Ovarian Toxicology

TARGET ORGAN TOXICOLOGY SERIES Series Editors A.Wallace Hayes, John A.Thomas, and Donald E.Gardner OVARIAN TOXICOLOGY Patricia B.Hoyer, editor, 248 pp., 2004 CARDIOVASCULAR TOXICOLOGY, THIRD EDITION Daniel Acosta, Jr., editor, 616 pp., 2001 NUTRITIONAL TOXICOLOGY, SECOND EDITION Frank N.Kotsonis and Maureen A.Mackey, editors, 480 pp., 2001 TOXICOLOGY OF SKIN Howard I.Maibach, editor, 558 pp., 2000 TOXICOLOGY OF THE LUNG, THIRD EDITION Donald E.Gardner, James D.Crapo, and Roger O.McClellan, editors, 668 pp., 1999 NEUROTOXICOLOGY, SECOND EDITION Hugh A.Tilson and G.Jean Harry, editors, 386 pp., 1999 TOXICANT-RECEPTORINTERACTIONS: MODULATION OF SIGNAL TRANSDUCTIONS AND GENE EXPRESSION Michael S.Denison and William G.Helferich, editors, 256 pp., 1998 TOXICOLOGY OF THE LIVER, SECOND EDITION Gabriel L.Plaa and William R.Hewitt, editors, 444 pp., 1997 FREE RADICAL TOXICOLOGY Kendall B.Wallace, editor, 454 pp., 1997 ENDOCRINE TOXICOLOGY, SECOND EDITION Raphael J.Witorsch, editor, 336 pp., 1995 CARCINOGENESIS Michael P.Waalkes and Jerrold M.Ward, editors, 496 pp., 1994 (Continued) DEVELOPMENTAL TOXICOLOGY, SECOND EDITION Carole A.Kimmel and Judy Buelke-Sam, editors, 496 pp., 1994 IMMUNOTOXICOLOGY AND IMMUNOPHARMACOLOGY, SECOND EDITION Jack H.Dean, Michael I.Luster, Albert E.Munson, and Ian Kimber, editors, 784 pp., 1994

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NUTRITIONAL TOXICOLOGY Frank N.Kotsonis, Maureen A.Mackey, and Jerry J.Hjelle, editors, 336 pp., 1994 TOXICOLOGY OF THE KIDNEY, SECOND EDITION Jerry B.Hook and Robin J.Goldstein, editors, 576 pp., 1993 OPHTHALMIC TOXICOLOGY George C.Y.Chiou, editor, 352 pp., 1992 TOXICOLOGY OF THE BLOOD AND BONE MARROW Richard D.Irons, editor, 192 pp., 1985 TOXICOLOGY OF THE EYE, EAR, AND OTHER SPECIAL SENSES A.Wallace Hayes, editor, 264 pp., 1985 CUTANEOUS TOXICITY Victor A.Drill and Paul Lazar, editors, 288 pp., 1984

Target Organ Toxicology Series

Ovarian Toxicology Edited by Patricia B.Hoyer Department of Physiology The University of Arizona Tucson

Boca Raton London New York Washington, D.C.

This edition published in the Taylor & Francis e-Library, 2005. “To purchase your own copy of this or any of Taylor & Francis or Routledge’s collection of thousands of eBooks please go to www.eBookstore.tandf.co.uk.” Library of Congress Cataloging-in-Publication Data Catalog record is available from the Library of Congress This book contains information obtained from authentic and highly regarded sources. Reprinted material is quoted with permission, and sources are indicated. A wide variety of references are listed. Reasonable efforts have been made to publish reliable data and information, but the author and the publisher cannot assume responsibility for the validity of all materials or for the consequences of their use. Neither this book nor any part may be reproduced or transmitted in any form or by any means, electronic or mechanical, including photocopying, microfilming, and recording, or by any information storage or retrieval system, without prior permission in writing from the publisher. The consent of CRC Press LLC does not extend to copying for general distribution, for promotion, for creating new works, or for resale. Specific permission must be obtained in writing from CRC Press LLC for such copying. Direct all inquiries to CRC Press LLC, 2000 N.W. Corporate Blvd., Boca Raton, Florida 33431. Trademark Notice: Product or corporate names may be trademarks or registered trademarks, and are used only for identification and explanation, without intent to infringe. Visit the CRC Press Web site at www.crcpress.com © 2004 by CRC Press LLC No claim to original U.S. Government works ISBN 0-203-50923-4 Master e-book ISBN

ISBN 0-203-57157-6 (Adobe eReader Format) International Standard Book Number 0-415-28795-2 (Print Edition)

CONTENTS

Contributors

vii

Foreword

xii

1

Ovarian physiology DIANE SUTER

1

2

Ovarian toxicity in small pre-antral follicles PATRICIA B.HOYER

17

3

Ovarian toxicity caused by pesticides CHRISTINA BORGEEST KIMBERLY P.MILLER DRAGANA TOMIC JODI A.FLAWS

41

4

Ovarian toxicity caused by endocrine disruptors PAUL F.TERRANOVA AND KARL K.ROZMAN

62

5

Phthalate toxicity in the ovary FRIEDERIKE C.L.JAYESTARA LOVEKAMP-SWAN AND BARBARA J.DAVIS

87

6

Hormonal control of ovarian function following chlorotriazine exposure: effect on reproductive function and mammary gland tumor development RALPH L.COOPER SUSAN C.LAWS MICHAEL G.NAROTSKY JEROME M.GOLDMAN AND TAMMY E.STOKER

96

7

The role of ovarian metabolism in chemical-induced ovarian injury ELLEN A.CANNADY AND I.GLENN SIPES

116

8

Placental induction of ovarian toxicity JENNIFER L.MARCINKIEWICZ

135

9

Chemoresistance in human ovarian cancer: possible roles of Xlinked inhibitor of apoptosis protein (XIAP) CHAO WU XIAO XIAOJUAN YAN HIROMASA SASAKI FUMIKAZU KOTSUJI AND BENJAMIN K.TSANG

150

vi

10

The epidemiology of ovarian cancer: the role of reproductive factors and environmental chemical exposure KATHRYN COE

175

11

Assessment of toxicant-induced alterations in ovarian steroidogenesis: a methodological overview JEROME M.GOLDMAN SUSAN C.LAWS AND RALPH L.COOPER

206

Index

227

CONTRIBUTORS

Christina Borgeest Department of Epidemiology and Preventive Medicine Division of Gender Based Epidemiology University of Maryland School of Medicine 660 West Redwood Street Baltimore, MD 21201 Ellen A.Cannady Eli Lilly and Company Drug Disposition Indianapolis, Indiana 46285 Kathryn Coe Arizona Cancer Center P.O. Box 245024 The University of Arizona Tucson, AZ 85724–5024 Ralph L.Cooper Endocrinology Branch Reproductive Toxicology Division National Health and Environmental Effects Research Laboratory United States Environmental Protection Agency Research Triangle Park NC 27711 USA Barbara J.Davis National Institute of Environmental Health Sciences (NIEHS) Laboratory of Women’s Health (LWH) 111 TW Alexander Drive P.O. Box 12233 Mail Drop B3–06 Research Triangle Park NC 27709

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USA Jodi A.Flaws Department of Epidemiology and Preventive Medicine Division of Gender Based Epidemiology University of Maryland School of Medicine 660 West Redwood Street Baltimore, MD 21201 USA Jerome M.Goldman Endocrinology Branch Reproductive Toxicology Division National Health and Environmental Effects Research Laboratory United States Environmental Protection Agency Research Triangle Park NC 27711 USA Patricia B.Hoyer Professor Department of Physiology Arizona Health Sciences Center The University of Arizona Tucson, AZ 85724 Friederike C.L.Jayes National Institute of Environmental Health Sciences (NIEHS) Laboratory of Women’s Health (LWH) 111 TW Alexander Drive P.O. Box 12233 Mail Drop B3–06 Research Triangle Park NC 27709 USA Fumikazu Kotsuji Department of Obstetrics & Gynecology Fukui Medical University Yoshidagun Fukui Japan 910–1193 Susan C.Laws Endocrinology Branch Reproductive Toxicology Division National Health and Environmental Effects Research Laboratory United States Environmental Protection Agency Research Triangle Park

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NC 27711 USA Tara Lovekamp-Swan National Institute of Environmental Health Sciences (NIEHS) Laboratory of Women’s Health (LWH) 111 TW Alexander Drive P.O. Box 12233 Mail Drop B3–06 Research Triangle Park NC 27709 USA Jennifer L.Marcinkiewicz Department of Biological Sciences Kent State University Kent, OH 44242–0001 USA Kimberly P.Miller Department of Epidemiology and Preventive Medicine Division of Gender Based Epidemiology University of Maryland School of Medicine 660 West Redwood Street Baltimore, MD 21201 USA Michael G.Narotsky Endocrinology Branch Reproductive Toxicology Division National Health and Environmental Effects Research Laboratory United States Environmental Protection Agency Research Triangle Park NC 27711 USA Karl K.Rozman Department of Pharmacology, Toxicology and Therapeutics University of Kansas Medical Center Kansas City Kansas 66160 Section of Environmental Toxicology GSF-Institute für Toxikologie Neuherberg Germany Hiromasa Sasaki Department of Obstetrics & Gynecology Fukui Medical University

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Yoshidagun Fukui Japan 910–1193 I. Glenn Sipes Department of Pharmacology The University of Arizona Yucson, AZ 85724 USA Tammy E.Stoker Gamete and Early Embryo Biology Branch Reproductive Toxicology Division National Health and Environmental Effects Research Laboratory United States Environmental Protection Agency Research Triangle Park NC 27711 USA Diane Suter Department of Biology Loyola University Chicago Chicago, IL 60626 USA Paul F.Terranova Center for Reproductive Sciences Department of Molecular & Integrative Physiology University of Kansas Medical Center Kansas City KS 66160 USA Dragana Tomic Department of Epidemiology and Preventive Medicine Division of Gender Based Epidemiology University of Maryland School of Medicine 660 West Redwood Street Baltimore, MD 21201 Benjamin K.Tsang Reproductive Biology Unit and Division of Gynecologic Oncology Department of Obstetrics & Gynecology and Cellular & Molecular Medicine University of Ottawa Ottawa Health Research Institute The Ottawa Hospital (Civic Campus) Ottawa Ontario Canada K1Y Y 4E9

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Reprint Request: Benjamin K. Tsang, PhD Ottawa Health Research Institute 725 Parkdale Avenue Ottawa, Ontario Canada K1Y Y 4E9 Chao Wu Xiao Reproductive Biology Unit and Division of Gynecologic Oncology Department of Obstetrics & Gynecology and Cellular & Molecular Medicine University of Ottawa Ottawa Health Research Institute The Ottawa Hospital (Civic Campus) Ottawa Ontario Canada K1Y Y 4E9 Nutrition Research Division Food Directorate Health Products and Food Branch Health Canada 2203C Banting Research Centre Ottawa ON Canada K1A A 0L2 Xiaojuan Yan Reproductive Biology Unit and Division of Gynecologic Oncology Department of Obstetrics & Gynecology and Cellular & Molecular Medicine University of Ottawa Ottawa Health Research Institute The Ottawa Hospital (Civic Campus) Ottawa Ontario Canada K1Y Y 4E9

