Reproductive Tissue Banking: Scientific Principles

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REPRODUCTIVE TiSq~ BANKING Scientific

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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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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 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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M. Lorraine Leibfried-Rutledge et al.

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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Alak, B. M., and Wolf, D. P. (1994). Biol. Reprod. 51, 879-887. Albertini, D. F. (1992). BioEssays 14, 97-103. Albertini, D., Wickramasinghe, D., Messinger, S. M., Mattson, B. A., and Plancha, C. E. (1993). In Preimplantation Embryo Development (B. D. B avister, Ed.), pp. 3-21. SpringerVerlag, New York. Almquist, J. O. (1978). N A A B Proc. 7th Tech. Conf. Artif. Insem. Reprod. pp. 33-37. Columbia, MO. Amann, R. P. (1970). In The Testis (A. D. Johnson, W. R. Gomes, and N. L. VanDemark, Eds.), Vol. 1, pp. 433-482. Academic Press, New York. Amann, R. P. (1987). J. Reprod. Fertil. Suppl. 34, 115-131. Amann, R. P., Hammerstedt, R. H., and Veeramachaneni, D. N. (1993). Reprod. Fert. Dev. 5, 361-381. Anderson, G. B. (1977). In Reproduction in Domestic Animals (H. H. Cole and P. T. Cupps, Eds.), 3rd ed., pp. 285-314. Academic Press, New York. Anderson, G. B. (1991). In Reproduction in Domestic Animals (P. T. Cupps, Ed.), 4th ed., pp. 279-313. Academic Press, New York. Anderson, L. D., and Hillenjso, T. (1982). Acta Physiol. Scand. 114, 623-625. Andrews, J. C., and Bavister, B. D. (1989a). Zoo Biol. Suppl. 1, 21-31. Andrews, J. C., and Bavister, B. D. (1989b). Gamete Res. 23. 159-170. Andrews, J. C., Howard, J. G., Bavister, B. D., and Wildt, D. E. (1992). Mol. Reprod. Dev. 31, 200-207. Archibong, A. E., Maurer, R. R., England, D. C., and Stormshak, F. (1992). Biol. Reprod. 47, 1026-1030. Arlotto, T. M. (1994). In Acquisition of Meiotic Competence in Bovine Oocytes and Resolution of a Model System for Study. Ph.D. dissertation, Ann Arbor Dissertation Service, Ann Arbor, MI. Arlotto, T. M., Schwartz, J.-L., First, N. L., and Leibfried-Rutledge, M. L. (1997). Theriogenology, in press. Armstrong, D. T. (1993). Theriogenology 39, 7-24. Armstrong, D. T., Holm, T., Irvine, B., Petersen, B. A., Stubbings, R. B., McClean, D., Stevens, G., and Seamark, R. F. (1992). Theriogenology 38, 667-678. Armstrong, D. L., Looney, C. R., Lindsey, B. R., Gonseth, C. L., Johnson, D. L., Williams, K. R., Simmons, L. G., and Loskutoff, N. M. (1995). Theriogenology 45, 162. [Abstract] Asher, G. W., Kraemer, D. C., Magyar, S. J., Brunner, M., Moerbe, R., and Giaquinto, M. (1990). Theriogenology 34, 569-578. Ashwood-Smith, M. J., Hollands, P., and Edwards, R. G. (1989). Hum. Reprod. 4, 702-705. Austin, C. R. (1951). Aust. J. Sci. Res. 4, 581-596. Austin, C. R. (1970). J. Reprod. Fertil. Suppl. 12, 39-53. Austin, C. R. (1975). J. Reprod. Fertil. Suppl. 22, 75-89. Austin, C. R., and Short, R. V. Eds. (1982). Reproduction in Mammals, Vol. 1. Cambridge Univ. Press, Cambridge. Avery, B., Jorgensen, C. B., Madison, V., and Greve, T. (1992). Mol. Reprod. Dev. 32, 265-270. Avery, B., Madison, V., and Greve, T. (1991). Theriogenology 35, 953-963. Bachvarova, R. (1985). In Developmental Biology. A Comprehensive Synthesis (L. Browder, Ed.), Vol. 1, pp. 453-524. Plenum, New York. Bachvarova, R. (1988). In Meiotic Inhibition, Molecular Control of Meiosis: Progress in Clinical and Biological Research (F. Haseltine and N. L. First, Eds.), pp. 67-86. A. R. Liss, New York. Bae, I.-H., and Foote, R. H. (1975). J. Reprod. Fertil. 42, 357-360. Baird, D. T., and McNeilly, A. S. (1981). J. Reprod. Fertil. Suppl. 30, 119-133. Baker, T. B. (1971). Adv. Biosci. 6, 7-23. Baker, T. B. (1972a). In Reproductive Biology (H. Balin and S. Blasser, Eds.), pp. 398-437. Excerpta Medica, Amsterdam.

