging process. If aging is prolonged, abnormalities in cleavage, karyotypes, and finally loss of fertilizing capacity are encountered. The in vitro maturation system is an effective method for studying age-related phenomenon since the selected time points can be very precisely timed. As shown in Figures 9A and 9B, success in embryo development is also impaired if cattle oocytes are fertilized or activated too soon after the start of the maturation process (SuskoParrish et al., 1991, 1994). In mice, if secondary oocytes are fertilized soon after ovulation, activation also does not occur and the chromatin of pene-

100" % total fertilization

80

% initial cleavage

60 C G

o

ormal fertilization

40

~

20

0

% M-BI/cleaved % M-BI/total

8

1'2

;8

2'0

2'4

2'8

3'2

Oocyte Age (h)

100 -

B

~ :

% activated % pronuclear

80 = "~

60

u

40

m_

----II---

% cleavage

----o--

% blastocysts

2O 0

16

20

24 age

30

42

F I G U R E 9 Loss of developmental competence of bovine oocytes during aging. Oocytes matured in vitro were either subjected to IVF (A) or parthenogenetic activation (B) at various

times after the start of culture. Ability of the fertilized or parthenogenic oocyte to reach the blastocyst stage of developmental is reduced as the oocyte ages. (A. Susko-Parrish et al., 1991. Biol Repro. 44 (Suppl. 1), 156. B. Susko-Parrish et al., 1994. Dev. Biol. 166, 729.)

Tissue M a t u r a t i o n in Vivo a n d in Vitro

59

trating spermatozoa is converted into a metaphase configuration (Kubiak, 1989). Earliest observation of expulsion of the first polar body may not mean that the oocytes have finished cytoplasmic maturation (Kubiak, 1989; Dominko and First, 1992). In both mice and cattle, oocytes artificially activated too early are either not activated or resume and complete the second reduction division but then undergo a metaphase III arrest (Kubiak, 1989; Susko-Parrish et al., 1994). If successful interaction of spermatozoa and oocyte occur within a set window of time after completing the first reduction division, those attempting to utilize the secondary oocyte for IVF, whether resulting from in vivo or in vitro maturation, must be cognizant of the age of the material being used. Well defined age-dependent curves have been demonstrated for only a few species of mammals. Details of these curves may vary in response to a number of factors such as the type and concentrations of gonadotropins used in the culture system for IVM. Concentration of supplemental gonadotropin may give a transitory delay in the timing of commitment to maturation (citations given previously) and thus change the optimum timing for gamete interaction. Age of female from which the oocyte was taken may also affect the time required to complete meiotic maturation, again potentially skewing the age postculture at which optimum sperm/egg interaction can occur. It is possible that a number of factors concerning the donor animal or in vitro culture system may affect the aging curves but these factors have not been examined to any extent. For optimum results in the production of offspring, the kinetics of maturation should be defined for a system and age-dependent response curves generated if possible. As stated previously, embryo production requires success at each step of sequentially linked processes. To make changes in an early step such as maturation and subsequently evaluate only embryo development as an endpoint does not allow determination of what was affected. Age of the donor animal itself may effect the success of maturation and the developmental process (Gosden, 1985a,b,c). Early in the study of gametes it was difficult to partition the source of failure between aging of the female gamete and the consequences of aging on the reproductive tract in terms of ability to support oogenesis or pregnancy (Gosden and Faddy, 1994). Use of short-lived species in reciprocal transfer studies implied that the reproductive tract accounted for the majority of pregnancy failures in aged recipients (Edwards, 1980; Gosden, 1985a). More recent studies using long-lived species and clinical data would also indict the aging oocyte as defective in some fashion (Speirs et al., 1983; Waiters et al., 1985; Edwards, 1986; Carnevale and Ginther, 1995), whether due to errors in its acquired developmental program accumulated during the growth phase in an aging female or to acquired genetic defects during aging of the animal. Thus it must be recognized that senescence of the female gamete can occur at many points in its life history.

60

M. Lorraine Leibfried-Rutledge et al.

D. C u l t u r e Systems for Oocytes from E a r l i e r Stages

of Oogenesis Primary oocytes from antral follicles that have attained meiotic competence are most often used as the female contribution in the various in vitro technologies developed for assisted reproductive purposes. If constrained to this stage, much genetic material is lost to the gene pool of a species and the value of in vitro technologies to medicine, animal agriculture, or species preservation will often be limited. For example, marker assisted genetic selection of animals for high genetic merit is contemplated in domestic animal production (Bishop et al., 1995). If oocytes from a fetus or prepuberal animal could be used to produce embryos, an evaluation of potential value of the animal and shortening of the generation interval for selection purposes could be accomplished before the animal has reached maturity (Betteridge et al., 1989; Georges and Massey, 1991; Gosden, 1992; Lohuis, 1995). Although these sources provide meiotically competent primary oocytes, production of embryos following fertilization has a low rate of success if these oocytes are placed in current IVM systems as previously discussed. Some initial success has been made in culturing growing oocytes from earlier stages of oogenesis until complete maturational competence or meiotic competence has been acquired, or in systems that use other than the isolated cumulus-oocyte complex removed from its follicle environment (Teller, 1996). A discussion of these systems will follow. 1. C u l t u r e o f Whole A n t r a l Follicles

Culture of the entire tertiary follicle for limited periods of time has been accomplished in several mammalian species (Tsafriri et al., 1972; Rush et al., 1973; Lindner et al., 1974; Moor and Trounson, 1977; Kruip et al., 1979; Fukui et al., 1987; Yoshimura et al., 1990). Since the purpose of these studies was not usually to produce offspring, the subsequent maturational and developmental capacity of the enclosed oocytes were not always evaluated. Whole preovulatory follicles of rats have been placed in organ culture and after application of gonadotropin, the enclosed oocytes have been induced to mature as evaluated by appearance of the chromatin configuration (Tsafriri et al., 1972; Lindner et al., 1974). Live young were produced in sheep after in vitro maturation of oocytes in whole follicle cultures (Moor and Trounson, 1977). Whole tertiary follicles of cattle and sheep have also been cultured using a perfusion system (Peluso and Hirschel, 1987, 1988; Skyer et al., 1987; Kruip and Dieleman, 1989). Both histological evaluation of the follicle wall and secretion of steroids were used as indicators of health of the somatic components of this system. The ability to induce maturation of the oocytes using gonadotropin in these follicles from larger, long-cycling species may be fraught with some difficulties since it would only be in the largest follicles that the appropriate receptor status would

Tissue M a t u r a t i o n in Vivo a n d in Vitro

61

be present. Entire ovaries of smaller laboratory species and even humans have been placed in perfusion culture with subsequent oocyte growth, maturation, ovulation, and sometimes live young produced following IVF and transfer to a recipient (Stahler et al., 1974; Janson et al., 1982; Kobayashi et al., 1983; Brannstrom et al., 1987; Brannstrom and Janson, 1991; Brannstrom and Flaherty, 1995). Organ culture has also been used to culture oocytes from larger species on hemisections of the follicle wall (Foote and Thibault, 1969; Tsafriri and Channing, 1975b; Moor and Trounson, 1977; Leibfried and First, 1980; Sirard et al., 1992). In this situation, meiotic arrest has been maintained, or if gonadotropin was added to the culture medium, meiotic resumption and completion to metaphase II was induced. These methods would seem to be needlessly cumbersome considering the ease of placing meioticaUy competent oocytes from tertiary follicles into culture. Yet we still are not quite sure what causes successful maturation of these primary oocytes such that production of developmentally competent oocytes is reliably obtained. As mentioned before, this is still a "black box" phenomenon. The oocyte and somatic components of the follicle undergo continuous interaction throughout the growth and differentiation of the follicle unit. The strategy behind using whole follicles or sections of the follicle wall to obtain maturation of the enclosed oocyte is that maintenance of the intercellular associations would yield an oocyte in which cytoplasmic maturation more closely resembles the in vivo situation. Also in situations where the material available is severely limited, these culture situations may give a higher probability of producing usuable embryos. At least in domestic animals, attempts to produce IVP embryos using single oocytes have not been as successful as using groups of oocytes. Maintenance or reestablishment of the cellular associations between oocyte and somatic components may improve the situation. Potentially promising results might be obtained if explant, organ culture, or tissue perfusion methods were reevaluated in light of current needs. 2. Culture o f P r i m a r y Oocytes in P r e a n t r a l F o l l i c l e s

Only in mice have offspring been born after culturing primary oocytes enclosed in secondary follicles (Eppig and Schroeder, 1989). While meiotically incompetent at the start of culture, these oocytes were subsequently capable of meiotic maturation and, following fertilization, embryogenesis. Unfortunately this procedure has a low success rate at present. It must be remembered that the secondary follicle unit in vivo is in the growth stage of folliculogenesis and oogenesis which is a very dynamic phase in the life history of the follicle unit. The follicle cells themselves are undergoing proliferation and differentiation into steroid secreting entities while the oocyte is very actively involved in synthetic activities pertaining to storage of the maternal developmental program. Beginnings have been made in several species to find methods of isolating preantral secondary follicles

62

m. Lorraine Leibfried-Rutledge et al.

and maintaining them in culture (Roy and Greenwald, 1985, 1989; Daniel et al., 1989; Jewgenow and Pitra, 1991, 1993; Nayudu and Osborn, 1992; Eppig and Teller, 1993; Roy and Treacy, 1993; Eppig, 1994; Figueiredo et aL, 1994a,b; Hirao, 1994; Hulshof et al., 1995; Teller, 1996). Much work is

needed at present to define the requirements of these growing follicles in respect to both metabolic and hormonal needs. Particularly in the growing follicle units of domestic animal species and primates where oocyte competency may not be completely attained until the follicle unit has become tertiary, it could be that a series of culture systems may need to be developed in which the secondary follicle will be cultured sequentially to obtain the desired results. Development of systems to culture the oocyte in secondary follicles to maturity will not only allow a practical goal to be attained, but will also greatly impact our meager understanding of oogenesis and folliculogenesis during this period. Primary oocytes are also found in both primordial and primary follicles in the ovary of adult animals and in fetal ovaries of many species. The first represents the nongrowing follicular component of the female and the most abundant follicle unit at most all stages of the female mammal's life history once follicle formation has occurred. There is scant information concerning why these follicle units begin to grow in vivo let alone methods of getting these to complete folliculogenesis in vitro. Some information does exist concerning methods of isolation, while there is at present few reports of attempts to culture these oocytes to maturity (Eppig and Schroeder, 1989; Greenwald and Moor, 1989; Torrence et al., 1989; Eppig and Teller, 1993; Figueiredo et al., 1994a,b; Spears et aL, 1994; Cain et al., 1995). Attempting to replicate such a complex process in vitro as represented by the complete process of folliculogenesis appears on the surface to be a daunting feat, yet the abundance of primordial follicles in the mammalian ovary and the wealth of genetic material this represents should not deter this enterprise from being undertaken by more laboratories. Very recently live birth resuited from the complete development in vitro of murine primary oocytes cultured in primordial follicles (Eppig and O'Brien, 1996). This is very encouraging. 3. C u l t u r e o f S t a g e s P r i o r to t h e P r i m a r y O o c y t e

Primordial germ cells and oogonia are found only during fetal stages of most mammals, except for the latter which in some species still exist in the neonate before folliculogenesis has been completed. Isolation of primordial germ cells has been accomplished for a number of laboratory and domestic animals. This is potentially a very exciting source of genetic material for use in both cryopreservation and assisted reproductive technologies. These cells appear to be pluripotent if not totipotent since they can be used in deriving cell lines that act like embryonic stem cells (Matsui

Tissue M a t u r a t i o n in Vivo a n d in Vitro

63

et al., 1992; Resnik et al., 1992) and have also been used in nuclear transfer immediately upon isolation (Lesimple et al., 1987; Tsunoda et aL, 1989; Ledda et al., 1996; Strelchenko, 1996). As stem cell lines, this genetic source

could be cultured to obtain an unlimited number of cells (Stewart, 1991) which then could be incorporated into a reproducing generation by either chimerizing with an embryo in order to obtain germ-line incorporation which has been accomplished in mice (Labosky et aL, 1994; Stewart et al., 1994) or potentially by creating an embryo and offspring with the process of nuclear transfer which has been attempted, sometimes successfully, with passaged stem cell lines derived from preimplantation embryos (Tsunoda and Kato, 1993; Campbell et al., 1996; Strelchenko, 1996). Since stem cell lines appear to contribute only to lineages descended from the inner cell mass, new individuals could theoretically be generated by placing stem cells inside trophoblastic vesicles derived from a closely related species and transferring to the recipient species that donated the trophoblast tissue. Interspecies chimeras made from trophoblast and inner cell mass of related species have resulted in live offspring in a limited number of situations (Rossant and Frels, 1980; Rossant et al., 1982; Fehilly et al., 1984; MeineckeTillmann and Meinecke, 1984; Papaioannou and Ebert, 1986; Polzin et al., 1987; MacLaren et al., 1993). The use of stem cells for nuclear transfer procedures still has not been highly successful although recent advances make this application more promising. Nuclear transfer itself is a technique that has the potential to increase the number of individuals produced from gametes or embryos that have been banked (First, 1991; First and LeibfriedRutledge, 1993), although within small breeding populations, it will not contribute to maintenance of genetic diversity. Utilization of primordial germ cells either as a potential source of stem cells or in nuclear transfer adds two more in vitro technologies that expand the range of female genetic material that can be salvaged from mammals. These two novel technologies avoid the need to mimic the completion of oogenesis in culture and hence may allow earlier practical usage of this source of genetic material. Aside from the need to bring to maturity the processes of nuclear transfer and stem cell production for a variety of mammalian species, probably the major deterrent to using primordial germ cells at this point is our lack of information concerning the time of gestation at which this gametogenic stage is found for more mammalian species than are currently known. Reproductive patterns in regard to oogenesis have been well described for species with biomedical or production value. Application of the burgeoning repertoire of in vitro technologies to exotics and endangered species as a means of preservation will need a united effort to accumulate and evaluate necessary histological materials and pertinent references from older and more obscure publications that will allow elucidation of reproductive patterns in more detail for a wider variety of mammals.

64

m. Lorraine Leibfried-Rutledge et al.

III. MATURATION OF SPERMATOZOA Spermatozoa are produced by the testes of male mammals after the onset of puberty and, in animals that exhibit seasonality, during the breeding season. The major component of the mature testis, both structurally and functionally, are the seminiferous tubules. Mature seminiferous tubule epithelium, the site of spermatogenesis, is composed of Sertoli cells, progenitor spermatogonia, and germ cells proper (Setchell, 1977; Setchell et al., 1994). Seminiferous tubules lie within testes as convoluted tubules separated by interstitial tissue comprised primarily of blood and lymph vessels and islands of Leydig cell. Leydig cells are LH-stimulated cells and are the primary androgen producing cells in the testis. Seminiferous tubules are not freely accessible to the circulatory system. A blood-testis barrier formed by tight junctions between the Sertoli cells markedly restricts permeability (de Kretser and Kerr, 1994), much as the follicle cells and differentiated granulosa cells do in the follicles of the ovary. Sertoli cells are responsive to both FSH from the pituitary and testosterone from Leydig cells. Androgens are essential for most stages of spermatogenesis. To sequester testosterone from Leydig cells, Sertoli cells produce androgen-binding protein which has high affinity for both testosterone and its reduced product, dihydrotestosterone. There are separate, structurally unique, androgen receptors in these cells which bind testosterone. Binding of testosterone and its reduction (by 5-a-reductase) is a prerequisite for biological action for all androgen-induced response. In addition to production of androgen-binding protein and androgen receptors, which have intracellular roles to play. Sertoli cells also synthesize and secrete a protein hormone called inhibin. Inhibin has systemic effects primarily at the level of the pituitary where it provides negative feedback, lowering, specifically, the circulating concentration of FSH. Once sexual differentiation has occurred in the fetus, the morphology of the testis remains little altered until prepuberal changes necessary for the future release of mature gametes has begun (White, 1974; Johnson, 1991). The undifferentiated germ cells or primitive spermatogonia, which have occupied the interior of the rudimentary seminiferous tubules since birth, now migrate to line the tubules in which a lumen soon begins to form. This is shown in Figure 10 along with a schematic diagram representing the stages of spermatogenesis. Primitive spermatogonia differentiate into stem cell or As spermatogonia which undergo a multiplicative phase followed by meiosis and spermiogenesis to yield mature spermatozoa. Primitive spermatogonia also give rise to reserve or Ao spermatogonia, often referred to as dormant spermatogonia, which will continue to proliferate into both Ao and As gametogenic forms during the male's reproductive lifetime and hence ensure the continuation of spermatogenesis after sexual maturity is reached in the male. The Ao is a slow dividing cell that may be kept in

65

Tissue Maturation in Vivo a n d in Vitro

~ ......~

. '

, ~

Indifferent cell

....

5perma-

t

9

~ '~,

b

u

l

~

~

B

i~.[~,'~'.'-[.,~i~'. .' ':

opermatms ,..~.:~:-._~~~ ~ '.~,.: ,:''" . 9 :.,. Secondary s p e r m a t o c y t e~.,r --..~~~~::~r

J. ~h~,~';~~.'.:::r,~.... ~ l ; ~ "Secondar ',:,: '~ 9 y sp'ermatocyte -.,,m. ~~.-a

.Primary s per matoc y t e . . . ~ b ~ ~ ~ : ~ Ili~~. ~"~',

u

'i

~i~?'~ .'

C~

;e~l

~

ermawgomum C

FIGURE 10 Generalized diagram of spermatogenesis as it occurs in the seminiferous tubules of the mammalian testes. Cross-sections of a seminiferous tubule prior to (A) and after puberty (B) are shown. A section of the tubule illustrating the stages of spermatogenesis is also depicted (C). (Arey, 1954.In Developmental Anatomy, W. B. Saunders Co., Philadelphia.)

reserve to repopulate the testis after massage damage or as a means of maintaining normal sperm production (Dym, 1983). As spermatogonia undergo a set number of mitotic divisions to yield primary spermatocytes which begin the meiotic process (Courot et al., 1970; Ortavant et al., 1977). There are still many questions to be answered concerning the differentiation of the spermatogonial lineages and which form actually leads to stem cell renewal in the testis (Dym, 1983; De Rooij et al., 1989; Meistrich and van Beek, 1993). After D N A replication and the early events of meiosis have taken place, the first meiotic division occurs, producing a cohort of secondary spermatocytes. These rapidly undergo the second reduction division yielding spermatids. Prior to this time these differentiating germ cells have been developing in cellular associations characterized by the presence of cytoplasmic bridges between lineal descendants of earlier spermatogonial stages (Burgos et al., 1970). Formation of the spermatid sees the disappearance of this specialized means of intercellular communication and also the beginning of spermiogenesis. It may be noted that while cohorts of oogonia in the female mammal are also joined by cytoplasmic bridges (Zamboni

66

m. Lorraine Leibfried-Rutledge et al.

and Gondos, 1968), this means of cellular communication is terminated shortly after the beginning of meiosis when folliculogenesis first becomes apparent (Ohno and Smith, 1964), usually during fetal existence or shortly after birth in a some species. Development of spermatozoa does not occur continuously along the entire length of the seminiferous tubules. Only certain developmental stages occur together in time and space, creating stages of maturity (Clermont, 1963, 1972). This process is less distinct in humans (Heller and Clermont, 1964) than in nonhuman species. This cycle of the seminiferous epithelium is composed of various numbers of stages in different species. In the rat, there are 14 stages. In man, there are 6 stages. The entire time course for the formation of mature human spermatozoa from spermatogonia requires four complete cycles of the seminiferous epithelium and takes between 64 and 80 days and constitutes one spermatogenic cycle (Table 2). The metamorphosis of spermatids into mature spermatozoa involves a very dramatic remodeling of the cellular morphology from a large, rounded form into a motile cell with well defined head and tail regions (Afzelium, 1972; Adelman and Cahil, 1989; Bearer and Friend, 1990; Dadoune, 1994) as illustrated in Figure 11. The complex process of spermiogenesis (Leblond and Clermont, 1952; Clermont, 1963; DeRooij et al., 1989) involves loss of a majority of the cytoplasm and its components such as RNA, endoplasmic reticulum, and ribosomes which severely limits the sperm's synthetic capabilities (Hecht, 1986). Formation of the acrosome from the Golgi apparatus, repackaging and condensation of the sperm DNA, and rearrangement of cell organelles such as mitochondria and centrioles with formation of tail filaments are also components of spermiogenesis (Bellve and O'Brien, 1983). After this extensive remodeling has been chiefly completed, the spermatozoa are finally shed into the lumen of the seminiferous tubules. Cell forms at each successive stage of spermatogenesis have been pushed toward the lumen until release occurs from the surrounding Sertoli cells with which they have been associated during differentiation. In brief, the entire development of the spermatogonia to fully morphologically differentiated spermatozoa and their subsequent release into the lumen of seminiferous tubules involves the processes of spermatogenesis, spermiogenesis, and spermiation. Spermatogenesis involves the production of haploid gametes from diploid progenitor cells through the process of meiosis. Cellular differentiation, proliferation of germ cells, and renewal of the progenitor cells are all part of spermatogenesis. Spermiogenesis is the process of morphological change from an essentially round cell type (round spermatid) to a flagellated cell with greatly reduced cytoplasmic volume (spermatozoa). Finally, the morphologically modified spermatozoa are released from the surrounding Sertoli cells into the lumen of the seminiferous tubules in a process termed spermiation.

Tissue Maturation in Vivo and in Vitro

67

MA/1JRATION PHASE

Fi

sheath

Mitochondrion

Acrosome1 Nucleus.

hondri

/~

Perforatorium or "rod"

11 A diagram of spermiogenesis in rat spermatids. Metamorphosis of the round spermatid begins in the testis and is completed during passage through more proximal regions of the epididymis. (Clermont, 1967. Archs Anat Micr. Morph. Exp. 56, 7.)

FIGURE

Spermatozoa are carried out of the testes and into the epididymis by fluid elaborated by Sertoli cells (Martan, 1969; Setchell et al., 1969; Waites and Setchell, 1969; Setchell, 1970, 1977) until they reach the cauda epididymis of this accessory reproductive organ where they are stored for a period of time prior to ejaculation. Both peristaltic contractions and cilliary movement by epithelial cells lining the male ductile system aid in movement of

68

M. Lorraine Leibfried-Rutledge et al.

Duration of Spermatogenic Cycles and Spermatogenesis in Some Mammals a

TABLE 2

Species

Duration of spermatogenic cycle (days)

Duration of spermatogenesis (days)

Boar

8.6

34.4

Cattle

13.5

49-54

Dog Human

13.6 16

54.4 64-80

Mouse

8.6

35

Rat

13

48-53

Sheep Stallion

10.5 12.2

49 49

a

Reference

Swierstra et al. (1974) Swierstra (1968) Berndtson and Desjardins (1974) Ortavant (1956) Foote et al. (1972) Clermont (1963) Heller and Clermont (1964) Schulze and Rehder (1984) Oakberg (1956) Clermont (1969) Leblond and Clermont (1952) Perey et al. (1961) Clermont and Harvey (1965) Huckins (1965) van Beek and Meistrich (1990) Clermont and Morgentaler (1955) Steinberger (1962) Clermont (1972) Clermont (1972) Amann (1981) Swierstra et al. (1974)

Modified from Sharpe (1994).

the spermatozoa. Final maturation and the capacity for fertility and motility are acquired during the passage from testes to cauda epididymis (OrgebinCrist, 1969; Bedford, 1975, 1979; Amann, 1987; Hammerstedt and Parks, 1987; Amann e t al., 1993). Spermatozoa complete maturation and acquire motility and the capacity to participate in fertilization during their transit through the epididymis. Table 3 shows the time interval required for sperm from some mammalian species to pass through the epididymis. During passage through the epididymis, the structure of the spermatozoa matures. Formation of the spermatozoal acrosome is completed (Bedford, 1963; Fawcett and Phillips, 1969; Jones, 1971). Cytoplasmic droplets retained on the spermatozoa from the processes of spermiogenesis and spermiation move posteriorly and detach (Hansel and McEntee, 1970). Subsequently, sperm are stored in the cauda epididymis. During this storage phase, if the frequency of ejaculation is low, the epididymis serves to eliminate "old" spermatozoa by phagocytosis and reabsorption. The electrolyte compositions of epididymal fluid from several species are compared in Table 4.

Tissue Maturation in Vivo and in Vitro

69

The Time Interval for Passage of Spermatozoa through the Epididymis in Some Mammalian Species

TABLE 3

Time of passage through the epididymis (days)

Species

Boar

9-14

Bull

8-11

Guinea Pig

14-25

Human Rabbit

12 7-10

Ram

14-20

Rat

12-22

Reference

Swierstra (1968) Hansel and McEntee (1970) Swierstra and Foote (1965) Koefoed-Johnson (1966) Swierstra (1968) Hansel and McEntee (1970) Koefoed-Johnson (1960) Swierstra and Foote (1965) Clubb (1951) Toothill and Yoning (1931) Rowley et al. (1970) Orgebin-Crist (1965) Amann et al. (1965) Swierstra (1968) Hansel and McEntee (1970) Amir and Ortavant (1968) Clubb (1951) Macmillan and Harrison (1955) Orgebin-Crist (1969)

N o r m a l m o r p h o l o g y of m a t u r e s p e r m a t o z o a varies dramatically f r o m species to species. G e o m e t r i c d i m e n s i o n s of a s p e r m a t o z o o n are e n o r m o u s l y i m p o r t a n t to successful c r y o p r e s e r v a t i o n , as e x p l a i n e d in C h a p t e r 6. Variations b e t w e e n s p e r m a t o z o a in an ejaculate m a y result f r o m inclusion of i m m a t u r e s p e r m a t o z o a , defective s p e r m a t o z o a l m a t u r a t i o n , or a b n o r m a l i ties of the m a l e r e p r o d u c t i v e tract.

TABLE 4

Characteristics of the Epididymal Fluid of Some Mammalian Species

Species

Sodium (mM)

Potassium (mM)

Calcium (mEq/liter)

pH

Osmolality (mOsm)

Boar Bull Dog Ram Rat Stallion

30 34 22 40 26 41

35 26 38 29 47 32

3.161-6 0.443,4,7 1.711 1.21,3,8 0.361,1~ 1.11

6.92 6.83 6.71 6.91 6.81 6.81

3261,2 3557 3171 3251,3 3321,9,1~ 3341

Note. Reference notations: 1, Jones (1978); 2, Einarsson (1971); 3, White (1973); 4, Crabo (1965); 5, Mann (1964); 6, Mann (1959); 7, Salisbury and Cragle (1956); 8, Quinn et al. (1965); 9, Levine and Marsh (1971); 10, Jenkins et al. (1980).

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More detailed descriptions of spermatogenesis and the various stages of differentiation of the male gamete may be found in basic reproductive textbooks or in some hallmark reviews (Salisbury and VanDemark, 1961; Parkes, 1962; Roosen-Runge, 1962; Hartman, 1963; Leblond et al., 1963; McKerns, 1969; Johnson et al., 1970; Hafez, 1974, Cole and Cupps, 1977; De Rooij et al., 1989; Dunbar and O'Rand, 1991; Bellve and O'Brien, 1983; Maul, 1989; Desjardins and Ewing, 1993; Spiter-Grech and Nieschlag, 1993; Eddy and O'Brien, 1994; Kierszenbaum, 1994; McLachlan et al., 1995). While a brief summary of this process is necessary for understanding the rest of this section, it is recommended that those investigators contemplating cryopreservation of male gametes become familar with all stages of differentiation of the spermatogenic process in order to take best advantage of various strategies for tissue banking and preservation of paternal genotypes. The process of spermatogenesis is designed to create a cell that can interact with the female gamete and participate in sexual reproduction. The male accessory reproductive tract and its secretions are specialized to maintain viability and final maturation and to aid in delivery of spermatozoa for their assignation with the oocyte (Kierszenbaum, 1994). Unique features of sperm physiology, metabolism, structure, and motility along with the specialized secretions of the male reproductive system in which they reside allow spermatozoa to fulfill its role in sexual reproduction. The roles of cells lining the reproductive tracts of both sexes as gametes and embryos pass through these organs and the fluids they secrete are vast. It has import for development of the tissue culture systems designed for gametogenic cells at various stages of differentiation. Although discussion of these factors is beyond the scope of this chapter, literature study concerning the in vivo environment of individual cell types and stages should be made in order to more fully understand the function and requirements of the cells intended for use in tissue culture or banking. The primary accessory glands of the male reproductive tract in mammals are: (1) the ampulla of the vas deferens, (2) the seminal vesicles, (3) the prostate, and (4) the bulbourethral glands. Not all of these glands are present in all mammalian species and some species have other or additional glands (Price and Williams-Ashman, 1961; Setchell et al., 1994). Fluids from these various glands combine to produce the composite seminal fluid that varies markedly from species to species. The composition of the fluids from these glands produced by several species has been reviewed (Price and Williams-Ashman, 1961; White and MacLeod, 1963; Mann, 1964; Quinn et al., 1965; Einarsson, 1971; Cragle et al., 1972; Sikorski, 1978; Mann and Lutwak-Mann, 1981; Brooks, 1990; Follas and Critser, 1992; Setchell et al., 1994). The contribution of each of these tissues to seminal plasma is highly variable. For example, fructose, the major glycolizable substrate for sperm, may have its primary source from either the seminal vesicles (man,

Tissue M a t u r a t i o n in V i v o a n d i n Vitro

71

mouse, ram, bull) or the coagulating glands (rat), while in the elephant, fructose appears to originate in both the ampulla and the seminal vesicles (Short et al., 1967). In still other species (dog) fructose is absent from the seminal plasma altogether (Hansel and McEntee, 1970). At the time of ejaculation, spermatozoa from the vas deferentia and the epididymides are mixed with the fluid secretions of the male reproductive tract to produce semen. The seminal plasma is the fluid, noncellular component of the semen. In the final ejaculate the seminal plasma is composed of secretions from the testes, epididymides, vas deferentia, and the accessory glands. During seminal emission, these fluid components are released in a specific temporal order so that the fluid from some accessory glands are released earlier and others later in the ejaculatory process. For example, in the human, prostatic fluid constitutes the first fraction of the ejaculate proper and contains the majority of the spermatozoa. Although it is common to assume that the spermatozoa exist in the "complete, mixed" seminal plasma and much effort has been directed toward defining the composition of the seminal plasma from many species, the sperm may not, in all situation, be exposed to this milieu. In the case where seminal deposition is directly into the female reproductive tract, the components from the various accessory glands may not completely mix; there fore the spermatozoa may not always be exposed to the chemical composition of the complete, mixed seminal plasma. In the special case where semen samples are collected using electroejaculation, the process of emission is stimulated in a manner which is unlikely to replicate the normal series of events which occur during normal ejaculation and the accessory glands may be stimulated in a different order or stimulated in a manner which results in abnormal amounts of fluid being released (Setchell et al., 1994). While issues related to "mixing" of the seminal plasma component in vivo or of the normalcy of seminal plasma composition resulting from electroejaculation are important and must be considered, in general it is reasonable and helpful to suppose that the normal environment for the spermatozoa at the time of ejaculation is the biochemical environment defined as the complete seminal plasma. The most commonly used spermatogenic stage for cryopreservation and in vitro technologies is the mature spermatozoon. For many mammalian species examined to date, this stage is both relatively abundant and has ease and economy of procurement to recommend it (Watson, 1978). Whether obtained as an ejaculate or from the caudal region of the epididymis, it has the advantage of being "ready to use" for either artificial insemination (AI), IVF, or cryopreservation. In mammalian species, mature ejaculated spermatozoa are motile but are not capable of fertilization (Bedford, 1983). In order to fertilize an oocyte, mammalian spermatozoa must undergo a sequential series of physiological changes known as capacitation (Austin, 1951; Chang, 1951; reviewed by Austin, 1975; Rogers, 1978; Bedford, 1983; Parrish, 1989; Yanagi-

72

m. Lorraine Leibfried-Rutledge et al.

machi, 1990; Florman and Babcock, 1990; Zaneveld et al., 1991). These changes include alterations in the plasma membrane (Bedford, 1983), hyperactivation of motility (Yanagimachi, 1970), and finally the acrosome reaction (Barros et aL, 1967; Fraser, 1987). Capacitation allows sperm to penetrate the egg investments and fuse with the oolemna of the oocyte. As discussed later, there are ways of circumventing this requirement using assisted reproductive strategies. Capacitation normally occurs as the sperm are transported through the female reproductive tract after mating or AI (Yanagimachi, 1981; Moore and Bedford, 1983; Katz et aL, 1989). Epithelial cells lining the reproductive tract or components of their secretions play a role in inducing capacitation in vivo (Parrish, 1989). Although sperm must transverse the entire reproductive tract after mating to effect sperm-egg union, high rates of fertilization can be obtained after deposition of sperm into the oviduct via surgery or laparoscopy. This implies that the oviduct by itself is competent to capacitate sperm (Austin and Short, 1982). The series of cellular and molecular changes comprising the mechanism(s) of capacitation have not been completely defined, nor have definitive roles for the various organs of the female reproductive tract, but accumulation of research findings (Parrish, 1991) indicates that there are multiple ways of inducing capacitation in vitro and perhaps in vivo. There is no universal protocol most effective in inducing capacitation and this needs be kept in mind when working with an individual species. Uses of mature spermatozoa in tissue banking to preserve the paternal genotype will be directly as frozen semen or via creation of embryos that can be frozen. The latter requires recovery of embryos from the female reproductive tract after natural mating or AI or production of embryos in the laboratory. Paternal genotypes cryopreserved as mature spermatozoa must eventually be thawed and also used in embryo production if expression of this genotype is desired. The succeeding discussion will therefore concentrate on in vitro strategies for utilizing mature spermatozoa to produce embryos and factors affecting sperm that may alter the success of this process. In this section of this chapter as elsewhere, discussion of cryopreservation protocols will be avoided since there are many excellent chapters in the book covering this topic for all stages of gametes and embryos for which in vivo and in vitro tissue maturation are detailed. A. S o u r c e s o f M a t u r e S p e r m a t o z o a Mature spermatozoa are found in both the cauda epididymis and the ejaculated semen (Amann et al., 1993). Examples of mature spermatozoa of several species taken from these two sources are shown in Figure 12. Notice the differences in architecture of the head region and length of the cell between species. Sperm from both cauda epididymis and ejaculate are

Tissue M a t u r a t i o n in Vivo a n d in Vitro

73

Mature spermatozoa from several species. All sperm were stained to reveal the acrosomal region (Bryan and Akruk (1977). Stain Technol. 52, 47) which appears pink or more reddish depending on species. Ejaculated spermatozoa from bull (A), stallion (B), and crane (D) semen are shown along with spermatozoa obtained from the cauda epididymis of a hamster (C). The bull spermatozoon indicated in (A) has lost its acrosome as indicated by the absence of staining over the head region.

FIGURE 12

capable of undergoing fertilization with the ensuing production of embryos that result in live-born offspring (epididymal sperm: Toyoda and Chang, 1974; Schroeder and Eppig, 1984; fresh ejaculated sperm: Edwards and Steptoe, 1978; Edwards et al., 1980; Bavister et al., 1984; Pavlok et al., 1989; frozen-thawed ejaculated sperm: Lu et al., 1987; Utsumi et al., 1991; Johnston et al., 1994). Sperm from both these sources can also be successfully cryopreserved for later use in AI or IVF. Of course the most frequent method used for obtaining spermatozoa is that of collecting ejaculates. This cannot be done for all species and focusing on just collection of ejaculates limits the salvage of paternal genetic material in case of unforeseen death of an animal, particularly of exotics or endangered species where expansion of the gene pool needs to be considered whenever possible. As in obtaining female genetic material, factors such as species, availability of male animals, value of the donor animal, and circumstances will all dictate method of collection and whether material obtained will be banked or used directly for producing embryos prior to cryopreservation. Keep in mind that the four divisions of mammals used here and elsewhere are merely for convenience and not due to surmised similarities of their reproductive material or its behavior. In fact the first three divisions (laboratory, domestic, and primate) probably represent potential models for approaching new species

74

m. Lorraine Leibfried-Rutledge et al.

in the last one (exotics and endangered species) since there is much accumulated material concerning the former groupings while efforts are ongoing to apply tissue banking and in vitro technologies to the latter. These divisions represent "use" classifications more than any other factor. As much variability in reproductive patterns exists within these categories as between them. Once the sperm suspension has been obtained, there are numerous established methods available for evaluating its characteristics given in standard references concerning semen evaluation for AI or cryopreservation (Gomes, 1977; Zaneveld and Polakoski, 1977; Chenoweth and Ball, 1980; Keel, 1990; Mortimer, 1990a; WHO, 1992; Mortimer, 1994). Depending on the use to which the specimen will be put, one or more semen characteristics such as appearance, volume, live-dead forms, motility, morphology, or sperm concentration may be evaluated after collection. This sets a baseline for the specimen and later comparison with post-thaw evaluation. Determination of cell concentration, at least, should be performed to most effectively make use of the material. This can be done either using a hemocytometer (Absher, 1973; Bearden and Fuquay, 1980; Mortimer, 1990b) or using photometric methods to evaluate optical density (Foote, 1974; Bearden and Fuquay, 1980; Davis and Katz, 1992).

1. Laboratory Species Spermatozoa are obtained from the most common smaller laboratory species such as rodents postmortem (Hafez, 1970; Oliphant and Eng, 1981). The caudal region of the epididymis is dissected free then the afferent and efferent regions are closed off. This is conveniently done with the use of a hemostat due to the small size of the tissue and the manner in which this region bends upon itself. The cauda is either punctured or cut in multiple areas and the contents exuded into a sperm extending medium or into a "mass" under oil. The mass can later be diluted into buffer or other medium designed for holding sperm. Retrograde flushing of the epididymides from rabbits and larger animals via the ductus deferens may be done to collect epididymal spermatozoa. In most smaller laboratory species it is extremely difficult to obtain ejaculated semen. Although electroejaculation can be done (Scott and Dziuk, 1959), the animal then must be killed since semen components left in the urethra coagulate causing later death of the animal due to urethral blockage and ensuing toxemia. The semen collected in this manner may also coagulate, negating any further use of the spermatozoa as discussed in Oliphant and Eng (1981). Seminal vesicles may be removed surgically prior to using electroejaculation in laboratory species to prevent this occurrence and death of the animal (Hafez, 1970). Ejaculated semen is usually collected from the larger laboratory animals as source of mature spermatozoa with the aid of an artificial vagina and the use of teaser animals or electroejaculation protocols. Ejaculated semen

Tissue M a t u r a t i o n in Vivo a n d in Vitro

75

is obtained from rabbits with the aid of an artificial vagina (AV; Oliphant and Eng, 1981) using either a doe or another buck as a teaser animal. Once a male has become trained to this procedure, it is a simple matter to routinely collect semen. Electroejaculation can also be performed successfully in this species. When using an AV to collect ejaculated semen, aspects of temperature, sanitation, ejaculate characteristics, and handling of the animal appropriate for the species must be considered. For common laboratory species used extensively in research, the methods are well documented (see Hafez, 1970). Ejaculated semen can be collected from both cats and dogs by electroejaculation, by using teaser animals or in the case of dogs, digital manipulation (Hafez, 1970; Platz and Seager, 1978; Oliphant and Eng, 1981; Concannon, 1991). Regard for the safety and comfort of the subjects and safety of the handlers should be considered when performing these procedures for all species. 2. D o m e s t i c A n i m a l s

Mature spermatozoa from domestic animals are normally obtained from ejaculated semen. Semen collection for a number of farm animals was reviewed by First (1971; see also Salisbury and VanDemark, 1961; Pickett, 1968; Gomes, 1977; Almquist, 1978; Ball, 1978; Hafez, 1974; Bearden and Fuquay, 1980; Evans and Maxwell, 1987). With the aid of an AV, semen can be obtained using a teaser animal to stimulate an ejaculation, after training to a dummy or after electroejaculation. The first two methods are recommended to ensure a high quality sample. Not all animals respond well to these methods so the latter procedure must be used. Electroejaculation can also be utilized to collect animals that due to health or other reasons cannot mount a teaser animal or a dummy (Martin, 1978). Electroejaculators can be bought that run off a variety of voltage sources. Rectal probes are designed for specific species and must be taken into consideration. Some species may be easily collected in this manner while in others some form of anesthesia should be considered prior to starting the process. Electroejaculation must be used cautiously as a means of semen collection since it can cause discomfort, and in some cases such as the stallion, death, so its use should be kept to a minimum. Collection of semen by any of these methods should be done after consulting experienced technicians to prevent injuries both to the animals and to the handlers. This will ensure that specimens will be of high quality and future usefulness. During ejaculation, some components of semen are added by organs of the accessory reproductive tract such as the prostate, seminal vesicles, bulbourethral glands, Cowper's glands, and ampullae. In some species such as the boar, the ejaculate may consist of several fractions. Some of these fractions may include a gel-like material which forms a plug in the female reproductive tract after mating. Anyone who has checked for the copulation plug in rodents to determine if mating has occurred will be familiar with this

76

m. Lorraine Leibfried-Rutledge et al.

phenomenon. Collecting semen through a filter or only catching the spermrich fraction avoids retaining this unwanted fraction in swine. Cleanliness and sterility of procedures and equipment are important issues both for potential use of semen in in vitro technologies and for prevention of disease transmission. The basic references previously cited concerning collection of semen discuss cleaning of both donor and equipment. Record keeping and health certification are other topics that should be researched for the species and setting for utilization of the semen obtained. Bulletins of AI and stud associations, zoological societies, and andrology societies should be consulted. If transport of semen is foreseen, either for import or export, regulations of the country chosen as destination must be carefully consulted. This is true for any living tissue being shipped. It is easy to obtain mature spermatozoa from the cauda epididymis of domestic animals. This method is rarely used due to the prevalence of methods for collecting ejaculated semen or due to the possibility of obtaining frozen semen. Under some conditions it may not be possible to conveniently keep mature males around. There are also cases where the freezing of semen has not been highly successful for a particular species, or the frozen semen is not readily accessible. In these cases the required portions of the male reproductive tract can be obtained from an abattoir. There is also the possibility that sperm can be taken from the cauda epididymis and used for tissue banking after death of an animal. Animal production currently involves such species as bison, deer, llamas, and other species normally considered as exotics. It may be worthwhile to salvage sperm for tissue banking from these animals upon their death since the total breeding population is already limited. To do this the entire cauda epididymis is dissected free, making sure that some of the associated corpus epididymis and a good length of the ductus deferens remain intact. The proximal end of the cauda is closed off. A small diameter needle with hypodermic syringe attached containing an extension buffer or other sperm holding media is inserted into the lumen of the ductus deferens. Once the needle is in place in the duct, it should be clamped so it does not slide out of the lumen. Fluid is gently forced from the syringe until the tubules are seen to swell in the cauda. An incision is made in the proximal caudal region of the epididymis while holding over a collection vessel. As fluid exudes, more sperm buffer can be flushed through to empty the contents of the epididymis. The resulting sperm suspension can be used in tissue banking or for in vitro technologies. 3. P r i m a t e s

Semen collection for nonhuman primates is most typically accomplished via penile or rectal electroejaculation (Lanzendorf et al., 1990; Gould and Mann, 1988) although there has also been some success with the use of an artificial vagina. A variety of techniques have been employed (Holt, 1986;

Tissue M a t u r a t i o n in Vivo a n d in Vitro

77

Gould and Mann, 1988) which may vary somewhat for individual species and typically require the expertise of a well trained, experienced handler. Semen collection for humans is most frequently by masturbation. A variety of materials are toxic to sperm, and care is required in selection collection vessels which support sustained sperm motility. The use of latex condoms (Jones et al., 1986) and lubricants (Goldenberg and White, 1975; Kaye et al., 1991) should be avoided when possible, since these products have also been shown to be detrimental to sperm function. The use of silastic condoms may be less detrimental to sperm (Zavos, 1985). Other methods of sperm collection have been employed for patients with ejaculatory dysfunction. For patients with retrograde ejaculation, bladder catheterization can be performed (Davis et al., 1993) to recover sperm. The viability of function of such sperm is often suboptimal. Electroejaculation can be employed to collect semen from individuals with spinal cord injury (Denil et al., 1992) and microsurgical epididymal sperm aspiration (MESA) has been used extensively to recover sperm from the epididymis of individuals with obstructive azoospermia (Temple-Smith et al., 1985; Tourney et al., 1994). Fine needle biopsy has also been used to recover sperm directly from the testis (Mallidis and Baker, 1994).

4. Exotics and Endangered Species The individual species will be of consideration when deciding which approach to take to collect mature spermatozoa from this grouping of animals. Death or sacrifice of an animal always allows flushing of the epididymis to obtain the paternal genotype. Viable cells can be collected for a surprising period of time after death. Sperm from exotics held in zoological parks should always be salvaged in this fashion since it is desirable to maintain as large a pool of genotypes as possible for future breeding purposes. Although in vitro technologies would probably make the best use of this resource, deposition of frozen-thawed semen at the tip of the horn or into the oviduct are two methods of AI that reduce the required number of sperm for a fertile mating and make this resource go further. Laparoscope-assisted AI using frozen semen has been successful in small domestic and exotic ungulates (Asher et aL, 1990; Wildt et aL, 1992b). As mentioned previously, both fertilization and production of offspring is possible using epididymal semen. It is advisable to verify cause of death of the male so future use of semen collected will not be vector for disease transmission. This applies to all methods of semen collection, since sanitation and health certification should always be taken into consideration. Training of exotics and rare specimens of their kind to teaser animals or dummies is usually not possible with animals that are infrequently handled. To employ valuable specimens as teaser animals would also expose both subjects to too much risk. For most male animals found in zoological parks, electroejaculation combined with anesthesia is probably the only

78

M. Lorraine Leibfried-Rutledge et al.

useful method of semen collection. This should only be considered under the direction of a veterinarian experienced in working with exotic species. B. C u l t u r e S y s t e m s f o r M a t u r e

Spermatozoa

There are three basic uses of culture systems when applied to mature spermatozoa. The first involves the diluents, buffers, and semen extenders used in both short- and long-term storage of male gametes. Since other chapters in this book address this topic as well as a wealth of previously published citations, it will not be examined any further. The second reason for designing media to be used with sperm is to induce capacitation. Since capacitation is required before sperm can penetrate oocytes, this aspect is normally a consideration when using spermatozoa in many in vitro systems. The last use of culture media as applied to sperm, is to allow sperm/egg interaction to take place. Although the latter two uses of sperm culture systems are often combined, this is not always so. Many similar factors come into play when selecting media to be used with a particular species of spermatozoa as discussed under culture systems for oocytes. Osmolarity, pH, gaseous atmosphere, energy substrates, macromolecular substrates, ionic components, culture conditions, etc., must all be taken into account for each new species. In essence, conditions in culture that stimulate the activity and prolong viability of spermatozoa as long as possible must be established. Factors inducing capacitation may then be applied with hopes of successful interaction with oocytes. The reader again is reminded that the sperm is a highly differentiated cell type with aspects that are often unique from routine cell culture lines. Standard references concerning sperm physiology and biochemistry provide an understanding of sperm biochemistry and physiology that is necessary to successfully maintain them in culture. 1. S p e r m - O o c y t e I n t e r a c t i o n s

Before more specialized uses of sperm in culture are discussed, a brief idea of the interactions occurring between the two parental gametes must be offered. Several excellent references covering the area of fertilization are available to enlarge one's working knowledge of the many cellular events comprising this event in an animal's existence (Gwatkins, 1977; Yanagimachi, 1988; Dunbar and O'Rand, 1991; Wassarman, 1991b). This interaction takes place in the proximal regions of the oviduct after natural mating or AI in the vast majority of mammals (Anderson, 1977; Gwatkin's 1977; Overstreet, 1983), although a few species do have intrafollicular fertilization (Mossman and Duke, 1973) as is found in teleosts. In many common multiple ovulators such as laboratory rodents or swine, contractions of the muscular walls of the oviduct compress the oocytes into an egg mass composed of cumulus cells and their secretions and the oocytes. Sperm

Tissue M a t u r a t i o n in Vivo a n d in Vitro

79

must pass through this unique microenvironment to unite with the egg. The zygote is often retained in this mass for a period of time after fertilization until secretions of the oviduct degrade the matrix which holds the mass together. In other species such as cattle or sheep, the cumulus cells with their hyaluronic acid matirx appears to be removed shortly after the expanded oocyte complex is picked up by the oviduct (Lorton and First, 1979; Thompson and Wales, 1987). The sperm has only to penetrate the zona pellucida prior to fusing with the oocyte membrane. Oviduct fluid and contact of spermatozoa with the ciliated epithelia cells of the oviduct creates a unique microenvironment for interaction between the sperm and the oocyte. There are examples of systems for capacitation and fertilization that utilize coculture with oviduct cells to enhance these reproductive phenomena (Ellington et al., 1991; Gutierrez et al., 1993). By the time spermatozoa have reached the surface of the zona pellucida, the nonliving investment directly surrounding the oocyte (Dunbar, 1983; Dunbar et al., 1991; Dean, 1992), they have most likely been capacitated so that interaction with a specific component of the zona pellucida will trigger the acrosome reaction (Bleil and Wassarman, 1983; Florman and First, 1988; Kopf and Gerton, 1991). Interaction of spermatozoa with the secondary oocyte during the process of fertilization is illustrated in Figure 13. The acrosome is a sac-like structure lying directly under the sperm plasmalemma and overlying the head region of sperm which is formed during spermiogenesis by coalescing of the Golgi apparatus (see previously cited references). This lysosome-like organelle contains several lytic enzymes capable of degrading glycoproteins and proteins and which aid the sperm, along with its own motility, in penetrating the investments surrounding the female gamete (Zaneveld et al., 1975; Gwatkins, 1977; Stambaugh, 1978). The acrosome reaction is caused by vesiculation and fusion of the sperm plasmalemma and outer acrosomal membrane which then allows release of the acrosomal contents (Yanagimachi, 1981, 1988, 1990). Release of the lytic enzymes effects sperm passage through the zona pellucida by digestion of its components in a localized region. In some species such as the guinea pig only acrosome reacted spermatozoa can bind to the zona pellucida and penetrate oocytes (Huang et al., 1981). In other species such as the mouse (Saling and Storey, 1979; Storey et al., 1984) and perhaps the golden hamster (Cummins and Yanagimachi, 1982; Gwatkin and Williams, 1977; Yanagimachi and Phillips, 1984), sperm must retain their individual acrosomes intact in order to be able to participate in the fertilization process; other sperm in the cohort lose their acrosome (undergo the acrosome reaction) and release acrosomal enzymes into the area, dispersing the oocyte vestments and allowing the acrosome-intact sperm to reach the zona pellucida. With Chinese hamster sperm (Yanagimachi et al., 1983) either acrosome-intact or acrosome reacted sperm are capable of binding to and penetrating oocytes (Myles et al., 1987).

80

M. Lorraine Leibfried-Rutledge et al.

Outer Acrosomal Membrane Plasma Membrane ~

Inner Acrosomal Membrane I Nucleus Head

Tail

Zone Pellucida -~ Per,v,tel Space I, ne ~ Plasma

~ d ~ing .~.~....~.:~:~.~.~..~ .... 3 .'..- ~., .'-~. .... ~,~ ~:'-.~,,--'~';-~,r"~,,":..~-, : ~ '~"~";'~:~~"~":~''~:~ . ~ " ~ Acros0me Reaction

~ ~:'~! ::~::~;~: ! ! ~ '~:i ,...~.,,,: ~ ~~~;,,~;.~ r/ ~ ~

Penetration

.~.!.-:~ ~..!.:,i;..!.:::;::!i:.;;::!~.,.:;::~'..:!.-~.;~. ii~~ .11 .'"..:"'.'.'.,'::.. ".,'.':.'.'."r '-:'.','.~-.."

..'"~.".~

'

~'.~;-.":,::~','.~;

Granules

.:.:.":.'.':,;.:.;.

:.

~!-.:ii~;i~'i,!ii!;:i:i:;!"~ii!:;;~:,i.;;:!~i~-ii2.i.::.

S S

~

... :.-:'.'....,-.-...-".':....,.:

j

# ~.:~;~'-.".~:~.,~i ~ :~'--';

~ ~ . i ~ i i l ;~ ~::~::~"

Cortical Reaction

Zone Reaction

Diagrammatic representation of sperm-oocyte interactions during fertilization in mice. The sequences of events includes attachment of sperm to eggs, binding of spermatozoa to the zone pellucida, accomplishment of the acrosome reaction, penetration of the zone, fusion of the gamete membranes, and the establishment of the block to polyspermy following the release of cortical granules which effect the zone reaction. (Wassarman, 1987. Science

FIGURE 13

2~s, 553.) Once in the perivitelline space, the spermatozoon is able to contact and fuse with the oolemna (Moore and Bedford, 1983). Interaction and fusion of the sperm plasmalemma with the cell membrane of the oocyte is most likely a receptor-mediated process. Whether the acrosome reaction is part of capacitation or a separate phenomenon has been a matter of debate. Accepting the acrosome reaction as an event enabled by capacitation and triggered by contact with the zone pellucida as suggested by Furuya et el. (1992) would be a sound, pragmatic viewpoint. Fusion of the two gametes also causes activation of the oocyte (Whitaker and Swann, 1993) and a cascade of events that are the beginnings of embryo development. It is not yet clear whether this is a receptormediated event or a result of components brought in by sperm (Belton and Foltz, 1995). Activation triggers the second reduction division in the oocyte, mechanisms that prevent polyspermy (Wolf, 1981; Wassarman, 1988b) including release of cortical granules (Cran and Esper, 1990) that modify the zone, and processing of paternal DNA (Zirkin et el., 1989).

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Release of the maternal developmental program and entry into mitotic cell cycles are also consequences of activation. The many fine reviews cited previously regarding sperm-egg interactions and fertilization should be consulted to increase one's understanding of the complexity of the process. The timing of sperm-egg interaction is a pertinent consideration for successful production of competent embryos. If maturation has not been completed prior to sperm penetration, the oocyte will not be activated (Kubiak, 1989). If the oocyte has become aged before fertilization then developmental competence of the embryo is impaired in various ways and severities depending on the extent of aging (see previous discussion of oocyte aging). Spontaneous activation of the oocyte may also occur during aging with release of cortical granules and the zona block to polyspermy which prevents sperm penetration. Penetration of the sperm prior to complete maturation of the oocyte or after aging can also result in polyspermy due to inability of the oocyte to raise a sufficient block to multiple sperm penetration. Interaction between the parental gametes is seen to occur at many levels. Oocyte investments can enhance sperm viability and motility and participate in causing sperm capacitation and passage to the oocyte membrane. Sperm induce activation of the secondary oocyte and release of the maternal developmental program. The interplay between cell types preceding and during fertilization must be kept in mind when selecting or designing culture environments for the production of embryos. Appraisal of the IVF system chosen should be a routine part of quality control in laboratories if the gametogenic material is not limiting. Failures in development or success of earlier treatments cannot properly be assessed unless verification that fertilization was not disturbed can be offered. Whole mounts of fertilized oocytes from hamsters and cattle are shown in Figures 14 and 15 along with abnormalities of fertilization encountered after IVF (Figure 15).

2. Systems f o r Capacitation and Fertilization Unfortunately there is no universal system for capacitating sperm. Many variations exist for each type of animal and also within a species. Successful capacitation of some species can be achieved in a number of ways, indicating either numerous backup or failsafe mechanisms or merely the ability to manipulate capacitation at a number of points in the cellular mechanism(s). An example of this is found in cattle where capacitation can be successfully induced in bovine spermatozoa in vitro using medium of high ionic strength (Brackett et al., 1982a), the glycosaminoglycan heparin (Parrish et al., 1988), calcium ionophores (Byrd, 1981; Whitfield and Parkinson, 1995), or agents that increase the intracellular concentration of cAMP (summarized by Gordon (1994) for a number of species). Capacitation systems may be as uncomplicated as allowing whole or extended semen to sit at room temperature for a length of time as utilized in some domestic

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In vitro fertilized hamster oocytes. Fresh oocytes were made into whole mounts after sperm-oocyte incubation and observed with Nomarski optics. The two polar bodies, pronuclei, and in one zygote, remnants of the fertilizing sperm's tail can be seen. Both zygotes would be considered normally fertilized.

FIGURE 14

species (summarized by First and Parrish, 1987; Gordon, 1994) or may require an inductive agent as seen in hamsters (Bavister and Yanagimachi, 1977; Leibfried and Bavister, 1981, 1982) and cattle (references previously cited). Within a family, capacitation systems may apply to several of the genuses it encompasses. A common method for inducing capacitation in bovine spermatozoa employs heparin as the inductive agent. This same system can be used to achieve capacitation of sperm from closely related species such as bison (McHugh et al., 1995), buffalo (Madan et al., 1994), gaur (Johnston et aL, 1994), banteng (Barnes et al., 1988), and yak (McHugh et al., 1995), as evidenced by ability to penetrate oocytes. Heparin-induced capacitation can also be applied to another ruminant, the goat (Cox et al., 1995). The source of mature spermatozoa may also influence the choice of capacitation protocol. In cattle, sperm from the cauda epididymis appear not to need an inductive stimulus for effecting capacitation, while sperm (First and Parrish, 1987) from ejaculated whole, extended, or frozenthawed semen require a chemical agent for successful capacitation. When beginning work with a new species it is recommended that the literature be searched for existing information concerning it or a closely related species to determine what media and methods will be selected to effect capacitation and or fertilization. High levels of some ions such as phosphates, calcium, and potassium may depress sperm motility and viability for a wide variety of species. Other media components such as glucose

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Common fertilization patterns observed in cattle oocytes after in vitro fertilization. An early stage of fertlization is shown in A. The oocyte chromatin is undergoing the second reduction division while the head region of the fertilizing spermatozoa is beginning to swell or decondense. The male pronucleus, two polar bodies, and tail remnants from the penetrating spermatozoa are shown in B, while the female pronucleus is not in this focal plane. Polyspermy is depicted in C, with two male pronuclei, female pronucleus, polar body, tail remnant between the upper two pronuclei, and third penetrating sperm with associated tail all visible. Tail remnant associated with the second male pronucleus is not in the same focal plane. The sperm showing little swelling of the head region may represent a late penetration and failure in the block to polyspermy. A well formed female pronucleus and a penetrating sperm that has not undergone nuclear processing is shown in D. Polyspermy and lack of male pronuclear formation as shown in C and D would be considered abnormal fertilization patterns.

FIGURE 15

inhibit or delay capacitation in species such as the guinea pig and cattle (Rogers, 1981; Parrish et al., 1989), while in others its presence may be neutral. Bicarbonate is reportedly required for mouse and rabbit sperm capacitation while calcium is essential for capacitation and acrosome reaction in all species studied (see capacitation reviews). Macromolecular supplements derived from body fluids have been thought to increase the effectiveness of capacitation protocols and maintain sperm viability in a variety of species (Miyamoto and Chang, 1973; Bavister and Morton, 1974; Hoppe and Whitten, 1974; Morton and Bavister, 1974; Meizel, 1978; Andrews and Bavister, 1989a; Dow and Bavister, 1989). More recently synthetic polymers have been substituted (Bavister, 1981b; Andrews and Bavister, 1989b; Keskintepe et al., 1995; Nagao et al., 1995) without decreasing the success rate in producing blastocysts. Few of these studies have attempted to produce offspring from the products. Incubation conditions must also be selected for a particular species when capacitating spermatozoa in vitro. Capacitation and therefore success of fertilization are known to be

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temperature-dependent processes (Mahi and Yanagimachi, 1973; Lambert, 1981; Lenz et al., 1983). The permissive temperature appears to have a narrow range and may approximate the core body temperature of the species (First and Parrish, 1987). Research the individual species as thoroughly as possible beforehand to prevent frustration and loss of material. Potential usefulness of a medium or capacitation protocol can be assessed prior to attempting fertilization. In fact it is suggested that this be done especially when the females' gametes are scarce and the most limiting factor in IVF systems. Visual assessment of sperm motility over time is an easy but effective way of determining if the selected medium will maintain sperm of a particular species. (See general references for semen evaluation in AI industry manuals, basic reproductive texts for domestic animals, and human clinical medicine training manuals.) To test quality of water used to make up media for use in gamete and embryo culture, the hamster sperm motility assay (Bavister and Andrews, 1988) is used by some laboratories including those with clinical emphasis. Both percentage motility and forms showing progressive motility are indicators of an acceptable medium. Timeexposure photography has been utilized for many years in the AI industry for objectively evaluating motility patterns of sperm from semen samples (Van Dellen and Elliott, 1978; Bearden and Fuquay, 1980). If capacitation is being evaluated then forms showing hyperactivation should be evaluated (Yanagimachi, 1981). Pattern of motility changes during capacitation but there are other less subjective ways of assessing capacitation. Aberrant patterns of motility are caused by cold shock and media that is not isotonic for semen and provide a means of evaluating both media and handling conditions. Mere oscillatory movement of sperm also provides indications of cellular damage, inappropriate media, or aged semen. Supravital or live-dead staining can also assist in evaluating the appropriateness of a medium for a species (Blom, 1950; Swanson and Bearden, 1951; Aalseth and Saacke, 1989). Effectiveness of an in vitro capacitation protocol can be evaluated by determining if the acrosome reaction has occurred or can be triggered in a high proportion of spermatozoa within a sample (Cross and Meizel, 1989; Bavister, 1990a). This can be evaluated in some species by using differential interference contrast microscopy to observe the change in morphology of the head region, which indicates that the acrosome reaction took place. A variety of staining methods that can also reveal whether the acrosome reaction has taken place has been developed (Bryan and Akruk, 1977; Didion and Graves, 1986; Didion et al., 1987, Ooba et al., 1991, Den Das et al., 1992; Christensen et al., 1994; Way et al., 1995). Many of these are used on sperm smears rather than on living cells. The acrosome reaction can also be triggered in capacitated sperm by agents such as lysophospholipids (Graham et al., 1986; Wheeler and Seidel, 1989) or zona extracts (Florman and First, 1988; Bleil, 1991), while these agents do not cause this to happen

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prior to capacitation, unless treatments are harshly applied in the case of the lysophospholipids. Alterations in distribution of cell surface molecules and binding patterns of agents that react with these molecules have also been used as correlates of capacitation. These latter methods have not been tested for a wide variety of species although part of capacitation is though to involve changes in cell surface components of spermatozoa (Oliphant and Eng, 1981; Yanagimachi, 1981). Ability of sperm to penetrate oocytes is the best indication that capacitation has occurred. If the female gamete is limiting it is sometimes possible to use an interspecies penetration test to characterize a suite of bulls in order to optimize conditions for capacitation and fertilization in readiness for the female gamete to become available (McHugh et al., 1995). Oocytes from a closely related species may be utilized as in the tribe Bovini where gaur, bison, banteng, and yak spermatozoa will penetrate the oocytes of domestic cattle (cited previously). The zonafree hamster oocyte can be penetrated by sperm from a wide variety of species (Yanagimachi et al., 1976; Brackett et al., 1982b; Yanagimachi, 1984; Berger and Horton, 1988; Fukui et al., 1988) and has been used to evaluate fertility of a specimen primarily in human clinical settings. Salt stored hamster oocytes and zonae (Boatman et al., 1988) are also used for this purpose, while these means of maintaining a ready supply of female gametes have been extended to other species for use in fertility evaluation (Andrews et aL, 1992). Even after capacitation and fertilization systems have proven effective and are being used routinely, it is recommended that subsamples of zygotes be used to assess penetration rates as a quality control procedure when sufficient material is available. It sometimes makes troubleshooting less difficult if cleavage or embryo production rates decline. Use of evaluation methods to ensure that each step of the process used to produce embryos in vitro is functioning adequately may appear to be a slow approach but will save material and frustration in the long run. Embryo production is the end result of a sequential series of reproductive events, and the final success rate will be no higher than that of the weakest or least efficient process. Conditions of culture for each event must be optimized individually to maximize the end result. 3. F a c t o r s A f f e c t i n g S p e r m Fertility

Just as the competency of the female gamete is influenced by a variety of factors, fertility of spermatozoa is also subject to a wide array of variables that affect the quality and fertility of male gametes. Frozen-thawed spermatozoa from single ejaculates of different males produce different levels of fertilization and embryo production when used for IVF (Leibfried-Rutledge et aL, 1987; Fukui et aL, 1988; Eyestone and First, 1989a; Hillery et aL, 1990; Donoghue et aL, 1992; Wildt et aL, 1992b; Totey et aL, 1993; Saeki et aL, 1995) and also differ in length of time required to complete capacitation (Parrish et al., 1986). Although we have not completely sorted this out from

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differences between ejaculates from an individual male, there is plenty of evidence from natural matings and AI to suspect that sire differences encountered in vitro are real. Unfortunately there are no foolproof measures for judging fertility of semen other than using it for AI or IVF. Knowledge concerning the reproductive patterns of the individual species and attention to the physical condition of the animal will aid in obtaining high quality specimens for use in tissue maturation or banking. Physical condition, nutrition, climatic influences, and age of the animal will influence quantity and quality of spermatozoa collected (reviewed by Amann, 1970; Leathem, 1970; Lodge and Salisbury, 1970; VanDemark and Free, 1970; Pickett, 1970; Faulkner and Carroll, 1974; Rattray, 1977; Lincoln and Short, 1980; Ortavant et al., 1981; Pelletier and Almeida, 1987). Effect of age of dairy bull sires on semen quality and fertility has been documented (Hahn et al., 1969). Depending on the longevity of a species, it is expected that a similar decline in fertility with advanced age may occur in many species. Although spermatozoa stored in the epididymis prior to ejaculation are continually voided into the urine and absorbed in the tail of the epididymis to be replaced by new cells from ongoing testicular production of sperm, the first ejaculate collected after long periods of sexual rest may contain degenerate forms. It is suggested that several ejaculates be collected to avoid this problem. Chronic disease states and genetic factors give rise to decreases in fertility. In small breeding populations the latter should be taken into account when selecting parentage for a mating or in tissue banking. Tissue banking and exchange of genetic material between institutions engaged in species preservation efforts will expand the breeding pool for captive specimens. Production of spermatozoa is a continuous process throughout the year in most of the species commonly used in research, commercial, or clinical settings. Many exotics and animals toward which species preservation efforts are directed have seasonal patterns of reproduction. In many of these species, the testes regress during the nonbreeding season and sperm production ceases. The seminiferous tubules come to resemble those of the juvenile animal. In other species partial depression of sperm production is observed according to an annual pattern. Tissue banking efforts must therefore be designed to take advantage of a sire during the maximum period of fertility. Again, knowledge of an animal's life history and reproductive patterns will allow the devising of scenarios that make best use of the gametogenic material available. Environmental factors are the mediators for species in which seasonal breeding is observed, but they will also affect the quality and fertility of semen production in animals who are normally considered to be continuous breeders or producers of spermatozoa. The extent to which environmental factors influence sperm production is related to species, breed, and latitude. The effects of ambient temperature, humidity, and day length on sperm production are mediated via neuroendocrine mechanisms or by direct el-

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fects on the testes. The type of forage available and specific compounds in food supplies in the wild can also be an indirect method for mediating seasonal effects of climatic conditions. While day length is probably the single most important determinant of the breeding season in animals subject to seasonal breeding, temperature may be the greatest factor in causing seasonal variations in sperm production and quality for those species normally considered to be continuous breeders. Sperm production is strictly temperature dependent and occurs successfully only below core body temperature (see previous reviews cited and textbooks of reproductive physiology). The anatomy of the scrotal sac and testes, surface position of arteries and veins of the testes, body location and countercurrent heat exchange mechanisms between the pampiniform plexus and testicular artery all contribute to thermoregulation of the testes to allow its spermatogenic function. Elevation of ambient temperature and hence elevation of testicular temperature will impair the spermatogenic process and damage sperm already produced. Any conditions such as fever, excesses of hair and wool, or extra fat deposition which impair thermoregulation of the testes will lower sperm production, increase the presence of abnormal forms, and generally lead to samples of lower quality and fertility. Again, knowledge of the life history of an animal, its specie's reproductive patterns and maintenance of good physical condition will lead to collection of high fertility semen and make best use of the genetic material. C. M t e r n a t i v e Uses o f S p e r m a t o g e n i c Stages i n V i t r o Despite all efforts, collection of mature spermatozoa capable of yielding high rates of fertilization after AI or IVF may fail. Azoospermia or oligospermia due to genetic, climatic, or other factors may be present. Specimens with an adequate number of cells may still be of low motility or viability or have many abnormal forms present. Depending on the situation, use of the specimens or males providing them is often still required. New methods for aiding infertile couples are being developed in clinical settings that extend the range of semen quality and spermatogenic stages that can be utilized to produce offspring. These will be discussed to widen the range of use scenarios for paternal genetic material either before or after tissue banking. A cautionary warning must be added at this point. First, if low fertility of a male is due to genetic causes, it is probably not reasonable to wish to perpetuate this defect in the offspring and population in general, particularly if the breeding population is small. Second, many of these procedures are more-or-less in their infancy with few live young produced to date. There is scant information concerning the consequences of these new procedures on the health of resulting offspring, and in the case of at least one of the techniques, nuclear transfer, data to suggest that an increased incidence of abnormal offspring are produced (Willadsen et al.,

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1991; Wilson et al., 1995; Garry et al., 1996). Although progeny produced from assisted reproductive techniques such as IVM, IVF, and embryo culture on the whole have appeared normal in the species tested, results reporting various abnormalities have been recurrent in the literature since early attempts to perform these processes in the laboratory. Microphthalmia was reported in 50% of the offspring in rats after IVF (Toyoda and Chang, 1974) and was observed again recently in an IVP calf (Farin and Farin, 1995) along with hydrocephaly. Since then physical abnormalities (Walker et al., 1992b; Farin and Farin, 1995; Hasler et al., 1995), altered birth weights (Bowman and McLaren, 1970; Holland and Odde, 1992; Reichenbach et al., 1992; Walker et al., 1992a,b; Mayne and McEvoy, 1993; Wang et al., 1994; Behboodi et al., 1995; Farin and Farin, 1995; Hasler et al., 1995; Spindle, 1995), increased neonatal morbidity (Thompson et al., 1992; Walker et al., 1992a,b; Behboodi et al., 1995; Hasler et al., 1995; reviewed by Walker et al., 1996), and skewing of the sex ratio (Tsunoda et al., 1985; Iwasaki et al., 1988; Avery et al., 1991, 1992; Xu et al., 1992; Yadav et al., 1993) have all sproadically appeared in the literature in species ranging from laboratory animals up to humans after one or more of the IVM-IVF-IVC protocols has been applied. At minimum this means that under laboratory conditions the processes involved in in vitro manipulations can be unknowingly perturbed such that development may be adversely affected even if visibly normal progeny are most often obtained. As we develop new techniques that allow us to impact the gene pool of various species, our awareness of how we impact it must also increase. As a particular technique becomes feasible, it should not prematurely be removed from the research setting and put into application. 1. Sperm Injection Mature spermatozoa can be injected into the cytoplasm of secondary oocytes with successful production of live offspring using ejaculated sperm (Hosoi et aL, 1988; Palermo et aL, 1992), epididymal sperm (Van Steirteghem et aL, 1993; Tourney et al., 1994; Kimura and Yanagimachi, 1995a; Tucker et aL, 1995), or testicular sperm (Schmoysman et aL, 1993; Ogura et aL, 1994; Bourn et aL, 1995; Devroey et aL, 1995; Kimura and Yanagimachi, 1995b; Silber et aL, 1995; Tucker et aL, 1995; Lanzendorf, 1995). If activation of the oocyte occurs or is induced after microinjection of the sperm, the paternal DNA can pariticipate in fertilization and the events of development. While human oocytes appear not to need an activation stimulus other than the cytoplasmic or membrane perturbation caused by microinjection, other species may require a separate activation protocol to trigger release of the maternal developmental program (Goto, 1993; LachamKaplan and Trounson, 1995). This may be affected by the presence or absence of a soluble sperm component that can cause activation of the oocyte (Stice and Robl, 1990; Swann, 1990, 1993) or perhaps there are

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species differences in how this oocyte-activating factor must be presented to the oocyte. Stage of spermatogenesis being used as source of paternal DNA may also determine whether artificial methods of activation are necessary (Ogura et al., 1994; Kimura and Yanagimachi, 1995a,b,c). Activation is also an age-dependent process as discussed previously. Age of the secondary oocyte after completing maturation must be taken into account. While young oocytes are difficult to activate parthenogenetically, older oocytes readily activate in response to a number of factors (Kubiak, 1989; Winston et al., 1991; Susko-Parrish et al., 1994; discussed by Kaufman, 1981). Aging or senescence of the secondary oocyte causes a decline in its developmental competence so that ability of the resulting embryos to initiate or maintain pregnancy or form a blastocyst is compromised (see earlier discussion on oocyte senescence). Timing for interaction of the gametes involved in fertilization are as important a consideration using the microinjection technique as in IVF. Technical considerations regarding sperm injection have been reviewed (Keefer, 1990; Cohen et al., 1992; Lanzendorf, 1995). Sperm microinjection is very economical in the use of paternal genetic material. A consideration to keep in mind is that the process of fertilization may also be a selection process designed to eliminate sperm that are not developmentally competent. We have little data concerning this possibility. Recently the birth of a human infant was reported using more immature stages obtained from testicular biopsy (Bourn et aL, 1995; Devroey et al., 1995; Silber et al., 1995; Tucker et al., 1995). Normal embryonic development has also been demonstrated in mice after the fusion or microinjection of nuclei from round spermatids obtained after testicular biopsy (Oguar et al., 1994; Kimura and Yanagimachi, 1995b). Mature spermatozoa capable of fertilization are normally found only in the tail of the epididymis or the ejaculate. Continuation of spermiogenesis occurs as the sperm passes out of the testes and through the proximal regions of the epididymis. Sperm properties and functions such as those involved in motility are acquired during this passage. Nonmotile sperm are not capable of penetrating the egg investments to fuse with the oocyte unless aided by assisted reproductive technology. The paternal DNA in less mature stages of male gametes might be competent to participate in development as long as the cell does not have to reach the oocyte on its own or cause activation, the other two sperm functions during fertilization. Very little work has been done to evaluate the developmental competence of cells at various stages of spermatogenesis. We know little about programming of the sperm DNA during spermatogenesis and when it is competent to participate in activation of the newly formed embryonic genome at the maternal-to-embryonic transition in the control of gene expression during development. It is safe to say that sperm injection using the secondary oocyte containing the maternal DNA must utilize a haploid male gamete with a 1C content of DNA or verification of protocols to ensure that separation of the sister

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chromatids has taken place prior to the first embryonic S-phase should be performed if secondary spermatocytes or even earlier stages are utilized. Reproductively competent mice have been born after the injection of nuclei from secondary spermatocytes into secondary oocytes (Kimura and Yanagimachi, 1995c). Premature condensation of the paternal DNA on a metaphase spindle was followed by the second reduction division of both parental sources of DNA after activation of the oocyte. Here again, age of oocyte used in the process will affect whether the paternal amount of DNA from cells that have not completed the meiotic process is reduced to a 1C amount prior to DNA synthesis of the first cell cycle in the zygote. A study using young or aged oocytes as recipients for a donated embryonic nucleus in nuclear transfer studies demonstrated that donated DNA was forced to undergo a reduction division after activation along with the oocyte's completion of meiosis II when maternal chromatin remained in the unaged oocyte. If older oocytes were used as recipient, the donated nucleus was merely remodeled into a pronuclear-like structure without nuclear envelope breakdown and reduction of the DNA content (Leibfried-Rutledge et al., 1992) although the maternal chromatin did complete meiosis. This has import for sperm injection work involving sperm stages prior to completion of meiosis. Generation of polyploid genotypes in embryos produced in this manner could result. One study has attempted to determine if nuclei from murine primary spermatocytes having a 4C chromatin complement will undergo two reduction divisions prior to syngamy when fused into oocytes (Ng and Solter, 1992). The results were not conclusive. Z Use o f Cells a t E a r l i e r S t a g e s o f S p e r m a t o g e n e s i s

As discussed previously under oogenesis, primordial germ cells of both sexes can be used immediately upon isolation in nuclear transfer protocols or to create stem cell lines which can be used to generate germ-line chimeras or in nuclear transfer. Although the efficiency of these technologies still needs to be improved, tissue preservation of this early gamete stage should not be ignored in banking paternal genotypes. Between fusion during nuclear transfer procedures and injection of whole cells or karyoplasts, paternal genetic material from almost every stage of spermatogenesis has been combined with the secondary oocyte in some fashion, at premei0tic, meiotic, and postmeiotic stages. Primordial germ cells (Tsunoda et aL, 1989; Ledda et al., 1996; Strelchenko, 1996), primary and secondary spermatocytes (Ng and Solter, 1992; Kimura and Yanagimachi, 1995c), spermatids (see the previous section), and immature and mature spermatozoa (see citations in the previous section) have donated genetic material for experimental manipulations with or without attempts to produce viable offspring. Primordial germ cells or primitive gonocytes of both sexes have been used as primary explants to combine with preimplantation embryos to make chimeric rabbit fetuses (Moens et al., 1996). Although chimerism into the

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fetal gonads was demonstrated, it was not clear if transmission through the germ line would have been possible or whether just somatic tissue elements of the conceptuses contained the gonial lineage. Recently in mice, male germ cell transplantation has been shown to be feasible using diploid spermatogonia. After transfer of spermatogonia from a fertile male into seminiferous tubules of a sterile male, spermatogenesis resumed and production of progeny with the donor genotype was demonstrated (Brinster and Zimmerman, 1994; Brinster and Avarbock, 1994). With techniques available for identifying and isolating spermatogonia and other stages of spermatogenesis (Bellve et al., 1977; Dym et al., 1995; Mays-Hoopes et al., 1995), banking of male gametes at any stage in their life history becomes a worthwhile objective. The range of scenarios for possible use of these cells broadens incessantly.

IV. PREIMPLANTATION EMBRYONIC DEVELOPMENT

The period of preimplantation embryonic development has been the subject of extensive study and can only be briefly summarized here. The reader is referred to previous references concerning the process of fertilization and recent reviews of embryonic development for more extensive discussion (Balinsky, 1981; Johnson, 1981; Davidson, 1986; Schultz, 1986a; Barnes and Eyestone, 1990; Maro et al., 1991; Pedersen and Bursal, 1994; Schultz et aL, 1995; Leibfried-Rutledge, 1996). When the spermatozoon unites with the secondary oocyte a one-celled embryo termed the zygote is formed. Fusion and entry of the sperm into the oocyte activates its developmental program which was stored during oogenesis (Schultz, 1986a; Santiago and Marzluff, 1989; Schultz and Heyner, 1992). The oocyte completes meiosis, paternal DNA is decondensed, and the sperm histones and other associated nuclear proteins are replaced by maternal forms (Wolgemuth, 1983; Poccia, 1989; Zirkin et al., 1989; Clarke, 1992; Perreault, 1992). Maternal genetic material is decondensed and repackaged in a nuclear membrane after completion of the second reduction division with expulsion of half the sister chromatids into the second polar body. The two nuclei resulting from these events are called pronuclei, one containing maternal and the other paternal haploid chromosomes (see Figures 14 and 15). The zygote now begins mitotic cell cycles as opposed to the meiotic cell cycles of the contributing parental gametes. Union of the two haploid gametes has restored the diploid state in the zygote. DNA is duplicated within each pronucleus and these two organelles migrate within the cytoplasm of the zygote until they are in close proximity. Chromosome condensation begins after completion of DNA synthesis and membranes of the pronuclei break down. Chromosome condensation continues to occur and microfilaments for the metaphase spindle of the first embryonic mitosis

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begin to form. Fully condensed chromosomes become attached by their teleomeres to microtubules of the spindle structure. Intermingling of the parental genetic material after release from their respective pronuclei and assembly on the metaphase spindle is termed syngamy. Separation of homologous chromosomes is effected by the events of anaphase and telophase followed by cleavage of the one-celled embryo into two blastomeres. The 2-cell embryo undergoes a period of repeated mitotic divisions and cleavage without growth. Actual mass and protein content both decrease during this period as does size of the blastomeres (McLaren, 1974; Anderson, 1991). Gene expression during these early cleavage divisions relies on maternal mRNA (Magnuson and Epstein, 1987; Telford et al., 1990; Schultz, 1993) that was accumulated and stored during growth of the primary oocyte after it exits the nongrowing pool of female gametogenic material in the ovary (Bachvarova, 1985, 1988). A major event in early development is the transition from maternal regulation of development to regulation by the embryonic genome (Flach et al., 1982; Bolton et al., 1984; Howlett and Bolton, 1985; Braude et al., 1988; Telford et al., 1990; Schultz, 1993, 1995; Schultz et al., 1995; Leibfried-Rutledge, 1996). As maternal stores of mRNA are degraded during early cleavage (Schultz, 1993), activation of the embryonic genome must occur to allow continued development. The period during which embryonic gene expression begins is termed the maternal to embryonic transition. Synthesis of new or embryonic mRNA is found as early as the 2-cell stage in short-cycl e species such as mice and hamsters and as late as the 8- to 16-cell stages in long-cycle species (reviewed by Telford et al., 1990). The multicelled embryo is called a morula. After a set number of cleavage divisions characteristic of a species and after activation of the embryonic genome, a process called compaction begins (Kidder, 1993). This takes place as early as the 8-cell stage in laboratory species having short estrous cycles such as mice, rats, and hamsters or can be delayed until after the fifth cleavage division or 32-cell stage as observed in cattle. Cells on the exterior of the embryo establish a definite polarity with cell adhesion and intercellular connections established on their apposed surfaces, while the numerous microvilli covering the surface of blastomeres in earlier cleavage stages are now confined to the free surface (Ziomak, 1987; Anderson, 1991; Maro et al., 1991). These polarized cells are called outside cells. The apolar cells, i.e., those blastomeres surrounded on all sides by other cells, are termed inside cells and may be visible smaller than the outside cells. These events comprise the first signs of determination and cell differentiation in preimplantation embryos (Wiley et al., 1990; Gueth-Halonnet and Maro, 1992). Channels for intercellular communication are also established (Kidder, 1987). When compaction is completed, fluid is pumped into the intercellular spaces in the interior of the morula (Benos and Biggers, 1981; Wiley, 1987; Pedersen, 1988; Watson, 1992; Guillomot et al., 1993). As embryonic

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cleavage continues a fluid filled cavity, the blastocoele, is created with a single layer of cells on the exterior and a grouping of smaller cells to one side of this fluid filled vesicle-like structure (Davies and Hesseldahl, 1971). This embryonic conformation is termed the blastocyst stage and is shown for cattle embryos in Figure 16. The outer layer of tissue is the trophoblast and is formed by the squamous trophoblast cells. The small grouping of cells found at one side of the central cavity are the inner mass cells. The inner cell mass and trophoblast cells represent the first cell lineages of the embryo. Trophoblast cells will give rise to placental tissue and embryonic membranes. Continued differentiation of inner cell mass cells leads to formation of the fetus. Experimental studies in mice would evince that outside cells of the morula were forerunners of the trophoblastic layer while inside cells gave rise to the inner cell mass. Individual blastomeres from cleavage stages prior to compaction appear to be totipotent or capable of expressing the complete developmental program through to production of live-born offspring. Determination and differentiation of early embryonic cells leading to a restricted potential for developing into placental or fetal tissue is characteristic of the period leading up to formation of the blastocyst (Ziomek and Johnson, 1982; Johnson and Ziomek, 1983). Experimental manipulation of blastomeres has suggested that restriction of developmental potential begins in the morula during compaction and is dependent

In vitro production ofbovine blastocysts. Oocytes from abattoir-derived bovine ovaries were matured, fertilized, and cultured in vitro. While blastocyst formation is first observed on Day 6 in this system, this photomicrograph of the microdrop culture taken on Day 8 after insemination shows expanded, hatching, and hatched blastocyst stages.

FIGURE 16

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on surrounding cellular contacts. Nuclear transfer of embryonic nuclei into cytoplasm of the secondary oocyte with the associated resetting of the developmental clock, would indicate that cellular potency is supported by nuclear potency. While donor nuclei taken from inside cells and cells of the inner cell mass are capable of directing development to blastocyst formation or live-born offspring, cells taken from outside cells or trophoblast are less successful (summarized by First and Leibfried-Rutledge, 1993). Understanding aspects of determination is useful in creating identical offspring from one embryonic genotype. These may be effective strategies for managing reproduction in individuals that are scarce or of preferred genotypes. Upon reaching the blastocyst stage of development, fluid accumulation in the blastocoele cavity continues so that expansion of the blastocyst and thinning of the elastic zona pellucida occurs. Whether due to expansion of the embryo or lytic secretions of the uterine epithelium as in rodents, the embryo eventually erupts from its glycoprotein shell in a process termed hatching (see Figure 16). Use of mammalian embryonic material in tissue banking or for in vitro tissue maturation almost exclusively utilizes preimplantation stages that are normally still enclosed by the zona pellucida, i.e., prehatching stages. Further discussion will therefore concentrate on the ontogeny of these developmental stages in culture. The preimplantation embryo exists in a free living state within the female reproductive tract. It is nourished to some extent by oocyte nutrient stores but primarily by secretions of first the oviduct (Nancarrow and Hill, 1995) and then the uterus until establishment of the fetal-maternal attachment and vascularization during placentation (Bazer et aL, 1986; Betteridge and Flechon, 1988; Concannon et al., 1989; Dey, 1995). The free living embryo undergoes cleavage and early events of differentiation as it passes through the oviduct and into the uterus as shown in Figure 17. Fertilization takes place in the ampulla of the oviduct. The cleavage stage at which an embryo enters the uterus is characteristic for a species (Harper, 1994; Taggart, 1994). Embryonic gene activation and compaction may be completed before entry into the uterus as observed in animals having short estrous cycles and brief gestation times. Long-cycle species often transition to embryonic gene expression in the oviduct and begin compaction when reaching the uterine lumen. In all mammals studied to date, blastocyst formation is accomplished in the uterine environment (Schultz et al., 1981). Both cleavage and success in embryonic initiation of pregnancy may be influenced by retardation or premature passage of the embryo through the reproductive tract (Glasser et al., 1994; Walker et al., 1996). Normality of the resulting offspring may also be compromised by entering the uterus prematurely. Components of female reproductive tract secretions such as metabolites, electrolytes, proteins, and characteristics like pH and osmolarity have been studied in common species for many years (Johnson and

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B~ Cow Migration of murine (A) and bovine (B) embryos through the reproductive tract. Free living stages of preimplantation embryo development are depicted in regard to location in the female reproductive tract where they are found for the individual species. Although not indicated here, the timing for the progression also differs greatly between the short-cycle vs long-cycle species.