FOREWORD

In recent years in the USA, there has been an increase in the number of working women, and a tendency for women to postpone the start of a family. These trends have heightened an awareness of the impact of environmental chemicals in the workplace on reproductive function. A variety of considerations can affect fertility in women who are older when beginning a family, and women with fertility problems may not discover them until their reproductive life span is waning. In addition to a generally reduced quality of oocytes with age, more years of exposure to environmental influences can also have a potential effect. A better understanding of the effect of these chemicals on ovarian function is of particular importance since the ovary is critical to normal reproduction. Reproductive function in women can be compromised by exposure to toxic chemicals. Reproductive toxicants can act via direct alterations in steroid hormone production (ovary) or by interference with steroid hormone action (hypothalamus, pituitary, reproductive tract). Alternatively, the effects of ovarian toxicants can result from ovarian failure caused by extensive oocyte destruction. As a result of extensive follicular damage, neuroendocrine feedback is disrupted, and circulating levels of the gonadotropins follicle stimulating hormone (FSH) and luteinizing hormone (LH) rise. Therefore, follicle destruction can ultimately disrupt endocrine balance by causing a reduction in ovarian production of estrogen and progesterone, and an elevation in FSH and LH. This book represents a compilation of chapters prepared by researchers who have substantially contributed to our understanding of the impact of xenobiotics and environmental factors on ovarian function. Additionally, issues associated with epidemiology and risk assessment testing as regards the ovary have also been addressed. It is hoped that this volume will prove equally interesting and helpful to scientists in the academic, industrial and regulatory settings. Patricia B.Hoyer, PhD Editor

1 OVARIAN PHYSIOLOGY Diane Suter

INTRODUCTION It is the hope of the author of this chapter that, for readers with expertise in toxicology, some detail or concept will spark a light to illuminate an intersection between ovarian physiology and toxicology. To that end, this chapter is broad but not particularly deep. The reviews and books cited at the end are excellent sources for those seeking more on a particular topic (Stouffer, 1987; Adashi and Leung, 1993; Hsueh and Schomberg, 1993; Fauser et al., 1999). DEVELOPMENT OF THE OVARY Molecular biology The ovary and the testis develop from the same primordial tissue. Only a few genes of importance in their embryonic development have been identified. The most famous is Sry, located only on the Y chromosome and therefore not present in genetic females. Its gene product is the testis-determining factor. Its functional counterpart is Dax-1, located on the short arm of the X chromosome. Its gene product can be thought of as the ovary-determining factor. SRY and DAX-1 proteins are mutually antagonistic. Two active copies of Dax-1 produce enough DAX-1 protein to stimulate the indifferent gonad to become an ovary, even in the case of abnormal presence of Sry. So, in the presence of no Sry and one or more develops into an ovary. If one Sry gene and one or fewer copies of Dax-1 are Dax-1, or in the presence of two Dax-1 genes, regardless of Sry, the indifferent gonad present, the indifferent gonad develops into a testis. The SRY protein is a transcription factor that acts as a switch, turning on expression of genes that cause embryonic differentiation of the testis. The DAX-1 protein encodes an orphan member of the nuclear receptor superfamily and may be a repressor of transcription. In the male, Sry and Dax-1 are both expressed in the genital ridge immediately before sexual differentiation, but Dax-1 expression

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soon ceases. In the female, Dax-1 is expressed and likely suppresses the activity of a variety of genes involved in testis formation. In addition, it acts to direct the development of granulosa cells, the first ovarian cells to develop, without which the other ovarian somatic cells and the oogonia cannot survive. The X-chromosome homolog of Sry is sox3. Its function is unknown, but mutations seem to be lethal, as no sox3¯ females have been reported. The only two other genes known to be involved in ovarian differentiation are encoded at the steel and white spotting loci in the mouse. The white spotting locus is thought to be the same as c-kit locus, a proto-oncogene. The protein encoded by this gene is the receptor for stem cell factor, the gene product of the steel locus. These two genes are necessary for survival of germ cells. Morphology The source of the somatic cells of the ovary is the genital ridge, a thickening of the mesoderm on the posterior wall of the celom. The genital ridge has a cortex and a medulla. In most species, the mature ovary is primarily of cortical origin, and the testis of medullary origin. During the indifferent stage, the medulla grows short spaghetti-like protrusions called cords or primary sex cords. In the testis, these develop, but in the ovary, they degenerate into the connective tissue of the ovarian hilus, the stalk that attaches the ovary to the body wall. In the cortex, a set of secondary cords develop. These secondary cords become the somatic cells of the ovary. Instead of retaining their cord-like appearance, the cords subdivide into clusters of cells. About this time, the germ cells arrive. The germ cells originate in a region of the embryo completely separate from the somatic cells. They originate in the yolk sac and are first apparent after about 1 month in the human embryo and 7 days in the mouse. They migrate first to the hindgut and then to the genital ridge, mitosing along the way. This mitosis has an interesting variation, however, in that cytokinesis is not completed, so the germ cells become clusters of interconnected oogonia. They arrive at the ovary at about 6 weeks in the human, and at about mid-gestation in the mouse. When they get there, a close association forms between the clusters of oogonia and the clusters of secondary sex cord cells. The secondary sex cord cells then completely engulf each individual oogonium with a single layer of cells, breaking the cytoplasmic connections between the oogonia and forming the nascent primordial follicles. A basement membrane, or membrana propria, is already present outside the layer of somatic cells. The germ cells are diploid at this point and are called primary oocytes. This packaging process is complete around the time of birth in rodents and about the end of the first trimester in humans. Once the primordial follicles form, the enclosed oocytes begin meiosis. Recombination occurs and the oocytes hypertrophy. The oocytes progress through meiosis I until the diplotene stage of prophase I. At that point, meiosis I is arrested and remains so until that particular oocyte is ovulated, weeks or

OVARIAN PHYSIOLOGY 3

decades after puberty. Thus, as long as they are in the ovary, the germ cells are diploid primary oocytes. ANATOMY In the mature ovary, all follicles are in the cortex and therefore near the surface of the ovary. An interesting anatomical exception is the horse, in which the cortex inverts and is largely surrounded by medulla. Only a small region of the surface of the equine ovary is made of cortex, and this is the only region of the surface from which ovulation can occur. As female reproductive cycles follow one after the other in overlapping waves, all of the structures described below are likely to appear simultaneously in a mature ovary. The least mature follicular structure is the primordial follicle, comprising a diploid primary oocyte arrested in diplotene I, a single surrounding layer of squamous granulosa cells, and a membrana propria surrounding the granulosa cells. No blood vessels traverse the membrana propria, even in the most mature follicles. The next stage in maturation of a follicle is the primary follicle. The granulosa cells are cuboidal rather than squamous, and a non-cellular layer, the zona pellucida, appears between the primary oocyte and the granulosa cells. The granulosa cells, however, send cytoplasmic processes through the zona pellucida, which touch microvilli on the primary oocyte and form gap junctions with the oocyte. The granulosa cells and the oocyte maintain constant biochemical contact with each other via these gap junctions throughout follicular development. In secondary follicles, also called pre-antral follicles, the granulosa cells divide and organize themselves into multiple layers surrounding the oocyte. A third cell type, the thecal cell, is recruited into the structure. Thecal cells, which differentiate from ovarian stromal cells, are spindle-shaped and surround the granulosa cells, outside the membrana propria. The thecal cells also proliferate, and substantial angiogenesis occurs, making the follicle a well-vascularized structure. The primary oocyte hypertrophies. The next stage of development results in a tertiary follicle, also called a Graafian or antral follicle. The granulosa cells further differentiate into four subspecialties. The corona radiata is the single layer of granulosa cells immediately adjacent to the zona pellucida. These are the granulosa cells that maintain gap junctions with the primary oocyte. Surrounding the corona radiata is the cumulus oophorus, a 3–4-cell-thick layer of granulosa cells that adhere closely to the corona radiata. At ovulation, the cells of the corona radiata and cumulus oophorus leave the ovary and travel down the oviduct along with the oocyte. The intercellular adhesions of the more distal granulosa cells begin to break down, and the granulosa cells secrete a serum-like fluid into the interstices. The fluid, called follicular fluid or liquor folliculi, accumulates and creates one large fluid-filled chamber called the antrum. In addition to water, electrolytes and serum proteins,

4 DIANE SUTER

the follicular fluid contains high concentrations of steroid and protein hormones secreted by the granulosa cells. The third variety of granulosa cell, the mural cells, cling to the membrana propria and maintain their intercellular adhesions, creating a sphere of granulosa cells surrounding the antrum and the oocyte/ corona radiata/cumulus oophorus complex. That complex does not float free in the antrum, but is attached to the mural granulosa cells by the fourth variety of granulosa cells, the stalk. The thecal cells comprise two subtypes, the theca interna and the theca externa. The theca interna has the cytoplasmic ultrastructure of steroidogenic cells, featuring many lipid droplets and extensive smooth endoplasmic reticulum. Not all follicles grow to maturity. Most, in fact, become atretic follicles. Atresia can occur at any of the above stages of development. Atretic follicles are defined anatomically by an irregular follicular shape, eosinophilia of the ooplasm, wrinkling of the oocyte’s nuclear membrane and degeneration of the granulosa cells by apoptosis. The dying granulosa cells can be identified with light-level histology by their pyknotic appearance, characterized by a shrunken, degenerating nucleus with dense, formless chromatin. They can also be identified with modern apoptosis-detecting kits. The number of apoptotic granulosa cells present at any one time, however, is very low, less than 1 percent. Thecal cells do not appear apoptotic. It is thought that they dedifferentiate back into stromal cells and then redifferentiate into thecal cells as other follicles enter the growth phase. When a follicle ovulates, only the oocyte, corona radiata and cumulus oophorus leave the ovary. The mural granulosa cells and the thecal cells stay behind. At the moment of ovulation, the follicular wall and its extensive vasculature break open. This process creates a transient structure, the corpus hemorrhagicum, a collapsed follicle in which the antrum has filled with blood. The presence of corpora hemorrhagica can be used as an assay to quantitate ovulatory sites in a recent ovulation. The corpus hemorrhagicum rapidly redifferentiates into a new structure, the corpus luteum. The name means “yellow body” in Latin, but in many species the corpora lutea are pink. Like the mature follicle, the corpus luteum is a temporary structure, enduring 2–21 days, depending on the species. It is a solid structure with no antrum. One corpus luteum appears per ovulating follicle. Although a more histologically homogeneous structure than the follicle, it does have more than one cell type. The two main functional cell types are defined by size. Large luteal cells, somewhat arbitrarily defined as being greater than 20 µm in diameter, have the ultrastructure of metabolically active cells: considerably dense rough and smooth endoplasmic reticulum, an extensive Golgi apparatus, and many electron-dense granules. The small luteal cells lack electron-dense granules but do have much smooth endoplasmic reticulum. At the end of its prescribed life span, the corpus luteum degenerates via apoptosis. The evidence of its past existence persists for some days in the form of a corpus albicans. This lingering structure appears white due to retention of connective tissue between degenerated cells.