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Baker, T. B. (1972b). In Reproduction in Mammals (C. R. Austin and R. V. Short, Eds.), pp. 14-45. Cambridge Univ. Press, London. Baker, T. G. (1982). In Reproduction in Mammals (C. R. Austin and R. V. Short, Eds.), pp. 17-45. Cambridge Univ. Press, London. Baker, T. G. (1987). In Germ Cells and Fertilization (C. R. Austin and R. V. Short, Eds.), pp. 1-13. Cambridge Univ. Press, Cambridge. Baldassarre, H., de Matos, D. G., Furnus, C. C., Castro, T. E., and Cabrera Fischer, E. I. (1994). Anim. Reprod. Sci. 35, 145-150. Balinsky, B. J. (1981). An Introduction to Embryology. Saunders, Philadelphia. Ball, G. D., Bellin, M. E., Ax, R. L., and First, N. L. (1982). Mol. CelL Endocrinol. 28,113-122. Ball, L. (1978). N A A B Proc. 7th Tech. Conf. Artif. Insem. Reprod., p. 57. Columbia, MO. Balmaceda, J. P., Pool, T. B., Arana, J. B., Heitman, T. S., and Asch, R. H. (1984). Fertil. Steril. 42, 791-795. Bar-Ami, S., and Tsafriri, A. (1981). Gamete Res. 4, 463-472. Bar-Ami, S., and Tsafriri, A. (1986). Gamete Res. 13, 39-46. Bar-Ami, S., Nimrod, A., Brodie, A. M. H., and Tsafriri, A. (1983). J. Steroid Biochem. 19, 965-971. Barnes, F. L., and Eyestone, W. H. (1990). Theriogenology 33, 141-152. Barnes, F. L., Balke, J. M. E., Eyestone, W. H., First, N. L., and Read, B. R. (1988). Theriogenology 29, 216. [Abstract] Barnett, D. K., and Bavister, B. D. (1996). Mol. Reprod. Dev. 43, 105-133. Barone, M. A., Wildt, D. E., Byers, A. P., Roelke, M. E., Glass, C. M., and Howard, J. G. (1994). J. Reprod. Fertil. 101, 103-108. Barros, C., Bedford, J. M., Franklin, L. E., and Austin, C. R. (1967). Membrane vesiculation as a feature of the mammalian acrosome reaction. J. Cell Biol. 34, C1-C5. Bavister, B. D. (1981a). In Fertilization and Embryonic Development in Vitro (L. Mastroianni, Jr., and J. D. Biggers, Eds.), pp. 41-60. Bavister, B. D. (1981b). J. Exp. Zool. 217, 45-51. Bavister, B. D. (1982). In In Vitro Fertilization and Embryo Transfer (E. S. E. Hafez and K. Semm, Eds.), pp. 13-29. MTP Press, Lancaster. Bavister, B. D. (1986). In Manipulation of Mammalian Development (R. B. L. Gwatkin, Ed.), pp. 81-148. Plenum, New York. Bavister, B. D. (Ed.) (1987). The Mammalian Preimplantation Embryo: Regulation of Growth and Differentiation In Vitro. Plenum, New York. Bavister, B. D. (1988). Theriogenology 29, 143-154. Bavister, B. D. (1990a). In Gamete Physiology (R. H. Asch, J. P. Balmaceda, and I. Johnston, Eds.), pp. 77-105. Serono Symposia USA, Norwell, MA. Bavister, B. D. (1990b). In Early Embryo Development and Paracrine Relationships (S. Heyner and L. Wiley, Eds.), UCLA Symposia on Molecular and Cellular Biology, New Series, Vol. 117, pp. 79-96. A. R. Liss, New York. Bavister, B. D. (1992). Hum. Reprod. 7, 1339-1341. Bavister, B. D. (1995). Hum. Reprod. Update 1, 91-148. Bavister, B. D., and Andrews, J. C. (1988). J. In Vitro Fert. Embryo Transfer 5, 67-75. Bavister, B. D., and Morton, D. B. (1974). J. Reprod. Fertil. 40, 491-493. Bavister, B. D., and Yanagimachi, R. (1977). Biol. Reprod. 16, 228-237. Bavister, B. D., Leibfried, M. L., and Lieberman, G. (1983). Biol. Reprod. 28, 235-247. Bavister, B. D., Boatman, D. E., Collins, K., Dierschke, D. J., and Eisele, S. G. (1984). Proc. Natl. Acad. Sci. USA 81, 2218-2222. Bavister, B. D., Rose-Hellekant, T. A., and Pinyopummintr, T. (1992). Theriogenology 37, 127-146. Bazer, F. W., Ott, T. L., and Spencer, T. E. (1994). Theriogenology 41, 79-93. Bazer, F. W., Vallet, J. L., Roberts, J. M., Sharp, D. C., and Thatcher, W. W. (1986). J. Reprod. Fertil. 76, 841-850.

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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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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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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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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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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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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

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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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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

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

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! 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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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

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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.

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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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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

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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-

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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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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. 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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 (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

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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

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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

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(Top) Simulations of cell volume excursion for mouse metaphase II oocytes exposed to various molar concentrations of DMSO at 20~ (membrane permeability values were Lp = 0.40; Ps = 1.3 E; and cr = 0.92 (Agca, unpublished data)). (Bottom) Simulations of cell volume excursion for mouse metaphase II oocytes pre-equilibrated with 1 M DMSO and abruptly exposed to either 0, 0.25, or 0.5 M sucrose at at 20~ (membrane permeability values were Lp = 0.40; Ps = 1.3E-3; and o- = 0.92 (Agca, unpublished data)).

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