FIGURE 17

Foley, 1974; Leese, 1988; Lippes et aL, 1991; Maguiness et aL, 1992; Gardner et al., 1996). Specificity of secretion in distinct regions of the tract and dependency on stage and hormones of the reproductive cycles have been well documented. The hormonal dependence and balance during early development are illustrated by the observation that exposure of the preimplantation embryo to elevated progesterone prematurely when it is transversing the reproductive tract and entering the uterus may have long-term effects that are manifest in the neonate (Fischer, 1992; Walker et al., 1992b, 1996). Recently we have begun to recognize the existence of many paracrine interactions between the preimplantation embryo and epithelial cells lining the female reproductive tract. Receptors and ligands, or their messages, are being characterized in embryos and epithelial cells for steroids, growth factors, insulin, and many other cytokines (Rizzino, 1987; Gandolfi et aL, 1989; Schultz et aL, 1992; Watson et al., 1992; Murphy and Barron, 1993; Heyner et aL, 1993; Schultz and Heyner, 1993; Simmen et aL, 1993; Gandolfi, 1994). Many articles are currently dedicated toward evaluating their effect on in vitro tissue maturation of oocytes and embryos. Their roles in embryo development and differentiation may soon be discerned (Sharkey, 1995). This will facilitate and improve design of culture media to sustain in vitro

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development of embryos. Currently no attempts have been made to determine whether the growth factors or cytokines improve pregnancy rates, neonatal viability, or normality after in vitro treatments. Interactions between embryos and the uterine environment are many and complex, not only for supporting early development and differentiation and later placentation but for the maternal recognition of pregnancy. At some stage during development of the mammalian embryo, the product of a fertile mating must signal its presence to initiate and/or maintain pregnancy, thereby preventing resumption of reproductive cycles (Bazer et al., 1986, Dey, 1995). Understanding this latter event has import for successful utilization of embryos used in tissue banking or resulting from in vitro tissue maturation of other forms of stored genetic material. Since eventual production of live offspring will most frequently be the goal of tissue banking efforts with gametes and embryos, this means that AI or embryo transfer (ET) to a recipient animal will be required. Successful ET is dependent on discerning the timing and factors involved in mechanisms for the maternal recognition of pregnancy (Guillomot, 1995). Pregnancy initiation in some species is induced by copulation (Hafez, 1970). Cycle extension is produced which is later maintained by the presence of viable fetuses until parturition. If a fertile mating is not effected, a state of pseudopregnancy is engendered which lasts until corpus luteum function terminates and reproductive cycles are resumed in the absence of embryonic signaling. In long-cycle mammals studied, the burden is on the embryo to emit a signal that prevents a subsequent reproductive cycle (Morton et al., 1983; Bazer et aL, 1986; Roberts, 1989; Thatcher et al., 1989). This first signalingmechanism causes pregnancy initiation and prevents regression of the corpus luteum (Thatcher et aL, 1986; Roberts et al., 1990; Bazer et al., 1994). Subsequent factors released from the embryo maintain gestation through to parturition. A minority of the mammals studied to ~date display an interesting variation in pregnancy initiation and prevention of reproductive cycles. The preimplantation embryo undergoes a period of arrested development known as diapause (Enders, 1963; Mead, 1993). Later signals from either the environment or the embryo causes initiation or maintenance of pregnancy to resume. These varied mechanisms for maternal recognition of pregnancy have been described for only a small fraction of mammals. Application of gene banking and assisted reproductive strategies to a wider variety of species requires a greater knowledge of these basic aspects of reproduction both for collection of appropriate embryonic stages and for obtaining successful outcomes to gestation. Specificity of embryonic stage in relation to location in the reproductive tract and hormonal balance thus have all been shown to affect the accomplishment and normality of embryo development and success of gestation. So precise are the numerous interactions that frequently successful embryo development and gestation cannot occur using the purebred embryo even

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between closely related species where the interspecies crosses may produce viable F~ progeny. This is unfortunate in many species preservation attempts where use of a more common member of a group would be desirable as a recipient for ET purposes. Even within the same tribe the maternal-fetal interactions may differ sufficiently so that successful gestation is not possible. Again using Bovini as an example, Bos taurus will carry purebred embryos of B. gaurus, B. mutus, and most likely B. javanicus through gestation. Although the interspecies cross using B. taurus and B. bison produces viable progeny, they may not be able to gestate a purebred embryo of the reciprocal species (Dorn, 1995; J. J. Rutledge, personal communication). Domestic cattle have a reduced frequency of calving the F1 cross, while bison cows support the species hybrid at apparently normal levels. It should be noted that while interspecies crosses within a genus are often possible, males resulting from such cross-breeding may be sterile or of low fertility while the female progeny are commonly fully fertile. We still have a long ways to go in solving the intriguing processes underlying differentiation and development of embryos. Yet sufficient basic biology is known to allow the merger of in vivo and in vitro tissue maturation with tissue banking to produce a variety of strategies serviceable in animal production, clinical settings, and species conservation. Although there is much we currently can do, it must be remembered that producing a new individual of a species results from a series of sequential events, each of which must be completed normally to allow success of the subsequent events. Even small deviations from the norm may have serious consequences for later developmental processes and even for the health of offspring produced in later life (Seamark and Robinson, 1995). With a limited understanding of the ontogeny of mammalian development, we have scant modes of assessing the resonance resulting from perturbations at earlier stages other than observing the success or failure of producing normal, viable progeny. Caution is warranted when applying a new process to a species and must engender a careful scrutiny of available models and a search for methods of determining normality at each earlier stage as thoroughly as possible. The embryo culture methods presented in the following discussion have current and continuing application. However, just as hormonal imbalances or asynchrony between embryonic stage and reproductive tract environment provokes aberrations in normality of offspring, factors in the tissue culture environment may produce the same abnormalities. With the numerous variations on developmental patterns observed for various species, there is no reliable way of predicting applications of results between systems. While the goals of tissue storage and assisted reproductive techniques are laudable and warranted, care in choosing applications is recommended until the consequences of possible perturbations to development are more completely known. This will take more effort directed toward completing experimental studies designed to compare di-

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rectly the results of modifications of culture systems using live birth as the endpoint. This will be a time intensive and laborious process since errors in in vitro technologies may occur as early as IVM or after embryo culture. A. I n V i v o S o u r c e s o f E m b r y o s

With the cautionary note ending the previous discussion, utilization of in vivo derived embryos in tissue banking at stages compatible with transfer

to a recipient is without doubt the most advantageous situation. Other than the possibility of damage due to cryopreservation and ability to maintain embryo-recipient synchrony during ET, other causes of perturbations to developmental success are minimized. Limiting the duration of embryo culture necessary to achieve developmental stages compatible with final application reduces the likelihood of additional factors compromising the developmental process and leads to greater chances of success after ET. Depending on the use scenario, this may not always be feasible and may limit the salvage of genetic material. In vitro embryo production is currently an alternative in a number of species. Pregnancy rates after ET using frozen-thawed embryos produced in this manner are lower than expected in at least one species studied (Leibo and Loskutoff, 1993). This again suggests that in vitro tissue maturation although having some success, still results in an embryo that is different from its in situ counterpart. Prior to consideration of embryo culture, methods available for obtaining embryos from the reproductive tract will be summarized. Methods of ET for the succeeding groupings of animals will be briefly mentioned since most use scenarios that will eventually require transfer back to a recipient to effect expression of the stored genetic material. 1. L a b o r a t o r y S p e c i e s

As in obtaining gametes, recovery of embryos from the tracts of the most common laboratory species usually occurs postmortem. Although it is possible to obtain embryos surgically, limitations on performing repeated surgery on animals due to animal welfare advisories and the low cost and plenitude of these animals renders this method rarely used. Dissection and flushing of the reproductive tract after death is normally done using a syringe and small diameter hypodermic needle that has been blunted (Hafez, 1970; Hogan et al., 1986). Some consideration should be given to the method used to bring about the demise of the subject since this may affect the embryo also. This is also true when collecting gametogenic material. If zygotes are required, this stage can often be found early after fertilization still trapped in the egg plug or mass as are fleshly ovulated secondary oocytes. After locating this mass in the ampulla, a tear is made in this region of the oviduct and the material expressed with gentle pressure. As in recovering all stages of embryos, knowledge of the region of the female

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reproductive tract containing the desired embryonic stage is necessary for recovering the genetic material as successfully and completely as possible. This has been well characterized for all common laboratory animals. Early preimplantation embryos from domestic cats and dogs are usually obtained by surgically flushing the oviducts (Hafez, 1970). Although the laboratory animals routinely encountered are polytocous, more use can be made of each animal sacrificed if a superovulation protocol is utilized prior to mating and collection of the embryos. Some species such as mice and hamsters will result in several-fold increases in embryos available for recovery, while rats yield a more mediocre response and guinea pigs do not appear to respond at all. As in all procedures, consulting available literature and experienced personae is advantageous. Factors such as age, strain, animal housing conditions, and season will all affect success of the superovulation protocol, fertility of the subsequent mating, and success of in vitro tissue maturation. This should be taken into consideration when planning acquisition of reproductive tissues for cryopreservation or other purposes. Two studies examining the seasonal response of gametogenic and embryonic tissue collected from mice and hamsters are shown in Figure 18. All animals were maintained under constant conditions with respect to temperature and lighting and fed a commercially available diet. In Figure 18A are shown data regarding the success of superovulation in hamsters over a 4-year period using animals housed in three different animal care facilities at the University of Wisconsin-Madison. Two of the hamster colonies were under the supervision of Dr. B. D. Bavister, Department of Animal Health and Biomedicine, University of Wisconsin, Madison, while the third was managed by one of the authors. Oocytes or early embryos were recovered from the oviducts. Frequency of animals responding to a standard superovulation protocol obviously showed a seasonal trend. Although response to superovulation was evaluated in this data set, number of eggs, fertility, and development in culture are also influenced by season. The murine study summarized in Figure 18B was done by R. L. Monson under the supervision of J. J. Rutledge, Department of Meat and Animal Science, University of Wisconsin, Madison. In it they examined the annual superovulatory response of an outbred mouse strain (ICR) housed under constant lighting and temperature conditions. Groups of 10 animals administered a standard superovulation protocol were killed weekly over a 50-week period and the zygotes harvested. Data for ambient temperature, day length, and barometric pressure were obtained from the National Meteorological Service during this period. The mean number of embryos recovered per animal ovulating was highly correlated with change in barometric pressure during the period of hormonal administration. Response to superovulation was depressed whenever the barometric pressure changed _+7.62 mm Hg. An inbred strain (C57b16), for which data are not presented, showed the same effect. Barometric pressure is known to

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Consecutive weeks (Feb-Dec 1989) FIGURE 18 Climatic effects on response of hamsters (A) and mice (B) to superovulation. Frequency of hamsters superovulating (positive response defined as animals with >20 eggs or embryos) in response to hormonal stimulation is plotted for 4 consecutive years in A. Range of animals observed in any 2-month period was 6 to 72 with an average of 30 per data point (Data courtesy of Dr. B. D. Bavister and M. L. Leibfried-Rutledge). Mean number of early embryos per ovulating female plotted along with change in barometric pressure during the period of hormonal administration is shown in plate B (Data courtesy of Dr. J. J. Rutledge and R. L. Monson).

influence expression of estrus, ovulation, and activity levels in rats and mice (Chang and Fernandez-Cano, 1959; Sprott, 1967) and calving in domestic cattle (Dvorak, 1978). Many reproductive phenomena may show distinct

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fluctuations in response to climatic conditions. Knowledge of such variables is necessary for correct design and interpretation of reproductive studies dealing with gametes and embryos even when we believe that all experimental conditions are being kept constant in the laboratory. One must also keep in mind that the superovulation protocol draws on a population of follicle units that includes follicles that might not have been selected for ovulation otherwise. Surgical methods are most frequently used for ET in laboratory species including dogs and cats (Hafez, 1970; Adams, 1982; Kraemer 1989; Hogan et al., 1986; Concannon, 1991; Pinkert, 1994). A fairly large volume of literature has accumulated in mice concerning the optimum stage of embryo and site of deposition in the reproductive tract that yields successful outcomes. While mice appear fairly forgiving in respect to these factors, embryos of hamsters are remarkably sensitive to handling prior to transfer (Farrel and Bavister, 1984) and appear to have a very limited window for embryo-maternal interactions before success in pregnancy is compromised (Adams, 1982). Nonsurgical transfer is infrequently used for ET and also AI in mice (Hafez, 1970) and other small laboratory species. With the ease and rapidity which surgical methods can be accomplished after practice, little attention has been paid to this method in the smaller laboratory species although it is possible. 2. D o m e s t i c A n i m a l s

Much of the focus for recovery of embryos in domestic animals has been slanted toward recovering the morula or blastocyst stages. These stages reside in the uterus and in cattle are effectively recovered by nonsurgical flushing of this organ. A thriving agricultural industry has sprung up around nonsurgical recovery of cattle genetics and has encouraged the flourishing of other assisted reproductive techniques such as superovulation to overcome the limitations of this monotocous species (Boland et aL, 1991; Armstrong, 1993; Dielman and Bevers, 1993), management of reproductive cycles to synchronize recipients for ET (Gordon, 1994), embryo cryopreservation (Niemann, 1991; Rall, 1992; Leibo and Loskutoff, 1993), and nonsurgical ET (Betteridge, 1977). In no other domestic species has embryo recovery been so accessible to exploitation. Equine morula and blastocyst stages can also be recovered by nonsurgical methods similar to those used in cattle (Betteridge, 1977). The size and anatomy of sheep and goats has made development of nonsurgical methods difficult and except for certain limited applications, there has been no overwhelming economic incentive to perfect them. Surgical flushing of the uterine horns toward the cannulated oviducts after exposure of the reproductive tract via a mid-ventral incision can be done with minimal trouble and high rates of recovery no matter what stage embryo desired (Betteridge, 1977). Later stage preimplantation embryos in pigs are also obtained surgically by retrograde flushing of the uterine

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horn after mid-ventral incision, but a cannula is usually inserted near the tip of the uterine horn since the anatomy of the junction prevents flushing of uterine stage embryos into the oviducts for collection. Some reports indicate that nonsurgical recovery of porcine embryos is possible if the length of the uterine horns are reduced surgically prior to collection. There has also been limited attempts to perform transcervical embryo recovery in swine (Hazeleger et al., 1989). These techniques have not been rapidly adopted in, but again there are only a few restricted situations where embryo recovery is utilized in pigs. As in many species, if the technique was available, management regimes would evolve to make use of the technique. Methods have been described for recovery of uterine preimplantation embryos in sheep in goats (Kraemer, 1989) which are currently receiving more attention, particularly for adaptation to smaller exotic ungulates. These techniques still require anesthesia. Collection of tubal embryonic stages from live domestic animals usually necessitates surgical intervention in order to flush the appropriate region of the reproductive tract depending on when a particular cleavage stage enters the uterus. Best recovery is realized if flushing is done from the uterotubal junction towards the infundibulum of the oviduct. The 8- and 16cell stages in cattle can actually be obtained from the uterus by nonsurgically flushing animals at the appropriate day postestrus. Since the embryo has recently entered the uterus at this point, it is still in the tip of the horn so rates of recovery are often low. Also in cattle, secondary oocytes, zygotes, and early cleavage stage embryos can be obtained from live animals by salphingectomy using a vaginal approach. This can be done quickly and efficiently with the animal in a standing position and only necessitates use of a spinal block. If a portion of the uterine horn near the uterotubal junction is also removed along with the ovary and oviduct using the eucrasure, embryos as late as the fifth round of cleavage division may also be recovered in this manner. Since this negates any future reproductive use of the subject, it will probably not be a technique of choice for tissue banking. There are more attractive nonsurgical methods for obtaining gametes and embryos that are compatible with continued reproduction of the donor animal. ET in cattle is easily done nonsurgically and is now used almost exclusively (Betteridge, 1977; Adams, 1982; Betteridge and Rieger, 1993; Hafez, 1993). An extensive volume of literature exists concerning techniques, preparation, and synchrony of recipients, etc. Again, similar techniques may be utilized in horses (Betteridge, 1977; Adams, 1982). In small domestic ruminants, ET is still most frequently performed surgically after exposure of the reproductive tract via a mid-ventral incision, similar to the technique used for embryo recovery (Betteridge, 1977; Adams, 1982). Transcervical and nonsurgical methods have been reported but are not in widespread use (Coonrod et al., 1986; Kraemer, 1989). Laparoscope-aided ET and AI

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is becoming very popular for small reminants (Wildt et aL, 1986). Although some attempts to do nonsurgical ET in swine have been made (Galvin et al., 1994), surgical methods are still most frequently used when required (Betteridge, 1977). The site of deposition in the uterus and relationship between estrous stage of the embryo donor and recipient is critical in this species, particularly if the embryos are maintained in holding medium for a period of time prior to transfer (Pope et aL, 1986; Blum-Reckow and Holtz, 1991). Although other farm animals give near optimum results for pregnancy rates after ET with exact synchronization between donor and recipient cycles, the best pregnancy rates in swine are produced if the recipient uterus is younger than that of the donor animal. As in other areas of reproduction, success rates after ET and embryo recovery are influenced by reproductive status of animals involved, seasonal factors, nutrition, climatic conditions, management, etc. It is suggested that the annual symposium issues for the International Embryo Transfer Society be consulted for updates on these influences. Many types of media have been used for flushing and holding the embryos recovered from the female reproductive tract. These range from simple physiological saline through phosphate-buffered saline modifications and more complex tissue culture media. Embryos do not usually spend long periods of time in these media prior to their utilization; therefore media considerations are simple including osmolarity, pH, a macromolecular supplement, some form of anti-microbial agent and a buffering system compatible with use in atmospheric conditions. If embryos will be exposed to the flushing media for longer periods of time, some thought should be given to adding a source of nutrients. These considerations are applicable to recovering any stage of embryo for all species. Recovered embryos are often transported to other locations for transfer either as fresh embryos or after cryopreservation and therefore care must be taken to prevent disease transmission (Stringfellow and Seidel, 1990). Protocols for donor animal health certification and handling of embryos to prevent them from being a vector for disease transmission differ by country as does regulations for bringing in biologicals of any sort. It is imperative that these be consulted if embryo import or export for any species is contemplated. 3. P r i m a t e s

Limited protocols have been developed for the recovery of embryos in primates (Kreitmann et al., 1981; Adams, 1982), perhaps due to the introduction of nonsurgical methods for oocyte recovery. The structure of the primate fallopian tube makes manipulations for early embryo recovery difficult, with the possible risk of impairing tubal function and reducing subsequent fertility. In contrast, nonsurgical methods of uterine flushing have been reported (Pope et al., 1980; Buster et al., 1985; Sauer et al., 1989; Formigli et al., 1990). The methods are limited by relatively poor recovery

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rates and the risk of possible unwanted pregnancy associated with retained embryos. The relative ease of ultrasound-guided oocyte recovery, accompanied by relatively high rates of in vitro fertilization, have thwarted interest in further development of this technique for primates. 4. Exotics and Endangered Species

Techniques to be utilized for embryo collection, transfer, and AI in these animals will employ some variation of the protocols discussed in the preceding sections despite the many anatomical variations that will be encountered between species (Bush et al., 1980; Wildt et al., 1986; Loskutoff and Betteridge, 1992; Lasley et aL, 1994; Del Campo et aL, 1995; Loskutoff et aL, 1995). Where possible, interspecies transfers should be considered to make the best use of small breeding populations. This has been performed successfully in a range of exotics using a closely related abundant relative for the embryo recipient (Stover et aL, 1981; Bennett and Foster 1985; Dresser et al., 1985; Kydd et al., 1985; Pope et aL, 1989; Flores-Foxworth et al., 1995). Since the basic reproductive patterns of many of these species will not have been completely described, factors such as relationship between embryo stage and maternal environment, feasibility of superovulation and estrous synchronization, etc., will have to be learned for the individual species. Reproductive management techniques as basic as heat detection used to estimate embryo or estrous stage may not be available if overt signs of receptivity are not manifest, manifest briefly, or are not recognized. Unraveling the interspecies variations in reproductive patterns will take time and pooling of resources considering the paucity of experimental or observational material available. Filling in the blank areas in our understanding is required for improving traditional animal breeding and propagation techniques and also for increasing the repertoire of reproductive strategies that conservation biologists can apply to any particular species. Even the in vitro production of embryos requires a knowledge of how endogenous and exogenous factors affect competency and fertility of the gametes and embryos in order to interpret the success or failure of experimental attempts to develop adequate culture systems. Although the task is daunting, the preliminary successes in applying assisted reproductive stragegies already obtained to a variety of species in a relatively short period of time should create a feeling of optimism toward the possibility of effective management of conservation efforts. Although searching of literature citations is of value in approaching each new species, knowledge of failed approches rarely make the literature. Since application of assisted reproductive strategies as applied to exotic species is burgeoning quickly, inquiries via the various electronic bulletin boards available to those interested in gametes and embryos represent the most effective mode of obtaining current information and helpful suggestions for approaching a novel species. Frequent contact with experienced personae is highly recommended.

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B. C u l t u r e o f M a m m a l i a n E m b r y o s The ability to culture mammalian embryos from early preimplantation stages to those most compatible with successful cryopreservation and simple ET by nonsurgical deposition into the uterus means that compaction and postcompaction stage must be attained in vitro along with the capability of supporting their viability in vitro to be immediately applicable to the broadest array of species. Embryo culture, as an assisted reproductive technology, grew along with the embryo transfer industry to support the goal of improving genetics of animals useful to man. A lovely review of the history of mammalian embryo culture written by one of the pioneers in this field is worthwhile reading for those interested in pursuing this area (Biggers, 1987). A thorough understanding of embryo biochemistry, composition of the maternal secretions the embryo encounters during preimplantation development, and the determinants of embryonic differentiation and gene induction would facilitate design of an ideal culture system for a species. Since only imperfect knowledge concerning these factors exists for any of the species studied to date, we continue trying to optimize both our understanding and the culture systems available. As more data regarding progeny are accumulated from utilization of cultured embryos, we are beginning to move our benchmarks for measuring culture success to ever later time points in an animal's life history. Where observing frequency of blastocyst formation was once considered sufficient indication of an adequate culture system or of a useful modification to an established one, ability to initiate pregnancy soon succeeded this. After learning that many types of experimentally created embryos could initiate a pregnancy, including mere trophoblastic vesicles, yet not maintain it for the full length of gestation, the benchmark was again shifted to include production of live offspring which are physically normal and vigorous. With the suggestion that perturbations during embryo development and growth may prognosticate the tendency toward certain health risks during adult existence, the benchmark for adequacy of a culture system may again be shifted. Unfortunately, the further displaced in time is the endpoint from the act of culture, the less abundant becomes the pertinent data. Manipulation of embryos in culture has resulted in skewing of expected birth weights, increased morbidity, higher mortality, skewing of the sex ratio, and physical abnormalities in various species and under a variety of conditions which are generally not completely defined (see previous citations given under alternative uses of spermatogenic stages). Several of the previous citations used in vitro produced embryos where in vitro maturation and fertilization may themselves be suspect for producing long-range effects on embryos. We have scant knowledge of how different culture systems for oocyte maturation, fertilization, and embryo development interact or whether these interactions have consequences for embryonic and fetal

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growth or postnatal health. Previous words of caution concerning premature application of in vitro technologies must be given earnest consideration depending on the setting for implementation. With this in mind, a discussion of various systems for culture of preimplantation embryos will be presented. The readers are advised to consult the many excellent reviews on culture of mammalian gametes and embryos that have already been cited for details on the many aspects of culturing mammalian embryos successfully. 1. Biological Incubators

Before low temperature storage and culture of mammalian embryos was workable, the possibility of inserting embryos of an economically useful species into the reproductive tract of an inexpensive laboratory animal was explored as a means of transport (Adams, 1982). The rabbit was able to incubate embryos from several species of farm animals satisfactorily for several days in its oviduct. Later the possibility of using live animals to incubate xenogenous embryos after surgical transfer into the oviduct was resurrected to circumvent the inability of culture systems to overcome the so-called culture block (Willadsen, 1982). Embryos arrest in development at a cleavage stage characteristic of a species if culture conditions are inadequate to support development and which coincides with the time when activation of the embryonic genome is expected (discussed by Schultz, 1986a; Telford et aL, 1990). Both rabbits and sheep were able to incubate embryos of other species in their oviducts through to blastocyst formation with subsequent production of progeny after transfer of the blastocysts to recipients of the appropriate species (Willadsen, 1982; Boland, 1984). One of the first studies that reported a pregnancy (Critser et al., 1986) with subsequent production of a live calf (Figure 19) after the in vitro maturation and fertilization of oocytes from a long-cycle species used the sheep oviduct as a biological incubator for culture of the zygotes to the blastocyst stage. Pregnancies were also reported after in vitro oocyte maturation of bovine oocytes from another laboratory in that same year (Hanada et aL, 1986) with two calves being born in late 1985, while "Falcon" was born in February of 1986. Use of a biological incubator has fallen from popularity with the rise of in vitro developmental systems for a wider array of species, a revisitation may be worthwhile in animals where limited material severely restricts experimentation. An interesting variation on biological incubators utilizes fertilized chicken eggs as host for mammalian embryos (discussed by Gordon, 1994). Just as there is no universal method for capacitating sperm for the purpose of IVF, no one culture system works well for all the species in which in vitro development has been attempted to date. There are myriad considerations for optimizing culture systems for a species such as osmolarity, electrolyte balance, temperature, pH, gaseous atmosphere, metabolites, cofactors, vitamins, macromolecular supplements, and finally mediators of endocrine, paracrine, and autocrine interactions. Considering

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19 This is one of the first offspring born after in vitro maturation and fertilization of oocytes from a long-cycle species (Critser et al., 1986). Ovaries supplying female gametes were obtained from an abattoir. IVF utilized frozen-thawed semen. Zygotes were blocked in agar and surgically placed into oviducts of a ewe. After 6 days, agar blocks were recovered, embryos dissected free, and single blastocysts transferred nonsurgically to recipient cattle.

FIGURE

the current concerns regarding normality of embryo development obtained from even the most commonly used culture systems and the amount of material required to test adequately the design of components, a ready alternative may be the use of biological incubators. There is one report in the literature (Behboodi et al., 1995) that indicates use of a biological incubator after IVM-IVF generation of zygotes reduced the frequency of the "large calf syndrome" compared to culture of the IVP embryos in vitro. 2. Coculture S y s t e m s

An early in vitro method that successfully overcame the culture block was to put embryos into an oviduct and place it into organ culture (Biggers et aL, 1962). With relatively successful results being achieved from organ culture and recognizing that the oviduct environment could support development through blastocyst formation, efforts were directed toward growing embryos in association with other cell types (Bongso et aL, 1990, 1991, 1993; Bongso, 1995). Epithelial cells of the reproductive tract were obvious candidates for initial studies and soon reports of live offspring produced from embryos subjected to coculture began appearing in the literature (Gandolfi and Moor, 1987; Rexroad and Powell, 1988; Rexroad, 1989;

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Eyestone and First, 1989b; Gandolfi et al., 1989; Ellington et al., 1990b). Oviductal cells (Gandolfi and Moor, 1987; Eyestone and First, 1989b; Bongso et al., 1989), uterine cells (Jiang et al., 1991; Weimer et al., 1993), cumulus cells (Kajihara et al., 1987; Goto et al., 1988; Fukuda et al., 1990), granulosa cells (Jiang et al., 1990; Plachot et al., 1993), and trophoblastic vesicles (Camous et al., 1984; Heyman and Menezo, 1987; Heyman et al., 1987) have all been utilized in coculture systems with variable success. Not only the cells themselves, but medium conditioned by their presence could also be used successfully to culture embryos (Eyestone and First, 1989b; Ellington et al., 1990a; Eyestone et al., 1991; Kobayashi et al., 1992). Since primary explants were most frequently used in these studies, the logical extension of coculture was to examine the potential of stable cell lines to support development of embryos as coculture components. This would prevent the cumbersome aspect of collecting, preparing, and maintaining explants when required for culture and eliminate the many variables associated with using in vivo tissue. More uniform and repeatable results might be expected. Passaged cell lines such as buffalo rat liver cells (Looney et al., 1994; Hasler et al., 1995), green monkey kidney cells (Vero cells: Menezo et al., 1990, 1992; Lai et al., 1992), their conditioned media (HernandezLedezma et al., 1993; Chen et al., 1994; Vansteenbrugge et al., 1994), and other cell lines (Goto et al., 1992; Takahashi et al., 1994) can also be used in coculture systems resulting in postcompaction embryos that have in some cases been shown to be developmentally competent. The properties of coculture systems that lead to developmental success are unknown (for discussion see Gardner, 1994; Bavister, 1995; Leese et al., 1995; Barnett and Bavister, 1996). Obviously the embryo is supported in development by surrounding tissues in its normal environment. Moreover these associations are continually changing as the embryo passes through the various regions of the female reproductive tract, leading to the development of appropriately staged coculture systems (Bongso et al., 1994). Coculture has not eliminated the potential for developmental anomalies or large birth weights and at this point in time we have no data to determine whether it reduces such possibilities. Use of cellular components or cell conditioned medium in research designed to study developmental processes or the effects of media constituents on these processes is also not advantageous in disclosing these interactions (Bavister, 1992). Yet coculture systems in conjunction with tissue banking efforts only require that culture be done at fairly high levels of success and that offspring resulting not be compromised to any extent greater than that currently found with other assisted reproductive techniques. As in the use of biological incubators, if offspring are the end objective and material is limiting, coculture systems may be an effective alternative in the application of assisted reproductive strategies. In the end, the final use of the genetic material and species application will dictate selection of a culture system at present. As basic research on

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requirements for successful culture of early mammalian embryos continues, the future should bring more defined culture systems that will work for a wider variety of species.

3. Defined Culture Systems As recently pointed out, there is no universally accepted definition for the use of defined media in conjunction with culture systems (Thompson, 1996). Try as we may to completely control the components of culture systems, there are many sources of potential contaminants and uncontrollable variables to which the embryo is exposed in vitro. Examples are the variable acetate and citrate levels of contamination in different lots of Hepes buffer (Abas and Guppy, 1995) and bovine serum albumin (fraction V; Gray et al., 1992), respectively, and their consequences for in vitro embryo development. Contaminants in commercial chemicals also indicates the need to assess various sources and lots of a particular constituent when used as an experimental treatment. If this is not done, then studies characterize only a specific lot or batch of a substance rather than its role as a constituent of culture media or as a factor in development. Many culture systems utilize oil overlays to prevent evaporation of medium when small volumes are used. This will change the effective concentration of both water-soluble constituents and those that are more hydrophobic. The popular use of membrane filtration to sterilize fluid components of culture systems can add contaminants to the media or again reduce the effective concentration of media additives by binding to the filter as in the case of steroids. The embryo itself modifies the original environment of the culture system by both metabolic processes and synthetic and secretory processes (Wiley et al., 1986; Lane and Gardner, 1992; Quinn et al., 1993; Gardner et al., 1994) so that we cannot think of even defined culture systems as unchanging, but rather must recognize their more dynamic nature (Rieger, 1992; Rieger et al., 1992; Martin et al., 1993). With the seemingly endless number of variables to be taken into account from source and quality of water used in making media (Nagao et aL, 1995) to the gas supply used to control the atmosphere of incubation, the concept of defined culture may be more of an ideal than a reality. Approximating this ideal effectively directs our thinking toward contemplation of the culture system as a unified whole and at minimum will increase efforts toward quality control measures and repeatability of methods. Using the nebulous working definition that defined culture systems support development without dependence on cellular components (recognizing that body fluids may still be used as source of macromolecular supplements), defined culture systems may be placed into two broad categories depending on whether the base medium utilized is complex or one of the simple physiological saline solutions previously mentioned (Table 1) that has a limited number of additional constituents added. With the increas-

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ing emphasis toward the addition of amino acids, vitamin mixtures, and other supplements to the simple salt solutions, the demarcation between these two categories is rapidly becoming blurred. There are many fine reviews covering the basic components of culture media, elements of a culture system, culture media available, and successful application of systems to a particular species (Bavister, 1987; Kane, 1987; Gardner and Lane, 1993; Gardner, 1994; Gordon, 1994; Johnson and Nasr-Esfahini, 1994; Bavister, 1995; Leese et al., 1995; Barnett and Bavister, 1996; Thompson, 1996). Our working knowledge of how such factors as pH, osmolarity, gaseous atmosphere, media constituents (i.e., amino acids, energy substrates, vitamins, growth factors and cytokines, protein supplements, lipids), and culture systems influence early development is greater for the mammalian embryo than for most gametogenic stages, except perhaps the mature spermatozoon. Despite this knowledge, we are a ways from being able to culture most mammalian embryos in completely defined systems with verification that embryo ontogeny is not compromised. Pertinent to this book is the observation that IVP embryos do not respond to current cryopreservation protocols as well as their in vivo recovered counterparts, and indeed may differ in other aspects (discussed by Wright and Ellington, 1996). Work is ongoing to optimize these systems for common species studied to date and to extend their use to new species. Little work has been done with assessing normality of fetal growth and development with the more highly defined systems. Scant to no data is available comparing this endpoint with products of natural matings or AI, between products of various culture systems or assessing how individual components impair or enhance fetal growth and differentiation. Choice of system will be influenced by many factors, including those pertaining to biology, use scenario and, unfortunately, oftentimes expediency. Although useful tools for research and current application, all the methods for embryo culture from use of biological incubators through to more defined systems should be approached with discretion. Much discussion exists concerning the best way to evaluate the outcome of embryo culture and in vitro embryo production in general. The acid test is of course, the production of live and normal offspring although this is not always feasible. Protocols for evaluating embryo viability both for experimental purposes and for selection of embryos for transfer and freezing have been attempted almost from the beginnings of embryo culture and transfer (Betteridge, 1977; Renard et al., 1978; Whittingham, 1978). A number of reviews discuss methods currently available (Rieger, 1984; Edwards, 1987; Overstrom, 1987; Butler and Biggers, 1989; Gerrity, 1992; Wassarman and DePamphilis, 1993; Rondeau et al., 1995; Overstrom, 1996). Again it must be emphasized that these will not predict normality of fetuses. The goal of these methods is to predict embryo viability and success of pregnancy initiation.

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V. CONCLUDING REMARKS Basic aspects of mammalian gametogenesis and early embryogenesis have been presented as they occur in vivo. In vitro tissue maturation systems which support portions of these biological processes have also been described. To serve the investigator possessing a serious intent to work in these areas with a broad perspective of the subjects and potentials for use, summaries and reviews have been relied on to provide a wealth of starting materials and alternative viewpoints. Other chapters in this book will indicate current mastery of preserving various reproductive tissues with subsequent application and indicate future perspectives. The material in this chapter, viewed against the background of possibilities for tissue banking, should enable the conception of many combinations of reproductive technologies that make use of both in vivo and in vitro tissue maturation to preserve genetic information for a variety of purposes. Basic principles of quantitative and population genetics should be taken into account to determine how gene banking can enhance the gene pools of current breeding populations and maintain sufficient genetic variability to overcome potential catastrophic situations. To achieve the most flexibility in terms of salvaging genetic material and planning future application scenarios, brief mention was made of novel technologies that have not reached maturity or have yet to be adapted for a broader array of species. With the increasing assembly of techniques possible and intensifying scientific and societal interchanges concerning goals engendering their utilization, human and animal health, production of animal products, and preservation of animal germplasm for future generations will be protected. REFERENCES Aalseth, E. P., and Saacke, R. G. (1986). Gamete Res. 15, 72-81. Abas, L., and Guppy, M. (1995). Anal Biochem. 229, 139-140. Abramson, F. D. (1973). Soc. Biol. 20, 375-403. Absher, M. (1973). In Tissue Culture: Methods and Applications (P. F. Kruse, Jr., and M. K. Patterson, Jr., Eds.), pp. 395-397. Academic Press, New York. Adams, C. E. (Ed.) (1982). Mammalian Egg Transfer CRC Press, Boca Raton, FL. Adashi, E. Y., Resnick, C. E., D'Ercole, A. J., Svoboda, M. E., and Van Wyk, J. J. (1985). Endocrinol. Rev. 6, 400-420. Adelman, M. M., and Cahil, E. M. (Eds.) (1989). Atlas of Sperm Morphology. American Society of Clinical Pathologists, Chicago. Adler, A., McVicker, R., Bedford, J. M., Aliani, M., and Cohen, J. (1993). J. Assist. Reprod. Gen. 10, 67-71. Afzelium, B. A. (1972). In Edinburgh Symposium on the Genetics of the Spermatozoon (R. A. Beatty and S. Gluecksohn-Waelsch, Eds.), pp. 131-143. Univ. of Edinburgh, Scotland. Aktas, H., Leibfried-Rutledge, M. L., and First, N. L. (1991). In Preimplantation Embryo Development (B. D. Bavister, Ed.), Abstract 25. Serono Symposia USA, Norwell, MA.

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Metabolic Support of Normothermia R o y H. H a m m e r s t e d t a n d J a n e C. A n d r e w s Department of Biochemistry and Molecular Biology The Pennsylvania State University University Park, Pennsylvania 16802

I. W H Y CARE ABOUT METABOLISM AT NORMOTHERMIC C O N D m O N S ?

A. A View of Integrated Cell Function Tissue storage represents a unique condition imposed upon cells unanticipated in their cell cycle. In fact, any storage environment imposed for technological reasons probably represents a demand to respond to conditions unanticipated anywhere in that cell's evolutionary history. If we assume that cell function (what we seek to preserve) evolved with design features to support it, an understanding of function leads to a need for the appreciation of the integrated (designed) systems used to support life. Metabolism is broadly defined as the sum of all processes by which an organism is produced, maintained, and destroyed. Thus, satisfying the goal of preservation of function leads to a need to preserve metabolic processes. Complete satisfaction of that goal is probably remote in that we have not (will not?) accumulated a complete description of all metabolic aspects of any cell. However, if we focus on core metabolic features, with special emphasis on "why" they are important rather than what exact pathway is used to get from "here to there," sufficient detail Reproductive Tissue Banking Copyright 9 1997 by Academic Press. All rights of reproduction in any form reserved.

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can be gleaned to allow design of appropriate tests to assess retention of basic functions. Two decades ago Daniel Atkinson (1977) wrote on the relation of energy metabolism to overall cell function. The following quote (pp. 225226) reflects some of the concepts he wanted to address. "In urging that metabolic function and molecular aspects of living organisms be considered as functionally interrelated products of evolutionary design, it would be difficult to improve on Darwin's words in one of the greatest and most personal passages in "The Origin of Species," written, of course, when virtually nothing was known of metabolism or the molecular constitution of organisms: When we no longer look at an organic being as a savage looks at a ship, as something wholly beyond his comprehension; when we regard every production of nature as one which has a long history; when we contemplate every complex structure and instinct as the summing up of many contrivances, each as useful to the possessor, in the same way as any great mechanical invention is the summing up of the labor, the experience, the reason, and even the blunders of numerous workman; when we thus view each organic being, how far more interesting~I speak from experie n c e ~ d o e s the study of natural history become!

When living things are considered in this light, the study of biology generally, and of biochemistry in particular, becomes a matter of attempting to unravel the functional interrelationships of a very intricately engineered mechanism." The challenge that had to be faced over the past 50 years in the study of metabolic features of cells is a reflection of a type of "biological uncertainty principle." The more we know about detail of metabolic features (accumulated by the classical biochemical approach of disrupt, isolate and characterize) the less we know about how each data bit fits into the overall schema of the cell. The latter information can be accumulated only from parallel analysis of intact cells, in the environment of interest, testing the role of each data bit in the functional design of a cell in that specific environment. An analogy is provided to illustrate a point. Your goal is to assemble teams of experts to help you to understand the features within an automobile responsible for its locomotion. The restriction is that you cannot use the same teams to disassemble the automobile to its constituent parts that are used to analyze automobile performance. In fact, let us add the restriction that the teams speak only distantly related languages and have very different approaches to life. Details of engines, drive trains, fluid and electrical systems accumulate; performance characteristics are defined. You now are asked to assemble all of this into a cohesive scheme on how the automobile converts chemical energy (fuel) into mechanical energy (motion) at less than 100% efficiency. What can you do? Measure fuel type and consumption rate? Measure quantity and identity of waste products? Probe with Xray and acoustic equipment to assess internal organization? Reasonable

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estimates of integration would accumulate, but t h e n . . . Fuel shortages dictate new assemblages! Market changes drive exports to new climates of extreme heat or cold! Fiberglass replaces metal, changing the relationship of body size and mass to drive train! Now what? Through careful examination of data available on both exterior and interior features a model can be assembled, and quantitative predictions can be made based upon the assumptions of the model. Externally induced changes violate those assumptions, and the model collapses and the cycle of study must begin again. The generation of new models is assisted by reflection upon the older models once their limits are understood. The same cycle of refinement occurs in biological studies, where core information of "general" value is sorted and modified to allow generation of refined estimates for specific use. In the case of reproductive tissues, we must begin with general knowledge accumulated by a multitude of investigators with many different cell types to provide a general framework and then customize this to the cell type of interest. A series of brilliant people attacked this problem over the years, with one estimate of their collective efforts being those huge metabolic charts (two-dimensional constructs, presented as if all aspects are phased in time) that were intended to describe life (three-dimensional constructs, with unique phases of function). Although students flounder now in this information, overwhelmed by detail, those people, by virtue of their unique backgrounds and broad experiences in a time when specialization was not rampant, understood. They thought like a cell (or at least attempted to do that). How do we pick up where they left off? How do we use those contributions to make sense of our system of interest? An initial approach might be to read the personal accounts of those who were there. Favorites include collections honoring Lipmann (Lipmann, 1971), Ochoa (Kornberg et al., 1976), Hans Krebs (Estabrook and Srere, 1981), Kornberg (Kornberg, 1989), and the autobiographical reflections found in each Annual Review of Biochemistry. The detail slowly takes perspective. Then, think about your cell of interest and its environment. What did it do? What does it have to do? Who are its neighbors? Any person interested in tissue or cells must develop a intuitive feeling for the system in order to move generalities from past studies of other systems to the system of their interest. After a while one develops a feeling of awe for the integrated system which has evolved to provide the flexibility necessary for the cell to withstand challenges and flourish in the environment to which it has adapted.

B. Effect o f Cell Storage o n Metabolic Balance Effect on "normal" metabolic processes must be considered when the cell is removed from its supporting environment in the intact animal, often subjected to temperatures below which it has adapted its functions, stored

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in media often incapable of adding all (most often, unknown) nutrients and removing waste products, warmed toward its host temperature, and then returned to an environment similar, but not identical, to that of the host. To paraphrase Hammerstedt et al. (1990), one should praise the survivors of such abuse rather than despair of our inability to readily extend apparently successful protocols from one biological system to another. Maintaining metabolic balance is central to cell and tissue survival through such manipulations, and this contribution focuses on critical factors that must be considered during the evolution of successful protocols. C. I n t e g r a t i o n o f T h e s e C o n c e p t s i n t o T h i s C h a p t e r The preceding description of the breadth of metabolism is useful to recognize the conceptual scope of the discipline, but applications to reproductive tissues need only a fraction of that material. This chapter will briefly outline the overall metabolic features associated with cells of the testis and ovary, emphasizing the abrupt transitions that occur in spermatogenesis, epididymal maturation, oogenesis, fertilization, and early development. Then, keeping with the concept that emphasis should be placed on cells likely to be stored/processed, key features of sperm, eggs, and embryos will be emphasized. A common feature of the listed cell types, the need for balanced synthesis and degradation of ATP, will be addressed in general terms. Detailed discussions illustrating that balance will be introduced by use of the most intensively studied cell type, sperm. The metabolism of sperm will be reduced to a description of the rate(s) of ATP turnover [reflecting rates of ATP synthesis and degradation]. Examples from the literature will illustrate differences associated with developmental state, species, and slight changes in temperature. A framework is supplied for use in constructing a similar analysis in other cells will be provided, with a summary of pertinent literature to begin such an analysis.

II. OVERVIEW OF METABOLIC NEEDS OF CELI~ OF REPRODUCTIVE INTEREST A. H e t e r o g e n e i t y in Metabolic R e q u i r e m e n t s Cells of reproductive interest probably have very different metabolic needs, reflecting their changing roles and degrees of specialization (Hammerstedt, 1981). On the male side, beginning in the testis, the demands of cellular multiplication, differentiation, and specialization set up a system with overall requirements similar to those for most somatic cells. In the terminal phases of testicular development, the cells pass from those with diploid, pluripotent capacity to more modest requirements of haploid cells

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completing differentiation. Upon spermiation and exit from the testis into the epididymis, most of the gross morphological changes are complete (along with the requisite complex metabolism needed to yield new structures) but the sperm are continually modified (Amann et al., 1993) during epididymal transit. The cells then reside in terminal aspects of the epididymis until delivery to the site of fertilization. Once puberty is passed, the process continues daily in most males until death. In females, rapid development and specialization, supported by complex metabolism, occurs early in life, leading to an ovary stocked with eggs "partially developed" and held in an arrested state. After puberty, a small subset of those eggs periodically are recruited for rapid terminal development with further metabolic needs to yield secondary oocytes which are released into the female tract (Hafez, 1993a). In the case of natural mating, where emission of sperm from the male into the female tract initiates the terminal phases of fertilization, the following changes occur to each haploid gamete as it prepares for and completes syngamy and enters into development as a unique diploid form. Sperm exiting the male by ejaculation mix with accessory gland fluids and are irreversibly changed. Interaction with female tract fluids initiate further changes, loosely termed capacitation (Austin, 1951; Chang, 1951), which have additional metabolic implications. The net result of these changes is residence within the female tract of sperm quite heterogeneous with regard to "readiness" for fertilization. Spermatozoal storage, perhaps associated with altered metabolic state, in the female tract occurs, with subpopulations held within specialized glands in some species (birds), or associated with oviductal involutions in others (Yanagimachi, 1994). Why the heterogeneity and/or the storage? Perhaps because transport of the oocyte down the female tract cannot be (at least, is not) synchronized precisely with sperm deposition, and this strategy allows for maximum probability of having an ovum interact with one sperm deposited during a copulatory event. Fertilization results in fusion of two haploid cells, each with relatively simplified metabolic needs, to form a diploid ovum. Subsequent cell divisions with associated biosynthesis of all necessary organelles and specialized proteins reintroduces complexity in metabolism in the reproductive cycle. While continuing down the female tract, cellular numbers increase and compaction occurs, leading to formation of the morula and then blastocyst. Unique and highly specialized, three-dimensional organizational features emerge. With implantation in the uterus, existence as a free-floating cell ceases and the complex nurture of embryonic development continues (Bazer et al., 1993). These sweeping generalities outline common processes, but speciesspecific differences in timing and/or mode of satisfying common requirements force critical inspection and reflection by any investigator wanting to incorporate the theme into the system of interest. A detailed listing of

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these differences is beyond the scope of this presentation, so simple illustrative examples will be used. Harvest of specialized, species-specific data from the recent literature can be achieved by careful computer search using species as a descriptor. For instance, a Medline search using descriptors of "Energy Metabolism" and 'Spermatozoa/Egg/Embryo" in their general (expanded) forms provides an entry point from which additional primary literature can be identified. B. S e l e c t i o n o f Cell T y p e s f o r D i s c u s s i o n This text is intended to introduce the reader to the range of factors to be considered in tissue banking of tissues of reproductive interest. This restricts the cell types to those most easily removed from donors and, except for cases where the animal is dying or gonads must be removed, restricts the focus to those cells which can be repeatedly obtained from the same donor. In all cases an alternative paradigm for sperm deposition from that of natural mating must be adopted. This is most often artificial insemination, either in a laboratory setting (in vitro fertilization with an isolated ova) or by deposition in an appropriately endocrine-primed female. An argument can be made for obtaining the most mature cell type possible, to allow maximum development in the intact animal before artificial technologies are introduced. For the male, ease of cell isolation was discussed (Hammerstedt, 1981) in context of obtaining sufficient cells for metabolic studies. Ejaculated sperm are most easily obtained, but in some cases (e.g., rodents) where ejaculates are difficult to obtain, cauda epididymal sperm might have to suffice. Thus, this survey will be restricted to those two sperm cell types, with the potential of using them for either artificial insemination into the female tract or for in vitro fertilization of isolated ova. For the female, artificially induced ovulation (Hafez, 1993a) can shed multiple oocytes for recovery and use from intact animals while ovariectomy (Hafez, 1993b) can be used to obtain large numbers of secondary oocytes for in vitro maturation to their fully competent state (Hafez, 1993b). Thus, the female haploid cell of interest to this discussion is the unfertilized oocyte. Finally, it is possible to complete in vitro fertilization to generate the diploid zygote, which, after appropriate incubation to allow further development, can be stored until deposited in the female tract for implantation and completion of development. m . SCOPE OF THIS REVIEW A. G e n e r a l B i o e n e r g e t i c P r i n c i p l e s Bioenergetic analysis is derived from first principles of thermodynamics. General textbooks provide a convenient summary of terms and concepts

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(Voet and Voet, 1990; Stryer, 1995). The reader is directed to Atkinson (1977) for an extended general discussion and Hammerstedt and Lovrien (1983) for a treatment restricted to sperm analysis. The treatment begins with the recognition that the tendency of a reaction to occur is indicated by the relative Gibbs free energies of input nutrient (e.g., glucose) and output product (e.g., lactate). If alternative products are formed (e.g., CO2), different reaction tendencies are predicted. Thus, all cells in the presence of glucose have: (a) the same overall potential to undergo that reaction and (b) no limits on choice of specific mode for carrying out that transformation of nutrient to product. Over time, it is reasonable to assume that many different modes for that transformation were tested, but for unknown reasons only a few of those choices have been retained in contemporary cells. This sets up a situation whereby the cell, if provided with suitable nutrients and having the genetic potential to produce necessary catalysts, can carry out a transformation of: Nutrient ~ Product + Energy. The flow of nutrient to product is assured by ability for each cell to make catalysts (via its repository of information in DNA and expression of that information via protein synthesis) as needed. These stepwise transformations (which constitute a metabolic pathway) provide for incremental changes in the structure of nutrient to one ever more similar to that of the final product. The unique compounds between initial nutrient and final product are termed metabolic intermediates. Each individual step has a characteristic Gibbs free energy relationship and an energy release or input. Metabolic intermediates can accumulate when the rate at which the product of one reaction is produced is faster than it is consumed by the next reaction. Thus, the flow of materials is uptake of nutrient from the surroundings, passage through a series of metabolic intermediates found within the cell, to excretion of product to the surroundings. Metabolic intermediates are critical to an understanding of cell function because of their multiple roles in cells. They are used for three purposes, independent of their roles in the specific flow of carbon to product: (a) energy storage (ATP/ADP), (b) source of reducing power (NADH/NAD, NADPH/NADP), and (c) service as critical intermediate(s) for biosynthesis of compounds not provided in the exterior environment. Cell types differ in their extent of use of these roles, with energy storage and reducing power universal in importance, and intermediates reflecting more specialized cellular needs. This organization of function, involving nutrient and products connected with metabolic intermediates serving multiple purposes, leads to the concept of metabolic stoichiometries (Atkinson, 1977). Examples will be provided by focusing on ATP related metabolism and its association to carbon flow. First, reaction or simple conservation stoichiometry must be satisfied. Any valid equation, for either a simple reaction or an extended

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Roy H. H a m m e r s t e d t a n d J a n e C. A n d r e w s

metabolic pathway, must have the same number of atoms on "both sides of the arrow":

C6H1206 ~ 2 C3H603

[1]

The second is obligate coupling stoichiometry. This results when interactions between metabolic sequences, often derived from shared use of metabolic intermediates, have stoichiometric relationships fixed by the chemical nature of the processes. A common example is the pairing of oxidation [removal of electrons] and reduction [addition of electrons] reactions, where oxidation cannot continue if the electrons are not deposited somewhere. This is evident in the overall balanced equation for complete oxidation of glucose and reduction of oxygen:

C6H1206 + 6

0 2 "--)

6

C O 2 -]--

6 H20

[2]

The relationship of the separate subsets of this equation is evident when we focus on the role of cofactors used in the separate processes in most mammalian cells. The oxidation is described by

C6H1206 + l0 NAD + + 2 FAD + 6 H20 6 CO2 + 10 N A D H + 10 H + + 2 FADH2

[3]

The paired reduction reaction, representing the respiratory chain, is illustrated by 10NADH+

10H + +2FADH2+602 10 NAD + + 2 FAD + 12 H20

[4]

Since the reaction has to be balanced, the cofactors reduced in one subset (Eq. [3]) are reoxidized in the second (Eq. [4]) and the sum is the overall balanced reaction (Eq. [2]). The overall oxidation-reduction principles (Eq. [2]) are a feature of chemistry and cannot be altered by evolution. To provide an example of use of this information, consider the case where an unknown cell suspension is subjected to metabolic assay and the moles of both O2 and glucose consumed over a period of time are measured. If mathematical comparison of the measured values were completed, and found to have a stoichiometric ratio of 1:6, the investigator could conclude that the only type of respiration-linked metabolism occurring in that cell is described by Eq. [2]. If a ratio of less than 6 were observed, that would indicate that carbons from glucose were being diverted to something other than CO2 and an incompletely oxidized form of carbon is accumulating somewhere. If a ratio of more than 6 were observed, it would indicate that something other than glucose is being oxidized at the same time that glucose is present and that an unknown source of electrons generated from that conversion is interacting with the respiratory chain. The last stoichiometry consideration is termed evolved coupling stoichiometry, and it will be illustrated by examples using ATP/ADP. The relation-

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ship of the adenine nucleotides to any balanced reaction is not fixed by any chemical necessity, but is a function of the evolutionary choice of the organism. Thus it cannot be predicted from any chemical consideration and can be estimated only by detailed analysis of the type of catalysts and cofactors in the cell. This reconstructed metabolic pathway is an estimation (best guess) of the most likely combination of steps involved in the transformation of nutrient to product. As an example, the moles of NAD and FAD indicated in Eq. [3] are derived from detailed evaluation of the specific reactions involved in some cells in the glycolysis (Embden-Meyerhof) pathway and tricarboxylic acid (Krebs) cycles. Not all cells accomplish the transformation in the same way. Thus, based upon results of >50 years of research, the accumulated data strongly suggest (but do not prove) that the evolved coupling stoichiometry for a cell using the Embden-Meyerhof pathway for conversion of glucose to lactate is shown in

C6H1206 + 2 ADP + 2 Pi--+ 2 C3H603 + 2 ATP + 2 H20

[5]

The overall equation is balanced and depends on the assumption that that specific mode of transformation is being used in the cell, and the inferred ATP yield for that transformation is 2 moles per mole glucose consumed. If another pathway were present, another estimate of ATP formed per mole of glucose consumed would be needed. The importance of these features are shown when the estimate for ATP yield from the combinations of Eqs. [3] and [4] are made. Note that Eq. [3] represents the oxidation process, with electrons deposited on the cofactors N A D H and FADH2. Reaction [3] cannot occur again until reaction [4] occurs. This requires that the reduced forms of cofactors (NADH and FADH2) interact with the mitochondria. Within the mitochondria the process of oxidative phosphorylation occurs, with the first name (oxidative) relating to the oxidation of the cofactors during respiration by transfer of electrons to oxygen and the second name (phosphorylation) relating to the synthesis of ATP from ADP + Pi. Thus, the exact relation of moles of ATP formed per mole of either N A D H or FADH2 oxidized depends on the specific mechanism used by the cell. While experts still discuss details of exact ATP yield per mole of either reduced form, we will use the ratios indicated in Eqs. [6] and [7]. The H20 appears twice as a reminder that the first is due to the oxidation and the second is due to the phosphodiester formation in the condensation of ADP and Pi: FADH2 + 1/2 02 + 2 A D P + 2 Pi --+ FAD + 1 H20 + 2 ATP + 2 H20

[6]

N A D H + 1 H + + 1/2 02 + 3 ADP + 3 Pi -+ NAD + + 1 H20 + 3 ATP + 3 H20

[7]

With these assumptions, any cell shown to have the balanced Eq. [1] would be described as Eq. [8] and have an inferred ATP yield of 38 moles ATP per mole glucose consumed:

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Roy H. Hammerstedt a n d Jane c. Andrews

C6H1206 + 6 02 + 38 ADP + 38 Pi--> 6 CO2 + 38 ATP + 44 H20

[8]

From these considerations, it becomes apparent that a detailed understanding of bioenergetics requires information on the metabolic rate of the intact cell under the conditions of interest: (a) quantitative information on the types of nutrients consumed, (b) amount of oxygen consumed, and (c) type and amount of products formed. From these data, one can work toward an understanding at the obligate coupling level. Detailed studies of organelles, enzymes, and cofactors within the cell will allow a first estimate of the type of metabolic pathway used to convert substrate to product. This will allow an estimate of the evolved coupling stoichiometry and a first approximation to the ATP yield as a result of that metabolic pathway. In cases where a metabolic intermediate can proceed down several different pathways, each with a different ATP yield, approximations become more difficult. For example, glucose 6-phosphate can be further metabolized by: (a) dephosphorylation to form glucose + Pi, (b) entry into the E m b d e n Meyerhof pathway of glycolysis, (c) entry into the pentose phosphate pathway, and (d) conversion to glycogen storage polymer. In these cases, more detailed analyses are necessary to allocate overall glucose consumption to each of its four possible metabolic fates.

B. C r i t i c a l Q u e s t i o n s to Be D e v e l o p e d i n T h i s P r e s e n t a t i o n Additional definitions must be provided to focus our comments for further discussion. In each case, that definition is followed by a critical question (in italics) of great importance to describing the metabolic status of the cell in its current environment. Normothermic is taken to mean temperature of the cells before isolation, or close to body temperature. What blend of metabolism satisfies cellular need, and what effects can be anticipated with slight changes in temperature? Metabolism, in its most general sense, is the sum of all chemical and physical events associated with "life." This is too broad for meaningful discussion so we have restricted our discussion to those reactions which account for general aspects of ATP turnover, the steady-state rates of ATP biosynthesis and degradation. What are the relative rates of these processes, and what can happen when rates of A TP synthesis and degradation are suddenly altered (termed metabolic mismatch)? The concept of ATP serving as metabolic currency was recognized over 60 years ago, now is introduced into the earliest levels of biological study, and yet remains misunderstood. The central facts that drive integrated ATP metabolism can be recognized if one takes an accountant's balance sheet view of the cell: (a) you cannot spend what you do not have, (b) cash flow is understood only by detailed analysis of modes of income and expense, and (c) a reserve must be retained to assure that cash flow can be sustained.

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What are the implications to cell survival when it is placed in a state of metabolic mismatch, where rates of A TP synthesis and degradation are not equal, especially when in all its previous evolutionary selection that imposed choice was never considered? Development of a balance sheet approach to the study of metabolism is slow (yielding minimal publications), repetitive (only the methodical and very precise need apply), and a bit out of favor (the peak in enthusiasm for such studies was 30 years ago). Thus, few completed examples exist for any cell type. The topic of integrated sperm metabolism has been carefully studied, when interested personnel could be found and funds could be solicited, in this laboratory over the past 25 years. As a result, that system provides a prototype of what can be done, and will be used to highlight important concepts. A compendium of reviews of data for other cell types is attached, providing a start for the interested reader to the status of less intensively studied systems. IV. OVERVIEW OF INTEGRATED ATP METABOLISM A. T h e ATP C y c l e The central reaction of metabolism is contained in Figure 1. ATP synthesis, derived by a host of different transformations of carbon compounds in the cellular environment, is accomplished by coupling oxidation/ reduction reactions to a few key enzyme systems. Thus, many pathways converge, with the end product being ATP synthesis. From this figure emerges three of the most important concepts to be made in this presentation: (a) ATP is never made unless A D P is present, or alternatively stated, ATP consumption sets the pace with ATP synthesis following; (b) the critical value in evaluating overall metabolism is the rate of this cycle (rate A D P + Pi Mode of synthesis is dictated by enzymes in cell and nutrient availability.

Consumption via multiple pathways d e p e n d s o n cell status, w h i c h in tum drives ATP synthesis.

ATP + H20 FIGURE 1 The ATP-ADP cycle. This sequence of reactions reflects the core of bioenergetic metabolism. Each reaction set is composed of subsets of reactions whose contributions to total ATP turnover depends on the cell type and nutrient availability. Details supplied in the text.

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Roy H. Hammerstedt and Jane C. Andrews

of ATP turnover), not the rate at which any given substrate is being removed from the cellular environment; and (c) if synthesis cannot satisfy demands of consumption, the cell cannot survive. These factors combine to set certain limits on analysis of cellular bioenergetic balance. Measurements of ATP, ADP, and AMP are useful for detecting the balance between ATP generation and consumption either by their direct comparison or via derived calculations such as energy charge (energy charge (EC) = [ATP] + 0.5[ADP] / ([ATP] + [ADP] + [AMP])). When cell suspensions evaluated under two different conditions have equivalent EC values, the assumption can be made that rates of ATP synthesis and use are balanced under those two conditions. No statement can be made about the absolute rate of ATP turnover. Measurement of any given pathway is of modest value, unless its absolute importance to total ATP synthesis is known. Consider a hypothetical study (representative of many examples in the literature) wherein the observation was made that cell type X under holding condition Y consumes less glucose per cell in the suspension after N hours of treatment. At least three interpretations can be made from these data, each having a unique impact on further studies with this system. (a) Is this a significant event in bioenergetic balance, indicating that ATP consumption has dropped? (b) Has a fraction of the cells in suspension lost viability, leaving fewer cells to consume glucose? (c) Is this a case where the cell has the same overall metabolic rate, but just chose to consume something else from its surroundings with no effect on bioenergetic balance? Nothing can be concluded without further information. If ATP status were known, and were equal in the two cases, option (b) can be excluded but no distinction can be made between (a) and (c). If total rate of ATP turnover were known, distinction between (a) and (c) can be made. Examples showing how something as modest as a change in mode of sample preparation can alter the relative importance of glycolysis to the ATP budget clearly illustrates the point (Hammerstedt, 1975b, 1981). Since rate of ATP consumption sets the pace for overall metabolic rate, it is critical that its absolute rate be known and interesting to know how that pool of ATP is distributed among various competing pathways. In general, rate of chemical reactions (i. e., metabolism) doubles with every 10~ increase in temperature for any cell type. Thus, metabolic rate for cells held at 37~ would be expected to be about 4• those of cells held at room temperature (17~ However, when washed ejaculated bovine sperm were compared at these temperatures, apparent glucose consumption rate increased five- to eightfold while overall total ATP turnover rate increased 3• (Inskeep and Hammerstedt, 1985). Why do the estimates differ? That will be discussed later in the presentation. However, the example is used here to introduce the fact that when only one metabolic feature (glucose

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consumption) is used as a marker to evaluate more general aspects of bioenergetic balance, errors in estimation can be made. B. M o d e s o f ATP G e n e r a t i o n U s e d By S p e r m The simplicity of spermatozoal metabolism relative to somatic cells, ova, or embryos is illustrated in Figure 2. Metabolic features of even complex cells can be reduced to three interdependent boxes (Figure 2a). Input of complex carbon compounds, plus oxygen, allows degradative metabolism to convert those materials to simpler molecules with side accumulation of ATP, NADPH, and 10-15 critical metabolic intermediates (e.g., phosphoenolpyruvate, oxaloacetate). The pathway used within that box depends on the enzyme content of the cell at that stage of its development and the menu of compounds in its surroundings. These accumulated metabolic intermediates then are used to synthesize monomeric materials not present in the cellular environment. Note the central roles of the ATP and N A D P H cycles in these interactions. Finally, the monomers are combined during growth and cell division to form the essential polymers of the cell. This complex metabolic scheme allows the cell to adapt to the wide variety of demands found during its life cycle. Detailed discussions have been preA ADP + Pi

Q

Input of fat, -] I protein, [_~ carbohydrate I and oxygen _1 I Output of " ~ CO2, 820 and waste

Input of q hexoses, C-3 [ - - p and C-4 a c i d s J

[i ~

Critical intermediates

1i

ATP + H20 NADP 11

L._~[

Q

NADPH + H + Critical intermediates

iI

r~ O oH

O

m

lJ

Degradative metabolism featured Glycolytic, with no glycogen or pentose cycle. TCA cycle is critical.

Output of " l ~ CO2, H20 and waste

Mitochondrial metabolism important. Mixture of endogenous and exogenous metabolism

Schematic representation of metabolism. (A) Metabolism of complex cells, such as an embryo or diploid germ cells. (B) The simplified metabolism of sperm. Modified from that of Atkinson (1977). Details supplied in the text.

FIGURE 2

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Roy H. Hammerstedt a n d Jane C. Andrews

sented on the role of these metabolic boxes in general metabolism (Atkinson, 1977) and the changes associated with spermatogenesis and epididymal maturation (Hammerstedt, 1981, 1993). The schematic representation of spermatozoal metabolism (Figure 2b) illustrates that the terminal phases of spermatogenesis "removed" two metabolic boxes, along with the NADP cycle, leaving only the degradative aspects. Input substrates are limited to a few materials (hexoses and 3Cand 4C- acids) that are metabolized by glycolysis, the tricarboxylic acid cycle (TCA cycle), and mitochondria to yield output waste products and ATP. That ATP is used to drive essential reactions of the cell (e.g., motility), completing the ATP cycle of Figure 1. This simplified metabolism has important effects, which must be considered during cell storage. Sperm are completely dependent on their surrounding environment to supply nutrients and remove toxic end products. Loss of N A D P H limits ability to repair damage induced by an adverse environment. They have a very limited ability to modulate ATP consumption, leading via the ATP cycle to a situation whereby even transitory loss of nutrients can lead to rapid loss of viability. Substrates to satisfy spermatozoal ATP generation can be derived from either of two sources, exogenous materials (taken from the surroundings through the cell membrane into the cell) or endogenous materials (mobilized from previously deposited stores within the cell membrane). This is important when testing cells for aspects of obligate coupling stoichiometry. If substrate and oxygen consumption ratio suggest that other sources of carbon are being oxidized in addition to the exogenous substrate, depletion of endogenous reserves is a likely possibility. The relative importance of each to the ATP cycle has been shown (Hammerstedt and Lovrien, 1983; Inskeep and Hammerstedt, 1983, 1985) for bull sperm, but not for any other cell within the scope of this review. The glycolytic pathway (Figure 3) is important to generating spermatozoal ATP in two ways. A few molecules of ATP are generated during conversion of glucose to lactate, and this mode of metabolism is of critical importance if the cell does not have access to oxygen. Many more molecules of ATP can be made by complete oxidation of the intermediate pyruvate in that degradation pathway to CO2 by the TCA cycle. The interaction between the ATP-consuming aspects of the pathway and ATPgenerating aspects in the terminal phases will be discussed later because of their important temperature dependence. C. M o d e s o f ATP C o n s u m p t i o n U s e d b y S p e r m 1. Value o f I n f o r m a t i o n to Cell Storage

Such information is of general intellectual interest when examining the ATP balance sheet and is of considerable practical value when considering

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optimum modes of cell storage. For example, assume that the required cell storage condition is one where it is impossible to provide sufficient nutrient to the cell to satisfy all ATP demand. If the relative importance of the ATP consuming pathways were known, and reversible inhibitors for them were available, ATP demand under storage could be reduced to maintain ATP balance under the mandated conditions. Implications of such information on design of storage containers were presented previously (Hammerstedt, 1993). 2. Allocation o f ATP to Various ATP Consuming P a t h w a y s A simple question was asked for the case of washed ejaculated bovine sperm incubated at 37~ (Hammerstedt et al., 1988). What is ATP used for? The answer for this very simple metabolic system is disconcerting and shows our ignorance of factors involved in metabolic balance in this relatively highly studied system. About half of total ATP is used to drive motility, with undetectable amounts assignable to ion balances at the plasma membrane. A later report (Nolan et aL, 1995) places an upper limit of a few percent for other membrane related phenomena. Thus, about half of all ATP consumption is directed to as yet unknown cellular processes. If the source of unknown ATP consumption were known and could be reduced during storage, demand for supply of nutrients and removal of metabolic end products would be minimized. 3. Differences in ATP Turnover between Cauda E p i d i d y m a l a n d Ejaculated Sperm Data summarized in Hammerstedt (1993) document that ATP turnover rate at 37~ for bovine ejaculated sperm is 2.5 x that of cauda epididymal sperm. Why? Estimates of motility for the cell types were equivalent, leading to the conclusion that: (a) an unknown ATP consuming system is "added" to sperm upon ejaculation or (b) motility of epididymal sperm is very efficient, leading to much less ATP demand for a given movement (viewed as unlikely, with no obvious mode of checking this possibility). This is presented as one example illustrating that choice of cell type for storage could have a considerable effect on the types of storage media and conditions needed for successful storage.

V. EXAMPLES OF METABOLIC BAIANCE OF SPERM UNDER NORMOTHERMIC C O N D m O N S

A. C o m p a r i s o n of ATP Turnover in Cauda Epididymal and Ejaculated Bull Sperm Data of Cascieri et al. (1976) illustrate the large changes in bioenergetic state associated with dilution of bovine sperm collected from cauda epidid-

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Roy H. Hammerstedt a n d Jane C. Andrews

ymidis into either seminal plasma or defined buffer; nucleotide ratios adjust within 1 min and motility is initiated shortly thereafter. When diluted cauda epididymal sperm are compared to ejaculated sperm, total rate of ATP turnover of the latter is increased about 3 x, driving motility at equivalent rates but with different wave forms and motility patterns (Inskeep and Hammerstedt, 1982; Inskeep et al., 1985). The following unexplained observation highlights the critical differences in the two cell types. Undiluted cauda epididymal sperm, suspended at a concentration of billions of cells per milliliter in their native cauda epididymal fluids, maintain high EC values for minutes to 1 hr after collection from the animal by indwelling catheter (Cascieri et al., 1976). This suggests that the cellular energy appetite is minimized or satisfied by that environment even after removal from the animal. In terms of the ATP cycle, synthesis and consumption are balanced in that environment. The following unpublished observations illustrate the effect(s) of dilution from that environment on cell function and regulation of the ATP cycle. Cauda epididymal sperm, previously diluted into either simple salts plus glucose buffer, seminal plasma, or cauda epididymal fluid, were concentrated by centrifugation and then resuspended in cauda epididymal fluid to concentrations approaching that in original native cauda epididymal fluid. Observations were: (a) the cells did not return to their native nonmotile state and (b) EC plummeted as the cells were unable to extract sufficient nutrients from the fluid to maintain ATP balance. This leads to the suggestion that irreversible alterations of sperm occur during the dilution process, independent of the media into which the cells are diluted. In terms of the ATP cycle, consumption exceeds supply and viability is lost.

B. ATP Turnover in Bull, Ram, and Ejaculated Rabbit Sperm Indirect measurements (based upon carbon balance and calculation) were used (summarized in Hammerstedt, 1981) to compare metabolic rate for ejaculated bull, ram, and rabbit sperm isolated for study by collection with an artificial vagina, washed in a standardized buffer, and subjected to a battery of simultaneous metabolic measurements. Sperm from all three species had equivalent estimated total rates of ATP turnover, but a striking difference was noted in the mode of providing that ATP. Bull and rabbit sperm used endogenous reserves of unknown materials to satisfy 30-50% of total ATP need, deriving the balance from the extracellular glucose supplied in the medium. In contrast, ram sperm derived none of their ATP from endogenous reserves and were totally dependent on extracellular glucose. In terms of sustenance of metabolism as part of a preservation protocol, ram sperm must be much more dependent on substrate supply than sperm from the other species.

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C. ATP D e m a n d s D u r i n g E p i d i d y m a l Storage Data for this section were chosen to illustrate the wide range in differences among species under conditions within the epididymal lumen and their resultant effects on metabolic rate. Crichton and colleagues investigated the conditions of the bat which might account for its capacity to store sperm, accumulated from a seasonal burst of spermatogenic activity before hibernation, within the epididymal tract for months during the hibernation period. The conclusion from those studies (Crichton et al., 1994) is that during the storage period within the male tract luminal fluid osmotic pressures are high (1500 mOsm), forcing a dehydration of the epididymal sperm and reduction of metabolic rate to very low rates. A unique structure of the bat epididymis (Crichton et al., 1993) provides the structural strength needed to withstand tubular pressures. When bats emerge from hibernation, the osmotic strength decreases toward "normal," resulting in a rehydrated cell ready to continue its path to syngamy with the oocyte. Amann et al. (1993) recently reviewed features of the more traditional modes of epididymal maturation; references to many additional reviews are found therein. A symposium on sperm preservation and encapsulation, covering a wide range of topics of interest to sperm storage in vivo and in vitro, was published (D'Occhio, 1993). There is considerable interest in elucidating the mechanism by which sperm are rendered quiescent during these periods of their existence. If such information were available and could be translated into design of unique storage containers and diluents, the problems associated with nutrient supply and metabolic end product removal would be minimized.

VI. EFFECT OF MODEST CHANGES IN TEMPERATURE ON ATP TURNOVER A. Effect o f I n c r e a s e d T e m p e r a t u r e o n M e t a b o l i s m o f Rooster S p e r m Fowl sperm provide an interesting system, in that they show a reversible temperature-dependent change in motility associated with slight changes in temperature. Complete primary references are cited within recent publications describing the possible molecular reasons for the following observations (Ashizawa et aL, 1994a,b,c). When suspended in synthetic diluents, the sperm become immotile if held at body temperature (40-41~ but recover motility when lowered to 30~ Apparent reasons involve a complex relationship between intracellular pH, temperature and C a 2+ content. The exact relation of these changes to bioenergetic balance is unknown, but the system provides a clear example of how modest changes in storage

156

Roy H. Hammerstedt and Jane C. Andrews

conditions, often not considered important by the investigator, can have profound effects on cell function. It also should alert the reader to the difficulties in relating in vitro observations to in vivo function. Fowl sperm are unique in that after deposition in the female tract they move into storage glands in the uterine/vaginal junction and remain in a quiescent form (reviewed by Bakst et al., 1994). Daily, a cohort of sperm is mobilized, exit the storage gland, and move up the oviduct to the site of fertilization at the infundibulum. All of this occurs at body temperature, illustrating the effect of cellular environment on those pathways (e.g., motility) of major bioenergetic importance. B. Effect o f D e c r e a s e d T e m p e r a t u r e o n M e t a b o l i s m of Bull Sperm Many storage protocols call for a slow reduction of temperature after collection to "ambient" temperature to avoid irreversible alterations to membranes. Detailed studies on the effects of these changes in overall bioenergetic balance illustrate the delicate relationship among pathways that can be altered by those changes. Extracellular glucose is taken into sperm, phosphorylated to form glucose 6-phosphate (Glc-6-PO4), and metabolized by the Embden-Meyerhof pathway to yield products for biosynthesis (rare in sperm), pyruvate for oxidation in the mitochondria, or excretion as lactate (Figure 3). The overall Embden-Meyerhof scheme, introduced 50 years ago (Burk, 1939), features consumption of ATP during metabolism at the hexose level and recovery at the triose level. The efficiency of this pathway in terms of ATP yield Mannose

I

Mannose

I

Man-6-P

I Glucose

~- 4Glucose O-r162

I

I

,

I

I

, I

FBP I O-r

I F rIu c t o s e

Fructose

-,,r L a c t a t e

I

, ,!11

I

Lactate

Schematic of glycolytic metabolism of sperm. The left section represents the phase of the reactions which consume ATP while the right section represents ATP repletion phases. Unless the phases are kept in synchrony, ATP consumption is in excess and overall ATP yield of the pathway will be decreased. Details supplied in the text.

FIGURE 3

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can be considered at three levels. First, thermodynamic factors establish the maximum ATP yield for the pathway from the difference in free energy between input hexose substrate and final product; this value is unlikely to be achieved. Second, evolved coupling stoichiometry ATP yield can be calculated. This is established by the nature of the individual enzymatic steps taken in the cell to convert substrate to product. For the Embden-Meyerhof pathway, two molecules of ATP are consumed at the "top" of the pathway and four molecules of ATP are provided at the "bottom" of the pathway. The overall stoichiometric yield is two ATP per mole of hexose consumed. The net (actual) ATP yield is the stoichiometric yield minus losses associated with the metabolic pathway chosen. Losses in this pathway are those involving phosphorylation-dephosphorylation pairs of reactions at the hexose level of the pathway. Glucose is phosphorylated by hexokinase to form Glc-6-PO4 at the cost of an ATP. If the Glc-6-PO4 moves forward in the pathway via the next enzyme (phosphoglucoisomerase) to fructose 6phosphate (Fru-6-PO4), the expenditure of that ATP can be considered productive. However, all cells have phosphatase enzymes that remove phosphate to regenerate the original hexose. If the phosphatase acts on Glc-6PO4 before the phosphoglucoisomerase does, the ATP was not productively used to move glucose down the pathway toward end product because glucose is set free to pass out of the cell and back into the media. Competition between kinases and phosphatases is termed substrate cycling and amounts to a potential use of ATP with no return to the cell for energetic reasons. Such competition is of considerable value to the regulation of overall flux through the pathway. Two other factors are important to this entry step into the pathway. First, the enzyme hexokinase can act on several substrates; preference is for either glucose or mannose over fructose. Second, a hexose can enter in one form (glucose) yet leave in alternative form (conversion from Glc-6-PO4 to Fru-6-PO4 followed by dephosphorylation to fructose). An additional substrate cycle exists in the pathway, Fru-6-PO4 interconversion with fructose 1,6-bisphosphate (FBP). Thus, there are two points at the top of the pathway where substrate cycling can occur. Hammerstedt and Lardy (1983) set up experiments with cauda epididymal bovine sperm to test for the effect of temperature of incubation (22 through 37~ on substrate cycling and net ATP yield. At the lowest temperature, using glucose as substrate, an unanticipated event occurred. Extensive substrate cycling occurred at the first point. ATP was expended as glucose was rapidly taken up to form Glc-6-PO4, which was transformed to Fru-6PO4, which was then dephosphorylated and fructose was released to the media. This had two important effects. First, this continued until all of the glucose was removed from the media and returned in the form of fructose. Fructose, previously excluded from entry by the preference of hexokinase for glucose, was then slowly metabolized. Thus, if one were to monitor glucose consumption only as a measure of bioenergetic demand, an erroneous conclusion

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would be reached, one where sperm have a high metabolic rate at 22~ Precise estimates of the rate of substrate cycling lead to the surprising conclusion that the rate was so high that the net yield of ATP between glucose and lactate was actually-0.6 mol ATP per mole hexose rather than the predicted stoichiometric value of 2.0. The cell under these conditions was saved from starvation by mitochondrial oxidation of a small portion of the total carbon consumed and oxidative phosphorylation. As the temperature was raised to 37~ extent of substrate cycling was relatively decreased and the net ATP yield from glucose from the pathway approached zero; again overall ATP accumulation was dependent on mitochondrial metabolism. If fructose was used as initial substrate at 37~ its slower entry via hexokinase resulted in lower substrate cycling and a net ATP yield per mole hexose between fructose and lactate was +0.6. Thus, cauda epididymal sperm did not yield ATP in the manner anticipated from generalized assumptions about the operation of the Embden-Meyerhof pathway. In a second report (Hammerstedt, 1983) bovine cauda epididymal and ejaculated sperm were directly compared with regard to extent of substrate cycling and ATP yield. Ejaculated sperm, regardless of temperature of incubation (i.e., flux through the pathway) exhibited much less substrate cycling than observed for cauda epididymal sperm when glucose was substrate. Net ATP yield for the path to lactate was about + 1 regardless of temperature; more ATP is made by mitochondrial oxidations. This documents yet another profound effect of mixing with accessory sex gland fluids and provides an example of how modification of some aspect of the cell (e.g., alteration of membrane bound hexokinase) can have dramatic effects on effectiveness of a total pathway. "Reasons" for such changes cannot be supplied, but it certainly appears to reflect a terminal processing event within the male which yields a much more effective system for recovering ATP for use later in the female tract. These examples establish that: (a) it is possible for addition of a highly preferred substrate (glucose) to sperm to have a detrimental effect on overall ATP yield under certain conditions, (b) the glycolytic pathway apparently functions to deliver carbons suitable for mitochondrial metabolism (and resultant ATP production) rather than for ATP generation per se; and (c) relatively small changes in temperature can have a large effect on balance within a metabolic pathway, leading to unanticipated deleterious effects on ATP yield. Inskeep and Hammerstedt (1985) extended these observations with bovine ejaculated sperm to evaluate effect of substrate on overall ATP yield. Temperature of incubation was varied from 20 to 35~ and sperm were given glucose or fructose (representing substrates capable of substrate cycling), lactate (entry into ATP yielding pathways via dehydrogenation to pyruvate and entry into the TCA cycle) or/3-hydroxybutyrate (dehydrogenation and direct entry into the TCA cycle). Incubations were conducted

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in a calorimeter so that contributions of endogenous metabolism could be evaluated. Results can be summarized as follows. Since the EC values were equivalent, measured ATP production rates equaled the rate of the ATP cycle. Increasing the temperature of incubation raised ATP cycle rates, but the fold increase was not identical for all substrates. For lactate, rate increased 2x between 20 and 35~ (2.2 to 4.5 /xmol ATP per hr per 108 sperm); for/3-hydroxybutyrate, rate increased 1.7x (2.0 to 3.5 tzmol ATP per hr per 108 sperm); and for glucose and fructose, rate increased 3.5x (3 to 9 txmol ATP per hr per 108 sperm). For all incubations, regardless of temperature or extracellular substrate provided, endogenous metabolism supplied between 1.5 and 2 txmol ATP per hr per 108 sperm. When glucose and lactate were compared as substrates at 35~ there was no difference in sperm motility. This indicates that ATP requirement for that critical attribute could be satisfied with a much lower overall ATP generation rate with some substrates (lactate) than others (glucose). Calculations provided in that presentation establish that the "excess" ATP produced using glycolytic substrates is largely consumed by substrate cycling during delivery of carbons from hexose to the TCA cycle.

VII. LITERATURE SURVEY OF METABOLIC NEEDS OF OTHER CEILS OF REPRODUCTIVE INTEREST

An exhaustive survey and critique of the existing literature is not presented because simple inspection of the literature revealed that no other cell type has been studied in sufficient detail to allow detailed metabolic calculations. As a result, the conclusions presented in Sections IV-VI for bull sperm stand as an isolated example of unknown value to an understanding of generalized metabolism of cells of reproductive interest. A simple representation of the literature is provided in Figure 4, where the total citations by time period are given from 1966 to 1995 for spermatozoa, oocytes, and preimplantation embryos (Figure 4A) and then the total data base over the period 1966-1995 was sorted by species (Figure 4B). Search criteria for Medline are presented in the figure legend and were set quite broad to identify the largest number of reports likely to contain data on metabolic rate of intact cells. There were very few examples where incubation conditions and technique were held "sufficiently" constant to allow direct comparisons across species, developmental state, or incubation melieu, and thus they do not provide information for detailed analysis and interpretation. Many-to-most are quite fragmentary in their approach and serve as an order-of-magnitude estimate of cellular features. Spermatozoal metabolism has been actively studied since 1970 with several hundred reports in the literature. Human sperm have the largest number of reports, but most provide general information without an inte-

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Literature citations covering metabolic features of spermatozoa, oocytes, and preimplantation embryos. A Medline database containing records from 1966-1995 was accessed and the primary search terms of spermatozoa, oocytes, or cleavage stage ova were used. These records then were further sorted by the single secondary qualifier of energy metabolism or ATP. All terms were exploded to maximize record recovery. Data are presented sorted by year (A) or by species (B). Data files are available on disc in format MS Word from the authors.

FIGURE 4

g r a t e d conclusion. This m a y be d u e to the fact that a m o d e s t n u m b e r of s p e r m can be o b t a i n e d in any single ejaculate, a n d r e p e a t e d s a m p l e s f r o m a n y single individual are difficult to schedule. Studies of s p e r m a t o z o a f r o m cattle a n d swine are also significant. O o c y t e m e t a b o l i c studies lag b e h i n d

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by 10-15 years, but the database is rapidly increasing now. Studies of X e n o p u s laevis, human, and mouse tissues predominate. General reviews by Adolph (1983) and Leese et al. (1993) are useful. Minimal data are available for preimplantation embryos from any species, with the recent reviews of Bavister (Bavister, 1995; Barnett and Bavister, 1996) containing useful summaries of the "practical side" of culturing preimplantation embryos for a variety of purposes. The detailed studies of bull and ram sperm described earlier were conducted at cell numbers of 50-100 million sperm per incubation (0.52 mg cellular protein) to provide a suspension consuming about 1 tzmol 02 per hr per incubation flask. Completion of an ATP balance sheet analysis (Section VIII) for other cells of interest will require an equivalent amount of metabolic activity per flask. Most sperm suspensions will have metabolic rates within an order of magnitude of the rate for bull and ram sperm, so the cell numbers recommended will serve as a guide to those types of studies. The studies of Brachet et al. (1975) provide an estimate of the number of X. laevis ooctyes needed for the analysis. A suspension of 100 oocytes will consume about 0.1 tzmol 02 per hr, so each vessel would have to contain -~1000 oocytes to allow detailed analysis of metabolic features. Waugh and Wales (1993) report that sheep, mouse, and cattle embryos produce 2-5 pmol CO2 per embryo per hr from glucose as a exogenous substrate. Using Eq. [2] as a guide, they should consume 2-5 pmol 02 per embryo per hr during that metabolism. Thus, 200,000-500,000 embryos per incubation flask would be needed, clearly showing why detailed studies are unlikely to be completed.

v m . A PRIMER FOR C O N S T R U C T I O N OF A N ATP B~CE SHEET

The objective of this section is to lead the reader through the steps outlined in Section III.A to proceed through the development of the simple conservation, obligate coupling, and then the evolved coupling stoichiometries for cell types of his/her interest. To conserve space, specific experimental details used for sperm studies will not be restated herein (Hammerstedt, 1975a,b; Cascieri et aL, 1976; Hammerstedt and Hay, 1980; Inskeep and Hammerstedt, 1982; Hammerstedt and Lardy, 1983; Hammerstedt, 1983; Inskeep and Hammerstedt, 1983, 1985; Hammerstedt et al., 1988). A summary chapter (Hammerstedt, 1981) provides an overview of concept and early results. A method of estimating glucose consumption introduced in the 1970s (Hammerstedt, 1975a) has flaws as outlined in detail in Hammerstedt and Lardy (1983) and should be used only after details of the early steps of glycolysis are understood.

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A complete study involves these considerations. First, sufficient cells (defined in terms of metabolic rate) must be available for use. An incubation medium must be chosen that is both compatible with requirements of metabolic measurements and useful for practical applications. Studies with whole cells should include: (a) estimation of oxygen consumption rate; (b) determination of substrate and product carbon balance, (c) estimation of contribution of endogenous reserves to metabolic rate, (d) partitioning of exogenous substrates among competing pathways within the cell, and (e) adenine nucleotide content. Analysis of disrupted cells is essential for estimates of enzyme and cofactor content. It is critical that a respirometer be rescued from deep reaches of departmental storage rooms so that simultaneous nutrient and oxygen consumption rates can be made. Initial calculations and definition of experimental conditions are essential. Valid estimates of nutrient and oxygen consumption require that each incubation contain cells sufficient to consume 0.5-1 tzmol 02 per hr. Cells used must be of sufficient purity to assure that metabolic measurements represent the major cell type only. Up to 10 parallel incubations are useful for sufficient replication and comparison. Choice of incubation medium is critical because it must: (a) satisfy ionic and osmotic requirements of the cell, (b) maintain pH during the incubation, and (c) contain a known (minimal) number of potential carbon-based compounds which could be potential exogenous substrates. This description of incubation and calculations is derived from studies of sperm, where simple metabolic fates of the following types were established for an initial exogenous substrate glucose: (a) extracellular glucose is metabolized to the level of pyruvate by the glycolytic pathway (no side reactions to glycogen or pentose phosphate pathway); (b) at the level of pyruvate, a bifurcation occurs; (c) some of the pyruvate is reduced to lactate and leaves the cell; (d) additional pyruvate is oxidized by the pyruvate dehydrogenase complex to form CO2 plus acetyl-CoA; (e) the acetyl-CoA has four possible fates of hydrolysis and exit from the cell as acetate, transfer to carnitine to form acetyl-carnitine, incorporation into other compounds (minimal), or oxidation by the TCA cycle to CO2. Allocation of carbon to these various metabolic products must be determined. Respirometer vessels contain a main chamber (to hold cells), a center well (to hold KOH to trap evolved CO2), and a side chamber (to hold H2SO4).Media containing cells and 14C-labeled substrate (assume glucose) are placed in the center well and the vessel is connected to the respirometer at the temperature of interest; define this as T = 0 min. About 20 min are allocated for temperature equilibration (metabolism takes place during this period) before 02 consumption measurements are initiated (T = 20 min). The incubation is continued for the desired period (e.g., Ttota I -- 60 min) at which time the incubation media are acidified by transfer of contents of the side chamber (this marks the end of total period of metabolism). Vessels

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are removed, 14C content of center well evaluated for CO 2 evolution, and contents of the main chamber evaluated for distribution of 14C among starting material and varied metabolic end products over total incubation period. A parallel incubation outside the respirometer can be run to determine adenine nucleotide content before and after the incubation. If the [14C]glucose is the only component in the buffer solution which can be metabolized (this assumption has to be tested in preliminary studies) then the fate of the exogenous substrate can be determined by measuring the distribution of 14C into metabolic products (e.g., CO2, lactate, acetate, acetylcarnitine, cellular components, other). From the specific radioactivity of the starting substrate, the number of moles of the various metabolic products derived from the exogenous substrate can be determined. The balance sheet approach demands that at least 90% (better >95%) of all the added ~4C be accounted for in terms of known components. The moles of 14CO2 produced are compared to the moles of 02 consumed as outlined in Section III,A for an assessment of the obligate coupling stoichiometry. From this calculation the potential role of other components (endogenous, if no other metabolically active components are in the incubation medium) in the balance sheet. The total amount of oxygen consumed (measured) can be allocated between: (a) that involved in metabolism of exogenous glucose (calculated from the observed 14CO2 measurement); and (b) that involved in the metabolism of unknown carbon compounds (difference between total and that for (a)). If significant endogenous metabolism is suggested, "best guesses" of the pathways involved must be made. Cellular content of key enzymes associated with the metabolism of the exogenous nutrient is determined and a "best guess" is made as to the pathways most likely to be involved in the transformation from starting material to product. If alternative pathways are likely to be competing for critical metabolic intermediates (e.g., glucose 6-phosphate), then additional experiments must be made to assess partitioning to those pathways. Adenine nucleotide content is used to assess the bioenergetic "stability" of the incubation over the period of the incubation. If this information is available, final calculations of the estimated ATP yield per N cells per unit of time can start. ATP yield from each metabolic transformation is calculated (e.g., glucose glucose -* 2 lactate; glucose -~ 2 acetate (or 2 acetyl-carnitine) + 2 CO2; glucose -~ 6 CO2). The sum of these values is the total ATP derived from the exogenous nutrient supplied. Endogenous metabolism is more difficult to estimate, but approximations can be made in the following manner. In general, all forms of preliminary metabolism for oxidative processes (e.g., those steps which generate either N A D H or FADH2) have modest ATP yields and can be ignored. Most of the ATP generated comes from the mitochondrial metabolism via oxidative phosphorylation and a standard conversion of 6/zmol ATP//zmol O2 consumed. The sum of the exogenous and endogenous metabolism is equal to

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the total rate of ATP synthesis. Thus, in terms of Figure 2, you have the relative importance of the individual "arrows" which constitute all of the ATP synthesis and the total rate of ATP synthesis. If the adenine nucleotide pool is held constant over the incubation period, then that calculated value is also equal to the rate of ATP degradation. Assessing the distribution of ATP to the various ATP demanding pathways is very difficult and no generalizations can be provided. The current best guesses are found in Hammerstedt and Lardy (1983) and Hammerstedt et al. (1988). Use of calorimetry (Inskeep and Hammerstedt, 1985) provides a useful extension for evaluation of endogenous metabolism and some estimate for ATP consumption (Hammerstedt et al., 1988) but its highly specific equipment requirements preclude use in routine studies.

IX. SUMMARY AND DEDICATION R.H.H. entered into the writing of this chapter with reluctance, driven only by the persistence of the editors and latent guilt. With time, marked by a mixture of procrastination and reflection, enthusiasm increased. Sperm have proven to be an excellent model for probing the relationship between various metabolic pathways and overall ATP demand because of their relatively simple modes of ATP use relative to somatic cells. This chapter provided a way of summarizing some of those relationships. Thoughts on how to approach these studies, and limits to interpretation, were provided by the chance to work with two eminent leaders in the field of bioenergetics. This effort is dedicated to their enthusiasm, encouragement, and leadership in the field. The first person, Henry Lardy, needs no introduction to the field of gamete preservation. The summer before he started graduate school in 1939, he and P. H. Phillips developed the first practical bull semen extender (Lardy and Phillips, 1939). That contribution was later recognized by receipt of the 1982 Wolf Prize in Agriculture. In the intervening years, and continuing to today, he repeatedly used sperm cells to probe a host of cellular features and, in total, they provide the backbone of our most of current understanding of that system. When combined with other ongoing projects with a host of somatic cells, he made major contributions in almost all aspects of the physiology and enzymology of ATP generating and utilizing pathways. The substrate cycling studies (Hammerstedt and Lardy, 1983) were developed during a sabbatical leave in his laboratory (1978-1979), and the concepts were further developed and discussed (Hammerstedt, 1983) in a symposium honoring his 65th birthday (Lennon et al., 1983). His role model for clarity in thinking, especially with regard to remembering that emerging data for any single pathway must be reviewed in context to the needs of the whole organism, turned

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those studies from interesting collection of analytical data to important contributions on the need for controlled integration of the distal aspects of an extended pathway. When asked a few years ago by a new graduate student what topic he would study if he were starting his career now, he replied, "Intermediary metabolism, no one else is!" The second person, Ef Racker, may be less familiar to readers in this field. To my knowledge, our interaction during a sabbatical leave in 1986 was his only professional entry into sex. Beginning in the 1940s, he too provided leadership to the field, making major contributions in almost all aspects of the area. Of special value to me were the major contributions in the field of oxidative phosphorylation, and the "purpose" on poorly described ATPase systems. He always found gold in what some would consider waste. As I completed my studies in his laboratory, we had a most interesting talk about my responsibility to help "reinvent the wheel" to assure that central facts were passed to the next generation. The topic was the introductory theme of this chapter: Cells make ATP only if they are consuming ATP. Seems simple enough, but that critical message gets lost in the ever increasing detail available on the metabolic pathways. At the end of the discussion, I was given the following assignment. Ef pointed out that Meyerhof had first pointed out that critical relationship (Meyerhof, 1945) with clearly designed experiments with cell extracts. Ef and Henry, plus others, developed the concepts very clearly over the next few decades. Delightful summaries of Ef's thoughts are found in his books (Racker, 1976, 1985) which should be read by all interested in these areas. To him, one of their goals was to provide a necessary recurring summary to avoid loss of the concepts of Meyerhof. He then pointed out that not many people cared about the topic any more, and that I had a responsibility every decade to keep the wisdom of the pioneers available to new entrants to the area. Ef's recent death has removed that guiding light from the area. Our publication (Hammerstedt et aL, 1988) took care of that decade, this chapter will take care of the 1990s, and volunteers are needed for the year 2000 and beyond.

REFERENCES Adolph, E. F. (1983). Respir. Physiol. 53, 135-160. Amann, R. P., and Hammerstedt, R. H. (1993). J. Androl. 14, 397-406. Amann, R. P., Hammerstedt, R. H., and Veeramachanent, D. N. R. (1993). Reprod. Fertil. Dev. 5, 361-381. Ashizawa, K., Tononaga, H., and Tsuzuki, Y. (1994a). J. Reprod. Fertil. 101, 265-272. Ashizawa, K., Katayama, S., Kobayashi, T., and Tsuzuki, Y. (1994b). J. Reprod. Fertil. 101, 511-517. Ashizawa, K., Wishart, G. J., Nakao, H., Okino, Y., and Tsuzuki, Y. (1994c). J. Reprod. Fertil. 101, 593-598. Atkinson, D. E. (1977). Cellular Energy Metabolism and Its Regulation. Academic Press, New York.

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Austin, C. R. (1951). Aust. J. Sci. Res. 4, 581-596. Bakst, M. R., Wishart, G. H., and Brillard, J.-P. (1994). Poult. Sci. Rev. 5, 117-143. Barnett D. K., and Bavister, B. D. (1996). Mol. Reprod. Dev. 43, 105-133. Bavister, B. D. (1995). Hum. Reprod. Update 1, 91-148. Bazer, F. W., Geisert, R. D., and Zavy, M. T. (1993). In Reproduction in Farm Animals (E. S. E. Hafez, Ed.), pp. 188-212. Lea and Fabiger, Philadelphia Brachet, J., Pays-de Shutter, A., and Hubert, E. (1975). Differentiation 3, 3-14. Burk, D. (1939). Cold Spring Harbor Symp. Quant. Biol. 7, 420-459. Cascieri, M., Amann, R. P., and Hammerstedt, R. H. (1976). J. Biol. Chem. 251, 787-793. Chang, M. C. (1951). Nature 168, 697-698. Crichton, E. G., Hinton, B. T., Pallone, T. L., and Hammerstedt, R. H. (1994). Am. J. Physiol. 267, R1363-R1370. Crichton, E. G., Suzuki, F., Krutzsch, P. H., and Hammerstedt, R. H. (1993). Anat. Rec. 237, 475-481. D'Occhio, M. J. (1993). Sperm Preservation and Encapsulation. CSIRO, Australia. Estabrook, R. W., and Srere, R. (1981). Curr. Top. Cell Regul. 18. Hafez, E. S. E. (1993a). In Reproduction in Farm Animals (E. S. E. Hafez, Ed.), pp. 114-143. Lea and Fabiger, Philadelphia Hafez E. S. E. (1993b). In Reproduction in Farm Animals (E. S. E. Hafez, Ed.), pp. 461-502, Lea and Fabiger, Philadelphia Hammerstedt, R. H. (1975a). Biol. Reprod. 12, 545-551. Hammerstedt, R. H. (1975b). Biol. Reprod. 13, 389-396. Hammerstedt, R. H. (1981). In "Reproductive Processes and Contraception" (K. W. McKerns, ed.), pp 353-392. Plenum, New York. Hammerstedt, R. H. (1983). In Biochemistry of Metabolic Processes (D. L. F. Lennon, F. W. Stratman, and R. N. Zahlten, Eds.), pp. 29-38. Elsevier Biomedical, New York Hammerstedt, R. H. (1993). Reprod. Fertil. Dev. 5, 675-690. Hammerstedt, R. H., and Hay, S. R. (1980). Arch. Biochem. Biophys. 199, 427-437. Hammerstedt, R. H., and Lardy, H. A. (1983). J. Biol. Chem. 258, 8759-8768. Hammerstedt, R. H., and Lovrien, R. E. (1983). J. Exp. Zool. 228, 459-469. Hammerstedt, R. H., Graham, J. K., and Nolan, J. P. (1990). J. Androl. 11, 73-88. Hammerstedt, R. H., Volont6, C., and Racker, E. (1988). Arch. Biochem. Biophys. 266, 111-123. Inskeep, P. B., and Hammerstedt, R. H. (1982). Biol. Reprod. 27, 735-743. Inskeep, P. B., and Hammerstedt, R. H. (1982). Biol. Reprod. 27, 735-743. Inskeep, P. B., and Hammerstedt, R. H. (1983). J. Biochem. Biophys. Methods 7, 199-210. Inskeep, P. B., and Hammerstedt, R. H. (1985). J. Cell. Physiol. 123, 180-190. Inskeep, P. B., Magargee, S. F., and Hammerstedt, R. H. (1985). Arch. Biochem. Biophys. 241, 1-9. Kornberg, A. (1989). "For the Love of Enzymes: The Odyssey of a Biochemist." Harvard Univ. Press, Cambridge. Kornberg, A., Horecker, B. L., Cornudella, L., and Oro', J. (1976). Reflections on Biochemist r y - I n Honor of Severo Ochoa. Permagon, Oxford. Lardy, H. A., and Phillips, P. H. (1939). Am. Soc. Anim. Prod. 32, 219-221. Leese, H .J., Conaghan, J., Martin, K. L., and Hardy, K. (1993). BioEssays 15, 259-264. Lipmann, F. (1971). Wanderings of a Biochemist. Wiley-Interscience, New York. Meyerhof, O. (1945). J. Biol. Chem. 157, 105-119. Nolan, J. P., Magargee, S. R., Posner, R. G., and Hammerstedt, R. H. (1995). Biochemistry 34, 3907-3915. Racker, E. (1965). Mechanisms in Bioenergetics. Academic Press, New York. Racker, E. (1985). Reconstitutions of Transporters, Receptors, and Pathological States. Academic Press, New York. Stryer, L. (1995). Biochemistry, 4th ed. Freeman, New York. Voet, D., and Voet, J. G. (1990). Biochemistry. Wiley, New York. Waugh, E. E., and Wales, R. G. (1993). Reprod. Fertil. Dev. 5, 123-133. Yanagimachi, R. (1994). In The Physiology of Reproduction (E. Knobil and J. D. Neill, Ed.), pp. 189-317. Raven Press, New York.