OVARIAN PHYSIOLOGY 5

A structure indicative of a pathological condition is the ovarian cyst, an antral follicle that neither ovulated nor underwent atresia at its appointed hour. Having a structure similar to healthy antral follicles except enlarged, often considerably so, ovarian cysts can be visualized via ultrasound. They cause acyclicity and lower back pain in humans, and because they secrete excess androgens, they also cause hirsutism and acne. The rest of the ovary is made up of stromal cells, and vascular and neural elements. Stromal cells are undifferentiated mesenchymal cells supporting the rest of the structures in the ovary. They are thought also to differentiate into thecal and luteal cells. Like all endocrine tissues, the ovary is highly vascularized. The exception is inside the membrana propria, where there are no blood vessels. In a mature follicle, a dense sphere of blood vessels, sort of a vascular “buckyball,” surrounds the membrana propria but does not traverse it. Despite this lack of vasculature, molecules such as glycoprotein hormones do reach the granulosa cells. Nonetheless, anything reaching the oocyte must pass first through the cells of the corona radiata. The innervation of the ovary is primarily sensory and sympathetic, with a modest amount of parasympathetic input. Stromal, follicular and luteal cells are innervated as well as the vascular smooth muscle. The innervation arises from the ovarian plexus and superior ovarian nerve. ENDOCRINOLOGY AND BIOCHEMISTRY Receptors and signal transduction Chemical messengers acting on or emanating from the ovary can have endocrine, paracrine or autocrine effects. Endocrine effects are those occurring at a distant site from where the messenger is secreted. Paracrine effects are those occurring at close range, on cells near the source of secretion. Autocrine effects occur at pointblank range, on the very cell from which a messenger molecule was secreted. Regardless of the range of action, hormones, growth factors and cytokines all exert their intracellular effects via signal transduction mechanisms. Most signal transduction mechanisms identified to date are active in the ovary. For an excellent review, see Findlay (1994). Two gonadotrophic glycoprotein hormones of the anterior pituitary, luteinizing hormone (LH) and follicle-stimulating hormone (FSH) are essential for ovarian function. Their receptors are a specialized subfamily of the superfamily of seven transmembrane domain G-protein-coupled receptors. They activate cAMP-protein kinase A pathways and inositol phosphate-protein kinase C pathways. The genes for these receptors have been cloned for several species. Along with the gene for the thyroid-stimulating hormone receptor, the DNA sequences have much similarity, indicating recent evolutionary divergence. The genes for the LH and FSH receptors are located very close to each other (in

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humans, on chromosome 2). Unlike others in the G-protein-coupled receptor family, these genes are large and have several introns. Also unlike others in the family, the extracellular N-terminal portion of the protein is very long and is the site of ligand binding. The transmembrane domains in the C-terminal half of the receptor protein are the sites of G-protein association. Receptors for LH are located in the theca interna, the late-stage granulosa cell, and the corpus luteum. Receptors for FSH are located in the granulosa cell. Receptors for the gonadotropins are internalized and down-regulated by their ligand. Some recycling of receptors occurs. The signal transduction pathways for steroid hormones are strikingly different from those for protein hormones. The receptors for steroid hormones belong to a superfamily of ligand-activated transcription factors. These receptors all contain an individualized ligand-binding domain, a highly conserved DNA-binding domain, an unconserved hinge region between them and an unconserved Nterminus. They are synthesized in the rough endoplasmic reticulum like other proteins and remain in an inactive state by virtue of their association with the heat shock protein (hsp90). They are translocated to the nucleus either before or after ligand binding, depending on the particular steroid. Ligand binding causes a conformational shift that induces dissociation of hsp90 and association of two identical receptorligand complexes into a homodimer. The homodimer, along with other requisite factors, binds to specific regions of DNA and induces their transcription. Steroids have endocrine, paracrine and autocrine actions, and most cells of mature follicles and corpora lutea express steroid receptors. Many growth factors are important in ovarian physiology. Receptors for many of these growth factors are tyrosine kinases (epidermal growth factor, basic fibroblast growth factor, insulin-like growth factor, nerve growth factor and ckit). Others are serine-threonine kinases (transforming growth factor β inhibin, activin). Receptors for prolactin are present in the corpus luteum and follicular thecal cells. They are in the same family as receptors for growth hormone, interleukins and erythropoietin. They have no intrinsic kinase activity but activate a soluble cytoplasmic kinase system known as Janus kinase signal transducers and activators of transcription (JAK-STAT). Steroids Soluble factors were identified as important mediators of reproductive function by Berthold in 1849, in the first controlled endocrinology experiment. These factors eventually proved to be steroids, small, lipid-soluble molecules with four common interlocking carbon rings and individualized residual groups that confer hormone specificity. They include the progestins, the estrogens and the androgens. Progestins, most notably progesterone, are 21-carbon steroids, synthesized in the follicle (mostly by the theca interna, but also by granulosa cells) and the corpus luteum. Androgens, including testosterone, are 19-carbon steroids. They are considered to be primarily male hormones, but they are

OVARIAN PHYSIOLOGY 7

synthesized in the ovary as obligate intermediates for the estrogens. Estrogens, most notably estradiol, are 18-carbon steroids synthesized primarily by granulosa cells, although the primate corpus luteum does synthesize estradiol. Detailed diagrams of steroidogenesis are available in any good endocrinology or reproductive biology text, such as Griffin and Ojeda (2000), Hadley (2000) or Johnson and Everitt (2000). Cholesterol is the synthetic precursor of steroids. Steroidogenesis comprises a progressive loss of carbons in a series of hydroxylation reactions. The enzymes involved in this process are in the superfamily of cytochrome P450 enzymes, including their associated electron-transport systems. The ovary can synthesize cholesterol from the simple 2-carbon molecule acetate, but the ovary gets most of its cholesterol by endocytosis of cholesterol from the blood. Granulosa cells have low-density lipoprotein (LDL) receptors in their plasma membranes and take up LDL-bound cholesterol via endocytosis. The cholesterol is esterified and stored in lipid droplets in the cytoplasm. The first step in steroidogenesis is the conversion of cholesterol to pregnenolone, catalyzed by the cholesterol side-chain cleavage enzyme. It is the rate-limiting step in steroidogenesis and P450scc is expressed in all steroidogenic cells. This enzyme is located on the inner mitochondrial membrane, and thus cholesterol must be transported across both mitochondrial membranes to initiate steroidogenesis. The steroidogenic acute regulatory (StAR) protein is responsible for chaperoning cholesterol across the mitochondrial membranes. StAR protein is short-lived (although its mRNA is not), conferring cycloheximidesensitivity upon steroidogenesis. The next enzyme in the steroidogenic pathway is 3β-hydroxysteroid dehydrogenase/∆5 → ∆4-isomerase, which catalyzes the conversion of pregnenolone to progesterone. Progestins, both pregnenolone and progesterone, are converted irreversibly to 19-carbon androgens by 17α-hydroxylase/17–20lyase (P45017α), which clips off a 2-carbon side chain. Androgens are converted irreversibly to estrogens by aromatase (P450aro), whicl cyclizes the A-ring, removing one carbon in the process. Another steroidogenic enzyme that merits mention is 17β-hydroxysteroid dehydrogenase/oxidoreductase, which catalyzes conversion of androstenedione to testosterone, a more potent androgen. It also catalyzes the conversion of estrone to estradiol, a more potent estrogen. Finally, 5α-reductase converts testosterone to 5α-dihydrotestosterone, an extremely potent androgen. The expression of 5α-reductase is inversely proportional to the expression of aromatase throughout the cycle. It serves as an alternative pathway for metabolism of androgens, rather than aromatization to estrogens. Furthermore, 5α-DHT may be luteolytic. The main steroidal product of the follicle is estradiol. Its synthesis is described by the two-cell, two-gonadotropin theory of steroidogenesis in the follicle. According to this well-supported theory, the theca interna expresses LH receptors, side-chain cleavage enzyme, 3β-hydroxysteroid dehydrogenase and

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17α hydroxylase/ 17,20 lyase, but not FSH receptors or aromatase. Granulosa cells express FSH receptors, side-chain cleavage enzyme, 3β-hydroxysteroid dehydrogenase and high levels of aromatase. The thecal cells synthesize androgens from cholesterol, which diffuse across the membrana propria into the granulosa cells, where they are aromatized into estradiol. This theory is supported by autoradiographic studies demonstrating the localization of radiolabeled LH to thecal cells and of FSH to granulosa cells. It is also supported by immunocytochemistry and in situ hybridization studies showing the localization of steroidogenic enzymes to the above mentioned cell types. Because of the presence of the membrana propria, it is possible to do a reasonably thorough job of separating thecal from granulosa cells in vitro, and subsequent incubation studies also support the idea that these two cell types work together to synthesize estradiol in the follicle. Proteins Protein hormones synthesized and secreted by the ovary include oxytocin and relaxin. Oxytocin is synthesized by the corpus luteum and participates in luteolysis by virtue of its ability to inhibit synthesis of progesterone. Relaxin is important in pregnancy. It increases endometrial aromatase activity, decreases myometrial contractility, induces cervical ripening and dilatation, and serves as a mammary gland growth factor. Other important ovarian proteins include the proteins of the zona pellucida, which are essential for proper fertilization. Proteins ZP-2 and ZP-3 are synthesized by the oocyte. ZP-3 is the sperm receptor and activates the acrosome reaction. Both are glycosylated and both are modified in the zona reaction to prevent polyspermy. Many of the proteins and peptides synthesized by the ovary are growth factors or cytokines, locally produced factors that exert autocrine and paracrine effects on proliferation and differentiation of ovarian structures. The number of these factors known to be expressed in the ovary is long and continues to grow. An excellent summary is presented in Findlay (1994). Foremost among them are the proteins of the inhibin family. Varying dimeric combinations of three protein subunits make either inhibin or activin. One of the three subunits has high sequence similarity to mullerian-inhibiting substance and transforming growth factor (TGF) β. Inhibin and activin are synthesized by granulosa cells and luteal cells. They behave in classical endocrine fashion: inhibin reduces secretion of FSH from the anterior pituitary and activin stimulates secretion of FSH. A third ovarian protein, follistatin, binds to activin, thereby inactivating it in the local ovarian environment. A theory of the paracrine effects of inhibin, activin and follistatin has yet to solidify. The second most well-characterized ovarian growth factor is insulin-like growth factor-1 (IGF-1). The ovary is one of the major producers of IGF-1 (human granulosa cells, however, secrete IGF-2 rather than IGF-1). It is secreted

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by granulosa and thecal cells. Its sites of action include the granulosa, thecal and luteal cells, as well as the oocyte. It acts synergistically with FSH to promote follicular growth. FSH increases expression of IGF-1 receptors. This synergy first stimulates granulosa cell differentiation, then estrogen and progesterone synthesis, and finally late expression of LH receptors on granulosa cells. It exerts a classical sulfation effect, stimulating production of heparan and dematan sulfate, possibly important for building the physical infrastructure of the antral follicle. In addition, thecal and granulosa cells also secrete several IGF-binding proteins (IGFBPs). Their production is inhibited by FSH. Atretic follicles do not produce IGF-1, suggesting that IGF-1 is necessary for follicular selection. Thus, FSH, IGFs and IGFBPs participate in a physiological balancing act that, depending on the particular balance, will result in maturation or atresia. Several growth factors have been demonstrated to play clear roles in ovarian function, even though their biochemistry and molecular biology is not as well characterized as those of inhibin or IGF-1. One of these is c-kit, mentioned above as being vital for the survival and migration of primordial germ cells. A second growth factor known to be essential to ovarian development is nerve growth factor (NGF). NGF stimulates innervation during development and perhaps maintains the strength of innervation during adulthood. It is synthesized by granulosa and thecal cells. Its receptors are located on these follicular cells as well as on innervating fibers. Ovarian cells and innervating fibers interact in stimulating follicular growth. A third well-characterized factor is maturation promoting factor (MPF), known to be the central trigger for resumption of meiosis at ovulation. It is a ubiquitous protein, made in the oocyte as somatic tissues, and is highly conserved across the animal kingdom. It consists of two components: a 34 Kd protein homologous to the cdc2+ gene product and cyclin B. Several other growth factors are known to be essential for ovarian function, but well-supported theories of their roles are not yet available. One is epidermal growth factor (EGF). EGF itself is not produced by the ovary, but granulosa cells do produce transforming growth factor (TGF) α and express TGFα receptors, which also bind EGF. These factors promote proliferation of granulosa and thecal cells, and are angiogenic. Furthermore, they inhibit expression of FSH receptors in granulosa cells, thus prolonging mitosis and delaying differentiation of granulosa cells. These facts suggest they play a role in the early growth phase of follicular development. A second in this category is basic fibroblast growth factor (FGF), which is produced by granulosa cells and stimulates cell proliferation. A counterpart to EGF/TGFα is TGFβ. It is synthesized by theca, granulosa and oocytes. It promotes differentiation rather than proliferation of granulosa cells. A cytokine known to be produced by the ovary is interleukin-1 (IL-1). IL-1, its receptor, and its natural antagonist are synthesized locally in the ovary. They appear in thecal cells before ovulation. An inflammation mediator, it is thought to be involved in ovulation, which may be an inflammatory-like process. A second factor that may behave as a cytokine in the ovary is tumor necrosis factor

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(TNF) α. It is synthesized by local macrophages and other immune cells, and also by granulosa cells and oocytes. Its roles are unclear: both growth-promoting and cell-killing effects have been observed. It promotes angiogenesis and inhibits steroidogenesis, suggesting a role in early follicular growth. In the corpus luteum, it inhibits steroidogenesis and induces prostaglandin F2α synthesis, suggesting a role in luteolysis. Enzymes The renin/angiotensin system is a classic endocrine system for regulation of blood pressure, as described in any basic physiology text. The ovary locally produces renin, angiotensinogen and angiotensin-converting enzyme. Angiotensin II is a potent vasoconstrictor and as such may promote atresia. It also, however, stimulates ovulation in mature follicles and promotes angiogenesis in corpora lutea. Two other enzymes, collagenase and plasminogen activator, are critical components of ovulation. They are synthesized by granulosa cells and break down the intercellular connective tissue in the follicle, reducing the tensile strength of the follicular wall at the time of ovulation. Eicosanoids Of the prostaglandins (PGs), PGF2α and PGE2 are the most important in the ovary. PGE2 is synthesized in the human ovary and is luteotrophic. PGF2α is synthesized in the follicle and is an important part of the triggering mechanism for ovulation. It also plays an important role in luteolysis in non-primates. THE OVARIAN CYCLE The ovary, via all of the hormones and other factors mentioned above, performs an intricate dance that creates reproductive cycles in females. Other than the ovary, the important participants in the dance are the anterior pituitary and hypothalamus. Protein hormones from the anterior pituitary—LH, FSH and prolactin—exert gonadotrophic effects on the ovary. Ovarian hormones— estradiol, progesterone, inhibin and activin—act in turn on the hypothalamus and anterior pituitary in negative and positive feedback fashions to create a cyclical pattern of follicular development, ovulation, luteal development and luteolysis. Follicular growth The first step in the dance is early follicular growth, during which many follicles develop from primordial follicles containing a single layer of squamous granulosa cells, to early antral follicles. There is some lack of uniformity of nomenclature here. Some biologists use the word “recruitment” to refer to this

OVARIAN PHYSIOLOGY 11

early period of growth, which can occur at any time, including before puberty. Some use it to mean a later process by which a small number of early antral follicles develop to mature antral follicles. I will follow the latter convention. In humans, unlike most other mammals, early follicular growth is quite protracted and begins several cycles prior to maturation of these follicles. This growth period is characterized primarily by proliferation of granulosa cells. Granulosa cells can divide in vitro in the absence of gonadotropins as long as intraovarian growth factors such as EGF, IGFs, bFGF, TNFα and TGFα are present. In vivo, however, experimental manipulation of gonadotropins suggests that tonic levels of FSH do play at least a facilitatory role in early mitosis in the granulosa cells of primary follicles. As would be predicted from the permissive role of FSH, the granulosa cells acquire FSH receptors and also steroid receptors. The granulosa cells divide until they are about 6–7 layers thick. The zona pellucida appears, next to the oocyte, and ZP-2 and ZP-3 are expressed. Angiogenesis begins even before theca cells appear. Later, there is extensive angiogenesis in the space between the granulosa and thecal cells. The follicular cells are mitotic and not yet differentiated. They do not yet express high levels of steroidogenic enzymes. The antrum begins to form. The corona radiata and cumulus oophorus remain tightly associated with the oocyte, surrounded by follicular fluid but attached to the mural granulosa cells by the stalk. The oocyte hypertrophies throughout this period but remains arrested in prophase I. Recruitment The second step is follicular recruitment, the entrance of some of the early antral follicles into a pool of pre-ovulatory follicles of which one or several, depending on the species, will ovulate. It is not known why, in any given cycle, some growing follicles are recruited and others are reserved for future rounds of recruitment. This process may be a stochastic one in which the follicles that are, by chance, the furthest along developmentally put the remaining follicles into a holding pattern until the more mature ones ovulate or become atretic. Many more follicles are recruited than are ovulated. Recruitment begins with negative feedback. As the ovary nears the end of the luteal phase from the previous cycle, circulating steroids and inhibin drop. This releases FSH from negative feedback. FSH levels rise and stimulate the transition of a cohort of follicles from the early antral to the mature stage. This transition is characterized first by the appearance of thecal cells and the acquisition of LH receptors by thecal cells. Expression of steroidogenic enzymes and a vastly expanded steroidogenic capability arises. Levels of estradiol in the blood rise. Estradiol and FSH act synergistically to promote this stage of maturation, including differentiation of granulosa and thecal cells, characterized by less mitosis, more steroidogenesis and more expression of gonadotropin receptors. Late in this step, expression of LH receptors by granulosa cells occurs. The appearance of LH receptors on granulosa cells is critical for the processes of luteinization and ovulation.

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Selection The third step is follicular selection, determination of which follicle (for monovulatory species such as humans) or follicles (for multiple-ovulating species such as rats) will ovulate. Timing is everything here. For example, monovulatory species like humans have a dominant (i.e., largest) follicle at any given day during follicular development, but it is not the same follicle every day. At some point, a critical series of events culminate in selection of one follicle (or a few, in multiple ovulators) and atresia for the rest. The acquisition of significant numbers of FSH receptors must coincide with a transient rise in FSH levels early in the follicular phase of the cycle. For follicles that are behind the times in generating FSH receptors, follicular atresia occurs. Those follicles that acquire enough FSH receptors respond to FSH by secreting estradiol and inhibin, which in turn inhibits FSH secretion, withdrawing trophic support from other recruited follicles. Again, the result is atresia for the also-rans. The synergy of endocrine FSH and paracrine/ autocrine estradiol allows the selected follicle(s) to continue to grow, despite the withdrawal of FSH’s gonadotrophic support, through enhanced sensitivity to FSH. The acquisition of LH receptors is also critical for successful selection. Having more LH receptors on granulosa cells than do other recruited follicles allows accelerated development, since LH and FSH, both acting through cAMP, have an additive stimulatory effect on granulosa cells that have both kinds of receptors. This synergy is enhanced by angiogenesis as well, as the follicles with the richest vascularization will be exposed to more circulating gonadotropins than other follicles. Atresia Not all Graafian follicles ovulate. For any follicle that does not ovulate, the fate is atresia, an apoptotic process. Atresia occurs even before birth in primordial follicles. It slows at puberty, but continues throughout reproductive life. Atresia is a phenomenon primarily of granulosa cells and oocytes. Thecal cells probably are able to dedifferentiate and redifferentiate into thecal cells in other follicles. Atretic follicles are poorly vascularized, and the lack of delivery of oxygen, nutrients and gonadotrophic factors as the volume of the growing follicle increases, may trigger apoptosis. Protein synthesis declines. The degeneration of the oocyte and pyknosis of the granulosa cells described in the anatomy section occur, and the dying cells are engulfed by invading macrophages. The mechanism is not well understood. Atretic follicles have a high ratio of testosterone:estradiol, suggesting that initiation of aromatase expression is essential for rescue from atresia. Growth factors implicated in the process include IGF-binding proteins and activin. Production of activin would tip the local environment away from inhibin (since inhibin and activin are assembled of the same subunits), and IGF-binding proteins would block the proliferative effect of IGF.

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Ovulation Ovulation relies on a positive feedback interaction between the ovary and the hypothalamic-pituitary axis to bring about the pre-ovulatory gonadotropin surge. Surges of LH and FSH trigger a cascade of events that result in meiotic maturation of the oocyte and physical rupture of the wall of the follicle. The ultimate, and physically obvious, step in meiotic maturation is germinal vesicle breakdown (GVB) or dissolution of the nuclear membrane that must occur in order for homologous chromosomes to segregate in completion of meiosis I. Microscopic observation of GVB is often used as an assay for meiotic maturation. The immature follicle contains a brake, a meiosis-inhibiting factor, the identity of which is not yet certain. A primary part of this braking mechanism is probably sufficiently high levels of cAMP. There is a complex signal transduction cascade that takes the oocyte to GVB. It begins with the pre-ovulatory surge of LH stimulating the granulosa cells. The granulosa cells then decrease their production of GVB-inhibiting substances and increase their production of GVB-stimulating substance(s). In Xenopus laevis, the stimulatory substance is progesterone. In mammals, the identity of the stimulatory factor from the granulosa cells is not certain. Subsequent early changes in the oocyte include increased cytoplasmic calcium, decreased cytoplasmic cAMP and increased cytoplasmic pH. Intermediate events have not all been delineated yet. The final signal transduction, however, is known to be increased MPF, a molecule known to be important in the cell cycle of all cells. It stimulates the transition from G2phase to M-phase, dissolution of the nuclear envelope, phosphorylation of histones that causes chromosome condensation and formation of the spindle apparatus. The first polar body is extruded, bringing the reduction division to completion. The oocyte proceeds to meiosis metaphase II and then enters a second meiotic arrest. Thus, after ovulation, the oocyte becomes a secondary oocyte. The secondary oocyte contains the cytoplasmic mRNAs that will be active in the early stages of development of the conceptus. Proceeding simultaneously with the above chemical process is the physical process of rupture of the follicular wall, necessary for transportation of the oocyte into the oviduct. An increased follicular volume occurs, but no increased intrafollicular pressure. The follicular wall distends as intercellular connections weaken. There is an increased permeability of blood vessels, leakage of erythrocytes and edema. Wall rupture is essentially an enzymatic process involving hydrolysis of intercellular proteins such as collagen. The components of the lytic system, including a plasmin-activating system, are all in place in a relatively mature follicle, but held in check by plasminogen activator inhibitor type 1 (PAI-1), present in granulosa cells. PAI-1 is suppressed by FSH during the pre-ovulatory surge. Also, LH and FSH stimulate prostaglandin synthesis, and prostaglandins in turn stimulate activation of collagenase and plasmin, and promote the vascular effects.