Pharmacological Interventions in V i t r o Armand

M. K a r o w

Department of Pharmacology and Toxicology Medical College of Georgia Augusta, Georgia 30912 and Xytex Corporation Augusta, Georgia 30904

I. INTRODUCTION Just as people in a city go about their daily lives without much thought to the supporting infrastructure of roads, utilities, and buildings, so people preserving tissues seldom give sustained thought to components of media supporting the tissue. The purpose of this chapter is to give attention to chemical components of tissue supporting media. These components are "additives" for modifying cellular function beyond that provided by basal support components such as electrolytes, metabolites, and osmolytes. Chemicals can beneficially alter cell function. In regard to reproductive cells, chemical intervention with cell function is achieved, for example, with hormones, motility stimulants, and cryoprotectants. Knowledge of such chemical intervention is usefully organized in a pharmacological manner. In this chapter basic pharmacological concepts will be presented which will then be illustrated by three classes of drugs of interest to persons banking reproductive tissues: gonadotropins, antioxidants, and cryoprotectants. II. GENERAL CHARACTERISTICS OF DRUG ACTION Chemicals with a pharmacological action, i.e., drugs, usually act upon a specific chemical site within cells, but some drugs have a nonspecific Reproductive Tissue Banking Copyright 9 1997 by Academic Press. All rights of reproduction in any form reserved.

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action upon cells. In either situation pharmacological action is directed at cellular function on the molecular level. Drugs are not chemically altered by their pharmacological action. The pharmacological action of all drugs is concentration-dependent; each drug has a characteristic, quantitative dose-response effect. "Specific" drugs usually interact with one of four classes of regulatory proteins within cells: 9 enzymes 9 transport molecules 9 ion channels ~ receptors Usually the regulatory protein is bound to the plasma membrane, but some are cytoplasmic proteins. The interaction is reversible. Target molecules respond to drug concentrations on the order of 10 -4 t o 10 -8 M. The term receptor was created by 19th-century pharmacologists (Ehrlich, 1913; Langley, 1909) and denotes regulatory proteins whose sole function is to serve as a recognition site, a transducer for a specific class of chemicals such as gonadotropins or steroids. Strictly speaking, a receptor has no function in the cell other than to initiate a series of chemical reactions in response to the stimulatory chemical, i.e., a ligand, hormone, or drug. If the ligand specifically activates or triggers the receptor, the ligand is an agonist. If the ligand inhibits receptor activation, the ligand is an antagonist. Ligands acting on a specific domain of a receptor generally have very similar chemical structures. Regardless of the original meaning of receptor, many people now use the term to indicate any protein that has a specific response to a ligand. Not all "specific" drugs are targeted to regulatory proteins. Some specific drugs interact with structural proteins such as tubulin or the peptidoglycans of bacterial cell walls. Other drugs classified as chemotherapeutic agents may specifically interact with nucleic acids. Drugs interacting with nucleic acid targets will become important as genetic pharmacology advances. "Nonspecific" drugs, on the other hand, do not bind with a specific target molecule. Nonspecific drugs include several major classes, e.g., chelators, general anesthetics, osmotic diuretics, cryoprotectants (CPAs), and disinfectants. All of the chemicals in a class have a similar effect but the chemical structures of individual agents in the class may be vastly different. Nonspecific drugs are usually active only at high concentrations, even multimolar. They act by a variety of mechanisms. In some cases these drugs act by colligative effects (to be discussed later); in other cases they may act by indiscriminant chemical reactions such as oxidation. All drugs, whether specific or nonspecific, may bind in a nonspecific manner to various molecules in a biological system. For example, many

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drugs bind nonspecifically to plasma protein such as serum albumin. Some drugs are highly soluble in fat. Nonspecific binding can affect the concentration of the drug free to act pharmacologically. All drugs have adverse effects, that is, effects other than the intervention desired when the drug is used. Adverse effects, sometimes called side effects or unwanted effects, range from disseminated physiological responses throughout the organism to frankly toxic effects capable of killing the target cell or even the organism. Some drugs, e.g., chemotherapeutic agents, are intentionally toxic for a select group of cells (Albert, 1965; Erhlich, 1913). Limiting drug toxicity to this group of cells is challenging. For example, the highly effective antifungal agent amphotericin B is also appreciably toxic when used to protect transplantable mammalian cells in vitro (Aguirregoicoa et aL, 1989; Brockbank and Dawson, 1993; Villalba et al., 1995). Toxicity is a function of dose, duration of exposure, and temperature. One measure of toxicity is therapeutic index, the ratio of a toxic dose or the lethal dose for 50% of the treated population (LDs0) to the effective dose for 50% of the treated population (EDs0). For example, when controlling for duration and temperature of exposure, the therapeutic index of a bactericidal drug used to protect cells in culture would be the concentration of the drug that kills 50% of the cultured cells divided by the concentration that provides 50% of the cells with a measure of protection from a specific bacterium. Therapeutic index is a useful statistic for any drug, not just chemotherapeutic agents. Therapeutic index of a drug is meaningful only for one specific effect of the drug; other effects of the drug will each have a different therapeutic index.

m . RECEPTOR-MEDIATED DRUG ACTION

The protein structure of many receptors is known. Some receptors have been chemically isolated. Chemically "pure" receptors, when inserted into artificial cells such as liposome membranes, respond to drugs in a manner similar to receptor response in living cells (e.g., Bagchi et al., 1990; Corthesy et al., 1988; Limbird, 1986; Reichert and Dattatreyamurty, 1989). For purposes of classification, let us consider two main classes of receptors: those bound to the plasma membrane and those that are soluble DNAbinding proteins. Two major functions of a receptor are ligand binding and signal transduction. It is speculated that these two functions are carried out on different functional sites within the receptor, a ligand-binding site and an effector site. The interaction of ligand with these sites defines the affinity of the drug for its receptor and the intrinsic activity of the drug acting on the receptor.

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A. Receptor Dynamics The chemical bond between ligand and receptor is specific for the interaction. Such interactions may involve any of the known chemical bonds: hydrogen, van der Waals, hydrophobic, ionic, and covalent. Multiple bond types are involved in the usual ligand-receptor interaction. The duration of the bond may be a fraction of a second to several days. The dynamics of interaction between drug and receptor can be described quantitatively and qualitatively. In general drug-receptor interactions adhere to the Law of Mass Action: the rate of a reaction is proportional to the concentration of the reactants (Langmuir, 1918). The assumption is made that a cell has a finite number of receptors (R) free to reversibly interact with the drug (D):

D+R~DR. The rate of the reaction will be governed by the concentration of D. Letting square brackets indicate the molar concentration of reactants and VA indicate the rate of association of the drug with free receptors, then VA = kl [D][R], with k~ being the rate constant for association. Similarly, letting VD indicate the rate of dissociation of the drug-receptor complex (DR), then

VD = k2 [DR], with k2 being the rate constant for dissociation. At equilibrium the rate of reactant association and the rate of product dissociation are equal, VA = Vo,

so that kl [D][R] = k2 [DR] or

[DI[R] _ k2 [DR] kl" The formation of DR subsequently yields some observable, measurable effect (E), i.e.,

DR--->E + R, but seldom will this E be an immediate, direct consequence of DR; this "reaction" is therefore a figurative black box illustrated in subsequent discussion of membrane-bound receptors. Except when the reaction is truly the immediate consequence of DR, a meaningful rate constant (k3) is elu-

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sive. Theoretically, maximal effect (EMAx) OCCURSwhen all free receptors are occupied by drug. If [D] ~ [R], the formation of E becomes the rate limiting step in drug-receptor interactions; since all available R is present as DR. The relationship between DR or E and D can be described as

[DR] = [R]TOTAL [D] KEO + [ D ] ' where KEG (equal to k2/kl) is the equilibrium binding constant. (KEG as used here is equivalent to Ko or Ko used elsewhere.) Kzo is expressed in units of concentration. There is no reaction (or E, effect) when [D] equals zero; there is half maximal E when [D] equals KEG, i.e., when half of the receptors are occupied by drug. The affinity of a drug for its receptor is the reciprocal of KEG, i.e., kl/k2. The efficacy of a drug is its ability to provoke a response; another name for efficacy is intrinsic activity. The potency of a drug, related to its EDs0, is a result of the combined effects of affinity and efficacy. The quantitative relationship between drug concentration, i.e., dose, and response is beneficially represented graphically (Gaddum, 1926). The mathematical expression of drug-receptor relationship is analogous to substrate-enzyme kinetics originally described by Michaelis and Menten (1913). Clark (1933) independently applied these kinetic relationships to drug-receptor interactions. Presentations of log [D] vs response, i.e., [E], gives a sigmoid curve useful in comparing potencies of various drugs. The sigmoid curve can be linearized by a Lineweaver-Burk (1934) or a Scatchard plot. The Lineweaver-Burk transformation is accomplished by assumptions that result in 1= E

KEG

j

1

EMAx[D] EMAX"

Graphic presentations (Figure 1) of linearizations illustrate KEO and EMAX and are also helpful in evaluating the effect of antagonists (Limbird, 1986), whether competitive or noncompetitive, on the drug-receptor interaction. Some ligands that are structurally similar to an agonist and act at the same site as the agonist actually inhibit the receptor; these are competitive antagonists. They have affinity for the site but lack efficacy. Other antagonists inhibit the receptor by acting upon sites other than that of the agonist; these are noncompetitive antagonists. Numerous computer programs (Kenakin, 1987) are available for analyzing pharmacodynamics from doseresponse data. Mathematical tools such as these facilitate identification of nonideal behavior of drug-receptor interaction.

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EMAx-

A

Effect Eo.5_

I

d

wfh

/

! Noncompetitive/ Antagony / / .,~comd~tilhe Effect -N-

1 I Effect I

C

B

d

/ ! KEQ Eo.o

[Dl,oo

1/[D]

Effect

FIGURE 1 Dose-response curves for receptor-specific drugs. (A) Magnitude of effect (E) of drug (d) plotted against drug concentration [D] expressed logarithmically. Examples of E: intensity of activity (sperm velocity, muscle contraction, electric potential, secretion) or frequency of response (rate of flagellation, oocytes per month). (B) A Lineweaver-Burk double reciprocal plot. Curve represents data shown in A. Also shown is the effect of d at various concentrations in the presence of a competitive antagonist. Alterations in the concentration of a competitive antagonist would rotate the curve about the intercept on the ordinate, i.e., 1/EMAx. Also shown is the effect of d at various concentrations in the presence of a noncompetitive antagonist. Alterations in the concentration of the noncompetitive antagonist would rotate the curve about the intercept on the abcissa, i.e., -1/KEo. The effect of the competitive antagonist is to decrease the affinity of d without changing its efficacy. The effect of the noncompetitive antagonist is to decrease the efficacy of d without changing its affinity; this is similar to the dose-response curve of a partial agonist. (C) A Scatchard plot of effect of agonist d at various concentrations. A Scatchard plot of [DR]/[D] on the ordinate vs. [DR] will give [R] at the abcissa intercept.

Drug-receptor interactions are usually more complex than the simple model of Clark (Kenakin, 1995a; Limbird, 1986). In real life a number of factors may decrease [D] at receptors relative to the dose applied to the tissue. Such factors include nonspecific binding of the drug to tissue components, degradation of the drug by tissue components, and even drug uptake by cells in the tissue. Furthermore, EMAXmay be achieved at [R] considerably less than the total number of target receptors. In these circumstances EMAX is attained when only a few of the available receptors are engaged by the drug. Even the exposure of the tissue and its cells to a particular drug may alter the responsiveness of the system to the drug. Alterations may occur in the number of receptors to the drug, or in the population of receptors in the "normal" conformation, or in transduction of receptor signal to effect. Terms used to describe such changes are upregulation,

supersensitivity, downregulation, desensitization, tachyphylaxis, tolerance, and drug resistance. Pharmacologists would like to know a receptor well enough to design agonists and antagonists for it. This would require intimate knowledge of receptor structure, location of pharmacological domains, position and orientation of electronic charges such as H-bond donors, and the influence of ligand charges on receptor sites. Recent advances in quantum chemistry, structural analysis, and computer simulation have facilitated the design of new ligands (Brann et al., 1995; Dean, 1986; Kenakin, 1987).

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B. Membrane-Bound Receptors S e v e r a l g e n e r a l classes of m e m b r a n e - b o u n d r e c e p t o r s h a v e b e e n identified. O n e w e l l - c h a r a c t e r i z e d r e c e p t o r - e f f e c t o r s y s t e m links a g o n a d o t r o p i n sensitive r e c e p t o r t h r o u g h G p r o t e i n to an e f f e c t o r system. O f t h e s e v e r a l g o n a d o t r o p i n s , special a t t e n t i o n will be g i v e n to follicle s t i m u l a t i n g h o r m o n e ( F S H ) a n d luteinizing h o r m o n e ( L H ) . T h e g o n a d o t r o p i n r e c e p t o r for t h e G p r o t e i n c o u p l e d s y s t e m is a m e m b r a n e - b o u n d p o l y p e p t i d e w i t h t h r e e d i s t i n q u i s h a b l e d o m a i n s : external, t r a n s m e m b r a n e , a n d i n t r a c e l l u l a r ( F i g u r e 2). S t r u c t u r a l l y , a glycosyl-

NH2 External Domain Cell ~" MembraneL

Pro,e,n \

ATP

O,yco0enes,s1 , / Steroidgenesis~ "-Cell Division j

FIGURE 2

/ /

Kinase ~

Idiesterasel

G protein-linked receptor. One example of membrane bound receptor systems is composed of a polypeptide receptor linked by G protein to an adenylyl cyclase second messenger system. The receptor linked to the G protein is always a single polypeptide chain that has three structural components: external domain, transmembrane domain, and intracellular domain. The external domain is the portion of the polypeptide that "begins" with the Nterminus and resides entirely in the extracellular space. There are six glycosylated moieties bound to the polypeptide external domain. An agonist (or antagonist) interacts with the external domain. A segment of the polypeptide chain, the transmembrane domain, completely penetrates the cell membrane seven times. In this domain amino acid sequences of each passage through the membrane are coiled to form an alpha helix, each symbolized by a cylinder. The polypeptide chain of the receptor also has a segment solely situated in the cytoplasm, the C-terminus of the intracellular domain. The intracellular domain interacts with the alpha unit of the G protein. The G protein consists of three units: alpha, beta, gamma. The beta and gamma units are situated entirely in the cell membrane, while the alpha unit is situated predominately in the cytoplasm. The alpha subunit can alternatively interact with the intracellular domain of the receptor or with adenylyl cyclase situated in the cell membrane. Adenylyl cyclase, activated by alpha subunit of G protein, catalyzes the conversion of ATP to cyclic AMP (cAMP). Cyclic AMP acts as a second messenger to activate protein kinase of the cellular effector system. The action of protein kinase is terminated by phosphodiesterase conversion of cAMP to AMP.

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ated polypeptide forms the linear external domain that subsequently becomes a series of seven alpha helixes embedded in the plasma membrane; depending upon the receptor, either the external or the transmembrane domain serves as the site for ligand interaction. The intracellular domain of the receptor is coupled to G protein. G proteins are a superfamily of ubiquitous membrane-bound heterotrimers (Gilman, 1984; Strader et aL, 1994). They mediate receptor-effector coupling by serving as a "draw bridge." For receptors sensitive to gonadotropins and for many other receptors, the effector mechanism linked by G protein is membrane-bound adenylyl cyclase which generates a second messenger, cyclic adenosine monophosphate (cAMP). For other receptors, however, G proteins may be linked to one of several other effector systems including phospholipase C (PLC), and phospholipase A2; each of these effectors produce their own second messengers. The G protein may also be able to direct a message from a single receptor to several second messenger systems (Gudermann et aL, 1992). G proteins consist of three subunits. The alpha subunit (39-46 kDa) serves as a shuttle between the receptor, the other two G protein subunits, and the second messenger system such as adenylyl cyclase. The alpha subunit provides the G protein with access to the cytoplasm and binds guanosine monophosphate (GMP) that subsequently is phosphorylated when G protein is activated by the receptor. Integral to the alpha subunit structure is a GTPase. When inactive, the alpha subunit of G protein is associated with beta-gamma subunits, 37 and 8 kDa, respectively. During activation the alpha subunit dissociates from beta-gamma subunits and both alpha and beta-gamma bind to target effector, e.g., adenylyl cyclase. Subsequently alpha subunit reassociates with beta-gamma subunits. Dynamics of this complex series of G protein reactions have been presented by Kenakin (1995b), Milligan (1993), and Ross (1992). A second messenger system acts inside cells as an intermediary effector to amplify a ligand signal (e.g., FSH or LH) received from the environment and then to relay the signal to the final effector. Each second messenger system utilizes a characteristic moiety to relay the signal; cAMP is one of these (Sutherland and Rail, 1958). This second messenger system consists of membrane bound adenylyl cyclase, any one of several protein kinases, and phosphodiesterase. Adenylyl cyclase, upon activation by the alpha subunit of G protein, hydrolyzes adenosine triphosphate to cAMP. In turn cAMP activates any one of several protein kinases (PK) (Francis and Corbin, 1994). Cyclic AMP-dependent protein kinase (cAPK), involved in the FSH/LH system, has been described by Dufau et al. (1977), Scott (1991), Su et al. (1995), and Taylor (1990). Active PK subsequently utilizes adenosine triphosphate (ATP) to phosphorylate the final effector process, e.g., glycogenolysis. The second messenger process is terminated by hydrolysis of cAMP to 5'-AMP by phosphodiesterase.

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The relationship between G protein and adenylyl cyclase is complex. The G protein superfamily consists of several members acting differently upon second messenger systems. Gs activates adenylyl cyclase; Gi and Go inhibit it. Similarly complex is the relationship between second messenger system and final effector. Interactions occur between various second messenger systems. Some of these have been described by Brown and Birnbaumer (1990) and Sternweis and Smrka (1992). The complexity offers numerous opportunities for pharmacological intervention in the recept o r - G protein-second messenger systems, especially for ligands that can cross the plasma membrane.

C. Nuclear Receptors Receptors for steroid hormones are intracellular phosphoproteins that regulate genetic transcription. These receptors form a superfamily that respond to a variety of ligands in addition to steroids: thyroid hormone, vitamin D, retinoic acid, 9-cis retinoic acid, isoforms of many of these ligands, and a group of "unknown" ligands for which receptors have been cloned! Current knowledge of these receptors is extensive and can only be summarized here. Authoritative reviews have been written by Brann et al. (1995), Evans (1988), and O'Malley et al. (1995). Also, the book edited by Moudgil (1994) is detailed. The presentation in this chapter is a coarse sketch of the biology of the estrogen receptor (ER) and the progesterone receptor (PR). ER and PR are phosphoproteins located in the nuclei of target cells at a concentration of 6000-10,000 per cell (Pickler et al., 1976; Spelsberg, 1976). These receptors bind their specific ligand with high affinity (KzQ of 10-8-10 -1~ M). Estrogen and progesterone are lipophilic and therefore can cross cell membranes with ease and are trapped in target cells by binding to their respective receptors. Each of these receptors itself has approximately 590 amino acids with a molecular mass of 66 kDa. The receptor, however, exists as a heteromeric complex with heat shock proteins (hsp), phosphokinase(s), and transcription factors. (Note: hsp are members of a family of ubiquitous molecular chaperones essential in vivo for correct assembly and folding of many proteins.) Steroid receptors are synthesized by ribosomes in extranuclear endoplasmic reticulum. The amino acid sequence for ER of humans and chickens is 80% identical. Alignment of amino acid sequences of ER from various species reveals five highly conserved functional "domains" (Krust et al., 1986). The N-terminal A/B domain is highly variable in the superfamily of receptors. This domain activates target genes. Of particular interest is C domain, the DNA-binding domain (DBD) of 70 amino acids responsible for receptor binding to DNA and for receptor dimerization. The DBD sequence includes two sets of four cysteines. Four cysteines in each set

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form coordination bonds with a zinc ion. Intervening amino acids form an alpha helix. These two helices of DBD, called "zinc binding motifs," are oriented perpendicular to each other. Zinc binding motifs contact the phosphate backbone of DNA (Schwabe et al., 1993). Distal to C region comes the variable D region serving as a hinge to allow receptor conformation. This region may also contain a "nuclear localization signal," amino acid sequences that enable the protein to pass through nuclear pores. The large hydrophobic E region is functionally complex, allowing for ligand binding, hsp association, dimerization, and additional nuclear localization. Finally, F region serves an unknown function and provides the C-terminus. Newly synthesized receptors become associated with hsp 90, hsp 56, and perhaps other hsp in the cytoplasm. These hsp seem to serve at least two functions: to give the receptor a conformation that will accomodate steroid binding and to transport the receptor to the nucleus. Pratt (1992) believes that hsp serve as transportosomes to move the heteromeric receptor complex along the cytoskeleton and into the nucleus. Formation of the heteromeric complex requires ATP. Estrogen and progesterone bind to their respective receptors in the nucleus. Hormone binding dissociates hsp 90 from the heteromeric complex. The ligand changes receptor conformation, activating the receptor so that it can bind specifically to DNA base pairs known as hormone response elements (HRE). Activation seems to require phosphorylation of the receptor, a process that may be mediated by cAMP protein kinase associated with the heteromer. Activated ER and PR bind to their HRE as homodimers (Beato, 1989). Binding of steroid-receptor complex to HRE may bend DNA in a manner essential to transcription (Nardulli et al., 1993). Gene activation may additionally be dependent upon the presence of transcription factors such as Pit-1 (Day et al., 1990)

IV. PHARMACOKINETICS: DRUG ACCESS To be effective a drug must arrive by some means at its site of action. A drug applied to a biological system may arrive at the site of action by diffusion, by transport systems, or by a combination of these means. The drug may also encounter barriers along the way. The drug may encounter processes that chemically transform the drug into another substance. Pharmacokinetics is the study of the processes of drug delivery and dispersion from the site of action. These processes importantly influence the intensity and duration of drug action. Net diffusion or flux (J) of a drug, i.e., a chemical solute, may be quantitatively expressed as moles of drug passing per second through a plane (A) of 1 cm 2 perpendicular to direction of flow. Diffusion is dependent upon three principal factors: cross-sectional area of the plane through which

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flow occurs, concentration gradient, and the diffusional coefficient. Concentration gradient (dc/dx) is the difference in drug concentration (moles) as a function of distance traveled (dx) in centimeters. Diffusional coefficient (F) considers the ease with which solute moves through the system. So, according to the Fick equation, J = -FA

dc

-~.

Diffusion has been presented reasonably by House (1974) and Solomon (1968). Diffusion of a drug is usually described as a steady-state system, that is a system in which there is no net movement except solute movement. In this situation F, the diffusional permeability coefficient, has the units of mole/ dyne/s. A steady-state system should be distinguished from bulk (volume) flow in which the diffusion coefficient for water is referred to as the hydraulic water-permeability coefficient (Lp) and may have units of cm3/dyne/sec. (Bulk flow is related to osmosis.) So for any given system, a permeability coefficient for diffusional flow (Fo) or for volume flow (Ff) can be studied. Diffusional permeability coefficient (Fo) considers the radius of the drug molecule (ro), viscosity of the solution (n), and absolute temperature (T). So, according to the Stokes-Einstein equation, BT

Fo - 6rrnro' where B is Boltzmann's constant (namely the gas constant divided by Avogadro's number giving 1.38 • 10 -23 joules/~ or 1.38 x 10 -16 erg/~ and rr is the geometric constant 3.14. Hypothermic conditions approaching 0~ will increase viscosity and decrease diffusion; alternatives to the StokesEinstein relationship must be considered to describe diffusion of small molecules at subzero temperatures (Easteal, 1990; Parker and Ring, 1995; Tyrrell and Harris, 1984). When solute must diffuse across a membrane, the system is greatly simplified because diffusion of most dissolved substances through a membrane is much slower than diffusion through solvent (Hill, 1928; Kushmerick and Podolsky, 1969; Waud, 1969); therefore diffusion time in water can be ignored. In other words, diffusion through a membrane is governed mostly by properties of the membrane (e.g., thickness, pore size) in addition to the concentration gradient. An example of this system is administration of a drug to cells (such as sperm) suspended in an aqueous medium in a laboratory container (Figure 3). This is a closed system; the system itself is incapable of adding or removing materials (such as excreting metabolites). Furthermore, it is a two-component system: extracellular and intracellular. Drug added to the extracellular compartment is separated from the intracellular compartment by a diffusion barrier, the cell membrane. If the mem-

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Passive diffusion across cell membrane. (A) A sphere bounded by a permeable membrane (such as a liposome or a cell model) placed in a container of physiological medium. The single sphere in the drawing represents the total homogenous (single cell) population that would be found in an experiment. A drug (D) introduced into the central compartment (extracellular space) will diffuse into the sphere (intracellular space). As the concentration of drug in the intracellular space (DI) rises, some of it will diffuse into the extracellular space. The equation for first-order kinetics provides [DE] at any moment in time (t) when the initial concentration [D]0 and the rate constant (k) are known. (B) The concentration of drug ([DE]; [DI]) as a function of time (t). At t = 0, D introduced into the extracellular space shown in A will be a maximum concentration, i.e., [DE] = [D]0, while there will be no drug in the intracellular space, i.e., [DI] = 0. With the passage of time an equilibrium is attained in the inward and outward diffusion across the permeable membrane so that [DE] = [DI]. Because intracellular volume is much smaller than extracellular volume, the change in [DI] with time will greatly exceed the change in [DE]. (C) Semilogarithmic plot of [DE] as a function of time (t). The slope provides the rate constant (k).

FIGURE 3

brane is permeable to the drug, Brownian movement, controlled by temperature, will propel drug molecules across the cell membrane. A net flow or flux of drug occurs from the compartment of higher concentration to that of lower concentration. Flux rate will be dependent upon drug concentration in both compartments, permeability barrier impedance, and the temperature (which influences the activity of drug molecules). If drug is added instantaneously as a bolus to the extracellular compartment, initially flux from the extracellular compartment into the intracellular compartment will be greater than flux in the opposite direction (Figure 3). The extracellular

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side will have a greater number drug molecules and therefore a greater number of molecules with sufficient energy to traverse the membrane. As drug concentration in the intracellular compartment approaches that of the extracellular compartment, the rate of drug flow into the the intracellular compartment decreases. Eventually drug concentration in both compartments will be equal and then flux in both directions will be equal. In a graphical plot of drug concentration ([D]) vs time (t), concentration in the extracellular compartment ([DE]) diminishes with time while concentration in the intracellular compartment ([DI]) increases. Any unit of concentration may be used, e.g., M, mg/ml. Rate of change continuously diminishes throughout the process, but the rate constants (kl, ka) of both inward and outward fluxes do not vary at any time. The rate constant is expressed in time -~ units. At any given instant in time [DE] is related to the initial concentration ([D]0) by the first-order expression [DE] = [D]oe -kt or by In [DE] -- In [O~okt or

log [DE] = log [D]-okt/2"3~ At equilibrium the forward and reverse rates must be equal. Factors affecting free drug concentration will affect drug flux. Such factors include adsorption to macromolecules and surfaces and chemical degradation. Numerous drugs adsorb to glass, plastics, and polymeric surfaces. Drug adsorption to proteins such as albumin is well known. Other drugs may be chemically unstable, undergoing degradation by hydrolysis or oxidation when placed in solution. Membrane factors also affect flux, and many of these membrane factors are altered by temperature. Unstirred water layers and membrane composition, surface area, and thickness (depth) are relevant factors. Unstirred water layers (also called boundary-layer) are regions of slow laminar flow parallel to the membrane surface; these can be rate-limiting in regard to drug diffusion (Wilson and Dietschy, 1974). Membrane surface geometry (plane, cylindrical, spherical) has profound effect (Kenakin, 1987). Cell membranes are usually convoluted. Membrane composition also has a major effect. The physical properties of cell membranes are dramatically altered by temperature, as discussed in Chapter 5 of this book. Volume contraction as in hypertonic conditions can alter the permeability of lipid membranes so that classical formalisms such as the Fick equation inadequately describe diffusion; changes in the lipid state (gel, fluid) also alter permeability (Biondi et al., 1992). The lipid characteristics of cell mem-

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branes retards the flux of ionic substances and therefore the pH of the bathing solution becomes a critical factor determining flux (Notari, 1987). pH of the medium as altered by temperature will affect drug solubility (Douzou, 1977; Taylor, 1981). Membrane thickness (depth) and pore size have obvious effects on flux. Flux will be impeded by long, narrow channels through the diffusion barrier. Bulk flow characteristics of water and CPAs across cell membranes at subzero temperatures are customarily predicted by equations of Kedem and Katchalsky (1958) and presented elsewhere in this book. These equations take into consideration the Lp coefficient of the membrane to CPA and the reflection coefficient. The reflection coefficient expresses interaction between CPA, water and membrane. Techniques for acquiring necessary data (Bernard et al, 1988; Curry et al., 1995; Gilmore et al., 1995; Karow, 1987; Mazur et aL, 1984; Walcerz et al., 1985) and making the calculations (Walcerz, 1995) have been published. In contrast to passive transport by diffusion across a membrane, the membrane may actively participate in the transport process. Cell membranes contain molecular structures called transporters that can transfer specific substances into or out of the cell. One of the best known transporters is the Na § § pump. Such transporters often require metabolic energy, usually in the form of ATP, and can work against concentration gradients. It may transport a ligand from one compartment to another without regard for an "equilibrium state." Molecules known to be actively transported include many electrolytes, sugars, amino acids and vitamins. Since there are a finite number of molecular pumps in a membrane, the system is capacity limited. If the total number of transferable molecules exceeds the number of transporters available for transfer, the system will be saturated; the rate of transfer will reach a maximum value that cannot be exceded without addition of more pumps. In this situation transport will obey zero-order kinetics, that is transport will be independent of concentration. However, if the number of molecules to be transported is substantially less than system capacity, the system will function according to first-order kinetics, at a rate determined by concentration. Obviously tissue in vitro contains several types of cells, each of which may transport a drug differently. A drug may enter one cell type by passive transport, another cell by active transport, and a third cell may actively extrude the substance. In this situation the apparent uptake of the drug by the tissue will be the sum of the individual rate constants. If there are two processes, each involving first-order kinetics, then [DE] = [Ol]oe-kt + [O2]o e-kt.

A semilogarithmic plot of the drug uptake can be resolved into two different compartments (Figure 4), a fast compartment and a slow compartment. Although in vitro systems are closed, the ability of specific cells to sequester

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F I G U R E 4 Drug transport into tissue. (A) Drug (D) introduced into the central compartment and transported into tissue compartments by first order kinetics (see Figure 3). Two different cell populations are represented by the hemispheres. The total transport process, e.g., the elimination of D from the central compartment is the sum of the first-order kinetic processes occurring throughout the tissue, whether passive or active transport. (B) Elimination of D from the extracellular compartment as a function of time (t). A semilogarthmic plot of measured values of [DE] at various times can be resolved into a fast component and a slow component by a technique known as compartmental analysis or "feathering." In this technique the straight line portion of the curve resulting from measured values is extrapolated (---) to the ordinate. This line represents the slow component and its intercept with the ordinate represents [D] in the slow compartment at t = 0, i.e., [D2]0, a theoretical value rather than a real value. [D2] values obtained from inspection of the slow component line can then be subtracted from the measured values of [DE] in order to give values of [D1] which can then be plotted ( - 9 - ).

drugs gives the appearance of drug elimination. Similarly, chemical transformation of a drug also gives the appearance of drug elimination. Interpretation of compartment analysis must be cautious. Processes going on may have nothing to do with fluid compartments. Processes may

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M. K a r o w

represent nonspecific binding, chemical degradation, or other phenomena. Absence of demonstrable compartments may reflect syncytia or cell-tocell coupling.

V. GONADOTROPIN MEDIATION OF FOI.I]CULOGENESIS A. Use o f G o n a d o t r o p i n s in Tissue Preservation FSH and LH are used in the preservation of reproductive tissues to support in vitro maturation of ovarian follicles. The role of gonadotropins in vivo and in vitro will be reviewed followed by a discussion of the structure of the ligands and their interaction with the receptor. A cursory review of in situ mammalian reproductive physiology in males and females gives perspective to the pharmacological action of gonadotropins in vitro. FSH and LH are synthesized and released from the anterior pituitary of males and females in response to gonadotropin releasing hormone (GnRH) synthesized in the hypothalamus. FSH in females acts on ovarian granulosa cells to stimulate follicular maturation, to form LH receptors (Channing and Kammerman, 1973), and to secrete estrogen. LH in females acts through follicular cells to cause follicle rupture and release of the cumulus oophorus. LH also causes the corpus luteum to produce progesterone. FSH in males acts on Sertoli cells in seminiferous tubules to promote spermatogenesis. FSH also induces formation of LH receptors in Leydig cells found in interstitial tissue surrounding seminiferous tubules. LH in males stimulates Leydig cells to synthesize testosterone. Thus it is obvious that these gonadotropins are essential for in vitro culture of testicular tissue, but this is beyond the purview here. The pharmacology of gonadotropins on folliculogenesis in vitro is contrasted with that in vivo. In immature female mammals, ovarian oocytes are found in preantral follicles, that is those follicles in which the oocyte is closely surrounded by two to three layers of granulosa cells. Although the oocytes have a zona pellucida external to the oolemma, the granulosa cells physically contact the primary oocyte through cytoplasmic processes transversing the zona. Various substances are exchanged by the oocyte and follicular cells through these cytoplasmic bridges (Buccione et al., 1990a; Heller et al., 1981). Periodically throughout mammalian reproductive life a cohort of preantral follicles will be recruited to become Graafian (antral) follicles. Stimulus for recruitment of specific follicles is unknown. Antrum development in mice requires 2-3 weeks; in humans, 10-12 weeks. Stimulus for early follicular development after recruitment includes estrogen and FSH. Estrogen alone causes granulosa cells to divide, to increase in their FSH receptors, and to synthesize cAMP. FSH promotes follicular growth, antral formation,

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and the proliferation of LH receptors in thecal, interstitial, and granulosa cells (Channing and Kammerman, 1973). The appearance of LH receptors in developing follicular cells is accompanied by a decrease in FSH receptors and FSH-responsive adenylyl cyclase. There are no LH receptors on the oocyte itself (Lawrence et al., 1980). During antral development, factors from granulosa cells maintain meiotic arrest (Leibfried and First, 1980). In a fully mature follicle, FSH triggers mucification and expansion of cumulus cells (Buccione et al., 1990b). This expansion of cumulus decreases communication between granulosa cells and the oocyte (Larsen et al., 1986) and may allow meiosis to procede to metaphase II. Ovulation occurs only from a mature ovarian follicle, a Graafian follicle, that has attained a requisite concentration of LH receptors (McFarland et al., 1989). The action of LH is mediated by cAMP (Hosoi et al., 1989, Mills, 1975). Preantral and antral follicles may be removed from ovaries and matured in vitro. Although preantral follicles are usually obtained from ovaries of immature animals, follicles taken from sexually mature animals can also be matured in vitro. During in vitro development of preantral follicles, oocyte growth and granulosa cell proliferation occur coordinately, sometimes even in absence of gonadotropin stimulation. Germinal vesicle oocytes encased in a cumulus mass and matured in vitro are more likely to be fertilized than are denuded oocytes (Schroeder and Epigg, 1984). Gonadotropins enhance spontaneous maturation of oocytes from human (Prins et al., 1987), bovine (Younis et aL, 1988), and felid (Johnston et al., 1989), but are unnecessary for oocytes from mice (Schroeder and Eppig, 1984) and cows (Sirard et al., 1988). The action of FSH on mouse granulosa cells has been demonstrated in vitro (Eppig, 1991; Erickson et al., 1979) and depends upon the activation of adenylyl cyclase and the production of cAMP (Knecht et al., 1981). The role of LH in the in vitro maturation of follicles is uncertain. In media for in vitro folliculogenesis the concentration range for FSH is usually 10 -9 to 10 -8 M (0.1 to 1.0/zg/ml); for LH, 10 -8 to 10 -7 M (0.5 to 50/xg/ml). In summary, FSH is a vital component to in vitro folliculogenesis, capable of producing fully matured oocytes that can be fertilized and implanted and gestate to partuition of live, normal offspring. The effect of FSH can be enhanced by supplements of LH, steroid hormones, growth factors, and other substances (Eppig et al., 1992; Gomez et al., 1993; Goodrowe et al., 1991; Harper and Brackett, 1993; Keskintepe et aL, 1995; Roy and Greenwald, 1989; Roy and Treacy, 1993; Spears et aL, 1994; Woodruff et al., 1993).

B. Chemistry of Gonadotropins LH and FSH are glycoproteins (Figure 5), structural homologs of thyrotropin (TSH), which is also synthesized in the anterior pituitary, and of

184 1

2

A r m a n d M. K a r o w

3

4

5

ALA PRO ASP VAL

6

V

GLN ASP

= N-linked oligosaccharide

A

I

7 8 9 10 20 28 30 CYS PRO GLU CYS THR LEU GLN GLU ASN PRO PHE PHE SER GLN PRO GLYALA PRO ILE LEU GLN CYS MET GLYCYS CYS PHE SER ARG ALA

I

I i

i

40 50 Y 59 TYR PRO THR PRO LEU ARG SER LYSLYS THR MET LEU VAL GLN LYS ASN VAL THR SER GLU SER THR CYS CYS VAL ALA LYS SER TYR ASN 52 I 60 70 V 82 84 [ ARG VAL THR VAL MET GLY GLY PHE LYS VAL GLU ASN HiS THR ALA CYS HIS CYS SER THR CYS TYR TYR HIS LYS SER-COOH 78 87 90

I

I

y

SER ARG GLU PRO LEU ARG PRO TRP CYS HIS PRO ILE ASN ALA ILE LEU ALA VAL GLU LYS GLU GLY CYS PRO VAL CYS ILE THR VAL ASN 1 5 10 15 20 25 30 ASN SER CYS GLU LEU THR ASN ILE THR ILE ALA ILE GLU LYS GLU GLU CYS ARG PHE CYS ILE SER ILE ASN

I

~

I THR THR ILE CYS ALA GLY TYR CYS PRO THR MET MET ARG VAL LEU GLN ALA VAL LEU PRO PRO LEU PRO GLN VAL VAL CYS THR TYR ARG 31 34 35 38 40 45 50 55 60 THR THR TRP CYS ALA GLY TYR CYS TYR THR ARG ASP LEU VAL TYR LYS ASP PRO ALA ARG PRO LYS ILE GLN LYS THR CYS THR PHE LYS

I

i

ASP VAL ARG PHE GLU SER ILE ARG LEU PRO GLY CYS PRO ARG GLY VAL ASP PRO VAL VAL SER PHE PRO VAL ALA LEU SER CYS ARG CYS 61 65 70 75 80 85 90 GLU LEU VAL TYR GLU THR VAL ARG VAL PRO GLY CYS ALA HIS HIS ALA ASP SER LEU TYR THR TYR PRO VAL ALA THR GLN CYS HIS CYS

I

I

I

GLY PRO CYS ARG ARG SER THR SER ASP CYS GLY GLY PRO LYS ASP HIS PRO LEU THR CYS ASP HIS PRO GLN LEU SER GLY LEU LEU PHE 91 95 100 105 110 115 I GLY LYS CYS ASP SER ASP SER THR ASP CYS THR VAL ARG GLY LEU GLY PRO SER TYR CYS SER PHE GLY GLU MET LYS GLU LEU

I

COOH

5 Amino acid sequence for human luteinizing hormone (hLH) and human follicle stimulating hormone (hFSH). Each hormone is composed of two independent glycosylated subunits: alpha and beta. (A) The alpha subunit of both hormones is identical, and is also the alpha subunit for human chorionic gonadotropin and human thyrotropin. Amino acid positions in A are numbered in absolute sequence for the human subunit. With the exception of four "missing" amino acids following position 4, the human alpha subunit is homologous with that of LH and FSH of other vertebrate species. The alpha subunit has two branched oligosaccharides N-linked to arginine at positions 52 and 78. The alpha subunit has five internal disulfide bonds linking cysteines at positions 7 and 31, 10 and 60, 28 and 82, 32 and 84, and 59 and 87. Cysteine pairs participating in each of these disulfide bonds are actually juxtaposed by virture of polypeptide folding, a topological feature that cannot be illustrated in sequence diagram. Alpha subunit determines affinity of the hormone (ligand) for the receptor. (B) Beta subunit of hLH (top row) consists of 121 amino acids; hFSH (bottom row), 111 amino acids. Amino acid positions of two human beta subunits are numbered in sequence for homologs of chorionic gonadotropin and thyrotropin from many vertebrate species. Beta subunit of hLH has an N-linked oligosaccharide in position 30. Beta subunit of hFSH has Nlinked oligosaccharides in positions 13 and 30. Beta subunit of both LH and FSH has six internal disulfide bonds linking cysteines at positions 9 and 57, 23 and 72, 26 and 110, 34 and 88, 38 and 90, and 93 and 100 ("determinant loop"). Beta subunit determines efficacy of the hormone (ligand) for its receptor (after data from Jameson et al., 1988; Lapthorn et al., 1994; Lustbader et al., 1993; Shome et al., 1988).

FIGURE

I

COOH

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chorionic gonadotropin (CG), which is synthesized by the placenta. The molecular mass of LH is approximately 28 kDa; FSH, 33 kDa. The binding affinity (KEQ) of hLH for its receptor is 1.2 x 10 -1~ M (Jia et aL, 1991); hFSH, 1.7 x 10 -9 M (Tilly et aL, 1992). Each of these glycoproteins is composed of two glycosylated polypeptide chains, alpha and beta subunits, noncovalently bound. Individually these two subunits are biologically inactive; they must be combined for each one to attain functional conformation. Within a species the alpha subunit of the four hormones is identical and is responsible for conformation required for receptor binding and for stimulation of receptor linked adenylyl cyclase. In fact the alpha subunit of one species can usually be combined with the beta subunit of another species without loss of hormonal efficacy (Liao and Pierce, 1970), but this is not always possible. The beta subunit of each hormone is structurally unique and is responsible for hormone-specific receptor response. The primary structures of the alpha subunit (Fiddes et al., 1979; Keutmann et al., 1978; Lustbader et al., 1993) and of hFSH ( Jameson et al., 1988; Shome et al., 1988) have been reported. The three-dimensional structure of hCG (Lapthorn et al., 1994) has corrected previous beliefs based upon assumptions and speculation. The human alpha subunit with 93 amino acids has a molecular mass of about 14 kDa. This subunit has oligosaccharide moieties coupled to asparagine, positions 52 and 78, through N-acetylglucosamine (Baenzinger and Green, 1988). Each oligosaccharide is composed of a branched quadrasaccharide chain (sequentially: N-acetylglucosamine, fucose, N-acetylglucosamine, mannose) with two or three terminal heterogenous oligosaccharide branches consisting of N-acetylgalactosamine, N-acetylgluosamine, galactose, and mannose in various sequences. Most of the branches are terminated with sialic acid residue on galactose or sulfate on N-acetylgalatosamine; these groups give the oligosaccharides a negative charge. Similar oligosaccharide moieties occur on the beta subunit, too. Alteration or removal of oligosaccharides increases the affinity of the ligand for the receptor and greatly reduces hormone efficacy at these sites (Sairam and Schiller, 1979; Sairam, 1989). Deglycosylation of a gonadotropin transforms it into a competitive antagonist inhibiting the receptor's ability to produce cAMP and, ultimately, steroids (Chen et al., 1982; Sairam and Bhargavi, 1985). In contrast to the oligosaccharide attached to the alpha subunit position 78, the oligosaccharide at position 52 is essential for signal transduction (Metzuk et al., 1989). The conformation of the alpha subunit is maintained internally by five disulfide cross-links in addition to the conforming association with the beta subunit. The only helical structure in the gonadotropin is formed by amino acids in positions 40-50 of the alpha subunit (Lapthorn et al., 1994). Five C-terminal amino acids of the alpha subunit are important to receptor binding (Lapthorn et al., 1994).

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Human beta subunit of LH has 121 amino acids; FSH, 111. In hLH there is one oligosaccharide group (position 30); in hFSH, two (positions 13 and 30). (Note: in LH of most vertebrates, the beta subunit oligosaccharide occurs in position 13.) These groups are structurally similar to those found on the alpha subunit. The conformation of the beta subunit is maintained internally by six disulfide cross-links (Lapthorn et al., 1994). A segment of the beta subunit wraps around the alpha subunit like a seatbelt clinched by the Cys 26-Cys 110 disulfide bond (Figure 5B), stabilizing the intact heterodimer (Lapthorn et al., 1994). It is thought that the beta subunit is more rigid than the alpha subunit and serves as the greater determinant of hormone conformation. Although specific amino acids are conserved at many positions within the beta subunit of LH, FSH, TSH, and CG, nonsimilar regions probably confer hormone specificity and participate in binding to the receptor. Amino acids 93-100 in the beta subunit form a loop held in place by a disulfide bond; this "determinant" loop is thought to be important in specificity of ligand-receptor interaction. Amino acids 40-46 in the beta subunit form a loop thought to be important in receptor binding. Commercially available gonadotropins are usually prepared from biological sources. FSH and LH derived from recombinant DNA technology are also available. When synthesized by cells, each subunit of the ligand is coded on genes located on different chromosomes and independently synthesized. During protein synthesis in gonadotropes of the anterior pituitary, glycosylated subunits combine in endoplasmic reticulum. Pituitary content of these hormones is probably turned over once or twice daily. The glycoprotein hormone synthesized by gonadotropes is essentially identical to circulating hormone. Although the serum concentration of hLH and hFSH is clinically reported in international units (U), for LH 1 mU/ml approximates 150 pg/ ml. 1 Therefore the normal serum concentration of LH in a premenopausal woman will range from 10 -8 M to a high of 10 -6 M at the time of ovulation. For FSH 1 mU/ml approximates 500 pg/ml. ~ The normal serum concentration of FSH in a premenopausal woman will range from 7.5 x 10 -9 t o a high of 6 x 10 -8 M at the time of ovulation. In vitro these hormones are effective at 10 -1~ M (Ryan et al., 1990). The circulating half-life of LH and FSH is about 1 hr. The half-life of these hormones is increased in proportion to their sialic acid content (Fiete et al., 1991; Morell et aL, 1971). Their clearance and excretion are dependent upon liver and kidney, but no information is available concerning metabolic biotransformation. In the presence of free alpha and free beta subunits in the circulation, the alpha subunit is much more resistant to biodegradation; the beta subunit is removed more 1 Based upon the Second International Standard Luteinizing Hormone, Pituitary (Code 80/ 552), and upon the Second International Standard Follicle Stimulating Hormone, Pituitary (Code 78/549), of the National Biological Standards Board of Hertfordshire, England, a World Health Organization Laboratory for Biological Standards.

Pharmacological Interventions in Vitro

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efficiently (Peters et al., 1984). In addition, endocytosis of hormonereceptor complex is common. About 10-20% of circulating hormones can be recovered unchanged from urine (PeppereU et aL, 1975).

C. Gonadotropin Receptors Ovarian receptors for FSH are found only on granulosa cells. Their number in vivo is fairly constant except for a rise at the end of pregnancy and a decrease in atretic follicles. Ovarian LH receptors are found in theca, interstitial cells, luteal cells, and granulosa cells where their numbers vary according to follicular maturation (Richards, 1980). The number of cellular sites for a specific receptor can be determined with radiolabeled ligands (Kenakin, 1987; Limbird, 1986). Gonadotropin receptors have been studied by direct binding of hormones containing 1251labeled tyrosine residues. This radiolabel does not impair biological activity (Catt et al., 1976). Furthermore, gonadotropin receptors solubilized and removed from testicular or ovarian cells retain functional properties remarkably similar to those of in vivo receptors (Reichert and Dattatreyamurty, 1989). Gonadotropin receptors are oligomeric hydrophobic glycoproteins. The FSH receptor appears to consist of four disulfide-linked monomers, each with a molecular mass of about 60 kDa (Reichert and Dattatreyamurty, 1989). The receptor from testicular tissue contains 669 amino acids; from ovarian tissue, 674 amino acids. The external region with the hormonebinding domain contains 331 (testis)-341 (ovary) amino acids. The amino acid sequence for both receptors has been determined for humans (Jia et al., 1991; Minegishi et al., 1991) and other mammals. The receptor has three domains (McFarland et al., 1989; Sprengel et a/., 1990): an external hormone-binding domain, a seven-membered transmembrane domain, and an intracellular domain (Figure 2). The external domain contains the N-terminal portion, 331 (testis)-341 (ovary) amino acids and six sites potentially involved in glycosylation (Minegishi et aL, 1989; Sprengel et aL, 1990; Tsai-Morris et al., 1990). Deglycosylation of the FSH receptor does not alter its ability to bind with FSH ligand (Dattatreyamurty and Reichert, 1992). Certain positions in the amino acid sequence of their external domain of FSH receptors are required for specific ligand bonding (Dattatreyamurty and Reichert, 1993; Leng et al., 1995). The intracellular domain is coupled to G protein (McFarland et al., 1989) and has the C-terminus.

VI. REACTIVE OXYGEN SPECIES (ROS) Reactive oxygen species (ROS) and their free radical products are significant contributors to health and disease of mammalian reproductive

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A r m a n d M. K a r o w

systems. In fact these chemicals are ubiquitous components of aerobic life. ROS, themselves not radical species, give rise to oxygen free radicals. In addition to being products of metabolism, they serve as regulatory signals (Burdon and Gill, 1993; Fialkow et al., 1994; Sundaresan et al., 1995), as agents in apoptosis (Hockenbery et al., 1993; Kane et al., 1993; Levine et al., 1994), and as a defense mechanism against infection (Chanock et al, 1994, Hyslop et al., 1995; Levine et al., 1994). Oxidative free radicals and ROS make significant contributions to normal mammalian reproduction as reviewed by de Lamirande and Gagnon (1994) and by Riley and Behrman (1991). These chemical species may contribute to ovarian physiology, ovulation, and corpus luteum formation. They influence spermatozoan structural integrity and function including motility, capacitation, and sperm-oocyte fusion. In the uterus they may participate in parturition and in creating a bactericidal environment. Phagocytic leukocytes in the reproductive tract produce ROS. When biological control of their chemistry fails, these agents give rise to pathological processes as reviewed by Freeman and Crapo (1982), Halliwell (1988), and by Tarr and Samson (1993). Loss of this control may occur in reproductive tissues in vitro and therefore special attention is given in this book to pharmacological intervention. To better understand the effect of ROS in reproductive tissues in vitro, literature discussing this effect in other mammalian tissues in vitro is included in this review. A. Source a n d B i o c h e m i s t r y o f ROS Oxygen free radicals include superoxide (O~'), the hydroxyl radical ('OH), nitric oxide ('NO), and peroxynitrite (ONOO-). ROS include hydrogen peroxide (H202) lipid peroxides, hypochlorous acid (HOC1), and singlet oxygen (02 electronically excited). Many of these species can be quantitively studied in living cells in vitro using electron spin resonance (ESR) technology (Tarr and Samson, 1993; Zweier, 1988). They arise in mitochondria as products of ATP synthesis by the cytochrome oxidase chain. During this complex process four electrons must be passed almost simultaneously in a variety of steps in the tetravalent reduction of 02 to H20. In this process 1-2% of the electrons produce O~'. Superoxide is also produced in other sites (e.g., microsomes, peroxisomes, cytosol, nuclear membrane, endoplasmic reticulum, plasma membrane) and by other chemical reactions, some of which are enzyme controlled (e.g., oxidation of reduced nicotinamide adenine dinucleotide phosphate (NADPH) to NADP+; oxidation of xanthine dehydrogenanse (XD) to xanthine oxidase (XO)). Production of O~" by membrane associated "respiratory burst oxidase" that catalyzes reduction of NADPH may be especially important in human spermatozoa (Aitken and Clarkson, 1987) and in phagocytic leukocytes (Chanock et al., 1994). Chemistry of O~" production from these several sources has been reviewed

Pharmacological Interventions in Vitro

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(Chanock et al., 1994; Halliwell, 1988; Tarr and Samson, 1993). Superoxide is relatively poorly reactive in aqueous media and crosses biological membranes much slower than H 2 0 . As mentioned a portion of O~"is utilized as a regulatory signal. Similarly, "NO, a product of arginine conversion to citrulline (Nathan, 1992), is a regulatory molecule known to be important to the physiology of immune, pulmonary, cardiovascular, and nervous systems; its role in reproduction is being investigated (Ben-Shlomo et al., 1994; Davidoff et al., 1995; Kugu et al., 1995; Shukovski et al., 1994; Welch et aL, 1995). Nitric oxide is normally "inactivated" by reaction with either O2 or O~" (Freeman, 1994). Endogenous reaction between O~" and "NO is fast, being 6.7 + 0.9 x 109 M/s (Huie and Padmaja, 1993), and produces significant quantities of ONOO- (Rubbo et al., 1994). Although ONOO- is oxidative and cytotoxic (Freeman, 1994), "NO limits these oxidation reactions through a feedback servoregulatory mechanism (Rubbo et al., 1994). Although O~" is not highly reactive, it must be disposed of; excess O~" and its products can be damaging to cells. Superoxide will form H202 spontaneously or catalytically by any of several superoxide dismutases (SODs). Reactivity of H202 is similar to that of O~', but diffusivity of H202 is much greater than O~'. Therefore H202 must be disposed of, too. In cells under most conditions H202 is rapidly converted to water by catalase or to other "nonreactive" products by peroxidases (especially glutathione peroxidase). H202 can also be converted to HOC1 by myeloperoxidase. The steady-state intracellular concentration of O~" is 10 -12 to 10 -13 M in liver and for H202, 10 -9 to 10 -7 (Chance et al., 1979). SODs, catalase, and peroxidases are considered to be detoxifying enzymes enabling cells to maintain O~" and H202 at physiological concentrations. SODs are metalloenzymes (32-134 kDa) found in almost all facultative and obligative aerobes. MnSODs are found in mitochondria; CuZnSODs, in mitochondria and cytosol of eukaryotes. Catalase is a 240-kDa heme enzyme containing iron and is found in peroxisomes and cytosol. Glutathione peroxidase is a metalloenzyme with selenium and is found in peroxisomes and cytosol. In addition to enzymatic detoxification of oxidative species, defense against high concentrations of oxidants include endogenous antioxidants (Table 1). Some antioxidants listed in Table 1 act by "scavenging" free radicals; that is they are oxidized by the radical and the product is subsequently reduced so that the original compound is regenerated (Keegan et aL, 1992). Many of these substances can be used pharmacologically to protect tissues in vitro from oxidative cytotoxicity. Of the water soluble agents in Table 1, ascorbic acid and glutathione seem to be most effective; of the lipophilic, alpha-tocopherol (vitamin E) is most effective. Defense against oxidative injury also includes cellular repair and special environ-

190

A r m a n d M. K a r o w TABLE 1

Sites of Major Natural Antioxidants

Tissue fluid Albumin Ascorbic acid (vitamin C) Bilirubin Ceruloplasmin Haptoglobin

Lactoferrin Superoxide dismutase Transferrin Uric acid

Cellular membranes Carotenoids (e.g., beta-carotene) Superoxide dismutase Tocopherols (e.g., vitamin E) Ubiquinol-10 Cytosol Ascorbic acid (vitamin C) Catalase Ferritin Glutathione

Glutathione peroxidase Glutathione transferase Superoxide dismutase

ments that may operate at low oxygen concentration (e.g., immature germ cells surrounded by nurse cells). Regardless of the means for converting O~" and H202 to relatively nonreactive species, on occassion these means are subverted or overwhelmed so that H202 is converted to highly reactive, cytotoxic "OH. The extreme reactivity of "OH essentially precludes its diffusion more than a few Angstroms from its origin. The hydroxyl radical is produced from H202 by Fenton and possibly Haber-Weiss chemistry that requires involvement of transition metals such as iron and copper. In Fenton chemistry, ferric ions and other transition metals come from proteins such as ferritin, transferrin, lactoferrin, myoglobin, hemoglobin and ceruloplasmin. Although transition metals are ordinarily tightly bound to protein, they are released when proteins react with O~', making metal ions available for Fenton reactions (Aruoma and Halliwell, 1987; Beimond et al., 1984; Vercellotti et al., 1985). Toxic effects of ROS and oxidative free radicals, especially'OH, include damage to cell membranes through lipid peroxidation, damage to nucleic acids, and protein oxidation. Biological membranes are particularly vulnerable to oxidative reactions because their phospholipids contain a significant proportion of esterified polyunsaturated fatty acids (Fuller et al., 1988). Hydroxyl radical initiates lipid peroxidation, a chain reaction propagated by formation of malondialdehyde (MDA), a toxic compound that crosslinks lipids and proteins (Gutteridge, 1988). (Formation of MDA is detected in laboratory studies through the thiobarbituric acid test). Furthermore

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phospholipid oxidation produces arachidonic acid, eicosanoids and prostaglandins (Riley and Behrman, 1991), reactions that generate additional oxygen free radicals (Egan et al., 1976). Deoxyribonucleic acid (DNA) damage from oxidants occurs in all cell types studied to date (Ames et al., 1993; Cochrane, 1991; Richter et al., 1988; Schraufstatter et al., 1986). DNA damage results in mutation of base sequence (Dionisi et al., 1975) and can cause cell injury or death. Damage in target cells is found at [H202] as low as 20/zM (Cochrane, 1991). Proteins also are damaged by oxidative events (Oliver et al., 1987; Stadtman, 1992). Phagocytic leukocytes produce high concentrations of oxidative free radicals and ROS as a normal component of their defense functions. A stimulus triggers a membrane-bound receptor linked to G protein to initiate a "respiratory burst" (Chanock et al., 1994; Rossi, 1986; Weisman et al., 1980). Respiratory burst requires transfer of intracellular Ca 2§ to active membrane protein kinase C, O2 uptake, stimulation of hexose monophosphate shunt, and production of O~" and H202. These oxidative species are then released from the cells. Respiratory burst is essential for effective microbicidal activity. Mammalian cells in vitro rendered hypoxic or hypothermic are predisposed to producing large quantities of O5" (Mack et al., 1991). Of several possible mechanisms by which ROS are generated under these conditions, one may involve calcium and mitochondria (Farber, 1981; Schanne et al., 1979). In contrast to extracellular Ca 2§ concentration of 10 -3 M, intracellular Ca 2§ concentration is normally maintained at 10 -7 M by a variety of ATPdependent pumps. During conditions of hypoxia or hypothermia, activity of these pumps are compromised and intracellular [Ca 2§ rises quickly. For example, intracellular [Ca 2§ rises 50% in i h in kidney cortex slices rendered hypoxic at 4~ (Trump et al., 1974); similar studies on reproductive tissues have not been reported. Although excess intracellular Ca 2§ is accompanied by increased production of O~" by mitochondria, the relationship is unknown. In addition to increased production of O~" by mitochondria in hypoxic or hypothermia conditions, these conditions decrease production of mitochondrial ATP, leading to further influx of Ca 2§ Also, oxidative species increase membrane permeability to Ca 2§ (Ungemach, 1985). In many tissues an increase in intracellular [Ca 2§ induced by hypoxia or hypothermia can also lead to excessive O~" by an extramitochondrial mechanism. This second mechanism is particularly insidious, producing free radicals when oxygen is restored to cells. Excess Ca 2§ converts XD to XO. XD and XO each catalyze breakdown of adenine nucleotide metabolites, specifically hypoxanthine to xanthine and xanthine to uric acid. Reaction with XO, in contrast to that seen with XD, requires NADPH and O2, and the reaction produces O~" (McCord, 1985). Ischemic increase in intracellular [Ca 2§ leads to an increase in [XO]. Also, during ischemia, as a result of ATP utilization [xanthine] increases without concomitant ATP regenera-

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A r m a n d M. K a r o w

tion; during 0 2 restoration the XO reaction involving high [xanthine] produces proportionate quantities of O~'. Time course for conversion of XD to XO is tissue specific within a species (Paler-Martinez et al., 1994): at 37~ the half-time for conversion ranges from 3.6 to 14 h; at 4~ 5 to 6 days (Southard et al., 1987). The importance of this reaction for reproduction tissues has not been reported. B. ROS a n d M a m m a l i a n S p e r m a t o z o a i n V i t r o Mammalian spermatozoa, like other mammalian cells, generate ROS (Aitken and Clarkson, 1987; Alvarez et al., 1987; Plante et al., 1994) and have chemical systems for controlling the concentration of ROS. In the male reproductive tract, the relative influence of enzymatic systems mediating free radicals varies by cell type (Bauche et al., 1994). ROS of mature spermatozoa presumably can originate from the rich complement of mitochondria (Gavella et al., 1992). ROS of mature spermatozoa are also generated by a membrane-bound NADPH oxidase (Aitken and Clarkson, 1987). ROS can be identified within the cells and within the seminal plasma. Spermatozoa, however, are surely not the only source of ROS in seminal plasma; granulocytes in the seminal plasma and possibly components of the male reproductive tract serve as additional sources. Furthermore, seminal plasma contains iron that is apparently free, not bound to ferritin or transferrin (Kwenang et al., 1987). Antioxidant systems are present in both spermatozoa and seminal plasma. SOD is found in both spermatozoa and seminal plasma (Alvarez et al., 1987; Menella and Jones, 1980). Similarly, catalase is found in both spermatozoa and seminal plasma of humans (Jeulin et al., 1989; Zini et al., 1993). Spermatozoa also have the glutathione peroxidase/reductase pair (Alvarez et al., 1987; Li, 1975; Menella and Jones, 1980). Other antioxidants found in seminal plasma include glutathione peroxidase/reductase (Alvarez and Storey, 1989; Kantola et al., 1988; Li, 1975) and a variety of ROS "scavengers'" glutathione, vitamins C and E, hypotaurine, taurine, and albumin (Alvarez and Storey, 1983; Chow, 1991; Dawson et al., 1992; de Lamirande and Gagnon, 1992b; Guerin et al., 1995; Holmes et al., 1992). In mammals, the relative influence of enzymatic systems disposing of free radicals and ROS in spermatozoa varies by animal species. SOD is the prime enzymatic mechanism in human spermatozoa even though glutathione peroxidase/reductase plays an active role (Alvarez and Storey, 1989). SOD is the prime mechanism in rabbit sperm; these have low glutathione peroxidase/reductase activity (Alvarez and Storey, 1989). Glutathione peroxidase/reductase is the prime mechanism in murine spermatozoa even though SOD is present (Alvarez and Storey, 1984, 1989). ROS have been implicated in normal capacitation of mammalian sperm. Capacitation (Austin, 1952; Chang, 1951) involves hyperactivation to propel

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sperm through barriers surrounding the oocyte; it also involves an acrosome reaction to cytolyze those barriers. Hyperactivation appears to be dependent upon sustained generation of O~', controlled by [SOD], and O~"conversion to H202 (Bize et al., 1991; de Lamirande and Gagnon, 1995; Oehinger et al., 1995); the acrosome reaction may be similarly dependent upon H202 (de Lamirande et aL, 1993). Physiological concentrations of ROS may also facilitate in the attachment of spermatozoa to oocytes (Aitken et al., 1989, 1991). Superoxide may be more influencial than H202 in sperm from some species of animals; H202 may be more influencial in others (Aitken et al., 1993; Alvarez et al., 1987; de Lamirande and Gagnon 1992a; Oehringer et aL, 1995). Each of these activities~capacitation and gamete fusion~are accompanied by an increase in spermatozoal intracellular [Ca 2§ (Fraser, 1995); research results suggesting a relationship between calcium influx and ROS generation in spermatozoa has been reported (Aitken et al., 1989; Oehninger et al., 1995). Another physiological role for ROS in mammalian semen is defense against infection. Phagocytic leukocytes present in semen generate ROS (Aitken et al., 1995; Aitken and West, 1990; Jones et al., 1979). Antioxidants in normal seminal plasma attenuate the inhibitory effect of ROS on sperm motility at leukocyte concentrations below 1-4 • 10 6 per milliliter. Spermatozoa placed in suspension free of seminal plasma are profoundly inhibited by leukocyte concentrations as low as 2 x 10 4 per milliliter (Aitken et al., 1995). Excess ROS damages spermatozoa by peroxidation of membrane lipids. Spermatozoa are particularly vulnerable to ROS by virtue of the high content of polyunsaturated fatty acids in their cell membranes (Jones et al., 1979) and the low concentration of detoxifying cytoplasmic enzymes (Alvarez et al., 1987; Alvarez and Storey, 1989). Membrane lipid peroxidation has been correlated with spermatozoal midpiece morphological defects (Rao et aL, 1989; Aitken et al., 1993) and with decreased motility (de Lamirande and Gagnon, 1992a,b; Griveau et al., 1995; Oehninger et al., 1995). The inhibitory effect of H202 on sperm motility has been known for many years (MacLeod, 1943; Tosic and Walton, 1950). High [ROS] appear to be the cause rather than the result of decreased motility of spermatozoa (Plante et aL, 1994). Furthermore, high [ROS] inhibit spermoocyte fusion (Aitken and Clarkson, 1987; Oehninger et al., 1995). The sensitivity of spermatozoal membrane to peroxidation is species specific (Alvarez et al., 1987), human being more sensitive than mouse or rabbit. Among humans, there is a large variability in lipid peroxidation measured in different ejaculates (Jones et al., 1979; Mann and LutwakMann, 1981; Rao et al., 1989) and within a given ejaculate. Within each ejaculate are some spermatozoa highly productive and highly conservative of ROS species; these fractions can be separated by discontinuous density gradients of Percol (Aitken and Clarkson, 1988; Zalata et al., 1995).

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Semen of infertile men with damaged or defective spermatozoa, in comparison to semen of fertile men, has [ROS] elevated about 40-fold (Aitken and Clarkson, 1987; Iwasaki and Gagnon, 1992; Krausz et al., 1994; Zalata et aL, 1995), an observation most likely due to elevated production of ROS by abnormal spermatozoa (Zini et al., 1993), but could be due to reduced concentrations of antioxidants (Jeulin et aL, 1989; Lewis et al., 1995). The suggestion has been made that [ROS] in semen might be used as a diagnostic indicator of infertility (Aitken et al., 1991; Rao et al, 1989; Zalata et aL, 1995). Laboratory procedures can initiate peroxidation of lipids and other molecule by ROS in reproductive cells. Iatrogenic causes of ROS production in reproductive cells may be speculative in cases such as anoxia, but in other situations such as centrifugation or cryopreservation it may be readily demonstrable. Whether spermatozoa or other reproductive cells in vitro experience anoxia is not known with certainty, but the effects of anoxia and subsequent reoxygenation with production of ROS has been demonstrated in other mammalian tissues. In vitro, kidneys rendered anoxic at normothermia for as little as 15 min will lose 80% of their ATP and 60% of their total adenine nucleotides (Pegg et al., 1981; Southard et al., 1977). The generation of ROS in various anoxic tissues reoxygenated has been measured (Southard et aL, 1987). Media components may also influence (enhance or inhibit) lipid peroxidation by ROS. The opportunity for unwitting enhancement is demonstrated by work suggesting that Hepes (4-(2-hydroxyethyl)-I piperazineethanesulfonic acid), a Good buffer similar to Tris (2-amino-2(hydroxymethyl)-l,3-propanediol), may stimulate formation of cytolytic concentrations of H202 (Bowman et al., 1985; Zigler et al., 1985). This effect of Hepes seems to be exacerbated by exposure to light (Zieger et al., 1991). The production of ROS by spermatozoa is increased 20- to 50-fold by repeated centrifugation and resuspension (Aitken and Clarkson 1988; Iwasaki and Gagnon, 1992; Zalata et al., 1995). This is observed even under mild conditions of 500g for 5 min. The mechanism by which centrifugation provokes ROS production is unknown. Seminal plasma and its SOD are usually removed after centrifugation (Aitken and Clarkson, 1988; Iwasaki and Gagnon, 1992; Zalata et aL, 1995), a process unlikely to account for elevated [ROS] although this possibility has not yet been ruled out by published experiments. Significant lipid peroxidation occurs in membranes of cryopreserved human spermatozoa (Alvarez and Storey, 1992; Bell et al., 1993). This peroxidation can be attributed to loss of SOD upon thawing (Lasso et aL, 1994). These results are consistent with the observation that cryopreservation can permeabilize plasma membranes with acute loss of vital intracellular molecules (Watson et al., 1992) and with delayed manifestations of

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injury (Holt and North 1984, 1986). There is no support for the hypothesis that post-thaw membrane leakiness is a result of freeze-induced membrane lipid peroxidation; lipid peroxidation inhibitors (albumin, hypotaurine, alpha-tocopherol) have no detectable effect in preventing post-thaw manifestations of injury to spermatozoa (Alvarez and Storey, 1993). Not all spermatozoa in an ejaculate are equally susceptible to these sublethal injuries (Lasso et aL, 1994).

C. Free Radical Scavengers Pharmacological opportunities to manipulate normal physiology of ROS are obvious, numerous, and largely ignored. Instead, much attention has been given to cellular protection from ROS cytotoxicity. Numerous compounds identified as free radical scavengers have been used in vivo and in vitro. Therefore the present discussion is focused on toxicological management of ROS found in reproductive tissues in vitro; correlative evidence from other tissues is presented. These drugs fall into two distinct groups: those that directly interact with free radicals and ROS and those that intervene to block generation of oxidative species or to treat consequences of injurious oxidative processes. In the first group, listed in Table 1, are endogenous agents and some exogenous agents, all of which are free radical scavengers. In the second group are drugs affecting enzymes (either glutathione peroxidase or xanthine oxidase), drugs chelating iron, or drugs blocking calcium channels. The full pharmacology of these drugs cannot be presented in this chapter; representative drugs are only mentioned. Catalase (4.5/xg/ml) added to bovine semen maintained at ambient temperature for 30-54 hr enhances fecundity; catalase added to refrigerated semen (5~ has no effect on fecundity (Shannon and Curson, 1982). Catalase and SOD added to samples of human spermatozoa free of seminal plasma protect the spermatozoa from ROS (Aitken et al., 1989), but catalase is much more effective (de Lamirande and Gagnon, 1992a). Catalase (8/zg/ml) totally counteracts toxic effects of ROS treatment, whereas SOD (1 mg/ml) cannot prevent temporary adverse changes in motility. In contrast to these results, SOD is much more effective than catalase in enhancing development of rabbit zygotes cultured in a protein-free medium with approximately 20% oxygen (Li et al., 1993). Alpha-tocopherol (vitamin E), a lipophilic antioxidant present in reproductive tissues, is an effective scavenger of free radicals and terminates lipid peroxidation. Dietary deficiency of vitamin E is associated with significant increase in peroxidation in rat testes (Lomnitski et al., 1991). Dietary vitamin E seems to significantly improve in vitro function of human spermatozoa (Kessopoulou et al., 1995). Vitamin E concentration in rat ovaries varies with luteal cycle phase (Aten et al., 1992).

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Alpha-tocopherol added in vitro to human spermatozoa that have been centrifuged and washed free of seminal plasma acts in a dose-dependent manner to prevent lateral diffusion damage in the plasma membrane but cannot completely reverse "OH disruption of the membrane (Aitken and Clarkson, 1988; Aitken et al., 1989). Alpha-tocopherol is difficult to solubilize for use in aqueous media, but a hydrophilic analog of it, 6-hydroxyl-2, 5, 7, 8-tetramethylchroman-2carboxylic acid (Trolox, Aldrich Chemical, Milwaukee, WI) also scavenges ROS (Castle and Perkins, 1986; Giulivi and Cadenas, 1993) and protects a variety of cells in vitro: human erythrocytes, human hepatocytes, and human ventricular myocytes (Wu et al., 1990); rat renal cells (Zeng and Wu, 1992); and rabbit corneal cells (Zeng et aL, 1995). Trolox penetrates biomembranes more rapidly than alpha-tocopherol and is a more effective scavenger (Castle and Perkins, 1986; Doba et al., 1985). Trolox as an antioxidant is not as effective as purpurogallin (Wu et al., 1991; Zeng and Wu, 1992; Zeng et aL, 1995). The effect of Trolox on reproductive tissues has not been reported. Purpurogallin (2,3,4,6-tetrahydroxy-5H-benzocyclohepten-5-one, Aldrich Chemical) is an amphipathic (i.e., both hydrophilic and lipophilic) antioxidant demonstrably cytoprotective in vitro of rat hepatocytes (Wu et al., 1991), rat renal cells (Zeng and Wu, 1992), and human erythrocytes (Sugiyama et al., 1993). Purpurogallin scavenges "OH (Prasad and Laxdal, 1994) and inhibits glutathione-S-transferase and glutathione reductase noncompetitively toward oxidized glutathione (Kurata et al., 1992). Butylated hydroxytoluene (2,6-di-tert-butyl-4-methylphenol) as an antioxidant at 0.1 mM is more effective in preventing iron-catalyzed lipid peroxidation of human spermatozoa than is vitamin E (Aitken and Clarkson, 1988). Butylated hydroxytoluene (BHT) is very lipophilic and readily permeates the spermatozoan plasma membrane. Interaction of BHT with cellular membranes may contribute to its ability to protect cells from ROS (Miura et aL, 1995). BHT can also disrupt several membrane-dependent functions of spermatozoa. At 1.0-10 mM, BHT suppresses both spermatozoal motility (de Lamirande and Gagnon, 1992a) and capacity for spermoocyte fusion, adverse effects not seen at 0.1 mM nor with equimolar vitamin E (Aitken and Clarkson, 1988). As noted in a previous chapter (Parks), BHT suppresses or inhibits manifestations of cold shock in spermatozoa of boars (Bamba and Cran, 1992; Hammerstedt et al., 1976), bulls (Killian et al., 1989), and rams (Pursel, 1979; Watson and Anderson, 1983). BHT also is beneficial to bull spermatozoa undergoing cryopreservation (Killian et al., 1989). A relationship between antioxidant effects of BHT and thermal protection by BHT for spermatozoa has not been established. Hypotaurine and taurine are effective in inhibiting lipid peroxidation of rabbit spermatozoa washed free of seminal plasma and also effective in preventing loss of spermatozoan motility from ROS activity (Alvarez and

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Storey, 1983). These antioxidants are present in seminal plasma and in oviductal fluids (Guerin et al., 1995). Taurine and hypotaurine do not enhance the survival of thawed bovine spermatozoa (Chen et al., 1993). Taurine does enhance development of rabbit zygotes cultured in a protein-free medium with approximately 20% oxygen (Li et aL, 1993). Bovine serum albumin is highly effective in inhibiting lipid peroxidation of rabbit spermatozoa washed free of seminal plasma and also effective in preventing loss of spermatozoal motility from ROS activity (Alvarez and Storey, 1983). Perhaps through a similar mechanism polyethylene glycol is able to inhibit lipid peroxidation of rat hepatocytes in the presence of iron in vitro (Mack et al., 1991). Several cryoprotectants, notably dimethylsulfoxide and mannitol, are known to protect a variety of cells (Green et al., 1986; Rubanyi and Vanhoutte, 1986; Shlafer et aL, 1982) including human spermatozoa from ROS (de Lamirande and Gagnon, 1992a). These seem to act as free radical scavengers. Reduced glutathione (GSH) is essential in the direct conversion of H202 to water by glutathione peroxidase (GPX). The activity of GPX in this conversion is subject to enzyme saturation. Inactivation of GPX occurs in the presence of H202 when endogenous GSH is completely converted to oxidized glutathione (GSSG). This has been demonstrated in murine and human spermatozoa (Alvarez and Storey, 1989). Theoretically supplemental GSH in media would help cells dependent upon GPX for defense against excess ROS, especially H202. A beneficial effect of GSH in vitro has been demonstrated for bovine oocytes (Yoshida et al., 1993), for mammalian renal cells (Leibach et al., 1974; Ploeg et al., 1988), and for human spermatozoa (de Lamirande and Gagnon, 1992a). GSH administered by injection to infertile men had a positive beneficial effect on spermatozoal motility kinetics and morphology in a placebo-controlled, double-bind, cross-over study (Lenzi et al., 1993). The mechanism of GSH in this clinical study and in many in vitro studies cannot be attributed to stimulation of GPX and H202 conversion without specific documentation. GSH is vital to cellular economy in many ways beyond its influence in H202 metabolism (Meister, 1983). Its physiological role in the mammalian testis (Peltola et al., 1992) and ovary (Aten et al., 1992; Okatani, 1993) has been reported. Other sulfhydryl compounds have been investigated as agents that intervene in ROS cytotoxicity. These include dithiothreitol (de Lamirande and Gagnon, 1992a; Rao and David, 1984) and N-acetylcysteine (Kakano et al., 1995). They may act as sacrificial targets for oxidation, sparing molecules with a vital function in cellular activity. Allopurinol, a potent inhibitor of XO, has been included in many media for maintaining cells in vitro (Toledo-Pereya et al., 1974; Paler-Martinez et al., 1994). Allopurinol seems to inhibit production of O~" by XO in mamma-

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lian tissue (Paller et al., 1984); but in the absence of conclusive studies, any beneficial effect might also be attributable to conservation of adenine nucleotides (Shim et al., 1992; Toledo-Pereya et al., 1974). Iron chelators have been reported to have a beneficial effect on spermatozoa (Aitken et al., 1993), cells in culture (Zieger et al., 1990), ischemic kidneys in vitro (Gower et al., 1989; Green et al., 1986), and even liposomes (Radi et al., 1991). These beneficial effects are observed for chelators such as ethylenediaminetetraacetic acid (EDTA) confined to the extracellular space and for chelators such as deferoxamine mesylate that enter the intracellular compartment (Zieger et al., 1990). These agents could act by blocking the generation of "OH by the Fenton reaction. Verapamil inhibits Ca 2§ influx into mammalian cells including spermatozoa (Anand et al., 1994) and seems to reduce ischemic injury (Gingrich et al., 1985), but whether it beneficially inhibits ROS generation is unknown. Verapamil may actually increase ROS generation in spermatozoa (Anand et al., 1994).

VII. NONSPECIFIC DRUG ACTION: CRYOPROTECTANTS

The purpose of this section is to introduce the concept of nonspecific drugs as illustrated by cryoprotectants (CPAs). (The reader is invited to contrast nonspecific drugs with specific drugs, discussed in other sections of this chapter.) Drugs that produce a desired effect on a biological system through multiple mechanisms are considered to be nonspecific drugs. CPAs are an example of nonspecific drugs; they enable cells to survive freezing (solidification) of water through a variety of mechanisms. Nonspecific drugs, therefore, do not achieve their effect by acting upon a specific receptor, enzyme, or gene. Typically members of a nonspecific class are unrelated chemically. Cryoprotection of cells is provided by alcohols (including glycols), amines (including amides), sugars, inorganic salts, and macromolecules (including proteins and polysaccharides); specific examples are given in Table 2. Regardless of their chemical diversity, CPAs have in common aqueous solubility and the ability to promote H-bond formation (MacFarlane and Forsyth, 1990). Other classes of nonspecific drugs have been previously mentioned. Nonspecific drugs are similar to specific drugs in that all have adverse effects in addition to the desired effect. No drug is absolutely nontoxic. Because many CPAs are used in multimolar concentrations they are considered to be nontoxic relative to specific drugs active in concentrations at a fraction of a mole. Macromolecular CPAs, however, are effective in millimolar to nanomolar concentrations.

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Examples of Freeze-Limiting Cryoprotectants (CPA) a

TABLE 2

Alcohol Adonitol Ethylene glycol; ethanediol Glycerol Mannitol Methanol Propylene glycol; 1, 2-propanediol Sorbitol Amines Acetamide Betaine Formamide Glutamine Lysine Proline Serine Sarcosine Taurine Inorganic salts Ammonium sulfate Magnesium sulfate Sodium acetate Macromolecules Albumin, serum Antifreeze glycopeptide Antifreeze peptide Dextran Hen egg yolk phospholipids Hydroxyethyl starch Pluronic polyols Polyethylene glycol Polyvinylpyrrolidone Sugars Glucose Lactose Maltose Sucrose Trehalose a

Dimethylsulfoxide is an important freeze-limiting CPA that does not fit into these chemical classes.

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Nonspecific drugs also are characterized by dose-response curves. Dose-response curves of specific drugs (Figure 1) are limited by ligandeffector saturation; dose-response curves of nonspecific drugs are limited by adverse reactions. The desired action of a nonspecific drug, e.g., cryoprotection, defines the class of drugs. Cryoprotectant usually connotes a drug that enhances post-thaw survival by limiting the crystallization of water; the focus of this section is on these drugs. There are also drugs that enhance post-thaw survival by an entirely different process: enhancing the crystallization of water. These too will be considered here; whether they will commonly be included in the class of CPAs is unknown. Historic classification of CPAs, i.e., those that permeate cells and those that do not, lacks functional significance. A. S o u r c e s o f C r y o i n j u r y Mechanisms of CPA action can be understood only in the context of sources of cryoinjury. A substantial portion of this book is devoted to this topic, especially in following chapters. A summary of cryoinjury is provided here. Two major factors are chiefly responsible for cryoinjury: low temperature and crystallization of water. Quantitatively the latter factor predominates but both are discussed. Reduction of cellular temperature causes nonuniform changes in rate constants of biochemical reactions. One well known example is the failure of ion pumps to maintain electrolyte gradients at reduced temperatures (Hochachka and Somero, 1984). Another important example is temperature-induced changes in physical properties of cell membranes (Drobnis et al., 1993; Morris and Clarke, 1987; Quinn, 1985) discussed by Parks in this book. Crystallization of water has greater consequences for cells than cooling. Focusing on crystallization is important, not confusing it with other means of solidifying water such as vitrification. Crystallization is a process in which molecules such as water are organized as a pure substance, ideally without inclusions, in a crystalline array. Freezing of an aqueous solution involves separation of its chemical components into crystals at the eutectic temperature (Karow, 1981; Franks, 1982). In contrast, vitrification of an aqueous solution involves solidification without crystallization. Crystallization may occur throughout a cellular system with ice in intracellular and extracellular spaces, or it may be restricted to the extracellular space. Intracellular crystallization usually results in disruption of cellular ultrastructure. Intracellular crystallization can be precluded by physical means (principally by controlling the rate of cooling). Crystallization limited to extracellular space results in dehydrated, shrunken cells; intracellular liquid water is lost to extracellular crystals. Loss of cell volume and concomi-

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tant distortion of cell membranes may be injurious during freezing (Meryman, 1968). Therefore both loci of crystallization potentially have adverse mechanical effects upon cells. Regardless of locus, however, crystallization of water desiccates or "dehydrates" in the sense of removing liquid-phase, solvent water. During crystallization, concentration of solutes rises in remaining solvent, allowing for chemical reactions between solutes. Concentrated solutes may be injurious during freezing (Lovelock, 1954; Pegg and Diaper, 1988). The fraction of extracellular water that remains unfrozen may be a factor critical to cell survival (Mazur et aL, 1981). (A quantitative distinction must be made in regards to removal of liquid water, a topic discussed by Crowe et al. (1990) and Potts (1994). By definition, freezing cannot remove unfreezable water, the hydration shell intimately associated with biomacromolecules. Profound drying does remove the hydration shell. Terminology regarding dehydration, desiccation, and freezing at this time is not so specific as to make necessary quantitative distinctions.) One major strategy for enabling cells to survive freezing involves treating cells with a CPA prior to freezing and controlling the cooling rate. The importance of CPAs was first appreciated by Polge et al. (1949). Both components of this strategy enhance post-thaw survival by managing crystallization. CPAs limit crystallization of water and thermal control directs ice locus.

B. CPAs L i m i t i n g F r e e z i n g CPAs may limit crystallization by a plethora of mechanisms acting alone or in concert. It is believed that cell survival is enhanced when CPAs quantitatively decrease intracellular ice formation, maintain cell volume, and limit macromolecular denaturation. Many of these mechanisms are discussed in detail by Karow (1969) and Shlafer (1981); they are summarized here. CPAs can quantitatively decrease intracellular ice formation by two mechanisms related to colligative effects of water: osmotic pressure and freezing point depression. All colligative effects, these and vapor pressure depression and boiling point elevation, occur because solutes decrease the chemical potential of solvent water. As explained by Andrews (1971, 1976), solutes dilute solvent with a resulting increase in entropy and decrease in solvent chemical potential. These four colligative properties, "bound together," are each predicated upon independent solute particles in solution. Since solute independence is inversely proportional to chemical potential (concentration), colligative properties are most easily demonstrated in dilute solutions. In the case of osmotic pressure (Air)

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Art = R*TAcs,

where Acs is the difference in solute concentration (mole) on two sides of a membrane permeable to water, impermeable to solute, R* is the gas constant (i.e., 8.31 joules/~ 1.99 cal/~ and T is the absolute temperature. A membrane of this kind is sometimes called a semipermeable membrane. In this situation Art is 22.4 atmosphere per mole (i.e., 22.4 bar/mole; 2240 kPa/mole). A rigorous presentation of osmotic pressure is presented by Hill (1979). Because many CPAs are used in high concentrations, their colligative behavior deviates from theory or "ideal." Rather than acting independently, CPA molecules actually interact with themselves, with water, and with other molecules in solution. These factors have been included in mathematical assessments of colligative properties (Fullerton et al., 1994; Keener et aL, 1995). CPAs quantitatively decrease intracellular ice formation by osmotically dehydrating cells, reducing the amount of intracellular water available for freezing. CPAs are osmotically active, some more than others; cell permeability to CPAs is less than that of water. Therefore, even "permeable" CPAs such as dimethylsulfoxide and glycerol have osmotic potential, enhanced when low temperature (10 to - 10~ decreases CPA kinetic activity. Use of "impermeant" CPAs to decrease intracellular ice formation must be sparing (by controlling CPA concentrations) because extensive dehydration will be lethal to cells. Most mammalian cells at freezing temperatures survive loss of 50% of cellular water; few if any survive loss of 90%. Extensive dehydration results in cellular structure collapse and chemical reactions enabled by increased concentration and molecule proximity. Some CPAs limit intracellular ice formation by colligative depression of the freezing point of water. In theory 1 mole of solute particles per kilograms of water depresses the freezing point of water -1.86~ This is because solute dilutes solvent water, limiting the amount of solvent accessible to crystal faces and causing melting of crystals (Andrews, 1976). Melting removes thermal energy from the liquid/crystal system and lowers the temperature until a new equilibrium between crystallization and melting is attained. Freezing of an aqueous solution changes the mole fraction of solutes remaining in the nonfrozen phase (Lovelock, 1953, 1954). The eutectic temperature may be calculated (Fahy, 1980; Pegg 1984, 1986). Multimolar concentrations of CPA increase viscosity of the biological system being cooled, inhibit crystallization entirely, and form an amorphous glass through the process of vitrification (Fahy, 1984). Some CPAs, namely antifreeze peptides and antifreeze glycopeptides (also called thermal hysteresis proteins), reversibly inhibit growth of ice crystals through a noncolligative mechanism (Burcham et al., 1986; Raymond and DeVries, 1977). The structure of these macromolecules allow

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their adsorption to ice crystals resulting in unstable highly curved growth fronts (Cho et al., 1992). An alternative mechanism has been proposed (Wilson, 1994). These peptides lower the local freezing point by the Kelvin effect (Knight et al., 1991), creating a difference between freezing point and melting points, i.e., a thermal hysteresis. Three types of antifreeze peptides differ in composition and structure. Type I, alpha-helical and alanine rich, has a molecular mass of 4-4.5 kDa (Chakrabartty et aL, 1989). Type II, beta-structured and cysteine-rich, has a molecular mass of 14-17.5 kDa (Ewart and Fletcher, 1993; Ng and Hew, 1992). Type III, with heterogenous amino acid composition, has a molecular mass of 6.5-7 kDa (Scott et al., 1988; Wang et al., 1995). The typology of type I (Sicheri and Yang, 1995; Yang et al., 1988), type II (Sonnichsen et al., 1995), and type III (Chao et aL, 1994; Jia et al, 1995; Sonnichsen et al., 1993) is known. Antifreeze activity of mixtures of these three types is independent of proportion of type; different antifreeze peptides neither inhibit nor potentiate each other's activity (Chao et al., 1995). Antifreeze glycopeptides are composed of tripeptide (Ala-Ala-Thr)n (n = 4-55) with galactose N-acetylgalactosamine bound to threonines and have a molecular mass of 2.6-34 kDa (DeVries, 1988). Antifreeze proteins enable a variety of animals (Duman et al., 1992; DeVries and Cheng, 1992), plants (Griffith et al., 1992; Urrutia et al., 1992) and microorganisms (Duman and Olsen, 1993) to avoid freezing. In plants they produce a freezing point in the range of minus 0.2-0.5~ in fish, minus 0.7-2.2~ in insects, minus 3-6~ In fish their serum concentration is 110 mM (Knight et al., 1991). Antifreeze peptides have been used to inhibit the damaging physical process of recrystallization (formation of larger crystals from smaller ones) during thawing of cryopreserved biological systems. (Arav et al., 1994; Carpenter and Hansen, 1992; Knight et al., 1995; Payne et al., 1994). Antifreeze peptides are commercially available. Other macromolecules such as dextran, hydroxyethyl starch, polyethylene glycol, and polyvinylpyrrolidone may be cryoprotective by binding water (Korber and Scheiwe, 1980; Korber et al., 1982) and promoting vitrification (Franks et al., 1977; Takahashi et al., 1988). They also seem to have a thermal hysteresis effect derived from excluded volume effects and Hofmeister effects (Collins and Washabaugh, 1985). These polymers are cryoprotective at a fraction of a mole. For example, hetastarch at a concentration of 0.2% protected the enzymatic activity of L-asparaginase over many freeze-thaw cycles while the enzyme frozen without hetastarch or with glucose or with lactose was denatured by freezing (Jameel et al., 1995). Hydroxyethyl starch effectively cryopreserves human erythrocytes (Sputtek and Rau, 1992) as does polyvinylpyrrolidone (Morris and Farrant, 1972); also, serum albumin is strongly cryoprotective for human erythrocytes (Morris and Farrant, 1972) and for nucleated cells (Knight et aL, 1977). Polyvinyl pyrrolidone is cryoprotective for bovine spermatozoa ( Jeyendran

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and Graham, 1980). Numerous other examples have been reviewed (Karow, 1969; Shlafer, 1981). Some CPAs (e.g., dimethylsulfoxide, glycerol) diffuse across cell membranes and exchange for cell water. This displacement of water by these CPAs, in addition to freezing point depression, decreases the possibility of intracellular ice and maintains cell volume during freezing (Karow and Shlafer, 1975). Maintaining cell volume may inhibit mechanical fracturing of cell membranes hardened by chilling. Ice crystal growth between cells and within cells may physically disrupt membranes. The possibility of beneficial interaction of CPAs to prevent biomacromolecular denaturation was suggested early on (Conner and AshwoodSmith, 1973; Doebbler and Rinfret, 1962; Karow and Webb, 1965; Karow, 1969), but demonstration came later. CPAs enable regulatory proteins, structural proteins, nucleic acids, and phospolipids to avoid freeze-thaw induced denaturation through several mechanisms. One mechanism is the enhancing of H-bonds in the hydration shell of macromolecules. Another is the stabilization of macromolecular topology by direct interaction with macromolecules as a substitute for the hydration shell. Topology (tertiary structure) and function of nucleic acids, phospholipids, polysaccharides, and proteins are determined in large measure by associated water and cosolvents (Kuntz and Kauzman, 1974). Cosolvents may increase structure of water (kosmotropes: Collins and Washabaugh, 1985) by hydrophobic effects and/or surface tension (Sinanoglu and Abdulmur, 1964; Sousa, 1995). A quantitative measure of hydrophobicity is provided by Black et al. (1979). In contrast to kosmotropes, chaotropes (Hamaguchi and Geiduschek, 1962) disrupt the structure of water and may denature biomacromolecules. For electrolytes, both inorganic and organic (e.g., peptides), kosmotropic/chaotropic activity is conceptualized by the Hofmeister series (Collins and Washabaugh, 1985; Hochachka and Somero, 1984; Hofmeister, 1888). Globular proteins are stabilized against denaturation by solutes (cosolvents) excluded from the hydration shell; many such compounds are CPAs (Timasheff and Arakawa, 1988). In the presence of CPAs (whether amines, alcohols, salting-out salts, or sugars), the relative affinity of globular proteins for water is greater than that for the cosolvent. Preferential exclusion of some cosolvents may occur at low temperatures (<25~ but fail at warmer temperatures. At lower temperatures hydrophobic bonding between cosolvent and protein is less likely and therefore exclusion is favored (Arakawa et al., 1990). The preferential exclusion of CPAs from a protein hydration shell represents a thermodynamic parameter encompassing all degrees of interaction between protein and CPA; under certain conditions stoichiomettic binding of CPA to protein may be measurable (Arakawa and Timasheff, 1984; Sousa, 1995). In contrast to the dehydration of freezing which does not remove unfreezable water from biomacromolecules, more vigorous drying will.