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A third aspect of ovulation is cytoplasmic maturation, the synthesis and storage of many proteins essential for early growth and development of the conceptus. Luteinization and luteal function Meanwhile, back in the ovary, a marvelous transformation of the follicular cells left behind occurs. Late in the process of follicular recruitment, granulosa cells acquire LH receptors. At ovulation, the oocyte, corona radiata and cumulus oophorus leave the ovary, but the rest of the granulosa cells and thecal cells remain. At ovulation, the membrana propria breaks down, allowing mixing of the granulosa and thecal compartments. Further angiogenesis occurs, including invasion of blood vessels into the granulosa layer. Expression of steroidogenic enzymes shifts to favor progesterone production, although the primate corpus luteum does produce significant amounts of estradiol. A new structure, the corpus luteum, is born. The corpus luteum has two cell types—large and small. A 20µm diameter is the arbitrary size cut-off between them. Large cells probably come from granulosa cells and small cells from thecal. Later, though, small cells likely become large cells, at least in some species. The number of small cells increases during the luteal phase. Small cells do not seem to undergo mitosis, however, suggesting that some small cells may arise from differentiating fibroblasts. The large cells are of greater secretory activity. They have all the cellular apparatus necessary to do so, including smooth and rough endoplasmic reticulum, Golgi and electron-dense granules. They make progesterone, oxytocin, relaxin and inhibin. In most species, only small cells respond to LH, even though both cells have LH receptors. Other cell types include vascular epithelial cells, immune and other blood cells, connective tissue cells and smooth muscle cells. The corpus luteum is a transient organ. It is kept alive by a luteotrophic complex of differing composition in different species. The presence of LH is an absolute requirement in most species, including primates. LH levels are in fact quite low during the luteal phase, due to progesterone’s negative feedback effects. The corpus luteum is, however, exquisitely sensitive to LH, due to the high concentration of high-affinity receptors for LH. Prolactin is an important component of the luteotrophic complex in some species. It is more important in rodents than in primates. In particular, it maintains the early corpus luteum, triggered by a neuroendocrine reflex arc initiated by the male’s stimulation of sensory neurons on the flanks of the female during mating. Progesterone is luteotrophic, as is estradiol in many species. Luteolysis The demise of this transient organ, called luteolysis, is an apoptotic process. It involves cessation of steroidogenesis and death of most luteal cells. An old saying is that “in most species, the corpus luteum is murdered by the uterus, but

OVARIAN PHYSIOLOGY 15

in primates, the corpus luteum commits suicide”. In primates, there is not an active luteolytic factor that anyone has found yet. Diminishing LH levels over the course of the luteal phase do occur, but this change does not seem to be sufficient for luteolysis. The primate corpus luteum becomes refractory to LH, but exactly how this occurs is not clear. Nonetheless, the combination of ebbing LH and reduced luteal sensitivity to LH brings about the demise of the primate corpus luteum. In non-primates, particularly ungulates, an early step in luteolysis is the loss of granules from large luteal cells, probably indicating the release of oxytocin. Ovarian oxytocin stimulates secretion of uterine PGF2α. PGF2α travels to the ipsilateral ovary and causes vasoconstriction and also directly inhibits progesterone production. Prostaglandin effects are mediated primarily via the large cells. PGF2α binds to large cells and inhibits their production of progesterone by a protein kinase C-mediated process. Some factors must also act in a paracrine fashion to inhibit progesterone production in the small cells as well. Increased cytoplasmic calcium in response to PGF2α starts the apoptotic process. Maternal recognition of pregnancy Pregnancy in mammals is absolutely dependent upon a steady source of progesterone. In some species, the placenta performs this function, but even in those species, another source of progesterone must be found until the placenta has time to develop. That source of progesterone is the corpus luteum. Hence, the conceptus must somehow signal the maternal physiology of its presence, lest luteolysis occur and cause spontaneous abortion. In the primate and a few other species, this phenomenon is accomplished by the conceptus’ secretion of a chorionic gonadotropin. This molecule is closely evolutionarily related to LH, binds to LH receptors in the corpus luteum, and causes maintenance of luteal function in early pregnancy in the absence of adequate luteotrophic support from LH itself. In some non-primates, including many ungulates, the conceptus secretes interferon-τ, which binds specifically to PGF2α. This binding process sequesters PGF2α in the uterus, preventing it from traveling to the ovary and triggering the luteolytic cascade. In many small animals such as rats and mice, gestation is short, and the reproductive cycle is extremely short. The maternal physiology, figuratively speaking, does not have time to wait around for the conceptus to get physically large enough to signal its presence before it has to make a decision about whether to go on to the next cycle. The evolutionary solution to this problem is pseudopregnancy, a state of luteal maintenance triggered by the physical act of mating. In response to coitus, the maternal anterior pituitary secretes pulsatile prolactin, which acts as a luteotrophic factor for 11 days. At that point, the placentae are large enough to secrete enough progesterone on their own to support the pregnancy for the remainder of gestation.

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CONCLUSION The cyclic nature of ovarian function entails involvement of myriad hormones, growth factors, signal transduction pathways and organismal feedback systems. As such, it is particularly vulnerable to toxic compounds, which need impact upon only one of these factors to disrupt ovarian function. To toxicologists, however, it is more than a vulnerable target: its intricacy and accessibility make it a valuable model for examining the impact of many toxic compounds. REFERENCES AND RECOMMENDED TEXTS Adashi, E.Y. and Leung, P.C.K. (eds) (1993) The Ovary. Comprehensive Endocrinology Revised Series, L.Martini (editor-in-chief). New York, NY: Raven Press. Fauser, B.C.J.M., Rutherford., A.J., Strauss III, J.F. and Van Steirteghem, A. (eds) (1999) Molecular Biology in Reproductive Medicine. New York: Parthenon Publishing Group. Findlay, J.K. (ed.) (1994) Molecular Biology of the Female Reproductive System. San Diego, CA: Academic Press. Griffin, J.E. and Ojeda, S.R. (2000) Textbook of Endocrine Physiology, 4th edn. New York, NY: Oxford University Press. Hadley, M.A. (2000) Endocrinology, 5th edn. Upper Saddle River, NJ: Prentice Hall. Hsueh, A.J.W. and Schomberg, D.W. (eds) (1993) Ovarian Cell Interactions: Genes to Physiology, Serono Symposia, USA, New York, NY: Springer-Verlag. Johnson, M.H. and Everitt, B.J. (2000) Essential Reproduction, 5th edn. Malden, MA: Blackwell Science. Stouffer, R.L. (ed.) (1987) The Primate Ovary, New York, NY: Plenum Press.

2 OVARIAN TOXICITY IN SMALL PREANTRAL FOLLICLES Patricia B.Hoyer

FOLLICULAR DEVELOPMENT Development and maturation of oocytes occurs within ovarian follicles. Successful ovulation requires appropriate follicular development, during which the follicle has passed through a number of distinct developmental stages (Hirshfield, 1991). Throughout the life of a mammalian female preceding each ovarian cycle, some follicles are selected to develop to maturity for ovulation and potential fertilization. The most immature stage of follicular development is termed primordial. This is the stage at which follicles first appear in the ovary of a developing female fetus. Development of a primordial (25 µm diameter) to an ovulatory follicle involves transitions through several stages as a pre-antral follicle (25–250 µm diameter; primordial, primary, secondary) and later as an antral follicle (>250 µm diameter; early antral and pre-ovulatory). The stages of follicular development toward ovulation involve a continuum of events, each providing further maturation of the follicular cells (Figure 2.1). Upon receipt of an as yet unknown signal for development, the primordial follicle is activated and becomes a primary follicle. As the follicle develops, there is proliferation of the granulosa cells surrounding the oocyte and acquisition of a layer of theca interna cells surrounding the granulosa layer. Follicles progress from the primary stage to the secondary stage when multiple layers of granulosa cells are present around the oocyte. When the follicle develops sufficiently, an antrum (fluidfilled space) develops within the granulosa cell layer. The antral follicle continues to grow, and at its most mature stage prior to ovulation is known as a Graaffian (pre-ovulatory) follicle. Resumption of meiosis in the oocyte occurs only at the time of impending ovulation. The exact mechanism for selection of a follicle for ovulation is not understood, but is believed to be under intra-ovarian control (Richards, 1980).

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Figure 2.1 Development of ovarian follicles. Primordial follicles are activated to grow and develop from primary through secondary and antral stages of follicular development until ovulation. Most follicles degenerate by the process of atresia before reaching the ovulatory stage. Those follicles which do ovulate luteinize to become corpora lutea which support pregnancy if fertilization occurs (Hoyer and Devine, 2001, with permission from CRC Press).

Fetal development Because of the nature of fetal development of ovarian follicles, women are born with a set number of oocytes, which cannot be regenerated later in life. During fetal development, primordial germ cells that are formed invade the indifferent gonad and undergo rapid hyperplasia. By one month of embryonic life, primordial germ cells have become established in the genital ridge, and oogonia proliferate by mitosis (Hoyer and Devine, 2001). During the period of mitotic proliferation of germ cells, somatic cells and supporting connective tissue develop and gradually become interspersed among the oogonia (Hoyer, 1997). Oogonia develop into oocytes in synchronous waves, once they stop dividing and enter into the first meiotic division. During this period, oocytes grow slowly and proceed to the diplotene stage of the meiotic prophase. The oocyte does not fully complete the first meiotic division at that time but becomes arrested until ovulation, if this should occur. Therefore, the number of oocytes at the time of birth is finite and comprises the total germ cell pool available to a female throughout her life. Around the time of birth, small individual oocytes within the ovary become surrounded by a few flattened somatic cells (pre-granulosa cells), and a basement membrane to form primordial follicles. Association of the granulosa cells with the oocyte at all times is critical for maintenance of viability, and follicle growth and development (Buccione et al., 1990).