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Protection of biomacromolecules from denaturation that will occur with vigorous drying requires molecules that will substitute for the hydration shell. In contrast to protein stabilization during conventional freezing, protein stabilization during vigorous drying requires bonding between CPA and polar residues on the macromolecules (Carpenter and Crowe, 1989). Under these conditions only carbohydrates are effective CPAs for proteins and of those tested, trehalose is most effective (Crowe et al., 1990) and is concentration dependent (Carpenter and Crowe, 1989). Chemical cryoprotection of cell membranes is provided by the same CPAs that prevent protein denaturation, but the mechanism is unclear. Chilling causes phase separation of phospholipid bilayers (Quinn, 1985) so that lipids form homogenous domains and proteins in the bilayers become aggregated. Phase separation induced by chilling is ameliorated by betaine, dimethylsulfoxide, glycerol, proline, sarcosine, sucrose, and trehalose (Quinn, 1985). At chilling temperatures in the absence of freezing, sucrose, and trehalose seem to be excluded from bilayers (Crowe and Crowe, 1991). Freezing causes fusing of phospholipid bilayers in addition to causing phase transition. These effects are ameliorated by betaine, dimethylsulfoxide, ethylene glycol, fructose, glycerol, maltose, proline, sarcosine, sucrose, and trehalose (Anchordoguy et al., 1987; Biondi et aL, 1992; Crowe et al., 1990; O'Leary and Levin, 1984; Park and Huang, 1992). Betaines may provide cryoprotection to liposomes by acting kosmotropicly to promote water structuring (Lloyd et al., 1994). Sucrose and trehalose seem to interact with the polar headgroup to protect frozen cells (Crowe and Crowe, 1991) while other CPAs may act on acyl chains in the bilayer (Anchordoguy et al., 1987; Crowe et al., 1990). Vigorous drying "denatures" phospholipid bilayers, ameliorated by sucrose and trehalose but not by dimethylsulfoxide, ethylene glycol, glycerol, or proline (Crowe et al., 1990). During drying trehalose may intercalate between phospholipid head groups and through H-bonds act as a water "substitute" (Crowe et al., 1990). Phospholipids per se are effective in preventing chilling injury to living cells, especially mammalian spermatozoa, and also have limited cryoprotective action to frozen sperm (Abdelhakeam et al., 1991a,b; Prins and Weidel, 1986). The discovery that yolk from hens' eggs (Phillips and Lardy, 1940) and its phospholipids (Graham and Foote, 1987; Kampschmidt et al., 1953) ameliorate chilling injury beneficially enhanced semen cryopreservation. It is believed that exogenous phospholipids are incorporated into cell membranes of chilled and of frozen and thawed cells including sperm (Cookson et al., 1984; Pace and Graham, 1974; Watson, 1981). Butylated hydroxytoluene is synergistic with phospholipids (Bamba and Cran, 1992; Graham and Hammerstedt, 1992). CPA adverse effects come in two varieties: osmotic and toxic. CPAs used in multimolar concentrations can have adverse osmotic effects on cells prior to freezing, during freezing and thawing, and following thawing. Also,

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CPAs at these concentrations can chemically interact with important biomolecules and thereby exert adverse effects. Dimethylsulfoxide exerts chemical toxicity even at subzero temperatures (Fahy and Karow, 1977). These two kinds of adverse reactions can be evaluated independently as demonstrated by studies on kidney slices (Fahy et al., 1987). An example of a CPA toxic effect on reproductive tissue is the effect of CPA dimethylsulfoxide on the metaphase spindle of unfertilized mammalian oocytes. The oocyte at ovulation has a well developed metaphase spindle in preparation for chromosomal separation in the second meiotic division. The function of the metaphase spindle is crucially dependent upon the microtubular structure of the spindle. Dimethylsulfoxide promotes microtubule assembly and polymerization in vitro (Algaier and Himes, 1988; Robinson and Engelborghs, 1982). Exposure of ovulated oocytes to dimethylsulfoxide (0.75-1.50 M) causes microtubular proliferation and spindle unravelling (Johnson and Pickering, 1987). With loss of the spindle, chromosomal dispersion occurs. These effects of dimethylsulfoxide on the ovulated oocyte are not fully reversible. Dimethylsulfoxide-induced spindle disruption accounts, in part, for chromosomal abnormalities including aneuploidy and polyploidy in thawed oocytes (Sterzik et al., 1992) and subsequent embryos (Bouquet et al., 1993). Cooling by itself also promotes microtubular and spindle disorganization (Aman and Parks, 1994; Magistrini and Szollosi, 1980; Pickering et al, 1990). Dimethylsulfoxide also causes a premature discharge of cortical granules in oocytes of humans and mice which results in zona hardening, blocking spermatozoal penetration ( Johnson, 1989; Pickering et al., 1991; Vincent et al., 1990). Protection against this zona hardening effect can be obtained by including fetal bovine serum (Vincent et al., 1990) or fetuin (a protein component of fetal bovine serum) in the medium (George and Johnson, 1993). Glycerol as a CPA may also be toxic to reproductive tissues. Evidence for this toxicity has been reported for ovulated murine oocytes (Hunter et al., 1995) and for porcine embryos (Dobrinsky and Johnson, 1994). Vitrification of porcine embryos causes actin polymerization in the cytoskeleton and also causes microtubular retention. Manifestation of these effects is dependent upon embryo age (5-7 days) and is observed after use of VS3a (6.5 M glycerol and 60 mg/ml bovine serum albumin in modified Delbecco's phosphate-buffered saline). Glycerol, like dimethylsulfoxide, stabilizes microtubules (Filner and Behnke, 1973). CPA adverse effects can be avoided by management protocols. Osmotic effects can be managed by controlling rates of concentration changes and by use of "counteracting" osmotic agents. Protocols for these procedures are discussed later in this book. Chemical toxicity, if a factor, can be managed by using several CPAs at less-than-toxic concentrations. Toxicity of at least one CPA, dimethylsulfoxide, can be managed by simultaneous use

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of compounds that specifically "neutralize" the toxicity of that CPA, e.g., acetamide (Fahy et al., 1987) or glucose (Clark et al., 1984). Chemical toxicity can be controlled in vitro to a certain extent by careful choice of physiological medium (Clark et al., 1984). CPA mixtures have been used to achieve synergistic effects or to reduce adverse effects. Cryoprotective effect of ethylene glycol and of glycerol for mouse morulae was substantially enhanced by combination with polyvinyl alcohol or with fetal calf serum (Gutierrez et al., 1993). Use of multiple CPAs is essential in vitrification, a practical procedure for pre-embryo cryopreservation (Kono and Tsunoda, 1988; Rail et al., 1987; Rail and Fahy, 1985; Rail and Wood, 1994; Scheffen et al., 1986). Vitrification solutions range in complexicity from combinations of dimethylsulfoxide, acetamide, 1,2-propanediol, and polyethylene glycol (Rail and Fahy, 1985) to glycerol and bovine serum albumin (Rail and Wood, 1994). Evaluating the contribution of components in a CPA mixture is facilitated by a statistic, tau (Chamberlain et al., 1989). Tau indicates the interdependence of mechanisms when several CPAs are used simultaneously. When tau is zero, CPAs are acting independently, when tau is greater than zero there is synergism, and when tau is less than zero there is antagonism between the CPAs or they have similar mechanisms of protection. Tau is negative for combinations of dimethylsulfoxide, glycerol, hydroxyethyl starch and propylene glycol (Kruuv et al., 1990). On the other hand, tau is zero when glutamine, an effective cellular CPA (Kruuv et al., 1988, 1992), is used with any one of these CPAs (Kruuv et al., 1990), indicating that glutamine and the second CPA act independently. C. CPAs E n h a n c i n g F r e e z i n g Intracellular ice is to be avoided; it is usually lethal as previously discussed in this chapter and elsewhere in this book. Initiation of extracellular crystals at high subzero temperature helps avoid formation of intracellular ice by cellular dehydration. Since liquid water has a higher vapor pressure than ice, intracellular water will exit cells and become incorporated in crystals. An additional factor, hypertonic solutions in the vacinity of ice crystals, will osmotically withdraw intracellular water during extracellular ice formation. In other words, initiation of crystallization at high subzero temperatures is usually beneficial to cell survival; supercooling is usually detrimental. Initiation of ice formation has, historically, been managed by physically touching the system to be frozen with a piece of ice (called "seeding") or with a metal rod chilled to a temperature below -70~ More recently initiation of ice formation has become possible by ice nucleating chemicals. Formation of extracellular ice is facilitated by ice nucleating protein (INP). INP was first isolated from bacteria (Maki et al., 1974). Subsequently, cells

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of other organisms including lichens, plants, insects, intertidal invertebrates, the frog Rana sylvatica (Storey et al., 1992), and the turtle Chrysemys picta (Storey et al., 1991) have been shown to synthesize INPs (Lee et aL, 1995). INPs nucleate ice throughout the temperature range of -2~ to -12~ Two classifications of ice nuclei have been proposed (Turner et al., 1990; Yankofsky et al., 1981). Amino acid sequences of three INP have been deduced from DNA sequences of three bacteria (Corotto et al., 1986). The three presumptive proteins are similar, each about 115 kDa and consisting of numerous repetitions of A l a - G l y - T y r - G l y - S e r - T h r - L e u - T h r in the center domain (Warren and Wolber, 1987). It is believed that N- and C-terminal domains are involved in assembly and stabilization of nucleation sites and that the center domain is the main site of ice interaction. Three-dimensional structures have been proposed (Kajava and Lindow, 1993; Mizuno, 1989; Warren and Wolber, 1987); these are consistent with current understanding of ice nucleation (Gavish et al., 1992; Lahav et al., 1993; Popovitz-Biro et aL, 1994). Membrane components, especially phosphatidylinositol, appear to enhance ice nucleation by INPs at high subzero temperatures (Kozloff et al., 1991). The chemical relationship of bacterial INP to INP synthesized by vertebrates is unknown.

VIII. CONCLUSIONS Chemical additives to biological media can have profoundly beneficial or detrimental effects on survival of cells maintained in vitro. Interaction between chemicals and cells are described by pharmacological principles. Many of these interactions may have catalytic characteristics described by receptor or enzyme kinetics. Other interactions may be purely physical. All interactions are influenced by concentration. The most meaningful descriptions of chemical-cellular interactions are those that include quantitative dose-response information. This chapter is presented as a paradigm of pharmalogical evaluation of several classes of drugs important to in vitro maintenance of reproductive tissue. It has, therefore, been a rather shallow, cursory review to indicate directions for thorough studies of specific drugs. This chapter has omitted important groups of drugs important to in vitro maintenance of reproductive tissues. An obvious omission has been those drugs enhancing spermatozoal motility. Other drugs of interest include those influencing cell fusion and those influencing genetics. Pharmacological intervention in reproductive biology is cost effective. Technology of assisted reproduciton and of micromanipulation are certainly welcomed additions to clinical practice, but they should be reserved for those occassions when highly effective less expensive procedures such as

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pharmacological interventions fail to accomplish the desired objective. One specific example relates to pregnancy with cryopreserved spermatozoa. Clinicians serving human patients reluctantly embraced cryopreservation of spermatozoa because these techniques early on compromised spermatozoal motility and fecundity. Improvement in these in vitro techniques using cryoprotectants have provided pregnancy rates similar to or higher than those in the general population. Such accomplishments may entice scientists to redouble efforts to utilize pharmacological means to improve the outcome of reproductive cells maintained in vitro. Most importantly, pharmacological studies will enhance fundamental understanding of cellular biology. Historically, pharmacology is responsible for discovering and elucidating receptors. Combined with cryobiology and physical chemistry, pharmacology may reveal profound knowledge of water in cellular systems. Certainly, pharmacology is a major factor in developing new means of cellular intervention: genetic medicine.

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van Blerkom, J. (1991). Cryopreservation of the mammalian oocyte. In Current Communications in Cell and Molecular Biology, Animal Applications of Research in Mammalian Development (R. A. Pedersen, A. McLaren, and N. L. First, Eds.), pp. 83-119. Cold Spring Harbor Laboratory Press, Cold Spring Harbor, NY. van den Eede, B. (1995) Investigation and treatment of infertile couples: ESHRE guidelines for good clinical and laboratory practice. Hum. Reprod. 10, 1246-1271. van Uem, J. F. H. M., Siebzehnrubl, E. R., Schuh, B., Koch, R., Trotnov, S., and Lang, N. (1987). Birth after cryopreservation of unfertilized oocytes. Lancet 1, 752-753. Wada, I., Macnamee, M. C., Wick, K., Bradfield, J. M., and Brinsden, P. R. (1994). Birth characteristics and perinatal outcome of babies conceived from cryopreserved embryos. Hum. Reprod. 9, 543-546. Watson, P. F. (1979). The preservation of semen in mammals. In Oxford Reviews of Reproductive Biology (C. A. Finn, Ed.), pp. 283-350. Oxford Univ. Press, London. Watson, P. F. (1990). Artificial insemination and the preservation of semen. In Marshall's Physiology of Reproduction (G. E. Lamming, Ed.), 4th ed., Vol II, pp. 747-896. Churchill Livingstone, London. Whittingham, D. G. (1971). Survival of mouse embryos after freezing and thawing. Nature 233, 125-126. Whittingham, D. G., Leibo, S. P., and Mazur, P. (1972). Survival of mouse embryos frozen to -196~ and -269~ Science 178, 411-414. Wildt, D. E., Byers, A. P., Howard, J. G., Weise, R., Willis, K., O'Brien, S., Block, J., Tilson, R. L., and Rall, W. F. (1993). Genome Resource Banking (GRB) action plan: Global need and a plan for the North American region. Presented at AAZPA Annual Meeting, Omaha, Nebraska, August, 1993. Wilmut, I., and Rowson, L.E.A. (1973). Experiments on the low temperature preservation of cow embryos. Vet. Rec. 92, 686-690. Yanagimachi, R., Lopata, A., Odom, C. B., Bronson, R. A., Mahi, C. A., and Nicholson, G. L. (1979). Retention of biologic characteristics of zona pellucida in highly concentrated salt solution: The use of salt-stored eggs for assessing the fertilizing capacity of spermatozoa. Fertil. Steril. 31, 562-574. Yokoyama, M., Akiba, H., Katsuki, M., and Nomura, T. (1990). Production of normal young following transfer of mouse embryos obtained by in vitro fertilization using cryopreserved spermatozoa. Exp. Anim. 39, 125-128.

H y p o t h e r m i a and Mammalian Gametes John E. Parks Department of Animal Science Cornell University Ithaca, New York 14853

I. INTRODUCTION The availability of viable, functionally normal gametes is an essential prerequisite to successful fertilization in mammals both in vivo and in vitro and is therefore critical to the implementation of a broad range of reproductive technologies such as artificial insemination, in vitro fertilization, embryo transfer, and genetic engineering. By eliminating the constraints of time and location on the availability of gametes and embryos, cryopreservation of these specialized and otherwise short-lived cells has become an essential complementary procedure in the application of most technologies requiring their use. The great success of commercial artificial insemination and embryo transfer in dairy cattle and to a lesser extent in other domestic animals has been possible in large measure because effective procedures were developed for cryopreserving semen and embryos, making it practical to store these materials indefinitely and transport them virtually anywhere in the world. Cryopreservation of gametes and embryos also plays an increasingly important role in assisted reproductive technologies designed to circumvent problems of human infertility and for preserving germ plasm from rare and exotic species of wildlife, companion animals, and valuable genetic strains of domestic and laboratory species. Reproductive Tissue Banking Copyright 9 1997 by Academic Press. All rights of reproduction in any form reserved.

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Although successful cryopreservation of gametes and embryos has been achieved for many species (Graham, et al., 1978; Leibo, 1992), post-thaw viability and fertility is reduced generally due to the cumulative effects of requisite steps in cryopreservation protocols. An essential step in all cryopreservation procedures is the initial temperature reduction from normal physiological temperature (normothermic) to around 0~ prior to freezing. This step is required to reduce normothermic metabolic activity which otherwise results in senescence of sperm or ova within a few hours depending on the species. Both gametes (Parks and Ruffing; 1992; Vincent and Johnson, 1992; Watson, 1990) and early cleavage-stage mammalian embryos (Niemann, 1991; Pollard and Leibo, 1994) are sensitive to this state of hypothermia with large species differences. Hypothermic sensitivity of mammalian sperm was first reported by Milovanov (1934) who observed that when sperm were cooled rapidly to near the freezing point of water, they suffered an irreversible loss of motility referred to as temperature shock, now more commonly referred to as cold shock (Watson, 1981). Sensitivity of mammalian oocytes and early embryos to hypothermia has been recognized more recently with the increased utilization of in vitro fertilization and related procedures in a variety of clinical, commercial, and research applications (Pollard and Leibo, 1994). Hypothermia may be defined simply as a subnormal physiological temperature. For this discourse, which focuses on mammalian gametes, hypothermia refers to the temperature range between normal body temperature (~37-39~ depending on the species) and near 0~ in the absence of ice crystal formation (Morris and Clark, 1981). The cellular injury or death observed after rewarming from hypothermic temperatures most often is attributed either to sensitivity to a specific temperature or temperature range (chilling injury) or sensitivity to cooling rate (cold shock) (Morris and Clark, 1981; Pollard and Leibo, 1994). Both forms of injury are observed in mammalian gametes. In the context of gamete cryobiology, hypothermia has a much different connotation than when applied to more complex organisms with the capacity to adapt to hypothermic conditions through altered metabolic pathways, synthesis of protective macromolecules, and modifications to membrane composition (Hazel, 1995; Morris and Clark, 1981). Gametes, particularly sperm due to their highly differentiated state, lack the biosynthetic machinery necessary to make such structural or metabolic adjustments to reduced temperatures (Watson, 1981; Hammerstedt et aL, 1990). Transcriptional activity decreases in fully grown oocytes and ceases after maturation (Wassarman and Albertini, 1994), and there is no evidence that oocytes are capable of adapting to hypothermic conditions other than through the overall reduction in metabolic rate. Because physiological adaptation does not occur to any significant extent, studies of hypothermia in gametes have focused largely on biochemi-

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cal and cytological effects of cooling, the relationship between these effects and cellular damage or viability, and in vitro approaches to preventing cellular injury. Progress has been made in each of these areas of study for both sperm and oocytes, but much remains to be determined about the fundamental basis for hypothermic damage and effective methods for precluding it. The purpose of this treatise is to provide an overview of hypothermic injury, its causes and prevention in mammalian gametes based on information accumulated during the 60 years since Milovanov (1934) first described cold shock sensitivity of mammalian sperm.

H. HYPOTHERMIA AND MAMMAHAN SPERM That the phenomenon of cold shock was documented first and has been studied exhaustively for decades in mammalian sperm is not surprising. First of all, sperm motility provides a definitive, functional, and easily measured endpoint for evaluating the effects of hypothermia. Furthermore, sperm are readily available in large numbers as a relatively pure, homogenous population of cells which facilitates compositional studies as well as experimental design and analysis. Most important, the clinical, commercial, and conservational ramifications of highly successful sperm cryopreservation procedures, which are lacking still for most species, provide sufficient incentive for elucidating the mechanisms by which cold shock and other factors contribute to cryoinjury. The dilemma with sperm hypothermia is that sperm from many species are especially sensitive to cooling, yet the catabolic nature of sperm metabolism necessitates a rapid reduction in metabolic rate, usually achieved by reducing temperature, to maintain cell viability for more than a few hours (Hammerstedt, 1993). Even at temperatures only slightly above freezing, metabolic activity continues so that cell damage and death are a function of both hypothermia and the intrinsic aging process. At ambient temperatures (>15~ effects of aging are probably more consequential than thermal effects. Below ~15~ effects of hypothermia become more important and the severity of damage is correlated directly with temperature differential and cooling rate (Watson, 1981; White, 1993). Mammalian sperm are most susceptible to hypothermic damage caused by rapid cooling in this temperature range. Damage can be reduced significantly by cooling sperm very slowly (<10~ but not eliminated entirely. Following slow cooling and rewarming, the nature of the damage observed resembles closely that which occurs during rapid cooling but is less extensive (Watson, 1981), suggesting that in addition to cold shock, chilling injury contributes at least in part to cell damage and loss of viability. Thus the etiology of chilling injury and cold shock in sperm likely have a common mechanistic basis with differences in the severity of damage

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being related to the ability of sperm to adjust at a particular cooling rate. Descriptions of the morphological, cytological, and biochemical effects of hypothermia on sperm have provided valuable insight into the probable mechanisms leading to cell injury and death.

A. O v e r v i e w o f M a m m a l i a n S p e r m Structure Mammalian sperm vary considerably in overall size and shape (Watson, 1985), but typically consist of three readily identifiable regions: the head, midpiece, and principal piece or tail (Figure 1). Key features of each region (see Eddy and O'Brien, 1994) are described here for later reference. The sperm head is composed primarily of the highly condensed, haploid nucleus which is surrounded in the anterior region by the acrosome, a membranebound organelle derived from the Golgi apparatus during spermiogenesis. The morphology of the nucleus and acrosome both contribute to the characteristic shape of the sperm head for individual species.

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outer acrosomal membrane

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The sperm midpiece is characterized by the presence of mitochondria typically arranged in an end-to-end helical configuration surrounding the base of the flagellum. As with other cell types, the mitochondria generate energy in the form of adenosine triphosphate (ATP) which drives the energy-requiring reactions of the sperm, including maintenance of ion gradients and locomotion but little biosynthesis (Hammerstedt, see Chapter 3). The axoneme of the flagellum in the midpiece consists of a typical "9 + 2" arrangement of microtubules surrounded by nine coarse outer fibers which appear to constrain the axoneme, thereby controlling the form of the flagellar beat (Eddy and O'Brien, 1994). The axoneme and coarse outer fibers extend beyond the midpiece to form the tail, which is surrounded by a fibrous sheath and may also influence the plane of the flagellar beat. The plasma membrane surrounds all three regions of the sperm. In the periacrosomal region, the plasma membrane lies in close apposition to the outer aspect of the acrosomal membrane with which it fuses during the acrosome reaction, a calcium-dependent exocytotic event induced at or around the time of sperm-binding to the zona pellucida of the oocyte (Yanagimachi, 1994). Release or activation of acrosin and other hydrolytic enzymes during the acrosome reaction is required for zona penetration during fertilization. B. Effects o f C o l d S h o c k o n M a m m a l i a n S p e r m Manifestations of hypothermic injury to sperm are many and varied and have been reviewed extensively in recent years (Watson, 1981, 1990; Watson and Morris, 1987; White, 1993). As one might predict, the irreversible loss of sperm motility, which is the hallmark of cold shock damage, appears to be ancillary to other forms of cellular injury. For example, vital staining techniques have been used to evaluate the effects of cooling on membrane permeability and demonstrate that the semipermeable property of the sperm plasma membrane is compromised by cooling based on an increased percentage of sperm staining (Watson, 1981; White, 1993). Corresponding to the increase in membrane permeability is the extensive loss of numerous intracellular and membrane components, including ATP, nucleic acids, phospholipids, several metabolic enzymes, potassium, and magnesium, and a diminished capacity to regulate intracellular calcium. Cold shock also impairs both anaerobic glycolysis and aerobic metabolism, limiting the ability of sperm to generate ATP required for motility and other cellular functions (Hammerstedt, 1993; Watson, 1981; White, 1993). Morphological, cytological, and ultrastructural evaluation of sperm following rapid cooling reveal extensive damage to the plasma membrane covering the anterior sperm head and the underlying acrosomal membrane, leading to disruption of the acrosome and loss of the ground substance contained within the acrosomal matrix (Watson, 1981; Watson and

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Plummer, 1985). Plasma membrane over the postacrosomal region and the midpiece are disrupted with effects on mitochondrial organization ranging from unaltered to severely damaged (Watson, 1981; Watson and Plummer, 1985). While there is some evidence that cytoskeletal elements of the sperm may be sensitive to cooling (Holt and North, 1991), the most severe structural damage is to the cellular membranes.

m . MEMBRANE ORGANIZATION AND THERMOTROPIC PHASE BEHAVIOR Collectively, the detrimental effects of cooling on spermatozoa point to the plasma membrane as the primary site of hypothermic injury. Therefore efforts to elucidate the molecular basis for membrane damage have focused intensively on the composition of sperm membranes, particularly as it relates to the biological and biophysical effects of cooling below normal physiological temperatures. A. O r g a n i z a t i o n a n d S t r u c t u r a l P r o p e r t i e s o f M e m b r a n e Lipids A general assumption is that membranes of the sperm including the plasma membrane conform to the fluid mosaic model proposed by Singer and Nicolson (1972) in which integral proteins are dispersed in as well as through a bilayer of polar phospholipids and glycolipids (Figure 2). Most phospholipids include hydrocarbon chains in ester or ether linkage to the 1- and/or 2-hydroxyl groups of glycerol (glycerophospholipids), phosphoric acid esterified to the remaining hydroxyl, and an alcohol (choline, serine, ethanolamine) in which the hydroxyl is esterified to the phosphoric acid. This configuration results in a polar or hydrophilic "headgroup" (i.e., phosphorylcholine) and one or two nonpolar or hydrophobic, hydrocarbon "tails" (Figures 2 and 3). Typically, the sn-1 hydrocarbon is saturated and the sn-2 chain exhibits varying degrees of unsaturation. Sphingomyelin is a common phospholipid with a configuration similar to that of the glycerophospholipids but without glycerol, rather with sphingosine representing one of the hydrocarbon tails and the other a fatty acid in amide linkage. Glycolipids contain polar, carbohydrate headgroups and nonpolar hydrocarbon chains based either on a glycerol backbone (as with glycerophospholipids) or on sphingosine (as with sphingomyelin, Figure 2). The fluid mosaic model has been advanced to include more recent concepts such as bilayer asymmetry, lipid polymorphism, and protein-lipid interactions (Cullis et aL, 1985; Hammerstedt et al., 1990; Quinn, 1989). Thus phosphatidylcholine and sphingomyelin are localized preferentially in the outer leaflet of the membrane bilayer while phosphatidylserine,

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phospholipid glycolipid ~ cholesterol 0 carbohydrate

protein Representation of the plasma membrane based on the fluid mosaic model of Singer and Nicolson (1972).

FIGURE 2

-ethanolamine, and -inositol are present in the inner leaflet. While most phospholipids form a bilayer configuration spontaneously in aqueous dispersion, some phospholipids and glycolipids (i.e., phosphatidylethanolamine, monogalactosyldiglyceride) prefer a non-bilayer (primarily hexagonal II) morphology (Figure 3) but generally are constrained to the bilayer configuration by association with bulk phospholipids and interaction with integral proteins. Aggregation of non-bilayer preferring phospholipids within the membrane occurs only transiently under normal physiological conditions as an intermediate step in membrane fusion (Hazel, 1995).

B. Thermotropic Phase Behavior of Membrane Lipids A major consequence of cooling membrane phospholipids is the thermotropic phase transition from a liquid-crystalline (L~) to a gel (L~) phase. In the liquid-crystalline phase, the hydrophobic core of the lipid bilayer is relatively disordered and phospholipids are permitted several modes of mobility including lateral diffusion within the plane of the bilayer and rotation about the long axis of the phospholipid (Cullis et al., 1985). A

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decrease in temperature is accompanied by an increase in the order in the hydrocarbon chains as rotation about carbon-carbon bonds is reduced and the chains become both extended and packed together in a parallel array, thus resulting in a more rigid membrane structure which constrains the mobility of individual components. Peak phase transition temperatures vary depending upon the molecular species, with phospholipids containing saturated hydrocarbon chains undergoing transition at higher temperatures than those with unsaturated chains (Oldfied and Chapman, 1972). The lower melting point of unsaturated phospholipids is attributed to the cis configuration of double bonds primarily in the sn-2 acyl position of naturally occurring phospholipids which creates a rigid kink in the hydrocarbon skeleton and thus precludes the cooperative packing possible with straighter saturated hydrocarbon chains (Figure 3). Cholesterol, the principal sterol in mammalian cell membranes, has a profound effect on phase behavior through its steric interaction with phospholipids. At temperatures above the phase transition, the sterol ring structure and hydrocarbon "tail" impose constraints on the motional freedom of phospholipid hydrocarbon chains, resulting in a more ordered bilayer (Demel and DeKruyff, 1976; Yeagle, 1985). As the temperature is lowered, the same steric hindrance prevents molecular packing required

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to form a gel phase (Figure 3). These opposing effects of cholesterol on phospholipid mobility and phase behavior result in a state of intermediate order under normal physiological conditions. An important consequence of thermotropic phase transitions during membrane cooling is lateral phase separation of higher melting-point phospholipids which become sequestered into gel-phase domains within the remaining liquid-crystalline phospholipids (Quinn, 1989). Thermotropic phase transition to the gel phase and lateral phase separation have profound effects on membrane structure and function, including increased permeability to cations and water due to packing defects at gel and fluid phase boundaries, segregation of integral membrane proteins by exclusion from gel-phase domains, reduced activity of membrane-bound enzymes, and decreased rate of lateral protein diffusion within the bilayer which interferes with diffusion-coupled processes (Hazel, 1995). Quinn (1985, 1989) has proposed a plausible and persuasive model for the molecular basis of membrane damage during cooling (Figure 4). In

Lateral phase separation model for hypothermic damage to membranes proposed by Quinn (1985, 1989). Figure adapted from that of Quinn (1985). See Figure 2 for symbols.

FIGURE 4

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Quinn's model, the membrane consists of both bilayer and non-bilayer preferring phospholipid species within an overall bilayer configuration as described above. As the membrane is cooled, the non-bilayer preferring phospholipids, which are generally higher melting point lipids, enter a gel phase within the bilayer, potentially altering critical protein-lipid interactions required for normal protein conformation and function. When cooled further, the non-bilayer preferring lipids undergo lateral phase separation within the bilayer, forming gel-phase domains and excluding proteins from their normal lipid environment. Upon rewarming, the domains of nonbilayer preferring lipids are no longer constrained to the bilayer configuration by lipid-lipid or lipid-protein interactions, resulting in localized membrane perturbation and loss of membrane integrity.

IV. SPERM MEMBRANE I/PID COMI~SITION Because of its profound influence on membrane structure and function during cooling, lipid composition of sperm membranes has been examined extensively and the details summarized in several reviews (Parks and Lynch, 1992; Watson, 1981; Watson and Plummer, 1985; White, 1993). Major lipid classes found in sperm (phospholipids, glycolipids, and sterols) are the same as those in membranes of other animal cells, but often include molecular species which are present in unusually high proportions relative to other cell membranes.

A. S p e r m P h o s p h o l i p i d C o m p o s i t i o n The predominant phospholipids in sperm are choline and ethanolamine phosphoglycerides and sphingomyelin, with serine- and inositol-containing phospholipids, phosphatidic acid, cardiolipin, and lysophospholipids found frequently as relatively minor membrane components. A characteristic feature of sperm membranes from most mammalian species is the extraordinarily high proportion of ether-containing choline and ethanolamine phospholipids (1-O-alkyl and 1-O-alkenyl) in which the 2-acyl hydrocarbon is almost exclusively a long-chain, polyunsaturated docosapentanoyl (22:5) or docosahexanoyl (22:6) moiety (22 carbons and 5 or 6 double bonds, Figure 5), making up 50-60% of total phospholipids in bull and boar sperm. The relevance of such a high percentage of phospholipids with this molecular configuration within the membrane is not known. Physiologically, the presence of phospholipids containing 22:5 and 22:6 may play a role in prefertilization changes in the plasma membrane or acrosomal membrane by providing a substrate for specific phospholipases resulting in localized concentrations of fusigenic lysophosphospholipids during sperm capacitation (Langlais and Roberts, 1985) or precursors for bioactive phospholipids

Hypothermia and Mammalian Gametes

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4,=

glycerylphosphorylcholine

.r

docosahexanoyl moiety (C22:6)

Molecular model of 1-O-palmitoyl-2-docosahexanoyl-3-glycerylphosphorylcholine, the prevalent molecular species in domestic animal sperm and present in sperm from other mammalian species. Figure adapted from that of Stubbs and Smith (1984).

FIGURE 5

such as platelet activating factor (Parks et al., 1990). Structurally, the high degree of unsaturation in the long acyl chains creates a number of turns in the hydrocarbon which increases the cross-sectional area of the molecule while effectively reducing its overall length (Stubbs and Smith, 1984). Nevertheless, this configuration does not reduce the phase transition temperature significantly below that of phospholipids containing a diunsaturated acyl moiety and the thermotropic phase behavior of phospholipids from sperm with a high proportion of long chain, polyunsaturated phospholipids does not appear to differ substantially from that of phospholipids containing a lower proportion of these phospholipids (see below).

B. Sperm Glycolipid Composition Glycolipid composition of mammalian sperm appears to be relatively simple and similar across species. The glycolipid fraction of bull and boar sperm, which is only 6-8% of total polar lipids and presumed to be enriched in the plasma membrane, is dominated by a single component, 1-O-alkyl2-O-acyl-3 sulfogalactosyl glycerol (SGDG) which has almost exclusively 16:0 as both its alkyl and acyl moieties (Ishizuka et al., 1973; Nikolopoulou et al., 1985; Selivonchick et al., 1980). When total glycolipids extracted from bull, boar, rabbit, and stallion sperm were fractionated further by thin layer chromatography, a single major component was detected which migrated the same distance relative to the solvent front (Ri) for each species (Parks

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J o h n E. Parks

and Lynch, 1992), suggesting that the SGDG of bull and boar sperm is the major glycolipid for all four species examined. Interestingly, the glycolipid fraction of avian (rooster) sperm also consisted of a single major component which migrated with a much lower RI than that of mammalian sperm but the molecular structure of this glycolipid component has not been identified (Parks and Lynch, 1992).

C. S p e r m Sterol C o m p o s i t i o n Sterol composition of sperm has been studied extensively because of its important role in regulating membrane structure and function, both relative to its effect on thermotropic phase behavior during cooling (Parks et al., 1981) and the concept that sperm cholesterol efflux is one of the important membrane modifications required for sperm capacitation (Langlais and Roberts, 1985; Parks and Ehrenwald, 1990). As is the case for most mammalian cells, cholesterol is the major free sterol in sperm, but other sterols are present as minor sperm membrane components in some species, including 24-hydroxycholesterol (desmosterol) which is an uncommon component of animal cells, and cholesterol sulfate (Langlais and Roberts, 1985). Cholesterol to phospholipid molar ratios, which determine the extent to which cholesterol modulates ordering within the membrane bilayer through steric interaction with phospholipids as described earlier, range from a low of 0.2 for boar sperm to 1.0 in human sperm (Darin-Bennett and White, 1977; Parks and Lynch, 1992).

V. RELATIONSHIP OF SPERM I/PID C O M P O S m O N TO COLD SHOCK With recent advances in our understanding of temperature effects on mixed lipid systems, the relatively comprehensive description of sperm membrane lipid composition across a variety of species should have some predictive value for determining the basis of cold shock sensitivity. Indeed, species differences in cold shock tolerance have been recognized for some time (see Watson, 1981; White, 1993) with boar sperm being extremely sensitive to cooling, sperm from other domestic animals (bull ram, and stallion highly sensitive, those from companion animals (cat, dog) more resistant, and sperm from man and rooster highly resistant. Two relationships between sperm membrane lipid composition and sensitivity to cooling are longstanding. First, the cholesterol to phospholipid molar ratio in mammalian sperm is positively correlated with cold shock resistance (Darin-Bennett and White, 1977), with boar sperm having the lowest ratio (0.2), domestic animals an intermediate ratio (~0.4-0.5), and rabbit and human the highest ratios (~.0.9-1.0). This correlation is consis-

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tent with the membrane condensing and phase transition-broadening effects of the sterol. The higher ratios for rabbit and human sperm are intriguing because they represent the highest ratios reported for membranes of mammalian cells (Yeagle, 1985). As cholesterol is generally enriched in the plasma membrane, the sperm from these species must have extraordinarily high cholesterol in the plasmalemma or an unusual distribution of cholesterol throughout the subcellular membranes of the sperm. It should be noted that this relationship does not extend to rooster sperm, which are tolerant to rapid cooling but have a relatively low cholesterol to phospholipid ratio (0.3, Parks and Lynch, 1992). A second correlation exists between cold shock and the unsaturation index of phospholipid-bound acyl chains. In this case, tolerance to cooling is negatively correlated with the degree of unsaturation (Watson, 1981; White, 1993) which is contradictory to the concept that a higher proportion of saturated phospholipids should maintain a greater degree of order within the bilayer at low temperatures. This apparent discrepancy may be related to several factors. First, as emphasized previously (Hammerstedt et al., 1990; Parks and Graham, 1992; Watson, 1981), the compositional analyses which serve as a basis for this correlation with unsaturation index were completed on total sperm lipids without regard for their regional or subcellular distribution or their distribution within a phospholipid class. Second, the unsaturation index is influenced largely by the polyenoic fatty acyl groups which predominate in domestic animal sperm and may affect ordering of the membrane differently than less saturated acyl species. For example, the melting point of docosahexanoic acid (22:6) is dramatically different from linolenic (-44.5~ versus -10~ but the peak thermotropic phase transition of phosphatidylcholine is relatively unaffected by an increase in sn-2 unsaturation above two double bonds (Stubbs and Smith, 1984). Furthermore, cholesterol has very little condensing effect on phosphatidylcholine containing 22:6 in the sn-2 position. Therefore, it seems most likely that the correlation between unsaturation index and cold shock resistance is incidental to more complex lipid-lipid or lipid-protein interactions resulting from the unique molecular configuration (i.e., decreased effective length and increased cross-sectional area) conferred on phospholipids by polyenoic sn-2 acyl chains. The high proportion of polyenoic acyl chains in sperm phospholipids also makes sperm highly susceptible to damage resulting from membrane lipid peroxidation (Aitken and Fisher, 1994; Alvarez and Storey, 1987; Jones et al., 1979). This type of damage does not appear to be related directly to cooling, but to the increased exposure of membranes to molecular oxygen during storage and the lost or decreased activity of protective enzymes at reduced temperatures (Alvarez and Storey, 1993; Lasso et al., 1994; Chapter 4, this volume).

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VI. DEVELOPMENTAL CHANGES IN COLD SHOCK SENSITIVITY During maturation in the epididymis, sperm undergo extensive membrane modifications required for the acquisition of fertilizing ability including a dramatic decrease in the content and altered composition of membrane lipids (Hammerstedt and Parks, 1987). These changes correspond to a decreased tolerance to rapid cooling (White, 1993), but not in a manner consistent with species differences. For instance, the cholesterol to phospholipid ratio either remains constant or increases as does the unsaturation index and the ratio of choline to ethanolamine phospholipids. These changes may be viewed as an overall net decrease in the ordering of the phospholipid bilayer required ultimately for the acrosome reaction, but with cholesterol they maintain plasma membrane integrity prior to capacitation (Parks and Hammerstedt, 1985). The relationship between these changes and hypothermic sensitivity are based largely on information from domestic animal sperm which contain low to intermediate levels of cholesterol and are sensitive to cooling. Whether a similar phenomenon occurs with sperm high in cholesterol remains to be determined. Also, it may be significant that these changes are occurring to sperm adapted to storage in the epididymis which is several degrees below normal body temperature (Setchell, 1978). Perhaps the ability of rooster sperm to resist cooling damage with a different complement of lipids than mammalian sperm is a consequence of adapting to the elevated temperature of abdominal testes in birds. An evaluation of sperm membrane lipid composition and chilling tolerance in a mammalian species with-abdominal testis such as the elephant might provide further insight into this relationship.

VII. THERMOTROPIC PHASE BEHAVIOR OF SPERM

MEMBRANE IJPIDS Direct and indirect measurements of thermotropic phase behavior in sperm membranes have been reported for several species. Freeze-fracture analysis of ram sperm cooled to 5~ demonstrated aggregation of intramembranous particles presumed to be integral proteins in the plasma membrane overlying the acrosomal region and tail (Holt and North, 1984). Aggregation, which was only partially reversed by rewarming to 30~ was attributed to lateral phase separation of membrane phospholipids. Similar effects have been reported for bull and boar sperm cooled to 0~ which was completely reversible at 38~ (DeLeeuw et al., 1988). Evidence for thermotropic phase transitions between 23 and 28~ has been presented for bull, boar, ram, rooster, and stallion sperm membranes and corresponding lipid extracts based on breaks in Arrhenius plots of

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ATPase activity (Breitbart and Rubinstein, 1983; Holt and North, 1985), fluorescence polarization (Canvin and Buhr, 1989; Holt and North, 1986), and differential scanning calorimetry studies (Parks and Lynch, 1992; Wolf et aL, 1989). Additional transitions have been detected at 17, 36, and 60~ in isolated plasma membrane from ram sperm suggesting the coexistence of liquid crystalline and gel phases (Holt and North, 1991; Wolf et al., 1989). Transition temperatures of 18~ for intact boar sperm (Drobnis et al., 1989) and 13~ for human sperm (Crowe et al., 1989) have been reported using Fourier transform infrared spectroscopy. Peak phase transition temperatures between 33.4 and 42.8~ have been reported for glycolipids from boar, bull, rooster, and stallion sperm (Parks and Lynch, 1992).

v m . SPERM MEMBRANE C O M P O S m O N BEHAVIOR OF MIXED LIPID SYSTEMS

R E I A T W E TO PHASE

Although some species differences in peak phase transition temperatures of sperm membrane lipids have been reported, these differences do not appear to be of sufficient magnitude to explain completely the dramatic differences in chilling sensitivity. More likely, it is the differential consequences of lateral phase separations during these transitions that result in membrane damage. Some relationships between sperm membrane lipid composition and cold shock sensitivity might be explained, however, by the phase separation model for membrane damage proposed by Quinn (1985, 1989). A central feature in this model is the fate of non-bilayer preferring phospholipids during lateral phase separation and their perturbing effect on bilayer structure, suggesting that a high proportion of nonbilayer preferring lipids, such as the ethanolamine phospholipids and SGDG glycolipid component of mammalian sperm, would increase chilling sensitivity. However, both boar sperm, which are highly sensitive to cooling, and human sperm, which are highly resistant, contain a high percentage of ethanolamine phospholipids. Other factors must then mitigate the species differences in segregation of non-bilayer lipids into gel-phase domains during cooling. One factor of obvious importance is the cholesterol to phospholipid ratio, which is known to modulate phase behavior in membranes. Cholesterol may be too low in boar sperm but high enough in rabbit and human sperm to prevent destabilizing effects such as aggregation of nonbilayer preferring lipids. Another factor which may affect susceptibility of the membrane to damage is membrane protein content. Protein in boar sperm plasma membrane is high relative to phospholipids when compared to other species (Nikolopoulou et al., 1985; Parks and Lynch, 1992). Because of the preferential association of non-bilayer lipids with proteins in the membrane, packing faults and other membrane perturbations resulting from lateral phase sepa-

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ration would be expected to increase proportionally with increasing protein content. In addition to structural defects, critical protein functions may be severely altered. For example, the inability of sperm to regulate intracellular calcium following cold shock results in elevation of intracellular calcium (Watson et al., 1985; White, 1993) perhaps due to altered activity of the outwardly directed calcium pump in the sperm plasma membrane (White, 1993). A quite different complement of membrane components appears to confer cold shock resistance on rooster sperm (Parks and Lynch, 1992). In this species, sperm cholesterol is low to moderate but so are non-bilayer preferring phospholipids and protein content. Furthermore, the glycolipid fraction appears to lack the high melting point component of boar and other mammalian sperm which may reduce both the extent of lateral phase separation and the proportion of non-bilayer preferring lipids segregating within the membrane. These observations emphasize the importance of not only the behavior of non-bilayer preferring lipids but their interaction with the complement of other membrane components during cooling. A composite comparison of membrane components summarized from the literature which influence membrane phase behavior in cold shock tolerant versus resistant species is presented in Table 1, The consequences of lateral phase separation according Quinn's model appear to be consistent with many of the morphological and biochemical effects observed with cold shock injury. Disruption and loss of membrane could be a direct effect of localized non-bilayer domains formed during cooling and, upon rewarming, leading to plasma membrane disruption or fusion of plasma membrane with the underlying acrosomal or mitochondrial membranes to which it is closely applied. Spurious fusion events might be Membrane Characteristics of Sperm from Species with High and Low Resistance to Cold Shock

TABLE 1

Species Low

High

Membrane feature

Boar

Human

Rooster

Protein/Phospholipid (w/w) Cholesterol/Phospholipid (mol/mol) Ethanolamine/Choline phospholipid Unsaturation index Tm phospholipids (~ Tm glycolipids (~

1.26 0.26 0.60 2.5 24.0 36.2

m 1.00 1.00 1.00 13.0

0.46 0.30 0.30 1.00 24.5 ND

Note. Tin, peak thermotropic phase transition temperature. ND, not detected between -IO~

and +70~

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exacerbated by cold-shock induced elevation in intracellular calcium, which is known to be a prerequisite for membrane fusion during the physiological acrosome reaction. Other documented effects of cooling damage, such as increased permeability, loss of membrane and intracellular constituents, and motility loss would logically follow the initial perturbation of the membrane.

IX. PROTECTION OF SPERM FROM HYPOTHERMIC EFFECTS Cold shock damage to sperm can be reduced dramatically in many species by cooling semen slowly (<-10~ to around 4~ However, more rapid cooling rates with improved sperm motility and fertility can be attained by including protective additives, especially lipoproteins and phospholipids, in semen diluents. The biochemical nature of these additives suggests that the plasma membrane, which is the primary site of cold shock injury, is also the site of action for protection against cold shock. A. P r o t e c t i v e A c t i o n o f Egg Y o l k a n d Its C o m p o n e n t s Since recommended by Phillips and Lardy (1940), egg yolk has become the most widely used semen diluent component for protecting sperm from cooling and freezing damage. The constituent of yolk which provides protection is the low-density lipoprotein fraction (Foulkes, 1977; Kampschmidt et al., 1953; Pace and Graham, 1974; Watson and Martin, 1975) of which phosphatidylcholine is the most effective component (Kampschmidt et al., 1953; Martin, 1963; Lanz et al., 1965; Simpson et al., 1986). Cholesterol affords bull sperm some protection against cold shock but is not as effective separately or in combination with egg yolk phosphatidylcholine as the phospholipid alone (Lanz et al., 1965; Parks et al., 1981). Interestingly, synthetic phosphatidylcholines varying in chain length and degree of unsaturation are not as effective as phosphatidylcholine from egg yolk (Graham and Foote, 1987). The differential effect may be related to the use of diacyl molecular species (identical acyl groups in the sn-1 and sn-2 positions of the glycerylphosphorylcholine) compared to the naturally occurring 16:0/16:1 or 16:0/18:2 phosphatidylcholine predominant in egg yolk. In the same study, nonsynthetic phosphatidylserine from bovine brain alone or in combination with phosphatidylcholines or cholesterol was highly effective in protecting bull sperm from cold shock. Butler and Roberts (1975) previously reported that phosphatidylserine but neither phosphatidylcholine nor-ethanolamine was effective in protecting boar sperm from cold shock, although the source and structural details of the molecular species used were not provided. Protection of sperm by lipoproteins and phospholipids and the nature of membrane damage due to cold shock has led to much speculation that

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the protective action of egg yolk lies in its ability to interact with the bilayer of the sperm plasma membrane. While these additives prevent cold-induced lesions in the sperm plasma membrane, the mechanism by which they interact with the sperm surface is equivocal. Foulkes (1977) reported that yolk lipoproteins remain associated with bull sperm and Simpson et al. (1986) observed that the protective effect of egg phosphatidylcholine is retained by ram sperm even after extensive washing. However, others have found that the protective effect of added lipids and lipoproteins is reversible upon washing sperm (Quinn et al., 1980; Watson, 1975). Evans and Setchell (1978) studied the interaction of phospholipids with bull, ram, and boar sperm and found that phosphatidylethanolamine and -inositol did not associate with sperm from these species. Added phosphatidylcholine associated extensively with washed and unwashed ejaculated boar sperm but with no protective effect. Association was minimal with ejaculated ram and bull sperm or testicular and epididymal ram and boar sperm. Phosphatidylcholine was never associated with more than 5% of washed or unwashed sperm for any of these species. Lipoproteins and phospholipids do not appear to adsorb or bind extensively to the sperm surface for most species (Evans and Setchell, 1978; Quinn et al., 1980; Streiner and Graham, 1993; Watson, 1975) or modify membrane phospholipid or sterol composition (Parks et al., 1981, Quinn et al., 1980; Streiner and Graham, 1993), nor do they influence thermotropic phase behavior (De Leeuw et al., 1988; Holt and North, 1984). The mode of action of added lipids in protecting sperm from hypothermic damage thus remains obscure, but apparently differs from that for plant protoplasts in which cooling tolerance is increased by fusion of phosphatidylcholine liposomes with the plasma membrane (Steponkus et al., 1988). Watson (1981) suggested that the beneficial effect may be exerted through calcium chelation as the protective effect of egg phosphatidylcholine is not additive with EDTA (Quinn and White, 1967). Shannon and Curson (1983) proposed that components of egg yolk competed with detrimental, possibly membrane destabilizing factors in seminal fluids for sites on the plasma membrane. B. Protective A c t i o n o f Milk

Whole cow's milk, skim milk, and reconstituted skim milk are all effective in protecting sperm membranes during cooling (Foote, 1978). The mechanism by which milk protects the sperm surface is even more enigmatic than that for egg yolk, however, as the protection appears to reside with the protein fraction rather than lipoproteins or phospholipids. While the ability of milk casein to chelate calcium or compete with detrimental seminal fluid factors on the sperm surface may be features in common with egg yolk, these mechanism remain speculative for both of these biological fluids.

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C. P r o t e c t i v e A c t i o n o f B u t y l a t e d H y d r o x y t o l u e n e (BHT) a n d Its A n a l o g s Hammerstedt et al. (1976, 1978) first reported that BHT protected membranes of ram and bull sperm from cold shock damage. Protection against cold shock has been reported also for the boar (Pursel, 1979) and ram (Watson and Anderson, 1983). The mechanism by which BHT protects sperm is not known but appears to be different from that of yolk and milk constituents as partitioning of BHT and its analogs into bull sperm membranes is required (Graham and Hammerstedt, 1992). These compounds partitioned selectively into membranes other than the periacrosomal plasma membrane and outer acrosomal membrane, but protected both mitochondrial membranes and the plasma membrane during cold shock. Differences in partitioning was attributed to lipid compositional differences among membrane compartments which may account for species differences in protection by BHT. These results suggest that protection from cold shock can be improved by targeting different sperm compartments with specific additives but also points out the complex interaction which can occur between additives and must be considered in semen diluents, such as partitioning of BHT between sperm membranes and egg yolk (Graham and Hammerstedt, 1992) or milkfat (Killian et al., 1989). D. A c q u i s i t i o n o f C o l d S h o c k R e s i s t a n c e i n B o a r

Sperm

Boar sperm are extremely sensitive to cooling to temperatures <-15~ but acquire resistance to cold shock when incubated at ambient temperature for 4-5 hr postejaculation (Butler and Roberts, 1975; Pursel et al., 1972, 1973). Acquisition of resistance is intrinsic to the sperm but is enhanced by seminal plasma (Pursel et al., 1973) or addition of phosphatidylserine (Butler and Roberts, 1975). Interestingly, yolk does not protect sperm and actually interferes with acquisition of cold shock resistance (Pursel et al., 1972). Little is known about specific changes during incubation which decrease sensitivity, although Moore et al. (1976) has suggested that basic seminal proteins which otherwise increase membrane permeability and thereby sensitivity to cooling dissociate from sperm during incubation.

X. CONCLUSIONS It is clear that sperm from most mammalian species examined are sensitive to hypothermia and, as for most cell types, the plasma membrane is the primary site of cooling damage. The precise mechanism by which injury occurs remains unclear, but increasing evidence suggests that thermotropic phase behavior of membrane lipids and its consequences play a role

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and that species differences in membrane composition may account for differences in sensitivity to hypothermia. Sperm from many species are protected from hypothermic injury by egg yolk and its components, milk, or other macromolecules, but the mechanism by which these additives protect is not known. A better understanding of the protective action of these substances would be helpful in developing more efficient and effective procedures for cooling semen. Strategies targeting multiple cellular components may also lead to improved sperm survival following cooling. For most species, hypothermia per se does not appear to be a formidable problem with the addition of protective additives. However, cooling may result in latent injury not manifested by conventional laboratory assays which generally are not reliable measures of fertility (Amann and Hammerstedt, 1993). For example, it is well established that motility of sperm cooled in protective medium can remain high for several days, but fertility decreases dramatically during the same period (Maxwell and Salamon, 1993; Watson, 1990). More subtle biochemical and biophysical changes in sperm and sperm membranes during cooling may be largely reversible upon rewarming but can be exacerbated by subsequent freezing and thawing where additional insults such as intracellular ice formation and elevated solute concentration are imposed. Improved methods for preventing hypothermic damage to sperm therefore depend on an improved ability to recognize and understand the changes which occur during cooling and the mechanisms by which these changes can be reversed or prevented.

Xl. HYPOTHERMIA AND MAMMAI~IAN OOCYTES The availability of developmentally competent oocytes is critical to the success of in vitro fertilization and related reproductive technologies. As with sperm, the fertile life of the oocyte in vitro is relatively short which often limits the implementation of in vitro methodologies. Many of these limitations could be circumvented with effective cryopreservation procedures, permitting the banking of germ plasm for a broad range of applications: preserving genetic lines of laboratory or domestic animals, recovering oocytes from ovaries salvaged from valuable individuals (i.e., genetically engineered or endangered) or from women anticipating the loss of gonadal function through surgery or other therapies, or simply increasing the availability of oocytes for research, clinical, and commercial applications such as in vitro sperm fertility assays, embryonic cloning, and genetic engineering. However, it is now generally accepted that procedures developed for embryo cryopreservation are far less effective when used for mammalian oocyte cryopreservation (Friedler et al., 1988; Niemann, 1991; Parks and Ruffing, 1992; Trounson, 1986), although live births from thawed oocytes have been reported from several species including the mouse, hamster,

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rabbit, cow, and human (see Fuku et aL, 1992; Parks and Ruffing, 1992). As with most cell types, cryoinjury appears to be cumulative with each procedural step (cooling, addition of cryoprotective agent, freezing, thawing). While hypothermia imposed during initial cooling may appear to be the least consequential step based on morphological appearance, dramatic effects of cooling on major cytological components of the oocyte (reviewed in Parks and Ruffing, 1992; Vincent and Johnson, 1992) and on fertilization and early embryonic development (Glass and Voelkel, 1990; Martino et al., 1996; Parks and Ruffing, 1992) suggest that cooling p e r se contributes substantially to the reduced developmental competence of cryopreserved oocytes at least in some species.

XII. OVERVIEW OF MAMMAI.IAN OOCYTE STRUCTURE Mammalian oocytes are extraordinarily large cells, ranging from ~80 to 120/zm in diameter when follicular growth is complete, depending upon the species. The oolemma or plasma membrane is surrounded by the zona pellucida, an extracellular glycoprotein matrix involved in sperm binding and penetration. Presence of other cytological features affected by hypothermia depend upon the developmental stage of the oocyte. Typically, oocytes are cryopreserved at either the germinal vesicle (immature) or at the metaphase II (mature) stage (Figures 6A and 6B). Immature oocytes are recovered from the antral follicles of the ovary where they remain arrested in prophase of the first meiotic division. They are characterized by a large prophase nucleus (the germinal vesicle), a dense, narrow band of filamentous actin subjacent to the oolemma, and an abundance of other organelles which make up the synthetic and metabolic machinery typical of most eukaryotic cells (mitochondria, endoplasmic reticulum, Golgi apparatus, etc.). In addition, the Golgi of the oocyte elaborates numerous lysosome-like vesicles, the cortical granules. Upon gonadotropin stimulation of the ovarian follicle or removal of the immature oocyte from the follicular environment, inhibitory factors which regulate meiotic arrest are removed. Nuclear progression and oocyte maturation then proceed to and become arrested at metaphase of the second meiotic division with expulsion of the first polar body, a daughter cell of the first meiotic division which contains only a residual amount of ooplasm. The mature oocyte is characterized by a large, peripheral spindle apparatus with microtubules extending from pole to pole and from each pole to the kinetochores of the bivalent chromosomes aligned along a metaphase plate. The spindle of mammalian ova is anastral and lacks centrioles but contains pericentriolar material (PCM) which serves as the microtubule organizing center for tubulin polymerization at each pole during spindle

250

John E. Parks

Cytological features of the mammalian oocyte and ovum. (A) Immature oocyte arrested in prophase of meiosis I. (B) Mature oocyte (ovum) following cumulus expansion and nuclear progression to metaphase of meiosis II.

FIGURE 6

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formation (Vincent and Johnson, 1992). Shape and orientation of the spindle vary with the species (Figures 7A and 7B). The cortical actin band persists and actin-containing microfilaments are distributed in the perinuclear and cortical ooplasm where they direct organellar distribution, microvillar structure, surface deformations associated with polar body extrusion, and eventually sperm engulfment. Golgiderived cortical granules migrate to the periphery of the oocyte just beneath the actin band where they are poised to undergo exocytosis at the time of fertilization. Release of the cortical granule contents, termed the cortical granule reaction, results in proteolytic modification of the zona pellucida which creates a physiological block to polyspermy (Wassarman and Albertini, 1994).

XIII. EFFECTS OF COOLING ON OOCYTE STRUCTURE Approaches to studying hypothermia in mammalian oocytes have been limited primarily to effects on morphology, cytology, and developmental competence. Compositional analyses and other biochemical approaches possible with sperm and other cell types are exceptionally difficult with oocytes because of the limited number of oocytes which can be recovered or harvested from an individual female. For example, the number of oocytes available for a single experiment typically ranges from only a few for humans to several hundred for mice and cattle on any given day. Thus, effects of hypothermia on special cytological features of the oocyte, especially those in arrested in metaphase II, have been the focus of many studies. Chilling p e r se does not appear to alter the morphology of oocytes from most species significantly (Parks and Ruffing, 1992), including highly sensitive porcine oocytes (Didion et al., 1990). Morphological appearance of cumulus-enclosed germinal vesicle stage pig oocytes, based on density of the cytoplasm, was not affected by cooling to 0~ even after 24 h of culture at 37~ Bovine oocytes appeared normal immediately after exposure to 0~ although some had a "yellowish" cytoplasm 22 h after fertilization in vitro (Martino et al., 1996). These observations suggest that chilling does not affect oocyte appearance directly, but latent chilling injury to the oocyte may be manifested morphologically at later developmental stages. A. Effects o f C o o l i n g o n t h e O o l e m m a Information on plasma membrane composition and thermotropic phase behavior is lacking for mammalian oocytes because the quantity of material required for meaningful analyses is limiting. However, it is a reasonable assumption that oocyte membrane structure and function is influenced by the effects of hypothermia on biophysical properties of the oolemma in

252

John E. Parks

/ /

"~.

J

~

~

'

spindle

pericentriolar ....... I ~ - - nlicrotnhules . PmerleCrena~ ri~ metaphaselIchromosome~---

B

pericentriolar material /

" ~ .

spindle

/micr~

NX

metaphase II chromosomes

C

g 0

O

metaphase II chromosomes

.

material

metaphase II chromosomes

Representationof the meiotic spindle apparatus and metaphase II chromosomes of murine and human ova and effects of chilling and rewarming on spindle organization. (A) Anastral, acentriolar metaphase II spindle of murine oocyte with pericentriolar material associated with the spindle and distributed in the cortical cytoplasm. (B) Human metaphase II spindle with pericentriolar material restricted to the spindle microtubules. (C) Effect of cooling on microtubule depolymerization and chromosome organization of mature mammalian oocyte. (D) Repolymerization of microtubules during rewarming. Degree of spindle reassembly depends upon availability of pericentriolar material (see text). Figure adapted from figure by Vincent and Johnson (1992).

FIGURE 7

Hypothermia a n d M a m m a l i a n Gametes

253

much the same manner as described for membranes of sperm and other cell types. Chilling alone does not appear to compromise the structural integrity of the oolemma in most species based on morphological evaluation (Sathananthan et al., 1987), yet subtle alterations in structure and function and latent injury to the membrane do occur in some species. This point was demonstrated dramatically by Didion et al. (1990) who reported that morphological appearance of immature, cumulus-enclosed porcine oocytes was normal in approximately 80% of oocytes cooled to 0~ but membrane permeability, based on distribution of vital stains, increased in all oocytes after cooling. While not necessarily a detrimental effect, hypothermia decreases permeability of the oolemma to water (hydraulic conductivity) and cryoprotective agents (Leibo, 1980; Hunter et al., 1992; Ruffing et al., 1993; Toner et al., 1990)

B. Effects of Temperature o n the Spindle Apparatus of Mature Oocytes Perhaps the most dramatic effect of cooling mature oocytes below body temperature is the disassembly of the meiotic spindle apparatus (Magistrini and Szollosi, 1980). The meiotic spindle is composed of microtubules which consist of a and 13 tubulin dimers in dynamic equilibrium between the free and the polymerized state. This equilibrium is extremely temperaturesensitive and favors net disassembly of microtubules below normal physiological temperatures. In mature mammalian oocytes, microtubules are located exclusively in the meiotic spindle (Vincent and Johnson, 1992). Sensitivity of the spindle to cooling as well as restoration of normal spindle morphology upon rewarming are influenced by final temperature, duration of exposure, and species (Vincent and Johnson, 1992). The majority of spindles in mouse, human, and bovine oocytes are reduced or abnormal within 10 min after cooling to 25~ (Aman and Parks, 1994; Pickering and Johnson, 1987; Pickering et al., 1990). Complete spindle disassembly increases in oocytes similarly exposed to 4~ (Figure 7C). Chilling effects on the spindle appear to be completely reversed in most mouse oocytes after rewarming to 37~ for 60 min, but spindle morphology in chilled human and bovine oocytes remains abnormal even after protracted rewarming (Figure 7D). The reduced ability of chilled human oocytes to reform a normal spindle after rewarming has been attributed to a deficiency in pericentriolar material which is required for nucleating tubulin polymerization (Pickering et al., 1990; Vincent and Johnson, 1992). The response of bovine oocytes to chilling and rewarming resembles that of human more than murine oocytes (Aman and Parks, 1992).

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John E. Parks

Disassembly or disorganization of the meiotic spindle is often associated with dispersion of the metaphase chromosomes and has been associated with increased chromosomal abnormalities such as aneuploidy and polyploidy (Glenister et al., 1987; Kola et al., 1988). Chromosome dispersion was observed in 8% of mouse oocytes cooled to 25~ for 10 min and in 14% of oocytes cooled to 4~ (Pickering and Johnson, 1987). The potential for chromosome dispersion is even greater with human oocytes in which transient cooling causes irreversible spindle disruption (Pickering et al., 1990). The consequences of these temperature effects include abnormal chromosome alignment and chromatid segregation with serious ramifications for in vitro fertilization and other assisted reproductive technologies in humans (Vincent and Johnson, 1992). C. Effects o f T e m p e r a t u r e o n the O o c y t e C y t o s k e l e t o n Microfilaments which make up much of the cortical cytoskeleton of the oocyte (see Section XII) do not appear to be modified directly by cooling in any significant, observable way. An exception to this generalization is found in mouse oocytes, in which a thick network of actin filaments is located in the cortex overlying the metaphase chromosomes and just beneath a microvillus-free region of the oolemma (Longo, 1987). An expansion of this microfilament network occurs in mouse oocytes cooled to 4~ (Vincent et al., 1990a) associated with an expansion of the microvillus-free area (Vincent and Johnson, 1992). This phenomenon has been attributed indirectly to spindle disassembly and the resulting chromosome dispersion, which causes withdrawal of microvillous actin associated with the chromosomes (Vincent and Johnson, 1992).

D. Effects o f T e m p e r a t u r e o n Cortical Granule Exocytosis Cortical granule exocytosis is a physiological response to the fertilizing sperm binding to the oolemma. Proteases released from the cortical granules into the perivitelline space modify the glycoprotein components of the zona pellucida responsible for sperm binding and penetration (Figure 8), thus preventing further binding and entry of supernumerary sperm and providing a block to polyspermy (Wassarman and Albertini, 1994). Cooling mouse oocytes to 4~ for ---5 min dramatically alters the zona pellucida, resulting in an irreversible decrease in chymotrypsin sensitivity and reduced fertilization rates (Johnson et al., 1988; Vincent et al., 1990b). Changes in the zona are associated with a decrease in cortical granules and do not occur in isolated zonae pellucida (Martinez et al., 1989; Vincent et aL, 1990b), suggesting that the effects of cooling on the zona pellucida are caused indirectly by premature cortical granule exocytosis (Vincent and Johnson, 1992).

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F I G U R E 8 Cortical granule reaction in mammalian ova. (A) Unactivated ovum with cortical granules located subjacent to the cortical actin band and oolemma. (B) Cortical granule exocytosis and consequent proteolytic modification of the zona pellucida (zona hardening). Direct and indirect effects of hypothermia have been implicated in reduced fertility of mammalian ova.

XIV. INTERACTION OF COOLING AND CRYOPROTECTIVE AGENTS Cryoprotective agents also have profound temperature-dependent effects on the special cytological features of the oocyte (see Parks and Ruffing, 1992; Vincent and Johnson, 1992). Dimethylsulfoxide (DMSO) at a concentration of 1.5 M appears to reduce the threshold concentration of free tubulin required for polymerization in mouse oocytes at 37~ resulting in the formation of multiple cytoplasmic asters. Over time, formation of cytoplasmic microtubules proceeds at the expense of the spindle, resulting in its disassembly and subsequent dispersion of the metaphase chromosomes. At 4~ the tubulin polymerizing activity of DMSO is reduced, presumably due to decreased permeability, to a level which decreases formation of cytoplasmic microtubules but counteracts cold-induced spindle disassembly (Johnson and Pickering, 1987; Magistrini and Szollosi, 1980), chromosome dispersion (Johnson and Pickering, 1987), and the incidence of aneuploidy (Glenister et al., 1987). Propanediol also prevents spindle disruption in mouse oocytes at room temperature but causes temporary loss of the spindle upon rewarming to 37~ (Van der Elst et al., 1988). Solvent effects of cryoprotective agents on the organization and distribution of microfilaments in mammalian oocytes is dependent upon species,

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type and concentration of cryoprotective agent, as well as equilibration temperature (Vincent and Johnson, 1992). For example, addition of 1.5 M DMSO at 37~ reduces the density of microfilaments in the region over the meiotic spindle of mouse oocytes and causes extensive chromosome dispersion (Vincent et al., 1990b,c). Both of these effects were reduced significantly when cryoprotectant was added at 4~ with the reduction attributed to decreased permeation of DMSO at the lower temperature. Cryoprotective agents also lead to premature cortical granule exocytosis and zona hardening when added at room temperature or above (Vincent and Johnson, 1992). Vincent et al. (1991) have proposed a mechanism by which the solvent effect of DMSO on the cortical actin band in combination with its transient osmotic effect during equilibration facilitates close apposition and fusion between the oolemma and cortical granules. As with microtubules and microfilaments, the reduced permeation of cryoprotectant at 4~ modulates the effect of both CPA and low temperature on cortical granule exocytosis possibly by reducing solvent and osmotic effects (Vincent and Johnson, 1992).

XV. EFFECTS OF HYPOTHERMIA ON FERTHffZATION AND DEVELOPMENT

The overall effect of chilling appears to have little effect on fertilization and subsequent development of rodent eggs and, in fact, mouse oocytes have been cryopreserved relatively successfully with conventional, ultrarapid, and vitrification procedures (reviewed by Parks and Ruffing, 1992). It is increasingly apparent, however, that oocytes from domestic animals are extremely sensitive to chilling between 0~ and seeding temperatures of down to -7~ The severe, damaging effect of cooling on porcine oocytes (Didion et al., 1990, see above) is well established. Fertilization rate of in vitro matured bovine oocytes was reduced significantly after only 2 min at 0~ (Parks and Ruffing, 1992) and cleavage rates decreased from over 60% to less than 20% when oocytes were cooled to 0 or -7~ (Niemann et al., 1993). Martino et al. (1996) recently reported that development to the blastocyst stage of in vitro matured and fertilized bovine oocytes was reduced to 50% of controls by exposing oocytes to 0~ for only 5 s, and developmental potential decreased with increasing exposure time. Results were similar when oocytes were cooled rapidly (>500~ or more slowly (~100~ Based on these observations, Martino et al. (1996) suggested that with holding times of several minutes prior to seeding and slow cooling rates associated with conventional cryopreservation procedures, chilling sensitivity alone can account for the low development rates obtained with frozen-thawed bovine oocytes.

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XVI. PREVENTING HYPOTHERMIC DAMAGE TO MAMMALIAN OOCYTES DURING CRYOPRESERVATION While the impact of chilling on oocyte structure and function is now recognized, relatively few innovative approaches have been successful in overcoming the problem. Rubinsky et al. (1990) reported that antifreeze proteins from Antarctic and Arctic fishes, which prevent the formation of damaging ice crystals in body fluids at below-freezing temperatures, protected the oolemma of porcine oocytes at hypothermic temperatures (4~ based on vital staining techniques. A mechanism has not been advanced by which these proteins interact with the oolemma to afford protection during chilling. Addition of lipoproteins or phospholipids, which protect mammalian sperm during rapid cooling (see above), has not been tested but is unlikely to be of great value because direct access to the oolemma is obstructed by the zona pellucida. Other cytological components such as the spindle apparatus may require relatively specific treatment to prevent hypothermic damage. For example taxol, a microtubule-stabilizing compound, promotes tubulin polymerization and might be added to oocytes to stabilize microtubules at hypothermic temperatures (Chu et al., 1993) then removed upon rewarming. Martino et al. (1996) proposed that because chilling injury to bovine oocytes is time-dependent, cooling at ultrarapid rates might preclude cellular damage by passing through the damaging temperature range rapidly enough to "outpace" the membrane and cytological changes associated with chilling injury. To achieve ultrarapid freezing rates (>3000~ oocytes were placed on electron microscope grids as described for Drosophila melanogaster embryos in <1/zl of an ethylene glycol-based vitrification solution and plunged directly into liquid nitrogen (Steponkus et al., 1990). Following in vitro fertilization, 15% of oocytes cooled using this technique developed to the blastocyst stage, a much higher rate of development than achieved previously with any other approach. These results demonstrate that it is possible to limit to some degree the chilling injury associated with more conventional freezing as well as ultrarapid and vitrification procedures where oocytes are packaged in 0.25-ml plastic straws and thus frozen at a slower rate.

XVII. CONCLUSIONS Hypothermia now appears to be one of the major contributing factors to the difficulty in cryopreserving mammalian oocytes. The large size and unique cytological features of the oocyte contribute to hypothermic sensitivity. While detrimental effects of hypothermia on the meiotic spindle ap-

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paratus, chromosome configuration, and cortical granules have been documented, the relative impact of these cytological disruptions on oocyte viability and developmental competence remains uncertain. Effects of hypothermia on the oolemma have not been studied as intensively, but it seems likely that the egg plasma membrane, as with that of sperm and other cells, is subject to perturbations associated with thermotropic phase changes. The interaction observed between cooling and cryoprotective agents on cytological damage to the oocyte supports the notion that detrimental changes associated with hypothermia are compounded by other requisite steps in a cryopreservation protocol, including cryoprotective agent addition and removal, freezing, and thawing. Recent progress in oocyte cryopreservation suggests that it may be possible to "outpace" the detrimental effects of hypothermia using ultrarapid cooling rates. These results are especially encouraging because they are not based strictly on morphological or cytological observations but developmental potential. REFERENCES Aitken, J., and Fisher, H. (1994). BioEssays 16, 259-267. Alvarez, J. G., and Storey, B. T. (1993). J. Androl. 14, 199-209. Alvarez, J. G., Touchstone, J. C., Blasco, L., and Storey, B. T. (1987). J. Androl. 8, 338-345. Aman, R. R., and Parks, J. E. (1994). Biol. Reprod. 50, 103-110. Amann, R. P., and Hammerstedt, R. H. (1993). J. Androl. 14, 397-406. Barros, C., Herrera, E., Fuenzalida, I., and Arguello, B. (1986). Gamete Res. 14, 149-158. Breitbart, H., and Rubinstein, S. (1983). Proc. llth World Congr. Fertil. Steril. 539. Butler, W. J., and Roberts, T. K. (1975). J. Reprod. Fertil. 43, 183-187. Canvin, A. T., and Buhr, M. M. (1989). J. Reprod. Fertil. 85, 533-540. Chu, B., Kerr, G. P., and Carter, J. V. (1993). Plant Cell Environ. 16, 883-889. Crowe, J. H., Hoekstra, F. A., Crowe, L. M., Anchordoguy, T. J., and Drobnis, E. (1989). Cryobiology 26, 76-84. Cullis, P. R., Hope, M. J., DeKruijff, B., Verkleij, A. J., and Tilcock, C. P. S. (1985). In Phospholipids and Cell Regulation (J. F. Kuo, Ed.), p. 3. CRC Press, Boca Raton, FL. Darin-Bennett, A., and White, I.G. (1977). Cryobiology 14, 466-470. Darin-Bennett, A., Poulos, A., and White, I. G. (1973). Aust. J. Biol. Sci. 26, 1409-1420. DeLeeuw, F. E., Colenbrander, B., and Vertkleij, A. J. (1988). XI International Congress on Animal Reproduction and Artificial Insemination, Dublin 1988, Vol. 3, p. 327. Demel, R. A., and DeKruyff, B. (1976). Biochim. Biophys. Acta 109-131. Didion, B. A., Pomp, D., Martin, M. J., Homanics, G. E., and Market, C. L. (1990). J. Anita. Sci. 68, 2803-2810. Drobnis, E. Z., Crowe, L. M., and Crowe, J. H. (1989). Cryobiology 26, 541. Eddy, E. M., and O'Brien, D. A. (1994). In The Physiology of Reproduction (E. Knobil, and J. D. Neill, Eds.), 2nd ed., pp. 29-77. Raven Press, New York. Evans, R. W., and Setchell, B. P. (1978). J. Reprod. Fertil. 53, 357-363. Foote, R. H. (1978). In Physiology of Reproduction and Artificial Insemination of Cattle (G. W. Salisbury, N. L. VanDemark, and J. R. Lodge, Eds.), 2nd ed., pp. 442-493. Freeman, San Francisco. Foulkes, J. A. (1977). J. Reprod. Fertil. 1, 1-2. Friedler, S., Giudice, L. C., and Lamb, E. J. (1988). Fertil. Steril. 49, 743-764.

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Fundamental Cryobiology of Mammalian Spermatozoa Dayong

Gao

Cryobiology Research Institute, Methodist Hospital of Indiana, Inc., Indianapolis, Indiana 46202; and Department of Mechanical Engineering, Indiana University--Purdue University, Indianapolis, Indiana 46206

Peter Mazur Fundamental and Applied Cryobiology Group, Biology Division, Oak Ridge National Laboratory 1, Oak Ridge, Tennessee 37831 John

K. C r i t s e r

Cryobiology Research Institute Methodist Hospital of Indiana, Inc. Indianapolis, Indiana 46206

I. THE IMPORTANCE OF AND NEED FOR CRYOPRESERVATION OF SPERMATOZOA Applications for the use of many cell and tissue types ex vivo have been developed over the past several decades (see Chapter 1). In the case of reproductive cells and tissues, utilization of spermatozoa in the context of artificial insemination (AI) has the longest history and is the most widespread (Critser and Linden, 1995). Prior to the development of cryopreser1Managed by Lockheed Martin Energy Research Corporation for the U.S. Department of Energy under Contract DE-AC05-96OR22464. Reproductive Tissue Banking Copyright 9 1997 by Academic Press. All rights of reproduction in any form reserved.

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Dayong Gao, Peter Mazur, and John K. Critser

vation, methods sufficiently effective to provide practical results were routinely used for AI. While the earliest verifiable reports using this approach date to the 18th century for nonhuman artificial insemination (Stone, 1980) and to the 19th century for human AI (WHO, 1987), routine use of this approach to assisted reproduction did not occur until the early part of this century (Stokes et al., 1944). During the early 1900s, use of AI rapidly expanded, so that by the 1930s-1940s, millions of horses, cattle, and sheep were being bred using AI. With such an emphasis on this approach to domestic animal breeding, there was a real need to develop methods to store semen for longer periods of time than could be achieved through addition of media components to extend the spermatozoa's viability and lowering the metabolic requirements of these cells by cooling them to temperatures above 0~ Some of the earliest attempts to successfully cryopreserve spermatozoa were conducted at this time, but it was not until Polge et al. (1949) succeeded in solving this intriguing problem with the application of a protecting agent (glycerol) for frozen sperm. This gave rise to the general use of cryoprotective agents (CPAs) (cf. Chapter 1). Since this time, spermatozoa from several mammalian species, especially bovine and human, have been cryopreserved effectively enough to be economical for subsequent use in AI or other assisted reproductive approaches. The cryopreservation of spermatozoa is also of interest for other reasons. Cryobiologically preserved sperm constitute one method of maintaining genetic diversity in endangered species, especially those in zoological parks which are candidate species for captive breeding programs (Ballou, 1992). Gamete cryopreservation is also a method of enhancing the distribution and preservation of lines that have desirable genetic characteristics, for example, several domestic species, strains of mice, and transgenic animals (Mobraaten et al., 1991; Watson, 1990). It is also extensively used in human reproductive medicine to treat infertility (Critser et al., 1987a,b; Mahadevan, 1980; Sherman, 1986). Recently, the possibility of transmission of the human immunosuppressive virus (HIV) from donor semen has resuited in the requirement that only frozen semen be used by therapeutic insemination using donor sperm programs (TID). The ability to store frozen semen allows sufficient time for seroconversion of donors so that possible carriers of the HIV may be detected (Mascola and Guinan, 1986). The empirical methods of cryopreservation developed in the 1950s are still used today. The motility of post-thaw cryopreserved sperm usually is 50% or less that of prefreeze motility and exhibits wide variability among individuals. In some domestic animal husbandry programs, notably in dairy cattle, AI using cryopreserved sperm results in the vast majority of all offspring produced (Iritani, 1980); however, in nondomestic bovids the use of similar cryopreservation procedures for AI are much less effective in producing calves (Schiewe, 1991). In other agriculturally important species (e.g., swine) success using cryopreserved sperm is still so low that it is not

Tissue Banking in Reproductive Biology

265

economically feasible to routinely use this approach (Crabo, 1991). With the empirical approach it took over two decades to develop methods of practical efficacy for freezing ram sperm, and even after four decades of study, the functional survival of cryopreserved porcine sperm is so low that it is of marginal utility (Johnson, 1985, 1989; Watson, 1990). The first reports of the successful freezing of mouse sperm did not appear until 1990 (Tada et al., 1990), and even these are contradictory and have not been repeatable in other laboratories (Critser et al., 1988b; Penfold et al., 1991; Watson, 1990). The cryosurvival of human sperm from the general population is low. The conception rates with the frozen-thawed human sperm may be lower than those with nonfrozen sperm (Corson et al., 1983; Richardson, 1980; Sherman, 1973). Some investigators, however, have found comparable clinical outcomes using either frozen or nonfrozen spermatozoa (Bordson et al., 1986; DiMarzo et al., 1990; Federation CECOS, 1989) when using selected donors or controlling for concentration. The low motility of cryopreserved mammalian spermatozoa and the often lower conception rates may be due to the fact that procedures for cryopreservation of many mammalian cell types, including sperm, have evolved empirically (Watson, 1979). This species variability is due, at least in part, to several factors: (1) the lack of a general knowledge of the fundamental cryobiology of spermatozoa from different species (little is known in this regard for any species except the human and a few domestic species), (2) a lack of knowledge regarding the basic reproductive biology of some species (e.g., wild and/or endangered species), and (3) technical difficulties in performance of AI (e.g., domestic sheep).

II. FUNCTIONAL ASPECTS OF SPERMATOZOA

A. Sperm Function Within the framework of this current chapter, we are concerned with sperm function in the context of cryopreservation and therefore we need to consider several fundamental issues regarding the way in which one plans to utilize the spermatozoa after cooling and warming. For example, if the semen/sperm sample is to be used for AI, the cryopreserved cells must retain essentially all the structural and functional integrity of a noncryopreserved sample (e.g., a high frequency of plasma membrane integrity, mitochondrial function, motility, acrosomal integrity) and have a relatively high concentration of structurally and functionally intact cells (e.g., 1-6 x 107/insemination). If the sample is to be used for in vitro fertilization, the same general characteristics must be maintained; but the frequency of maintaining those characteristics might be significantly reduced (e.g., lower

266

Dayong Gao, Peter Mazur, and John K. Critser

percentage motility or acrosomal integrity) and the concentration of cells retaining these functional characteristics may be reduced by one or two orders of magnitude (e.g., 1 • 105) and still the sample may function well in terms of initiating a high frequency of fertilization and embryo development. In the case of intracytoplasmic sperm injection (ICSI), the sperm sample may have extremely poor andrologic characteristics (e.g., low viability, low or no motility) with an extremely low concentration (10 3 to 10 4) and still provide the sperm nucleus required for assisted fertilization and genetic recombination. In the ultimate situation, lyophilized hamster sperm, with no viability in terms of cellular integrity, have been injected into hamster oocytes, resulting in apparently normal fertilization and embryo development (Katayose et al., 1992). As a brief review, various portions of some mature mammalian male reproductive tracts are illustrated in Figure 1. The characteristics of these male reproductive tracts are listed in Table 1. The structure of a testis (testicle) is demonstrated in Figure 2, which is composed of a large number of coiled seminiferous tubules where the sperm are formed. The sperm then empty into the epididymis, and thence into the vas deferens, which enlarges into the ampulla of the vas deferens immediately before the vas enters the body of the prostate gland. The different timing of morphological differentiation of the testis of some mammalian species are indicated in Table 2. The distinct characteristics of seminal plasma of some mammalian species are described in Table 3. The spermatogenesis, spermiogenesis, and spermiation are summarized schematically in Figure 3. Although morphology of mature mammalian spermatozoa varies dramatically from species to species (Figure 4) they have the similar structure and basic components as shown in Figures 5-7. B. A s s a y s o f Sperm Function Historically the primary assay of sperm function is the use of insemination and measurement of pregnancy initiation (Graham, 1978). In addition, the current standard in vitro assays of sperm function are those which measure the basic structural and functional aspects of sperm physiology: (1) motility; (2) plasma membrane integrity; (3) mitochondrial function; (4) acrosomal integrity; (5) sperm-egg interactions including sperm binding to the zona pellucida, penetration of zona-free oocytes, and fertilization of intact oocytes (Critser and Noiles, 1993; Henry et aL, 1993; Mortimer, 1985). 1. I n s e m i n a t i o n a n d P r e g n a n c y I n i t i a t i o n

In general, AI using frozen-thawed sperm is routinely used for only a very few species, most notably dairy cattle (but relatively few beef cattle are bred using AI) and humans. In cattle, efficacy is commonly expressed in terms of 60- to 90-day nonreturn rates, which is a percentage of the females which, between 60 and 90 days after insemination, did not show

267

Tissue Banking in Reproductive Biology

Seminal vesicle

Prostate

\

/

Cowper's gland

Bladder Vas deferens

Bladd Preputial gland

Testicle ""

Epididymis

Vas deferens

IBoarl

I Catl Seminal vesicle

Semina vesicle

Prostate Ampulla

~ A I

Cowper's gland

Bladder

Was deferens

Vas deferens

Cowper's gland Epididymis Epididymis .

.

.

.

[ Bull I

.

.

.

.

.

.

.

Testicle

I Man I

F I G U R E 1 Schematic diagrams of some mature mammalian male reproductive tracts. In general, the mammalian male reproductive tract consists of two testes, two epididymides, each with its vas deferens, and the accessory reproductive glands. Redrawn with permission from Setchell et al. (1994).

behavior estrus, suggesting an ongoing pregnancy. Such nonreturn rates are generally reported in the range of 60-70%; however, subsequent calving rates (i.e., the percentage of those animals which actually have live offspring) is in the range of 40-60% (Salisbury et al., 1978). As high as these

268

Dayong Gao, Peter Mazur, and John K. Critser

Characteristics of the Male Reproductive Tract of Some Mammalian Species

TABLE 1

Vas

Species

deferens Seminal Bulbourethral ampulla vesicles Prostate glands Reference

Human

+

+

+

+

Bull

+

+

+

+

Ram

+

+

+a

+

Stallion

+

+

+

+

Boar

_{_a

+

+

+

Mouse

+

+

+

+

Rat

+

+

+

+

Cat

+a

_

+

+

Dog

-

-

+

Elephant + Dolphin -

+ -

+ +

+ -

Price and Williams-Ashman (1961) Eckstein and Zuckermann (1956) Price and Williams-Ashman (1961) Eckstein and Zuckermann (1956) Price and Williams-Ashman (1961) Eckstein and Zuckermann (1956) Hafez et al. (1974) Price and Williams-Ashman (1961) Eckstein and Zuckermann (1956) Price and Williams-Ashman (1961) Eckstein and Zuckermann (1956) Hafez et al. (1974) Price and Williams-Ashman (1961) Eckstein and Zuckermann (1956) Price and Williams-Ashman (1961) Eckstein and Zuckermann (1956) Price and Williams-Ashman (1961) Eckstein and Zuckermann (1956) Price and Williams-Ashman (1961) Eckstein and Zuckermann (1956) Short et al. (1957) Price and Williams-Ashman (1961)

a Scattered lobules of gland tissue.

efficacy m e a s u r e s are in d o m e s t i c dairy cattle, it is i m p o r t a n t to state that use of very similar a p p r o a c h e s for s p e r m c r y o p r e s e r v a t i o n in n o n d o m e s t i c bovids yields results which are overall, m u c h lower (Schiewe, 1991). In h u m a n s it has b e e n r e p o r t e d that, in the n o r m a l p o p u l a t i o n , a b o u t 2 0 - 3 0 % of h u m a n o v u l a t o r y cycles will result in p r e g n a n c y if couples h a v e u n p r o t e c t e d i n t e r c o u r s e two to t h r e e times p e r w e e k ( L e r i d o n , 1977). Collectively, studies in which the efficacy of n o n c r y o p r e s e r v e d s e m e n was e v a l u a t e d e s t i m a t e fecundability rates (the m o n t h l y probability of conception) at 12-27%. In contrast, estimates of the efficacy of c r y o p r e s e r v e d s e m e n r a n g e s f r o m 5 to 17% (see Critser and L i n d e n , 1995, for review).

2. Motility B e c a u s e m a m m a l i a n s p e r m are a motile cell type and, u n d e r nonassisted conditions, h a v e an a b s o l u t e r e q u i r e m e n t for motility to be able to fertilize an oocyte; this attribute has b e e n widely m e a s u r e d as an e n d p o i n t of functional integrity. Until fairly recently s p e r m motility was m o s t often assayed using direct, microscopic a s s e s s m e n t (e.g., placing s e m e n o n t o a p r e w a r m e d

Tissue Banking in Reproductive Biology

269

F I G U R E 2 The epididymis is located on the testis and is comprised of the ductuli efferentes (efferent ducts) and the ductus epididymis (epididymis). Reproduced with permission from Follas and Critser (1992).

microscope slide and observing the specimen magnified 100 to 400 times). The type of information which can be obtained using this approach are: (1) a subjective estimate of the percentage of the spermatozoa that are motile (have flagellar activity) (i.e., the number of motile sperm/the total T A B L E 2 Timing of Morphological Differentiation of the Testis of Some Mammalian Species

Species

Gestational length (days)

Testicular differentiation (days)

Human

270

42-45

Cattle

270-290

41-42

Mouse

18-21

12

Rat

21

13-14

Reference

Gondos (1980) Jirasek (1971) Pelliniemi and Niemi (1969) Sitteria and Wilson (1974) Gondos (1980) Vigier et al. (1976) Merck (1972) Gondos (1980) Price and Ortiz (1965) Merck (1972) Feldman and Bloch (1978) Jost et al. (1974) Lording and de Kretser (1972) Warren et al. (1973)

TAaLE 3 Characteristics of the Seminal PIasma of Some Mammalian Species Sodium (rnEq/liter)

Porussium (rnEq/liter)

25-30

123-368

22-103

2-10

300-2000

65-165

20-70

Dog

1-25

4-450

56-124

Human

2-6

2-250

100-133

2000-5000 30-800

Species

Ejaculate volume (m!)

Boar

150-500

Bull

Ram

0.7-2.0

Stallion

30-300

Sperm concentration (X l@/rnlj

pH

Osmoluliry (inOsm)

1-3

7.3-7.9

317-339

10-40

6.4-7.8

274-392

0.4-0.9

6.1-7.0

312-322

17-27

7-30

7.2-7.6

2%-312

45

19

5

5.9-7.3

2%-376

30

17

10

7.2-7.8

301

8

Calcium (rnEq/liter)

Reference Hansel and McEntce (1970) Setchell et al. (1990) White and MacLeod (1963) Hansel and McEntee (1970) Setchell et al. (1990) White and MacLeod (1963) Hansel and McEntee (1970) Setchell et ai. (1990) White and MacLeod (1963) Hansel and McEntee (1970) Setchell et al. (1990) White and MacLeod (1963) Hansel and McEntee (1970) Setchell et al. (1990) White and MacLeod (1963) Hansel and McEntee (1970) Setchell et al. (1990) White and MacLeod (1963)

Tissue Banking in Reproductive Biology

A

~

Leydig Spermat?-

271

Capillary

gonia ~ ,

Primary spermatocyte

~

Spermatozoa~.~.

~ ~'~ ~ ~spermotocyte ) ~'i~ (l~'ff ~ Spermatids l!imn~

B

Stem cells

-

ALong _ APale _

Differentiating Spermatogenesis spermatogonia

" ~~B

ADark Diploid

""

chromosome content

Meiotic division

Spermatocytes

Spermiogenesis

Spermatids

Spermiation

i Sa Sbl

Sb2 Sc Sdl Sd2

Haploid

chromosome content

~.~'~

The Sertolicells line the seminiferous epithelium which provide the necessary and functionalmilieuin which spermatogenesis, spermiogenesis, and spermiation occur. Reproducedwith permission from Follas and Critser (1992).

FIGURE 3

structural

number of sperm) • 100; (2) the percentage of spermatozoa which demonstrate progressive motility (i.e., the number of sperm which actually change their location/the total number of sperm) • 100; (3) a subjective score of the velocity of the progressively motile sperm (using a scoring system of, for example, 0-4, where 0 represents no progressive motility and 4 represents all sperm rapidly moving forward) (Critser and Noiles, 1993). More recently, quantitative methods for determining sperm motility have been utilized. Initially, cinematic techniques were used which first filmed micrographs of sperm movement that were "played back" and al-

272

Dayong Gao, Peter Mazur, a n d John K. Critser

Man

~uinea

ull

pig

Dog

Cat

Omithorhynchus and Tachyglossus (monotremes) Chinese hamster

Perameles

(bandicoat)

The morphologyof mammalianspermatozoavaries dramaticallyfrom species to species. Redrawn with permission from Austin (1965).

FIGURE 4

lowed the "frame by frame" progression of cells to be assessed. Later, computer-assisted systems were developed incorporating digitial image capture and analysis in real time (Katz and Davis, 1987; Mortimer, 1990; Mortimer and Mortimer, 1990). Using these techniques, sperm motility and other motion characteristics can be quantitatively assessed. While subjective assessments of sperm motility have been widely used as a standard assay to estimate sperm function; correlations between motility and fertilization or fertility have been low (Graham, 1978; Peng et al., 1987). However, more complex analyses utilizing multiple motion characteristic measurements (e.g., motility, velocity, amplitude of lateral head displacement [a measurement of side to side sperm cell movement]) which do have a high predictive value of fertilization can be developed (Critser et al., 1989; Critser and Noiles, 1993). 3. Plasma Membrane Integrity

After motility, plasma membrane integrity is likely the most commonly used assay of sperm viability. While there are several approaches to measuring sperm plasma membrane integrity, these methods generally fall into one of two categories: supervital staining or hyposmotic swelling (Critser and Noiles, 1993). In the general case of supervital staining, the underlying premise is that live cells possess intact plasma membranes through which

Tissue Banking in Reproductive Biology HEAD

I

I

273

FLAGELLUM

I

i z.Connecting piece Mitochondrial . sheath

--

"

'

--

-

g,

_,_._

"i~_

MIDDLE PIECE PRINCIPAL PIECE

i

END PIECE

General features of the mammalian sperm. The head of the spermatozoon is attached to the connecting piece of the flagellum. The other regions of the flagellum are the middle piece, the principal piece, and the end piece. The middle piece contains the mitochondrial sheath, while the principal piece contains the fibrous sheath. Longitudinal and crosssectional views of the principal piece and a segment of fibrous sheath are indicated by arrows. Redrawn with permission from Eddy and O'Brien (1994).

FIGURE 5

m a c r o m o l e c u l a r size stains cannot pass, resulting in unstained cells being classified as "viable," while stained cells are interpreted as having one or m o r e lesions in their plasma m e m b r a n e s and are classified as "nonviable." Variations on this general approach include the use of " p e r m e a b l e " fluorescent compounds such as fluorescein diacetate ( F D A ) or carboxyfluorescein diacetate ( C F D A ) which pass through an intact cell m e m b r a n e and are cleaved by intracellular esterases which cause the c o m p o u n d to both fluoresce green and b e c o m e " i m p e r m e a b l e " and therefore sequestered within the cell (Noiles et al., 1991). In cells without an intact plasma membrane, these esterases are no longer present and therefore the c o m p o u n d is not modified or sequestered. Using either a fluorescent microscope or a fluorescence-activated cell sorter (FACS), the sperm can then be monitored for fluorescence and counted as "viable" if they fluoresce. Counterstains may be added with either visible light stains (e.g., nigrosin with eosin)

274

Dayong Gao, Peter Mazur, and John K. Critser

F I G U R E 6 Structure of the head of guinea pig and human sperm. The plasma membrane of the principal segment domain and equatorial segment domain overlies the outer membrane of the acrosome. The inner acrosomal membrane, in turn, overlies the nuclear envelope. The acrosome is thinner in the equatorial segment region than in the principal segment region. Redrawn with permission from Eddy and O'Brien (1994).

or fluorescent compounds (e.g., propidium iodide with CFDA) to make interpretation easier. In the case of fluorescent compounds, multiple stains can be used to monitor different aspects of the cell's integrity, so, for example, plasma membrane integrity and acrosomal integrity can be measured simultaneously (Tao et al., 1993a). The other general approach to evaluating plasma membrane integrity is to assay the maintenance of membrane semipermeability by testing the Outer Doublet

Plasma Membrane

Outer = _ _ _

Inner Arm

Inner Doublet

F I G U R E 7 The axoneme in the end piece of the sperm flagellum. Nine outer doublets of microtubules surround a central pair of microtubules. The outer doublets consist of microtubules A and B. Inner and outer dynein arms extend from the A microtubule toward the B microtubule of the adjacent pair, forming a 9 + 9 + 2 configuration. Redrawn with permission from Eddy and O'Brien (1994).

Tissue Banking in Reproductive Biology

275

cell's ability to change its volume when exposed to anisomotic conditions (Critser and Noiles, 1993). Cells shrink in response to hyperosmotic conditions due to the exosmosis of water from the cell and, reciprocally, cells swell when placed into hyposmotic conditions. Because increases in sperm volume are much easier to detect than decreases in volume, a hyposmotic swelling (HOS) test was designed to measure the sperm's ability to respond osmotically (Jeyhendran et al., 1984; Zaneveld and Jeyhendran, 1988). Spermatozoa are placed into a hyposmotic environment, insufficient to cause lysis, but sufficiently hyposmotic to cause clear swelling. Examination of the swollen sperm using phase contrast microscopy allows easy detection of those responding. Using this assay, it is possible to evaluate whether sperm have intact plasma membranes and are, therefore, "viable." 4. A c r o s o m a l S t a t u s

Transmission electron microscopy was initially used to assess the acrosomal status of mammalian spermatozoa (Figure 8) and is still the standard against which other methods are evaluated (Fawcett, 1970; Koehler, 1978; Phillips, 1972; Talbot and Chacon, 1980). However, this approach is very slow, time consuming, and expensive. In some mammalian species (e.g., the boar), the acrosome can be visualized directly using phase microscopy in unstained preparations (Pursel and Johnson, 1972, 1974). However, in many mammalian species, including the human, the acrosome is relatively small and cannot be readily visualized at the light microscopic level unless stained. The first was the "triple stain" method utilizing trypan blue as a supravital stain, rose bengal to stain the acrosome, and Bismarck brown as a contrast stain (Talbot and Chacon, 1981). Subsequently, several investigators have used a number of approaches including chlortetracycline (Fraser and Herod, 1990; Lee et al., 1987), polyclonal (Cross et al., 1986) or monoclonal antibodies (DubovaMihailova et al., 1991; Fenichel et al., 1989; Gallo et al., 1991; Homyk et al., 1990; Kallajoki et al., 1986; Wolf, 1985), and fluorescein-conjugated lectins (Liu and Baker, 1988; Mortimer et al., 1987, 1990). 5. In Vitro S p e r m - E g g I n t e r a c t i o n s

As mentioned previously, the ultimate assay of sperm function is the ability to participate in fertilization. However, assessment of fertilization and pregnancy initiation in vivo is often technically difficult, and inherently expensive and time consuming. To address this, several in vitro assay systems have been developed in order to obtain a measure of the spermatozoa's ability to interact with an oocyte at various levels. One can perhaps best view these levels from the perspective of the cumulus-oocyte complex. In a simplified model, the cumulus-oocyte complex presents a series of barriers through which the sperm must successfully traverse in a particular sequence: (1) the cumulus (and corona) matrix, (2) the zona pellucida and the oocyte plasma membrane (the oolemma). In some species, notably the domestic

276

Dayong Gao, Peter Mazur, and John K. Critser

Transmission electron micrograph of human spermatozoa undergoing the acrosome reaction. (A) Unreacted spermatozoon (PM, plasma membrane; OAM, outer acrosome membrane; IAM, inner acrosomal membrane) (original magnification, • (B) Reacting spermatozoon, exhibiting vesicles formed by the fusion of the plasma membrane with the underlying outer acrosomal membrane (• (C) Fully reacted spermatozoon (original magnification, x40,000). The fusion point of the plasma membrane along the anterior limit of the equatorial segment is indicated by arrows. This fusion implies that the sperm is intact and a true acrosome reaction has occurred. Reprinted with permission, from Wiley-Liss, Division of John Wiley and Sons, Inc., from Suarez et al., Gamete Research (1986).

FIGURE 8

Tissue Banking in Reproductive Biology

277

cow, the cumulus is lost quickly after ovulation and, for the most part, is not present as a barrier to sperm. Assay systems have been developed to examine whether sperm from a given source can pass through either a single barrier (e.g., the zona pellucida or the oolemma) or a combination of barriers (e.g., cumuluszona-oolemma in combination or the zona-oolemma only). In vitro fertilization (IVF) assays are designed to determine the sperm's ability to pass through either all three barriers (the cumulus-zona-oolemma) or the latter two (the zona and oolemma). These assays systems are commonly used to assess fertilizing ability of spermatozoa in domestic (Parrish, 1991) and rodent species (Carron and Saling, 1991; Saling and Storey, 1979). However, for ethical reasons, IVF is rarely used to assess sperm function in humans, although in some clinical settings, results of IVF treatment cycles are often useful in assessing sperm function. The zona-free hamster assay (also called HEPA [hamster egg penetration assay], SPA [sperm penetration assay], HEPT [hamster egg penetration test], or HOP [hamster oocyte penetration]) was developed to determine the ability of sperm to fuse with an oolemma. The assay was developed by Yanagimachi (1972) for use with guinea pig sperm but has been adapted for use with many different species including the human (Yanagimachi et al., 1976), the bull (Bird et al., 1989; Brackett et al., 1982), the boar (Berger, 1990a,b; Imai et al., 1979), the stallion (Brackett et al., 1982), the domestic goat (Berger, 1990b), the domestic cat (Howard et al., 1991), the Siberian tiger (Byers et aL, 1987; Post et al., 1987), the leopard cat (Howard and Wildt, 1990), and the gorilla (Lambert et al., 1991). The assay is based upon the anomaly that zona-free oocytes from the golden hamster will allow sperm from other species to penetrate. Therefore, this system allows the measurement of the spermatozoan's ability to undergo capacitation, the acrosome reaction, membrane fusion (sperm-oocyte membrane fusion), incorporation of the sperm into the oocyte, and sperm nuclear decondensation (Critser and Noiles, 1993). In general, the assay is performed by obtaining ovulated oocytes from golden hamsters, removing the cumulus using hyaluronidase, and then removing the zona pellucida with trypsin and then coincubating the sperm to be assessed versus a known control sperm source with the zona-free hamster oocytes (Coetzee et al., 1990; Johnson et al., 1990). However, the zona-free hamster penetration assay does not assess the sperm's ability to bind to and penetrate the zona pellucida. The hemizona assay (HZA) was developed to measure this aspect of human sperm function (Burkman et al., 1988). The assay is performed by coincubating spermatozoa from an infertile or unknown source versus a known fertile control, with zonea which have been microbisected. The relative binding of the unknown and known fertile sperm samples are then compared (Critser and Noiles, 1993). This assay has been applied to some nonhuman primates

278

Dayong Gao, Peter Mazur, and John K. Critser

including the lowland gorilla (using human zonea) (Lanzendorf et aL, 1992) and cynomolgus monkeys (using monkey zonea) (Oehninger et al., 1993).

6. Temporal Aspects of Sperm Function In general, the control against which cryopreserved semen samples or sperm preparations have been judged is a noncryopreserved aliquot of the same sample. However, it is important to note that the majority of the cohort of spermatozoa in a given ejaculate or initial preparation normally undergo a series of postejaculation changes which are required before those sperm acquire the capacity to participate in fertilization. These series of changes are collectively described as the process of capacitation. Prior to capacitation, the spermatozoa cannot fertilize an oocyte. Therefore, some comparisons of the functional integrity of sperm (i.e., assays of sperm-egg interactions) between noncryopreserved sperm and cryopreserved sperm are temporally dependent. For example, Critser et al. (1987) compared the ability of noncryopreserved and cryopreserved human sperm to penetrate zona-free hamster oocytes, a common test of sperm function (Yanagimachi, 1972; Yanagimachi et al., 1976), at various times postejaculation or postthaw. They found that the value for the highest frequency of penetration was not significantly different between the two groups; but the cryopreserved sperm penetrated with high frequency immediately upon exposure to oocytes, while the noncryopreserved sperm required the usual 18-20 hr to capacitate prior to achieving their highest penetration rate (Figure 9). These types of data lead to the hypothesis that cryopreservation of spermatozoa causes nonlethal damage which alters the sperm plasma membrane in a manner which, at least phenomenologically, accelerates the capacitation process (Watson et aL, 1992; Watson, 1995) (Figure 10). Such an "accelerated" effect does not necessarily mean that the cells are more or less functional (in terms of being able to fertilize an oocyte); but do suggest that the timing of sperm-egg interaction will be altered. However, if this timing alteration is not adjusted for in practice, the cryopreserved sperm may produce low results (fertilization).

m . FUNDAMENTAL CRYOBIOLOGY OF MAMMAI.IAN SPERMATOZOA A. C u r r e n t T h e o r y o n Cell C r y o i n j u r y The major steps used in cell cryopreservation can be summarized as follows: (1) adding cryoprotective agents (CPAs) to cells before cooling, (2) seeding samples at or a few degrees below the freezing point of the cell suspension (i.e., spontaneously or artificially inducing ice crystals in the cell suspension to avoid the supercooling) and cooling the cells toward

Tissue Banking in Reproductive Biology

100

-

90 80 o C

7O

279

/ zoa

-

~ ~

60

Fresh

C

50 cD 40 IX. 3O C

,o

20

~',(

I

0

""" ....~ .... spermatozoa

I

6

I

12

I

I

18 24

I

30

I

36

I

42

I

48

Time (h)

FIGURE 9

The penetration of zona-free hamster eggs by fresh or cryopreserved human

spermatozoa after incubation in media for up to 48 hr. Modified and reproduced with permission from Critser et al. (1987b).

a low temperature at which the cells are stored, (3) warming the cells, (4) removing the CPAs from the cells after thawing the sample. Injury to cryopreserved cells has been shown to be caused by each or a combination of the above steps. 1. C r y o i n j u r y d u r i n g Cooling a n d W a r m i n g P r o c e s s e s

Contrary to popular belief, the challenge to cells during cryopreservation is not their ability to endure storage at low temperature; rather, it is the lethality of an intermediate zone of temperature ( - 1 5 to -60~ that cells must traverse twice: once during cooling and once during warming (Mazur, 1963). As cells are cooled to about -5~ both the cells and surrounding medium remain unfrozen and supercooled. Between - 5 and about -10~ ice forms (either spontaneously or as a result of seeding the solution) in the external medium but the cells' contents remain unfrozen and supercooled; presumably because the plasma membrane blocks the growth of ice crystals into the cytoplasm. The supercooled water in the cells has, by definition, a higher chemical potential than that of water in the partially frozen extracellular solution, and thus water flows out of the cells osmotically and freezes externally. The subsequent physical events in

280

Dayong Gao, Peter Mazur, a n d John K. Critser Environment

Sperm Development

Epididymal spermatozoa

Epididymal fluid !

!

I

I

Seminal fluid ! !

Cervical

i

I

I

i

Ejaculation

!

I ....

I

|11,

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media

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Follicular fluid F I G U R E 1 0 Diagram of the proposed effects of cooling and cryopreservation on the postthaw function of spermatozoa; assumed recovery of spermatozoa in similar state than that when frozen ( ..... ); actual recovery of live spermatozoa in a more reactive state than that when frozen ( . . . . ). Redrawn with permission from Watson (1995).

the cells depends on the cooling rate. If cells are cooled too rapidly, water is not lost fast enough to maintain equilibrium; the cells become increasingly supercooled, eventually attaining equilibrium by freezing intracellularly (Mazur, 1963, 1990). In most cases, cells undergoing intracellular ice formation (IIF) are killed (Farrant, 1970, 1977; Fujikawa, 1980; Mazur, 1977a, 1984; Muldrew and McGann, 1994; Rapatz et al., 1963; Steponkus and Wiest, 1979; Stowell et al., 1965; Trump et al., 1965). If cooling is sufficiently slow, the cells will lose water rapidly enough to concentrate the intracellular solutes sufficiently to eliminate supercooling. As a result, the cells dehydrate and do not freeze intracellularly. However, if the cells are cooled too slowly, they will experience a severe volume shrinkage and long time exposure to high solute concentrations before eutectic is achieved, both of which may cause cell injury. Lovelock (1957) proposed that the increased concentration of solutes and cell dehydration have deleterious effects on the lipid-protein

Tissue Banking in Reproductive Biology

281

complexes of cell membranes, weakening them and increasing lipid and phospholipid losses. The cell is rendered permeable to cations and swells, eventually rupturing. Levitt (1962) proposed that loss of water from the protoplasm brings protein molecules into apposition, presenting the opportunity for the formation of new chemical bonds previously too distant and rigidly structured in hydrated form to permit combination. Thawing would have a disruptive force on the new combination, permitting unfolding and denaturation. Karow and Webb (1965) explained slow freezing injury as a consequence of the extraction of bond water from cellular structures for incorporation into ice crystals, denuding proteins of lattice-arranged bound water essential to cell integrity. The possible effects that may cause cell injury as a result of concentration of solutes have been characterized collectively by Mazur et al. (1972) as "solution effects." They suggested that solution effects on cells are greatly enhanced in a slow cooling process during which the time of the exposure of cells to a highly concentrated solution is prolonged. They also indicated that hyperosmotic stress may cause a net leak/influx of nonpermeating solutes; when cells are returned to isosmotic conditions, they swell beyond their normal isotonic volume and lyse. Meryman (1970) proposed that slow freezing in most cells is a result of decrease in intracellular volume beyond a critical volume. As the cell reduces in size in response to increasing osmolarity, the compression of contents enhances the resistance to further shrinking. This results in a hydrostatic pressure difference across the cell membrane, incurring cell membrane damage. Steponkus and Wiest (1979) invoked a maximum cell surface area hypothesis: the cell shrinkage induces irreversible membrane fusion/change, and hence the effective area of cell membrane is reduced; when returned to isotonic condition, the cells lyse before their normal volume is recovered. Some cells could be shown to exhibit a reversible inhibition of shrinkage at some point during a progressive increase in extracellular concentration, implying the development of membrane stress (Williams, 1979). Alternative theories have embraced such mechanisms of injury as macromolecules denaturation from dehydration or phase changes of membrane lipids (Fishbein et al., 1978). Pegg et al. (1988) invoked a "packing effect," that is an inverse dependence of cell survival upon the proportion of the initial sample volume that is occupied by the cells. Mazur and colleagues, however, have proposed that a considerable portion of the damage, at least in human erythrocytes, is due to the reduction in the size of the unfrozen channels which results in increased cell-ice or cell-cell contacts (Mazur and Cole, 1985). Therefore, either too high or too low a cooling rate can kill cells, although the mechanisms underlying cell damage are different. Based upon this, an optimal cooling rate for cell cryosurvival should exist between the "high" and the "low" rates. This has been confirmed experimentally. As

282

Dayong Gao, Peter Mazur, and John K. Critser 70

~b~MOUSE EMBRYOS EM CELLS .

C

5O

~ 5 0 -

9

10-

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,,._ 9

o

o

0(31

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.

. .

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.

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-

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F I G U R E 11 Relationship between survival and cooling rate in five types of cells. Stem cells refer to mouse marrow stem cells. Reproduced with permission from Mazur (1976).

shown in Figure 11, cell survival plotted as a function of cooling rate produces a characteristic inverted U-shaped curve. It is also clear from the above theory that whether a given cooling rate is too "high" or "low" for a given cell type depends on the ability of water to move across the cell membrane. Differences in water permeability account largely for the magnitude of difference in optimal cooling rates for different cell types (Figure 11). The rate of exosmosis of water during freezing can be described by four simultaneous equations (Mazur, 1963). The first relates the loss of cytoplasmic water to the chemical potential gradient between intracellular supercooled water and external ice, expressed as a vapor pressure ratio, dV/dt = ( L p A R T In pe/Pi)/~l,

[11

where V is the volume of cell water, t is time, Lp the permeability coefficient for water (hydraulic conductivity), A the cell surface area, R the gas constant, T temperature, r the molar volume of water, and Pe and Pi the vapor pressure of extracellular and intracellular water, respectively. The change in this vapor pressure ratio with temperature can be calculated from the Clausius-Clapeyron equation and Raoulrs law: d ln(pe/Pi)/dT = L f / R T 2 -

[N2~/(V + N2~)V]dWdT.

[2]

Here, N2 is osmoles of intracellular solute, and Lf is the latent heat of fusion of ice. Time and temperature are related by the cooling rate (B), which, if linear, is given by dT/dt = B.

[31

Finally, Lp is related to temperature by the Arrhenius relation, Lp = L g e x p [ - E a / R ( 1 / T - 1/Tg)],

[4]

Tissue Banking in Reproductive Biology

283

where Lg is the permeability coefficient to water at a known temperature Tg, Ea is its activation energy, and R is the gas constant in cal/mol-degree. To avoid intraceUular freezing, the water content of the cell given by the above four equations must, before reaching the intracellular icenucleation temperature (usually between - 5 and -40~ have approached the equilibrium water content given by V = ~ M i / [ e x p ( L f / R T - L f / R 2 7 3 ) - 1],

[5]

where Mi is the initial osmolality of the extracellular solution. The above quantitative expressions permit one to calculate the extent of supercooling in cells as a function of the cooling rate provided one knows or can estimate the permeability of the cell to water (Lp), its temperature coefficient or activation energy (Ea), the osmoles of solute initially in the cell, and the ratio of the cell surface area to volume. The results of such calculations are commonly expressed as plots of the water content of a cell as a function of temperature relative to the normal or isotonic water volume. Figure 12, for example, shows computed water loss curves for mouse ova. The calculated extent to which a cell becomes supercooled is the number of degrees that any given curve is displaced to the right of the equilibrium curve at a given subzero temperature. In the example shown, mouse ova cooled at 4~ will be supercooled by 15~ as the temperature passes through -20~ Three biological parameters have special influence on the position and shape of these curves, namely water permeability coefficient (Lp), its 1.0W I--

~ 0.8d _.1 W

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.

,, 0.60 W

j

[ 2 * C / m i n ; E = 17' kcal/mole]

.

0.4.

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~ 0.2. J W

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!

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FIGURE 12 Computed kinetics of water loss from mouse ova cooled at 1 to 8~ in 1 M DMSO. The curve labeled EQ shows the water content that ova have to maintain to remain in equilibrium with extracellular ice. The other solid curves, labeled 1 to 8~ were computed assuming the activation energy E of Lp to be 14 kcal/mol. The dashed curve shows the effect of changing E to 17 kcal/mol. Reproduced with permission from Mazur (1984).

284

Dayong Gao, Peter Mazur, and John K. Critser

activation energy (Ea), and the size of the cell, or more properly its surface to volume ratio (A/V). An increase in Lp produces the same effect as a comparable decrease in cooling rate. The effect of cell size is the opposite. An increase in size reduces the cooling rate required to produce a given probability of intracellular freezing. Changes in these two parameters shift the positions of the curves. Changes in the activation energy, Ea, have a major effect on the shape of the curves. For instance, the dashed line in Figure 12 shows the effect of changing Ea from 14 to 17 kcal/mol. All the parameters needed to compute the curves can be estimated from permeability measurements that do not involve freezing (measurement made at above 0~ temperature). However, once computed, the curves can be used to estimate the probability of intracellular freezing as a function of cooling rate. Cells that have dehydrated close to equilibrium, prior to reaching their ice-nucleation temperature, will have a zero probability of undergoing intracellular freezing. Cells that are still extensively supercooled when cooled to their nucleation temperature, and therefore still hydrated, will have a high probability of undergoing intracellular freezing. A cell that has survived cooling to low subzero temperatures still faces challenges during warming and thawing which can exert effects on survival comparable to those of cooling (Mazur, 1984). These effects depend on whether the prior rate of cooling has induced intracellular freezing or cell dehydration. In the former case, rapid thawing can rescue many cells, possibly because it can prevent the harmful growth of small intracellular ice crystals by recrystallization. Even when cells are cooled slowly enough to preclude intracellular freezing, the response to warming rate is often highly dependent on the freezing conditions and cell type and is difficult to produce a priori.

2. Preventing Injury during Slow Freezing While the avoidance of numerous and large intracellular ice crystals is necessary for cell survival, it is not sufficient. Most cells also require the presence of cryoprotective solutes or agents. The role of glycerol as an effective CPA for sperm and human erythrocytes was discovered nearly 50 years ago by the group in Mill Hill, England (Polge et aL, 1949). It remains one of the more effective and commonly used CPAs to date, although others have since emerged and are, in some cases, more effective, namely dimethyl sulfoxide, ethylene glycol, methanol, propylene glycol, and dimethylacetamide. The most important characteristics of all these solutes is that they are, for the most part, readily able to permeate the cells and they are relatively nontoxic in concentrations approaching 1 M or more. The primary basis for their protective action is that they lower the concentration of electrolytes during freezing and therefore decrease the extent of osmotic shrinkage at a given temperature. The extent of protection depends primarily on the molar ratio of the CPA to endogenous solutes

Tissue Banking in Reproductive Biology

285

inside and outside the cells and the general protective mechanism of action is thus colligative. The effectiveness of a given CPA for a given cell type usually depends on the permeability of that cell to that solute and its toxicity. While this class of CPAs must permeate to protect, their permeation before freezing and their removal after thawing generates osmotic volume changes, which themselves can be damaging (Gao et al., 1995; Mazur and Schneider, 1986; Schneider and Mazur, 1988), a topic that will be discussed in more detail in Section IV. The second class of CPAs is composed of nonpermeating solutes. This includes sugars and higher molecular weight compounds such as polyvinylpyrrolidone (PVP), hydroxyethyl starch (HES), polyethylene glycols (PEG), and dextrans. In most cases, these solutes will not protect in the absence of a permeating CPA, but will often substantially augment the effectiveness of a permeating CPA or permit the use of a lower concentration of permeating CPA. All of this discussion refers to the role of CPAs during slow equilibrium freezing at low enough cooling rates to prevent IIF. A diametrically different approach to cryopreservation is to combine very high concentrations of certain solutes with high cooling rates to induce the cell cytoplasm to form a glass (i.e., vitrify) rather than to crystallize. Several of the CPAs that are effective in ameliorating slow freeze injury also act to promote glass formation, but the required concentrations are much higher, e.g., 68 M. (Fahy, 1984). Vitrification procedures have not been applied to sperm and consequently will not be discussed further here.

B. S p e r m a t o z o a as a Model Cell Type for F u n d a m e n t a l Cryobiology Research In previous studies to date, cells have generally been considered simple compartments of cytoplasmic solution enclosed by a semipermeable plasma membrane. The emphasis has been on how cell survival is related to the physical responses of the whole cell to the physical-chemical events involved in the cryopreservation process. Survival has generally been defined in terms of intactness of the plasma membrane (e.g., lack of hemolysis in the red cell) or the ability of the cells to undergo subsequent growth and development (e.g., hematopoietic stem cells or embryos). Intactness of the plasma membrane is obviously necessary for functional cell survival. In some cases, it is sufficient that freezing conditions not produce demonstrable plasma membrane damage. In other cases (e.g., granulocytes; Armitage and Mazur, 1984a), intactness of the plasma membrane is not sufficient. Findings like the latter emphasized that cells are not simply bags of cytoplasm bound by a plasma membrane. Rather, within the bounds of the plasma membrane are other membrane-bound structures and organelles essential to cell function. Little is known about how these intracellular

286

Dayong Gao, Peter Mazur, and John K. Critser

structures and organelles respond to freezing, partly because it is difficult to assay their state and function in situ in an unambiguous fasion (McGann et aL, 1988). The spermatozoan is an especially good model for investigating the cryobiological role of both intracellular structures and the plasma membrane because sperm possess two clearly defined and measurable characteristics, motility and the acrosome reaction phenomenon, both of which depend on both functional integrity of organelles and the plasma membrane. An especially attractive feature of the acrosome reaction endpoint in cryobiology is that it involves membrane fusional events (Yudin et al., 1988). Investigation of the effects of freezing on such a reaction may provide an experimental model for the growing view that membrane fusion plays a major role in cryobiological injury (Mazur and Cole, 1985, 1989). To be fully functional, sperm must reach the site of fertilization and the oocyte, they must capacitate, undergo the acrosome reaction, penetrate the zona pellucida, and fuse with the plasma membrane of the oocyte. Freezing could interfere with or ablate the capacity of the sperm to undergo one or more of these steps (Critser et aL, 1987a,b; Wheeler and Seidel, 1986). While a motile spermatozoan is not necessarily a fully functional cell, a nonmotile sperm or one exhibiting a damaged plasma membrane is almost certainly nonfunctional (in the context of unassisted fertilization). Consequently, multiple levels of biological function must be considered in assessing the role of the biophysical parameters that have been shown to be critical to cell survival.

C. C r y o b i o l o g y o f M a m m a l i a n

Spermatozoa

1. Effect o f Cryoprotective Agents (CPA$)

Survival of cells subjected to cryopreservation depends not only on the presence of a permeating CPA but also on the concentration of the CPA. For example, the percentage of mouse bone marrow stem cells that survive freezing after cooling at the optimum rate increases dramatically as the concentration of glycerol increases from 0.4 to 1.25 M (Leibo et aL, 1970). The results are similar for other cells (Mazur, 1984). One of the problems with the cryopreservation of sperm is that they generally will not tolerate glycerol concentrations of 1 M and above. In fact, the sperm of many species (e.g., bull, boar, and human) are damaged by exposure to glycerol (Watson, 1979). Sherman (1964a) reported that glycerol exposure caused a reduction in human sperm motility. Critser et al. (1988a) have demonstrated that exposure of human sperm to 7.5% glycerol for as little as 15 min causes a dramatic reduction in sperm motility, accounting for approximately 50% of the motility loss observed after 24 h post-thaw (Figure 13). Historically, bull spermatozoa were exposed to medium containing

Tissue Banking in Reproductive Biology

287

o hr

80

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60

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60

90

120

TIME (rain) of Exposure F I G U R E 13 Percentage motility (mean ___SEM) for 12 donors (n = 12) for seminal plasma control ( e - - e ) and HSPM treated (G---O) samples at 0, 15, 30, 60, and 120 min posttreatment at either 0 or 24 h. Motility was lower (P < 0.05) in the HSPM-treated group at both 0 and 24 h. There was a treatment by time interaction (P < 0.05). Means with different letters are significantly different (P < 0.05) within hour. Reproduced with permission from Critser et al. (1988b).

glycerol for an extended period of time, which was referred to as "equilibration time." However, Berndtson and Foote (1972a,b) reported that glycerol was able to permeate bull sperm at either 25 or 5~ within 3 to 4 min, and found further that maximum post-thaw motility occurred when the sperm were exposed to glycerol for the shortest time measured (10 sec). These data suggest that bull spermatozoa are either highly permeable to glycerol or, unlike other cells, permeation is not required for protection. What then is the role of "equilibration time"? Is it, as some have proposed, a matter of allowing for protective membrane alterations (Watson, 1979)? It is important to note that human sperm differ from the sperm of most domestic species and do not show enhanced cryosurvival with increasing delay between addition of CPA and freezing (Sherman, 1964). Karow et al. (1992) found that the survival of human sperm in vitro at 3~ was significantly enhanced by 20 mM potassium (K +) in Tyrode's solution relative to Ty-

288

Dayong Gao, Peter Mazur, and John K. Critser

rode's solution with less K +. A metabolizable sugar such as glucose was essential to maintain sperm viability in K+-free media. The addition of raffinose to media containing glucose improved motility of sperm stored at 3~ for 6 hr. The detrimental effect of glycerol to sperm can be alleviated to a certain extent by reducing the temperature at which the cryoprotectant is added in both bull and human sperm cells (Critser et al., 1988a; Sherman, 1963). To what extent the sensitivity of sperm to glycerol reflects the osmotic consequence of its addition and removal, or to what extent it reflects chemical toxicity, is a current subject of investigation (see Section III.C.5).

2. Effect o f Cooling Rate: Cooling to the Freezing Point Most procedures, across species, utilize an initial slow cooling rate between body temperature or the temperature at which the sample is collected (often ambient temperature) and +5~ (Foote, 1975; Graham, 1978; Sherman, 1973; Watson, 1979). In general, this is due to the fact that some species of mammalian spermatozoa are sensitive to temperature changes in this range, with abrupt cooling (a high cooling rate) causing a high frequency of the cells to become irreversibly damaged. This phenomenon has been termed "cold shock" (Watson, 1981). The sensitivity of sperm to cold shock varies among species; bull, ram, boar, and stallion sperm are highly sensitive, dog and cat sperm are moderately sensitive, and rabbit and human sperm are relatively resistant (Critser et al., 1988a; Watson, 1981). The resistance of human sperm to cold shock is of particular importance in the context of developing new approaches to human sperm freezing. It is also an important example of the differences in fundamental cryobiology of cell types among species, highlighting the fact that simple modifications of procedures developed for freezing of domestic animal sperm are unlikely to yield optimal results in other species. 3. E f f e c t o f Cooling Rate: Cooling below the Freezing Point As in other cells, plots of the survival of sperm versus the cooling rate during the freezing are in the form of an inverted "U" (Figure 14) (Fiser and Fairfull, 1984; Henry et al., 1993; Kincade et al., 1989; Watson and Duncan, 1988). However, the numerical value of the optimum rate for the cryopreservation of sperm is less clear than in many other cells. In general, the survival of sperm is considerably less sensitive to cooling rate than that of most other cells; optimal cooling rates for sperm have been reported between 1 and 170~ This broad range may be a result of multifactor interactions existing among cooling rate, warming rate, and CPA concentrations. These interactions may also account for the fact that even at the socalled optimum cooling rate, survival is generally lower than in many other cell types.

289

Tissue Banking in Reproductive Biology i

i

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(A) Mean plasma-membrane-intact human sperm [(% carboxyfluorescein diacetate positive and propidium iodide negative sperm after thawing/% carboxyfluorescein diacetate positive and propidium iodide negative sperm before freezing) • 100]. The cooling rates were 0.1, 1.0, 10, 175, and 800~ (please note logarithmic scale). Specimens warmed at either 1.0~ (O) or 400~ (V). Means with different superscripts are different (P < 0.05) within warming rate (n = 11). (B) Mean post-thaw motility [(% motile sperm after thawing/% motile sperm before freezing) • 100]. The cooling rates were 0.1, 1.0, 10, 175, and 800~ (please note logarithmic scale). Specimens were warmed at either 1.0~ (0) or 400~ (V). Means with different superscripts are different (P < 0.05) within warming rate (n = 11). (C) Mean mitochondrial functional sperm [(% rhodamine 123 positive and propidium iodide negative sperm after thawing/% rhodamine 123 positive and propidium iodide negative sperm before freezing) x 100]. The cooling rates were 0.1, 1.0, 10, 175, and 800~ (please note logarithmic scale). Specimens warmed at either 1.0~ (0) or 400~ (V). Means with different superscripts are different (P < 0.05) within warming rate (n = 11). Reproduced with permission from Henry et at (1993).

F I G U R E 14

Initial reports of bull sperm freezing (Polge and Rowson, 1952) used relatively slow cooling rates (l~ to -10~ 3 to 4~ from - 1 0 to - 3 0 or -40~ This approach was generally used until the mid-1960s with the advent of pelleting semen on solid carbon dioxide and the use of plastic

290

Dayong Gao, Peter Mazur, and John K. Critser

straws frozen over the surface of liquid nitrogen (Cassou, 1964). These techniques produced cooling rates of approximately 100 and 170~ respectively. For human sperm, optimal cooling rates have been reported to range between l~ (Freund and Wiederman, 1966) to 16 to 25~ min (Sherman, 1963; Trelford and Mueller, 1969). The generally low sensitivity of sperm survival to cooling rate and the wide range of reported optima is puzzling. It may be a reflection of the use of suboptimal concentrations of glycerol and a masking of damage by the use of rapid thawing. The puzzle is not likely to be solved until the effect of cooling rate on osmotic shrinkage and intracellular freezing is known. The question is often asked as to the length of time that cells can be kept in the frozen state without damage. The question is probably moot if the storage temperature is below -120~ and is certainly moot at -196~ (liquid nitrogen). Below -120~ chemical reactions cannot occur in human-relevant times. At -196~ no thermally driven reactions can occur in less than geologically relevant times. The reactions that can occur are the slow accumulation of direct damage from ionizing radiation, but this becomes significant only after centuries of storage (Mazur, 1984).

4. Warming and Thawing The effects of warming rate on cell survival depend on the prior cooling rate and on the cell type. For human sperm there is relatively little information regarding the optimal warming rates. Previous reports used a wide variety of imprecise methods yielding a wide variety of rates. These include thawing in ambient air, in water baths with temperatures ranging between 5 and 37~ in coat pockets, and thawing the samples in the investigator's hands (Mahadevan, 1980). A more comprehensive study was conducted by Mahadevan (1980) in which warming rates between 9.2~ and 2140~ min utilizing coat pocket, hands, air (5, 20, or 37~ or water baths (5, 20, 37, 55, or 75~ were examined in combination with a standard freezing rate of 10.0~ from +5 to -80~ The results of that study indicated that slower thawing in 20 or 35~ air resulted in more optimal cryosurvival of human sperm in terms of both motility and supravital staining; although there were significant differences in post-thaw motilities only between the fastest 2 rates (1837 and 2140~ and the other 12 rates. Additionally, Mahadevan (1980) examined the interaction between freezing and thawing rates. Slow thawing was reported optimal for samples frozen slowly; however, there was little effect of thawing rate when a fast freezing rate was employed.

5. Fundamental Cryobiological Characteristics o f Mammalian Spermatozoa Although there is great diversity in the cryobiological response of sperm among different mammalian species, and even within a species, cryosurvival requires that cell freezing and thawing be carried out within certain bio-

Tissue Banking in Reproductive Biology

291

physical and biological limits defined by cryobiology principles. Namely: (1) Cells must be frozen in such a way that little or none of their water freezes intracellularly. In addition, they must be warmed in such a way that any unfrozen intracellular water remains unfrozen during warming, or that small ice crystals that form during cooling remain small during warming (Mazur, 1984). (2) Even when these conditions are met, most cells will not survive unless substantial concentrations of CPAs are present and the solutes permeate. These solutes must be introduced before freezing and removed after thawing in ways that do not exceed osmotically tolerable limits. Their concentrations also must not be toxic (Critser et al., 1988a; Fahy, 1986; Meryman, 1974). Although these general limits are necessary, they may not be sufficient, for a number of possible reasons. One is that cells may be injured by factors such as cold shock or chilling injury that have nothing to do with ice formation or CPA damage. Another reason is that cell viability limits are defined primarily in terms of an intact plasma membrane that retains normal, semipermeable properties. It is possible that conditions that allow the plasma membrane to "survive" may not allow the "survival" of critical organelles such as the acrosome or the mitochondrial-axonemal system responsible for motility. With the progress in understanding mechanisms of injury to cells during cryopreservation, various physical modeling and mathematical formulations have been developed in cryobiology research to simulate a cell's response to environmental change during cryopreservation process and to predict optimal cryopreservation conditions (Mazur, 1963; Mazur, 1990; Muldrew and McGann, 1994; Pitt and Steponkus, 1989; Toner et al., 1990). For example, Mazur's Eqs. [1]-[4] have been utilized to predict the degree of intracellular supercooling, cell water content and efflux, as well as probability of intracellular freezing as a function of cooling rate. The ability of these predictive models has been confirmed using data collected on cell types ranging from plant protoplasts to mammalian embryos (Gao et al., 1995; Mazur et al., 1984; Mazur and Schneider, 1986; Muldrew and McGann, 1994; Pitt and Steponkus, 1989; Toner et al., 1990). Application of these models to a given cell type requires the determination of a series of cell cryobiological characteristics, including (1) osmotic behavior of the cells, cell volume and surface area, as well as osmotically inactive intracellular water fraction; (2) temperature dependence of cell membrane permeability coefficients to water and the CPA as well as reflection coefficient of cell membrane to the CPA; (3) temperature dependence of the limits of osmotic volume excursions tolerated by cells; and (4) intracellular ice formation temperatures. These characteristics of mammalian sperm have not been investigated until recently. Determination of these characteristics for the mammalian spermatozoa is an ongoing project in fundamental cryobiology research, which will be reviewed in the following sections with an emphasis on how to use them to optimize cryopreservation conditions.

T-LE Species

4

Morphologic Dimensions of Some Mammalian Spermatozoa

Head, L/WD ( p m ) Midpiece, Y D I ( p m ) Principal piece, L/DI ( p m j Total length ( p m ) Surface area (pdj Cell volume

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8.3L 0.8N

46.3

10

44

59.4

136‘

31

8,9

212c

34‘

1.8

-

60-62

Note. L , length; W, width; D, depth; DI, diameter. References: 1, Hansel and McEntee (1970); 2, Morgenthal (1967); 3 , Nishikawa ez al. (1951); 4,Du el al. (1991); 5, Niwa and Mizuho, (1964); 6, Noiles er al. (1995); 7. Du er af. (1994~);8, Cummins and Woodall (1985); 9, Cummins, J. M. Personal observations, cited in Cummins and Woodall (1985). Kleinhans et al. (1992). Gilmore et al. (1995). Calculated from values in the references cited using the following equations:

Volume Area

= =

-

A . r n . [rnr . (rnd12)’ + p l . (pd12)* + rl . (rd12)*] (Du et al., 1994b); 0.73 X B X L = (0.73) . (6.45) . (3.9) = 18.36 (van Duijn, 1957).

Dott (1975). ‘Noiles, E. E., Mazur, P., Benker, F. W., Du, J., Kleinhas, F. W., Amann, R. P., Critser, J. K., unpublished data. f Gilmore et al. (1996). g Du ef al. (1994b), Theriogenology. Hammerstedt et al. (1978). ‘ O’Donnell (1969). Minks et al. (1996), submitted manuscript. Estimated from values in Cummins and Woodall (1994).

t3

W

w

294

Dayong Gao, Peter Mazur, and John K. Critser

a. Osmotic Behavior o f Spermatozoa

i. Determination of Sperm Volume and Surface Area Sperm volume and surface area are difficult to measure because of the irregular shape of the sperm. The techniques used to measure sperm volume include optical dimensions (van Duijn, 1957), electronic counter (Brotherton and Barnard, 1974; Gilmore et al., 1995), volume exclusion (Ford and Harrison, 1983), and electron paramagnetic resonance (Hammerstedt et al., 1978). Recent electronic counter measurements (Gilmore et al., 1995) indicated that human spermatozoa at isotonic condition (286 mOsm) have an average volume ranging from 28.5 to 33.9/zm 3. Electron paramagnetic resonance measurements indicate a total intracellular water volume of 20 _ 2.9/zm 3 (Kleinhans et al., 1992). The surface area of human sperm has been estimated to be 120/zm 2 (van Duijn, 1957). Average dimensions of several other mammalian spermatozoa have been also determined, including mouse, bull, stallion, boar, rat, cat, and dog (see Table 4). ii Osmotic Response to A n i s o s m o t i c Conditions An underlying assumption in the equations that are used to describe the kinetics of osmotic cell volume responses is that mammalian spermatozoa behave as linear osmometers. This means that cell volume at osmotic equilibrium is a linear function of the reciprocal of the osmolality of nonpermeating solutes in the external medium. The equation describing this response is [6]

V/Viso --- Miso )< (1 - Vb/Viso)/M + Vb/Viso,

where V represents cell volume; M, extracellular osmolality; Miso, isosmolality; Viso, cell volume at isosmotic condition; and Vb, osmotically inactive cell volume. The osmotic response of sperm from a variety of species has recently been shown to be ideal over a broad range of solute concentrations (Curry et al., 1994, 1995; D u e t aL, 1993, 1994a, 1995; Gilmore et aL, 1995). Examples of electronic counter measurements on human sperm osmotic behavior are shown in Figure 15 (Boyle van't Hoff plots) (Gilmore et al., 1995). The intercept on the volume axis is the cell volume at infinite extracellular osmolality, and it indicates that approximately 50% of the total isotonic human sperm volume is osmotically inactive (i.e., solids and osmotically inactive water). b. Sperm Water Permeability Coefficient Energy (Ea)

(Lp) and

its Activation

i. Determination of Lp using "Time-to-Lysis" Method Determination o f Critical Tonicity. To determine the permeability of plasma membranes to water, one approach is to expose cells to a hypotonic

Tissue Banking in Reproductive Biology

295

1.2

0.8

~ ....~"'~

T= 22 ~ Vb 50% I

~ 0

>

=

0.4 1.6-

I

t

1.2 U

0.8 ,.d <

0.4 / 0 Z

T= ll~ Vb = 41%

J

I

I

I

I

1.6 1.2

0.8 ~ 0.4 0.0

T= 0~ ~=52%

l

I

I

1

0.5

1.0

1.5

2.0

285/mOsm

F I G U R E 15 B V H plots of human sperm at 22, 11, and 0~ T h e sperm samples were exposed to four different osmolalities: (1) 900 m O s m / k g , (2) 600 m O s m / k g , (3) 285 m O s m / kg, and (4) 145 m O s m / k g . The samples were allowed to equilibrate for 3 min before analysis (mean _ S E M ) (n = 5 at each temperature). Reproduced with permission from Gilmore et al. (1995).

solution containing only nonpermeating solutes (e.g., salts) and determine the kinetics of their subsequent swelling and time to lysis. The first step is to determine the lytic volume of the sperm. Since sperm behave as ideal osmometers, this is determined indirectly by determining the hypotonic concentration that results in 50% cell lysis, i.e., the critical tonicity (Noiles et al., 1993; Watson et al., 1992a,b). When mammalian sperm are exposed to hypotonic media of a range of concentrations, so-called "fragility curves" are generated (i.e., curves of percentage of membrane-intact cells vs concentrations). From these curves the critical tonicity can be estimated. Figure 16 shows fragility curves for sperm from several mammalian species (Watson et al., 1992a). The fragility curves fall into two classes. One, exemplified by fowl and human sperm, shows a high percentage of intact sperm until the external osmolality is reduced to below 100 mOsm; then the percentage drops rapidly, most likely due to osmotic lysis. The other class, exemplified by bull, boar, and ram sperm, shows an initial drop at higher tonicities, a

296

Dayong Gao, Peter Mazur, a n d John K. Critser

,0o t "

........

.

......

........

,,

,o

_q

-

.=._, a0 /

i ]

=

i

1

~~ 1 o

0

" ]

~ , "

"

-

----II---

:

Human

n=lO

=

- .~...~

R~.-8

--o-100

.

.

. 200

.

.

300

Osmolality (mOsrn) F I G U R E 1 6 The survival of spermatozoa under hyposmotic solutions.Reproduced with permission from Watson et al. (1992b).

plateau, and then a second drop to complete lysis at low tonicities. The first drop is of unknown cause, but is believed not to have an osmotic genesis (Watson et al., 1992a). The second drop occurs at about the same hypotonicity as in fowl and human sperm and is, therefore, believed to represent osmotic damage. It is interesting to note that the differences in the damages at high hypotonicities among different species appears to correlate with the species' sensitivity to cooling damage above 0~ (i.e., cold shock sensitivity; Watson et al., 1992a). Determination o f Lp and Its Ea. Using the value of the critical tonicity for sperm, one can conduct time-to-lysis experiments, and from these times, compute water permeability coefficient (Lp) (Noiles et al., 1993; Watson et al., 1992b). Values of Lp for sperm of several mammalian species have been determined using this approach and are listed in Table 5. By measuring the Lp of sperm at various temperatures, one may also obtain estimates of the activation energy (Ea) of Lp, assuming an Arrhenius relationship between Lp and temperature (i.e., Eq. [4]) (Gao et al., 1992; Gilmore et al., 1995; Noiles et al., 1993). ii. Determination of Lp using Electronic Counter Another commonly used method for determination of Lp involves two steps: Step 1, measure kinetics of cell volume change after cells are exposed to an anisosmotic condition; Step 2, fit computed kinetics of cell volume change to the experimentally measured kinetics using Lp as an adjustable parameter. The Lp value that generates the best fit is considered to be the water permeability coefficient of the cell membrane (Gilmore et al., 1995). A common method of following cell volume changes is that of optical microscopy, but this technique is generally applied to spherical cell types and is not feasible in cells with irregular shape like sperm or human erythrocytes. Another prob-

Tissue Banking in Reproductive Biology

297

Estimates of the Water Permeability (Lp) and Its Temperature Dependence (Ea) for the Spermatozoa of Several Mammalian Species

TABLE 5

Species

Lp (tzm/min/atm)

Temperature (~

Human

2.5 2.4 1.5 1.8 1.84 1.5 1.0 1.1 1.0 10.8 26.0 8.47 0.63

30 22 8 0 22 -1 -3 -5 -7 22 22 25 25

Human Human

Bull Stallion Ram Rabbit

Ea (kcal/mol)

Reference

Noiles et al. (1993) 3.92 3.48

7.48 3.00 N/A 1.06 17.8

Gilmore et al. (1995) Noiles et al. (1993)

Watson et al. (1992b) Noiles et al. (1992) Curry et al. (1995) Curry et al. (1994)

lem in determination of the Lp of sperm is that water permeability of spermatozoa is so high that osmotic water equilibration occurs in seconds. As a result, it is difficult to measure the kinetics of the water-flux-induced volume change. In 1994, Gao et al. investigated the effects of initial intraand extracellular osmolalities on the kinetics of cell volume change (Gao et al., 1994a). They found that the osmotic water equilibration time and maximum cell volume change are greatly increased with an increase in the ratio of initial intracellular osmolality to initial extracellular osmolality. This finding is helpful for optimal experimental design to ensure that the kinetics of sperm volume change are measurable within both time and space. Based on this study, Gilmore et al. (1995) exposed human sperm to relatively hyposmotic conditions and kinetics of water-flux-induced human sperm volume change were first time measured using an electronic counter (Coulter counter), a cell-shape independent method. The Lp and associated Ea of human sperm were determined (Gilmore et al., 1995). The measured values of Lp and Ea are consistent with those determined using the timeto-lysis method (Table 5). Like human erythrocytes, the activation energy of Lp is substantially lower than that of other cells. The high water permeability and low activation in human erythrocytes has recently been shown to be a result of the presence of specific water channels formed by a transmembrane protein, CHIP28 (Preston et al., 1992). However, efforts to identify an analogous membrane water channel in sperm have thus far been unsuccessful (Liu et aL, 1995). c. P e r m e a b i l i t y Coefficient o f Sperm to CPA (PcpA) a n d Its Activat i o n E n e r g y (Ea) The time-to-lysis method can also be used to measure

298

Dayong Gao, Peter Mazur, and John K. Critser

the permeability of sperm to CPAs such as glycerol. In such a case, one determines the time to lysis of sperm suspended in a solution that is hypertonic with respect to glycerol, but strongly hypotonic with respect to nonpermeating salts. The method used was derived from that developed for bovine and human red cells by Mazur et al. (1974) and Mazur and Miller (1976). Human sperm permeability coefficient (PcPA) to glycerol has been determined over a temperature range of 30 to 0~ using the time-to-lysis method (Gao et al., 1992). At 22~ the PCPA values in 0.5, 1.0, 1.5, and 2.0 M glycerol are 1.90, 2.21, 1.98, and 1.81 x 10 -3 cm/min, respectively. The activation energy calculated from an Arrhenius plot of the PCPA values (using 1 M glycerol) versus the reciprocal of the temperature (K), is 11.76 kcal/mol. These values of PCPA and their Ea have been confirmed using electron spin resonance (ESR) techniques (Du et al., 1992). The ESR value for PCPA (1 M glycerol) at 20~ is 1.3 x 10 -3 cm/min with an Ea value of 11.4 kcal/mol between 30 and 0~ ( D u e t al., 1992). Based on the linear irreversible thermodynamic theory (Kedem and Katchalsky, 1958), water-CPA coupled transport across the cell membrane can be mathematically formulated as 1 d Vdt( t ) = I Lp {(Csalt i Jv = Ac e i Csalt) + o" (C~p A -

C~pA)}RT

[7]

1 ONcPA _ CCPA (1 - tr)Jv + PcPg (C~pA -- C~pA)RT, dt

[8]

and JCPA -- Ac

where Jv represents total volume flux (~3/(/~m2 min)); V, cell volume (/zm3); t, time (min); N, mole number of solute; Ar cell surface area (/zm2); Lp, water permeability coefficient of cell membrane (/zm/min/atm); C, concentration of solute (osmolality); JCPA, CPA flux across the cell membrane; e, extracellular; i, intracellular; R, universal gas constant (0.08207 liter x atm/ (mol x K)); T, temperature (K), PCPA, CPA permeability coefficient of cell membrane (/zm/min); and Ccpg, the ave_rage of extracellular and intracellular CPA concentrations (osmolality); Ccpg, (C~pA- C~pg)/[ln (C~pA/ C~pg)]; and tr, reflection coefficient of cell membrane to the CPA. The tr is a measure of selectivity of cell membrane allowing a given solute to move across the membrane (Kedem and Katchalsky, 1958). The value for tr ranges for 0 to 1. In extreme cases, tr = 0 if the cell membrane is nonselective for solvent (e.g., water) and solute to move across the membrane. Intracellular concentrations of impermeable solute (salt) and permeable solute (CPA) can be calculated as follows (Mazur and Schneider, 1986), C~alt(t)--Cealt(O) ( V ( 0 ) - Vb - VCPA N~pA(0)) V(t) - Vb -- VCPA N~pA(t)

and,

[9]

Tissue Banking in Reproductive Biology

299

N~pA(t) C~pA(t) = ( V ( t ) -

[10]

Vb ---- ~--'~CPAN ~ p A ( t ) ) ' m

where Vb represents osmotically inactive cell volume (/zm3); VCPA, partial molar volume of CPA (/zm3/mol); N, mole number; and 0, initial condition (t = 0). Initial conditions for V(0), C~alt(0), C~pA(0), N~pA(0) are known based on each experimental condition. Using the above equations and the Coulter counter-based technique, Gilmore et al. (1995) have recently determined the PCPA in human sperm for several CPAs and the value of Lp in their presence. Several conclusions can be drawn from the results summarized in Table 6: (1) The values of the PCPA differ among the four CPAs, with the values for ethylene glycol being the highest and dimethysulfoxide (DMSO) the lowest (the low value for DMSO is somewhat surprising). (2) Except for DMSO, all the values are among the highest reported for mammalian cells. (3) In spite of the high values, the ratio of water permeability expressed as Pf (= Lp) to solute permeability, PCPA, is 500-1000 (120 for ethylene glycol). The high ratios for the first three are also reflected in the high values of the reflection coefficient tr. (4) The values of Lp are significantly reduced by the presence of the CPAs. d. Intracellular Ice Formation Temperatures Intracellular ice formation temperature of human sperm has been investigated by determining their survival (motility and membrane integrity) after cooling at a slow cooling rate to various subzero temperatures followed by rapid cooling to -196~ and then warming slowly. No unique nucleation temperature was identified. Instead, intracellular freezing appears to occur over a temperature range of - 3 0 to -40~ (Critser et al., 1991), consistent with a recent TABLE 6 Comparison of Human Sperm Membrane Permeabilities at 22~ to CPAs and to Water in the Presence of CPAs, as Well as the Associated tr Values

None Glycerol Propylene Glycol DMSO Ethylene Glycol

Concentration (molar)

Lp P~ (lxm rain -1 atm -1) (10 -2 cm/min)

1M 1M

1.84 _ 0.06 0.77 +_ 0.08 1.23 __+0.09

249 _ 8 104 _ 11 166 _ 12

0.21 ___0.010 0.23 ___0.01

0.93 _ 0 0.95 _ 0

1M 2M

0.84 _ 0.07 0.74 ___0.06

112 _ 9 100 _ 8

0.080 _ 0.002 0.79 ___0.07

0.98 _ 0 0.77 +__0

PCPA (10 -2

m

a Lp expressed as a filtration coefficient or velocity, Pf = RTLp/Vw. b Mean +_ SEM.

cm/min)

tr

300

Dayong Gao, Peter Mazur, and John K. Critser

report by Pitt et al. (1992) investigating this phenomenon in rye protoplasts and bovine oocytes. Using cryomicroscopy it was found that the seeding temperature (i.e., a subzero temperature at which ice crystals are artificially induced (seeded) in a cell suspension) is very important for the cell survival (Gao et al., 1994b). For example, in a 5-tzl human sperm suspension with 1 M glycerol, seeding at -16~ caused approximately 50% cell motility loss, while seeding at -22~ caused almost 100% cell motility loss. A seeding temperature between - 3 and -8~ was recommended. It is thought that the lower seeding temperatures are damaging because they almost instantaneously confront a highly supercooled cell with extracellular ice and thus enhance the probability of intracellular ice formation. Seeding is nearly always omitted in standard cryopreservation procedures for sperm. However, the results indicate that it should be included. It has been included in many other cell types like mouse embryos (Leibo and Mazur, 1978). The probability of intracellular freezing from this source can be reduced to near zero if seeding is carried out above -8~ e. S p e r m Tolerance Limits for V o l u m e Excursion Viability of mammalian sperm is very sensitive to osmotic stress and the associated cell volume excursion. As described in Section III.C.5.b, when extracellular osmolality is decreased below the critical hyposmolality, over 50% of sperm will lose membrane integrity. Moreover, previous studies (Gao et al., 1993; Curry et al., 1994) indicated that sperm are also highly sensitive to posthyperosmotic treatments and that the osmotic injury of the human sperm is dependent upon their degree of volume excursion rather than the nature of the solutes (sucrose, NaC1, or glycerol). Gao et al. (1995) determined the tolerance limits of human spermatozoa to swelling in hyposmotic solutions (TALP diluted with water) and to shrinkage in hyperosmotic solutions (TALP with sucrose) using motility and membrane integrity as endpoints, respectively. These anisotonic solutions contained only membrane-nonpermeable solutes with a range of osmolality from 40 to 1200 mOsm. Spermatozoa were exposed to these solutions for 2 s to 30 rain and then returned to isotonic TALP (286 mOsm) medium. Sperm plasma membrane integrity was measured by dual fluorescent staining and flow cytometry, and sperm motility was assessed by computer assisted semen analysis (CASA), before, during, and after the anisosmotic exposure. The results indicate that (a) motility was much more sensitive to anisotonic conditions than membrane integrity, and (b) motility was substantially more sensitive to hypotonic than to hypertonic conditions (Figures 17 and 18). Based on these experimental data, osmotic injury as a function of human sperm volume excursion (swelling or shrinking) was determined. As shown in Figure 19, the volume excursion upper limit is 1.1 times (or 110% of) isotonic volume, and the lower limit is 0.75 times (or 75% of) isotonic volume if 5% motility loss is chosen as a criterion.

Tissue Banking in Reproductive Biology

301

100-

~

n=8

80-

I" ~ ~ I: b

\

..

0

t

40-

\ \

'

.,

"~k.

\ W

13. CO

20O. "0 ....... 0 ............ 0

0 -

I

I

I

I

I

I

I

0

200

400

600

800

1000

1200

OSMOLALITY (mOsm) 1 7 A comparison of human sperm motility (% mean ___ SEM, n = 8) after a 5-min exposure to the various hypo- and hyperosmotic solutions of nonpermeating solutes before (O) and after (Vq)the return to near isotonic conditions (273-343 mOsmol). Reproduced with permission from Gao et al. (1995).

FIGURE

1009080-

.< "-"

70-

w

60

~ w

5()-

>" _J z

40-

0.

30-

10I

I

I

1

I"

1

I

0

200

400

600

800

1000

1200

1400

-OSMOLALITY (mOsm)

18 Membrane integrity (CFDA and propidium iodide stain) (% mean _ SEM, n = 8) of human spermatozoa which were abruptly (one-step) returned to near isotonic conditions (273-343 mOsmol) after they had been exposed to different anisosmotic conditions for 5 min. Reproduced with permission from Gao et al. (1995).

FIGURE

302

Dayong Gao, Peter Mazur, a n d John K. Critser

100 n=8

8O

.J I0

60

4O

20-

0 0.50

1

1

1

1

i

1

0.75

1.00

1.25

1.50

1.75

2.00

2.25

RELATIVE HUMAN SPERM VOLUME

FIGURE 1.9 Postanisosmoticsperm motilityrecoveryas a function of relative spermvolume (normalized to the isotonic sperm volume of 1) in different anisosmotic equilibrium states. Human spermatozoa were abruptly (one-step) returned to near isotonic conditions after exposure to anisosmotic conditions for 1 min. Reproduced with permission from Gao et al. (1995).

f. H o w to Use D e t e r m i n e d Cryobiological Characteristics to Optimize Cryopreservation Procedures i. O p t i m i z a t i o n o f C P A A d d i t i o n and Removal Procedures Like other cells, sperm require equilibration with nearly molar concentrations of CPAs to survive freezing. Removal of the CPAs from sperm after thawing is also required to minimize potential chemical toxicity of the CPA to cells. Addition and removal of CPAs have dramatic osmotic effects upon cells. Cells exposed to molar concentrations of permeating solutes undergo extensive initial dehydration followed by rehydration and swelling when the solutes are removed. Unless precautions are taken, this shrinkage and/or swelling can be extensive enough to cause cell damage and death (Gao et al., 1995; Mazur and Schneider, 1986; Schneider and Mazur, 1988). Experiments to distinguish between damage resulting from the degree of sperm dehydration during glycerol addition and damage associated with rapid swelling during glycerol removal have demonstrated that, at least in terms of plasma membrane integrity, rapid addition is not a cause of damage as long as the glycerol concentration remains below 3 0 s m , but rapid removal results in a significant loss of membrane integrity (Gao et al., 1993). This damage associated with glycerol removal can be significantly reduced by using a multistep dilution procedure (Gao et al., 1993). When glycerol addition and removal rates have been examined in the context of motility, similar, but more exaggerated results have been found: the rate of addition

Tissue Banking in Reproductive Biology

303

has minimal effect on motility, while the rate of removal has a marked effect (Gao et al., 1995). These data suggest that one ought to be able to use the PCPA and the osmotic volume equations to design protocols for the addition and removal of CPAs that hold volume excursions within the tolerated range and thereby minimize osmotic damage even when the CPAs are present in higher than usual concentrations. The CPA used to test this concept in human sperm was 1 M glycerol. The procedure was to use the measured glycerol permeability coefficient to compute (using Eqs. [7]-[10]) the volume excursions produced by adding and removing glycerol in a variable number of steps. The data in Figures 17 and 18 were then used to predict the effects of the maximum volume excursions on membrane motility and integrity, respectively. Finally, the predictions were compared with the actual membrane integrity or motilities of sperm subjected to given addition and removal protocols. Two forms of step-wise addition and removal were used in both the computer simulations and the experiments. In one, a glycerol solution or a PBS diluent were added in steps of fixed volume (FVS). In the other, volumes of glycerol or diluent were varied in such a way that each step produced a given increment or decrement in the molarity of the glycerol (FMS). Figure 20 shows the calculated sperm volume excursion during a one-step or four-step addition of glycerol to achieve a final 1.0 M glycerol concentration at 22~ A one-step addition of glycerol to spermatozoa was predicted to cause approximately 20% sperm motility loss because the minimum Volume which the cells would achieve during this glycerol addition is approximately 72% of the isotonic cell volume, i.e., below the lower volume limit of 75%. In contrast, the prediction was that a four-step FMS glycerol addition would prevent significant sperm motility loss. A four-step FVS addition was predicted to produce a lower minimum volume and, therefore, be more damaging than a four-step FMS procedure. As shown in Figure 21, the number of steps used to remove glycerol was predicted to have even more dramatic effects on motility. A singlestep dilution was calculated to produce a 160% increase in volume and motilities of less than 30%. In contrast, the maximum volume produced by an eight-step FMS dilution never exceeds the upper volume limit and therefore is predicted to have essentially no adverse effect on motility. Figure 22 compares the effects of four-, six-, and eight-step FMS dilutions. The four-step dilution may be marginally damaging (approximately 10% motility loss), while the six-step dilution, like the eight-step, should be essentially innocuous. A given number of FVS dilution steps are computed to produce higher maximum volumes than that same number of FMS dilutions and therefore be more damaging. The predicted consequences of eight-step dilutions by the two approaches are illustrated in Figure 21. The number of dilution steps is predicted to be more important than the time interval between

304

Dayong Gao, Peter Mazur, a n d John K. Critser

1.1 W ,--I

........

.....

9 9

1.0

O >

n," w 0.9 D..

......

-r" W

ONE-STEP ADDITION

.......

4-STEP ADDITION (FIXED MOLARIT'Y)

z <

~

ONE-STEP ADDITION

4-STEP ADDITION (FIXED VOLUME)

..J ~_. O

0.8

>

I-<~ 0.7

UJ

ii,i

..................................................

~

.......................................................................................... 40%............

w

......................................

0.6

n,"

95%

....................................................

20% ......................................................................

j

I

=

I

0

20

40

60

l

J

80 0

TIME

=

I

I

1

20

40

60

80

(sec)

FIGURE 20 (Left) Calculated relative sperm volume (normalized to the isotonic sperm volume of 1) as a function of time after 1 M glycerol was added to spermatozoa by either one-step or four fixed-molarity steps. (Right) Calculated relative sperm volume (normalized to the isotonic sperm volume of 1) as a function of time after 1.0 M glycerol was added to spermatozoa by either one step or four fixed-volume steps. The estimates of percentage motility recovery as a function of sperm relative volume were obtained from Figure 19 and are indicated in the diagrams. Reproduced with permission from Gao et al. (1995).

successive steps. This is a consequence of the high permeability of the sperm to glycerol. Because of the high permeability, the sperm are predicted to reach osmotic equilibrium within 20 s after the alteration in glycerol concentration produced by a given step. An alternative approach to removing CPAs without causing excessive cell swelling is to transfer CPA-loaded cells to a medium that contains a nonpermeating solute (usually sucrose) but no CPA. The presence of the sucrose prevents an excessive initial spike in cell volume swelling during initial dilution; i.e., it acts as an osmotic buffer. The cells are left in the sucrose solution for a time long enough to permit the efflux of most of the CPA. At that point they can then be transferred to isotonic saline without undergoing a volume increase above isotonic. This so-called "sucrosedilution" procedure was proposed by Leibo and Mazur (1978) for mammalian embryos and it is now used routinely for that cell type. The higher the concentration of sucrose, the smaller the initial volume increase that cells undergo. However, the higher the sucrose concentration, the more the cells shrink during the subsequent efflux of CPA. Too high concentration may eliminate damage from the former, but may result in damage from the latter. A low concentration of sucrose will protect against the latter, but may

Tissue Banking in Reproductive Biology 1.7

/ 1.6

305

ONE-STEPDILUTION

--

W

0_J > 1.5-

.....................................................................................................

30% #

rY UJ EL 1.4 03 Z < "1" iii

~

v

._1 I-

o

1.3-

.....................................................................................................

~; UJ EL 03

46%

rY'

.......EIGHT-STEPDILUTION( FIXEDVOLUME)

1.2-

/

W nf'

95%

1.1.0 ; . ]"["t ~i . i~. '....... . ' ./"I' ';. "'"EiGHT:STE;' . ' . ' . ' .ii l DiLUTiON( FIXEDMO~'Riii:il ~ ....... 0

20

40

60

80

100

TIME (sec)

120

140

160

FIGURE 21 Calculated relative sperm volume (normalized to the isotonic sperm volume of 1) as a function of time after 1.0 M glycerol was removed from spermatozoa by one-step, eight fixed-molarity steps, or eight fixed-volume steps. The estimates of percentage motility recovery as a function of sperm relative volume were obtained from Figure 19 and are indicated in the diagrams. Reproduced with permission from Gao et al. (1995).

result in damage from the former. In embryos, intermediate concentrations (typically 0.75 M sucrose) minimize damage from both causes. However, that may not be the case with human sperm. Our data have shown that the upper tolerated hypertonicity for maintenance of motility is 600 mOsm. If that concentration of sucrose is used, sperm loaded with 1 M glycerol are predicted to initially swell to 125% of isotonic volume upon transfer to the sucrose solution (Figure 23), and Figure 19 indicates that that degree of swelling will produce over a 40% loss in motility. Experiments confirmed the above theoretical predictions. The percentage motility of human spermatozoa after one-step or four-step FMS or FVS addition of glycerol is shown in Figure 24. A one-step addition resulted in approximately 19% sperm motility loss, while the four-step FMS or FVS addition reduced motility by only 6 or 9%, respectively. Figure 25 shows the effects of different glycerol removal procedures on motility loss. Onestep removal of 1 M glycerol produced a loss motility of more than 70%. In contrast, with an eight-step FMS dilution, the loss was only 8%. An eight-step FVS removal was intermediate (38% loss). The loss in motility after a two-step removal of glycerol using sucrose as an osmotic buffer was

306

D a y o n gGao, Peter Mazur, a n d John K. Critser 1.20

FOUR-STEPDILUTIONt

1.15 ~

1.10 .............................................................................................................................

1.05 1.00 ~11

m =. Z ,r

o

1.20

TEP DILUTION ....................................................................

1.15 1.10 1.05

1.00 [-, ,J r~ 1.20

o

1.1,.5 1.10 1.05 1.00

0

20

40

60

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Calculated relative sperm volume (normalized to the isotonic sperm volume of 1) as a function of time after 1.0 M glycerol was removed from spermatozoa by four, six, and eight fixed-molarity steps. Straight lines in this figure indicate the upper volume limit, 1.1, below which >95% of spermatozoa can maintain their motility. The four- or six-step dilution results in a cell volume excursion causing >5% motility loss. Reproduced with permission from Gao et al. (1995).

FIGURE 22

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.

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Calculated relative sperm volume (normalized to the isotonic sperm volume of 1) as a function of time after 1.0 M glycerol was removed from spermatozoa by two steps using a "hyperosmotic buffer" solution. Step l, 1.0 M glycerol was removed from spermatozoa by one-step exposure of spermatozoa to 600 mOsmol hyperosmotic (salt + sucrose) solution without glycerol. Step 2, spermatozoa in the 600 mOsmol solution were returned to isotonic condition (286 mOsmol) in one step. Reproduced with permission from Gao et aL (1995).

FIGURE 23

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Experimental results: motility recovery (% mean ___SEM, n = 15) of human spermatozoa after addition of 1.0 M glycerol. (1) Original spermatozoa in isotonic condition; (2) after four fixed-molarity step addition of 1.0 M glycerol; (3) after one-step addition of 1.0 glycerol; (4) after four fixed-volume step addition of 1.0 M glycerol. Reproduced with permission from Gao et al. (1995).

FIGURE 24

57%. T h e s e e x p e r i m e n t a l results a g r e e d well with t h e p r e d i c t i o n s g e n e r a t e d from computer simulation. It s h o u l d b e n o t e d t h a t t h e definition a n d d e t e r m i n a t i o n of " s p e r m i n j u r y " is d e p e n d e n t u p o n t h e assays used. S p e r m motility was u s e d as a

Experimental results: motility recovery (% mean _ SEM, n = 15) of human spermatozoa after removal of 1.0 M glycerol from spermatozoa. (1) Original spermatozoa in isotonic solution, (2) after one-step removal of 1.0 M glycerol, (3) after eight fixed-molarity step removal of 1.0 M glycerol, (4) after eight fixed-volume step removal of 1.0 M glycerol, (5) after two-step removal of 1.0 M glycerol using a "hyperosmotic buffer." Reproduced with permission from Gao et al. (1995).

FIGURE 25

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Dayong Gao, Peter Mazur, a n d John K. Critser

standard of sperm viability in the study by Gao et al. (1995) because of its relatively high sensitivity to cell volume excursion and the requirement of sperm motility for functional viability. If sperm membrane integrity was chosen as the endpoint for evaluating sperm viability, as shown in Figure 18, different cell tolerance limits would be obtained. One can readily repeat the same procedures to predict the extent to which spermolysis is caused by the different glycerol addition/removal procedures. For example, it was found from Figure 18 that over 85% of spermatozoa maintained membrane integrity when they were returned to isotonic conditions after had been exposed to anisosmotic conditions ranging from 90 to 700 mOsm. The corresponding sperm volume excursion range was 0.7-2.1 times the isotonic sperm volume. From Figures 20 and 21, it was found that one-step addition and one-step removal of 1.0 M glycerol might result in a minimum relative sperm volume of 0.72 and maximum volume of 1.68; respectively, which did not exceed the sperm volume excursion range (0.7-2.1 times relative volume) for maintaining over 85% sperm membrane integrity. Based on this information, one can predict that the majority (>85%) of spermatozoa would maintain membrane integrity even using one-step addition and onestep removal of glycerol. Again, this prediction was consistent with experimental results (Figure 26). As can be seen by a comparison with the preceding paragraph, membrane integrity is more tolerant of volume excursion than is motility.

Experimental results: membrane integrity (CFDA and Propidium iodide stain) (% mean _ SEM, n = 15) of human spermatozoa after addition and/or removal of 1.0 M glycerol. (1) Original spermatozoa in isotonic solution, (2) after four fixed-molarity step addition of 1.0 M glycerol, (3) after four fixed-molarity step addition and eight fixed-molarity removal of 1.0 M glycerol, (4) after one-step addition of 1.0 M glycerol, (5) after one-step addition and one-step removal of 1.0 M glycerol. Reproduced with permission from Gao et al. (1995).

FIGURE 26

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Since the hypothesis proposed in the present study was confirmed by experimental data, the procedures used for testing the hypothesis provides a methodology for predicting optimal protocols for CPA addition/removal. This methodology has the following advantages in comparison with an empirical approach.

An Understanding of Basic Cell Biological Properties. Cell volume excursion limits are biological properties of a given cell type, which can be readily determined using anisosmotic solution containing only nonpermeating solutes (e.g., sucrose). These limits can then be generally used to optimize procedure for the addition and removal of CPAs. Water and CPA permeability coefficients of cells are also biological properties of a given cell type. Knowledge of these coefficients in combination with information on the tolerated limits permits one to design optimal procedures for addition and removal of various concentrations of CPAs at given temperatures. We have mentioned that the maximum concentrations of glycerol used in most procedures for freezing sperm are below 1 M, and we have indicated that for many cells, such concentrations are suboptimal. The advantages of using a relative high CPA concentration are (a) cell cryosurvival rate is less dependent upon the cooling and warming rate (Mazur, 1984); and (b) the cells may possibly be vitrified at a feasible cooling rate. Osmotic Damage vs Toxicity. Recently, Gilmore et al. (1995) determined human sperm permeability coefficients to DMSO, propylene glycol (PG), ethylene glycol (EG), and glycerol (G) using the electronic counter technique. The results indicated that EG has a highest permeability of the four. Figure 27 shows volume increase in human sperm subjected to onestep removal of these four CPAs. Because of high permeability of EG, predicted maximum volume of the sperm during one-step removal of 1 M EG does not exceed 110% of isotonic volume, an innocuous value. One assumption in these analyses is that the injury in response to exposure to anisosmotic conditions is due solely to the osmotic volume excursions of the cells. A second is that the CPA plays no role in cell survival other than its contribution to the osmotic response. It is possible, however, that chemical toxicity of the CPA could also be a factor. The approach used here actually provides a powerful way of testing that possibility. Toxicity is usually reduced at lower temperatures. If one determines the permeability coefficient to the CPA at that lower temperatures, one could use the procedures described herein to reoptimize the protocols to minimize osmotic damage from the addition and removal of the CPA at the lowered temperature. Plasma membrane integrity is substantially more resistant to osmotic volume excursion than is motility. This suggests that motility is affected by the osmotic volume responses of cellular structures (other than the plasma

310

Dayong Gao, Peter Mazur, and John K. Critser 1.7

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F I G U R E 2 7 Calculated volume changes in human spermatozoa after one-step removal of 1 M (top) and 2 M (bottom) difference C P A s : D M S O ( . . . . ), ethylene glycol ( . . . . ), glycerol ( . . . . ), propylene glycol ( ..... ).

membrane) that are directly or indirectly involved in motility. One possibility is the mitochondria.

ii. Optimization of Cooling and Warming Rates The data obtained in Sections III.C.5.a-e provide the information necessary to compute kinetics of water loss and the degree of intracellular supercooling during freezing process as a function of cooling rate and initial CPA concentration (Mazur, 1990). With this information plus information of temperatures at which human sperm undergo the nucleation of intracellular ice (Section III.C.5.d), one can compute the relationship between cooling rate and intracellular freezing. Theoretically, any relatively slow cooling rates that ensure less than 2~ intracellular supercooling and produce over 90% loss of isotonic intracellular water before the cells have cooled to the intracellular ice nucleation temperature should prevent intracellular freezing (Mazur, 1990). The highest of these rates will be chosen as the optimal cooling rate because

Tissue Banking in Reproductive Biology

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higher cooling rates reduce the length of exposure to highly concentrated extracellular solution and hence minimize toxicity and damage from osmotic shrinkage (Mazur, 1990). In a number of cells, the cooling rates just below those calculated to produce intracellular ice are also the optimum cooling rates that produce maximum survivals experimentally (Mazur, 1984). That is not the case in human and bull sperm. Because of their very high Lp, low Ea of Lp, and high surface to volume ratio, computations indicated that sperm cells should not undergo intracellular freezing unless cooled at rates considerably greater than 10,000~ (Noiles et al., 1993; Watson et aL, 1992b). The experimental observation, however, is that the motility of sperm falls when they are cooled faster than 100~ Three possible explanations exist to reconcile this discrepancy. Either the estimates of Lp and its Ea and the resulting calculations are incorrect or mechanisms other than intracellular ice formation are involved in the cellular damage between 100 and 800~ With respect to the first possibility, the calculated 10,000~ cooling rate for intracellular freezing is based on the measured Lp of sperm in the absence of CPAs, whereas in the freezing experiments, the sperm were cooled in a 7.5% (0.85 M) glycerol solution (Noiles et aL, 1993; Watson et al., 1992b). Gilmore et al. (1995) have determined that the presence of 1 M glycerol reduces L v by 40% (Table 6). This reduction in Lp would reduce the calculated critical cooling rate to 7000~ but that is not nearly enough to account for the discrepancy. Another possible explanation relates to the values of the Ea of Lp used in the calculations. The position and shape of the calculated shrinkage curves during freezing are highly sensitive to the value of Ea used. Since the values of Ea at subzero temperatures during freezing have not and probably cannot be measured in sperm, the assumption has been made that the value remains the same below zero as it is above 0~ If Ea below 0~ is substantially higher than Ea above 0~ the critical cooling rate would be substantially lower than presently calculated. In this context, Noiles et al. (1993) noted that the Ea of Lp for human sperm shows a marked discontinuity below 0~ in the Arrhenius plot. These observations form the basis for our current hypothesis that the large discrepancy between the high value for the predicted optimal cooling rates and the much lower observed optimal cooling rates is due in part to the affect of glycerol on L v and, in part, to one or more additional discontinuities in human sperm water permeability coefficient at low temperatures. The third possible reason for the discrepancy is that injury is a consequence of internal freezing in critical organelles like the mitochondria or acrosomes rather than a consequence of the freezing of the cytoplasm in general. The probability of ice formation with a given cooling rate in the latter would be determined by the Lp and Ea of the plasma membrane. But the probability of ice formation within organelles would be determined by the Lp and Ea of their semipermeable

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Dayong Gao, Peter Mazur, and John K. Critser

membranes, and there are reasons for believing that the Lp of the organelle membranes may be much lower than that of the sperm plasma membrane.

D. F u t u r e R e s e a r c h A r e a s

From a survey of this chapter, it should be apparent that research in fundamental cryobiology of mammalian spermatozoa has just recently been initiated, and many questions and problems need to be answered. First, unlike several other cell types (e.g., human erythrocytes, mouse oocytes, bone marrow progenitor/stem cells), spermatozoa show an extreme sensitivity to osmotic challenge which may be the most important determinant of the proportion of cells surviving cryopreservation. This is probably a major barrier for any attempt to use higher concentrations of a given cryoprotectant to increase and stabilize the cryosurvival rate (Mazur, 1984). This osmotic damage is especially enhanced when sperm are subjected to any high influx across the cell membrane under either hyposomotic or posthyperosmotic treatments (Gao et al., 1994b, 1995). The high flux is due to extremely high water permeability of spermatozoa. The mechanism of this influx-induced injury is not clear. The underlying mechanism of high water permeability is still unknown. A recent NMR study (Baker et al., 1995) suggested that protein channels for water transport should exist in the membrane of human sperm. However, such potential channels have not been identified, and CHIP28 protein is not the water transport channels in the human sperm (Liu et al., 1995). In practice, new devices for additionremoval of cryoprotectants and new cryopreservation media need to be developed to prevent osmotic injury, stabilize sperm membrane, and inhibit metabolism and further development toward capacitation. Ethylene glycol appeared to be a better candidate than glycerol in preventing severe osmotic volume excursion of human sperm (Gilmore et al., 1995). Another important cryobiological property for a given cell type is the intracellular ice nucleation (IIN) temperature(s) and probability of IIN as a function of cooling rate, concentration, and type of cryoprotectant. For relatively large cell types, IIN may be observed using cryomicroscopy as "a sudden flashing or darkening" of the interior of a cell during the cooling process. However, IIN in spermatozoa cannot be viewed using cryomicroscopy because of the small cell size, relatively low water content, and highly condensed mass of nucleus in the sperm head region. Novel approaches are required to quantify the IIN probability in sperm. When these questions and problems described above are solved, optimum cryopreservation conditions for spermatozoa will be defined, providing cryopreserved spermatozoa much the same as fresh spermatozoa for use in medical research, artificial insemination, and clinical treatment of human infertility.

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ACKNOWI~DGMENTS The authors thank J. William for assistance with preparation of illustrations and C. Minks-Willoughby for assistance in preparing the manuscript. The research described was supported, in part, by Methodist Hospital of Indiana, grants from the National Institutes of Health (RO1-HD25949, RO1-HD30274 and K04-HD00980), Oak Ridge National Laboratories (ORNL 321064-0429), U.S. Department of Agriculture (NRICGP 93-37203-9272, 9537203-2232), and a NATO Collaborative Research Grant (CGR 920170).

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The Cryobiology of Mammalian Oocytes John

K. Critser

Cryobiology Research Institute Methodist Hospital of Indiana, Inc. Indianapolis, Indiana 46202

Yuksel Agca Cryobiology Research Institute Methodist Hospital of Indiana, Inc. Indianapolis, Indiana 46202

K a r e n T. G u n a s e n a Cryobiology Research Institute Methodist Hospital of Indiana, Inc. Indianapolis, Indiana 46202

I. INTRODUCTION Although oocyte preservation has been a goal of reproductive medicine and cryobiology for several years, to date the oocytes of almost all mammalian species studied have proven recalcitrant. Because viability with spermatozoa and embryo cryopreservation techniques is sufficiently high to satisfy applied needs, until recently, a critical driving force to understand the underlying cryobiological fundamentals of oocytes has been lacking. However, with increasing efforts directed toward improvements in follicular recruitment and in vitro maturation in many species, there is an associated increased probability of supernumerary oocytes which would require preservation until a subsequent cycle to maximize clinical safety and efficacy. In addition, in the context of human reproductive medicine, there is an increase in the activity directed toward the development of "donor Reproductive Tissue Banking Copyright 9 1997 by Academic Press. All rights of reproduction in any form reserved.

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oocyte" programs. With the initiation of these and similar programs, the possibility of disease transmission (e.g., HIV and the hepatitis B and C viruses) from use of contaminated donor material has resulted in the recommendation from the American Society for Reproductive Medicine (ASRM) and the American Association of Tissue Banks (AATB) that only gametes from "disease screened" donors be used in such programs. The ability to store frozen human oocytes would allow sufficient time for seroconversion of donors so that possible carriers of the HIV could be detected (Mascola and Guinan, 1986), as is currently done for sperm donors. Cryopreservation offers the opportunity for preserving oocytes of women who are at risk of losing ovarian function through pelvic disease, surgery, or clinical treatment involving radio- and/or chemotherapy. In addition, women who are in their late reproductive or menopausal years, who have hereditary diseases, abnormal oocytes, or who have had a history of difficulty with oocyte retrieval for in vitro fertilization (IVF) treatment may also benefit from oocyte banking. Cryopreservation of oocytes circumvents many ethical and legal objections to human embryo cryopreservation. It is also reported that in several European countries (for example, Austria, Germany, Switzerland, Denmark, and Sweden), cryopreservation of embryos is either banned or under very strict limitations (Jones, 1990; Knoppers and Bris, 1993; Wood et aL, 1984). Because of the relatively short life of human oocytes, the cryopreservation of oocytes also provides the potential to supply a large amount of viable and developmentally competent oocytes for use in clinical programs (e.g., in vitro fertilization and other assisted reproductive technology (ART) programs). Although embryos from most mammalian species can be cryopreserved with relatively high efficacy, corresponding success is extremely low for most mammalian oocytes. In the context of nonhuman species, the oocytes from laboratory animals, such as mice, could be banked in order to preserve specific strains for experimental purposes, without the high costs of perpetuating these strains through continuous breeding. Among species used for animal agriculture (e.g., cattle, pigs, and sheep), cryopreserved oocytes may play an important role in developing improved breeding programs. There is also a need for cryopreserving oocytes from domestic livestock animals with high economic or genetic value. There are many cases in which genetically valuable cows, ewes, mares, etc., have impaired reproductive abilities which could be addressed using oocyte collection via ultrasound-guided transvaginal retrieval. These oocytes could be either fertilized in vitro and immediately transferred or cryopreserved as embryos. However, if appropriate methods were developed, the oocytes could be directly cryopreserved, allowing more flexibility in their utilization for breeding programs. In large measure, the current inability to successfully cryopreserve oocytes of most mammalian species is due to the fact that the fundamental

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cryobiological factors that determine viability or death after the several steps involved in freezing of the gametes of essentially all mammalian species remain largely unknown. While empirically derived protocols which allow sperm and embryos to be effectively frozen in some species have been developed, the majority of species' gametes (and some embryos) cannot currently be efficiently cryopreserved. These fundamental characteristics are often species- and cell type-specific and need to be investigated on this basis. For example the high intracytoplasmic lipid content of pig oocytes and embryos and in vitro produced bovine embryos have recently been found to negatively influence cryopreservation (Pollard and Leibo, 1994) and removal of the lipid following centrifugation has resulted in higher post-thaw survival rates in pig (Nagashima et al., 1995) and bovine in vitro produced cleavage stage embryos (Ushijima et al., 1996). In the specific case of oocytes, this cell type presents with a relatively complex subcellular structure within which many of the subcellular components are particularly temperature and osmotically sensitive. In addition, the developmental stage of the oocyte affects the cell's cryobiological properties and therefore, the optimal procedures needed to effectively preserve these cells. For example, in the course of oocyte maturation and subsequent fertilization, changes occur in the plasma membrane which alter the permeability characteristics to water and to cryoprotectant agents. In addition, the distribution of organelles in the cytoplasm, such as the cortical granules, mitochondria, cytoskeletal elements, and nucleolar organization, also change during maturation and fertilization. The oocyte is evolutionarily situated as no other cell type in hosting the series of events which occurs during fertilization. These events, in which two gametes initially coexist, then undergo syngamy with subsequent ontologic development, are highly dependent upon maintenance of structural and functional integrity at many levels including, but not limited to, an intact plasma membrane, cortical granule vesicles, mitotic spindles (in the metaphase II (MII) stage), and other cytoplasmic organelles (Albertini and Rider, 1994). It has been reported that cumulus cells and tranzonal processes have a metabolic role and also play an important part in development of immature oocytes (Brower and Schultz, 1982; Heller et al., 1981). Cyclic adenosine monophosphate (cAMP), a regulator of oocyte maturation (Magnusson and Hillensjo, 1977), may originate from cumulus granulosa cells, since increasing cAMP levels within cumulus cells is associated with increasing cAMP levels in oocytes (Bornslaeger and Schultz, 1985). Because this interaction is mediated by the follicle cell processes, osmotic stress during addition and removal of a cryoprotectant solution might have an adverse effect on the development of oocytes (see Figure 1). Therefore, as a cryopreservation protocol is designed for oocytes, all these parameters must be taken into consideration in order to obtain optimal cryosurvival.

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John K. Critser, Yuksel Agca, a n d Karen T. Gunasena

1 Electron micrographs of oocytes. (A) Control oocyte. Cortical granules are subjacent to the oolemma (x18,750). (B) Oocytes treated in 1.5 M DMSO at 37~ for 20 min. Notice cortical granules surrounded by membrane in the subzonal space (original magnification: x31,000). (Reproduced with permission from Vincent et al., JRF 89, 253-259, 1990, Fig 2.)

FIGURE

II. THE HISTORY OF OOCYTE CRYOPRESERVATION The cryobiology of mammalian oocytes has a more recent and less successful history than that for either spermatozoa or embryos. Sherman and Lin (1958) investigated the survival of unfertilized mouse oocytes during cooling and warming. Then nearly 20 years later, Leibo et al. (1975) and Parkening

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333

et al. (1976) began investigation of the fundamental cryobiology of mouse oocyte, leading in 1977 to the first report of successful IVF and development of live offspring from cryopreserved mouse oocytes (Whittingham, 1977). Subsequently, occasional reports of success have appeared for several mammalian species, including the human (Chen, 1986; Van Uem et al., 1987). However, a comprehensive understanding of oocyte cryobiology remains to be developed and issues related to low fertilization need to be resolved before routine application of oocyte cryopreservation can occur. Efforts have been made to cryopreserve immature (germinal vesicle; GV) and mature (MII) oocytes with or without cumulus granulosa cell complexes from several mammalian species using either slow cooling or ultrarapid freezing protocols in the presence of commonly used permeating cryoprotectants (e.g., DMSO, propanediol) sometimes supplemented with nonpermeating solutes (e.g., sugars). However, most of these investigations have focused on the cryopreservation of mature (MII) oocytes. These previous experiments have indicated that oocytes are exquisitely sensitive to nonphysiologic conditions in many ways. In general, MII oocytes from all species studied to date are very sensitive to both nonphysiologic temperatures and to anisosmotic conditions. These conditions manifest as a loss of one or more requisite structural and/or functional components (i.e., microtubules, microfilaments, cytoplasmic organelles, zona pellucida glycoproteins, plasma membrane integrity). Therefore, it has been concluded from several studies that there is no simple cryopreservation protocol that can be used for cryopreserving oocytes from all species and all developmental stages (Table 1). In reality, if this issue is approached from an application point of view, because the in vitro maturation process of oocytes has not been well established for many mammalian species (including human, mouse, domestic cat and dog, and many endangered species), in some cases we may have to cryopreserve these oocytes at the GV stage until in vitro maturation protocols are available. For example, patients undergoing chemotherapy treatment or those who have had an oophorectomy may have no other choice to rescue gametes other than cryopreserving at the GV stage (Van Blerkom, 1991). There are advantages and disadvantages for cryopreservation of oocytes at either developmental stage and for the techniques used. Therefore, available technologies and developmental stages of oocyte have to be matched for successful post-thaw survival.