OVARIAN TOXICITY IN SMALL PRE-ANTRAL FOLLICLES 19

Primordial follicles Primordial follicles consist of a small oocyte surrounded by a single layer of fusiform-shaped granulosa cells. The small oocyte contained in these follicles is arrested in prophase of the first meiotic division, is non-dividing as follicles develop, and does not resume meiotic division until just prior to ovulation. Primordial follicles provide the pool for recruitment of developing follicles; therefore, they are a fundamental reproductive unit within the ovary. In humans, 1–2 pre-ovulatory follicles develop approximately every 28 days, whereas, in rats, 6–12 follicles develop every 4–5 days (Richards, 1980). The primordial follicles from which pre-ovulatory follicles develop are mainly located near the ovarian cortex and represent the pool for recruitment. Regular waves of the onset of development of primordial follicles is a continuous process from birth, until ovarian senescence occurs (Richards, 1980). Primary follicles Some primordial follicles leave the quiescent state as soon as they are formed, and some are dormant for months or years. The first sign of oocyte growth in primordial follicles is alteration of surrounding squamous (flattened) granulosa cells into cuboidal-shaped cells (Hirshfield, 1991). Once the follicle makes this transition from primordial to primary, other structural changes occur such as development of the zona pellucida and acquisition of the theca layer. The zona pellucida is composed of a glycoprotein matrix to provide protection for the oocyte as well as to provide attachments for the specialized inner layer of granulosa cells, known as cumulus cells. At this stage, another layer of specialized somatic cells begin to proliferate and form a shell outside the basement membrane enclosing the oocyte and granulosa cell layer. These cells appear in concentric rings surrounding the follicle and are designated theca interna cells. Theca cells provide two important functions: (1) attachment of arterioles for the development of an independent blood supply and (2) secretion of progestins and androgens to regulate follicle development (Hirshfield, 1991). Secondary and antral follicles The oocyte contained in a secondary follicle has a large, spherical nucleus (germinal vesicle) that grows in proportion to the growth of the oocyte, and the follicular cells continue to proliferate and form a second layer around the oocyte. As the secondary follicle continues to develop, the layers of granulosa cells surrounding the oocyte increase rapidly to reach follicular diameters of up to 250 µm. Gap junctions are formed between individual cells in the granulosa cell layer to facilitate intercellular transport of nutrients and metabolites to the oocyte (Hoyer, 1997). At that point an antrum begins to form within the granulosa cell layer and this fluid-filled cavity enlarges as the final stages of follicular

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development are reached. The number of follicles that reach the antral stage is quite small. In women, only one follicle per menstrual cycle is usually chosen as the dominant follicle destined for ovulation. A number of autocrine, paracrine and endocrine signals begin to influence follicular development in these stages. At that point, an antrum begins to form within the granulosa cell layer and this fluid-filled cavity enlarges as the final stages of follicular development are reached. The number of follicles that reach the antral stage is quite small. In women, only one follicle per menstrual cycle is usually chosen as the dominant follicle destined for ovulation. The somatic cells acquire receptors for follicle stimulating hormone (FSH) to enhance follicle growth, and they develop steroidogenic capacity for synthesis of androgens, estrogens and progesterone. Collectively, primordial, primary and secondary follicles are less than 250 µm in diameter and are referred to as pre-antral follicles, with primordial and primary being considered small pre-antral follicles. Atresia Throughout the reproductive life span of a female, the total number of primordial follicles selected to develop for ovulation is small compared to the total population. Instead, the vast majority are lost to attrition in various stages of development by a process called atresia. Ovarian content of oocytes is dynamic and fluctuates with age. The total number of oocytes peaks during embryonic development. In humans, the peak number of oocytes ever present, about seven million, occurs at five months gestation, at birth the number has dropped to two million, 250,000–400,000 at puberty, and no functional oocytes remain at menopause (Hirshfield, 1991; Hoyer, 1997). During the lifetime of a woman, ovulation only accounts for 400–600 oocytes. Therefore, of the primordial follicles a female is born with, greater than 99 percent will be lost by atresia at various stages of development. CONSEQUENCES OF FOLLICULAR DESTRUCTION Follicle-specific effects For chemicals that destroy ovarian follicles, the stage of development at which the follicle is targeted determines the impact that exposure to the chemical will have on reproduction (Hoyer and Sipes, 1996). Damage to large growing or antral follicles can cause a disruption of cyclicity by impacting on ovarian steroid production and ovulation. Chemicals which selectively damage large growing or antral follicles only temporarily interrupt reproductive function because these follicles can be replaced by recruitment from the greater pool of primordial follicles. Thus, these chemicals produce a readily reversible form of infertility that is manifest relatively soon after exposure. Conversely, chemicals which

OVARIAN TOXICITY IN SMALL PRE-ANTRAL FOLLICLES 21

extensively destroy oocytes contained in primordial and primary follicles can cause permanent infertility and premature ovarian failure (early menopause in women) since once a primordial follicle is destroyed, it cannot be replaced. Destruction of oocytes contained in primordial follicles may have a delayed effect on reproduction until such a time that recruitment for the number of growing and antral follicles can no longer be supported (Generoso et al., 1971; Hooser et al., 1994). Table 2.1 Long-term effects of 30d dosing of female B6C3F1 mice with VCHa,b

Notes a Summarized from Hooser et al. (1994). With permission from Annual Review of Pharmacology and Toxicology. b Data expressed as % control mice. c Day after onset of dosing (30d). d Determined by vaginal cytology. e Different from control mice, p < 0.05.

Although direct destruction of small ovarian pre-antral follicles may not immediately alter circulating hormone levels, the long-term result is disruption of negative feedback at the level of the hypothalamus and pituitary in response to loss of ovarian hormone regulation. FSH produced in the anterior pituitary regulates follicular development, and is under a negative feedback regulation of release by ovarian hormones, such as estrogen, progesterone and inhibin (Hedge et al., 1987). A primary effect at the ovarian level might cause a later disruption of this regulatory axis and FSH levels should increase due to loss of negative feedback from the ovarian hormones. In a long-term study, female B6C3F1 mice were treated with the occupational chemical, 4-vinylcyclohexene (VCH) for 30 days (age 28–58 days) and then observed for one year (Hooser et al., 1994; Table 2.1). Although a greater than 90 percent loss of oocytes in small follicles was measured by 30 days, FSH levels were only first observed to be increased above control animals at 240 days. Therefore, ovarian changes preceded the rise in circulating FSH levels. Also at 240 days, vaginal cytology still displayed evidence of ovarian cyclicity in VCH-treated mice. However, by 360 days (from the onset of 30 days of dosing), unlike control animals, complete ovarian failure had resulted in VCH-treated mice, as determined by increased circulating levels of FSH, lack of cyclicity, the complete absence of ovarian follicular or luteal structures and marked ovarian atrophy. From this study, it was concluded that the

22 PATRICIA B.HOYER

ovarian failure that occurs long after cessation of dosing with VCH is an indirect consequence resulting from extensive depletion of small, pre-antral follicles. Menopause Ultimately, if the ovary is depleted of primordial follicles, ovarian failure occurs. Ovarian failure (menopause in women) is associated with the cessation of ovarian cyclicity. The average age of menopause in the United States is 51, and this is a direct consequence of depletion of the follicular reserve. Menopause has been associated with a variety of health problems in women. These include increased incidences of osteoporosis, cardiovascular disease, depression and Alzheimer’s disease (Christensen et al., 1980; Paganini-Hill and Henderson, 1994; Sowers and La Pietra, 1995; Oparil, 1999; Dhar, 2001). Additionally, in laboratory animals, premature ovarian failure is associated in the long term with an increased incidence of ovarian neoplasms (Hoyer and Sipes, 1996). A variety of environmental factors have been highly correlated with early menopause in women (Beverson et al., 1986; Cooper et al., 1995). Therefore, as a woman ages, her overall health is significantly affected by the onset of menopause, and this can be further impacted by environmental factors to which she has been exposed. Because of these health risks, the subject of this chapter is chemicals that can cause premature ovarian failure by destruction of small ovarian follicles. OVOTOXICITY IN SMALL PRE-ANTRAL FOLLICLES Ionizing radiation Rapidly dividing primordial germ cells and oogonia present during fetal development in all species are highly sensitive to destruction (Dobson and Felton, 1983). Destruction of oocytes contained in ovarian primordial follicles can be caused by a variety of environmental chemicals (Hoyer and Sipes, 1996). Additionally, exposure to irradiation is known to produce rapid destruction of oocytes contained in primordial follicles, followed by increased follicular atresia, stromal hypertrophy and loss of ovarian weight (Dobson and Felton, 1983). In animal studies, Mattison and Schulman (1980) noted that pre-natal exposure to ionizing radiation also affects the number of oocytes and reproductive capacity of female offspring. More recently it was shown in pre-pubertal female mice that exposure to irradiation caused degeneration of ovarian primordial and primary follicles by causing apoptosis in granulosa cells, oocytes or both (Lee et al., 2000). These effects are suspected in humans because of reports of amenorrhea and sterility in women undergoing therapeutic irradiation (Chapman, 1983; Damewood and Grochow, 1986).

OVARIAN TOXICITY IN SMALL PRE-ANTRAL FOLLICLES 23

CHEMOTHERAPEUTIC AGENTS Cylcophosphamide (CPA) is an alkylating chemotherapeutic agent. Since their early use as antineoplastic drugs, alkylating agents have been associated with ovarian failure. Animal studies have confirmed this effect by observing follicle destruction. CPA significantly reduced the number of primordial and antral follicles in C57BL/6N and DBA/2N mice and Sprague-Dawley (SD) rats (Shiromizu et al., 1984; Plowchalk and Mattison, 1991). CPA is a precursor for the active form of the drug that must be bioactivated to exert its functional activity (Brock, 1967). The active metabolites include 4-hydroxy-CPA, aldophosphamide and phosphoramide mustard, which is thought to be the active anticancer agent (Boddy and Ratain, 1997; Boddy and Yule, 2000). Phosphoramide mustard is also thought to be responsible for the ovarian follicle destruction caused by CPA (Plowchalk and Mattison, 1991; Anderson et al., 1995). The ovary does not appear to metabolize CPA, rather metabolism is thought to occur in the liver with uptake of the reactive metabolites from the blood (Anderson et al., 1995). Miller and Cole (1970) studied the ovaries in mice treated with CPA in low doses for 1 year, and found reduced numbers of oocytes (especially primordial) and corpora lutea with no effect on other tissues such as the kidney, spleen, thymus or lymph nodes. Estrous cyclicity was destroyed and cysts/tumors were observed in the ovarian germinal epithelium. In a short-term study, susceptibility to CPA was greatest in primordial follicles in exposed C57BL/6N and D2 mice and SD rats (Shiromizu et al., 1984). Accordingly, in a 5–6-week study in Balb/c mice, CPA destroyed primordial follicles but left larger follicles and ovulation, mating and pregnancy rates intact (Meirow et al., 1999). Plowchalk and Mattison (1992) observed a time- and dose-dependent relationship between CPA and ovarian toxicity by looking at changes in ovarian structure and function. In C57BL/6N mice given a single i.p. injection of CPA (75, 200, or 500mg/kg), primordial follicle numbers were significantly reduced to 73, 42 and 38 percent of controls, respectively. The loss of primordial follicles was essentially complete in three days, and the estimated ED50 (concentration that produced 50 percent follicle loss) was 122mg/kg body weight. From these results it appears that premature ovarian failure in women treated with CPA is likely to be via destruction of primordial follicles. Interestingly, in a study in rats, it was shown that a single injection of CPA caused damage in growing and antral follicles, but spared an effect on primordial follicles, because cyclicity was disrupted within a week, but had been restored within 2 weeks of the one-time exposure (Jarrell et al., 1991). Therefore, the impact of CPA on ovarian follicles may be species dependent.