111. THE CURRENT STATUS OF MAMMALIAN OOCYTE CRYOBIOLOGY Although cryopreservation of mammalian oocytes, with maintenance of normal developmental potential, is currently considered to be generally unavailable, success has been achieved for both the mouse and the rabbit (A1-Hasani et al., 1989; George and Johnson, 1993; Schroeder et al., 1990;

1 C r y o p r e s e r v a t i o n o f O o c y t e s and O u t c o m e s f r o m S e v e r a l M a m m a l i a n Species at D i f f e r e n t D e v e l o p m e n t a l Stage U s i n g Various Methods

TABLE

Species

Stage o f d e v e l o p m e n t

Composition of CPA medium

Cooling method

Viability assay

Success rate ( % )

References

Bovine Bovine Bovine Bovine Bovine Bovine Bovine Bovine Bovine Hamster Hamster Hamster Hamster Hamster Hamster Hamster Rat Rat Rat Rabbit Rabbit Rabbit Sheep Human Human Human Human Human Human Human

GV IVM GV IVM GV MII IVM IVM IVM MII MII MII MII MII MII MII GV MII MII MII MII MII GV MII MII MII MII MII MII GV

D AP(213) DAP(213) 2 M PROH 2 M PROH 1.8 M EG + 0.2 M S 1.8 M EG + 0.2 M S 5.5 M E G + 1 M S 1M G DAP(213) 1.5 M P R O H 1.5 M P R O H + 0.1 M S 1.5 M P R O H 1.5 M DMSO 6 M DMSO D A P (213) 1.5 M DMSO 1.5 M DMSO Polyethylene oxide 400 1.5 M DMSO 1.5 M DMSO 2 M P R O H + .5 M S 1.5 M P R O H + 0.5 M S 1.5 M DMSO 1.5 M DMSO 1.5 M P R O H 1.5 M PG 3 M P R O H + .25 M S 1.5 M DMSO 1.5 M DMSO 1.5 M P R O H + .2 M S

Ultrarapid Ultrarapid Slow Slow Slow Slow Ultrarapid Slow Ultrarapid Rapid Slow Slow Slow Ultrarapid Ultrarapid Slow Slow Slow Slow Slow Rapid Slow Slow Slow Slow Slow Rapid Slow Slow Slow

IVF IVF IVF, ET IVF IVF IVF IVF IVM IVF, ET SP IVF IVF IVF IVF SP IVF GVBD Flourescent IVF IVF ET IVF, ET IVM IVF IVF IVF, ICSI IVF IVF PTMS IVM

1.1 4.9 1.1, 2 preg 16.2 7 1.7 34 16 10, 2 preg 94 58.7 31 41 94 39 11.8 48 Viable 47% 64 9 74, 18 16 46 47 13, 46 70 53 18 58.3

Fuku et al. (1992) Fuku et al. (1992) Fuku et al. (1992) Fuku et al. (1992) Otoi et al. (1995) Otoi et al. (1995) Martino et al. (1995b) Lim et al. (1991) Hamano et al. (1992) Tobback et al. (1991) Todorow et al. (1989) Todorow et al. (1989) Todorow et al. (1989) Wood et al. (1993) Critser et al. (1986) De Mayo et al. (1985) Pellicer et al. (1988) Angelova et al. (1991) Kasai et al. (1979) Siebzehnrubl et al. (1989) Vincent et al. (1989) A1-Hasani et al. (1989) Sulieman et al. (1990) Siebzehnrubl et al. (1989) A1-Hasani et al. (1988) Kazem et al. (1995) A1-Hasani et al. (1988) Hunter et al. (1991) Trounson (1986) Toth et al. (1994a)

Human Human Human Human Human Human Human Human Human Human Human Human Human Mouse Mouse Mouse Mouse Mouse

MII MII MII MII MII MII MII MII MII MII MII MII MII MII MII MII MII MII

1.5 M PROH PROH + S 1.5 M DMSO 1.5 M G 1.5 M PROH 1.5 M PROH 1.5 M DMSO 4.2 M DMSO 2.8 M DMSO 3.5 M DMSO 1.5 M DMSO 1.5 M DMSO 1.5 M DMSO 1.5 M PROH 1.5 M PROH 1.5 M DMSO 1.5 M DMSO 1.5 M PROH

Mouse Mouse Mouse Mouse Mouse Mouse Mouse Mouse Mouse Mouse Mare Monkey Pig

MII MII MII MII MII GV GV GV GV GV GV MII GV

1.5 M DMSO DAP(213) DAP(213) VS1 DAP(213) 1.5 M DMSO 1.5 M DMSO 1.5 M DMSO 25% PROH + 1 M S DAP(213) 40% EG + 18% 1.5 M DMSO 17.5% PG + 2.5% G + 0.05 M S

+ S + S + .25 M S + .06 M S + 0.5 M S

+ S

Slow Slow Slow Slow Slow Slow Rapid Rapid Rapid Rapid Slow Slow Slow Slow Slow Slow Slow Slow

PTMS IVF IVF IVF PTMS PTMS PTMS PTMS PTMS PTMS ET ET ET PTMS IVF IVF IVF IVF

60 21 59 23 64 46 25 48 30 60 2 preg 1 preg 1 preg 4 27 17 48 35

Ultrarapid Ultrarapid Ultrarapid Ultrarapid Ultrarapid Slow Ultrarapid Slow Ultrarapid Ultrarapid Ultrarapid Slow Ultrarapid

PTMS, B IVF, ET IVFC IVF IVF, ET GVBD IVF IVM, IVF IVM Viability IVM IVF IVM

32, 15 84, 25 55.4 75 78, 46 5 28 74, 37 22 90 17 27.6 24.5

Trounson (1986) VanBlerkom and Davis (1994) Todorow et al. (1989) Hunter et al. (1991) Gook et al. (1993a) Gook et al. (1993b) Trounson (1986) Pensis et al. (1989) Pensis et al. (1989) Pensis et al. (1989) Chen (1986) Van Uem et al. (1987) Diedrich et al. (1987) Gook et al. (1993a) Todorow et al. (1989) Todorow et al. (1989) Carroll et al. (1990b) Hernandez-Ledezma and Wright (1989) Sathananthan et al. (1988) Kono et al. (1991) Shaw et al. (1991) Shaw et al. (1990) Nakagata (1989) Carroll et al. (1990a) Van der Elst et al. (1990) Van der Elst et al. (1990) Zhiming et al. (1990) Van Blerkom (1989) Hochi et al. (1995) De Mayo et al. (1985) Rubinsky et al. (1991)

Note. GV, germinal vesicle; MII, metaphase II; IVM, in vitro matured. IVM, in vitro maturation; IVF, in vitro fertilization; IVMF, in vitro maturation, fertilization; SP, sperm penetration; ET, embryo transfer; GVBD, germinal vesicle breakdown; PTMS, post-thaw morphologic survival; IVFC, in vitro

fertilization culture; ICSI, intracytoplasmic sperm injection; HB, hatching blastocytes; B, blastocytes. S, sucrose; G, glycerol; EG, ethylene glycol; DMSO,

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Vincent et al., 1989). Because we are at this temporal threshold in which methodology exists for a few species, but do not yet understand the fundamental science sufficiently to allow us to develop applications to other important species or to achieve high efficiencies, it would seem that this area is well positioned to be investigated in this context in which both application and research are priorities. The critical issues that present themselves with oocyte preservation are at the organelle/subcellular level and therefore are generally more complex than, for example, embryo freezing (at least as it is currently viewed). To date, several factors have been found to be important for developing optimal cryopreservation protocols for oocytes and also several parameters have been reported. Cooling affects cytoskeletal elements (e.g., spindle fiber integrity) and cortical granules; disruption of the former is likely to lead to aneuploidy, while disruption of the latter is likely to lead to zona hardening (George and Johnson, 1993; Parks and Ruffing, 1992; Vincent and Johnson, 1992). In addition, cryoprotectants affect the organization of the microtubule system in mouse oocytes; it was reported that DMSO has a profound effect on microtubules (Johnson and Pickering, 1987). Also, genetically altered (polyploidy) embryos have been obtained from frozenthawed mouse oocytes or oocytes exposed to DMSO before freezing and it has been suggested that cryopreservation may induce DNA damage (Bouquet et al., 1993). Alterations of zona pellucida glycoproteins, especially ZP2, is reported to be responsible for zona hardening in mouse oocytes (Moiler and Wassarman, 1989). A reduced in vitro fertilization rate due to changes in zona pellucida and premature exocytosis of cortical granules has also been reported in mouse oocytes (Carroll et aL, 1990a, Vincent et al., 1990) (see Figure 2). Exposing mouse and human oocytes to DMSO and propanediol before freezing has also resulted in significant premature exocytosis of cortical granules (Schalkoff et al., 1989). Ultrastructural changes of vitrified-warmed bovine immature and mature oocytes have also been examined and it was concluded that cortical granule kinetics is one of the most important elements affecting fertilizability of bovine oocytes. Germinal vesicle oocytes were also found to be more sensitive to the cryoprotectant agent than MII stage (Fuku et al., 1995). Chemical and physical changes in the oocyte plasma membrane during maturation have been reported (Ashwood-Smith et aL, 1988). The effects of cryopreservation on cellular organization in immature and mature mouse and human oocytes have been examined (Gook et al., 1993a; Van Blerkom and Davis, 1994). These groups reported normal nucleii and cytoplasm in frozen-thawed immature oocytes that were capable of maturation and implantation. On the other hand, matured human and mouse oocytes have been associated with an increase in the frequency of aneuploidy. Most recently, it has been proposed that designing a strategy for the cryopreservation of mammalian oocytes requires recognition of the contri-

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337

FIGURE 2 Electron micrographs of oocytes. (a) Control oocyte. Cortical granules are subjacent to the oolemma (x18,750). (b) Oocytes treated in 1.5 M DMSO at 37~ for 20 min. Notice cortical granules surrounded by membrane in the subzonal space (original magnification: x31,000). (Reproduced with permission from Vincent et al., JRF 89, 253-259, 1990, Fig 2.)

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J o h n K. Critser, Yuksel Agca, a n d Karen T. Gunasena

bution of cytoskeletal elements to events associated with both cell cycle progression (organelle movement, spindle morphogenesis) and somatic cell interaction at the level of the plasma membrane (Albertini, 1995). Cryopreservation is a multistep procedure in which oocytes are exposed to an anisosmotic solution (cryoprotectant) before cooling. In each step, oocytes experience drastic volume changes due to different osmotic pressures between the intracellular and extracellular solutions which cause water and cryoprotectant transport across the oocyte membrane (Mazur et al., 1984). These changes in cell volume affect several parameters that play a role on cryosurvival of oocytes, including integrity of the plasma membrane and subcellular structures. Cells generally demonstrate an ideal osmotic response and this type of ideal osmotic response has been characterized (by the Boyle Van't Hoff equation, where cell volume is a linear function of 1/osmolality) for mature mouse oocytes (Hunter et al., 1992a) and immature and mature bovine oocytes (Ruffing et al., 1993). Oocyte osmotic tolerance limits have to be known for each species and developmental stage of oocyte in order to avoid excessive shrinkage and swelling and also to predict optimal addition and removal of cryoprotectants (Gao et aL, 1996) (see Figure 3).

IV. THE CRYOBIOLOGY OF VARIOUS MAMMAIJ&N SPECIES OOCYTES A. M o u s e O o c y t e s In the area of oocyte cryopreservation, by far the most thoroughly studied species to date has been the mouse. Johnson and colleagues (George and Johnson, 1993; Vincent and Johnson, 1992) have recently published a method for mouse oocyte (MII) cryopreservation which results in high (79%) maintenance of cytoskeletal elements, including spindle fiber integrity and nonhardened zona (e.g., chymotrypsin digestible) and therefore, by implication, nondisrupted cortical granules. This procedure differs from previous methods primarily in that it utilizes cryoprotectant (1.5 M DMSO) addition and removal at 4~ rather than the typically used room temperature. However, it has been shown (Pickering et al., 1990; Sathananthan et al., 1988) that the mouse oocyte is fundamentally different from the human oocyte in its capacity to reverse the disruption of the meiotic spindle fibers and "repolymerize" in an appropriate manner. Therefore, although a "workable" cryopreservation approach may well have been developed for the mouse, this same approach when applied to other species, including the human and bovine, fails to maintain at least nuclear integrity. However, these data for mouse oocyte do provide an important foundation upon which to initiate oocyte cryopreservation procedures; but they also raise several questions regarding the cryobiology of such a procedure, such as

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the permeability of the cell to cryoprotectants at this low temperature and (as indicated above) the extension of these data to other species. B. B o v i n e O o c y t e s Recent rapid improvements in ovum pick-up techniques, in vitro maturation, fertilization, and culture have generated great interest in cryopreservation of bovine oocytes. It is apparent that effective cryopreservation of bovine oocytes will certainly enhance the utilization of oocytes from animals with high genetic value (Pieterse et al., 1991).

340

J o h n K. Critser, Yuksel Agca, a n d Karen T. Gunasena

Live births have been reported from frozen-thawed immature and mature bovine oocytes using a slow cooling procedure in the presence of 2 M 1,2-propanediol and it was also reported that the fertilization rate of vitrified-warmed (DAP213) oocytes was lower than that of slowly cooled cohorts at both developmental stages (Fuku et al., 1992). Hamano et al. (1992) also obtained similar low fertilization rates after using the same vitrification protocol with in vitro matured oocytes. It has been previously reported that chilling of immature and mature bovine oocytes down to 0~ is reduced to subsequent development to blastocyst stage to <5% (Martino et al., 1995a). Martino et al. (1995b) later tried to overcome this problem by exposing in vitro matured oocytes to 5.5 M ethylene glycol plus 1 M sucrose for <30 s before plunging into liquid nitrogen in order to minimize duration of exposure to low temperature with a maximum development to blastocyst of 14%. However, the exact nature of damage to oocytes during the cryopreservation process has just recently been taken under investigation. Although many reports are in abstract form (Arav et al., 1995; Leibo et al., 1995; Zeron and Arav, 1996), important steps have been taken to illuminate the fundamental cryobiologic properties of bovine oocytes. Hyttel et al. (1989) reported abnormal changes to the ultrastructure of bovine oocytes during maturation and fertilization. Based on this report, an investigation of bovine oocytes exposed to vitrification solution (DAP213) was undertaken to identify the nature of any intracellular damage using transmission electron microscopy (Fuku et al., 1995). According to this study, normal microvilli originated from oocyte plasma membrane and extended to the foot processes in the zona pellucida, and abnormal microvilli were associated with degeneration and detachment of foot processes. While cortical granules in normal immature (GV) oocytes were peripherally located and largely clustered, in abnormal oocytes they were dispersed along the oolemma. During maturation, cortical granules (GV) migrated and aligned along the oolemma. In abnormal conditions, cortical granules were released prematurely into the previtelline space and did not disperse close to the oolemma. While small vesicles were normally scattered throughout the ooplasm except in peripheral regions, in abnormal oocytes, vesicles migrated into the ooplasm, became confluent, and ruptured. While normal mitochondria have a continuous membrane and their matrix is dense and homogenous, the cristae are delineated. Abnormal mitochondria showed extensive vacuolization, absence of most of the cristae, and reduced matrix density. The phase transition during cooling has been investigated in membrane lipids of GV and MII stage bovine oocytes (Afar et al., 1995). It was reported that while the lipid phase transition of the membrane lipids for GV stage oocytes is 13-20~ no phase transition was observed in MII stage oocytes. Also cooling the GV and MII stage oocytes down to 4~ and staining with fluorescein diacetate has shown no significant changes in

The Cryobiology of Mammalian Oocytes

341

membrane integrity between the immature or mature stage. Electrofusion of nucleated GV oocytes with anucleated MII oocytes showed chilling resistance (membrane integrity) after cooling to 4~ (Arav et al., 1995). Based on this investigation, one of the antioxidants, butylated hydroxytoluene (BHT), which was reported to protect ram and bull sperm plasma membrane from cold temperature (Hammerstedt et al., 1976), was also used to stabilize GV oocytes before cooling to 4~ and it was concluded that the protection provided by BHT against chilling injury depends upon the cooling condition and duration of exposure at low temperatures (Zeron and Arav, 1996). As previously discussed, polymerization of microtubules and microfilaments requires physiologic temperature. Temperatures below this appear to have an adverse effect on organization of the cytoskeletal elements. Bovine oocytes are also very susceptible to low temperature like the other mammalian oocytes. Effects of cryoprotective agents and cooling on the cytoskeleton and meiotic spindle apparatus of bovine oocytes have also been investigated (Richardson and Parks, 1992; Williams et al., 1992) and it was concluded that microtubule dynamics were perturbed by both cooling and the cryoprotectant used. Osmotic characteristics of the plasma membrane to water and intracellular ice crystal formation kinetics of bovine oocytes from different developmental stage (GV, MII, IVF) have also been very well documented (Ruffing et al., 1993). Osmotic behavior in the presence of NaC1 as a function of time and intracellular ice crystal formation temperature in the presence of glycerol, ethylene glycol, and propylene glycol as a function of cooling rate has been investigated. It was concluded from these experiments that differences exist in oocyte from different developmental stages and it was suggested that these factors have to be considered in developing cryopreservation protocols (Table 2). C. Rat O o c y t e s Rat oocytes are one of the less investigated lab animal gametes in terms of their cryobiologic properties. One of the reasons for this is that procedures such as superovulation and oocyte collection are not well defined compared to mouse, bovine, and human. It has been previously reported that viable rat oocytes could be obtained post-thaw after slow cooling in the presence of polyethylene oxide (Angelova et al., 1991). However, in this report viability was determined only by a membrane integrity assay using fluorescein diacetate. In another investigation, morphologic and functional properties of rat immature oocyte cumulus complexes were examined after cryopreserving oocytes in the presence of DMSO; and it was reported that oocytes with more cumulus cell layers survive better (Pellicer et al., 1988).

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Factors Affecting the Cryosurvival of Mammalian Oocytes at Different Developmental Stages Using Either Equilibrium or Nonequilibrium Methods

TABLE 2

Equilibrium freezing

Nonequilibrium freezing

Immature (GV)

Advantages

Advantages

Osmotic shock to plasma membrane, microvilli, and follicle cell processes may be minimized (Pellicer et al., 1988).

Disadvantages

Cold shock injury to plasma membrane may be avoided (Arav et al., 1995) Intracellular ice crystal formation may be avoided (Kono et al., 1991).

Disadvantages

Chilling injury to plasma membrane may be increased (Arav et al., 1995). Intracellular ice crystal formation may occur (Mazur, 1984). Risk of damage from high solute concentration (Mazur, 1984). Cytoskeletal elements (microfilaments) may be disrupted due to lower temperatures (Vincent et al., 1990).

Osmotic shock to plasma membrane, microvilli, and follicle cell processes may be increased due to high concentration of cryoprotectants (Fuku et al., 1995; Younis et al., 1996). Distribution of cytoskeletal elements may be increased due to the high concentration of cryoprotectants (Johnson and Pickering, 1987).

Mature (MII)

Advantages

Advantages

Osmotic stress during cooling to plasma membrane may be avoided,

Intracellular ice crystal formation may be avoided (Rall and Fahy, 1985).

Premature exocytosis of cortical granules (zona hardening) may be avoided due to lower concentrations of cryoprotectants (Johnson and Pickering, 1987).

Meiotic spindle fibers may be protected due to short exposure to low temperature (Aigner et al., 1992).

Disadvantages Intracellular ice crystal formation may occur (Mazur, 1984). Risk of damage from high solute concentration (Mazur, 1984).

Disadvantages Osmotic stress to plasma membrane may increase due to increasing concentrations of cryoprotectants (Joly et al., 1992). continues

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343

TABLE 2 - - Continued Equilibrium freezing

Meiotic spindle fibers may be disrupted due to long exposure to intermediate temperatures (Aigner et al., 1992). Premature exocytosis of cortical granules (zona hardening) may be increased due to low temperature (Vincent et aL, 1990).

Nonequilibrium freezing

Premature exocytosis of cortical granules (zona hardening) may be increased due to high concentrations of cryoprotectants (Johnson and Pickering, 1987). Cytoskeletal elements (microtubules and microfilaments) may be disrupted due to high concentrations of cryoprotectants (Joly et al., 1992).

D. H u m a n Oocytes Comparison of the cytoskeletal systems of murine and human oocytes has indicated significant differences in the organization in their respective responses to cooling and exposure to cryoprotectant compounds. In terms of the cytoskeletal structure the mouse has been shown to have a "barrelshaped" anastral spindle, while the human has a peripheral anastral spindle pointed at each pole (Pickering et al., 1990). In terms of the effects of cooling on the spindle, human oocytes are much less robust than mouse oocytes in their ability to recover normal spindle morphology after cooling. This is thought to be due, at least in part, to the organization of the microtubule nucleation between the two species. The organization of the spindle requires both the chromosomes, which cause a local reduction in the threshold for microtubular polymerization, and the pericentriolar material (PCM) to nucleate microtubule polymerization (Pickering et al., 1990). It is the PCM at the spindle poles that give the spindle its shape. The mouse differs from the human in that it has many foci of PCM throughout the cortex in addition to that located at the spindle poles. Therefore, when the human spindle is disrupted (e.g., cooled or exposed to cryoprotectants) the PCM may "drift" away and upon restoration of conditions that favor microtubule polymerization, it is possible that little or no PCM will be available for the process. The result would be clumps of chromosomes surrounded by radiating microtubules, which is exactly what Pickering and colleagues (1990) have found. With the advent and subsequent rapid growth of human ART programs, there has been a parallel increase in the desire to safely and efficaciously cryopreserve human oocytes. Relatively early in the development of such methods, there was a realization that applying "standard" cryopreservation approaches to mammalian oocytes was fraught with several serious problems, as introduced above. Clearly, the problem of most concern was the

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J o h n K. Critser, Yuksel Agca, a n d Karen T. Gunasena

matter of meiotic spindle depolymerization with associated increased likelihood of aneuploidy. Trounson (1986) initially suggested this concern; however, in the same year Chen (1986) and A1-Hasani et aL (1986) reported the first live births from cryopreserved human oocytes. Subsequent investigations have found continued low clinical success and further concern for potential sublethal damage during cryopreservation. A1-Hasani et al. (1986) cryopreserved 283 human oocytes from 48 patients and found that only 30 oocytes were fertilized (with a high incidence of polyploidy) and from those only 2 pregnancies were initiated. In that same year, Siebzehnruebl et al. (1989) cryopreserved 38 unfertilized oocytes; only 14 were fertilized, 7 of which reached syngamy prior to transfer to 10 patients, resulting in a single live birth. Kola et al. (1988), following Trounson's earlier concern (1986) of the possibility that materials and methods associated with the standard cryopreservation approach applied to oocytes could result in a high frequency of aneuploidy, tested this hypothesis in the mouse with resulting data supporting this hypothesis. Investigation of the mechanisms involved with this low fertilization and high frequency of aneuploidy followed. Pickering et al. (1990) studied and detailed the spindle fiber depolymerization with reduced temperature and reported that this was an irreversible event. Pickering et aL (1991) found further that exposure of oocytes to DMSO at high temperatures (37~ but not low temperatures (4~ resulted in low fertilization rates. The use of 1,2-propanediol as a cryoprotectant for mouse oocytes has been investigated (Joly et al., 1992) but found to both disrupt the spindle fibers (although low concentrations caused more disruption than high concentrations) and (at high concentrations) cause cytoplasmic blebing. However, when 1,2-propanediol was used with both mouse and human oocytes, similar adverse effects were observed in the mouse oocytes (4% cryosurvival and 0% fertilization) but not with human oocytes (64% survival, no IVF performed) (Gook et al., 1993a). Hunter et al. (1991) exposed human oocytes to either glycerol or DMSO, as cryoprotectant solutes, and found that only 3 of 13 oocytes cryopreserved with glycerol fertilized normally, while 5 of 15 oocytes with DMSO fertilized normally. Of all oocytes, only one (with DMSO) developed beyond the 2 pronuclei stage. Gook et aL (1994) further evaluated cryopreservation of human oocytes with 1,2propanediol. They reported 51% post-thaw survival of metaphase II oocytes with no "stray" chromosomes and with normal karyotypes after fertilization. A more recent report by Gook et al. (1995) demonstrated parthenogenetic activation of human oocytes after exposure to cold (i.e., cryopreservation), but not after exposure to propanediol, previously shown to induce parthenogenetic activation of mouse oocytes (Shaw and Trounson, 1989). These findings confirm clear species differences that must be considered when developing clinical protocols.

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345

Toth et al. (1994a,b) have evaluated the cryosurvival of immature (prophase I) human oocytes from stimulated (1994a) and unstimulated patients (1994b). In patients undergoing controlled ovarian hyperstimulation (1994a), cryosurvival was 58%, with subsequent rates of maturation and fertilization that were not different than fresh control oocytes. In contrast, oocytes from unstimulated ovarian tissue (1994b) were more variable in both frequency of cryosurvival and subsequent completion of nuclear maturation. In this later study, two cryopreservation protocols were compared. In protocol I, cryosurvival was low (15.6%) with moderate frequency of maturation (58.3%). Protocol II showed reciprocal efficiencies, with higher cryosurvival (43.4%) and a reduced frequency of nuclear maturation (27.3%). The net effect was that there was no overall difference in the two protocols in the cumulative frequency of maturation of cryopreserved oocytes. However, these overall rates of maturation of cryopreserved oocytes were quite low (9.1% Protocol I; 11.8% for Protocol II). Routine clinical application of these technologies will require significant improvement of efficiencies. Subsequent to this succession of empirically derived data, there have been two reports of the investigation of the basic cryobiological properties of human oocytes. The first (Hunter et aL, 1992a) examined the water permeability of both "flesh" and failed-to-fertilize oocytes and found that human oocytes have oolemma water permeability characteristics very similar to those of mouse oocytes (0.48/~m/min/atm at 20~ with an activation energy of 9.5 kcal/mol. The second (Fuller et aL, 1992) examined the permeability of mouse and human oocytes to 1,2-propanediol and water simultaneously. The results of this study indicated that human oocytes are about twice as permeable to 1,2-propanediol than mouse oocytes (Table 3). While the fundamental cryobiology of this cell type has begun to be understood, only the "tip of the iceberg" has been addressed. If oocyte cryopreservation is to become a safe and efficacious clinical application in the rapidly growing ART field, an in depth understanding of the biophysical factors which lead to success or failure must be developed.

V. VITRIFICATION In the context of cryopreservation, vitrification is an alternative to conventional equilibrium freezing. Freezing involves the precipitation of water as ice, resulting in the separation of water from the solutes collectively composing the solution. During this process, at least two types of damage can occur to biologic materials in the solution: (1) formation of intracellular ice (when the sample is cooled "too" quickly and water is "trapped inside the cells" and (2) damage due to the enormously high solute concentrations generated when water precipitates as ice (Mazur, 1977,1984,1990). Alterna-

TABLE 3 C o m p a r i s o n of Plasma M e m b r a n e Permeability Characteristics to W a t e r ( t p ) and C r y o p r o t e c t a n t (Ps) and Their Interactions (or) with O o c y t e s from Several Species

Species

Stage

Solute

Concentration (mOsm)

Temperature (~

Lp (txm" min 9atm)

Ps (txm/s)

or (unitless)

References

Human Human Human Human Mouse Mouse Mouse Mouse Mouse Hamster Bovine Bovine Bovine Bovine Bovine Bovine Bovine Bovine Monkey Goat Goat

MII MII MII (fresh) MII (aged) MII MII MII MII MII MII GV IVM IVF GV IVM GV IVM GV IVM GV MII

PROH a DMSO b NaC1 NaC1 PROH PROH DMSO DMSO NaC1 NaC1 NaC1 NaC1 NaC1 DMSO DMSO EGc EG Gd NaC1 PROH PROH

1691 1688 920 920 1691 1691 1688 1688 920 500 709 709 709 1688 1688 1640 1640 1390 1200 1740 1740

20 3 20 20 20 20 20 22 20 20 20 20 20 22 22 22 22 20 0 20 20

1.08 0.43 0.40 0.64 2.20 0.81 0.81 0.65 0.48 0.80 0.45 0.84 0.55 0.70 1.14 0.50 0.83 0.54 0.23 0.97 0.60

0.82 0.16 N/A N/A 0.48 0.31 0.24 0.23 N/A N/A N/A N/A N/A 0.36 0.48 0.22 0.37 N/A N/A 0.14 0.20

0.68 0.82 N/A N/A 0.71 0.78 0.80 0.85 N/A N/A N/A N/A N/A 0.86 0.90 0.94 0.76 N/A N/A N/A N/A

Fuller et al. (1992) McGrath et al. (1995) Hunter et al. (1992a) Hunter et al. (1992a) Fuller et al. (1992) Fuller et al. (1992) McGrath et al. (1992) Gao et al. (1996) Hunter et al. (1992a) Shabana and McGrath (1988) Ruffing et al. (1993) Ruffing et al. (1993) Ruffing et al. (1993) Agca (unpublished data) Agca (unpublished data) Agca (unpublished data) Agca (unpublished data) Myers et al. (1987) Younis et al. (1996) LeGal et al. (1994) LeGal et al. (1994)

a 1-2, Propanediol. b Dimethylsulfoxide. c Ethylene glycol. d Glycerol.

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347

tively, vitrification involves the solution composition remaining unchanged during cooling and water does not precipitate out of the solution (i.e., ice crystals do not form) (Fahy, 1989). Vitrification, in the context of cell and tissue cryopreservation, is the transition of aqueous solutions from the liquid state to the glass state (solid), bypassing the crystalline solid state. That is to say, as the temperature is decreased, "the viscosity of the sample becomes greater and greater until the molecules become immobilized and the sample is no longer a liquid, but rather has the properties of a solid" (Fahy, 1989). However, to achieve vitrification, these solutions, in which the biologic material (e.g., cells or tissue) is suspended, must have very high concentration of cryoprotectant additives. Loading a biologic system (cells and tissues) with these high concentrations of cryoprotectants has been both biologically problematic and technically difficult (Rall, 1987). Although Luyet first investigated the possibility of using a vitrification approach to cryopreserving biological systems (see Luyet and Gehenio, 1940), Rail and Fahy (1985) reintroduced this concept with their work in the area of vitrification of early mammalian embryos. The two primary factors which govern the success of equilibrium approaches to cryopreservation also govern the success of vitrification approaches: (1) cooling rate and (2) solute effects, or reciprocally, cryoprotectant concentrations. In the case of equilibrium cryopreservation methods a cooling rate which is "too fast" will result in intracellular ice formation and, usually, cell death (Mazur et al., 1972). However, in this same context, a cooling rate which is "too slow" will cause the cells to be exposed to the high solute concentrations which form when water precipitates as ice and concentrates the cells and solutes (electrolytes, etc.) in the unfrozen channels (Mazur, 1984; Mazur et al., 1972). To avoid damage due to these high solutes, cryoprotectants are added to the cell suspension to maintain the solutes at a concentration tolerated by the cells and a given cooling rate and therefore time of exposure. In the context of vitrification, these two parameters (cooling rate and solute concentration) affect the outcome somewhat differently. In general, to vitrify a solution either a very high cooling rate or a very high solute concentration is needed. In practice, for the vitrification of biological samples, there is a practical limit to achievable cooling rates and there is a biological limit to the concentration of solutes (cryoprotectants) which the cells will tolerate. Therefore a balance is sought which generally seeks to maximize the cooling rate and minimize the cryoprotectant concentration. In their initial work, Rall and Fahy used a solution composition of 20.5% (w/v)/DMSO, 15.5% (w/v) acetamide, 10% (w/v) propylene glycol, and 6% (w/v) polyethylene glycol in a modified DPBS. This solution, called VS1 (vitrification solution 1) and later derivatives of VS1 using glycerol as the primary permeating cryoprotectants (e.g., VS2, VS3, and VS3a) were subsequently used by a number of other investigators in the general area of

348

John K. Critser, Yuksel Agca, and Karen T. Gunasena

embryo cryopreservation (Ali and Shelton, 1993; Rail et al., 1987; Scheffen et al., 1986; Schiewe et al., 1991) with generally good results. However, previous attempts to vitrify mammalian oocytes have met with mixed results. Using the VS1 solution of Rail and Fahy, Critser et al. (1986) investigated the possibility of vitrifying hamster oocytes and found a lower survival of vitrified oocytes than those frozen using conventional freezing approaches. Kono et al. (1991) used VS1 to vitrify mouse oocytes and found, in part, that a high frequency of oocytes survived morphologically (---80%) and could be fertilized in vitro (~-85%) but relatively few (--~50%) were able to develop in vivo (with an overall development to live young of only 25%). Similar results were achieved independently by Nakagata (1989). More recently, Wood et al. (1993) examined the use of equilibration of mouse and hamster oocytes in 1.5 M DMSO with subsequent brief exposure to 3.9 M and then cooling in 6.0 M DMSO. Shaw et al. (1992) examined the effect of time of exposure of mouse oocytes to VS1 prior to cooling and found that shortened exposure times resulted in higher cryosurvival rates, with an optimal exposure time of only 15 s. Subsequent IVF resulted in 32% of oocytes developing to the blastocyst stage. Similar to the results achieved by Kono et al. (1991) and Nakagata (1989) and Shaw et al. (1991), Wood et al. found a relatively high initial survival and fertilization rate, but a relatively low in vivo development rate of those embryos derived from vitrified oocytes. The reason for this high incidence of in vivo loss of vitrified mouse oocytes is currently not understood (Wood et al., 1993). However, this may be related to the previously discussed adverse affects that cryoprotectants (DMSO or 1,2-propanediol) have been reported to have on oocyte structural and functional integrity (e.g., depolymerization of the meiotic spindle, hardening of the zona pellucida, and parthenogenetic activation [cf, Johnson and Pickering, 1987; Shaw and Trounson, 1989; van der Elst et aL, 1988, 1992]). Figure 4 illustrates an example of a phase diagram (temperature vs concentration) of a typical cryoprotectant. Detailed discussion of the regions on Figure 4 can be found elsewhere (Fahy et al., 1984; Sutton, 1991). Although dimensions on the figure are arbitrary, a similar phase diagram is expected for the media which would be used for vitrification of oocytes. In the vitreous state at concentrations below the intersection of the glass transition temperature (Tg) and ice nucleation temperature (Th), crystallization can be circumvented. It should be noted that there is a critical cryoprotectant concentration needed for vitrification, and for some cryoprotectants, this minimum concentration (which will be referred to as Cv here) may result in either osmotic or true chemical toxicity. There are basically three ways of preventing solute toxicity and reducing Cv. Mehl (Sutton, 1991) argues that substituting the OH group of an alcohol with an amino group increases the glass-forming ability of the alcohol. Of

The Cryobiology of Mammalian Oocytes

il

349

ii111!i IV

A

o

-40

0

IJJ

rt"

i.--

.< LU O.

-80

%%% "

UJ p-

-120

_

Tg

I

...-"

I ! I 20 4.0 CONCENTRATION

::::::::

!il}iiiil ~--...--~

s)BuE ii!i!!il :"F:":,

i

6O (%w/w)

Genericsupplemented phase diagramcurves representingmelting temperature (Tin), glass transition temperature (Tg), ice nucleation temperature (Th), and cryptic nuclei (formed during cooling) during warming (Td). Reproduced from permission from Fahy et al., 1984, Cryobiology 21:407-426.

FIGURE 4

the amino alcohols investigated so far, 2-amino-2-methl-l-propanol is the most efficient glass former. The second approach to reducing Cv is to increase the hydrostatic pressure on the solution. It was shown by Kanno et aL (1975) that the homogenous nucleation temperature is depressed by applying high hydrostatic pressure. This added bonus in using high pressures is that Tg increases with increasing pressure as a result of decreasing fluidity caused by compression (MacFarlane et al., 1986). Thus, exposure to high hydrostatic pressure results in a decrease in Th and an increase in Tg, the net effect of which is to move to lower concentrations. For example, the homogenous nucleation temperature of a 35% aqueous DMSO solution is -80~ but exposure to 1300 atm pressure allows the solution to vitrify (MacFarlane, 1987). On the negative side, the application of high hydrostatic pressures may have damaging effects. Dog kidneys can survive a 20-min exposure to 1000 atm (Karow et aL, 1970), whereas rabbit kidneys are severely damaged after 20 min at 500 atm. However, on the plus side, high pressure is required only during the vitrification process itself; subsequent storage would be at atmospheric pressures. The third scheme for reducing Cv is to include a nonpermeating polymeric material (Fahy et aL, 1984). This can be a compound too large to permeate the cell membrane, which thus stays in the extracellular space. Cells naturally contain high concentrations of proteins, which are known to help vitrification. The implication here is that a greater concentration

350

John K. Critser, Yuksel Agca, a n d Karen T. Gunasena

of cryoprotectant is required to vitrify extracellularly than intracellularly. The addition of a high molecular weight polymer such as polyvinylpyrrolidone (PVP) may be sufficient to vitrify the extracellular milieu with the same concentration cryoprotectant as is required intracellularly. Fahy has shown that in certain cases a polymer can reduce Cv by an average of 7% and if this is combined with the effect of pressure, Cv is reduced by up to 24% (Fahy et aL, 1984). A tissue that has been vitrified and stored at liquid nitrogen temperatures must be warmed to ambient temperature before it can be used. During warming the tissue is susceptible to devitrification and recrystallization. Warming protocols based on thermal conduction cannot be used at this stage because heat diffusion via conduction mode may cause crystallization (Sutton, 1991). To ensure a uniform and fast warming rate throughout the biological material, electromagnetic (or microwave) heating is used. However, selection of the correct frequency is vital since the tissues may be simply "cooked." Currently, various radio and ultra high frequencies are under investigation at powers up to 1 kW (Sutton, 1991). Warming of up to 1200 K/min has been claimed by using radio frequency heating (Ruggera and Fahy, 1989). The crucial point is to identify the frequency by closely matching it with the dielectric properties of cryoprotectant solutions, thus obtaining more uniform temperature field distribution (Rachman et al., 1990). In light of these data, an approach which seeks to utilize vitrification as an alternative to conventional freezing will necessarily need to be concerned with the potential increased concentrations of cryoprotectant solutes used. Therefore, experiments investigating these effects are of critical importance to the further development of vitrification approaches.

VI. SUMblARY In several mammalian species, the ultrastructure of the immature (GV) and mature (MII) stage oocyte--cumulus cell complexes have been described (bovine [Hyttel et aL, 1989; Van Blerkom et aL, 1990];human [Motta et al., 1995; Van Blerkom and Henry, 1988; Van Blerkom et al., 1990], mouse (Pickering et a/.,1990), and rats (Zernicka-Goetz et aL, 1993]). There are subtle, but potentially critical, subcellular differences which exist among oocytes from different species. Additionally, fundamental changes in the structure and function of the oocyte occur as they develop from the GV stage (in which the chromosomes are well protected in their condensed form), to the MII stage (where the chromosomes and connected spindle fibers freely exist in the cytoplasm). There is an acute need to develop an understanding of how these differences among species and during oocyte maturation relate to their fundamental cryobiological characteristics.

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REFERENCES Aigner, S., Van der Elst, J., Siebzehnrubl, E., Wildt, L., Langh, N., and Van Steirteghem, A. (1992). The influence of slow and ultra rapid freezing on the organization of the meiotic spindle of the mouse oocyte. Hum. Reprod 7, 857-864. Albertini, D. F. (1995). The cytoskeleton as a target for chill injury in mammalian cumulus oocyte complexes. Cryobiology 32, 551-552. [Abstract] Albertini, D. F., and Rider, V. (1994). Patterns of intercellular connectivity in the mammalian cumulus-oocyte complex. Microsc. Res. Tech. 27, 125-133. A1-Hasani, S., van der Ven, H., Diedrich, K., and Krebs, D. (1986). Successful in vitro fertilization of frozen/thawed human oocytes. In Workshop on Embryos and Oocytes Freezing (Reports), pp. 25-39. Collection Foundation Marcel Merieux, Annenc. [As cited in A1-Hasani et al., Hum. Reprod. 2, 695] A1-Hasani, S., Diedrich, K., van der Ven, H., and Krebs, D. (1988). Cryopreservation of human and rabbit oocytes. Cryobiology 25, 584-585. [Abstract] AI-Hasani, S., Kirsch, J., Diedrich, K., Blanke, S., van der Ven, H., and Krebs, D. (1989). Successful embryo transfer of cryopreserved and in vitro fertilized rabbit oocytes. Hum. Reprod. 4, 77-79. Ali, J., and Shelton, N. (1993). Vitrification of preimplantation stages of mouse embryos. J. Reprod. Fertil. 98, 459-465. Angelova, P. A., Zvetkova, E. B., Dikov, A. L., Baleva, K. P., and Neronov, A. Y. (1991). Diagnosis of the viability of rat oocytes after cryopreservation. CR Acad. Bulgare Sci. 44, 81-83. Arav, A., Stefaneli, E., Leslie, S. J., and Crowe J. H. (1995). Phase transition temperature and chilling sensitivity of immature, in vitro matured and electrofused immature and in vitro mature bovine oocytes. Cryobiology 32, 570. [Abstract] Ashwood-Smith, M. J. (1986). The cryopreservation of human embryos. Hum. Reprod. 1, 319-332. Ashwood-Smith, M. J., Morris, G. W., Fowler, R., Appleton, T. C., and Ashhorn, R. (1988). Physical factors are involved in the destruction of embryos and oocytes during freezing and thawing procedures. Hum. Reprod. 3, 795-802. Bornslaeger, E. A., and Schultz, R. M. (1985). Regulation of mouse oocyte maturation: Effect of elevating cumulus cell cAMP on oocyte cAMP levels. Biol. Reprod. 33, 698. Bouquet, M., Selva, J., and Aureoux, M. (1993). Cryopreservation of mouse oocytes: mutagenic effects in the embryo? Biol. Reprod. 49, 764-769. Brower, P. T., and Schultz, R. M. (1982). Intracellular communication between granulosa cells and mouse oocytes: Existence and possible nutritional role during oocyte growth. Dev. BioL 90, 144. Carroll, J., Depypere, H., and Matthews, C. D. (1990a). Freeze-thawed-induced changes of the zona pellucida explains decreased rates of fertilization in frozen thawed mouse oocytes. J. Reprod. Fertil. 90, 547-553. Carroll, J., Whittingham, D. G., Wood, M. J., Teller, E., and Gosden, R. G. (1990b). Extraovarian production of mature viable mouse oocytes from frozen primary follicles. J. Reprod. Fertil. 90, 321-327. Chen, C. (1986). Pregnancy after human oocyte cryopreservation. Lancet 1, 884-886. Critser, J. K., Arneson, B. W., Aaker, D. V., and Ball, G. D. (1986). Cryopreservation of hamster oocytes: Effects of vitrification of freezing on human sperm penetration of zona free hamster oocytes. Fertil. Steril. 46, 277-284. DeMayo, F. J., Rawlins, R. G., and Dokelow, W. R. (1985). Xenogenous and in vitro fertilization of frozen thawed primate oocytes and blastomere separation of embryos. Fertil. Steril. 43, 295-300.

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Diedrich, K., AI-Hasani, S., and Van der Ven, D. (1988). Successful in vitro fertilization of frozen-thawed rabbit and human oocytes. Ann. N.Y. Acad. Sci. 541, 562-570. Fahy, G.M. (1989). Hand-Out for Mini-Lecture on Vitrification: Pre-Meeting Tutorial, June 10, 1989. 6th Annual Meeting, Society for Cryobiology. Fahy, G. M., MacFarlane, D. R., Angell, C. A., and Meryman, H. T. (1984). Vitrification as an approach to cryopreservation. Cryobiology 21, 407-426. Fuku, E., Kojima, T., Shioya, Y., Markus, G. J., and Downey, B. R. (1992). In vitro fertilization and development of frozen-thawed bovine oocytes. Cryobiology 29, 485-492. Fuku, E., Liu, J., and Downey, B. R. (1995). In vitro viability and ultrastructural changes in bovine oocytes treated with a vitrification solution. Mol. Reprod. Devel. 40, 177-185. Fuller, B. J., Hunter, J. E., Bernard, A. G., McGrath, J. J., Curtis, P., and Jackson, A. (1992). The permeability of unfertilized oocytes to 1,2-propanediol: A comparison of mouse and human cells. Cryo-Letters 13, 287-292. Gao, D. Y., Benson, C. T., Liu, C., McGrath, J. J., Critser, E. S., and Critser, J. K. (1996). Development of a novel microperfusion chamber for determination of cell membrane transport properties. Biophys. J. 71, 443-450. George, M. A., and Johnson, M. H. (1993). Cytoskeletal organization and zona sensitivity to digestion by chymotrypsin of frozen-thawed mouse oocytes. Hum. Reprod. 8, 612-620. Gook, D. A., Osborn, S. M., and Johnston, W. I. H. (1993a). Cryopreservation of mouse and human oocytes using 1,2-propanediol and the configuration of the meiotic spindle. Hum. Reprod. 8, 1101-1109. Gook, D. A., Osborn, S. M., Bournem H., and Johnston, W. I. H. (1993b). Fertilization of human oocytes following cryopreservation. Normal karyotypes and absence of stray chromosomes. Hum. Reprod. 9, 684-691. Gook, D. A., Osborn, S. M., Bourne, H., and Johnston, W. I. H. (1994). Fertilization of human oocytes following cryopreservation; normal karyotypes and absence of stray chromosomes. Hum. Reprod. 9, 684-691. Gook, D. A., Osborn, S. M., and Johnston, W. I. H. (1995). Parthenogenetic activation of human oocytes following cryopreservation using 1,2-propanediol. Hum. Reprod. 10, 654-658. Hamano, S., Koikeda, A., Kuwayama, M., and Nagai, T. (1992). Full-term development of in vitro matured, vitrified, and fertilized bovine oocytes. Theriogenology 38, 10851090. Hammerstedt, R. H., Amann, R. P., Rucinsky, T., Morse, P. D., Lepock, J., Snipes, W., and Keith, A. D. (1976). Use of spin labels and electron spin resonance spectroscopy to characterize membranes of bovine sperm: effect of butylated hydroxytoluene and cold shock. Biol. Reprod. 14, 381-397. Heller, D. T., Cahill, D. M., and Schultz, R. M. (1981). Biochemical studies of mammalian oogenesis: Metabolic cooperativity between granulosa cells and growing mouse oocytes. Dev. Biol. 84, 455. Hernandez-Ledezma, J. J., and Wright, Jr., R. W. (1989). Deep freezing of mouse one-cell embryos and oocytes using different cryoprotectants. Theriogenology 32, 735-743. Hochi, S., Fujimoto, T., and Oguri, N. (1995). Viability of immature horse oocytes cryopreserved by vitrification. Theriogenology 43, 236. Hunter, J. E., Bernard, A., Fuller, B., Amso, N., and Shaw, R. W. (1991). Fertilization and development of the human oocyte following exposure to cryoprotectants, low temperature and cryopreservation: A comparison of two techniques. Hum. Reprod. 6, 14601465. Hunter, J. E., Bernard, A., Fuller, B. J., McGrath, J. J., and Shaw, R. W. (1992a). Measurement of the membrane water permeability (Lp) and its temperature dependence (activation energy) in human fresh and failed-to-fertilize oocytes and mouse oocytes. Cryobiology 29, 240-249.

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Hunter, J. E., Bernard, A., Fuller, B. J., McGrath, J. J., and Shaw, R. W. (1992b). Plasma membrane water permeabilities of human oocytes: The temperature dependence of water movement in individual cells. J. Cell Physiol. 150, 175-179. Hyttel, P., Greve, T., and Callesen, H. (1989). Ultrastructural aspects of oocyte maturation fertilization in cattle. J. Reprod. Fertil. 38(Suppl.), 35-47. Johnson, M. H., and Pickering, S. J. (1987). The effects of dimethylsulphoxide on the microtubular system of the mouse oocyte. Development 100, 313-324. Joly, C., Behini, O., Boulekbache, H., Testart, J., and Bernard, M. (1992). Effects of 1,2propanediol on the cytoskeletal organization of the mouse oocyte. Hum. Reprod. 7, 374-378. Jones, H. W., Jr. (1990) Cryopreservation and its problems. Fertil. Steril. 53, 780-784. Kanno, H., Speedy, R. J., and Angell, C. A. (1975). Supercooling of water to -96~ under pressure. Science 189, 880-881. Karow, A. W., Liu, W. P., and Humphries, A. L., Jr. (1970). Survival of dog kidneys subjected to high pressures: Necrosis of kidneys after freezing. Cryobiology 7, 122-128. Kasai, M., Iritani, A., and Chang, M. C. (1979). Fertilization in vitro of rat ovarian oocytes after freezing and thawing. Biol. Reprod. 21, 839-944. Kazem, R., Thompson, L. A., Sirikantharajah, A., Laing, M. A., Hamilton, M. P. R., and Templeton, A. (1995). Cryopreservation of human oocytes and fertilization by two techniques: In vitro fertilization and intracytoplasmic sperm injection. Hum. Reprod. 10, 2650-2654. Knoppers, B. M., and Bris, S. L. (1993). Ethical and legal concerns: Reproductive technologies 1990-1993. Curr. Opin. Obstet. Gynaecol. 5, 630-635. Kola, I., Kirby, C., Shaw, J., Dave, A., and Trounson, A. (1988). Vitrification of mouse oocytes results in aneuploid zygotes and malformed fetuses. Teratology 38, 467-474. Kono, T., Kwon, O. Y., and Nakahara, T. (1991). Development of vitrified mouse oocytes after in vitro fertilization. Cryobiology 28, 50-54. LeGal, F., Gasqui, P., and Renard, P. J. (1994). Differential osmotic behavior of mammalian oocytes before and after maturation: A quantitative analysis using goat oocytes as a model. Cryobiology 31, 154-170. LeGal, F. (1996). In vitro maturation and fertilization of goat oocytes frozen at germinal vesicle stage. Theriogenology 45, 117-185. Leibo, S. P., McGrath, J. J., and Cravalho, E. G. (1975). Microscopic observation of intracellular ice formation in mouse ova as a function of cooling rate. Cryobiology 12, 579. [Abstract] Leibo, S. P., Martino, A., and Pollard, J. W. (1995). Chilling injury of mammalian oocytes, zygotes, and embryos. Cryobiology 32, 551. [Abstract] Lim, J. M., Fukui, Y., and Ono, H. (1991). The post-thaw developmental capacity of frozen bovine oocytes following in vitro maturation and fertilization. Theriogenology 35, 12251235. Luyet, B. J., and Gehenio, P. M. (1940). Life and Death at Low Temperatures. Biodynamica, Normandy. MacFarlane, D. R. (1987). Physical aspects of vitrification in aqueous solutions. Cryobiology 24, 181-195. MacFarlane, D. R., Scheirer, J., and Smedley, S. I. (1986). Pressure coefficients of conductance and of glass transition temperatures in concentrated aqueous LiC1, LiI and A1C13solutions J. Phys. Chem. 90, 2168-2173. Magnusson, C., and Hillensjo, T. (1977). Inhibition of maturation and metabolism of rat oocytes by cyclic AMP. J. Exp. Zool. 201, 139. Martino, A., Pollard, J. W., Nakagawa, A., and Leibo, S. P. (1995a) The kinetics of chilling sensitivity of bovine oocytes cooled to non-physiological temperatures. Theriogenology 43, 272. [Abstract] Martino, A., Songsasen, N., and Leibo, S. P. (1995b). Development into blastocysts of bovine oocytes cryopreserved by ultrarapid cooling of very small samples. Cryobiology 32, 565. [Abstract]

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Cryopreservation of Multicellular Embryos and Reproductive Tissues Sharon Paynter, Angela Cooper, Non Thomas, and B a r r y F u l l e r Department of Obstetrics and Gynaecology University of Wales College of Medicine Heath Park, Cardiff CF4 4XN, Wales

I. INTRODUCTION The importance in infertility treatment of successful cryopreservation of single cell suspensions of gametes (spermatozoa or oocytes) has been addressed in preceeding chapters. However, there is an equal need for successful freezing of multicellular systems in other contexts within the same overall strategy. Probably the most challenging requirement is the need to cryopreserve intact reproductive tisssues, particularly ovarian tissue, as a source of oocytes for fertilization (either outside the body in an artificial culture system or after grafting of stored tissue into a suitable host). As described later, ovarian tissue comprises a complex structure of reproductive cells and supporting stromal cells, which normally interact in specific ways to produce competent gametes. There are a variety of clinical, ethical, and economic reasons why it would be advantageous to preserve and manipulate ovarian tissues in vitro to provide material for infertility treatment. In addition, the manipulation in culture of preimplantation embryonic stages (ranging from one-cell fertilized oocytes through to multicellular blastocyst stages) requires similar attention to devise successful cryopreservation protocols, although in these circumstances the degree of complexity of cell-cell interaction is not so great. In Reproductive Tissue Banking Copyright 9 1997 by Academic Press. All rights of reproduction in any form reserved.

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this chapter we have set out to describe the philosophies behind current approaches to cryopreserving these multicellular tissues and have reviewed what is currently known about the success of these strategies. We have also drawn attention to the areas where information is lacking and which might highlight effort for future studies to improve cryopreservation protocols.

II. CRYOPRESERVATION OF REPRODUCTIVE TISSUE

The successful cryopreservation of ovarian tissue would have many benefits for infertility treatment. A direct application of such a technique would be in overcoming infertility in cancer patients rendered iatrogenically infertile by harmful treatments such as chemotherapy and radiotherapy which indiscriminately destroy diseased as well as healthy cells. Currently, patients about to undergo cancer treatment have a number of options regarding the preservation of their gametes. Stimulation by gonadotrophins resulting in the production of many oocytes could be undertaken and oocytes collected. However, the success of hormonal stimulation in these patients is unpredictable and when time is limited it may be difficult to obtain sufficiently large numbers of oocytes. Once collected, they could either be frozen directly or fertilized and then frozen. Although the fertilization of human oocytes following cryopreservation has met with limited success to date with only a few live births reported from frozen oocytes (Chen, 1986; Van Uem et al., 1987), the alternative option of freezing embryos (whilst overcoming the practical difficulties of freezing oocytes) raises a number of ethical considerations, especially if the patient does not already have a committed partner. The proposed alternative would be to remove an ovarian wedge biopsy prior to cancer therapy and cryopreserve it indefinitely until required by the patient. The cryopreservation of an ovarian biopsy would allow the storage of many potential gametes at different stages of development. It would be of benefit to cryopreserve immature oocytes since mature oocytes are arrested in metaphase II (i.e., they have their chromosomes lined up on the meiotic spindle) and are consequently very sensitive to changes in temperature. When required, the tissue could be thawed and either grafted directly into the host or cultured in vitro to yield oocytes for fertility treatment. Although grafting of frozen-thawed tissue has led to success in animals (Gosden et al., 1993, 1994) and may have advantages in that the in vivo environment is optimal for growth and development of oocytes, it also has a number of flaws. In many patients it might be difficult to reestablish normal tubo-ovarian relationships and, in cancer patients, metastatic cells, present in the vasculature may survive the freeze-thaw process and reimplant. Also, the applications for tissue donation between individuals are limited because of an immune reaction to the graft, itself requiring further drug treatment. The alternative option of culturing the tissue in

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order to release mature oocytes which could be fertilized is more practical. The resultant embryos could then be replaced into the patient when required and once successful cancer therapy has been completed. The successful "banking" of ovarian tissue would also provide a potential source of "third party" donor oocytes for in vitro fertilization (IVF) patients who are unable to produce their own oocytes. Other clinical applications may include the storage of ovarian tissue from women who have numerous healthy immature oocytes within their ovaries but are unable to mature the oocytes successfully in vivo, as in the condition known as Turner's Syndrome. Tissue could be removed from these patients, stored, and matured in vitro under optimum culture techniques. A . S t r u c t u r e a n d P h y s i o l o g y o f the

Ovary

The structure and function of the ovary have been presented in detail earlier and a brief review is all that is necessary at this point merely to emphasize how the many different cell types within the tissue must survive cryopreservation if it is to be of any clinical use. The functional unit of the ovary, the primordial follicle, is composed of an oocyte surrounded by granulosa cells. During maturation the oocyte increases in diameter and proceeds through meiotic division, the whole process being controlled by hormones and growth factors produced by the surrounding granulosa and thecal cells. In addition a number of locally produced proteins such as inhibin and activin are involved in regulating follicle growth. The delicate interaction and timing of all these factors ensures that in a typical month only one follicle reaches maturity and only one oocyte is released for fertilization. The exact control mechanism whereby a dominant follicle is recruited has yet to be elucidated and suggestions include a local environment where growth conditions are optimal and where gradients of the numerous factors involved are neither too high nor too low. Once such a follicle has become established positive feedback mechanisms might apply with the follicle increasing in size until it eventually ruptures. It is therefore obvious that to mimic these exact conditions in vitro will be difficult since the ovary is a complex structure requiring a high degree of physiological and metabolic interdependence between the many specialized cell types for normal function. If cryopreservation of ovarian tissue is to be successful it will be essential to maintain the delicate communication between all these elements especially if fertility is to be re-established using thawed tissue. In addition, if mature oocytes are to be produced from ovarian tissue after cryopreservation culture conditions must be established which will reproduce those pertaining in vivo. B. H i s t o r i c a l R e v i e w o f C r y o p r e s e r v a t i o n

of Ovarian Tissue

A rather comprehensive study on fundamental research concerned with cryopreserving ovarian tissue was carried out during the 1950s by a team

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at the National Institute for Medical Research (Mill Hill, London) headed by Sir Alan Parkes (Parkes, 1956, 1957, 1958). Prior to this, attempts at culturing or implanting rat ovaries which had been frozen as 1 mm 3 pieces in liquid air without the presence of a cryoprotectant (CPA) had failed to produce vaginal cornification in the recipient animals (Payne and Meyer, 1942) and, although Lipschtitz had managed to achieve endocrine activity in grafted guinea pig ovaries which had been stored at low temperatures, negative results were obtained when ovaries were stored below 0~ (Lipschtitz, 1928). When experimental ovaries are returned to an oophorectomized animal, vaginal cornification is often used as an indicator that estrogen production, and therefore ovarian function, has been restored to recipient animals. However, although estrogen production may be reestablished in the grafts, the oocyte is considerably more sensitive to changes in temperature than surrounding tissues and as such, estrogen activity alone is not sufficient to determine whether the ovarian graft has been fully successful. One of the most significant events in the history of cryobiology came in 1949 with the discovery that glycerol conferred cryoprotecting properties to fowl spermatozoa (Polge et al., 1949) and this finding allowed major developments to be made in cryopreserving a variety of tissues. The first positive results concerning freezing ovarian tissue came with a study by Smith (1952) which showed that rabbit granulosa cells could be grown in culture after slow cooling to -79~ either in serum, 15% glycerol-serum, or in 15% glycerol-saline. A series of studies were then conducted using 15% glycerol-saline and a slow-cooling regime in an attempt to cryopreserve pieces of rat ovarian tissue. Parkes and Smith (1953, 1954) exposed ovarian tissue (1 mm 3 pieces) to CPA for 1-2 hr at room temperature before transferring it to tubes to be frozen to either -79~ or -190~ The slow-cooling method using the Lovelock apparatus (Polge and Lovelock, 1952) enabled tissue to be gradually cooled and proved to be more successful than rapid-cooling methods. It was established during the course of the experiments that longer exposure to glycerol caused harm to the tissue and that the number of grafts becoming established after cryopreservation using glycerol-serum was greater than those frozen with glycerol-saline. Although functional activity of the grafts was assessed by examination for the cyclical vaginal cornification, histological appearances of the tissue following grafting showed that a proportion of the ovarian cells were destroyed and that oocytes at all stages of development were particularly sensitive. In these studies grafts were made either into the same individual (autograft) or into an individual of the same species (originally designated as "homograft" but now more commonly referred to as an allograft) and the number of successful "takes" with autografts was higher than with allografts. Autografts are known to be readily vascularized and obviously do not provoke an immune response; allografts, however, do succumb to

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lymphocyte infiltration caused by the "allograft reaction" and have delayed vascularization with eventual graft atrophy. These observations from animal experimental work are important indicators for human treatment and should be treated as a cautionary note for the grafting of ovarian tissue between individuals (unless immune-modulating drugs are also used). Deanesly (1954) followed a protocol similar to that of Parkes and Smith allowing the tissue (half a rat ovary) to equilibrate for 1-2 hr prior to slow cooling to -79~ Histological evaluation of tissue at the time of implantation showed damage to be extensive, with the healthy follicles to be found only at the exterior of the tissue, suggesting that tissue toward the interior of the organ had received little cryoprotection (see below). The type of damage observed was similar to that seen in the glycerolexposed controls, although in this case, more ova and follicles were shown to persist than seen in thawed material. However, despite this obvious tissue damage, examination of recipients in order to check the return of the estrous cycle showed most grafts to be successful and histological assessment of grafts postimplantation showed some ova and follicles to have survived the freeze-thaw process. The first successful cryopreservation study on mouse ovarian tissue was conducted by Parkes (1956), again using the same freezing protocol. Autografting was followed by examination for vaginal cornification as an indicator that the graft had become established. Using this as a measure Parkes demonstrated no difference between oestrogen production in fresh, glycerol-exposed or frozen tissue. Further papers by Green et al. (1956), Deanesly and Parkes (1956), Deanesly (1957), and by Parkes (1957, 1958) comprehensively review the literature and discuss the variables involved in freezing and thawing ovarian tissue using glycerol in order to achieve a viable graft. Three methods of investigation were used in most studies in order to assess experimental success: the proportion of animals in which a graft had become established, the average latent interval, and the survival time of the graft. To summarize, the most successful protocols used quartered rat tissue which was exposed to 15% glycerol, shown to be more effective than 10 or 5%, although concentrations higher than 15% had previously been found to be ineffective (Parkes and Smith, 1953), possibly because of toxic effects of the CPA. Equilibration of tissue for 1-2 hr at room temperature was followed by slow cooling at a controlled rate and subsequent thawing by rapidly plunging into water at +40~ Endocrine activity quickly developed with glyceroltreated tissue but grafts which had been cryopreserved in diethylene glycol, monoethyl ether, methanol, or carbowax all failed presumably because the substances had failed to protect against freezing damage at -79~ or because of toxicity. Various other factors investigated included the exposure time which could be reduced from 1 hr to 15 min without affecting the tissue recovery, and in fact longer incubation periods (such as overnight exposure) proved lethal to the tissue. A rapid cooling rate was investigated

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and was most damaging, whereas a two-stage method with a "hold" period at -20~ seemed to be most effective. Changes in the pH of the CPA solutions used, either above 7.8 or below 5.8, also decreased the number of graft takes. A landmark paper by Parrott in 1960 investigated the freezing of mouse ovarian tissue in detail, again slow cooling tissue to -79~ after exposure of tissue for 30-40 min to 12% glycerol in horse serum. She investigated the grafting by assessing: (1) the proportion of recipient mice fertile after grafting, (2) the length of time that mice remained fertile, and (3) the number of oocytes remaining in the grafts recovered at autopsy. This thorough investigation allowed both oocyte survival and endocrine function to be assessed following cryopreservation. Although some live young were produced from frozen-thawed ovarian tissue grafts, Parrott showed that the number of oocytes that survived was low and that the reproductive life of the females bearing the grafts was curtailed in each of the four strains of mice used. Parrott's results agreed with the work of Parkes in that oocyte destruction was shown to be increased when the concentration of glycerol was reduced or if the tissue was exposed for longer periods. These results agreed with those of Deanesly (1954, 1957), who also demonstrated that glycerol solutions had a damaging effect upon oocytes within ovarian tissue, with 80% of the oocytes in the tissue destroyed by exposure to a solution of 15% glycerol at room temperature; however, when this was followed by freezing and thawing no survivng oocytes could be demonstrated. In 1976, St~ihler et al. published an investigation into the cryopreservation of human ovarian tissue. Ovaries were obtained during the follicular phase from oophorectomy patients aged between 30 and 41 years who showed normal menstrual cycles and ovaries were immediately flushed in ice-cold heparinized perfusion medium prior to perfusion of the cryoprotectant via a canula into the ovarian artery. Tissue was cooled rapidly to -18~ and rapidly thawed by perfusing a warm solution containing bovine serum albumin (BSA). Investigations of oxygen and glucose absorption showed that some degree of protection by CPA from freezing damage was conferred to the tissue but the CPA solution failed to protect the tissue from lethal damage when cooling was continued to liquid nitrogen temperatures which would be necessary for long-term storage. Although successful cryopreservation of ovarian tissue would have great benefits for the treatment of infertility, another application would be in the possible conservation of endangered species. A paper by Candy et al. (1993) reported on the cryopreservation of ovarian tissue from the common marmoset. Tissue was exposed to 1.5 M dimethylsulfoxide (Me2SO) in M2 medium supplemented with 10% fetal bovine serum prior to slow cooling (0.3~ to -40~ and storage in liquid nitrogen. Rapid warming was followed by step-wise dilution to remove the Me2SO and

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tissue was transplanted beneath the kidney capsule of immunologically incompetent bilaterally ovariectomized mice. Tissue was removed between 7 and 28 days later and histological examination showed follicles at all stages of development, suggesting successful cryopreservation and grafting. Using a similar protocol, Harp et al. (1994) reported on the cryopreservation of whole mouse ovaries in 1.4 M Me2SO in Tyrode's salts. Exposure at room temperature for 5 min was followed by cooling at 0.5~ to -55~ and immersion into liquid nitrogen at -196~ After rapid thawing (15~ min) the tissue was washed in Tyrode's salts to remove the CPA and tissue transplanted. Vaginal cytology showed that grafts had become established, suggesting vascularization and viability of transplanted ovarian tissue within 1 week. Histological evaluation revealed that 63% of the ovaries cryopreserved demonstrated progressive maturation with many primordial and primary follicles as compared to 75% of controls which demonstrated similar progression. Most impressively, using the same freezing regime (1.5 M Me2SO followed by slow cooling) Gosden et al., (1994) successfully demonstrated the restoration of fertility to sheep and the birth of a lamb after grafting ovarian tissue which had been stored at -196~ Tissue slices were slow-cooled at a rate of 2~ to -7~ followed by manual seeding. The temperature was then lowered by 0.3~ to -40~ and thereafter by 10~ to -140~ followed by plunging into liquid nitrogen. Samples were allowed to thaw at room temperature before transfer to a water bath to complete the thawing process. The tissue was then grafted back into the same animal. Examination of samples 1 week after grafting showed that none of the developing follicles had survived but there were small groups of primordial follicles which appeared to be normal and viable. Follicles seemed to be generally less abundant after frozen storage, although it was difficult to be sure because of the variation between individual grtafts. It was also very difficult to comment on hormone concentrations as these varied considerably. A different method of freezing ovaries was investigated by Miyamoto and Sugimoto (1994) who cryopreserved rat ovaries using a vitrification technique. The CPA was removed by step-wise dilution in sucrose solutions. Histological examination of follicles showed those near the surface appeared well-preserved but degenerative changes were observed throughout the tissue such as pyknosis, vacuolation, and cell swelling. Although the tissue showed partial viability it was accepted that the slow-cooling method was better. Throughout these studies some investigators had pointed out the need for a quantitative study to assess different cryopreservation protocols. Therefore, we conducted a histological investigation of frozen-thawed pig and mouse ovarian tissue (Thomas et al., 1995a). Tissue was frozen in different CPA solutions and compared to exposed and normal controls.

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Freeze-thaw damage was estimated by attributing a grade to each individual follicle in each section based on damage and degree of shrinkage of granulosa and thecal cells surrounding the oocyte. Three different CPAs were investigated (1.5 M MezSO plus 10% fetal calf serum (FCS), 1.5 M 2,3-butanediol, 10% glycerol). Examination of all frozen specimens showed a range of follicular damage which could not be attributed to a particular area of the tissue. In Figure 1 are shown photomicrographs of normal ovarian tissue (Figure 1A, showing a large antral cavity, surrounded by closely adherent layers of granulosa cells, and containing an oocyte) and types of pathologies noted after cryopreservation. Mild damage (Figure 1B) was expressed as separation of layers of granulosa cells surrounding the antral cavity, while nuclei of individual granulosa cells were pyknotic. More severe damage (Figure 1C) was accompanied by cavities developing in the normally contiguous layers of cells in the granulosa and theca. Results indicated that tissue frozen in butanediol showed maximal damage while those in MezSO minimal. Control samples had least damage but exhibited slight shrinkage due to the action of the fixative. Attempts to circumvent the problems of cryopreserving intact ovarian tissue have also been made by taking the approach of isolating primary follicles immediately and then cryopreserving these for later use, in the hope that by dealing with smaller functional units, problems inherent in cryopreserving tissue masses (see later) may be avoided. Mouse ovarian tissue was dissociated by enzyme digestion so that isolated primary follicles could be frozen in 1.5 M MezSO (Carroll et aL, 1990). Following cryopreservation the follicles were cultured in collagen gels initially in vitro and then subsequently in vivo beneath the kidney capsule of ovariectomized mice. Following hormonal stimulation the collagen gels were removed from the mice and the resultant oocytes isolated, fertilized, and returned to pseudopregnant mice, resulting in birth of live young. A similar study (Carroll and Gosden, 1993) again investigated mouse primordial follicles which, after slow cooling in MezSO, showed good viability using a trypan blue dye exclusion test. These frozen-thawed cells were then suspended in plasma clots and transplanted to the ovarian bursas of host animals and produced estrogenic activity and normal offspring. In a more recent report Chung et al. (1994) demonstrated the in vitro growth of frozen-thawed human and bovine ovarian follicles. Follicles were isolated by enzymatic degradation and cryopreserved with propane-l,2-diol solution. Following thawing follicles were cultured and showed increased diameter and protein synthesis during the growth period investigated. To summarize, success has been made over recent years in achieving live young from frozen-thawed tissue grafted into recipient animals. However, tissue damage is known to be considerable with the number of offspring produced and the reproductive life of the animals both shown to be reduced. For clinical use the need would be to culture frozen-thawed tissue in vitro

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so that resulting oocytes or embryos could be individually returned to the recipient. This would allow donation between individuals as well as conserving fertility in cancer patients and would obviously avoid any immune response consequent upon tissue grating. As mentioned earlier major developments are being made in the area of in vitro growth of ovarian tissue and once model systems have become established it should pave the way for the successful growth of frozen-thawed tissue.

m . FUNDAMENTAL ASPECTS OF OVARIAN TISSUE CRYOPRESERVATION The success of any cryopreservation procedure used to freeze individual cells depends upon carefully controlling heat and mass transfer (Mazur, 1970), so that movements of water and CPAs can be effected under conditions which avoid osmotic and chemical toxicities or damaging ice formation. These physical events can be described in mathematical terms, once certain constants have been defined, and this allows greater examination of the combined effects in the protocol or modeling to provide information which permits predictive (rather than empirical) experiments. In complex tissue structures values for many of these physical constants have either not been derived or have only recently beome available. Nevertheless, working within the limits of this current knowledge, some mathematical models which allow a deeper insight into transfer of heat, water, and CPAs in three-dimensional biological structures have been proposed, and these are discussed in the following section. A. P h y s i c a l P a r a m e t e r s o f Ice F o r m a t i o n in T i s s u e s

The physicochemical events which occur during freezing and thawing of biological systems have been well documented over the past 30 years (Mazur, 1970; Farrant, 1977), and are discussed elsewhere in this book (Chapter 6). To briefly restate the principles, cooling a biological system (with inherently high water content) below its equilibrium freezing point will result in nucleation and growth of ice crystals, and since biological membranes act to constrain nucleation in intracellular compartments, ice forms initially in the extracellular environment. On further cooling, the extracellular ice phase will grow, excluding solutes and thus increasing the osmolality of the extracellular solution. As cooling proceeds these changes will displace the thermodynamic equilibrium between extracellular and intracellular environments, and the system will attempt to regain equilibrium by increasing the osmolality of the intracellular fluid. This can be achieved by several mechanisms including efflux of water from the cell, influx of solute, or formation of intracellular ice. Which mechanisms pre-

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1 Light micrographs of sections of murine ovarian tissue. (A) Normal tissue showing a large antral follicle, A, containing an oocyte, surrounded by continuous layers of cells of the granulosa oophora and theca, B. (B) Tissue following cryopreservation using Me2SO, showing mild damage with separation of the layers of granulosa cells, C. (C) Cryopreserved tissue showing severe damage with gaps between the layers of granulosa, D, and theca, E.

FIGURE

dominate in a given situation will depend upon the permeability of cell membranes to water and solute, diffusivities of solutes in intra- and extracellular compartments, solution nonideality (including effects imparted by any added CPA), the time-temperature history, and cell volume to surface

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FIGURE I--Continued

area ratio. Several mathematical models have been proposed to describe the sequence of events in individual cells during freezing of cell suspensions, and these have been refined as and when experimental measurements for each important factor (such as cell membrane water permeability) have been verified experimentally (Levin, 1979; Mazur et al., 1984; Mazur and Schneider, 1986), such that there is now good agreement between predicted responses and experimental observations for many cell types. However, when moving to describe the freezing process in intact tissues, there are problems to be faced which are dictated by the biological structure. Inevitably in tissues, the cells will be packed at high density, with significant cell-cell and cell-matrix interactions, while the extracellular "fluid space" (comprising extracellular spaces, lumens of blood vessels, lymph ducts and other fluid-filled channels) is small relative to the volume of the combined intracellular compartment. These physical constraints will combine to affect heat and mass transfer in ways not encountered during freezing of dilute cell suspensions in small volumes, where the spatial location of extracellular ice in the suspensions is generally not considered to be of great importance; on the other hand, in tissues which may depend upon specific three-dimensional organization for normal function (as for example, the relationship between cells of the corona radiata and the developing follicle in the ovary), the potential disruption resulting from extracellular ice may be fundamentally important for post-thaw function (Hunt et aL, 1982). Nevertheless, advances have been made in understanding the process of freezing and thawing in intact tissues, and models have been proposed to describe the events (Hayes and Diller, 1983; Rubinsky, 1988). Rubinsky

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in particular has made several contributions in this area (Rubinsky and Cravahlo, 1984; Rubinsky et al., 1987; Rubinsky and Pegg, 1988). A description of one such model will be given in order to highlight the main factors currently perceived to influence the freezing process in tissues and to indicate what experimental investigations might be required to apply such an approach to ovarian tissue. It will be obvious from the descriptions of ovarian structure in this chapter that the ovaries of adult patients or females of other large mammalian species are relatively large (in terms of the volume of tissue which might be considered for freezing) and the cryopreservation of the intact ovary is rarely considered. The most optimistic reports on cryopreservation of ovarian tissues from large animals have concentrated upon the use of tissue fragments or slices (Gosden et aL, 1993; Candy et aL, 1993, 1995; Thomas et al., 1995a,b). Ovaries from small mammals (e.g.mouse) have been cryopreserved whole (Harp et al., 1994), which effectively represent similar approximate tissue volumes (2-4 mm 3) as fragments from large ovaries. This still represents a large volume of tissue with many cells linked by extracellular compartments and thus cannot be considered as a homogenous matrix during freezing. Several experimental observations have confirmed that ice will form preferentially in extracellular "fluid spaces," notably vascular spaces (Rubinsky et al., 1987), and that ice propagates through these spaces in the general direction of the temperature gradient across the sample. Ovarian tissue has an abundant vascular and capillary network, and there is no reason to assume that this will behave differently from other studied tissues, although direct experimental evidence has not yet been forthcoming. Ice can propagate through the vessel "fluid spaces" as cooling proceeds, while the water in the surrounding cells is compartmentalized in small volumes defined by membranes and will remain in a supercooled state while ice formation initially progresses. Consequently the cells will dehydrate as water moves down its chemical potential gradient to form ice in the fluid space, resulting in expansion of these small ice-filled spaces observed during direct cryomicroscopic observation of freezing in tissue (Rubinsky et aL, 1987). For purposes of modeling heat and mass transfer in tissue, it is necessary to define the "functional freezing unit." In physiology, several models have been proposed to describe mass transport in tissues under normal conditions (House, 1977; Hempling, 1988). The evidence and assumptions indicate that mass transport takes place predominantly in small blood vessels (of the size of capillaries), where the ratio between the surface area of vessel fluid space to that of the volume of surrounding tissue is much larger than in larger blood vessels. Because of the relatively uniform distribution of capillaries in a tissue, the tissue is modeled as a series of identical repeat units, based on the capillary plus surrounding cells, and this is commonly referred to as a "Krogh cylinder" after Krogh (1919), who developed the

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concept. The dimensions will affect the analysis, but usually these are taken as the diameter of the capillary, with the tissue space defined as the space between adjacent capillaries. In the specific case of freezing, the length of the capillary is not relevant, since there is no vascular flow, but for modeling it can be assumed that the tissue unit is cuboid in shape. Other assumptions are that the Krogh cylinder is small relative to the temperature gradients in the tissue (particularly at the relatively slow cooling rates used) and thus is at a uniform temperature at a given time during freezing and that the tissue component can be represented as a single compartment containing a solution of solutes in water, initially at osmotic equilibrium with the vessel fluid space. It is also assumed that the solute concentration in the tissue and fluid space can be represented by one averaged value, that this is also true for the mass transfer properties of the interface between tissue and fluid space, and that this interface is permeable to water but not solutes. The mass transfer process across the tissue/fluid space interface can be modeled by applying any one of a number thermodynamic realtionships, such as the Kedem-Katchalsky model of nonequilibrium thermodynamics (Kedem and Katchalsky, 1958). In the present example, Rubinsky used the simpler Fick's law, as .iv = P ( C v -

Ct),

[11

where ]~ is the volumetric flow of water from tissue to vessel fluid space per unit surface area, while Cv and Ct are solution molar concentrations in vessel and tissue, respectively. P represents the water permeability of the interface, comprising cell membranes and any extracellular matrix. As the Krogh cylinder is considered a closed unit during freezing, and since mass in the unit must be conserved, the flux of water from the tissue component into the vessel space must be accompanied by an equal change in volume of the tissue unit, Vt: jv = 1/VS d v t

d---7

[21

Also, since the interface between tissue and vessel space is impermeable to solute, the amount of tissue solute cannot change, and thus the molar concentration of solute in the tissue (Ct) can be evaluated at any specific time during freezing as a function of the starting concentration (Cto) and the volume at that selected time, by Ct = Cto Vt~ -- Vns V t - Vn s '

[31

where Vto is the initial tissue volume and Vns is the nonosmotically active volume of the tissue compartment. The molal solute concentration (Mv) can be predicted (as can the amount of ice formed in the vessel space)

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from the unique thermodynamic relationship between the temperature at a given time and the phase diagram for the ice-saline mixture (assumed to be predominantly sodium chloride). T = 273.15 - 1 . 8 6 o v M v ,

[41

where o is the osmotic coefficient and v the dissociation coefficient for the solute. Rubinsky (1988) combined these equations [1]-[4] to yield a differential equation for mass transfer in the Krogh cylinder as a function of change in temperature with time during cooling, dvt(t) _ P S v dt

[

T(t) - 273.15

1.86ov

Vto

ns]

+ Cto C t ( t ) - Vns "

[5]

In order to derive an equation for the balanced volume changes between tissue compartment and vessel space, the geometrical relationships of the Krogh cylinder can be added in, as

]

dr(t) = _ p [ T(t) - 273.15 - V__ns :_ .aX__ + Ct~ ( A S ) 3 - V n s - 7rr(t)2 A S ] ' dt [ 1.86ov

[6]

where the volume of the cuboid unit V = ( A S ) 3, volume of tissue unit Vt = V -7rr 2 AX, and space volume Sv = 27rrAX, with initial vessel space radius r(0) = ro and the maximal radius rmax = [(z~tX3 -- Vns ) / ~ ] 1 / 2 . These relationships reinforce the importance of the dimensions of the functional freezing unit in the tissue, the permeability of the interface to water, and time-temperature relationships within the cooling procedure. There are indeed further physicochemical parameters which need to be considered. These relate to problems of heat transfer under the nonlinear conditions introduced at the boundary between solid and liquid domains in the tissue as freezing progresses. Solidification processes such as freezing are also intimately linked to energy change resulting in the latent heat of phase transformation; thus to completely derive energy equations for the freezing process, account must be taken of energy within the system resulting from both sensible heat and latent heat. The assumptions and derivations required to produce linearized energy equations from these nonlinear conditions are beyond the scope of the current discussion and can be found elsewhere (Rubinsky, 1988). Their relative importances are likely to change as larger volumes are considered in the feezing process and under different time-temperature relationships. Freezing in bulk systems (of the magnitude of intact human ovaries) will be significantly affected by latent heat released and nonplanar interfaces as the ice front progresses. Even in small tissue pieces, it should be understood that the heat transfer properties of the freezing vial and suspending medium will have an impact on heat transfer.

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Bald (1993) has used finite element analysis to model temperature change based on the Biot modulus (Bi), Bi = h R / K ,

[7]

where h is the surface heat transfer coefficient, R the relevant dimension of the sample container (vial), and K the thermal conductivity of the contained sample. Even within a "typical" cryopreservation vial (containing approximately 1 ml of medium suspending cells or tissue pieces), small but significant differences in time-temperature profiles can be predicted and measured experimentally. The thrust of these discussions has been to reveal the complexities of tissue freezing, and the need for further studies in many areas in relation to ovarian tissue. In reality, the need is for parallel effort on both experimental verification of the numerical models derived from the biophysical principles, and refinement of the predictive models as more data are fed back from direct observations in "biological" samples. In ovarian tissue, there is at present little or no information on the important physical constants which will impact on the freezing process, and thus it is not surprising that there is room for improvement in current approaches to cryopreservation. Preliminary studies on one aspect of ovarian tissue cryobiology (the permeation of cryoprotectants~see below) suggest that in this one respect, ovarian tissue behaves in a fashion broadly similar to that of other tissues such as liver (Fuller and Busza, 1994), which have been more extensively modeled (Rubinsky, 1988), but even in this one aspect some differences in kinetics were noted. Thus much more information is required before the freezing process in ovarian tissue can be comprehensively predicted. Before moving on from freezing, one additional comment on ovarian tissue is required. The discussion so far has centered on ovarian tissue and the probability of ice formation within capillaries during freezing. In addition, the large fluid spaces of the antral cavities would seem equally good candidates for nucleation and growth of extracellular ice. There is no direct evidence, but from the earliest to the most recent reports of cryopreservation of ovarian tissue (Green et al., 1956; Gosden et al., 1993) it has been noted that the large antral follicles (several orders of magnitude larger than capillary lumens) are very susceptible to damage during freezing. Thus ice formation in antral cavities is one specific characteristic of ovarian tissue which may need to be considered in describing the freezing process. B. P e r m e a t i o n

of Ovarian Tissue by Cryoprotectants

One major step in any cryopreservation protocol which will have an impact on the effects of freezing in ovarian tissue is the degree of protection afforded by cryoprotectants. From the earliest studies of Deanesly (1954, 1957) and Green et al. (1956), empirically derived protocols for exposing

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ovarian fragments to CPAs were applied, derived from early work on cell freezing and from experimental observations. Even in these studies, it was realized that a balance had to be achieved between prolonging the exposure time to CPA solution to achieve good tissue permeation, versus the toxic (chemical and/or osmotic) effects of the CPA which increased with time of exposure at above-freezing temperatures (Deanesly, 1954, 1956). The problem has always been to measure CPA permeation in solid tissues. The methods of direct microscopy applied to single cells (see Chapter 6) are not applicable to tissue pieces. Elford (1970) undertook extensive investigations into measurements of CPA permeation in smooth muscle fragments using radiolabeled Me2SO. However, such methods require homogenization and tissue destruction to facilitate measurement. More recently, we have applied aH nuclear magnetic resonance (NMR) spectroscopy to measure permeation of tissue water by Me2SO in tissues. This is a noninvasive technique, which, at least in theory, could be used on intact tissue subsequently used for other tests such as histology. By exploiting the difference in chemical shifts between the protons of water and the CPA molecule, it is possible to measure the molar concentration of Me2SO in tissue water, and the method can be equally applied to measuring efflux of CPA from preloaded tissue samples. At commonly used field intensities, Me2SO displays a characteristic single peak in the proton NMR spectrum. The relative intensity of the water peak to that of Me2SO (measured as integrated areas) under fully relaxed conditions represents the molar concentration of protons in water to those of Me2SO, and can be given by

(CMe2SO- VMe2SO)]}- 1, [8] where R is the proton molar ratio, VH20 and VMe2SO are the partial molar R = 3.0(VH20/VMezSO){IO00/[IO00-

volumes, and CMe2SOis concentration in M. Using this technique, studies on permeation of cylindrical pieces of liver (chosen as a model for compact tissues) by Me2SO were performed at 0~ During exposure to CPA concentrations of up to 3 M, it was demonstrated that over the course of practically useful times of exposure (up to 2 hr)equilibration to only about 60% of the theoretical maximum was achieved (Fuller and Busza, 1994). Even after exposure for extended periods (up to 24 hr), only 75% equilibration was achieved. When the method was recently applied to study CPA permeation into ovarian tissue, Me2SO was selected since this was the agent used in the recent reports on cryopreservation of ovarian tissues in sheep (Gosden et al., 1993) and primates (Candy et al., 1995). The conditions of exposure were those used in the freezing protocols (exposure of ovarian tissue pieces for up to 20 min at 0~ with 1.5 M Me2SO), and porcine ovaries were chosen as a large animal model relevant in size and structure to the human ovary. The results showed that permeation under these conditions was relatively slow, with only 45% of the theoretical maximal equilibration achieved after 20 min (Thomas et al., 1995b). We have also recently

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investigated uptake of MeeSO into human ovarian tissue, and results were very similar (Figure 2), confirming that the porcine model is very appropriate for near-clinical studies. The data summarized in Figure 2 show the uptake and elution profiles (achieved by transferring the tissues, loaded with Me2SO for 20 min into fresh, cold CPA-free buffer). There was exponential uptake of CPA into the tissue pieces, reaching approximately 50% of theoretical maximum at 20 min, while after elution, this was reduced to 1% by 60 min. Thus there remained a significant proportion of "NMR- visible" ovarian tissue water which was inaccessible to CPA. This could reflect intracellular compartments such as intramitochondrial spaces (Garlid, 1979), where membranes may be so closely apposed to each other as to stearically "hinder" adjacent water molecules, rendering a fraction of the water unable to act as solvent for CPA. However, it could also represent the fact that diffusion was so restricted by the density of cell packing that only the surface layers of the ovarian tissue pieces could be penetrated by the Me2SO. There is some support for this latter explanation, because when NMR spectroscopy was used to study permeation of CPA into other tissues 0.8

0.9

y = 0.76971-0.5958 lxlog(x) r 2 =0.95455

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0.7-

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0.6 0.6 0.5

a) x o e-

0.4

ffl e--

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y=O.O7506+O.47958xlog(x) r2=0"9223 ! I

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10

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Log Time (min) F I G U R E 2 Measurements of (a) influx and (b) efflux of Me2SO into human ovarian tissue using 1H NMR spectroscopy. Tissue pieces (4 mm 3) were exposed to 1.5 M Me2SO in buffer. Conditions were as described in Thomas et al. (1995b). After 20 min, only approximately 50% of theoretical maximal equilibration had been achieved.

ii11 100

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(isolated small arteries) where continuous perfusion was used to introduce the agent, complete equilibration of the NMR-visible water was achieved in a comparatively short time (20-30 min) at 0~ (Bateson et al., 1994). Continuous perfusion is a much more efficient method for introducing CPA than surface diffusion, and the data suggest that in reality all tissue water is available to act as a solvent for CPA, which is in agreement with early studies using a radiolabeled tracer method (Elford, 1970). Nevertheless, it is apparent that the empirically selected methods for exposing ovarian tissues to CPA require considerable further investigation if optimal concentrations of CPA, sufficient to protect viability in all parts of the tissue fragment, are to be achieved even before systematic freezing studies are undertaken. Another approach to using NMR technology to study cryoprotectant permeation in tissues has been developed recently by Fyfe, Isbell, and colleagues (Fyfe et aL, 1994), who have applied NMR imaging to provide simultaneous measurements of both location and concentration of solvents in tissues in a noninvasive fashion. Signal intensity at each pixel location in the NMR image is related to the concentration of protons (and thus the solvents that contain them) at the corresponding site in the sample. Signal intensities are also dependent upon other physical NMR parameters within the imaging protocol, including relaxation times (T1 and T2) of excited nuclei, the pulse repetition time, and echo time, which affect the contrast of the acquired image. Isbell has determined the relevant parameters in model solutions of Me2SO/water at a variety of temperatures and in tissue slices exposed to different solvent concentrations (Isbell, 1996). There was a linear correlation between Me2SO concentration and signal intensity at both 1 and 20~ in spite of the predicted problems of contrast expected with short relaxation times. The technique has yet to be applied more extensively to measuring tissue CPA uptake, but offers the promise of providing real-time spatial information on degree of permeation which would be very valuable in ovarian tissue studies.

IV. CRYOPRESERVATION OF PREIMPLANTATION EMBRYOS

The ability to cryopreserve preimplantation embryos (preembryos) from both animal and human sources has helped to overcome some of practical concerns, as well as the ethical and moral dilemmas encountered in IVF and other assisted conception techniques. The purpose of this section is to explain the stages of development of an embryo (from fertilization to preimplantation) which are available for manipulation and cryopreservation, then to review the theoretical aspects and history of cryopreservation, and finally summarize by the methods and current opinion on embryo

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cryopreservation in both human infertility treatment and in animal breeding. The observation that embryo preservation could be undertaken successfully in several animal species has lead to the establishment of substantial commercial industries involving cattle and sheep embryo banks (Trounson, 1986). This industry now uses embryo banking extensively in animal breeding programs, in order to reduce the cost of maintaining expensive animal colonies in cases where animal usage is sporadic or when large numbers of animals are being held pending future use (Bernard, 1991) and to improve cost efficiency in large animal husbandry. The cryopreservation of animal embryos is also being used to maintain embryo banks of unique genetic information which can be held at low temperature indefinitely to prevent genetic drift and to maintain stable inbred strains for future research. Mouse embryo banks have also been extensively developed for the genetic conservation and breeding of rapidly proliferating mouse strains (Trounson, 1986) along, more recently, with storage of transgenic strains (Pomeroy, 1991). The cryopreservation of these valuable embryos not only protects them from environmental catastrophes but is also an economical method of storing lines for future detailed analysis (Pomeroy, 1991). The success of these techniques has allowed genetic material and species to be moved around the world and has also allowed indigenous and exotic breeds to be preserved by development of rare breed embryo banks (Fahning and Garcia, 1992). In clinical practice the experimentation undertaken on small laboratory animals has played an essential role in transferring methods and protocols to human IVF technology (Trounson and Wood, 1993). Superovulation techniques used in IVF raise the dilemma that large numbers of oocytes and embryos may be produced and cryopreservation of these would offer the following clinical advantages (Gelety and Surrey, 1993): (i) reduce the risk of multifetal gestation by limiting the number of embryos replaced in the initial stimulating cycle; (ii) increase the number of potential replacement cycles without additional egg retrievals, improving the overall pregnancy rate; (iii) decrease the potential risk of severe ovarian hyperstimulation syndrome by substituting frozen embryo transfer in unstimulated cycles; (iv) create potential for pregnancy following the birth of a child resulting from IVF-embryo transfer without additional oocyte retrieval. Embryo cryopreservation is now firmly established as a routine and important component of IVF and assisted conception techniques for the resolution of human infertility (Trounson, 1990; Titterington et al., 1995). This allows any supernumerary embryos to be cryopreserved for some time

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before a subsequent second attempt at replacement should the first attempt fail (VanSteirteghem et al., 1992). Despite the establishment of these methods in routine IVF, there are some disadvantages in that many programs are still struggling to achieve the first pregnancy from these cryopreserved embryos (Garrisi and Navot, 1992). However, the world results on success from frozen embryo transfer (VanSteirteghem and Vanden Abeel, 1990) indicate that 56.6% of embryos are recovered from frozen with an overall 4.1% implantation rate with a pregancy rate of 11.5% of each replacement. This is somewhat similar to that of fresh embryo transfer, although some abnormalities and malformations have been reported (VanSteirteghem et aL, 1992). Hence further research needs to be undertaken to refine methods and protocols within this field and facilitate the reproducibility of the technology. A. D e v e l o p m e n t o f t h e P r e - e m b r y o The development of an embryo begins after fertilization of the mature oocyte with a spermatozoon. The process by which spermatozoa meet the oocyte involves a very complex and intricate sequence of events which has been discussed in an earlier chapter and also by Harper (1994). With regard to IVF, briefly, the mature oocytes are aspirated from follicles of superovulated individuals and placed in culture with the spermatozoa (Paulson, 1993; Trounson and Wood, 1993). In different species the main features of embryogenesis are very similar; however, the timing of development is very different. In Hemicentrotus, Drosophilia and Zenopus the first 24 hr after fertilization can produce free living larvae composed of up to 60,000 cells, whereas in other species, e.g, mouse and human, the embryo is still only at the 2-cell stage after this time and division continues very slowly with no increase in mass as it moves along the oviduct for implantation (Hogan et aL, 1986). During development the embryo forms two cell lineages (the trophoectoderm and primitive endoderm) which form the basis of the placenta and extraembryonic yolk sac which are required for successful interaction with the mother (review: Pederson and Burdsal, 1994). The scope of this section is to investigate only the development of the preembryo, that is from fertilization to implantation.

1. Stages of Pre-embryo Development At fertilization the mature oocyte is arrested in the second meiotic metaphase having extruded the first polar body. When the spermatozoan reaches the oocyte, the acrosome reaction occurs, allowing the sperm to enter the zona pellucida of the oocyte. The head, midpiece, and a large part of the sperm tail are all incorporated into the cytoplasm, the midpiece contributes the paternal centrioles and mitochondria to the zygote but these are diluted by the oocyte cytoplasm.

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The fusion with spermatozoa causes depolarization of the membrane and displacement of bound calcium into the cytoplasm of the ovummthe zona reaction. These events occur within minutes to ensure that further spermatozoa can no longer fuse with the surface of the membrane, thus reducing the chances of polyploidy (review: Yanagimachi, 1994). The resumption of meiosis occurs 2-3 hr after fertilization with reactivation of meiotic metaphase and extrusion of the second polar body. After approximately 4 h, the two haploid sets become surrounded by distinct membranes and are termed pronuclei. The first mitotic division occurs 18-21 hr after gamete fusion. The ovum achieves syngamy and becomes an embryo with a cleavage furrow and ultimately two cells. At this point the embryo starts to control its own subsequent development (Johnson and Everitt, 1980; Pederson and Burdsal, 1994). Mouse embryos when grown in culture have retarded development postfertilization with cleavage progressing 2 or more hours slower than in vivo (Hogan et aL, 1986). Human embryos also develop more slowly in vitro, dividing at the interval of 16-24 hr during early cleavage (Balakier et aL, 1993; Figure 3). During mid to late cleavage in placental mammals the blastomeres of the embryo begin a process of compaction, at this stage blastomeres seem to lose their identity and merge into a single mass (Agostoni, 1993; Pederson and Burdsal, 1994). Gap junctions now appear between the outer blastomeres and the process of epithelial differentiation occurs, culminating in the formation of the trophectoderm in the blastocyst stage (Agostoni, 1993; Wiley et al., 1990). The final morphogenetic event in the pre-embryo is the formation of the blastocyst cavity, the blastocoel. Fluid transport by trophoectoderm cells leads to compartments being formed in the blastocyst with an inner mass and outer epithelium (Biggers et al., 1988). The cell number at blastocoel formation varies greatly between species: 16-cell stage in hamster and in pig, 32-cell stage in mouse, 64-cell stage in sheep and human, 128-cell stage in rabbit (Trounson and Wood, 1993). During this blastocyst expansion asynchrony between cell divisions increases, and in the mouse at Day 4-4.5 the blastocyst hatches from the zona pellucida (Pederson and Burdsal, 1994). At this time the inner cell mass has differentiated into an outer layer of primitive endoderm surrounding an inner core of primitive ectoderm and is ready to implant. 2. Embryo Culture

Embryos from both human and animal species when placed into culture often become arrested during the early stages of cleavage. This is particularly apparent with the culture of embryos from domestic farm animals, which are usually cultured to later development stages before implantation (Meyers et al., 1994). In the human situation several hypotheses have been put forward to explain the low rate of success in IVF programs, and these include:

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fertilised pronucleate oocytes

J

Day 1

J

Day 2

2- to 4-cell stage

8- to 16 cell stage

[(y~.~

{(f~~

Day 3

compaction and morula formation

Day 4

cavitation

Day 5

blastocyst formation and expansion

O

Day 6

f'x irmercellmass~ t r o p h e e t o d e r m

A schematicof the chronologicaldevelopmentof the humanembryofollowing fertilization on Day 1.

FIGURE.3

(i) cleavage and viability reduction of mammalian preimplantation embryos when grown in conventional in vitro systems (ii) asynchrony between embryo and uterine environment at the time of transfer (iii) the possible efflux and/or expulsion from the uterus associated with poor localization of the replaced embryo (Schillaci et al., 1994).