24 PATRICIA B.HOYER

Polycyclic aromatic hydrocarbons Many animal studies have demonstrated ovotoxic effects of polycyclic aromatic hydrocarbons (PAH). Cigarette smoke contains high levels of the PAHs, 9:10dimethyl-1:2-benzanthracene(DMBA),benzo[a]pyrene (BaP) and 3methylcholanthrene (3-MC) (Mattison and Thorgeirsson, 1978; Vahakangas et al., 1985). Krarup (1967, 1969) reported that DMBA depletes oocytes and produces ovarian tumors in mice. Subsequently, these effects have also been reported for 3-MC and BaP. In mouse studies, oocyte destruction was shown to occur in response to these three chemicals (Mattison, 1979). The three PAHs destroyed oocytes in small follicles in SD rats and in D2 and B6 mice, within 14 days following a single i.p. injection (Mattison, 1979). Under these conditions, mice were more susceptible to ovotoxicity than rats. Oocyte destruction in primordial and primary follicles was observed in mice treated with DMBA, BaP and 3-MC (Mattison and Thorgeirsson, 1979). The relative toxicities for primordial follicles were DMBA > 3MC > BaP. Furthermore, a direct relationship between the dose of PAHs and destruction of primordial follicles has been shown in the mouse ovary. Daily oral exposure during pregnancy in mice between 7 and 16 days of gestation with high doses of BaP also caused complete sterility of the female offspring (Mackenzie and Angevine, 1981). Pregnant mice exposed to a lower dose (10 mg BaP/kg) gave birth to offspring with severely compromised fertility. In both studies the litters of exposed mothers were smaller in number and size, compared with controls. PAHs are not directly ovotoxic, but require metabolic activation to reactive metabolites. Ovarian enzymes involved in the biotransformation (i.e. aryl hydrocarbon hydroxylase and epoxide hydrolase) of PAHs have been identified in mice humans (Bengtsson et al., 1988). Therefore, oocyte destruction by PAHs may and rats (Mattison et al., 1983), monkeys (Bengtsson and Mattison, 1989) and involve distribution of the parent compound to the ovary where ovarian enzymes metabolize the compound to reactive intermediates (Mattison et al., 1983). These metabolites are capable of covalent binding to macromolecules such as DNA, RNA and protein (Sims and Grover, 1974). However, the direct intracellular target for ovotoxicity has not been determined. B6 mice were more susceptible to BaP than D2 mice (Mattison and Nightingale, 1980); yet, both strains were equally susceptible to the arene oxide metabolite of BaP (Sims and Grover, 1974). Furthermore, inhibition of PAH metabolism with αnaphthoflavone prevented oocyte destruction observed in mice (Mattison et al., 1983). These observations indicate the PAHs must undergo bioactivation to arene oxides to produce their ovotoxic effects.

OVARIAN TOXICITY IN SMALL PRE-ANTRAL FOLLICLES 25

Occupational chemicals 1, 3-Butadiene 1,3-Butadiene (BD) is an industrial compound used in the synthesis of polymers, resins and plastics (IARC, 1986). BD is also released as a by-product from the production of plastics and is found in automobile exhaust, gasoline vapors and cigarette smoke (NTP, 1984). BD and the related olefins, isoprene and styrene are also released during the manufacture of synthetic rubber and thermoplastic resins and the estimated annual occupational exposure of US employees is 3,700– 1,000,000 people (IARC, 1986). These chemicals have also been reported in cigarette smoke and automobile exhaust. Chronic animal inhalation studies have shown that carcinogenesis caused by BD is higher in mice than rats. At lower doses, female mice exposed daily by inhalation for up to two years exhibited ovarian atrophy, granulosa cell hyperplasia and benign and malignant granulosa cell tumors (Melnick et al., 1990). Thus, ovarian effects of BD appear to occur at lower concentrations than are required to produce effects in other tissues. Because of the possibility of epoxidation of these butadiene-related compounds, they have the potential to be ovotoxic and carcinogenic. Metabolites of BD were used in one study in female B6C3F1 mice dosed daily for 30 days. 1, 3-Butadiene monoepoxide (BMO, 1.43 mmole/kg) depleted small follicles by 98 percent and growing follicles by 87 percent compared with control animals (Doerr et al., 1995). At 0.14 mmole/kg 1,3-butadiene diepoxide (BDE) depleted small follicles by 85 percent and growing follicles by 63 percent. The results of this study support that a diepoxide formed in the metabolism of BD is more potent at inducing follicle loss. Additionally, isoprene was reported to be ovotoxic, whereas, styrene and its monoepoxide did not reduce mouse ovarian follicle numbers (Doerr et al., 1995). The results of these studies demonstrated direct ovarian targeting of the ovary by the diepoxide of BD and provided evidence that this is the ovotoxic form of the chemical. 4-Vinylcyclohexene (VCH) VCH is a dimer of BD which forms spontaneously in the manufacture of butadiene (Keller et al., 1997). VCH is also used as an intermediate in the manufacture of flame retardants and plasticizers, as well as a solvent in the manufacture of VCH diepoxide (VCD). Exposure to VCH is likely to be in the occupational and/ or industrial setting. Workers could potentially be exposed to VCH during the production of BD-based rubber, rubber vulcanization in the manufacture of shoe soles, tires and other rubber products, as well as in the manufacture of flame retardants and insecticides. However, production policies in these venues have mandated that these chemical processes are performed in closed vessels. Thus, human exposure to VCH is limited, with the exception of accidental spills and leaks (IARC, 1994). Nonetheless, air concentrations of VCH

26 PATRICIA B.HOYER

have been measured in the workplace. For instance, levels of 0.03-0.21 mg/m3 were measured in an Italian shoe plant, levels of 0-0.003 mg/m3 were measured in an Italian tire factory, and levels of 0.24-0.43 mg/m3 were measured in an American tire curing room (Rappaport and Fraser, 1977; IARC, 1994). After these short-term air concentration studies, the American Conference of Industrial Hygienists in 1992 established 0.4 mg/m3 as a threshold value for VCH exposure in an 8-hour period. Currently there are no epidemiological studies to relate human exposures to adverse effects. However, in animal studies, ovarian damage caused by VCH and its related epoxide metabolites has been demonstrated by a variety of exposure routes, including dermal, oral, inhalation, and intraperitoneal injection (NTP, 1989; Smith et al., 1990; Grizzle et al., 1994; Bevan et al., 1996). It is, therefore, important to consider the potential risks for human exposure. The occupational chemical, VCH and its diepoxide metabolite, VCD, have been well characterized for their ability to cause selective loss of primordial and primary ovarian follicles in mice and rats (Smith et al., 1990; Springer et al., 1996a; Kao et al., 1999). This selective follicle loss has been induced in neonatal rats (post-natal day 4), rats that were immature at the onset of dosing (d28), and in adult rats (d58) (Flaws et al., 1994; Devine et al., 2002). But, the studies have largely characterized this follicle loss in d28 rats. The manner in which VCD exerts such a selective, targeted response is the subject of ongoing research. However, it is felt that this, in part, is because it affects meiotically arrested cells in small follicles that are directly exposed to circulating distribution of the chemical. Earlier structure-activity studies determined that the diepoxide metabolite of VCH, VCD, is the active form for inducing follicle loss in both mice and rats (Smith et al., 1990; Doerr et al., 1995). 2-Bromopropane One of the most recently identified ovarian toxicants is 2-bromopropane (2BP) (NTP, 2002). Concern was raised over the toxicity of this chemical in 1996 due to adverse reproductive problems in workers in a Korean factory (Kim et al., 1996). Subsequent clinical investigations in Korea as well as experimental research in animals demonstrated adverse effects on hematopoiesis and the reproductive system in both males and females, in humans and rats (Park et al., 1997; Takeuchi et al., 1997). 2BP has been used as a cleaning agent, an intermediate for various chemical syntheses, and a replacement for chlorofluorocarbons. Because of the concerns raised in the Korean factory, 2BP use has been limited to some extent in the US. However, it is also a contaminant of 1-bromopropane which is widely used as a solvent for the manufacture of a variety of chemicals in the industrial setting (NTP, 2002). Human exposures, therefore, would predominantly occur in industrial settings and could be through either dermal contact or inhalation. Data on environmental levels for humans or animals are not available (NTP, 2002). However, the similarity of effects

OVARIAN TOXICITY IN SMALL PRE-ANTRAL FOLLICLES 27

observed in humans and in experimental animals provides greater credibility to 2BP-induced reproductive effects. There is evidence that male and female germ cells are the ultimate targets of 2BP. Reduced numbers of early stages of spermatogonia (stage 1) were determined to be the initial morphological alteration in response to a single subcutaneous injection of 1355 mg/kg 2BP administered to rats (Omura et al., 1999). Similarly, Yu et al. (1999) demonstrated that primordial follicles of rats were the first ovarian follicle stage to be affected by a single 8-hour inhalation exposure to 3000 ppm 2BP. Additionally, loss of ovarian follicles of all types was seen in female offspring of SD rats when mothers were exposed during gestation and lactation (Kang, 2002). To date, neither the mechanism nor the cellular or molecular target by which 2BP induces germ cell loss has been elucidated. Other ovotoxic agents The alkylating agents 1,4-di(methanesulfonoxy)-butane (Myleran), trimethylenemelamin (TEM) and isopropyl methanesulfonate (IMS) have been shown to destroy oocytes in small follicles in SECXC57BL/F1 mice following a single i.p. injection (Generoso et al., 1971). This destruction was observed within three days of dosing with TEM and IMS, and within 14 days with Myleran. Daily oral administration of nitrofurazone over two years caused ovarian lesions including development of benign mixed tumors and granulosa cell tumors in mice (Kari et al., 1989). The results of an in vitro mutagenicity study in E.coli using a number of industrial and laboratory chemicals demonstrated a high correlation between alkylating activity and increased mutagenicity (Hemminki et al., 1980). In addition to the chemicals discussed so far, Dobson and Felton (1983) reported a variety of other compounds that were capable of producing significant primordial follicle loss in mice. These chemicals included methyl and ethyl methanesulfonate, busulfan and urethane. Additionally, of a number of fungal toxins and antibiotics tested, procarbazine HCl and 4-nitroquinoline-1-oxide were ovotoxic. Finally, dibromochloropropane, urethane, N-ethyl-N-nitrosourea and bleomycin demonstrated primordial follicle killing, with bleomycin being the most potent. In general, all of these ovotoxic chemicals are also known to possess mutageniccarcinogenic effects. Thus, these studies have further provided a correlation between ovotoxicity and subsequent development of tumorigenesis. How these two events are linked is not clearly understood at this time. The polychlorinated biphenyl compound, 3,3,4,4-tetrachlorobephenyl (TCB), has been shown to be teratogenic in the mouse and embryolethal in the rat, as well as having transplacental ovarian toxicity in the mouse (Ronnback, 1991). Follicles in all stages of development were reduced 40-50 percent in female offspring at 28d of age when mice were exposed in utero on day 13 of gestation. Interestingly, during a 5-month period of testing, this extent of follicular damage did not adversely affect reproductive function in these offspring. Finally, the chlorinated organic chemical hexachlorobenzene has been shown to cause