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The low pregnancy rate still achieved in assisted reproduction technologies indicates that IVF methodologies appear to be flawed in biological as well as technological aspects (Cohen and Wiemer, 1992). In human IVF spare embryos, when placed into culture, do have poor rates of cleavage to blastocyst; however, embryos generally are replaced at the four- to eightcell stage to avoid this later loss, giving an overall low pregnancy and "take home baby" rate (Bongso et al., 1989). An improvement in embryo culture and in vitro conditions could provide the way forward in improving the success rate. Other ways of manipulating the embryos derived from standard culture have been investigated; these include assisted hatching of the embryo to aid in implantation (Cohen and Wiemer, 1992; Alikani and Cohen, 1992), but these will not be discussed further. Three basic systems are currently used in most human IVF laboratories to support the human gamete and early embryo. The "culture tube" and "organ culture dish" systems are essentially termed open systems in which the culture is undertaken in a large volume of medium. Another culture system uses microdroplets of media under paraffin oil in a nonhumidified, 5% CO2 atmosphere. Early embryo research studies used only the simplest culture medium (e.g., physiological saline) in an effort to maintain embryo homeostasis in vitro, but little progress on embryo viability was made (Thibodeaux and Godke, 1992). The addition of serum, buffers, and amino acids to culture media have shown some improvement, but the task remains a significant challenge. However, there is still controversy as to the optimum preparation (Cohen et al., 1990). Attempts to improve culture conditions for IVF have focused on formulation and selection of appropriate culture media and additives, gas atmosphere, and the stage of embryo development (Bongso et al., 1990). Improvements in culture systems for these early stage embryos are imperative for enhancing embryo viability, thus improving the pregnancy rate (Meyers et al., 1994). In recent years the most notable progress made in this area of research has followed the use of "helper" cells to coculture early stage embryos to hatched blastocyst (Thibodeaux and Godke, 1992). These types of coculture systems are based on embryological experience with early stage embryos from larger domestic species (Bongso et al., 1991). The principle theory behind the use of coculture is that it could mimic more closely the developmental support provided by the oviduct in vivo. This could enable the culture period of human embryos to be prolonged, allowing embryos to be transferred at the developmental stage at which they would normally enter the uterus (Carneige et al., 1995). Coculture systems have been used successfully with farm animals to increase the rate of blastocyst formation during extended culture in vitro (Myers et al., 1994). It is pertinent to note that high embryo transfer rates >60% have been observed in domestic animals after replacement of blastocyst stage embryos in the uterus (Bongso et al., 1990). Improved development and pregnancy rates have been ob-

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served in sheep embryos (Gandolifi and Moor, 1987), ovine embryos (Rexroad and Powell, 1988), and bovine embryos (Xu et al., 1990), all of which were cocultured with species-specific oviductal cells. In the human situation, however, it does not seem ethical to use primary cultures of animal cells; therefore, human ampullary and endometrial primary cultures and cell lines have been developed (Bongso et al., 1990). The cell specificity requirement has not been proved and many workers are investigating established cell lines, conditioned media, and even components of the basement substratum to produce the same positive results in animals (Meyers et al., 1994; Schillaci et aL, 1994; Carneige et aL, 1995). These methods may eventually lead to replacement of blastocyst stage embryos --4-5 days after oocyte recovery with the possible increase in pregnancy rates, as was observed in domestic animals.

B. F u n d a m e n t a l A s p e c t s o f C r y o b i o l o g y i n P r e - E m b r y o s Similar to the descriptions of the biophysics of freezing in ovarian tissue outlined previously in this chapter, considerations of heat and mass transfer will dictate how embryos respond to cryopreservation procedures. In fact, detailed studies on the measurement of important parameters such as cell membrane permeability to CPAs or water, and the temperature relationship of these phenomena are available for mammalian embryos, largely from the groups of Mazur, Leibo, and co-workers (Rail et al., 1983; Mazur et aL, 1984; Mazur and Schneider, 1986; Mazur, 1990). Four coupled equations have been proposed to describe the events during cooling of embryos as the ice phase grows and cells dehydrate by osmotic forces, as outlined by Mazur (1990). In his first equation, he described the loss of supercooled intracellular water down the chemical potential gradient of the residual unfrozen solution, exposed as a vapor pressure ratio, d Vdt- =

(LpART In Pe) v~

[9]

where V is the volume of intracellular water (um3), t is time (min), Lp is the permeability coefficient for water (/zm min -1 atm -1), A the cell surface area (/zm2), R the gas constant (/zm 3 atm mo1-1 deg -1), T the temperature (Kelvin) and v~ the molar volume ofwater (18 • 102/zm3/mol). The symbols pe and pi refer to the vapor pressure of water in the extracellular and intracellular solutions, respectively. Mazur's second equation is used to calculate the change in this vapor pressure ratio with temperature during cooling, d ln(pe/Pi)/dT = Lf RT 2 -[Ntv~ + Ntv~

9dv'/dt

[10]

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where Nt is osmoles of solute in the cytoplasm and Lf is the latent heat of fusion of water (5.932 • 1016/zm3 atm mol-1). The third equation relates time and temperature as a cooling rate which, if linear, is given by dT d--t-= B.

[11]

Finally, the change in water permeability coefficient (Lp, also known as hydraulic conductivity) with temperature is given by Mazur's fourth equation, Zp-

L~ exp{-

E * / R ' [ ( 1 / T ) - (1/T8)]},

[12]

where L~ is the known hydraulic conductivity at temperature Ts, E* is the activation energy of water permeability in cal/mol, and R' the gas constant in deg -1 mo1-1. Using these equations, Mazur (1990) has calculated the changes in volume of cell water in eight-cell mouse embryos during cooling under a variety of conditions and demonstrated good relationships between the degree of cell dehydration under conditions where sufficient time is available for the process to approach equilibrium (effectively, during cooling at rates of < - I ~ min-~), avoidance of intracellular ice nucleation and ultimate survival of frozen embryos. However, there remain small discrepancies in the relationships, particularly when considering rapid cooling regimes (see later) with steps introduced to achieve appropriate cell dehydration. Many areas remain to be fully investigated, including the possible nonlinearity of E* for hydraulic conductivity at low subzero temperatures, and similar effects of high osmolalities in the residual unfrozen fraction on Lp. Nevertheless, there is a remarkable degree of coincidence between the observed and predicted outcomes of freezing in mammalian embryos when using Mazur's approach. A similar approach has been used by Schneider and Mazur (1986) to measure mass transport of CPA into preimplantation embryos (in this case, for bovine 7-day embryos) and the osmotic consequences of transmembrane permeation of the neutral solute glycerol. The coupled equations to describe the process were derived from Mazur's original study in red blood cells (Mazur and Miller, 1976) and later by Jackowski et al. (1980) for mouse oocytes. The first equation relates the flux of glycerol across the cell membrane to the solute permeability coefficient, ds

dt = Pg~ycZ[Me/lO00- S(t)Vw(t)],

[13]

where Pglyc is the permeability coefficient for glycerol (cm/min), A is the surface area of the cell, M e and S(t)Vw(t) the difference in molalities between external and internal glycerol concentrations, respectively.

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The second equation is used to allow for the osmotic adjustment for cell water volume (assumed to be instantaneous) so as to maintain osmotic equilibrium between intracellular anql extracellular solutions as the embryos are exposed to molar concentrations of glycerol, Vw(t) = [M~iso + lO000~(t)s(t)]/(M e + oeMe),

[14]

where the volume of cell water at time (t) is [Vw(t)], and at the same (t) the total intracellular osmolality is [Mi~o + lO000~(t)s(t)], the total external osmolality is M e + O e, Miso and M e are respectively the osmolalities of nonpenetrating solutes in the cell under isotonic conditions and in the extracellular solution, and 0 is the osmotic coefficient of the permeating solute (in this case, glycerol). Using these equations, best-fit curves were generated for embryos exposed to glycerol at 23~ where cell volumes changed initially by shrinkage as the embryos were exposed to the hypertonic glycerol solution, followed by re-expansion over time as glycerol permeated the intracellular compartments, achieving osmotic equilibrium between intra- and extracellular solutions with time. A value for P of 2.2 x 10 -3 cm/min at 23~ was derived. Knowledge concerning the biophysical principles underpinning events during cryopreservation of embryos have in fact developed in parallel to practical studies on freezing which were based on empirical assumptions derived from observation of survival or destruction of embryos under different cryopreservation regimes. In the next sections are given historical perspectives of the development of these methods for embryo freezing.

V. APPROACHES TO EMBRYO CRYOPRESERVATION

A. T e c h n i q u e s U s i n g S l o w C o o l i n g Embryo freezing by slow cooling with prior equilibration of CPA is the method developed independently by Wilmut (1972) and Whittingham et al. (1972) and which has been applied successfully to various species of embryo. CPA equilibration is achieved either by transfer through graded solutions or by gradually increasing the cryoprotectant concentration, resuiting in a final concentration of 1-2 M. The time and temperature of equilibration is dependent upon the CPA used; the aim being to reduce CPA toxicity while minimizing osmotic stress. Embryos are then cooled to around -7~ at which temperature ice formation is induced (seeded) by touching the outside of the vial with a precooled "cold-sink" such as forceps cooled in liquid nitrogen. The embryos are held at this temperature for 10-15 min to allow dissipation of the latent heat of crystallization. This seeding process prevents spontaneous ice nucleation throughout the following controlled cooling.

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Samples are cooled slowly, at a rate of less than l~ At such cooling rates ice growth occurs extracellularly, increasing the extracellular solute concentration. This in turn causes osmotic dehydration of the cells (Mazur et al., 1984). Once sufficient cellular dehydration has been achieved (by cooling to temperatures between - 3 0 to -80~ the embryos can be cooled rapidly without lethal intracellular ice formation. Following rapid cooling the embryos are stored in liquid nitrogen. If slow cooling is terminated at - 3 0 to -40~ (Willadsen, 1977), rapid warming (300~ is required since small innocuous intracellular ice crystals are likely to have formed during the second, rapid cooling, which may then grow rapidly during slow warming. If slow cooling is continued to -60~ or lower, the cells will be more shrunken with highly concentrated intracellular concentrations preventing intracellular ice formation. Rapid warming in this case may result in osmotic damage, hence such embryos should be thawed at a rate of 5-15~ CPA removal can be achieved using stepwise dilution of CPA or a sucrose dilution technique in which the sucrose acts as an osmotic buffer (Leibo and Mazur, 1978; Leibo, 1984) allowing CPA to be removed without excessive cellular water uptake.

B. Techniques Using Rapid Cooling While slow or "equilibrium" freezing has been applied successfully to embryos, the method is time consuming and, in order to be reproduced reliably, really requires expensive rate-controlled cooling equipment. Twoor three-step rapid freezing techniques have proved successful for mouse embryos (Kasai et al., 1980; Wood and Farrant, 1980); here embryos are exposed to 1.5-2 M Me2SO, seeded, then transferred to -20~ for 1015 min and either plunged in liquid nitrogen (two-step) or held for 10 min at -100~ before being transferred into liquid nitrogen (three-step). Thawing is rapid in a 30-40~ water bath (350-500~ With such techniques it is thought that some intracellular ice nucleation may occur during transfer to liquid nitrogen and hence warming should be rapid to prevent ice crystal growth. Kasai et al. (1980) found that cooling by direct transfer of embyros to -100~ was injurious and concluded that the step at -20~ was essential to achieve some degree of cellular dehydration before rapid cooling. More recently, the need for cellular dehydration during cooling has been avoided by prior dehydration using impermeable solutes such as sucrose (Renard et al., 1984; Massip et aL, 1984) and/or by only brief exposure to higher (3-4.5 M) concentrations of permeating CPA (Sz611 and Shelton, 1986a,b; Takada et al., 1984a; Trounson et al., 1987). In these cases it is thought that the intracellular concentration of CPA, together with the concentrating effect on endogenous cytoplasmic solutes brought

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about by partial dehydration, is sufficient to allow intracellular vitrification, whereby the cell contents solidify into a "glassy" amorphous state without ice crystal structures. Again warming is rapid to prevent crystallization. A method for vitrification of the entire embryo suspension was devised by Rall and Fahy in 1985. This approach requires CPA concentrations sufficiently high to avoid crystallization and to solidify, as a glass, at practicable cooling rates. Such concentrations are in excess of 6 M and can create problems of toxicity, hence combinations of CPAs are often used in order to reduce toxicity of each of the individual CPAs, as well as combinations of permeating and nonpermeating CPAs. Complete equilibration with CPA is not essential even for vitrification (Rall, 1987). Partial equilibration of embryos can be achieved by performing initial exposure with low concentrations of CPA at room temperature, followed by further additions at lower temperatures. Lower temperatures reduce CPA toxicity and also restrict movement of CPA into the cells. Hence cells remain shrunken, concentrating CPA already within cells as well as concentrating cytoplasmic solutes sufficiently to facilitate glass formation. Having been exposed to CPA, embryos are cooled rapidly, the rate of cooling for vitrification being relatively unimportant, provided only that it is sufficiently rapid to prevent crystallization. Direct plunging into liquid nitrogen may, however, cause fracturing of the glass which can, in turn, cause embryo damage; hence it is recommended to cool in liquid nitrogen vapor prior to plunging in liquid nitrogen (Rail, 1987). Although visible ice formation is avoided with this technique, the formation of very small ice nuclei is almost inevitable. On warming, the presence of such nuclei, plus the formation of additional nuclei, render the vitreous solid susceptible to devitrification, i.e., crystallization. The thermal events which take place during cooling/rewarming can be identified using differential scanning calorimetry to detect the enthalpy changes associated with various transitions. A plot of heat evolution versus temperature can be generated on which the glass transition phase is evident as an endothermic step and the formation of ice is associated with an exothermic peak followed by endothermic melting of that ice (MacFarlane, 1986). If the rate of warming is sufficiently high, however, devitrification can be prevented. The rates of cooling and warming required for vitrification depend upon the CPA used. Again, stepwise or sucrose dilution techniques are required to remove CPA from cells. C. R e s u l t s o f E m b r y o C r y o p r e s e r v a t i o n

1. Slow Cooling Techniques Slow or "equilibrium" cooling methods of mammalian embryo cryopreservation were first described in the mouse by Wilmut (1972) and Whit-

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tingham et al. (1972). Since that time many similar methods have been applied to different species, these have been reviewed in detail by Fahning and Garcia (1992). In summary, to date successful results based on pregnancy rate have been obtained with cryopreserved cow, sheep, goat, rabbit, and horse embryos. Post-thaw survival has been shown to be largely dependent on initial embryo quality, developmental stage, and species. With sheep embryos a slow cool protocol using 1-1.5 M glycerol has been shown to be the most effective (Bilton and Moore, 1983) with blastocyst stage showing the best survival (Nieman, 1986). These protocols use a stepwise addition of glycerol, with seeding at -7~ followed by cooling at -0.3~ to -30~ before plunging into liquid nitrogen. The embryos are thawed rapidly followed by a stepwise removal of the CPA. Pregnancy rates in the range of 35-55.5% can be achieved (Wierbowski et al., 1984). Early stage sheep embryos can be preserved by a slow cool protocol using either 1.5 M MezSO, ethylene glycol, or propane-l,2-diol (Fahning and Garcia, 1992), with some success. With goat embryos the best results have been achieved using 1-1.4 M glycerol (Wang et al., 1988; Rong et al., 1989). With bovine embryos no difference in embryo survival was observed using either 1.4 Mglycerol or 1.5 M MezSO (Bilton and Moore, 1979; LehnJensen and Greve, 1980). Propane-l,2-diol in a slow cool protocol has been used successfully with early stage bovine embryos(Fahning and Garcia, 1992). A direct transfer method has also been designed for bovine embryos, which eliminates the need for an embryologist at the time of the embryo transfer procedure. The method involves either placing the dilution solution in the straw with CPA (Voelkeil and Hu, 1992) or freezing with 1.36 M glycerol and 0.25 M sucrose in PBS (Massip and VanderZwalmen, 1984). With the latter method a pregnancy rate of 51.8% has been achieved. Equine embryos have been frozen at Day 6 with success using 11.3 M glycerol (Cztonkowska et al., 1985; Slade et al., 1984; Takada et al., 1984b; Yamamanto et al., 1982). The protocols involve a stepwise addition and removal of CPA and slow cool to -33~ prior to plunging in liquid nitrogen. The pregnancy rate that has been achieved is 53% at 50 days post-ovulation. Limits in freezing these embryos have been in part due to the lack of acceptance of these principles from large horse breeding organizations. Cryopreservation of porcine embryos has produced by far the greatest challenge to investigators wishing to preserve embryos from farm animals. Nagashima et al. (1989) have reported post-thaw survival of expanded and hatched blastocyst after a slow cool protocol with 1.5 M MezSO to -20~ prior to plunging in liquid nitrogen. Low viability of blastocysts and hatched blastocysts has been reported following slow cooling with a variety of CPAs plus 5 or 10% egg yolk; one live birth was reported following cryopreservation of a hatched blastocyst in the presence of 10% glycerol plus egg yolk (Fujino et al., 1993). The reason for this low success could be due to the

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high lipid content of these embryos (Polge and Willadsen, 1978). This seems to have been confirmed by a recent report of successful preservation of porcine embryos following removal of their cytoplasmic lipid droplets (Nagashima et al., 1995). Normal offspring have been obtained from these lipid depleted two- to four-cell-stage embryos following cryopreservation. Cryopreservation of supernumary human embryos from IVF clinics have benefitted from these studies on laboratory and farm animals. The first reported pregnancy from the transfer of a cryopreserved embryo in humans was that by Trounson and Mohr (1983). They used a slow cooling protocol with Me2SO and glycerol based on successful cryopreservation in laboratory animals (Whittingham, 1977; Trounson et al., 1982). Greater success was achieved using Me2SO (Trounson and Mohr, 1983). The method utilized a stepwise addition and removal of up to 1.0 M for glycerol and 1.5 M for Me2SO. The embryos were cooled to -6~ at 2~ and then ice crystallization was initiated and the embryos held for 20-30 min before being cooled at 0.3~ to -39, -60, or -80~ and then transferred into liquid nitrogen. Embryos were rapidly thawed, if plunged at -39 or -60~ or slowly thawed, at 10~ if plunged at -80~ Zeilmaker et al. (1984) also reported successful pregnancies with the Me2SO protocol, i.e., 1.5 M Me2SO, seeded at -5~ then cooled to -40~ at -0.3~ before plunging into liquid nitrogen. Rapid thawing was undertaken followed by stepwise dilution of the Me2SO. The method with Me2SO has been found to be useful with human embryos of all stages of development (Gelety and Surrey, 1993), particularly four- to eight-cell stages. From these initial studies many papers have been published using different CPAs at different stages of embryo development. Pronuclear and early cleavage embryos have been most successfully frozen using a slow cooling protocol with propane-l,2diol at 1.5 M and with 0.1 M sucrose. The protocol involved slow cooling to -30~ after seeding at -6~ followed by plunging into liquid nitrogen; a rapid thawing and stepwise dilution of CPA was then undertaken (Lassalle et al., 1985; Testart et al., 1986; Mandelbaum et al., 1988). This method has now become the one of choice in many IVF clinics all over the world (Garrisi and Navot, 1992; Wang et aL, 1994) and appears to be the most successful (Testart et al., 1990). Slow cooling with propane-l,2-diol is associated with survival rates of 88% for pronucleate oocytes (Testart et al., 1986), but with more advanced cleavage stage embryos survival rate is greatly reduced (Freidler et aL, 1988). Use of glycerol has been reported with blastocyst stage embryos (Cohen et aL, 1985; Fehilly et al., 1985; Trounson, 1990). Slow cooling is undertaken with 8-10% glycerol (Gelety and Surrey, 1993), however, glycerol needs to be added very slowly and embryos are generally transferred through 1, 2, 4, 6, to 8% (v/v) glycerol in buffered medium. On thawing the glycerol needs to be removed in the same way (Trounson, 1990). Blastocyst freezing offers the advantage that there will be better synchrony between the uterine

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environment and the thawed replaced embryos (Fehilly et al., 1985). The ability to culture embryos to blastocyst may act as a quality control method thus allowing greater success in freezing this stage of embryo. With this selection of freezing protocols it would seem that all stages of human pre-embryos can be preserved provided the correct method is used for the correct development stage. However, there is no conclusive evidence about which development stage when frozen produced the most favorable outcome with successful pregancies (VanSteirteghem & Vanden Abbeel, 1990; Gelety and Surrey, 1993).

2. Rapid Cooling Techniques Since the initial report of successful vitrification and subsequent recovery of intact mouse embryos (Rall and Fahy, 1985), live births following vitrification have been achieved with a number of species of embryo including rabbit (Kasai et al., 1992a), rat (Kono et al., 1988), cattle (Rall, 1992; Tachikawa et al., 1993) and sheep (Ali and Shelton, 1993b). The initial vitrification solution (VS1) consisted of a combination of CPAs, namely MeESO (2.6 M), acetamide (2.6 M), propane-l,2-diol (2.6 M), and 6% polyethylene glycol (PEG). With stepwise addition and dilution of the CPA mixture embryos survived both rapid and slow cooling provided it was in the presence of at least 85% VS1 and the warming rate was 300~ or higher, warming at 10~ resulted in devitrification and embryo death. The initial study involved eight-cell mouse embryos and has since been shown to produce live young (Rall et aL, 1987). The VS1 solution has also proved to be an efficient vitrification media for mouse two-cell embryos (Friedler et al., 1987) and for mouse blastocysts although survival of the latter has been reported to be lower than that of eight-cell embryos (Hsu et aL, 1986; Kono and Tsunoda, 1987; Matsumato et al., 1987). Live young have also been produced from rat blastocysts vitrified in VS1 (Kono et al., 1988). High morphological survival of mouse morula (92%) has been achieved with a slight modification of the VS1 solution to 2 M Me2SO, 1 M acetamide, and 3 M propane-l,2-diol. Of these morula, 83% went on to blastocyst and 45% normal young were produced following embryo transfer (Nakagata, 1993). However, morphological survival of mouse blastocysts was only 13.3%. A wide range of CPAs, alone and in various combinations, have been investigated as vitrification solutions. Propane-l,2-diol, which vitrifies at lower concentrations than MeESO, has been used with eight-cell mouse embryos (Rall, 1987) and bovine blastocysts (Tachikawa et al., 1993); however, propane-l,2-diol appears to be toxic at concentrations necessary for vitrification (Ali and Shelton, 1993b). Greater success has been achieved following vitrification of embryos in high concentrations of glycerol, which is less toxic than propane-l,2-diol. High survival was reported following

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vitrification in the presence of 6.5-6.85 M glycerol with eight-cell mouse embryos (Lieham et aL, 1990; Rail, 1987; Rail and Wood, 1994) and Day 7 bovine embryos (Rail, 1992). Recently attention has focused on the use of ethylene glycol. Studies have found this to be the least toxic of methanol, Me2SO, glycerol, propane1,2-diol, and butanediol for all stages (one cell-blastocysts) of mouse embryos (Ali and Shelton, 1993c; Valdez et al., 1992). At concentrations needed for vitrification (8 M) ethylene glycol is toxic; hence lower concentrations (40%) have been used in combination with Ficoll and sucrose. Using such a combination vitrified mouse morulae and blastocysts gave 94-100% survival when CPA exposure was optimized (Kasai et al., 1992b; Zhu et al., 1993). Development of blastocysts to live young following embryo transfer was as good as controls (Zhu et al., 1993). Rabbit morulae vitrified in the above solution showed 100% recovery and 65% development to term on transfer to recipients (Kasai et al., 1992a). High survival (74-89%) has also been reported for bovine blastocysts (Mahmoudzadeh et al., 1995; Tachikawa et al., 1993), however, when CPA exposure was extended from 2 to 3 min or longer the survival rate dropped to 7-0% (Tachikawa et al., 1993). Combinations of CPAs have been investigated for vitrification in an attempt to reduce toxicity by using a nontoxic concentration of each CPA. A combination of ethylene glycol and glycerol has been found to be the least toxic for mouse embryos and sheep compacted morulae (Ali and Shelton, 1993b). The concentration of each CPA within the cocktail, together with the addition strategy, can greatly influence survival, complete loss of survival of sheep embryos (Day 6) being reported following exposure to 5.5 M ethylene glycol + 2.5 M glycerol (Ali and Shelton, 1993a,b), whereas with stepwise addition of 6 M ethylene glycol + 1.8 M glycerol in vitro survival was 88.2%. Mixtures of glycerol and propane-l,2-diol have been shown to give better results with mouse embryos than the equivalent concentration of either CPA alone (Scheffen et al., 1986). High survival of bovine blastocysts (Kuwayama et aL, 1992) and rabbit morulae (Papis et aL, 1993) have been reported using this combination, survival of sheep embryos being less successful (Sz611 et al., 1990). Finally, a CPA mixure of 20% ethylene glycol + 20% Me2SO + 10% butane-l,3-diol (Valdez et al., 1992) gave a development rate in vitro for mouse blastocysts not significantly different from that of nonvitrified controls (45% vs 60%). Little success has been achieved with cryopreservation of porcine embryos using vitrification methods. Porcine expanded and hatched blastocysts have been exposed to mixtures including Me2SO, acetamide, and propane1,2-diol as well as ethylene glycol alone and in combination with propane1,2-diol. Viabilities were less after exposure to CPA than for controls with no differences between CPA mixtures. Stepwise addition of CPA was found beneficial; however, following four-step addition and vitrification viability was reduced by 60-100% (Yoshino et al., 1993). Twenty-six percent survival

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of pig two-cell embryos has been reported following rapid cooling in the presence of antifreeze glycoproteins from antarctic fish (Rubinsky et al., 1992). Successful vitrification of a number of embryo species at various stages of development has been achieved. The degreee of success is primarily dependent upon the type of CPA used and its method of addition and dilution. Cooling and warming rates should be sufficiently fast to avoid extensive devitrification, although a small amount of ice formation will be tolerated in some cases. If these factors are optimized it should prove possible to cryopreserve all embryo types; however, the intrinsic characteristics of embryos such as those of the pig may limit effective application of current procedures.

3. Cryopreservation of Micromanipulated Embryos Recent developments in embryo micromanipulation have made it necessary to cryopreserve embryos that have had deliberate damage subjected to their zona pellucida (Depypere et al., 1991). The zona pellucida is the glycoprotein coat that surrounds the oocyte and is very important during fertilization and preimplantation development of the embryo (Naito et al., 1992). Micromanipulations are currently used in zona drilling, zona cutting, microinjection of sperm, and embryo biopsy. One of the major areas is in preimplantation diagnosis; this involves invasive techniques which damage the zona pellucida (Grossman et aL, 1994). Preimplantation diagnosis of biopsied embryos requires that genetic analysis of the blastomere and embryo transfer are all carried out within 3-4 days. This does not allow enough time to perform any repeated or lengthy diagnostic test. In these cases cryopreservation offers the means by which embryos can be arrested while genetic analysis continues. Zona-free or zona damaged embryos are also produced when chimeric embryos are being developed, and this is a useful technique for the preservation of animals on the verge of extinction. A chimera of the endangered animal can be made with a related species, which leaves the rare genes in the chimera (Nagashima et al., 1991). Manipulation can also be used in the artificial production of twins in domestic animals, by embryo bisection (Landa, 1990), which involves cryopreservation of the embryo after disrupting the zona (Grossman et al., 1994). In all these micromanipulation techniques it is essential to be able to successfully cryopreserve the resultant embryos. The protocols utilized fall into two main types; one is based on a slow cooling protocol with 1.5 M propane-l,2-diol (Lassalle et al., 1985) which has been used successfully with zona intact embryos and the others involve rapid freezing protocols. Liu et aL, 1993 using the slow cooling protocol with 1.5 M propane1,2-diol showed that is was possible to remove up to three blastomeres from an eight-cell embryo, cryopreserve the biopsed embryo, and still get survival. They concluded that an embryo with a hole in the zona could

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survive freezing and thawing and develop both in vivo and in vitro in a way similar to frozen-thawed zona intact embryos. Using the same protocol survival has also been shown for zona-free and zona-slit embryos (Grossman et al., 1994; Sandalinas et aL, 1994 and Thompson et al., 1995) Wilton et al. (1989) found success using an ultrarapid protocol with 4.5 M Me2SO and 0.25 M sucrose in mouse embryos biopsied for preimplantation diagnosis. Other rapid cooling protocols utilized include the use of 30% (v/v) glycerol plus 20 (v/v) fetal bovine serum and 50% (v/v) 2 M sucrose for zona-free mouse embryos (Landa, 1991); this involved a microdrop technique directly into liquid nitrogen. With this technique most success was achieved using eight-cell zona-free embryos. Glycerol has also been reported along with propane-l,2-diol, in a vitrification method (Nasgashima et aL, 1991), for chimeric and half embryos. The method uses 25% glycerol and 25% propane-l,2-diol as the vitrification solution, followed by a direct plunge into liquid nitrogen. In all cases warming was rapid followed by a stepwise dilution of the CPA. This method proved to be successful for freezing half embryos without the zona and chimeric embryos. The presence of the zona pellucida appears not to be necessary for cleavage to occur in vitro. It would appear, however, that the stage of embryo development and the number of cells exposed to the stress of cryopreservation could play an important limiting role. Cryopreserved twocell embryos largely show disaggregation of the blastomeres after thawing, but eight-cell zona-free embryos survive successfully (Grossman et aL, 1994). With the zona-damaged embryos it would appear that slitting the zona is worse than piercing and that the larger the hole the more susceptible they are to freeze-thaw damage (Thompson et aL, 1995). The ability to cryopreserve this type of embryo will pave the way for future research into embryo development and more intricate methods in preimplantation diagnosis.

VI. SUMMARY In summary, it can be stated that cryopreservation of reproductive tissues is an area of growing interest. Banking of multicellular embryos in a variety of species has made a significant impact on reproductive biology. Much has also been learned since the early studies some 30 years ago, and there is now a greater understanding of the fundamental principles of the physical effects of freezing, particularly in ovarian tissue. There may be an equivalent need for cryopreservation of testicular tissue if there are further developments in methodologies for infertility treatment, but this remains to be seen. Since compiling this chapter a report has appeared describing a pregnancy using sperm extracted from a cryopreserved testicular biopsy, employing a glycerol-based CPA and slow cooling (Fischer et al., 1996).

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Certainly, the impetus for luther studies on ovarian tissue will grow following a recent decision by a group in the United Kingdom to cryopreserve ovarian tissue from a young female patient undergoing ablative surgery. However, the research community must work hand-in-hand with those concerned with the developing debate about ethical and legal implications of such new reproductive technologies, so that maximum benefit can be assured.

ACKNOWLEDGMENTS Parts of the work described in this text were supported by MRC (UK) and the WellBeing Charity.

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van Blerkom, J. (1991). Cryopreservation of the mammalian oocyte. In Current Communications in Cell and Molecular Biology, Animal Applications of Research in Mammalian Development (R. A. Pedersen, A. McLaren, and N. L. First, Eds.), pp. 83-119. Cold Spring Harbor Laboratory Press, Cold Spring Harbor, NY. van den Eede, B. (1995) Investigation and treatment of infertile couples: ESHRE guidelines for good clinical and laboratory practice. Hum. Reprod. 10, 1246-1271. van Uem, J. F. H. M., Siebzehnrubl, E. R., Schuh, B., Koch, R., Trotnov, S., and Lang, N. (1987). Birth after cryopreservation of unfertilized oocytes. Lancet 1, 752-753. Wada, I., Macnamee, M. C., Wick, K., Bradfield, J. M., and Brinsden, P. R. (1994). Birth characteristics and perinatal outcome of babies conceived from cryopreserved embryos. Hum. Reprod. 9, 543-546. Watson, P. F. (1979). The preservation of semen in mammals. In Oxford Reviews of Reproductive Biology (C. A. Finn, Ed.), pp. 283-350. Oxford Univ. Press, London. Watson, P. F. (1990). Artificial insemination and the preservation of semen. In Marshall's Physiology of Reproduction (G. E. Lamming, Ed.), 4th ed., Vol II, pp. 747-896. Churchill Livingstone, London. Whittingham, D. G. (1971). Survival of mouse embryos after freezing and thawing. Nature 233, 125-126. Whittingham, D. G., Leibo, S. P., and Mazur, P. (1972). Survival of mouse embryos frozen to -196~ and -269~ Science 178, 411-414. Wildt, D. E., Byers, A. P., Howard, J. G., Weise, R., Willis, K., O'Brien, S., Block, J., Tilson, R. L., and Rall, W. F. (1993). Genome Resource Banking (GRB) action plan: Global need and a plan for the North American region. Presented at AAZPA Annual Meeting, Omaha, Nebraska, August, 1993. Wilmut, I., and Rowson, L.E.A. (1973). Experiments on the low temperature preservation of cow embryos. Vet. Rec. 92, 686-690. Yanagimachi, R., Lopata, A., Odom, C. B., Bronson, R. A., Mahi, C. A., and Nicholson, G. L. (1979). Retention of biologic characteristics of zona pellucida in highly concentrated salt solution: The use of salt-stored eggs for assessing the fertilizing capacity of spermatozoa. Fertil. Steril. 31, 562-574. Yokoyama, M., Akiba, H., Katsuki, M., and Nomura, T. (1990). Production of normal young following transfer of mouse embryos obtained by in vitro fertilization using cryopreserved spermatozoa. Exp. Anim. 39, 125-128.

Genome Resource Banking I m p a c t on Biotic C o n s e r v a t i o n and Society

D a v i d E. Wildt Conservation and Research Center National Zoological Park Smithsonian Institute Fort Royal, Virginia 22630

I. I N T R O D U C T I O N Cryopreservation of biomaterials is an emerging "tool" that has enormous implications for the assessment, conservation, and sustainable use of natural resources, food animals, and crops. In turn, the ability to preserve and then successfully use tissues has a profound effect on human society, perhaps superseding scientific or economic impacts. We are dealing with a tool that, when used judiciously, can substantially and positively affect us as a society. Perhaps the most provocative advantages include the ways that humans protect and conserve other species and the extant gene diversity in the biosphere. The latter is referred to as "conservation biology" or the science of dealing with scarce resources which includes biomaterials collected from animals and plants, wild and domesticated. This chapter considers how cryobiology offers the opportunity to manage genetic resources while improving biological viability. The issues are diverse and, because the field is in its formative years, opportunities for debate and controversy abound. We will be referring frequently to the concept of genome resource banks (or GRBs), defined as the systematic and organized collection, storage, and use of biological materials. Discussion will largely be related to reproductive tissues, in part because of the focus Reproductive Tissue Banking Copyright 9 1997 by Academic Press. All rights of reproduction in any form reserved.

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of this book, but also because germplasm and embryos are the fundamental bioingredients for perpetuating species and species' well-being. Nonetheless, the consequences of formulating and using a GRB go far beyond reproduction, and, thus, reference will be made to preserving other tissues, blood products, and DNA so useful for a host of other taxonomic, genetic and disease issues. Because of an amazing lack of fundamental information, discussion will center on livestock, other domesticated species, and a few wild mammal counterparts. However, whenever possible, the reader will be reminded of the vast biodiversity on this planet and the need to consider the consequences of effective tissue cryopreservation as it pertains to all wild mammals, fishes, invertebrates, microorganisms, and plants.

II. AN INTRODUCTION TO BIODIVERSITY This chapter is preceded by a vast amount of fundamental information on the scientific and technical aspects of tissue preservation. Authors (and many readers) of this text not only are experts in the science of cryobiology, but also understand explicitly how their craft applies to their specific field of interest. In most cases, this involves an individual species or elucidating a specific mechanism using a common laboratory animal as a research model. The author expects that many cryobiologists have considered, abstractly or peripherally, the idea that their field has something to offer in protecting other life forms. Few of us who have written grant proposals have avoided the temptation of interjecting a sentence or two indicating that "projected new knowledge gained from these studies may have major applications to future work to save endangered species." In general, however, most cryobiologists (and in fact most biologists) do not understand the biodiversity crisis, its complexities, or implications in terms of quantity and quality of life and life forms on this planet. Further, conservationists in general have focused upon the larger issue of mass habitat (and thus mass species) loss with less attention on the topic of genetic diversity, that is, ensuring that extant species, subspecies, populations, and individuals maintain sufficient genetic vigor to remain reproductively viable. Preserving genetic and biological diversity also goes beyond what we usually consider as conventional conservation targets (i.e., wild animals and plants). As discussed below, organized efforts are in place for the systematic storage of biomaterials from traditionally farmed crop plants and animals. These programs are the foundation for feeding and sustaining the world's exploding human population (ironically the force behind most extinctions of other life forms). Last, it is humans themselves that benefit from the technology, in terms of achieving personal fertility goals, preserving their genetic potential, and ensuring that the biosphere is healthy, genetically dynamic, and a worthy habitat for their descendants.

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The biodiversity crisis has spawned a host of eloquent advocates for awareness and change including E. O. Wilson, Peter Raven, and Michael Soul6, among others. These individuals are true pioneers in the sense that, for the first time, they argue the need for organized and practical approaches to conserving biodiversity. The tactics must be multidimensional, and Soul6, who is neither a cryobiologist nor reproductive physiologist, has suggested that one path to biotic survival is "suspended ex situ programs, including germplasm and seed bank" repositories (Soul6, 1991). However, before giving the impression that cryobiology is the cure-all for preserving life on Earth, let us face reality by addressing three issues that every cryobiologist (and every individual interested in the societal implications of the technology) must first understand. The first concerns why we should even bother to preserve and protect biological and genetic diversity. The second deals with the overall complexity of the problem, and the third centers on how germplasm, seed, and tissue cryopreservation could complement existing or planned global or local conservation schemes. The author readily admits that many of the following ideas and concepts were extrapolated from the brilliant treatises of others. The objective here is to consolidate and interrelate these many thoughts and ideas for the purpose of familiarizing the nonconservation biologist with the larger-scale theme and the potential of cryobiology for playing a contributing role.

m . w H Y CONSERVE BIO- AND GENETIC DIVERSITY? E. O. Wilson (1992) has stated that "biological diversity is the key to the maintenance of life as we know it." An accurate rate of species extinctions will never be known, but this same author and others (for a review, see Soul6, 1991) have predicted the loss of as many as 50,000 species annually with this extermination rate likely to continue for at least several more decades. The consequence, as bluntly put forth by Wilson, is an "extinction spasm" not experienced since the demise of the dinosaurs. As citizens of planet Earth, we now have all the natural resources that we will ever havemthese either can be used rationally and sustainably or they can be destroyed, never to be recovered again. There are three oft-cited reasons for why we should care about preserving biodiversity (Raven, 1994). The first is that biodiversity is Earth's life support system, including regulating local climate and atmospheric quality, absorbing pollutants, protecting watersheds, and generating and maintaining soils. Functional ecosystems capture and transform solar energy into the biochemical constituents that allow life to proceed for all species, including humans. The second reason is purely economic~the realization that we routinely use and depend upon organisms daily for our foods, medicines, chemicals, fiber, clothing, structural materials, energy, and recreation.

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Throughout history, humans have used about 7000 kinds of food plants of which only 20 now supply 90% of the world's food resources; just three (wheat, maize, and rice) provide more than half (Munk, 1993). Alarmingly, it appears that agriculture's steady gains in global production are not fully meeting rising demand as a result of soaring human population. By the year 2050, global demand for food may be three times greater than it is today (Anonymous, 1994a). Moreover, during the past two decades food production growth rate actually declined significantly (Anonymous, 1994a). Furthermore, efforts to increase food production have come at a high cost because many local, heterogeneous plant varieties (with specific resistance to parasites and diseases, with unique nutritional qualities, or with as yet undiscovered, valuable traits) have been lost forever. It also is a cold, hard fact that 98% of U.S. crop production is based on species that originate elsewhere (Oldfield, 1989). Plants and other organisms are natural biochemical factories (Raven, 1994). Plants are the source of everyday medicines for more than 60% of the world's populace. Various organisms also produce the resources from which the majority of the Western society's pharmaceuticals are derived. Even a decade ago the top 20 medicines in the United States had their origin in natural products. So it is both reasonable and likely that the remaining 250,000 kinds of plants in the world (most are unstudied) harbor a wealth of potential for combating human disease (Wilson, 1992). Frequently cited examples include taxol (found in the western United States yew tree) which is a drug with high promise against breast and ovarian cancer, and artemesin (discovered from traditional Chinese medicine) which is the only therapeutic agent effective against all strains of Plasmodium causing malaria, a finding no doubt of interest to the quarter of million people afflicted annually with this malady. (For an excellent review on the medicinal value of biodiversity, the reader is referred to Oldfield (1989).) Biodiversity offers other economic benefits. United States state and national parks alone attract up to 800 million visitors per year (largely to enjoy the variety of landscapes the parks and forests protect) while 54 million Americans fish and 19 million hunt for sport, spending together more than $32 billion dollars annually (U.S. Congress, 1987). The third justification is that, because humans are the dominant species on this planet, we have an absolute moral responsibility to protect every cohabitant (Regan, 1986; Taylor, 1986; Norton, 1987). Although an immeasurable, esoteric justification, it is one deeply believed across most cultures and by all but the most cynical (Maybury-Lewis, 1992). This same obligation also naturally extends to the purely esthetic; that is, because the world is a magical, wondrous web of life, it deserves our attention and protection. Many other reasons for preserving biodiversity exist in the literature, ranging from the importance of charismatic carnivorous predators (like tigers and killer whales) for sustaining the natural food chain to the biochemical versatility of bacteria and fungi for environmental remediation and

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industrial production of enzymes, antibiotics, and alcohol (for an excellent perspective, see Wilson, 1992). Particularly exciting will be the continued melding of molecular technology with the availability of diverse biota, especially the microorganisms representing an infinite variety of unknown genes of enormous potential. For example, the bacterium-derived DNA polymerase (known as Taq) has played a key role in the global application of the polymerase chain reaction (PCR) while simultaneously revolutionizing the field of molecular biology. The ultimate benefactors from protecting biodiversity are humans, rural industries (agriculture, forestry, aquaculture/ mariculture, horticulture), and manufacturing (pharmaceuticals, food, energy, construction). Given that the process remains scientifically based, then a natural by-product will be a massive increase in fundamental knowledge that will drive an ever-expanding positive and practical application cycle. The final product will be an ever-improved ability to sustain our natural resources, heritage, and cultures while enhancing the quality of life. Although most popular and scientific attention has focused on species or ecosystem conservation, the underlying challenge of preserving gene variation and the evolutionary potential within species, populations and individuals should never be forgotten or set aside as a secondary component. Although the sheer loss of species is based on estimates from indirect evidence, it is certain that thousands of species (domestic and wild, animals and plants) are suffering population declines, resulting in losses in genetic variation (Heywood, 1992). A reduction in genetic variation can result from genetic drift, demographic bottlenecks, founder effects, and inbreeding. The latter is a mating scheme actually used in the past to perpetuate certain desirable traits/genotypes in livestock and laboratory species. The problem then (as now) is that inbreeding eventually results in the fixing and expression of deleterious (and even lethal) alleles, exposing the population to a host of undesirable characteristics. In inbred cattle, two commonly compromised traits are fecundity and thriftiness of neonates. Likewise, a landmark study quantified the impact of lost genetic diversity in zoo-bred species (Rails et aL, 1979); an alarming increase in neonatal and juvenile mortality was associated with inbreeding (documented via parentage analysis). Other studies, including in our laboratory, have found a direct relationship between monomorphic genotypes and teratospermia (many structurally malformed sperm in the ejaculate) of various felid (cat) species living in zoos or isolated wild habitats (for reviews, see Wildt, 1994; Wildt et al., 1995b). Perhaps the most profound proof that inbreeding depression occurs in nature involves the Florida panther (Felis concolor coryi), a remnant population of mountain lion, or cougar, living in isolation in southern Florida. These few remaining cats not only ejaculate an extraordinary proportion of pleiomorphic (malformed) sperm (circa 90%+/ejaculate) (Barone et aL, 1994), but also have a high incidence of cryptorchidism (one or both testicles

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retained), cardiac defects, and seroprevalence to numerous disease pathogens (Roelke et aL, 1993). The Florida panther will long stand as a model for illustrating the deleterious effects of lost gene diversity on the demise of a natural population. In the context of this chapter as a whole, tissue banking, or a GRB, will be useful not only for helping to "insure" genes from secure wild populations, but (as discussed below) for eventually moving genes among fragmented populations to avoid inbreeding depression. All in all, it can easily be argued that preserving biological and genetic diversity provides society with major lifestyle, health, and economic benefits while maximizing options and minimizing regrets for the future. Despite the complexities, worldwide interest in biodiversity has grown profoundly as demonstrated by the signing of the Convention on Biological Diversity at the 1992 Earth Summit in Rio de Janeiro in Brazil. In a general context, the Convention formally recognized the global biodiversity crisis and began to catalyze worldwide dialogs and partnerships that hopefully will translate into conservation actions. The Convention was ratified by the requisite 30 countries in September 1993 and became international law in December of the same year. The focus is not simply on preserving habitat or ecosystems, but on "reversing the alarming trends that destroy the biological capital of the Planet" (Chasek, 1994). More than 65 countries now comprise the "Parties" to the Convention and have established working groups that have begun serious debates about conservation priorities, sustainable use of biodiversity, biosafety, international cooperation, technology transfer, financial mechanisms, and funding needs. Despite the usual glacial speed and political pitfalls of such international activities, the Convention on Biological Diversity is important, in part, because it has increased societal awareness. It also has started genuine debate, with the Parties discussing those issues that will affect accessibility to biological resources, technological capacities, and training and research opportunities. There also will be direct impacts on those of us interested in coordinating animal and plant biomaterials and ex situ conservation. For example, the Parties are discussing intellectual property rights, regulations governing the collection and dissemination of biomaterials, equitable sharing of benefits with indigenous peoples and local communities, the role of basic research and biotechnology, and innovative measures of mobilizing funding at national and international levels (Anonymous, 1993). Already, the Parties have established that genetic resources lie within the sovereign jurisdiction of individual nations (Anonymous, 1995). Although some countries are moving toward formulating legal policies to regulate access to and utilization of their biological materials, few models exist, and capacities for enforcing such regulations will remain weak for some time.

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IV. H O W COMPLEX IS THE TASK OF CONSERVING BIOLOGICAL AND GENETIC DIVERSITY? Although the benefits of conserving biological and genetic diversity are enormous, the challenges are truly staggering. Almost every conservation biologist agrees that we do an abysmal job of actually protecting and saving resources and gene diversity. Lest we believe the problem only affects others, our "developing nation counterparts," a brief review of U.S. progress is noteworthy. More that 4000 taxa in this country languish to be considered for formal listing as "endangered," largely due to an underfunded bureaucracy. However, other regions are faced with ongoing and pending disasters and an even greater overall challenge, in part, because developing countries contain at least 80% of the global biodiversity, more than 75% of the world's human population, but ironically only about 6% of the world's scientists (Raven, 1994). Soul6 (1991) has elegantly and effectively summarized the various tactics available for combatting the biodiversity crisis. He has described a "biospatial hierarchy" of protective classifications, including whole ecosystems (or landscapes), communities, species, populations, banked germplasm, and genes. Most conservationists prefer conserving from the top of the hierarchy, that is, protecting entire ecosystems as they hopefully are composed of all the other hierarchical components. This strategy, in place for more than a century, involves establishing protected areas (usually with high floral and faunal diversity) (Wright, 1993) and then safeguarding by limiting human access and use, usually by proscription and enforcement. The result has been some 8000 protected areas worldwide, comprising about 4% of the Earth's land surface. Nonetheless, there is rapidly growing disillusionment with this approach. Because of a natural insularity, these reserves tend to become biologically impoverished over time, especially when composed of species that require huge home ranges or are exquisitely sensitive to habitat fragmentation (e.g., species usually at the top of the food chain). There are other difficulties, including enormous costs and the lack of means to actually ensure existing biological richness (although designated as protected areas, often no resources exist to pay for the protection). Finally, there is the fact that 96% of the planet's biodiversity still remains unprotected (Wright, 1993). As a result, there is an important societal issue emerging known as community based conservation (Wright, 1993). That is, that "the protected-area approach, dependent on centralized power and top-down planning, has often robbed rural communities of their traditional user rights over forests, waters, fisheries, and wildlife, without offering appropriate renumeration. It has obliged poor people who are resident in contested landscapes to bear most of the costs of conservation, while larger societal interests reap most of the benefits. The real trouble

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with the protected-area strategy is that it tends to omit humanity from the realm of nature and from the enterprise of nature conservation." In the larger scheme of conservation, these attitudinal shifts argue the need to rethink traditional ways of preserving biodiversity. There obviously is a growing theme that we need to develop partnerships (globally, regionally, and especially locally) while sharing knowledge and technology without becoming authoritative or regulatory (also addressed later in this chapter). However, these changes in philosophy also support the notion that we need to address protection at every level of Soul6's biospatial hierarchy, including exploiting biotechnologies like tissue banking for understanding and conserving individual species and genes. However, if the GRB concept is ever to become reality, then the community based conservation concept perhaps will dictate how tissue banking actually contributes to biodiversity and gene diversity protection. The complexity of conserving biological and gene diversity cannot be understood without a clear comprehension of human and related societal/ economic impacts. Again, Soul6 (1991) helps us understand the tangle of factors by claiming that loss and fragmentation of habitat, overexploitation, spread of alien species and diseases, air/soil/water pollution, and climate change are important, but only proximal causes. There is an even deeper layer indicating that biotic attrition is rooted in various contemporary human conditions. These factors are worthy of note and include: 1. Ever-expanding human growth patterns. Raven (1994) has provided a provocative description of human population impacts, the first spurt occurring coincident with the onset of the agricultural age (8000 to 11,000 years ago). From 5 million people then, rapid growth occurred thereafter to about 130 million people 2000 years ago, 500 million by 1650, 2.5 billion by 1950, and an amazing 5.5 billion today. Projections are a doubling to at least 10 billion people within the next 50 years (Ehrlich and Ehrlich, 1981, 1990), an overconsumptive population unlikely compatible with certain natural phenomena like large, flee-living predators, annual bird migrations, or simply the basic continuation of protected areas. 2. Poverty. People in developing countries naturally strive to improve lifestyle, the inevitable result being that wild habitat will be co-opted for farming, mining, industry, and other uses. It is difficult to expect people to be concerned about conservation when the primary objective is feeding and providing for one's family. 3. Misperception of time scale. We expect conservation action to occur quickly. However, in reality there are two problems. Governments naturally move slowly, and the success of specific conservation projects often is not immediately apparent, perhaps being realized only over long time periods, sometimes centuries. Additionally, managers naturally tend to plan scenar-

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ios in terms of their own 20- to 40-year careers, not into the long-range future. 4. Anthropocentrism. Interestingly, while we profess to be concerned about conservation, we give little to the cause. Financial funding for conservation entails only 1.5% of all donations supporting environmental groups and efforts. Corporations traditionally have focused support on short-term gain, "win-win" and non-conservation-oriented projects, and society, in general, still fails to recognize or believe in the utilitarian benefits of conservation. 5. Cultural transitions. The transition from an agriculture-based to an industrial-urban society for significant segments of the global community results in too little time or attention devoted to conservation, especially when the target can be an enhanced standard of living. 6. Economics. Obviously, continued population growth mandates accelerated exploitation of resources. The problem is exacerbated by industrial nations encouraging developing regions to harvest lowland forests, mangroves, and reef habitats not so much to support the local economy but as a cheap resource for their own, already developed regions. 7. Policy implementation. Momentum of the population explosion and resulting societal instability prevent implementing and enforcing reasonable policies, especially in developing countries. As emphasized by Soul6 (1991), recent wars in at least 14 African countries have led to the deterioration or total destruction of national parks and the loss of at least one regional seed bank (Somalia).

V. A ROLE FOR OUR SCIENCE IN CONSERVATION BIOLOGY Discussion to this point may set a depressing tone, suggesting that the task is enormously complex, if not overwhelming. Nonetheless, the author sees the glass half-full, rather then half-empty and the need rather aweinspiring. If we trickle down the task to our immediate scientific expertise, one might even askmis there a place for me or my discipline (cryobiology, physiology) in the field of conservation biology? The answer is a resounding yes, and the author has addressed the need for a new breed of conservation biologist in another venue (Wildt et al., 1995b). The real question is not if, but h o w our type of science can contribute. As already addressed in an earlier paper (Wildt et al., 1995b), the real need is for an improved understanding of the fundamental biology of every species. For example, despite the existence of more than 4500 mammalian species on this planet, fewer than 100 have been studied intensively, and most of these have been either economically valued (i.e., livestock), laboratory models (e.g., rodents and nonhuman primates), or the highly charismatic. There also is no doubt that species specificity will play a role in our ability to apply reproductive

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techniques (assisted reproduction) to practical applications (Wildt et al., 1992a). This should be no surprise, given the common knowledge that, for example, sperm and embryo cryopreservation protocols vary markedly among livestock and laboratory species. Therefore, a primary role for cryobiologists and physiologists is to characterize basic normative traits and mechanisms for new species of interest. As will be discussed later, an equally important role will be working in partnership with others to better define how genome banks can be formulated and implemented to ensure that there is a true conservation impact.

VI. GENERAL TYPES OF CONSERVATION NEED The paths to biotic survival are as diverse and treacherous as understanding the forces mandating the need for action. Some have been alluded to earlier, especially the need for in situ or natural management activities, usually large-sized, protected areas. Another in situ version is the "extractive reserve" that provides a level of self-sustainability because certain within-reserve resources are harvestable in a sustainable manner (i.e., rubber, nuts, fruits). Finally, in situ conservation can include: (1) protected habitat that may support indigenous as well as reintroduced species and (2) degraded habitat that is being highly managed for the purpose of restoration. There are also two types of ex situ conservation systems, the one most commonly considered being the zoo, botanical garden, aquarium, and private or commercial farm (for genetically valuable livestock). Zoos in particular should be highlighted because of their focus on intensive genetic and demographic management of high priority species. For example, in North America more than 70 endangered species (of diverse global origins) are bred in captivity, but strictly for maximizing genetic diversity. This is achieved through a highly structured "specimen plan" approach known as a Species Survival Plan (or SSP), each of which is administered via the American Zoo and Aquarium Association. Analogous management plans (with different names) exist in other world regions. While effectively an ex situ approach, these plans also promote and support on-the-ground conservation programs for the same species in their home ranges. The concept is unique for two reasons, the first being that it is "grass-roots" with participation and activities led by a committee of experts, usually representing various zoos. The second is that the goal is not simply to preserve a species, but to do it on the basis of computer-generated matchmaking, thus ensuring that offspring are genetically valuable, viable, and capable of contributing to a self-sustaining, reservoir population. The sophistication of the genetic management (based on the principles of small population biology and an overall relatedness factor known as mean kin-

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ship) (Ballou, 1991; Ballou and Lacy, 1995) surpasses that used for any other propagation program, including the breeding of cattle where the final goals are simpler to achieve (i.e., the widespread distribution of genes from only a few superior individuals that will enhance growth, feed efficiency, and milk production). The zoo model is important because it reaffirms our earlier argument for the need to maintain all extant genetic variation from small populations to allow for evolutionary potential and to avoid potential inbreeding potential. As will be discussed, this is one of the primary reasons that the GRB concept will be so important in conserving rare animals and plants. The second ex situ system involves the long-term preservation of biomaterials including germplasm, embryos, tissues, blood products, and DNA, that is, a GRB. Most of the subsequent discussion will focus on the impact of this approach on perpetuating valuable genes and ultimately species. However, as will be demonstrated, GRBs have the potential of contributing at all levels of the conservation hierarchy, including in situ approaches.

VII. CONSERVATION OF CROPS AND LIVESTOCK The traditional driving forces behind most efforts to conserve genes, of course, have been economics and a desire to ensure food security for human populations. Therefore, it is not surprising that most technical advances in low temperature storage, assisted breeding and production have been with crops and livestock. Clearly it is far beyond the scope of this chapter to discuss the history and societal implications of this agri-industrial revolution. Nonetheless, it is worthwhile to briefly review in-progress programs that affect the direction and expansion of growth and, most importantly, the utilitarian value of all such resources as we enter the twenty-first century. From a global perspective, plant germplasm enthusiasts are more successful and organized than their counterparts concerned with animal germplasm. This is due, in part, to the technical ease in storing most plant biomaterials (dry at ambient temperature). However, an organizational structure also exists. There is a Consultative Group on International Agricultural Research (or CGIAR), a consortium of 43 public and private sector donors that jointly support 17 International Agricultural Research Centers (or IARCs) (Brown, 1994). The latter generally are located in developing countries and have two primary functions, the first being management of germplasm resources (largely seed banks of traditional crops) and the second being basic research (e.g., detailing pedigrees of new varieties that may reveal the contribution of landraces and folk variety genes, thereby helping generate new commercial varieties). Also under the umbrella of CGIAR is the International Plant Genetics Resource Institute

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(IPGRI), an autonomous, international, scientific organization that is the decision-making body of the CGIAR, developing programs and recommending policies to facilitate ex situ conservation including promoting an international network of activities to ensure collection, conservation, documentation, evaluation and use of germplasm. The IARC genebanks are seen as vast repositories holding about one-third of ex situ crop germplasm or more than 500,000 accessions representing more than 3000 species (Putterman, 1994; Serageldin, 1994). Such a resource has provoked worldwide debates on international property rights, accessibility, and ownership. Much of this concern has been alleviated by CGIAR's recent signing of a landmark agreement with the United Nations Food and Agricultural Organization (FAO), thereby placing these collections into a trust (Anonymous, 1994b). The result is that the CGIAR centers will not seek any intellectual property rights or claim ownership of the germplasm accessions, but will manage and administer the materials to the benefit of the international community. FAO, in turn, will enable centers to adhere to agreed standards and assist in making germplasm available to users. These points are emphasized here because they reflect the kinds of difficult issues that society will face as large-scale GRBs are developed for all kinds of genes (domestic and wild, plant and animal). In the United States, there is a National Genetic Resources Program (NGRP) within the Agricultural Research Service of the U.S. Department of Agriculture. The NGRP "deals with the maintenance and enhancement of the program for the collection, preservation and dissemination of genetic material of importance to American food and agricultural production" (Shands and Duvick, 1994). This program is an extension and outright replacement of the original National Plant Germplasm System and now encompasses plants and other life forms including animals, insects, microbes, and aquatic species. Despite lofty ideals and goals, the birthing of the NGRP in 1990 was not without controversy (Anonymous, 1990a), and, moreover, program expansion to animals and other taxa has not been supported by more federal funding. Nonetheless, the NGRP has been viewed as a way for the U.S. Congress to address and include agricultural genetic resources in response to the escalating concern and awareness within the general public about the loss of biodiversity. One of many responsibilities of the NGRP is the continued support of the National Seed Storage Laboratory in Fort Collins, Colorado, frequently referred to as the "Fort Knox" of plant germplasm (Anonymous, 1990b). This repository protects the genetic heritage of native food species while also having the unenviable task of supporting abandoned and closed collections now requiring rescue and maintenance. Most crop genebanks throughout the world also contain small quantities of seed collected from various wild species, including wild relatives of crops, but few of these are systematically selected or bred. More encouragingly,

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regional, national, and international networks of botanical gardens have developed systematic programs for ex situ rare plant conservation. The best known of these in the United States is the Center for Plant Conservation (CPC). In existence since 1984, CPC is a consortium of 25 voluntarily associated botanical gardens and aboreta that have placed rare, native plant conservation, research, and education as high institutional priorities. The CPC's National Collection of Endangered Plants now stands at 494 taxa, about half of which are wild collected. Two hundred sixteen of these plants are federally listed as endangered or threatened. Of the nearly 500 taxa in this living collection, 139 have been placed in long-term cold storage at the NGRP's National Seed Storage Laboratory (NSSL) in Fort Collins under terms of a cooperative agreement between CPC and NSSL. This part of the Center's collection represents nearly 3.5 million seeds, with more than one-third of these now in cryopreservation. CPC also organizes rare plant task force meetings throughout the United States, writes imperiled plant action plans, produces guidelines and policies associated with a range of rare plant issues, and contributes publications on rare plant genetics and reintroduction. The native plant collections in the more than 1500 botanic gardens and aboreta worldwide represent an even greater assemblage of plant diversity held outside of nature (in excess of tens of thousands of species) (Heywood, 1992). Many of these gardens, like the 25 CPC institutions, are increasingly sensitive to the conservation problems associated with small-sized accessions derived from limited gene pools (Williams, 1991; Center for Plant Conservation, 1991). Despite current limitations, it is significant that the International Union for the Conservation of Nature and Natural Resources (IUCN, recently renamed the World Conservation Union) assisted in establishing the Botanic Gardens Conservation Secretariat to encourage and coordinate such conservation activities internationally. Separating itself from the World Conservation Union in 1991 and renaming itself Botanic Gardens Conservation International (BGCI), this organization now is composed of more than 300 member institutions in 67 countries. Like CPC, but at an international level, BGCI is involved in and promotes a range of off-site plant conservation activities, including e x situ conservation of wild plant germplasm, long-term seed storage, micropropagation, genetic assessments, reintroduction, and conservation education. Among other activities, this group has developed a handbook that provides general directions for number and extent of populations to sample, sample size, collection/storage techniques, and repository documentation (Akerody and Jackson, 1995). BGCI has predicted that botanical gardens worldwide have the capacity for long-term storage of as many as 25,000 species, mainly in the form of seed and for the purposes of conservation, breeding, and reintroduction programs and as insurance against possible wild extinctions (Heywood, 1992).

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Conventional animal agriculture has, of course, benefited from the large-scale distribution of genetic material (predominately sperm and embryos from genetically valuable individuals) in making "better" cattle and to a lesser degree other domesticated livestock species. Collection and storage are based strictly on economic traits and the production of healthy, vigorous stock, not on preserving vast and diverse gene diversity which is the goal, with wildlife species conservation (see below). The impact of using cryopreserved sperm and artificial insemination in cattle is impressive (U.S. Congress, 1987; Seidel and Seidel, 1989) and is one of the many reasons why Americans enjoy the best quality/lowest-cost beef and dairy products in the world. Despite these successes, there appears to be some recognition of need to expand national and international efforts beyond simply providing germplasm for strict production purposes alone. One related by-product has been the establishment of the National Animal Germplasm Program under the umbrella of its NGRP (see above). This program hopes to contribute by not only identifying and evaluating germplasm and establishing repositories, but also by developing an information storage/retrieval data bank and by mapping livestock and poultry genomes. The latter activity, in particular, could have important implications for identifying specific genes valuable for conservation or for producing economically friendly transgenics. Research also is focusing on technologies related to collection, processing, culture, and cryopreservation of spermatozoa and embryos and isolation, culture, and cryopreservation of embryonic stem cells. Priority species for the program include cattle, swine, sheep, goats, chickens, and turkeys. Not all livestock genotypes are common, and there is significant interest in what are called "rare breeds," often considered primitive and shaped by both natural and human selection. Their value comes from being largely unchanged from ancestral stock as well as adapted to a specific environment or use, frequently on the periphery of conventional agriculture. One result has been the emergence of Rare Breeds International, a nonprofit global agency chartered in 1991, which "encourages exchange of information among national, regional and non-governmental domestic animal genetic conservation organizations" (Christman, 1994). Its constituency includes a worldwide membership challenged with generating sufficient support for preserving genetic diversity of many breeds that fail to occupy a secure role in commercial production. It is estimated that half the domestic animal breeds in Europe have disappeared in this century. Further, of the FAO's data bank on about 2800 breeds representing seven domesticated species worldwide (Lohuis, 1994), as many as 30% are at high risk of extinction, especially those indigenous to developing regions. Some experts already have called for large-scale cryopreservation efforts of germplasm to stem the loss of these unique genotypes (Cunningham, 1992; Loftus, 1993).

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v m . CONSERVATION OF LABORATORY ANIMAl.S, INVERTEBRATES, AND MICROORGANISMS Studies of animal "models" in the laboratory (mice, rats, hamsters, guinea pigs, rabbits, cats, dogs, among others) have produced much of what we currently know about disease processes in humans. Almost always these animals are characterized as having a uniform or specific genotype that (1) ensures that the researcher is using a strain that gives repeatable results or (2) expresses a trait that allows a particular biological mechanism, malformation, dysfunction, or disease to be studied precisely. Although the unique genetic backgrounds are invaluable, the proliferation of genotypes (enhanced by transgenic technology) stresses the ability to maintain enormous numbers of living animal colonies. Continued availability means continuous breeding and increased need for new housing and expanded management programs; these are complicated by complex mating requirements of congenic, alloantigenic, and recombinant inbred strains. The burden of stock maintenance then inevitably challenges managers with the tortuous decision whether to eliminate marginally significant strains, knowing well that these unique genotypes may be needed in the future when new research problems or diseases surface. Cryotechnology has been recognized as a partial solution, and several programs have emerged in the United States (largely at the Jackson Laboratory in Bar Harbor, Maine, and the National Institutes of Health in Bethesda, Maryland) that focus on the systematic collection and storage of rodent (mouse, rat, and rabbit) embryos and, occasionally, spermatozoa. However, neither of the programs has sufficient resources to address the explosion of new laboratory genotypes deserving attention and protection. In the United States, there also is national interest in developing a conservation program for destructive and beneficial insects, including establishing centralized insect stock centers under the aegis of the NGRP (Bram, 1993). The challenges here are immense, in part, because alternatives to colony-rearing of invertebrates, including cryopreservation of sperm, ova, embryos, and immature or adult stages, have largely gone unstudied. Therefore, the priority emphasis for these taxa will be fundamental research. The ability to successfully freeze, store, and thaw insect sperm would be the most immediately feasible method of germplasm preservation, especially for certain species in which artificial insemination has been developed (Bram, 1993). For example, research with honey bee sperm already has resulted in low-level survival following liquid nitrogen storage. In contrast, embryo cryopreservation will be more challenging because of complications associated with large size, a naturally high lipid content and complex embryonic membranes that inhibit penetration by cryoprotectants. Nonetheless, the partial success of cryopreserving Drosophila embryos using vitrification

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(Steponkus et al., 1990) demonstrates that these biological impediments will only be transitory, given sufficient basic research. Most of society fails to appreciate that the biomedical and health science industry, environmental and bioremediation technology, and our food industry are highly dependent upon microorganisms which constitute approximately 25% of the Earth's total biomass (Jong, 1988). The economic value of microbial resources is estimated to be tens of billions of dollars (Cunningham, 1994). There is no doubt that the premier organization for acquiring, authenticating, maintaining reference cultures, related biomaterials and data and distributing these to the world research community is the American Type Culture Collection (ATCC), currently located in Rockville, Maryland. The not-for-profit ATCC maintains more than 60,000 cultures of algae, protozoa, bacteria, bacteriophages, cell lines, hybridomas, fungi, yeasts, recombinant DNA materials, viruses, and plant tissue cultures. These biomaterials, supplied to qualified scientists in government, industry, and education, find a multitude of uses, including: (1) assaying antibiotics, vitamins, amino acids, vaccines, organic chemicals, and foods; (2) controls for clinical diagnostics; (3) production of pharmaceuticals, foods, and beverages; (4) environmental clean-up and waste management; (5) pest control; (6) energy production and fermentation; (7) genetic engineering; (8) systematics; and (9) disease diagnosis and toxicity testing. Of added significance is the long-term, systematic experience of ATCC as an organization responsible for safely maintaining and tracking enormous numbers of different kinds of biological materials. In this context, ATCC has emerged as a kind of "bureau of standards," especially in terms of a genome banking organizational structure, security, and informatics (computerization of databases providing ready access to information on stored biomaterials). However, even given this impressive record and the obvious benefits of microorganisms to science and society, there continues to be insufficient funding to ensure microbial germplasm collections in perpetuity (ATCC Board of Directors, 1994).

IX. GRBs FOR Wl[LDLIFE CONSERVATION---ADVANTAGES FOR THE ENDANGERED "OTBOE"

The idea that we should be helping to protect biodiversity by using cryopreservation of germplasm is not new. The U.S. National Academy of Sciences declared in 1978 that "what is done for domestic species (e.g., sperm and embryo freezing) should be done for all species" (Anonymous, 1978). Other venerable organizations including the U.S. Agency for International Development, the U.S. Congress' Office of Technology Assessment and the National Science Foundation have made similar proclamations (for a review, see Wildt et al., 1993). The reason for this endorsement is that

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the advantages could be profound. In earlier papers, we have identified at least seven major advantages of GRBs for contributing to conservation (for reviews, see Wildt, 1992, and Wildt et al., 1993). In this venue, however, the author takes a more illustrative approach, specifically how the GRB could contribute to in situ and ex situ management of an endangered, hypothetical species, Extincionus approximus, the common name being "otboe" (on-the-brink-of-extinction). Otboes are large creatures that normally bear single young. There perhaps are 1000 otboes living in nature with 100 individuals maintained in ten zoos scattered throughout North America, Europe, and Southeast Asia (the latter being the native range). The otboe has been studied occasionally in nature by a few field ecologists who are sufficiently confident about estimating wild population size to become alarmed that the species should receive more attention. Of particular concern is the limited nature of the otboe's home range. All animals live in one of four distinct wildlife parks, each of which is known for its extraordinarily high ambient daily temperature and an abundance of parasites. Even under these rather extreme circumstances, the free-living otboe appears to thrive, except for one ever-increasing problem. All of the wildlife parks are facing human pressures, especially withinpark fragmentation because more roads are being built to provide goods and services for a rapidly growing population of native people living on the park peripheries. Otboes have been bred in zoos, in fact, with sufficient success that curators from each of the regions have developed a species management plan. As a result, the lineage of each captive animal is known, allowing pedigrees and an international "studbook" to be developed. However, there is a problem; all 100 individuals in the existing captive population are derived from a total of three original pairs (or founders), originally caught in the wild. Therefore, the genetic diversity of the zoo-maintained otboes is likely to be quite narrow. Nonetheless, to ensure proper identification, each individual otboe has a unique tattoo that corresponds to a subcutaneously implanted transponder "chip" that can be read electronically. The collection curators in the three regions are beginning to communicate by fax and electronic mail about improving the chances of preserving this endangered species. Their partners include both the field ecologists as well as the authorities managing the national parks who are supportive of a zoo program as a reservoir of genetic diversity. Together, these people have begun a "grass-roots" network and a "recovery team," the first step in successful single-species conservation. They also have had sufficient foresight to initiate action before the species has become irrevocably doomed. Finally, they are willing to consider any and all ideas; that is, they are willing to maximize their options to avoid future regrets. Collectively, they decide that there are two immediate objectives: (1) to help preserve the natural, in situ population and (2) to ensure that the captive population remains genetically healthy and becomes self-perpetuating. They set a goal

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of maintaining at least 90% of the existing genetic diversity in the current world population of otboes for the next 100 years. The recovery team is approached by a young reproductive biologist/ cryobiologist, Dr. Iwanta Dothis, from the range country of the otboe. Dr. Dothis has been studying the basic biology of the species in her local zoo for about 5 years. She has been able to collect sperm by electroejaculation and finds that sperm motility and acrosomal status is reasonable after thawing spermatozoa previously stored in liquid nitrogen. Further, by studying behavior and monitoring hormonal metabolites in the urine of females on a daily basis, she has documented the length of the estrous cycle and estimated the time of ovulation. With this knowledge, she even has been able to produce two pregnancies and two normal young using artificial insemination. Dr. Dothis recommends that the recovery team develop a global GRB for the otboe. What would be the advantages?

A. Advantage 1: Easier and Cheaper M o v e m e n t of Genetic Material The GRB would serve as an adjunct to the global need to move animals and animal biomaterials among the living populations. Healthy animal populations are genetically diverse. Otboes are terrific candidates for experiencing genetic challenges that confront small populations (e.g., inbreeding depression). Their numbers are restricted, and they are widely scattered among zoos within and across different continents. Further, the genetic base of the captive specimens likely is too narrow to maintain 90% of the extant genetic diversity for the next 100 years, so an infusion of genetic vigor from the wild would be advantageous. To maintain the targeted level of genetic variation for the zoo population, as well as to bolster this gene pool by sampling from the wild, the recovery team has two options. It can translocate living animals or, alternatively, move germplasm and then use assisted reproduction, in this case, artificial insemination. However, the GRB also is useful to the free-living population, largely because there would be no need to remove more animals from the wild to support the zoo breeding programs. On the contrary, the recovery team is uninterested in obtaining living wild otboes for the captive breeding program, but rather prefers to collect surplus germplasm containing all the available genes for invigorating the genetically stagnant zoo populations. The wild otboes remain in nature where their presence will help protect native habitat. As studies progress, it may become apparent that certain wild populations also lack adequate genetic diversity. Therefore, the GRB could contribute to sustaining a healthy wild population, by the periodic capture of free-living otboes for artificial insemination with sperm from vigorous, outbred males. The rapid release of these females into native habitat (before becoming adapted to human management) could result in infusions of new genes into

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the fragmented wild populations. Finally, the potential of an otboe GRB is bolstered even further, because scientists have some fundamental information on its basic reproductive biology and, in this case, even have demonstrated the potential of assisted reproduction by producing offspring by artificial insemination. Therefore, the chances of success are high. B. A d v a n t a g e 2: I n c r e a s e d E f f i c i e n c y i n Captive Breeding; More Animals Become Successful Breeders The GRB will be useful for improving the efficiency of the captive otboe breeding program, which now is a reservoir of valuable genes. Because otboes are being managed strictly to maximize genetic diversity, the curators will identify desirable animal pairings (e.g., Animal A should be bred with Animal B). However, there is no guarantee that these individuals will be sexually compatible. On the contrary, animals can (like people) show individual preferences for sexual partners. Therefore, it is not unusual that the labor and cost of shipping animals long-distance to achieve the ideal mating ends frustratingly (at least on the part of the curators) in sexual failure (on the part of the otboes). Therefore, if otboes (or any endangered wildlife species) are to be bred strictly on the basis of mean kinship, there always will be a need for assistance, in this case, artificial insemination. As already identified under Advantage 1, certainly the cost and safety risk of transporting otboe sperm will be far less than those of shipping the entire animal. Additionally, there are animal welfare implications for using germplasm, completely eliminating the stresses associated with long-distance truck or airplane transport of a stress-sensitive wild species.

C. Advantage 3: Reduced Genetic Problems An otboe GRB will extend the generation interval of individual animals indefinitely. As long as viable sperm remains in the repository, the genes do not die with the individual. Rather, they can be rederived and infused into the living population at any time, 5, 20, or more than 100 years from now. This approach of reintroducing "original" genes over time also would decelerate natural losses in diversity as a result of genetic drift.

D. Advantage 4: Fewer Space Problems The GRB will help managers resolve one of the greatest challenges of zoos today, that is, contributing to real conservation in the face of too little space to protect too many species, subspecies and populations. Thousands of species like the otboe deserve captive management attention, but zoos using conventional approaches can adequately manage fewer than 1000

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total species for conservation purposes (Conway, 1986). The problem does not resolve itself by building more or larger zoos. Rather, the issue becomes how to make current zoos more efficient within finite space restrictions. To achieve our targeted 90% genetic diversity maintained for the next 100 years may require hundreds of animal housing spaces, and our otboes will be competing with other equally endangered species for this precious resource. A GRB could help reduce space needs. For example, it has been calculated that as many as 300 spaces are needed in a given region (like the United States) to meet a similar genetic goal for large-sized carnivore species. If the captive population was partially managed using artificial insemination and frozen semen, then only 160 spaces per species would be needed (Leslie Johnston, personal communication) because the genes of selected, genetically valuable males now would be available from liquid nitrogen storage, rather than living animals. The goal is n o t to preserve all otboes in liquid nitrogen, creating a frozen otboe zoo. After all, the living population is the recipient for all cryopreserved germplasm and the producer of all offspring, and there is increased susceptibility to random events (like a sudden skewed sex ratio) if the captive breeding population becomes too small. However, the GRB will facilitate meeting the recovery team's genetic goals while making zoos more efficient, largely by minimizing space resources needed per species and then redirecting newly available space to other species in crisis.

E. Advantage 5: Preserved Extant Genetic Diversity This GRB will help insure extant genetic diversity as well as the preservation of the species itself. Otboes survive, but in fragmented wild and zoo habitats. These small populations are particularly susceptible to events beyond genetics, such as acute and devastating environmental catastrophes or outbreaks of disease. We all have become more aware of the significance of such perturbations, ranging from oil spills to wars to viral epidemics. The fact is that most of the Earth's biodiversity, including the otboe, lives in habitat sensitive to epizootics and drastic shifts in social and political structure. The organized and systematic sampling of biomaterials from freeliving otboes would help preserve all existing and truly "wild" genetic diversity in a living archive. Once captured, these materials could be maintained in perpetuity as insurance and as a tool for maintaining healthy freeranging and zoo populations. F. A d v a n t a g e 6: A R e s o u r c e f o r O t h e r B i o m a t e r i a l s (i.e., B l o o d Products, Tissue, and DNA) The GRB offers other biomaterials (beyond germplasm and embryos) useful for phylogenetic, systematic, and disease studies. The organized

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plan to collect otboe spermatozoa provides an invaluable opportunity for simultaneously obtaining blood and tissue, biologicals that can be used for harvesting serum, plasma, blood cells, and DNA and for originating longlasting tissue/cell cultures. The value of these materials for basic and applied research are enormous. For example, we could, for the first time, understand the relationship between the otboe and other purported species in the genus Extincionus. Quantifiable amounts of genetic diversity could be determined for every captive and wild population, helping drive management decisions about genetic restorations or augmentations achievable through the breeding program. Biomaterials could be used to understand the processes that underlie observed patterns of diversity, such as gene flow, selection, and mating. Blood samples could be screened for various clinical chemistries, thus providing a data bank on species norms, information useful for helping to resolve health problems in individual otboes maintained in captivity. As importantly, resulting data from blood samples collected longitudinally over time could be used to identify the onset and cause of disease epidemics, speeding remedial actions. Finally, these biomaterials could be used by other researchers outside the conventional mainstream of conservation biology. For example, perhaps there is a scientist at a local university interested in the resistance of otboe red blood cells to a parasite-borne pathogen that affects humans living under similar tropical conditions. The GRB provides a central resource repository to respond to such requests that normally swamp already overly busy zoo veterinarians. The request can be satisfied without the need to anesthetize or restrain the animal again. The product may in fact benefit humans, but the by-product (in this case new data on otboe red blood cells) generated by colleagues in academia will increase our fundamental knowledge, and any and all such information ultimately could benefit species preservation.

G. Advantage 7: E c o n o m i c s A GRB provides as-yet unknown opportunities for improving general agri-economy and quality of life, especially in developing countries. In the case of the otboe, we know that the species is especially temperatureand parasite-resistant, and perhaps the isolation and eventual incorporation of these gene complexes into the genome of local livestock species could generate more meat or dairy products from healthier animals. Although theoretical now, these and other relevant technologies will be available in the early 21st century. Other practical advantages will be evident, and some already are useful today. For example, there are several wild cattle species thriving in the forests of Southeast Asia and known to occasionally produce hybrid calves with common indigenous domestic cattle. The resulting genetic vigor, combined with natural disease

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resistance, makes wild cattle species attractive resources for boosting the quality of local meat-producing stocks. A GRB could be established for the purpose of not only insuring native wild cattle stocks, but also for enhancing animal agriculture through the shared use of germplasm with local cattle producers. The above scenario, although fictional, is not unrealistic. The world's wildlife parks and zoos have many species facing similar small population sizes and pressures, forcing managers to make the same difficult decisions. The result has been a tremendous coming together of managers, curators, wildlife authorities, and researchers to identify priorities and resources, including the development of individual species management plans. The various plans have different names based on the global region of origin. We already have indicated that North America has more than 70 Species Survival Plans or SSPs. Although acronyms abound among and within regions, all such plans have a similar goal to manage these small populations responsibly, usually for the specific genetic goal cited in the illustration. These management committees also have formulated ways for scientists to become involved, usually as members of a taxon advisory group (or TAG). Among meeting other objectives, the TAG is responsible for optimizing captive space availability and ensuring that every avenue, including assisted reproduction and genome resource banking, is explored. Likewise, technology is fast becoming an inextricable component of wild species management. The literature is filled with examples of the utility of molecular biology for sorting out systematic and phylogenetic issues and resolving questions about degree of genetic variation and even parentage in natural populations (for reviews, see O'Brien, 1994a,b) Assisted reproductive technology also has advanced to the degree that artificial insemination is close to being a helpful tool for managing certain species. For example, in our laboratory alone, offspring have been produced from the black-footed ferret (Mustela nigripes), Eld's deer (Cervus eldi), cheetah (Acinonyx jubatus), tiger (Panthera tigris), puma (Felis concolor), snow leopard (Panthera uncia), clouded leopard (Neofelis nebulosa), ocelot (Felis pardalis), and leopard cat (Felis bengalensis) by artificial insemination (for a review, see Wildt et al., 1995b). For some species (ferret, Eld's deer, cheetah), the results have been highly repeatable, and in these same three species (as well as the ocelot and leopard cat) we have produced young from frozen-thawed spermatozoa (Wildt et al., 1992b). The widespread, successful use of both animal and plant biomaterials and related biotechnologies will increase exponentially as the potential is realized and as the basic biology of highly endangered species becomes better understood. Even so, given that we knew all the relevant details about all the relevant biota deserving the added protection of a GRB, how would we proceed? This issue currently is receiving the most attention.

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X. ORGANIZATIONAL PLANNING FOR EFFECTIVE WILDLIFE GRBs The concept of GRBs for wildlife conservation have languished for more than two decades as no more than an almost futuristic idea. Enormous tasks like these frequently tend to overwhelm effective organization. Additionally, whenever stakeholders are entering a new arena (in this case one involving rare and valuable resources), there usually is a diversity of opinions on how to proceed. One organization, however, the Conservation Breeding Specialist Group (or CBSG) of the IUCN's Species Survival Commission, has initiated a global dialog. CBSG is one of more than 100 IUCN-sanctioned specialist groups and is a "neutral" catalyst and facilitator with an excellent record in helping regions worldwide develop effective conservation plans for animals, plants, and entire habitats. The CBSG network is formidable, with more than 600 members and a history of species recovery workshops in more than 40 countries on six continents. One of CBSG's many objectives is to develop new tools for conservation, especially for addressing difficult issues that may have become bogged down because of bureaucratic complexities or personal agendas. For example, CBSG uses scientific, standardized criteria to answer oft-cited questions about which species or taxa deserve highest priority conservation attention. CBSG has pioneered a first-cut analysis technique termed the Conservation Assessment and Management Plan (or CAMP) that allows objective criteria to clearly identify those species requiring critical and immediate attention (Ellis and Seal, 1995). This kind of workshop is conducted with the active participation of the "world's experts" who then make the appropriate recommendations for in situ and ex situ action, including more management and research. CBSG also has developed the Population and Habitat Viability Assessment (or PHVA), another type of workshop that considers all the factors impacting a particular species, subspecies, or population (e.g., environment, demography, genetics, human pressures) (Seal, 1993). By identifying the variables and weighing their impact on the basis of known information (from the field or from ex situ programs) and by using computer simulation modeling, workshop participants can objectively identify a plan that will avoid extinction and allow species or habitat recovery. Like the CAMP, PHVA workshops frequently recommend specific research actions that provide guidance to scientists as well as to appropriate funding agencies. CAMP and PHVA workshops provide proactive tools for wildlife authorities, zoo experts, researchers, landowners, and other stakeholders to begin forging interactive, cooperative activities. As most recently discussed by Westley and Vredenburg (1996), CBSG workshops provide a "social" domain for multiple stakeholders (most with diverse viewpoints) to work together interactively (often in small working groups) to reach cooperative consensus. This formation of strong alliances, partnership bonds and "buy-

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in" from group work focusing on a common goal, appears to be one of the most positive characteristics of current strategic planning for conservation. The concept, like the SSP program (discussed earlier), is grounded in grassroots discussion and action. Hierarchical decisions are discouraged, in fact, disallowed in CBSG workshops, and final recommendations originate from within the group rather than any single individual. This brief discussion of the facilitative role of CBSG and its related interest in exploring new tools for conservation is appropriate to its recent focus on GRBs for wildlife conservation. CBSG's objective has been to provoke international awareness, debate, and discussion (Wildt and Seal, 1995), to disseminate specific information to all interested parties, and to facilitate regional meetings on the banking of wildlife biomaterials (one example was a regional workshop in southern Africa) (Bartels and Wildt, 1994). CBSG members also have formulated a resolution statement on the value of such repositories that has been submitted to I U C N ~ t h e World Conservation Union (Table 1). Most importantly, CBSG has produced the idea of a GRB "Action Plan," a written document that guides the stakeholders in the future of a particular species or taxon through the many minefields associated with banking activities. To date, the need for written action plans has been identified as a high priority, primarily for three reasons. First, the systematic collection, storage, and controlled use of biomaterials is a major undertaking. There are many complicated, interactive factors dictating the success (or failure) of such a program, and these must be recognized, documented, and discussed. Participants in these exercises have been amazed at the variety of issues requiring attention. Second, it is important that these documents contain all relevant details because one of the major goals will be the shared used of limited (endangered) resources. This in itself provokes many concerns about equability and the proper use of biomaterials. Third, GRB efforts have a financial cost, and it is likely that a detailed plan (much like a research grant proposal) will be necessary to secure longrange funding support. In advancing the action-planning concept, the first accomplishment was generating actual guidelines for writing such a Plan (Table 2). Ten sections of an action plan were identified: (1) summary; (2) justification; (3) current knowledge of life history and natural reproduction; (4) current knowledge of assisted reproduction; (5) studbook and regional collection plan status; (6) status in the wild; (7) accessibility of existing animal resources for contributing to the GRB; (8) type and amount of germplasm to preserve in relation to genetic management; (9) technical germplasm collection, storage, use and ownership; and (10) resources and funding. Table 2 illustrates some of the intricate details required in considering the development of an effective, conservation-oriented repository. Of particular interest has been Section 8, dealing with the type and amount of biomaterials to collect, store,

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T A B L E 1 P r o p o s e d I U C N - W o r l d Conservation U n i o n S t a t e m e n t on A n i m a l G e n o m e R e s o u r c e Banking for Species Conservation ( D R A F F )

Problem statement The IUCN-World Conservation Union holds that the successful conservation of species requires integrated management efforts to sustain available genetic diversity. These efforts included programs to protect and manage animal populations within their natural, native habitat (in situ conservation) as well as supporting programs that manage populations, individuals, gametes, and/or embryos outside of natural environments (ex situ conservation). The IUCN-World Conservation Union recognizes that, although habitat protection is the most desirable, first approach for conserving biological diversity, supportive intensive management programs are essential in many cases. Such programs can deal effectively with short-term crises and with maintaining long-term potential for continuing evolution. The IUCN-World Conservation Union further recognizes that the efficiency and efficacy of intensive conservation efforts can be increased many fold by applying recent advances in reproductive technology. These include assisted or "artificial" breeding and the low temperature storage (banking) of viable animal germplasm, namely spermatozoa, oocytes, and embryos. Germplasm banks (more broadly defined as genome resource banks): (1) offer a high degree of security against the loss of diversity and, therefore, entire species from unforeseen catastrophes; (2) minimize depression effects of genetic drift and inbreeding; and (3) provide a powerful method for managing the exchange of genetic diversity among populations. Ancillary conservation benefits include banks for basic and applied research including repositories of serum, DNA, and cultured cell lines from germplasm donors that permit studies on disease status, detection of microbial antibodies, pedigree determination, taxonomic status, geographical differentiation of populations, and cellular physiology. The IUCN-World Conservation Union also recognizes that the establishment of genome resource banks must be matched by developing strategies for use as a genuine and practical conservation asset for supporting natural breeding. Furthermore, genome resource banks should follow specific, scientifically developed guidelines consistent with an international standard, thus ensuring their use as a meaningful, practical, ethical, and cost-effective conservation tool. The Conservation Breeding Specialist Group (CBSG) of the IUCN's Species Survival Commission is charged with exploring novel approaches to assisting in the conservation of biodiversity and genetic diversity. Since 1991, the CBSG has been developing and refining strategies for the practical implementation of genome resource banks. These activities have included: (1) publication of scientific manuscripts on the utility of this new conservation approach; (2) development of a comprehensive Action Planning process (with explicit guidelines) to ensure that all such repository programs have conservation application; and (3) identification and coordination of a global network of people and resources dedicated to the systematic formation of genome resource banks. Recommendations The IUCN-World Conservation Union regards the development of genome resource banks as a valuable component of integrated conservation programs. Therefore, the IUCN-World Conservation Union recommends that the CBSG continue to pursue developing the framework for international coordination of this type of program based upon agreements to cooperatively manage species for demographic and genetic diversity. (continues)

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TABLE I--Continued

To achieve this recommendation: 1. Genome resource banking programs, where appropriate, should be incorporated directly into the framework of other conservation action strategies including Conservation Assessment and Management Plans (CAMP process), Population and Habitat Viability Assessments (PHVA process), global/regional collection planning, and recovery plans for restoring species to natural situations. 2. Genome resource banks should be developed only in the context of systematic, written and detailed Action Plans, thereby ensuring that there is a defined conservation goal associated with the collection, storage and use of animal biomaterials to support natural breeding. The development of an integrated plan with clear conservation goals is the single most important consideration prior to initiating banking activities. 3. The CBSG, when requested, should assist taxon Specialist Groups, propagation groups for species, regional conservation programs and others in developing genome resource banking strategies and specific Action Plans. The development of the Action Plan resides with those groups with specific responsibilities for in situ and ex situ conservation of specific taxa, species, and populations. The CBSG will support these activities by interlinking global/regional groups interested in genome resource banking, providing specific information on banking strategies, and integrating information on: (a) reproductive and genetic histories of ex situ and in situ populations; (b) efficiency of reproductive/genetic technologies; (c) approaches for achieving genetic management goals; (d) types of biomaterials requiring storage; (e) appropriate protocols for banking and using biomaterials; (f) ethical issues related to biomaterials ownership/distribution; (g) concerns about disease and regulation; and (h) areas requiring further research. 4. A globally standardized, record-keeping database should be developed for cataloging, pooling, and managing data and transfers of banked materials. It is highly desirable that these biomaterials are linked to individually identifiable source animals to ensure meeting the objective of assisting in managing genetic diversity. Note. Source: C B S G News (1994) 5, 28-29.

and use in the face of genetic management objectives. This subject was so important that the CBSG facilitated a workshop on the Genetic Aspects of Genome Resource Banking, attended by population biologists, geneticists, reproductive physiologists, and cryobiologists (Wildt and Seal, 1994), which, in turn, has resulted in other scientific observations (e.g., Johnston and Lacy, 1995). These fora and the resulting documents are, for the first time, addressing controversial issues about the kinds and amounts ofbiomaterials to collect in the context of small population management as well as how and over what time frame these biologicals should be used to effectively impact conservation. Of particular significance have been three major findings. First, there was consensus that at least two banks should be developed for each species, one designated an "in-perpetuity" repository (for use only when the species approached extinction) and the other for routine management of living animals in the ex situ and in situ populations. Second, whenever possible and appropriate, there is a need to systematically sample the free-living popula-

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Factors to Be C o n s i d e r e d in D e v e l o p i n g a G e n o m e R e s o u r c e B a n k ( G R B ) for a Taxon/Species: Guidelines for Writing a G R B A c t i o n Plan

TABLE 2

I. II. III. IV. V. VI. VII. VIII.

Summary Justification Current knowledge of life history and natural reproduction Current knowledge of assisted reproduction ISIS, studbook, and regional collection plan status Status in the wild Accessibility of existing animal resources for contributing to the GRB Type and amount of germplasm to preserve in the context of management and genetic goals (computer modeling) IX. Technical germplasm collection, storage, use, and ownership X. Resources and funding

Specific factors to consider and information to be included for written section on I. Summary Synopsis (1 page) providing brief description of justification, goals, and overall conservation plan in the context of a GRB. II. Justification A. Provide specific short- and long-term goals for the GRB. B. Describe in detail how a GRB will contribute to conservation ex situ and/or in situ of this taxon/species including, if appropriate, usefulness to sustainable development. III. Current knowledge of life history and natural reproduction A. Assemble information on sexual maturity, reproductive senescence, seasonality, duration of the reproductive cycle, induced versus spontaneous ovulation, time of ovulation, duration of pregnancy/incubation, postpartum estrus, clutch interval, litter size/clutch, and embryonic and postnatal mortality. B. Indicate reproductive success as influenced by genetic, nutrition, disease, and management events. C. Describe extent of technology available for monitoring/managing animal health and provide any available evidence for vertical transmission of diseases. IV. Current knowledge of assisted reproduction A. Indicate prior success at stimulating ovarian activity and/or estrous activity using exogenous hormones and drugs. B. Indicate prior success at monitoring hormonal status using circulating blood hormones or hormonal metabolites measured in voided urine, feces, or saliva. C. Indicate prior success at cryopreserving sperm, oocytes, and embryos based upon in vitro function assays. D. Indicate prior success at artificial insemination with fresh or frozen-thawed sperm. E. Indicate prior success at embryo transfer using fresh or frozen-thawed embryos. F. Indicate prior success at in vitro fertilization using fresh or frozen-thawed gametes. G. Indicate prior success at oocyte maturation followed by in vitro fertilization and transfer using fresh or thawed embryos. H. If no information is available on species of interest, indicate prior success in each area in a closely related species. (continues)

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TABLE 2-- Continued

V. ISIS, studbook, and regional collection plan status A. Provide information on total number of males and females in ISIS, global, and/or regional studbooks and, when appropriate, in private collections. B. Indicate demographic distribution of populations and individuals. C. Provide priority of the species in the context of the global/regional taxon master plan. D. Identify founders and founder lines. E. Prioritize individual donors including providing location, age class, and reproductive history/current status. VI. Status in the wild A. Indicate known or predicted animal numbers in various geographic regions. B. Provide relevant population and habitat viability assessment (PHVA) results, including the status of in situ management programs. C. Indicate prior success (if any) at reintroduction of captive born animals to the wild. VII. Accessibility of existing animals for banking A. Identify and indicate accessibility of wild populations and individuals (in situ and ex situ).

VIII. Type and amount of germplasm (and other biological materials) to preserve A. Define short and long-term management and genetic goals. B. Describe how the banking program will meet stated management and genetic goals. C. Using computer modeling, calculate the minimum number of available individuals (beginning with founders or founder lines) to meet plan objectives. D. Identify materials to be stored (i.e., sperm, oocytes, embryos, cell lines, blood cells, tissues, DNA, bodily fluids [serum, milk, urine, saliva]). E. Using computer modeling, calculate amount of material needed from available, individual founders over a specific interval to meet plan objectives. IX. Technical germplasm collection, storage, use, and ownership If no or limited technical information is available, proceed to conduct research to satisfy the following needs A. When appropriate and needed, identify generic animals available for basic research purposes. B. Develop safe and effective methods for collecting germplasm, including anesthetic procedures, gamete collection, ovulation induction and estrous synchronization. C. Establish baseline gamete/embryo norms (i.e., sperm numbers, sperm morphology, quality grades for embryos) and in vitro assays for determining biological viability. D. Develop procedures for ensuring known health status of donors. E. Conduct comparative studies examining the impact of various cryobiological factors upon post-thaw viability of required biological materials. 1. cryoprotectant solutions and prefreeze processing/equilibration; 2. normal microbial flora associated with collected germplasm; 3. prefreeze quality of germplasm; 4. freezing method including cooling techniques; 5. storage conditions including temperature requirements and storage containers; 6. warming conditions; 7. post-thaw processing in preparation for use, especially cryoprotectant dilution. (continues)

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TABLE 2m Continued

F. Determine adequate post-thaw viability by: 1. gross morphology/quality; 2. in vitro function assays (i.e., sperm longevity, oocyte penetration tests, embryo development in culture); 3. in vivo function assays (i.e., established conceptions in conspecifics or closely related taxa following artificial insemination, in vitro fertilization, and/or embryo transfer). G. Establish criteria for the minimum acceptable viability of germplasm (and other biological materials) after thawing to meet management/genetic needs (i.e., minimum numbers of motile, undamaged sperm, or minimum embryo quality grade capable of resulting in conception). H. Determine potential for using frozen germplasm with other reproductive biotechniques, including interspecific embryo transfer, sperm microinjection, zona piercing, cloning, sexing, assisted embryo hatching, and ultrasound-assisted aspiration and deposition. I. Understand the impact of disease(s) on the effectiveness and safety of banking and using biological materials. J. Assemble information on research resources (i.e., current investigators/ institutions and new/ongoing research findings) available in a computerized database. If cryopreservation technology is available, proceed to formal banking A. Indicate that optimal technology is to be used based upon previous empirical studies. B. Describe how health status of donors is to be determined to prevent disease transmission via movement of germplasm or other biologicals. C. Assemble the information necessary to identify precisely all stored aliquots of each biological material in a central database system. D. Establish and describe a labeling procedure containing key/coded information that is placed upon each stored sample container. E. Identify primary and secondary (back-up) locations for stored materials and the database. F. Describe quality control program to be used ensuring that the following is included: 1. periodic post-thaw viability checks of frozen materials; 2. a system for routine examination of donors for disease; 3. multiple alarm systems to monitor security from unauthorized access and to ensure proper function of all low-temperature refrigerators and safety equipment; 4. back-up power generators to ensure continuous operation of alarm systems, safety equipment, and low temperature freezers; 5. back-up storage space on-site in the event of individual freezer failure. For use of stored materials A. Describe a plan that allows frozen materials to contribute to conservation and genetic management, including information on: 1. individuals and institutions allowed access to the biological materials; 2. how biological materials will be released; 3. how the various biological materials will be used; 4. the geographic region of use including the potential problems associated with import/export restrictions (i.e., disease transmission); 5. proper storage and handling of biological materials after release. (continues)

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TABLE 2n

Continued

B. Indicate the circumstances under which stored materials will be provided. C. Indicate the circumstances under which stored materials will be provided free of charge or sold. D. Indicate the strategy, preferably a computerized database, for assembling and disseminating follow-up information on the usefulness of the distributed materials. For ownership of stored materials A. Determine ownership (individuals or partnerships) of frozen biological materials (i.e., institution owning donor, the taxon specialist group, or country of animal origin). B. Determine ownership of offspring resulting from the use from stored germplasm. C. Define how patents resulting from research using this material will be handled. X. Resources and funding A. Define personnel resources and expertise available for each phase of the banking process. 1. Cryobiologists; 2. Gamete biologists; 3. Embryologists; 4. Veterinarians; 5. Population biologists; 6. Molecular geneticists; 7. Registrars (database specialist); 8. Captive breeding specialists; 9. Field biologists; 10. Representatives of taxon/species coordination and management groups B. Define facilities resources (including buildings, equipment, supplies) for: 1. personnel charged with the systematic collection of materials; 2. primary storage site; 3. secondary storage site(s); 4. the computerized database; C. Identify sources of short- and long-term funding for: 1. personnel charged with the systematic collection of materials; 2. primary storage site; 3. secondary storage site(s); 4. the computerized database; 5. distributing stored materials. D. Identify plan to assure the transferability of the stored collection if those responsible are unable to maintain the bank in perpetuity. Note. Source: CBSG News (1992) 3, 29-31.

tion to capture all extant diversity. Third, using computer simulations, it was determined that the utility of GRBs is more powerful than originally expected, to the extent that even the occasional use of frozen sperm and assisted reproduction could greatly enhance the ability of managers to achieve genetic goals (Ballou, 1992; Johnston and Lacy, 1995). To date, CBSG has made one additional contribution, developing a prototype action plan using a highly endangered species, the tiger (Wildt et al., 1995a). The tiger was chosen, in part, because of its precarious

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status, especially in the wild, but also because of its charisma that attracts substantial attention to the cause and process. The purpose has been to illustrate how the Action Plan would be designed and implemented while highlighting the numerous details required of those interested in initiating a GRB for any animal or plant species. Controversial issues range from justifying need to dealing with questions of ownership and accessibility to biomaterials in the bank. It also is emphasized that the Plan is a "living" document, that is, it is not to be considered rigidly regimented. On the contrary, all good strategic planning should remain structured, but sufficiently flexible to deal with progressive change and the need to include new stakeholders. For example, at the time of this writing, the Tiger GRB Action Plan focuses on assisting conservation of tigers maintained in North American zoos. However, to be truly effective for global conservation, the Plan needs participation from all regions, particularly those comprising the range countries. Therefore, the Plan is being augmented by including Indonesia, Australasia, Europe, and other interested collaborators through the development of formal memoranda of cooperation. CBSG's wide-ranging dialogs about the ramifications of GRBs and their prototype action plans illustrating how the actual process should proceed are a positive first step, certainly preferable to random, spurious collections without clear conservation goals. The CBSG sees the GRB as a potentially effective conservation tool, but only if the organizational context involves multiple stakeholders, strong science, and a commitment to proactive, global cooperation.

XI. SCI1ENCE AND SOCIETAL NEEDS TO ACHIEVE

BIOTIC CRYOCONSERVATION There can be no doubt that above all we, as a society and unified scientific community, must first understand the crucial need for genetic resource conservation. Given that, then the natural question becomes where do we go from here? The answer is to first identify needs and then logical priorities and strategies followed by implementation. As discussed, CBSG has pioneered prioritization tools and certainly increased awareness and stimulated interest and large-scale debates about GRBs. Because these discussions largely have occurred in regional and even global fora, there are some rational, even obvious, needs requiring first-order attention. The following is offered as food for thought and comment.

A. Knowledge and Support First, there is a need for more knowledge and support at every level. Perhaps a helpful analogy is that the current common use of

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cryopreserved sperm and embryos in livestock production did not occur overnight. Success was built upon decades of basic studies that were followed by years of applied research, all of which involved hundreds of animal scientists working in the world's finest university laboratories. These pioneers had access to thousands of "experimental cattle" and millions of dollars of federal, state, and commercial funding, almost assuring eventual success. Likewise, the current boom in combating human infertility largely can be attributed to the biomedical community's ability to respond to an instinctive human need, the ability to reproduce. Hence, thousands of fertility clinics have emerged and hundreds of thousands of children have been conceived by assisted reproduction, including by using cryopreserved sperm and embryos. Again, the driving force was a desire to improve the human health condition, but with this, there was major financial gain by entrepreneurial physicians and especially the biomedical industry. Two points then are relevant. First, these successes largely were derived from a massive amount of basic biological information that eventually was transformed into an applied impact (offspring production). Second, success was achieved on the back of enormous financial support for both long-term basic and applied research. Neither of these crucial building blocks (basic data or research support) are available for most species comprising the planet's biodiversity. They are inextricably interwoven and in their absence there is little that conservationists, including cryobiologists, can do to protect or ensure extant biological and genetic diversity. The research targets are vast and, for the cryobiologist, the obvious focus is upon low-temperature biology issues. What are the mechanisms allowing germplasm and embryos from diverse taxa to be cryopreserved and then regenerated successfully? In most cases, this question cannot be answered; for example, the common fish embryo (certainly one of the most diverse and plentiful life forms on earth) has yet to be successfully frozen for the first time. Even in common species in which cryobiological characteristics are thought to be understood (e.g., the mouse embryo), strain-specific variances are observed in embryo survival to standard cryopreservation techniques (Schmidt et al., 1985, 1989). This can be interpreted to mean that many factors, including genotype within a species, can interact to significantly influence the ability to cryopreserve biomaterials. Further, we still must contend with the perplexing problem of germplasm that appears viable upon thawing, but somehow fails to result in young. Is the problem due to currently undetectable damage within the spermatozoon or embryo or to our failure to create a suitable in utero environment in the host female? Certainly related to almost every issue are the needs for more fundamental knowledge about reproduction and genetics. It boggles the mind to consider the sheer number of known (ignoring the unknown) species that have gone completely

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unstudied in these two crucial fields. Yet reproduction and genetics are symbiotic and inextricably linked to successful collection, storage, and use of germplasm. It has been demonstrated time and time again that systematic and invasive and noninvasive approaches can be used to characterize in detail the reproductive biology of almost any species (including wildlife) (Lasley and Kirkpatrick, 1991; Brown et al., 1994), and there now are many examples of using assisted reproduction to produce viable offspring. Likewise, the explosion of technology in molecular genetics has been highly useful for refining taxonomic classifications and phylogenetic relationships, measuring genetic variation, and resolving issues related to paternity/kinship (O'Brien et al., 1994a,b). All of these will be immeasurably useful in gene banking and follow-up. Research priorities for reproductive and molecular characterizations have been defined (Wildt and Seal, 1988), and now the real challenge is in defining the high priority species requiring the most GRB attention. To summarize, there is the fundamental need to know what is out there, its character, its usefulness, and how to protect it. Therefore, we must continue to advocate and seek support for basic research in conservation biology at all levels. This should involve novel and expansive ideas like the proposed National Institute for the Environment (currently before Congress) that would promote and fund taxonomic/biogeographic surveys, the causes of extinction and the maintenance of ex situ and in situ genetic resources, including germplasm conservation (Blockstein, 1990; Hubbell, 1993). Regardless of source, the financial commitment must be long-term because the goal will be managing and protecting biodiversity in perpetuity.

B. Cooperation and Sharing Perhaps the second most important goal is developing cooperative links at all levels (global, regional, national, and local), scouring all communities for interest, active participation, and support. This first step involves identifying the nodes in the ever-expanding conservation network. Especially important will be the sharing of the benefits of biological diversity. Because developing countries hold most of the world's bio-richness, it is imperative that emerging regions benefit most, largely by receiving compensation to become self-sufficient in conservation (in general) and in cryoscience and genome resource banking. This means industrialized regions providing training and all types of resources for program development within the range country and within the locale of interest, partnerships if you will, in community-based conservation initiatives. Only then, by this interactive, on-the-ground sharing process can we go far beyond our oft-cited, in-spiritonly claim of doing conservation.

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C. Birthing GRBs The third most important need is deciding what to preserve and how best to establish and manage effective GRBs. Identifying preservation targets is the simpler of these two issues. It is essential that systematic and quantitative measures be used to select species for inclusion, and, as discussed earlier, tools have been developed by the IUCN Species Survival Commission's CBSG to deliver first-cut prioritization. It then becomes the regional and local responsibility of the collective managers (ex situ and in situ) for a given species or taxon to determine the usefulness and implementation of the banking process. To reiterate, a GRB should be established only in the face of a clear conservation need as determined by all the stakeholders in the future of the species. In the ideal world with unlimited resources one might envision major regional genebanks comprising huge building complexes and many curatorial staff caring for a vast array of living faunal and floral biomaterials. Perhaps we should strive for this lofty goal of multi-life-form, centralized GRBs, but even staunch advocates that have preserved orthodox seeds from crucial food plants for decades continue to struggle for financial stability of their centers. It may be that centralized repositories will be too difficult to establish for logistical, legal, safety and economic reasons. More debate is required. But for now, the preservation of most rare forms (like germplasm from endangered species) should occur on a much more modest level. Certain organizations (for example, conventional crop seed banks) should expand current activities to include more diversity, in terms of both rare species and additional gene diversity. Other institutions (for example, museums) should consider expanding collections to include frozen, but living, specimens, thereby meeting (yet cleverly expanding upon) their archival mission. For the vast majority of endangered species, however, there is a paradox, a need for immediate action, but in the face of no readily identifiable structure from which to coordinate needed activities. Perhaps the first priority should be to develop a model program. An excellent example is the Center for Plant Conservation which not only is coordinating conservation activities among botanical gardens but also is developing cooperative agreements with other resources like the National Seed Storage Laboratory. For this reason, we advocate the grass-roots approach, like that described earlier in which a GRB Action Plan is developed among the major conservation/scientific players at the taxonomic or species level. Surely progress will be slow initially, but demonstrating a few successes will lend credence to the mission and accelerate activities at all levels, including funding. Given the current absence of major, centralized facilities that can deal with the sheer number of life forms needing attention, this approach also seems the most practical. Further, substantial progress has been made

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in regional collection planning and in developing intensive species management strategies, especially ex situ. In some cases, these efforts are linked to appropriate range countries and free-living animal conservation programs. The cooperative strategy has been forged, and the benefits are becoming apparent. Therefore, it only is natural to superimpose gene banking on the entire process, providing yet more strength to a chain that links science to true conservation. Further, it should be re-emphasized that the only avenue to success is one paved with the intention of globally conserving species, intimately involving the range country, and achieving all of this via a cooperatively produced, written Action Plan. Collecting scarce and valuable resources tends to breed territoriality and raise control issues, so writing an Action Plan explicitly states the sharing process and deals with controversial issues of ownership, accessibility, and proprietary interests. Choosing the participants in the Plan is crucial. Scientists play an important role, but not to the exclusivity of others, especially species mangers (ex situ and in situ). These "stewards" must buy into the process and be involved at all levels, because without manager cooperation, a GRB either fails to exist or simply fades into a worthless warehouse of stagnating materials. As with cattle and humans, successful use of cryopreserved germplasm from other life forms can become inevitable and routine, given that we adhere to basic tenets involving scientific principles, cooperation, and thorough written plans. Perhaps the dream of expansive, high-rise frozen repositories deep within the regions of biological richness can be achieved one day. However, for now we must strive to produce an array of successful models that have both conservation applicability and excitement potential for promoting the cause. D. Specific R e s o u r c e s If the action planning approach is to be used, then we must be vigorous in identifying resources and ensuring that biological materials are collected, stored, and used appropriately. Only a handful of zoos are seriously considering systematic collections for real conservation. There needs to be more interinstitutional communication and cooperation within regions followed by connectedness among all regions of the world, perhaps through the development of a global GRB network. The CBSG's ongoing efforts to identify and encourage linkage of interested parties within and among regions is a first step. Eventually and ideally, there should be a society formed that deals with all the implications of conserving genetic resources. Participants should go far beyond the usual animal and plant conservationists (who usually deal with domestic or wild stocks) to exploit academic and commercial talents. Universities in particular should become involved at all levels, from conducting basic science and field collections to develop-

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ing new storage techniques and offering facilities/resources to the international network. Because initial efforts are to be grass-roots, these GRB programs (at least initially) must rely on already available facilities and personnel which means that university resources are crucial. Therefore, there is even more incentive for partnerships as zoos and botanic gardens tap into neighboring university systems as well as commercial industries, such as bull stud services. Two-way training will be mandatory in making this linkage effective. GRB experts will need to share and transfer technology to wildlife managers, but academicians also will require training. As emphasized throughout this chapter, conservation biology is a complex science. A n appreciation for (1) multidisciplinary, integrative approaches to solving holistic problems, often in a politically charged atmosphere, (2) networking and crossinstitutional/regional cooperation, and (3) dealing with unusual, stresssensitive species (sometimes considered local, national, or even global treasures) must become a standard component of the academic partner's repertoire. Within this resources category, there is a need to set standards for safe and effective monitoring, quality control, and long-term support of these specialized accessions. Surely, we should learn from and exploit those protocols already proven useful for securing common agricultural germplasm and other kinds of collections. For example, the American Type Culture Collection's programs for ex situ management of microorganisms is an outstanding model of large-scale, multi-life-form, safe storage, tracking, and distribution. Standard operating procedures must adhere to the highest quality control standards and at least a two-site storage scheme (in the event of catastrophe). There also is a need to come to grips with quarantine issues, that is, developing the detailed policies, procedures, and cooperative arrangements that ensure safe, yet rapid and efficient acquisition, storage and use of biomaterials. Quarantine policies must be biologically based without regard to economic or political considerations. Crucial partners will be veterinarians, wildlife and regulatory authorities, among others, who will help prevent the GRB process from transferring disease pathogens that could contaminate wild or domestic stocks. In this context, further research will be mandatory to develop improved methods of pathogen detection and germplasm treatment and to reduce or eliminate risks of transfer. Biosafety protocols, which will include laws as well as rules, are a high priority to ensure that these new technologies continue to benefit (rather than threaten)global biodiversity. E. D a t a b a s e s The value of any rare biological material (originating from a living animal or plant or its part, product, germplasm, or by-product) is directly

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related to fundamental knowledge about the particular specimen. Excitement about technology can overwhelm common sense, easily allowing us to ignore the basic need for identifying and tracking information. Poor data translates into worthless biomaterial. Therefore, the intrinsic value of the specimen can be maintained only by having ready access to all useful information, including (among others) type, quality, source, owner, and ancestry. Interestingly, computer software for GRBs is not yet available, at least of a kind that will allow biomaterials to be useful for worldwide conservation. Nonetheless, th e need is immediate, and extensive experience (largely based upon zoo-management programs) indicates that these relational databases could be developed and available in the near future. An important and obvious player will be the International Species Information System (ISIS), an international nonprofit membership organization based in Minneapolis, Minnesota, that currently tracks living specimens maintained in zoos and primate centers (Seal et al., 1976; Flesness et al., 1984, 1995). ISIS presently monitors origin, provenance, pedigrees, birth and death dates, sex, translocation information, and clinical and autopsy data on more than 850,000 specimens of 6000 taxa representing 495 institutions in 54 countries. An essential component of this live animal GRB and the network connecting the participating institutions is the computer software tools. However, generating this global GRB database system will not be simple, because in the ISIS experience, local perspectives vary markedly and definitely will influence local data-recording objectives (Nathan Flesness, personal communication). Adequate data quality and integrity can be established only through a broad-scale integrated information system that returns both short- and long-term valuable services to individual GRB collections. For this reason, the highest priority is to develop minimum specimen data standards followed by the central formulation of applications software that will ensure the ability to pool data among banks and share information across continents, regions, countries, and locales. Therefore, an immediate goal should be to begin developing adequate GRB information systems by building on the existing network and software for the ultimate purpose of assuring that documentation is meticulous and readily accessible.

XII. SUMMARY This chapter has argued to the at-large scientific community that biodiversity is important and that science and technology, specifically cryobiology and assisted reproduction, can contribute to its protection and propagation. In addition to providing obvious economic and lifestyle benefits, the vast wealth of genetically healthy species on this planet offers the scientific community a "laboratory" for exploring a host of wondrous, fascinating,

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comparative mechanisms in the life sciences. However, the laboratory is being depleted and with it vast, undiscovered knowledge, some of it perhaps key to our own survival. Effective conservation takes advantage of all reasonable opportunities. The author has attempted to make a strong case for cryotechnology having enormous potential for assisting in the preservation of single species, e x s i t u and in truly wild habitats. This argument largely is based on the many provocative advantages of genome banking as well as the significant advances recently made in reproductive physiology (especially assisted reproduction), molecular biology and veterinary medicine. Each of these fields will contribute to and benefit from systematic genome resource banking. The need now is for hard-nosed promotion of the utility of these science partnerships and for developing the resources to meet the objectives identified near the end of this chapter. In this context, we must become our own most vocal and enthusiastic public and policy advocates. One does not have to be a "conservationist" by career or training, only sufficiently intrigued about applying strong science in a collegial spirit for a highly worthwhile cause. We can continue to play a passive role or begin to become part of the rapidly growing network of scientists, zoo managers, and wildlife authorities that already are excited about cooperative conservation biology.

ACKNOWLEDGMENTS The author thanks W. F. Rail, U. S. Seal, J. Ballou, R. Lacy, L. A. Johnston, and N. Flesness, who continue to provide many valuable ideas and stimulating dialog and debate on the emerging concept of genome resource banks for wildlife conservation. The author is especially indebted to W. F. Rail, N. Flesness, S. Ellis, A. P. Byers, L. A. Johnston, U. S. Seal, and B. A. Meilleur for valuable comments on an early draft of the manuscript. Dr. Meilleur of the Center for Plant Conservation was especially generous in providing new information on flora conservation. Finally, much of the discussion on biodiversity was driven by the inspirational writings of M. Soul6, P. Raven, and E. O. Wilson.

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Anonymous (1994a). World Resources, 1994-95, report by the World Resources Institute in collaboration with the United Nations Environmental Programme and the United Nations Development Program, pp. 107-111. Oxford Univ. Press, New York. Anonymous (1994b). Consultative group signs landmark agreement to place CGIAR genebanks under FAO trusteeship. Diversity 10, 4-5. Anonymous (1995). Regulating access to genetic resources. S.E. Asian Zoo Assoc. Newsl. 6,13. ATCC Board of Directors. (1994). A resolution for sustained support for germplasm collections. Diversity 10, 50. Ballou, J. D. (1991). Management of genetic variation in captive populations. In The Unity of Evolutionary Biology, Fourth International Congress of Systematics and Evolutionary Biology (E. C. Dudley, Ed.), pp. 602-610. Dioscorides Press, Portland. Ballou, J. D. (1992). Potential contribution of cryopreserved germplasm to the preservation of genetic diversity and conservation of endangered species in captivity. Cryobiology 28, 19-25. Ballou, J. D., and Lacy, R. C. (1996). Identifying genetically important individuals for management of genetic diversity in pedigreed populations. In Population Management for Survival and Recovery (J. D. Ballou, T. Foose, and M. Gilpin, Eds.), in press. Columbia Univ. Press, New York. Barone, M. A., Roelke, M. E., Howard, J. G., Anderson, A. E., and Wildt, D. E. (1994). Reproductive characteristics of male Florida panthers: Comparative studies from Florida, Texas, Colorado, Chile and North American zoos. J. Mammal 75, 150-162. Bartels, P., and Wildt, D. E. (Eds.) (1994). Genome Resource Banking for Conservation in Africa. IUCN/SSC's Conservation Breeding Specialist Group, Apple Valley. Blockstein, D. (1990). Committee formed to create "NIH for the environment." Diversity 6, 44. Bram, R. A. (1993). National Insect Genetic Resources Program faces preservation challenges. Diversity 9, 63-65. Brown, F. (1994). Serageldin credited with CGIAR renaissance. Diversity 10, 5-8. Brown, J. L., Wasser, S. K., Wildt, D. E., and Graham, L. H. (1994). Comparative aspects of steroid hormone metabolism and ovarian activity in felids, measured non-invasively in feces. Biol. Reprod. 51, 776-786. Center for Plant Conservation. (1991). Genetic sampling guidelines for conservation collections of endangered plants. In Genetics and Conservation of Rare Plants (D. A. Falk and K. E. Holsinger, Eds.), pp. 227-238. Oxford Univ. Press, New York. Chasek, P. (1994). Stage is set for Bahamas to host Bioconvention's conference of the parties. Diversity 10, 16-17. Christman, C. J. (1994). Rare Breeds International hosts conference on animal genetic conservation. Diversity 10, 20-21. Conway, W. G. (1986). The practical.difficulties and financial implications of endangered species breeding programs. Intl. Zoo Yrbk. 24/25, 210-219. Cunningham, I. S. (1992). Developing countries the focus of new FAO initiative to protect animal genetic diversity. Diversity 8, 5. Cunningham, I. S. (1994). National Academy of Sciences releases long-awaited landmark report on global genetic resources. Diversity 10, 33-37. Ellis, S., and Seal, U. S. (1995). Conservation Assessment and Management Plan Workshop Reference Material Packet. IUCN/SSC Conservation Breeding Specialist Group, Apple Valley. Ehrlich, P. R., and Ehrlich, A. H. (1981). Extinction: The Causes and Consequences of the Disappearance of Species. Random House, New York. Ehrlich, P. R., and Ehrlich, A. H. (1990). The Population Explosion Simon and Schuster, New York. Flesness, N. R., Garnatz, P. G., and Seal, U. S. (1984). ISIS: An international specimen information system. In Databases in Systematics (R. Allkin and F. A. Bisby, Eds.), pp. 103-112. Academic Press, London.

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Flesness, N. R., Lukens, R., Porter, S., Wilson, C., and Grahn, L. (1995). ISIS and studbooks, very high census correlation for the zoo populationmA reply to Earnhardt, Thompson and Willis. Zoo BioL, 14, 509-518. Heywood, V. H. (1992). Efforts to conserve tropical plants: A global perspective. In Conservation of Plant Genes (R. P. Adams and J. E. Adams, Eds.), pp. 1-14. Academic Press, Inc., San Diego. Hubbell, S. P. (1993). Reinventing our environmental research enterprise. NIE Netw. News, July, pp. 2-3. Johnston, L. A., and Lacy, R. C. (1995). Genome resource banking for species conservation: Selection of sperm donors. Cryobiology 32, 68-77. Jong, S.-C. (1988). Microbial germplasm. Beltsville Symposium on Biotic Diversity and Germplasm Preservation: Global Imperatives, p. 9. Lasley, B. L., and Kirkpatrick, J. F. (1991). Monitoring ovarian function in captive and freeranging wildlife by means of urinary and fecal steroid metabolites. J. Zoo Wildl. Med. 22, 23-31. Loftus, R. (1993). World watch list for domestic animal diversity released by FAO and UNDP provides "early warning system." Diversity 9, 34-36. Lohuis, C. (1994). Scientific interaction hallmark of 5th World Livestock Congress. Diversity 10, 22-23. Maybury-Lewis, D. (1992). Millennium: Tribal Wisdom and the Modern World. Viking, New York. Munk, L. (1993). On the utilisation of renewable plant resources. In Plant Breeding: Principles and Prospects (M. D. Haywood, N. O. Bosemark, and A. Romagosa, Eds.), pp. 500-5227. Chapman & Hall, London. Norton, B. G. (1987). Why Preserve Natural Variety? Princeton Univ. Press, Princeton. O'Brien, S. J. (1994a). A role for molecular genetics in biological conservation. Proc. Natl. Acad. Sci. USA 91, 5748-5755. O'Brien, S. J. (1994b). Genetic and phylogenetic analyses of endangered species. Annu. Rev. Genet. 28, 467-489. Oldfield, M. L. (1989). The Value of Conserving Genetic Resources. Sinauer Associates, Sunderland. Putterman, D. (1994). Premium put on equity issues at Biodiversity Convention's first Conference of the Parties. Diversity 10, 12-16. Rails, K., Brugger, K., and Ballou, J. (1979). Inbreeding and juvenile mortality in small populations of ungulates. Science 206, 1101-1103. Raven, P. (1994). Why it matters. Proceedings of the First Meeting of the Conference of the Parties on Conservation of Biological Diversity. Regan, D. H. (1986). Duties of preservation. In The Preservation of the Species: The Value of Biologic Diversity (B. G. Norton, Ed.). pp. 195-220. Princeton Univ. Press, Princeton. Roelke, M. E., Martenson, J. S., and O'Brien, S. J. (1993). The consequences of demographic reduction and genetic depletion in the endangered Florida panther. Curr. Biol. 3, 340-350. Schmidt, P. M., Hansen, C. T., and Wildt, D. E. (1985). Viability of frozen-thawed mouse embryos is affected by genotype. Biol. Reprod. 32, 507-514. Schmidt, P. M., Monfort, S. L., and Wildt, D. E. (1989). PMSG source influences fertilization and fresh or thawed embryo development but the effect is genotype-specific. Gamete Res. 23, 11-20. Seal, U. S. (1993). Population and Habitat Viability Analysis (PHVA) Workshop, Reference Material Packet. IUCN/SSC Conservation Breeding Specialist Group, Apple Valley. Seal, U. S., Makey, D. G., and Murtfeldt, L. E. (1976). ISIS: An animal census system. Intl. Zoo Yrbk. 16, 180-184. Seidel, G. E., and Seidel, S. M. (1989). Analysis of applications of embryo transfer in developing countries. Theriogenology 31, 3-16.

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van Blerkom, J. (1991). Cryopreservation of the mammalian oocyte. In Current Communications in Cell and Molecular Biology, Animal Applications of Research in Mammalian Development (R. A. Pedersen, A. McLaren, and N. L. First, Eds.), pp. 83-119. Cold Spring Harbor Laboratory Press, Cold Spring Harbor, NY. van den Eede, B. (1995) Investigation and treatment of infertile couples: ESHRE guidelines for good clinical and laboratory practice. Hum. Reprod. 10, 1246-1271. van Uem, J. F. H. M., Siebzehnrubl, E. R., Schuh, B., Koch, R., Trotnov, S., and Lang, N. (1987). Birth after cryopreservation of unfertilized oocytes. Lancet 1, 752-753. Wada, I., Macnamee, M. C., Wick, K., Bradfield, J. M., and Brinsden, P. R. (1994). Birth characteristics and perinatal outcome of babies conceived from cryopreserved embryos. Hum. Reprod. 9, 543-546. Watson, P. F. (1979). The preservation of semen in mammals. In Oxford Reviews of Reproductive Biology (C. A. Finn, Ed.), pp. 283-350. Oxford Univ. Press, London. Watson, P. F. (1990). Artificial insemination and the preservation of semen. In Marshall's Physiology of Reproduction (G. E. Lamming, Ed.), 4th ed., Vol II, pp. 747-896. Churchill Livingstone, London. Whittingham, D. G. (1971). Survival of mouse embryos after freezing and thawing. Nature 233, 125-126. Whittingham, D. G., Leibo, S. P., and Mazur, P. (1972). Survival of mouse embryos frozen to -196~ and -269~ Science 178, 411-414. Wildt, D. E., Byers, A. P., Howard, J. G., Weise, R., Willis, K., O'Brien, S., Block, J., Tilson, R. L., and Rall, W. F. (1993). Genome Resource Banking (GRB) action plan: Global need and a plan for the North American region. Presented at AAZPA Annual Meeting, Omaha, Nebraska, August, 1993. Wilmut, I., and Rowson, L.E.A. (1973). Experiments on the low temperature preservation of cow embryos. Vet. Rec. 92, 686-690. Yanagimachi, R., Lopata, A., Odom, C. B., Bronson, R. A., Mahi, C. A., and Nicholson, G. L. (1979). Retention of biologic characteristics of zona pellucida in highly concentrated salt solution: The use of salt-stored eggs for assessing the fertilizing capacity of spermatozoa. Fertil. Steril. 31, 562-574. Yokoyama, M., Akiba, H., Katsuki, M., and Nomura, T. (1990). Production of normal young following transfer of mouse embryos obtained by in vitro fertilization using cryopreserved spermatozoa. Exp. Anim. 39, 125-128.

Implications of Tissue Banking for Human Reproductive Medickne Armand

M. K a r o w

Department of Pharmacology and Toxicology Medical College of Georgia Augusta, Georgia 30912, and Xytex Corporation Augusta, Georgia 30904

I. INTRODUCTION Reproductive tissue technologists in human medicine are generally confident that their efforts as health care providers do no harm. From the numbers of satisfied patients, technologists may even feel confident that their efforts are beneficial. However, in medicine today nonmalfeasance and beneficence cannot be assumed; they must be proven continually. Reproductive health care has special responsibilities to persons and for society. Reproductive health is an essential component in communication of love and in creation of families. It is essential to continuation of societies. For these reasons technology affecting human reproductive medicine has long had potential for fundamental repercussions in society. Social importance of reproductive activities is reflected in its deeply religious connotations ascribed by most cultures as noted throughout human history. The introduction of anesthesia to American obstetrical practice was met with vigorous resistance by persons who believed it was God's will for humans, conceived in sin, to be born in pain. More recently the introduction of reliable contraceptives was met with alarm because it separated the psychological and physiological components of reproduction and interfered with God's will for human families. However, beyond the religious and psychosoReproductive Tissue Banking Copyright 9 1997 by Academic Press. All rights of reproduction in any form reserved.

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cial impact of human reproductive technology, these technologies have the potential for major economic, demographic, and therefore political impacts. Now tissue banking presents additional opportunities for controlling human reproduction in ways that many people find troubling. The perceived threat posed by these new opportunities is magnified because they are synergistic with other reproductive technologies being developed independently and simultaneously. This chapter will consider the influences of precedence on current practices of tissue banking in human reproductive medicine. This approach enables speculation upon direction and destinations of tissue banking. As in banking animal reproductive tissues, banking human reproductive tissues offers direct access to gametes and their genes. Gamete technology today in medicine is used primarily to assist infertile patients. Although access to gametes provided by reproductive technologies is presently more important to human medicine than is the means of influencing their genetics, this may change. This chapter will look at how reproductive tissue banking and genetics in human medicine has not only contributed to society, but also has been shaped by society. Contributions of the technology to medicine are simple to enumerate. However, impact of the technology on society is far more significant than simply numbers of patients helped and numbers of dollars generated. There is a vague unease that these technologies could change human heredity, devalue human life, or give rise to coercive social programs. These social anxieties have influenced the direction of research in human reproductive technologies. This can be demonstrated through a review of social policy development pertaining to reproductive medicine and genetics. This review of policy will be limited to an American perspective. American values generally embrace widely divergent personal beliefs expected of a diverse, multicultural society. American values are also strongly influenced by a national policy favoring private enterprise that creates a free market economy. For these reasons an American perspective may differ considerably from those based upon other values. However, this perspective focuses on a general phenomenon, namely a reciprocal influence of applied science and social policy on the development and implementation of each other. The choice to present only one country was made to illustrate the intensity of response to reproductive technology at a variety of social levels in one social system, not to suggest that American behavior or values should be a paradigm for emulation. In fact, American behavior is influenced by events and experiences of all members of the global community. A study of social reaction to changes in reproductive processes occurring in other societies would reveal an appropriate intensity of concern, different from America's. A comparative study of interactive social responses to reproduc-

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tive technology might be interesting but is far beyond the scope of this chapter. Changes provoke anxiety and profound changes may provoke fear. A change in an activity as fundamental as reproduction is profound. Fear of reproductive technology run amok originates in historical reality. As recently as the 20th century, humans have witnessed cruel behavior from presumably well meaning, civilized people. During the first half of the 20th century American state laws and courts permitted involuntary sterilization of tens of thousands genetically "unfit" persons (feeble-minded, paupers, criminals) (Kevles, 1985). By the middle of the century American attitudes turned to revulsion by this and other coercive eugenic programs occurring elsewhere (Kevles, 1985). These overt practices and revelation of condoned but covert medical research activities conducted in America influenced reproductive medicine and clinical genetics in the last half of the 20th century. During the decade of 1971-1980 advancements in reproductive medicine and genetics produced cataclysmic events. By 1973, molecular biologists became alarmed that technology would soon give them the tools to alter the genetics of living organisms (Berg et al., 1974). In the same year, the United States Supreme Court in Roe v. Wade ruled that each woman has the right to abort her pregnancy. On 25 July 1978 Louise Brown, the first baby conceived by in vitro fertilization (IVF), was born (Edwards and Steptoe, 1980). In 1980 the U.S. Supreme Court in D i a m o n d v. Chakrabarty ruled that genetically engineered life forms could be patented. These events were accompanied by a plethora of learned discussions at universities, trade associations, independent institutes, local and state governments, and all three branches of the American federal government. At the federal level, study groups (e.g., Congressional Office of Technology Assessment created in 1972 by Public Law 92-484) were formed to assess new biological sciences. Their recommendations led to policy decisions that directed the development of genetic and human reproductive technology. To place in context these decisions regarding reproductive technologies, let us first define several major reproductive technologies utilized in human medicine and quantify their economic impact in America. Then social responses to these technologies will be examined and the development of genetic technologies that have been or may be incorporated in reproductive medicine will be reviewed. These two examples permit conclusions to be drawn concerning the social impact of reproductive technologies generally, including reproductive tissue banking. II. REPRODUCTIVE TECHNOLOGY SERVING MEDICINE

Moving into the 21st century, reproductive tissue banking offers human medicine three general gametic technologies discussed extensively else-

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where in this book. The technologies can maintain the viability of human sperm or oocytes in vitro for several hours. They can maintain the viability of a zygote for several days. With difficulty, athey can cryopreserve sperm, oocytes, or zygotes for years. The clinical significance of the ability to maintain human gamete viability in vitro is presently limited. The primary usefulness of the ability is in regard to providing sperm for insemination and providing oocytes to be used for IVF and for oocyte transfer. It is also useful in preparing gametes for cryopreservation. Technology is available to assist defective sperm in fertilization. These existing technologies in clinical practice are believed to be innocuous to gametes and their offspring. Technology is sought, but not available, to directly "enhance" defective sperm. Technology for genetic diagnosis including sex selection of sperm is in development. The clinical significance of maintaining the viability of human zygotes (preembryos) in vitro is substantial. This technology gives access to blastomeres for genetic diagnosis. This technology permits preembryo maturation to a stage appropriate for embryo transfer or for cryopreservation. Additional technology is needed for assessment of preembryo "quality" while in vitro.

The potential significance of cryopreserving human gametes and embryos is underdeveloped but may be large. In reality there is great variability between men in the post-thaw quality of their sperm. Although some sperm from most men will survive thawing, the actual efficiency of the process is less than satisfactory in regard to having fertile thawed material suitable for cervical or intrauterine insemination. A primary function of a human sperm bank currently is to recruit donors who have "freezable" sperm, namely, about 1 in every 10 men interested in being a sperm donor. In a similar manner, only a portion of human zygotes or pre-embryos survive thawing; and mature human oocytes, those at metaphase II at the time of ovulation, seldom survive thawing. For this reason, great interest is given to the potential of successfully cryopreserving immature oocytes.

m . ECONOMIC IMPACT OF REPRODUCTIVE TECHNOLOGY IN AMERICA

These technologies are being clinically applied to assist the reproductive objectives of Americans. Let us consider here the economic impact of technologies for assisting patients in becoming pregnant and then separately consider genetic technologies. Of American couples in the reproductive years about 85% wanting a child can conceive and deliver a baby. Many of those who cannot have babies experience severe traumatic anxiety of childlessness. For this reason

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many policy makers recognize infertility as an abnormal condition if not a "disease." Infertility is defined as a situation existing when a woman 15-44 years old does not become pregnant after 12 months of "unprotected" intercourse. The definition of infertility includes two major groups: therapeutic sterility (as a consequence of voluntary surgical sterilization and of iatrogenic sterilization) and "natural" or involuntary infertility. About 8.5% of married American couples with wives in this age range are deemed to be infertile. The percentage has remained constant since 1965, but the actual numbers of infertile Americans has increased because of population increases (Mosher and Pratt, 1990). The total number of infertile Americans in 1994 was approximately 10 million. Of the total number of infertile Americans, about half have become sterile as a result of therapy (Mosher and Pratt, 1990). Of the remaining 5 million infertile Americans (either childless or impaired fecundity) about 55% are women. About 2.3 million Americans annually will seek some sort of professional service, including counseling, regarding their infertility. These services, costing $2 billion annually, enable infertile patients to give birth to 150,000-250,000 thousand babies. Other infertile patients annually adopt about 50,000 children born outside of their family (National Committee for Adoption, 1989). In considering patients seeking infertility services, from a social perspective they can be classified as requesting or requiring either gametes from an intimate sexual partner or gametes from someone other than an intimate sexual partner. (This does not include those who will adopt children or who will choose to remain childless.) The adjective autologous indicates use of gametes from an intimate sexual partner. The adjective homologous indicates use of "substitute" gametes, that is, gametes from someone other than an intimate sexual partner. Autologous assisted reproductive technology obtains in situations in which a spouse stores gametes for a postponed pregnancy. A spouse undergoing cancer chemotherapy or other procedure potentially imparing his or her gametes might request gamete storage. Autologous gamete storage requires cryopreservation and is a relatively uncommon procedure in American medicine, but it is not an unusual or rare procedure for American clinical reproductive banks. Technology is also available to assist in the fertilization of defective autologous gametes. Annually about 172,000 American women undergo therapeutic insemination; of these about 50% used her husband's sperm, producing 35,000 children (U.S. Congress, 1988a). Autologous insemination is highly preferred over the alternative of using an anonymous fertile donor. In a survey of 701 adult Americans in the general population (a survey not limited to persons trying to become pregnant), 78% of the women would not want to use an anonymous donor (Stein, 1994); these figures corroborate earlier surveys of university student attitudes (Dunn et al., 1988; Miller, 1974).

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Autologous insemination could be used to enable couples to choose the sex of their offspring. Technology is being developed to separate sperm with the X-chromosome from sperm with the Y-chromosome (Johnson et aL, 1993). (Alternative methods for selecting the sex of a conceptus include DNA analysis of a blastomere removed prior to embryo transfer, or chromosome analysis of fetal tissue. These alternatives then involve possible destruction of the pre-embryo, embryo, or fetus not of the desired sex.) Although in vivo insemination with defective sperm is usually ineffective in achieving a pregnancy, IVF with such sperm can sometimes fertilize oocytes, or fertilization might be accomplished by instrumentally inserting a sperm directly into the oocyte. More than 70% of the general population approves use of autologous IVF for married couples who cannot have children any other way (Miller, 1974; Stein, 1994). About 38,000 American women undergo variants of IVF procedures annually (American Fertility Society, 1994). Each monthly attempt typically costs a couple $5,000-10,000, including necessary drugs (Simpson and Carson, 1992; U.S. Congress, 1988b). Statistically 17% of couples using IVF technologies actually take home a baby (American Fertility Society, 1994). Although autologous procedures are preferred by Americans, many infertile persons are willing to use homologous procedures as an alternative. Homologous procedures have the potential of allowing patients to have the experience of pregnancy (if possible and desired) and to have offspring with genes from one of the social (rearing) parents. Homologous procedures may involve the use of a sperm donor, an egg donor (or both), or even a surrogate to carry the fetus to term. (The noun surrogate is used to indicate any woman who allows her uterus to be used for gestation of a fetus intentionally created for rearing by one or more persons other than herself.) About 86,000 American women annually begin therapy with donor semen producing 30,000 children (U.S. Congress, 1988a). Donor semen is cryopreserved in order to conduct testing necessary to avoid disease transmission. About 1800 women annually use donor oocytes, and about 230 others use surrogates (American Fertility Society, 1994). There is a substantially low approval rate from the general population for homologous procedures with the lowest for homologous procedures involving an anonymous surrogate (Stein, 1994). The number of American physicians providing infertility services is 11,000 (U.S. Congress, 1988a). Several hundred human sperm banks operate in America; the exact number is unknown because no compulsory national registry exists; 77 are registered within California (1995). It is reasonably estimated that a majority, perhaps 90% of American sperm banks are operated by physicians for the benefit of his/her patients. Other sperm banks serve an extended geographic area, sometimes internationally.

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IV. SOCIAL ISSUES IN REPRODUCTIVE TECHNOLOGY Procedures in reproductive technology, whether autologous or homologous, are fraught with ethical, legal and social issues. The issues may be grouped into five general categories: (1) family values, (2) moral value of humans, (3) property rights, (4) access to health services, and (5) pursuit of knowledge. Although analysis of any one issue is beyond the scope of this chapter, it is reasonable to indicate the breadth of issues and to refer the reader to analysis published elsewhere.

A. F a m i l y Values The composition of an American family today is highly variable. At a minimum it consists of two adults (heterosexual or homosexual) or one adult and a child. An American family could include more than two adults and children from more than two parents. Each family member has a set of values or perceptions about human relationships within a family. Usually these perceptions include ideas about adult roles, male and female, within family activities. Usually there are perceptions about children in the family. Persons and other components of this intimate setting become symbols, taking on meanings beyond physical reality. Homologous reproductive procedures inject another adult participant into intimate family activities, and this is potentially disruptive. The third party is needed, yet is an intruder into symbolic relationships. The importance of personal fertility is perceived differently by men and by women (Mahlstedt, 1985; Newton and Houle, 1993). Men sometimes confuse fertility with virility; virility is more important for male self-esteem. For women fertility with its implications for motherhood is highly important for self-esteem. When personal fertility is diminished, men and women both grieve the disillusionment (Mahlstedt, 1985; Nachtigall et al., 1992; Newton and Houle, 1993; Wright et al., 1991). Family relationships can be altered profoundly by reproductive technology involving only autologous gametes. Because viable gametes can be cryopreserved for years, it is possible for these gametes or their embryos to survive their donor. Autologous sperm have been stored for posthumous use (Shapiro and Sonneblick, 1986; Wadlington, 1983), and autologous embryos have been orphaned (Smith, 1985-86). Sometimes courts have had to decide the disposition of these materials. The use of "posthumous" gamete may be influenced by the legal rule against perpetuities (Staff, 1987). "Posthumous" offspring complicate inheritance rights, especially those of other offspring from the autologous gamete donor (legal parent) (Hecht v. Superior Court).

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The use of homologous gametes can similarily alter family relationships. Use of a gamete donor or a surrogate calls into question traditional roles of husband and father, wife and mother. Use of homologous gamete donors especially challenges exclusivity of spousal and of parental relationships. To avoid these challenges gamete donors, notably semen donors, have historically remained anonymous and therefore absolutely outside the family construct (Karow, 1993). This is consistant with principles of personal privacy and confidentiality granted by the United States Constitution in the Fourteenth Amendment. Additionally this anonymity protects the donor from possible financial or legal responsibilities to the offspring. Lifting of anomymity could affect relationships between members of the extended family, especially "grandparents" of the offspring (Snowden, 1983). Also, use of a gamete donor gives rise to the possibility of unwitting consanquineous union between offspring from different families of one donor, that is, a type of incest (Hajnal, 1960; Jacquard and Schoevaert, 1976). Although children conceived without medical technology may not know the identity of one or both genetic parents, the wisdom of donor anomymity is being reconsidered in view of the possible benefit that offspring might derive from knowing genetic parents (Baran and Pannor, 1989; Haimes, 1992). Nevertheless, relatively few couples undertaking donor insemination intend to tell the child of his/her genetic origin (Klock and Maier, 1991). The reason for this in part is parental fear of rejection by persons including family and/or offspring. The number of children conceived with donor semen who seek their genetic father is not known, but only about 1% of adoptees seek either genetic parent (National Committee for Adoption, 1989). Does a person have a right to know his/her paternal origins? Does a gamete donor or surrogate have a right to know his/her offspring? If so, does this right supercede a right to privacy? The issue of access to medical records on patients, offspring, and donors is presented by the Office of Technology Assessment (U.S. Congress, 1993). A child conceived with gametes taken from a deceased donor or a fetal donor (Hartshorne et aL, 1994) would never have the possibility of knowing the donor socially, but the possibility of having genetic information could exist. Even though rearing parents may obscure the social reality of their gamete donor, the gamete bank may preserve in perpetuity their confidential medical records on each donor. Furthermore, the gamete bank may take from each donor diploid cells, such as lymphocytes, to cryopreserve in perpetuity the chemical genetic record that could be read using the results of the Human Genome Project (discussed below). Such data, written and chemical, could be accessed by offspring, rearing parents, or other responsible persons knowing the donor's identifying code assigned by the gamete bank. In addition to the shaping influence the family has on the person that will develop from the pre-embryo, other societal components will have a powerful influence on that person just as they do on all family members.

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For example, attitudes of family members toward homologous reproductive procedures will be influenced by persons outside of the nuclear family. This includes authority figures such as professional providers of reproductive services, religious leaders (Fletcher, 1954; Ramsey, 1970; Rosner, 1972, Schenker, 1985; Vatican, 1987), law makers (Dickens, 1994), and other influencial persons in the larger community. The values of these persons may be different from that of a family seeking homologous reproductive services. These kinds of social influences on the child and on other persons will be mentioned again in this chapter.

B. Moral Value o f Being H u m a n Consider now the value of an individual, human dignity, the worth of a person. Rules for protecting the rights of humans derive from cardinal ethical principles (Beauchamp and Childress, 1994; National Commission, 1978) of beneficience, justice, and personal autonomy. They are used to strike a balance between the rights and freedoms of the individual with those of society. These rights, however, are given to individuals by their society. Some rights take precedence over others. Codified personal rights (in contrast to property rights) are known as tort law. Formal rules for obtaining informed consent from research patients in America developed from the Belmont Report (National Commission, 1978) and are codified (Public Law 99-158; 45 CFR 46, 1991). From these documents rules evolved for informing all people, including patients, gamete donors, and surrogates, about medical alternatives. Risks and benefits of new technologies must be thoroughly assessed and explained to a person who then has the right to refuse the procedure. It is almost impossible for a governmental agency to impose a medical procedure on a person or group of persons simply because "it is for the good of society." Removal of human tissues or cells such as spermatozoa or oocytes from a body, whether living or dead, usually requires informed consent. Tissues can be removed only when consent is obtained from a person competent to provide informed consent, that is, the person must understand the consequences of tissue donation, have legal dispositional authority for the tissue, and be independent rather than subservient and vulnerable to coercion. The person must be told of risks of tissue acquition, use of the tissue, and that he or she may withhold permission at anytime without jeapordy. Informed consent to use fetal tissues for research or other purposes must be obtained from the women aborting a fetus. The potential for commercial exploitation of the removed tissue must be explained to the donor (Moore v. Regents). The appropriateness of fertilizing gametes, whether autologous or homologous obtained from human fetuses (Hartshorn et al., 1994) or deceased persons is doubted by many people because such donors are unable to provide contemporaneous consent to the fertiliza-

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tion. Note, however, that some believe that genes belong to the global community rather than to individuals; therefore, they believe consent of donor is unnecessary. Although Americans are not expressly granted a right to procreate, legal scholars believe that substantial precedence provides this right (Charlesworth, 1993; Flannery et al., 1979; Robertson, 1986 and 1994). The right includes use of medical technologies and even of surrogates. On the other hand, American society is probably not required to use public resources to assist its citizens in procreating. In 1973 the U.S. Supreme Court in R o e v. W a d e determined that any American woman could authorize by herself termination of her pregnancy through fetal abortion. This decision was predicated on the right to privacy provided by the Fourteenth Amendment to the Constitution. She may undergo an abortion at anytime up to the point at which the fetus "is potentially able to live outside the mother's womb . . ." The Supreme Court determined that until this time the fetus is not a "person" within the context of the Constitution. The Court, however, has determined that individual state governments may refuse to provide public resources for abortions. So an American woman may use her own personal finances to have her fetus removed for any reason including birth control or sex selection. Public policy such as R o e v. Wade is broad, allowing considerable latitude for individual ethical choice. Various polls (Dionne, 1989; Lamanna, 1984) show that more than two-thirds of Americans believe abortion to be unethical, but agree that the woman should be allowed to make the decision. An overwhelming majority believes that abortion should be available in cases of severe fetal abnormalities, incest, or rape. In 1986, 3.8 million Americans were born and 1.5 million elective abortions were performed (National Committee for Adoption, 1989) A few Americans, the exact number is unknown, have exhibited intensely strong emotions against the R o e v. Wade decision. Such persons have been willing to spend many hours in public demonstrations in an effort to persuade others not to use clinics that perform abortions. Some have gone beyond legally sanctioned demonstrations to illegal acts including murder of clinic personnel. Equally emphatic, on the other side, is the determination of persons seeking abortions to utilize such clinics despite demonstrations and personal risk. The fertilized human egg, whether in vivo or in vitro, clearly is genetically human and has the potential of becoming an infant by birth (Ethics Committee, 1994; Grobstein, 1990; Robertson, 1986 and 1994). However, this genetic potential does not grant legal rights to the fertilized egg. In fact under American law, the fertilized egg has no legal status or rights. Just as human development from a fertilized egg is progressive rather than instantaneous, the attainment of human rights is progressive. A newly born

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infant has limited rights, e.g., the right to life but not the right to vote. Biological development and maturation conveys full human rights. Although the fertilized egg and the pre-embryonic cell mass prior to implantation do not have rights, they are entitled to special respect (Ethics Committee, 1994). It is possible that this respect precludes the commodification of pre-embryos. This is an extension of American family law that precludes trade in infants; parents have responsibility for their children but do not own them. At still another level, the U.S. Constitution through its Thirteenth Amendment precludes property rights in humans, i.e., slavery.

C. Property Rights in Personal Tissue Concepts of personal tissue ownership have broad implications. They affect perceptions of human dignity, life, and death. It is not a foregone conclusion that people own their bodies (Andrews, 1986; Bray, 1990; Scott, 1981; Moore v. Regents). Neither tort law nor property law considers the suitability for trade of bodies and human tissue such as reproductive cells. Dispositional authority over human gametes, both sperm and oocytes, and over their products of fertilization is vested in their donors (Davis v. Davis). The donor may convey, transfer, or relinquish that authority to a tissue bank. Gamete donors are encouraged to make binding agreements about the disposition of these cells, including the in vitro product of fertilization, as precaution against unanticipated situations such as death, divorce, or donor unavailability. Presumably a surrogate has temporary dispositional authority over any pre-embryo in utero. No American statute, state or federal, prohibits the sale of blood or reproductive tissues. (Sale of specified organs is a felony under the National Organ Transplant Act (NOTA; Public Law 98-507, 1984), but regenerative tissues are not considered. Also, NOTA permits assessment of professional fees associated with organ "harvesting," but no payment may be made to the donor or his/her family.) State statutes (Uniform Anatomical Gift Act; Uniform Commercial Code) and common law usually characterize payments made to blood donors as compensation for services rather than the sale of a commodity. The primary legal reason for characterizing these transactions as involving services rather than commodities is to avoid the specific performance provisions of the Uniform Commercial Code and to avoid liability for transmission of infectious diseases. It is not known whether these provisions accorded to blood donation would be extended to gamete donations. Presumably most American blood donors and semen donors believe they are being paid for their tissue. Unresolved legal issues obtain to products of fertilization. These living cells have no legal right of existence and no legal status as discussed earlier.

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D. A c c e s s to H e a l t h Services Issues of persons' access to health services in reproductive tissue banking is beyond the scope of this chapter. It deals with the cost and availability of treatment for infertile patients. This issue as it pertains to reproductive medicine is thoroughly discussed by Charlesworth (1993), by Coddlington et al. (1990), and by Reagen (1992). E. Pursuit o f K n o w l e d g e It is questionable whether governmental authorities might absolutely prohibit certain kinds of research that might produce "unacceptible" knowledge. In the late 20th century, moratoria were placed on American research involving recombinant DNA and involving human IVF. When molecular biologists realized early in the 1970s that they were close to making a gene, that is, taking part of the DNA from one organism and inserting or recombining it with DNA of another organism, they became anxious that results could be detrimental to life on earth. Discussion of these concerns in several conferences and in journals (e.g., Berg et aL, 1974) led the Director of the National Institutes of Health (NIH) to form an advisory committee on October 7, 1974. The main responsibility of the committee was to recommend "guidelines to be followed by investigators working with potentially hazardous recombinants." In the absence of any guidelines several communities followed the lead of the city fathers of Cambridge, Massachusetts, and temporarily banned research using recombinant DNA. The NIH Recombinant DNA Molecular Program Advisory Committee (RAC) issued its first guidelines in 1976. Although these guidelines officially controlled only research funded by NIH, they were voluntarily adopted by American industry and by many foreign governments. With acquisition of experience the original guidelines have been gradually relaxed over time. A similar political process was created to control research involving human IVF. As human IVF became imminent, the federal Department of Health, Education and Welfare (DHEW; predecessor to Department of Health and Human Services) decreed on August 8, 1975, that No application or proposal involving human in vitro fertilization may be funded by the Department or any component thereof until the application or proposal has been reviewed by an Ethics Advisory Board.

After the 1978 birth of Louise Brown, two federal panelsma DHEW Board (1979) and a Presidential Commission (1983)mconcurred that research on human IVF could be ethically acceptible. Nevertheless the executive branch created a de facto moratorium on human IVF research by not appointing the Ethics Advisory Board necessary to review submitted research proposals. In large measure this moratorium was a political response to groups objecting

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to wanton experimentation on "human" life and the subsequent destruction of the life. The moratorium was ended by the NIH Revitalization Act signed by President Bill Clinton on June 10, 1993 (Public Law 103.43, Sec 492A). However, later, on December 2, 1994, President Clinton instructed NIH not to provide federal funds for research creating human pre-embryos to be used in research. In 1995 Congress prohibited NIH from funding research that would result in creation of human embryos, effectively blocking human IVF research. So American public pressure for many years has blocked NIH from funding clinical research in advanced reproductive technologies. The U.S. Constitution through its First Amendment provides virtually unlimited freedom of expression. However, the Supreme Court has long recognized that expression can be regulated in proportion to its impact upon people or the environment (Emerson, 1979; Flannery et al., 1979). In this regard, limitations provided by RAC and by informed consent are consitutionally appropriate. Limitations on other kinds of biomedical research might be upheld by the Court. Furthermore, the Constitution grants Congress authority "to regulate commerce with foreign nations, and among the several states" (Article I, section 8), a provision the Court has interpreted extremely broadly, permitting regulation of any activity affecting commerce including scientific development.

V. AMERICAN REGULATION OF REPRODUCTIVE TECHNOLOGIES All human societies control the behavior of their members. In its most basic form this control is exerted through public opinion and social norms. Society provides incentives, both positive and negative, to attain conformance. This control, essential to social order, provides individuals and groups with rights and responsibilities and protects the weak from exploitation by the strong. Enacted law provides a minimal level of enforceable standard of conduct; ethical principles provide guidance for a voluntary, higher standard of conduct. The operation of American tissue banks is regulated informally and formally (Karow, 1991). Informally, certain voluntary organizations such as the American Society for Reproductive Medicine (Birmingham, AL) and the American Association of Tissue Banks (McLean, VA) make recommendations in regard to ethical conduct and professional standards. Regulations of tissue banks generally exist to ensure safety rather than efficacy of tissues. Recommendations regarding the safety of patients and tissue donors enhance public confidence in tissue banks. Tissue efficacy is determined in large measure by medical application, a different regulatory province in America. Other recommendations limit competition especially from

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newcomers to tissue banking. In sum, current codified regulations of human reproductive tissues wisely do not address many of the major issues presented in this chapter. Conformance to such codes is voluntary and is usually more costly from an operational perspective than is nonconformance. Although voluntary codes of conduct are not directly enforceable by legal sanctions, retribution can be exacted for infractions by exclusionary action, public exposure, and civil lawsuits. Thus, in the end, conformance may actually be cost effective. In addition to informal regulation, tissue banking activities in America are subject to enforceable state laws. A few states have been authorized by their legislative bodies to regulate tissue banking activities such as donor selection; tissue acquisition, processing, and storage; tissue distribution; and personnel qualifications. Laboratory evaluation of donors and tissues is regulated additionally by states and also by the federal government (e.g., Clinical Laboratory Improvement Act of 1988). The Fertility Clinic Success Rate and Certification Act of 1992 (Public Law 102-943) requires fertility clinics to report their success rate in performing various procedures. These reports, to be filed with the federal Centers for Disease Control (Atlanta, GA), must comply with specified reporting standards and will be available as public documents. Funds for implementation of this law have not been granted by Congress as of 1995; therefore its implementation is in limbo. Truth in advertising by tissue banks is regulated by the Federal Trade Commission and could be regulated by the Food and Drug Administration. As of 1995 the federal government does not specifically regulate reproductive tissue banks although the Food and Drug Administration has legal authority to do so and regulates other transplantable human tissues. Voluntary and compulsory regulation of reproductive tissue banks in America occurred in response to concern that human semen transmits human immunodeficiency virus (HIV-1, i.e., HTLV-III). The federal Centers for Disease Control recommended in 1988 that human semen be quarantined for 180 days. The quarantine was thought to protect the inseminated recipient from lethal infection with HIV. At the time of the recommendation no direct test for the virus was available. Since the development of detectable human anti-HIV antibodies might require 60 days, infectious disease authorities in the government recommended that a serum sample be taken at the time of semen donation. If serum was negative for HIV-antibodies, semen would be cryopreserved and held in 180-day quarantine after which a second serum sample would be taken for antibody testing. Stored semen would be released for clinical use only if the second serum sample tested negative. There have been no documented instances of HIV-transmission from semen processed in this recommended manner. Regardless of its societal benefits, there are potentially negative effects of regulation, especially compulsory regulation. Cost of regulation can

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financially inhibit tissue bank growth. Regulation can inhibit innovation. Regulation can be excessive so that the cost/benefit ratio is adverse. Regulation can be applied capricously by persons in authority. Also, regulatory agencies can be self-perpetuating.

VI. SOCIAL INTERACTION WITH GENETIC TECHNOLOGY W O R K I N G T H R O U G H REPRODUCTIVE MEDICINE Reproductive processes are critical to continuation of genetic heritage. Unlike other physiological systems, reproductive processes would be superfluous in the biological life of the organism in the absence of genetics mechanisms. Reproductive technologies including tissue banking provides scientists and clinicians access to gametes with the potential of influencing genetic expression. This influence might be obtained by selecting specific cells in the reproductive process either before or after fertilization. This influence might be obtained by chemical control of nucleic acids. Gene expression or nucleic acid sequences might be altered beneficially. Humans have perhaps 100,000 genes. Genetic abnormalities are relatively common. Of all neonates 3-5% have a recognizable genetic disease. Genetic abnormalities may account for 33% of all pediatric admissions to hospitals and for about 25% of neonatal deaths (Centers for Disease Control, 1989). There may be 5000 genetic diseases (McKusick, 1992; Scriver et al., 1995). Everyone is heterozygous for about 5 lethal genes. About 60-70% of all concepti are never implanted or are spontaneously aborted because of genetic or chromosomal abnormalities (Bou6 and Bou6, 1973; Hertig et al., 1959; Leridon, 1973). Substantial progress has been made toward development of technology that could selectively alter genes in human gametes. In 1970 H. G. Khorana and associates (Agarwal et al., 1970) reported the first synthesis of a gene. In 1973 a gene, inserted in a bacterium by S. N. Cohen and H. W. Boyer, was shown to be functional (Cohen et al., 1973); this process using recombinant DNA was patented in 1980. Also in 1980 the U.S. Supreme Court decided that the Commissioner of Patents and Trademarks could issue patents for genetically engineered organisms ( D i a m o n d v. Chakrabarty). Since that time patents have been issued for a variety of genetically altered animals including mammals. Some people (Rifkin, 1985) strenuously object to patents being issued for genetically altered life forms. On September 14, 1990, a gene was inserted in somatic cells of a child born without the ability to synthesize adenosine deaminase, an enzyme necessary for immunocompetence. The child was cured (Anderson, 1992; Culver et al., 1991). Technology for directly altering gamete genetics will become possible (Anderson, 1992; Shinsheimer, 1975; Wivel and Waiters, 1993).

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Already procedures are available and utilized for selecting a baby's sex. This has been done for centuries in some cultures by infanticide. In America the R o e v. W a d e decision made sex selection based upon diagnostic analysis of fetal tissues possible through abortion. Now the sex of a zygote can be determined during IVF prior to embryo transfer (Handyside et aL, 1990). Technology is developing to determine whether an individual spermatozoan bears the X- or the Y-chromosome (Johnson et al., 1993). Sex selected sperm could be used to individually fertilize an egg through "intracytoplasmic sperm insertion" (ICSI) procedures. Persons might want to choose the sex of a child in order to avoid having a child with a sex-linked disorder. Or they might want to choose a preferred sex. The social consequences of widespread availability of sex selection has been predicted (Coombs, 1977; Etzioni, 1968; Pebly and Westoff, 1982) and more data are becoming available (Khatamee et al., 1989). Ethics of fetal sex identification have been debated (Anon, 1993; Fletcher, 1979 and 1980). A 1973 survey (Miller, 1974) of 88 female university students (ages 18-23) revealed that, given the opportunity, 36% would select the sex of their children, and 31% said they might but were not certain. The fact is, there has not been a great rush to use available sex selection techniques. Perhaps its utilization will increase substantially when its cost can be reduced below $1000 (Simpson and Carson, 1992). With the advent of cryopreservation of human sperm (Bunge and Sherman, 1953; Bunge et al., 1954), it was realized that the technology could be used for eugenics. In fact a Nobel laureate, Herman J. Muller (1961), actively promoted its use in a eugenics scheme; J. K. Sherman did, too (1973). The scheme called for storing semen from "superior" men and making it available only to "superior" women. Although many hundreds of sperm banks have been established in the United States since 1953, fewer than five promote this eugenic objective; most American reproductive health providers encourage gamete recipients to select a donor from donor "panels." It must be remembered, however, that sperm banks select for donors having "freezable" sperm. Ethics of altering germ plasm genetics have been debated (Anderson, 1992; Blank, 1981; Grobstein, 1990; Neel, 1993; President's Commission, 1982; U.S. Congress, 1984; Wivel and Waiters, 1993). There are at least three levels of intervention in germplasm genetics. The first involves prevention or correction of a genetic abnormality. The second involves changing from one anticipated normal characteristic to a preferred characteristic. The third type of intervention involves enhancement of a normal characteristic. Many persons fear that such technology, particularly the third type, is inappropriate. Any process for altering a specific gene requires accessing that gene. Until establishment of the Human Genome Project, specific molecular structure and specific chromosomal location of most human genes were

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unknown. The objective of the project, begun in 1988 with financing primarily by the U.S. government's NIH and Department of Energy (DOE), is to localize and identify the nucleotide sequence of every DNA component of the human genetic blueprint, the human genome. It is expected that the project will be completed before the year 2005. Although the idea of mapping genes to chromosomes is many decades old, tools for completing the task in humans did not become available until the late 1970s. In 1911 E. B. Wilson was able to locate the gene for color blindness on the X-chromosome. Subsequently a few more genes were located on the X-chromosome and on autosomes through the statistical technique of linkage analysis. Identification of genetic markers useful in linkage analysis was accelerated by techniques that fused mouse and human cells (Weiss and Green, 1967), that cut DNA into identifiable segments (Smith, 1979), that copied the segments (Cohen et aL, 1973), and that enabled identification of nucleotide sequences in those segments (Maxam and Gilbert, 1977; Sanger et al., 1977). With these tools the project was proposed (Botstein et al., 1980; Solomon and Bodmer, 1979) and a pilot study (Donnis-Keller et al., 1987) demonstrated its feasibility. Whether to organize and fund the task of mapping the human genome was considered. DOE with an interest in radiation effects on human genes, a large budget, and well equipped laboratories at Los Alamos, New Mexico, and Berkeley, California, convened a major conference in March 1986. In June 1986 the private Cold Spring Harbor Laboratory directed by James Watson devoted its annual symposium to the "Molecular Biology of H o m o sapiens." Both the U.S. Congress's Office of Technology Assessment and the National Research Council of the U.S. National Academy of Science conducted studies of the economics of mapping the human genome; their reports were published in 1988. Human Genome Project became a reality in 1988 as a cooperative venture between NIH and DOE. NIH agreed to lead the project and appointed James Watson (1990) as project director. DOE appointed Charles Cantor (1990) director of its component. Both agencies initially committed tens of millions of dollars annually to the project, and the combined budget exceeded $150 million by 1991. About 3% of NIH funds for the project were committed annually to studies of ethical, legal, and social issues (Cook-Deegan, 1994). Early during the Human Genome Project, Craig Venter, an NIH researcher, suggested that his employer should patent the genes it was deciphering. Although this idea was disapproved by James Watson, the Director of NIH decided that a request for a gene patent would be submitted (Healy, 1992). This action provoked international controversy (Cook-Deegan, 1994). The Commissioner of Patents rejected the request as unpatentable, but a novel process for "purifying" or for using a gene is deemed patentable.

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Deciphering the human genome in America has not been exclusively supported by public funds. For example, The Institute for Genomic Research (TIGR) in Gaithersburg, Maryland, has sequenced nucleotides in most exons (i.e., genes that code for proteins), ignoring introns or "junk" DNA in the genome. The function of most of the exons, at the time they were sequenced, was unknown. Another group, Human Genome Sciences Inc. (HGS) in Rockville, Maryland, has rights to commercialize TIGR's sequences. Other American commercial interests also are investing many millions of dollars to gain special access to genomic data (Carey, 1995).

VII. CONCLUSION Gamete technologies provoke considerable societal and personal anxiety because they challenge traditional, significant behavioral patterns. These technologies attain social implications beyond demographic and economic statistics. The symbolic component of human gametes and embryos adds an ethical or moral value to the social implication of these technologies. American responses to these technologies illustrate the intensity of concern at various social levels ranging from personal and family to international. Although the overt American response is uniquely American, the intensity of feeling about these matters is found in human communities globally. Beyond opportunities reproductive technologies offer for managing growth of human groups, they also cause people to reconsider basic beliefs about reproductive behavior and, consequently, responsibilities of persons for each other. These technologies have caused Americans and many other people to ponder anew concepts of fatherhood, motherhood, and childhood. Is it acceptible for a single woman, even a lesbian, to become pregnant with donor semen in order that she will not have to share parenthood with a man? Is it acceptible for a couple to use a surrogate in order to avoid problems and risks of pregnancy? And does this couple have the right to refuse to accept the baby? Or may a couple use any available means to ensure that their offspring will be born with specific genetic characteristics? Who can exercise these options. . . only wealthy and influential persons? Should offending science be prohibited in the laboratory? Will these technologies explode into social catastrophe? Several crucial groups must concur for successful introduction of significant new technology. The technological community including funding sources must agree that development of technology will be cost effective, will do no harm, and will even be beneficially effective. There must also be popular concurrence including federal leadership (executive and legislative), news media, and religious organizations. Although judicial support is an effective alternative to leadership from the executive and legislative branches of government, judicial support is much more difficult to manage.

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Even when all other groups concur on the importance and advisability of developing new revolutionary technology, public acceptance can be agonizingly slow as illustrated by hesitant community acceptance of personal computers; this rate of change may be repeated in regards to human genetics. History indicates these social processes of evaluating technology protect society from self-inflicted harm; the American community as a whole entity ended its early experiment in eugenics, and it also chose to delay introduction of recombinant technology into genetic engineering. The reflective process and social debate on reproductive technologies has begun in corridors of power and in workplaces and in livingrooms. The intensity of debate will surely increase as technology offers more social options. Debate will help people find a path into the future that is right for their society. Progress of debate will be facillitated by reason expunged of inflammatory rhetoric. Where new technologies will go, of course, is difficult to predict. However, making predictions is a characteristic human behavior with well known limitations. People are more successful with prophecy about the near future. We are more successful when considering actual, concrete possibilities. We are more successful when considering events with immediate impact upon our personal situation in contrast to events that will only affect other persons. With these limitations in mind, I will risk a few speculations about America. Reproductive technologies can enhance freedom of personal fulfillment. They can replace reproductive obstacles and despair with opportunities and hope. They are consistent with American diversity and can be provided at a cost acceptible to many Americans. As long as these technologies do not adversely affect lives, their use will be permitted. Reproductive technologies affect persons yet to be born, but genetics of unborn children have always been affected by spousal choice of biological mates. Society accepts the freedom of adults to select sexual partners and will also allow potential parents to proactively influence the genetics of a wanted child. Society need not fear that new reproductive technologies will pervert human development. Reproductive process creates human organisms; society creates human beings. Although parents provide a child's genetic resources, society provides the environment which creates persons from human neonates. Society created the person of each parent, and it will create the person of their children, regardless of the reproductive process, natural or assisted by technology. Society evolves and matures in its understanding of its resources. At one time humans feared supernatural dragons of land, sea, and air. Maturity and science conquered this fear and harnessed resources of nature. Humanity is now cautiously moving toward an era in which, for the first time, persons may constructively participate in biological creation.

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Just as people have a choice to avail themselves of contraceptives during lovemaking or anesthesia during childbirth, some Americans will choose to have control over additional aspects of their personal reproductive activities and will pay to make it available. Not many will seek such control, at first. More will try the new technologies when their benefits have been proven and when they become less expensive, less controversial, and less risky in regard to biological efficacy. Society will observe the effect of new reproductive technologies on people and will adapt accordingly. Americans will deem reproductive freedom to be an affirmation of personal development. Despite controversies Americans will eventually embrace many new reproductive technologies as an enhancement of humanity.

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Index

AATB, s e e American Association of Tissue Banks Abortion, s e e R o e v. W a d e mcrosome evaluation, 273-275 reaction, 286 structure, 274 Activation energy, s e e Energy of activation Adenosine triphosphate, 159, 161,163-164 ATP cycle, 149-151 consumption, 173-174, 176, 180 consumption by spermatozoa, 152, 159 energy charge, 150, 152, 159 energy storage, 145 formation, 147-148, 157, 188, 191,233 formation by spermatozoa, 151-152, 156, 159 balance sheet, 144-148, 161-164 Adenylyl cyclase, 173-175, 183, 185 Adoption, infant, 445, 448 AFP, s e e Antifreeze protein AFS, s e e American Society for Reproductive Medicine

Ageing, s e e Senescence Agonist, 168; s e e a l s o Drug; Receptor AI, s e e Insemination, artificial A1-Hasani, S., 15 Albumin, serum bovine, 47, 197 human, 47 Alpha-tocopherol, s e e Tocopherol American Association of Tissue Banks, 5, 330, 453 American Fertility Society, s e e American Society for Reproductive Medicine American Society for Reproductive Medicine, 5, 330, 453 American Type Culture Collection, 414, 434 American Zoo and Aquarium Association, 408 Antagonist, 168, 171-172; s e e a l s o Drug; Receptor Antifreeze glycopeptide, s e e Antifreeze protein Antifreeze protein, 202-203, 257, 334-335

465

466

Index

Antioxidant, 47, 190, 195-198; s e e a l s o s p e c i f i c agents, (e.g., catalase, superoxide dismutase) Arrhenius relation, 282, 296, 311 ART, s e e Technology Artificial insemination, s e e Insemination, artificial Ascorbic acid, 189-190 ASRM, s e e American Society for Reproductive Medicine Assisted reproductive technology, s e e Technology, reproductive Atkinson, D., 140 A T P , s e e Adenosine triphosphate Axoneme, 275

Barometric pressure, 99-101 B HT, s e e Butylated hydroxytoluene Biodiversity, s e e a l s o Genome resource conservation, 405-406, 408-414 definition, 399-400 importance, 401-403 problem, 401,403-404, 406-407 Bioenergetics, s e e Adenosine triphosphate, Metabolism Biot modulus, 373 Blastocyst, 92-94, 101, 105-106, 256-257, 379 Botanic Gardens Conservation International, 411 Boundary-layer, 179 Boyle van't Hoff plot, 294-295, 338 Brown, L., 9, 443, 452 Butanediol, 366, 390 Butylated hydroxytoluene, 196, 247, 341

Calcium, 244-246 Calorimetry, 164, 386 cAMP, s e e Cyclic adenosine monophosphate Cantor, C., 457 Capacitation, s e e Spermatozoa Catalase CDC, s e e Centers for Disease Control and Prevention Cell membrane, s e e Membrane; Oolemma Cell volume analyzer, 294, 297, 299 Center for Plant Conservation, 416, 432 Centers for Disease Control and Prevention, U.S., 454

Chang, M. C., 8 Chen, C., 15 Chilling injury, s e e Cold shock Cholesterol, 236-237, 240, 242-245 Chorionic gonadotropin, 185-186 Chromatography, 239-240 Chromosome, 254, 336, 343-344 Clark, A. J., 171 Clausius-Clapeyron equation, 282 Clinical Laboratory Improvement Act, 454 Clinton, Bill, 453 Cold shock, 84, 196, 230-231,239, 245, 247-248, 251,253, 256, 288, 336, 341 Colligative properties, 201-202 Compartmental analysis, 180-182 Computer-assisted semen analysis, s e e Spermatozoa, evaluation Consanquinity, unwitting, 448 Consent, informed, 449 Conservation, s e e Biodiversity Conservation Assessment Management Plan, 421 Conservation Breeding Specialist Group, 421-429, 432-433 Constitution, U.S., 448, 450-451,453 Consultative Group on International Agricultural Research, 409-410 Convention on Biological Diversity, 1992, 404 Cooling rate effect, biological, 347, 280-281 optimal, 281,288-290, 310-311 Corpus luteum, 96, 182 Cortical granule, 254-256, 336, 340 Coulter counter, s e e Cell volume analyzer CPA, s e e Cryoprotectant Cryobank, s e e Embryo bank; Genome resource bank; Sperm bank Cryoinjury, 200-201,366 Cryopreservation, s e e s p e c i f i c cell o r tissue, (e.g., Embryo; Oocyte; Ovary; Spermatozoa) history, 8, 11,264, 361-364 Cryoprotectant, s e e a l s o s p e c i f i c agent, (e.g., dimethylsulfoxide; ethelene glycol; glycerol) classification, 198-200 history, 264 ice nucleating protein (INP), 207-208 macromolecule, 203-205 mechanism of action, 201-205, 284-285, 290-291,347, 349-350

Index

pharmacokinetics (administration; removal), 287, 298-299, 300-309, 374-376, 383-385 phase diagram, 348-349 toxicity, 205-207, 255, 309, 336, 348-349, 363-364, 374 vehicle, 287 viscosity, 202 Crystallization, water biological effect, 201,336, 342-343, 367-373, 384-385 intracellular, 200, 280, 284, 299-300, 310-311,340 physics, 200, 203, 207-208, 284, 312, 350 Culture system biological "incubator", 106-107 co-culture, 53, 107-109, 381-382 microdroplet, 52-53, 381 perfusion, 61 Cumulus granulosa, 275, 331,333, 341 Cumulus oophorus, 182 Cyclic adenosine monophosphate, 173-174, 182-183, 185, 331 Cytochrome oxidase chain, 188 DAP213, see Dimethyl sulfoxide; Vitrification Darwin, C., 140 Deoxyribonucleic acid, 16, 89, 175-176, 191,336, 452, 455 Department of Agriculture, U.S., 410 Department of Energy, U.S., 457 Department of Health, Education, and Welfare, U.S., 452 Dextran, 285 D i a m o n d v. C h a k r a b a r t y , 443, 455 Diapause, 96 Diffusion (J; J u), 176-180, 298, 371 Diffusion coefficient, see Permeability coefficient Dimethylsulfoxide, 197, 202, 204, 255, 283, 299, 309-310, 333-335, 338-339, 344, 346, 348-349, 364-366, 374-375, 387-389, 392 DMSO, see Dimethylsulfoxide DNA, see Deoxyribonucleic acid Donor, human gamete evaluation, 454 fetal, 448 living, 445-446, 448 nonanonymous, 448 oocyte, 13

467

posthumous, 447, 449 rights, 449-451 spermatozoa autologous, 6-7 homologous, 5-7 Drug adverse effect, 169 affinity, 172 efficacy, 172 intrinsic activity, 169, 171 nonspecific vs specific, 167-169, 198 potency, 171 Drug-receptor interaction, 170-173

Earth Summit, 1992, 404 Edwards, R., see Brown, L. Electron paramagnetic resonance, 294 Electronic cell volume analyzer, see Cell volume analyzer Embryo access, 98-104 bank, 377-378 bovine, 383-384 cryopreservation, 10-12, 382-389, 391-392 culture, 379-382 metabolism, 151, 159-161 Embryogenesis, in vivo, 25, 91-98 Embryogenesis, in vitro, 378-380 Energy charge, see Adenosine triphosphate Energy of activation, 282-283, 296, 383 Epididymis, 68-69, 242, 267-268 Equilibrium freezing, see Crystallization Erhlich, P., 168-169 Estrogen, 182 Ethics, 11, 16-17, 449 Ethylene glycol, 299, 309-310, 312, 334-335, 340, 346, 390 Eutectic temperature, 280

Fahy, G. M., 347 Family, human, 446-449 FDA, see Food and Drug Administration Feathering, see Compartmental analysis Federal Trade Commission, U.S., 454 Fertility Clinic Success Rate and Certification Act, 454 Fertilization, in vitro, 6, 12, 59, 277, 452-453 Fertilization, in vivo, 24-25, 78-81, 143

468

Index

Fick equation, 177, 179, 371 Flavin adenine dinucleotide, 146-147, 163 Flow, s e e Diffusion Flow cytometry, s e e Fluorescence-activated cell sorter Fluorescence-activated cell sorter, 272, 300 Fluorescent vital dyes, 270-272 Flux, s e e Diffusion Follicle stimulating hormone binding affinity, 185 concentration, human serum, 186 effect, females, 52, 182-183 effect, males, 64, 182 pharmacokinetics, 186-187 receptor, 173-175, 187 structure, 183-186 synthesis commercial, 186 i n v i v o , 182 Follicullogenesis, in v i v o , 14, 26-30, 56-57, 182-183, 361 Folliculogenesis, i n v i t r o , 60-63, 183 Food and Agricultural Organization, 410, 412 Food and Drug Administration, U.S., 5, 454 Fragility curve, 295, 296 Free radicals, s e e Reactive oxygen species Freezing, s e e Crystallization; Vitrification FSH, s e e Follicle stimulating hormone G-protein, 173-175, 187 Gamete, s e e a l s o Oocyte; Spermatozoa commerce, 451 property, 449, 451 Gametogenesis, s e e Folliculogenesis; Oogenesis; Spermatogenesis Gel (L~) phase, s e e Lipid Gene, human abnormality, 455 commercial, 458 disease, 455 ethics, 456 lethal, 455 number, 445 patent, 443, 455, 457 property, 450 therapy, 455 Genetic diversity, s e e Biodiversity Genome resource bank, 7-8, 12-13, 264, 399, 400, 406, 409, 416-420, 422-424, 425-428, 432-435

Germ cells, primordial, 24-26, 61-62, 90-91 Germinal vesicle, s e e Oocyte Gibbs free energy, 145 Glucose, 49, 82-83, 145, 147, 150, 159 Glutathione, 189, 190, 196-197 Glutathione peroxidase, 189, 192, 197 Glycerol, 2, 202, 204, 264, 284, 286-288, 297-299, 302-311,362-363, 366, 383, 385, 387-389, 392 Glycolipid, 238-239, 241 Glycolysis (Embden-Meyerhof), 147-148, 152, 156-157, 233 GnRH, s e e Gonadotropin releasing hormone Gonadotropin, 49-52, 173, 249; s e e a l s o Chorionic gonadotropin; Follicle stimulating hormone; Luteinizing hormone Gonadotropin releasing hormone, 182 Graafian follicle, 182-183 Granulosa cell, 182-183, 187, 361-362 Growth factor, 52, 95-96

Heat shock protein, 175-176 Heating, electromagnetic radiation, 350 Hepatitis virus, s e e Sexually transmitted disease Hexagonal II configuration, s e e Lipid HIV, s e e Sexually transmitted disease Hofmeister effect, 203-204 Hormone response element, 176 HRE, s e e Hormone response element hsp, s e e Heat shock protein Human genome project, 448, 456-457 Human Immunodeficiency Virus, s e e Sexually transmitted disease Hunter, J., 4 Hydraulic coefficient, s e e Diffusion coefficient Hydraulic conductivity, s e e Permeability coefficient Hydrogen peroxide, s e e Reactive oxygen species Hydroxyethyl starch, 285 Hypothermia, 156-159, 200, 230, 248

Ice, s e e Crystallization; Vitrification ICSI, s e e Intracytoplasmic spermatozoa injection

Index

Infertility, human incidence, 445-446 treatment, 445-446 cost of treatment, 445-446 INP, see Cryoprotectant Insemination, artificial efficiency, 3, 264-265 history, 4-5, 263-264 incidence, bovine, 3 incidence, human, 5, 6 incidence, porcine, 4 incidence, porcine, 3-4 regulation, 5, 453-455 International Agricultural Research Center, 409 International Plant Genetics Resource Institute, 409-410 International Species Information System, 435 International Union for the Conservation of Nature and Natural Resources, see World Conservation Union Intracytoplasmic spermatozoa injection, 7, 88, 266 Intracellular ice formation, see Crystallization Inverted U, see Cooling rate, optimal I V F , see Fertilization

Jackson Laboratory, 413

Kedem-Katchalsky kinetics, 180, 298, 371 Krebs citric acid cycle, see Phosphorylation, oxidative Krough cylinder, 370-373

Langley, J. N., 168 Langmuir, I., 170 Lardy, H., 164-165 Lateral diffusion in lipid bilayers, 235 Leydig cell, 64, 182 LH, see Luteinizing hormone Lineweaver-Burk plot, 171-172 Lipid bilayer, 234-238 gel (L~) phase, 235, 237 hexagonal II, 235-236 liquid-crystalline (L~) phase, 235, 237 phase transition, 340-341

469

Lipid peroxide, see Peroxidation Lipoprotein, 245-246 Luteinizing hormone binding affinity, 185 concentration, human serum, 186 effect, females, 52, 182-193 effect, males, 64, 182 pharmacokinetics, 186-187 receptor, 173-175, 187 synthesis commercial, 186 in vivo, 182 structure, 183-186 Luyet, B., 347

Mass Action, Law, 170 Me2SO, see Dimethylsulfoxide Medium, culture, 41-52, 83-84, 103, 109 Meiosis, 249, 379 Membrane, cell, 173, 177-180, 188, 190, 193, 196, 200-201,204-205, 234-235, 244-245, 251,270-273, 280, 285 Membrane fusion (oocyte-spermatozoa), 277 Metabolism, see also Adenosine triphosphate; Glycolysis definition, 139, 148 intermediates, 145 in v i v o vs in vitro, 139-142, 156 Metaphase II, 249 Meyerhof, O., 165 Microtubule, 249, 252-253, 255, 341,343 Milk, 246 Milovanov, 230-231 Mitochondria, 147, 151-158, 188, 191, 232-233, 249, 250, 340 M o o r e v. R e g e n t s , 449, 451 Morula, 92-93, 101 Mouse, transgenic, 7 Muller, H. J., 456

National Genetic Resources Program, 410-413 National Institute for the Environment, 431 National Institutes of Health, U.S., 413, 452-453, 457 National Plant Germplasm System, see National Genetic Resource Program National Seed Storage Laboratory, 410-411,432

470

Index

Nicotinamide adenine dinucleotide, 146-147, 163 Nicotinamide adenine dinucleotide phosphate, 151-152 Nonequilibrium freezing, see Vitrification Nonreturn rate, 266 Nuclear magnetic resonance spectroscopy, 374-376 Nuclear transfer, 94 Nucleation, ice, 312

Office of Technology Assessment, U.S., 443, 457 Oocyte cryopreservation bovine, 334, 339-341 hamster, 334 history, 331-333 horse, 335 human, 13, 15, 330, 334-335, 343-345 monkey, 335 mouse, 335, 338-339 porcine, 331,335 rabbit, 334 rat, 334, 341 sheep, 334 germinal vesicle stage, 249, 336 in vitro

acquisition technique, 32-40 maturation, 40-42, 53-54 primary, 54, 61-62 metabolism, 151, 159-161 structure, 249-252, 331,350 viability test, 334-335 Oogenesis, 24, 26, 29-31, 54-59, 143, 249, 331 Oolemma, 80, 182, 249, 250-253, 258, 275, 336, 345 Ovary cryopreservation, 15, 360-361 structure, 361 Oxidation, 146-147 Oxygen, 46 Oxygen free radicals, see Reactive oxygen species

Parkes, A., 362-363 Parrott, D. M. V., 364 Parthenogenesis, 58, 89, 344 Pericentriolar material, 249, 252-253, 343

Perivitelline space, 250 Permeability coefficient, 177, 253, 282-283, 294-297, 298-299, 311,345-346, 382-383 Peroxidase, see Glutathione peroxidase Peroxidation, lipid, 183-195, 197, 241 Pharmacodynamics, see Drug-receptor interaction Pharmacokinetics, 176-182 Phase separation, lateral, 237-238, 243-244 Phase transition, thermotropic, 235-237, 239, 241-244, 247-248, 251-253 Phosphodiesterase, 174 Phospholipase, 174 Phospholipid, 234, 236-247, 280 Phosphorylation, oxidative, 147, 158, 162, 233; see also Adenosine triphosphate Plasma membrane, see Membrane; Oolemma Polge, C., 2, 264 Polyethylene glycol, 285, 389 Polyvinylpyrrolidone, 285, 350 Population and Habitat Viability Assessment, 421 Pregnancy, 96, 266 Property, tissue, 449 Propylene glycol, 299, 309-310, 333-355, 387-392 Protein, membrane, 243-244, 312 Protein, milk, 246 Pseudopregnancy, 96

Quinn, P., 237-238, 243

Racker, E., 165 Rail, W. F., 347 Raoult's law, 282 Rare Breeds International, 412 Reactive oxygen species analysis, 188, 190 biochemistry, 188-191 biological effect in v i v o , 188, 190-192 cellular site, 190 definition, 187-188 hydrogen perioxide, 188-190, 193-194, 197 hydroxyl radical, 188-190 hypothermia, 191 hypoxia, 191, 194

Inaex

infertility, 194 nitric oxide, 188-189 oocyte, 193, 197 peroxynitrite, 188-189 phagocyte, 188, 191 physiological action, 187-189, 191-193 scavengers, 195-198 spermatozoa, 192-198 superoxide, 188-193 toxicity, 190-195 Receptor estrogen, 175-176 G protein-linked, 173 gonadotropin, 173 function, 168-169 nuclear, 175-176 progesterone, 175-176 steroid, 175-176 Recrystallization, s e e Crystallization Reduction, 146 Reflection coefficient, 180, 298-299, 346 Regulation, government, 264, 330 Respirometery, 162-163 R o e v. W a d e , 443, 450, 456 ROS, s e e Reactive oxygen species

SART, s e e Society for Assisted Reproductive Technology Scatchard plot, 171-172 Seeding, 256, 384-385, 388 Semen, 70-71, 276; s e e a l s o Spermatozoa Senescence, 231 Sertoli cell, 64-65, 182, 269 Serum, fetal, bovine (calf), 47 Sex selection, 445, 456 Sexuality, human, 447 Sexually transmitted disease, 5, 264, 329-330, 454 Sherman, J. K., 331-332, 456 Smith, A. U., 362 Society for Assisted Reproductive Technology, 11 SOD, s e e Superoxide dismutase Sodium-potassium pump, 180 Solution effects, 281 Spallanzani, L., 2 Species survival plan, 408, 420 Sperm bank, human American number, 5-6, 446, 456 regulation, American, 5, 453-455

471

Spermatogenesis, 24, 64-68, 70, 142-143, 182, 269 Spermatozoa, s e e Cold shock acquisition, 72-78 assessment (evaluation), 248, 268-270, 300 capacitation, 71-72, 79-85, 238, 277-279 cryopreservation, 2-8 culture, 81-85 epidydimal, 154-155 evaluation, function, 74, 86, 266-279, 300 membrane, 238 metabolism, 152-159 structure, 79, 232-233, 270-275 volume, 291-294 Spermiation, 269 Spermiogenesis, 269 Spindle, meoitic, 248, 338, 340, 343-344 chilling, 251-253 dimethylsulfoxide, 255-256 STD, s e e Sexually transmitted diseases Stem cells, s e e Germ cells, primordial Steptoe, P., s e e Brown, L. Steroid, 50-53; s e e a l s o Cholesterol; Estradiol; Estrogen; Progesterone; Testosterone Stoichiometry, 145-146, 157, 161 Stokes-Einstein equation, 177 Storage temperature, 290 Supercooled water, s e e Water, liquid Superoxide dismutase, 189, 192, 194-195 Surrogate reproduction, 446 Syngamy, 80-81, 91,331,379

Taxol, 257 Taxon advisory group, 420 Technology, reproductive, s e e Insemination; Fertilization; Intracytoplasmic sperm injection classification, 443-444 economic impact, 444-446 physician number, 446 regulation, American, 453-455 Testis, 64, 87, 267 (fig), 268 (fig) Testosterone, 64, 182 Thaw, 385 Thecal cell, 183 Therapeutic index, 169 Thermal hysteresis protein, s e e Antifreeze protein

472

Index

Tocopherol, 189-190, 195-196 Toxicity, drug, 169 Transport, active, 180-182 Transporter, 180 Tricarboxylic acid cycle, s e e Phosphorylation, oxidative Trophoblast, s e e Embryogenesis

United States Department of, s e e Department of Agriculture; Department of Energy; Department of Health, Education, and Welfare; s e e a l s o National Genetic Resources Program; National Institutes of Health

Viscosity, 347 Vitamin E, s e e Tocopherol Vitrification, 200, 285, 349 embryo, 385, 389-392 oocytes, 340, 342-343 ovary, 365 Vitrification solution, s e e Cryoprotectant, 347-348

Warming rate, 284, 290, 310-311,350 Water, freezing, s e e Crystallization; Vitrification Water, liquid, supercooled, 280, 283 Water-permeability efficient, s e e Diffusion coefficient Watson, J., 457 Whittingham, D. G., 8, 332 Wilson, E. B., 457 World Conservation Union, 411,421, 423-424, 432

Xanthine dehydrogenase, 188, 191-192 Xanthine oxidase, 188, 191-192

Yolk, egg, 245-246, 257

Zona pellucida, 27, 46-47, 79, 94, 182, 233, 249, 250-251,254-257, 275, 277, 336, 379, 391 Zoo, s e e Biodiversity