28 PATRICIA B.HOYER

destruction of primordial follicles in cynomolgus monkeys at doses that did not produce evidence of systemic or hepatic effects (Jarrell et al., 1993). MECHANISMS OF OVOTOXICITY How ovotoxic effects of environmental chemicals are produced is generally not well understood, but might be due to one of several possible mechanisms. Oocyte destruction can result from a toxic chemical directly impairing oocyte viability. Conversely, because oocytes at all stages of follicular development are surrounded by granulosa cells, these mechanisms might also be indirect, involving alterations within the granulosa cell, which compromise its ability to maintain viability in the oocyte (Buccione et al., 1990). Lastly, environmental chemicals might cause follicle loss by accelerating the overall rate of atresia, the normal mechanism by which the majority of follicles degenerate during development. In determining mechanisms involved in ovotoxicity, it is first important to determine whether selective or distinct follicular populations are targeted. In studies investigating the mechanism(s) by which VCD is ovotoxic, rats were dosed daily for 30 days. Morphological evaluation of ovaries from control and treated rats revealed that significant loss of small follicles had occurred in follicles in the primordial, primary and secondary stages (Figure 2.2). However, at a shorter time of dosing, reduced numbers of only primordial and primary follicles were seen on day 15, yet the number of secondary follicles was unchanged. Thus, these findings supported that primordial and primary follicles are directly targeted by VCD and that the loss of secondary follicles seen on day 30 was the result of fewer primordial and primary follicles from which to recruit. Two long-term studies were conducted in mice and rats (age d28) which were dosed daily for 30d with VCH (mice) or VCD (rats) and evaluated at different time points for up to 360d after the onset of dosing for effects on reproductive function (Hooser et al., 1994; Mayer et al., 2002). These studies established that premature ovarian failure could be induced in mice and rats as a result of the selective depletion of the primordial follicle pool. Taken together, the reports related to mechanisms of cell death during ovotoxicity suggest that dose and duration of exposure impact the outcome. This provides further rationale for designing animal studies using low-dose repeated exposure to more closely mimic the nature of human exposures. Cell death The ultimate event associated with follicular destruction is cell death. This is the natural fate of the majority of ovarian follicles because only a selected few follicles that develop will ever be ovulated (Hirshfield, 1991). This process, called atresia, can occur in follicles in all stages of development and can be morphologically distinguished from healthy follicles. Follicular atresia in rats

OVARIAN TOXICITY IN SMALL PRE-ANTRAL FOLLICLES 29

Figure 2.2 Reductions of small follicle numbers by repeated dosing with 4vinylcyclohexene diepoxide (VCD). Female Fischer 344 rats (age d28) were dosed daily with vehicle control (open bars) or VCD (80 mg/kg, i.p., closed bars) for 15 days or 30 days. Ovaries were collected and processed for histological counting of primordial, primary and secondary follicles. *p350 microns in diameter) to respond to eCG, a hormone with FSH and LH-like activities. Clearly at the time of hCG injection, the pre-ovulatory follicles were not of the same quality as controls (lower LH and FSH binding). Previous studies indicated that TCDD may interfere with follicular rupture and that may very well be the case (Petroff et al, 2001). However, the subnormal pre-ovulatory follicles at the time of hCG injection must be also considered as a possible explanation for the reduced number of ova shed. Administration of TCDD after the pre-ovulatory follicles had matured may provide insights as to whether or not this environmental toxicant interferes with follicular rupture as well. A large volume of data indicates a major role of estradiol in enhancing follicular development in the rat (Richards, 1980; Farookhi and Desjardins, 1986; Richards et al, 1987; Tonetta and diZerega, 1989; Wang and Greenwald, 1993; Robker and Richards, 1998; Drummond and Findlay, 1999). Several studies have also demonstrated antiestrogenic properties of TCDD in nonovarian tissues (Astroff and Safe, 1988, 1990; Romkes and Safe, 1988; Kharat and Saatcioglus, 1996; Tian et al, 1998a, b). Whether or not the growing stage of follicles (>350 microns) also depends on estradiol for further development is unknown. However, since such follicles’ response to eCG was altered, it is conceivable that the antiestrogenic properties of TCDD were responsible for this effect, which would suggest a role for estradiol in this critical growing stage of the follicle.

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Direct effects of TCDD on gonadotropin-stimulated ovarian steroidogenesis in the rat are unclear because different studies reported a variety of effects. In vitro, TCDD decreased FSH-stimulated granulosal aromatase activity and reduced CYP450scc and aromtase mRNA in rat granulosa cells (Dasmahapatra et al., 2000). However, a report using enriched populations of granulosa and thecainterstitial cells as well as whole ovarian dispersates (mixtures of thecainterstitial cells, granulosa cells and other ovarian cell types) from immature rats revealed no in vitro effect of TCDD on basal and FSH- and LH-stimulated steroidogenesis (Son et al, 1999). Two studies reported in vitro effects of TCDD on FSH (Hirakawa et al, 2000a) and LH (Hirakawa et al, 2000b) receptors in rat granulosa cells. In vitro, FSH increased FSH and LH receptor mRNA and both were reduced dose-dependently by TCDD. 8-Bromo-cAMP also increased FSH receptor mRNA in granulosa cells and this was inhibited by TCDD indicating a post-cAMP site of TCDD action. The rate of FSH receptor mRNA gene transcription was also reduced by TCDD, but the stability of the FSH receptor mRNA was not affected. However, the stability of LH receptor mRNA was decreased. In vitro effects of TCDD have been consistently observed on human granulosallutein cells collected from patients undergoing in vitro fertilization (Enan et al., 1996a,b). TCDD decreased protein kinase A activity, and progesterone and estradiol secretion. Another study also confirmed that TCDD reduced estradiol secretion in the human granulosa luteal cell model; however, with additional culture of the cells and addition of an androgen precursor, the reduced estradiol returned to control values (Heimler et al., 1998). A summary of the in vitro effects of TCDD on granulosa cells is given in Table 4.2. Another potential site of TCDD action is the hypothalamic-pituitary axis since the LH and FSH surges are blocked in adults (Li et al., 1995a,b) and in the immature gonadotropin-primed model (Petroff et al, 2001). However, the mechanisms by which TCDD blocks the LH/FSH surges are still unclear. Circulating levels of estradiol in the intact gonadotropin-primed immature female rat are similar to control levels prior to the LH surge (Gao et al., 2000a). It is well known that estradiol during the pre-ovulatory period acts as a critical positive feedback hormone stimulating the secretion of LH and FSH to “surge” levels that ultimately induce rupture of the pre-ovulatory follicle (s). If TCDD is indeed acting as an antiestrogen, then it likely reduces the effectiveness of estradiol during this critical positive feedback period. Indeed this appears to be the case since an exogenous long-acting estrogen, estradiol cypionate, overcame the inhibitory effects of TCDD in blocking of the LH and FSH surges (Gao et al., 2001). In fact, a circulating estradiol level 8–10 times higher than normal had to be maintained in the preovulatory period in order to overcome the inhibitory effects of TCDD. There was no demonstrable inhibitory effect of TCDD on the pituitary since exogenous gonadotropin-releasing hormone (GnRH) induced significant secretion of LH and FSH in TCDD-treated rats during the pre-ovulatory period (Gao et al., 2000b).Although exogenous GnRH significantly increased LH

70 PAUL F.TERRANOVA AND KARL K.ROZMAN

Table 4.2 In vitro effects of TCDD on granulosa cells

and FSH secretion beyond control values, ovulation was only partially restored indicating that TCDD also had a direct inhibitory effect on the ovary. Indole-3-carbinol (I3C), a component of cruciferous vegetables, is a weak agonist of the AhR (Bjeldanes et al., 1991) although other studies have reported antagonistic activity towards the AhR (Chen et al., 1996). A recent report using an immature gonadotropin-primed rat model for inducing synchronized follicular development and ovulation was used to test I3C’s ability to block ovulation (Gao et al., 2002). Immature rats were given daily doses of I3C ranging from 0 to 1.5 g/kg/day. The initial dose of I3C was given one day prior to 5IU equine chorionic gonadotropin. I3C blocked ovulation dose-dependently at the expected time and this coincided with reduced concentrations of LH and FSH in sera at the time of the expected proestrus surges. Administration of hCG that mimicked a normal pre-ovulatory surge of LH partially restored ovulation indicating that I3C may have had direct effects on the ovaries in blocking ovulation in addition to its ability to block the surges of LH and FSH. I3C given by gavage is subjected to acidic conditions in the stomach and is converted to various oligomers that are thought to mediate the biological effects of I3C (DeKruif et al, 1991; Wortelboer et al, 1992). One of the oligomers, 3,3'diindolylmethane (DIM) has structural similarities to TCDD (Cashman et al, 1999), is capable of binding to the Ah receptor, and inducing cytochrome P450 (Vang et al., 1990; Bjeldanes et al, 1991; Jellnick et al, 1993). However, DIM had no effect on ovulation in the immature rat model (Gao et al., 2002). Thus, the similarity in actions between I3C and TCDD may be the result of their capability to bind to the Ah receptor, but a lack of effect by an AhR agonist such as DIM raises questions about the generalizability of the AhR hypothesis. I3C and its oligomers exhibit antiestrogenic activity like TCDD in a variety of assays (Kociba et al, 1978; Wattenberg and Loub, 1978; Stoewsand et al, 1988; Bradlow et al, 1991; Gierthy et al, 1993; Grubbs et al, 1995), although some of its reaction products can bind to the estrogen receptor exhibiting weak estrogenic activity (Liu et al., 1994; Riby et al, 2000). Unlike some of the I3C metabolites, TCDD does not bind to the estrogen receptor but it may interfere with signal transduction of estrogens (Safe, 1995). Nevertheless, an antiestrogenic action of I3C may account for its ability to block the LH surge on expected proestrus. It is

OVARIAN TOXICITY CAUSED BY ENDOCRINE DISRUPTORS 71

Figure 4.5 Direct and indirect effects of Ah receptor agonists hypothalamicpituitary axis and ovary leading to blockage of ovulation.

on

the

clear that the inhibitory action of TCDD is due to its antiestrogenicity since a long acting estrogen, estradiol cypionate, effectively overcomes the ability of TCDD to block the LH surge (Gao et al, 2001) but this is not known for I3C. Direct and indirect effects of Ah receptor agonists on the hypothalamic-pituitary axis and ovary leading to blockage of ovulation are shown in Figure 4.5. Tamoxifen (TAM), a well-known antiestrogen, with high affinity to bind to the estrogen receptor blocked ovulation very similar to that of TCDD and I3C in the gonadotropin-primed immature rat model (Gao et al., 2002). However, unlike with I3C and TCDD, exogenous hCG given on expected proestrus completely overcame the inhibitory effects of TAM on ovulation (Gao et al., 2002) or TCDD (Petroff et al, 2001). Thus, it appears that the effects of TAM occur solely at the level of the hypothalamic-hypophyseal axis whereas I3C and TCDD, each, blocked ovulation at the level of the ovary as well as at the hypothalamichypophyseal axis. Atrazine, a neuroendocrine-ovarian axis disruptor Atrazine, a chloro-5-triazine herbicide, has been studied extensively as an ED because of its wide spread use in the United States. It has been reported that atrazine increases the incidence of spontaneous mammary tumors in female Sprague-Dawley (SD) rats (Stevens et al., 1994). The original reports of endocrine disruption by atrazine appeared in 1994, and attempted to explain the differential effects in inducing mammary tumors in SD rats but not in Fischer 344 rats (Eldridge et al., 1994; Stevens et al., 1994; Wetzel et al, 1994). In short-

72 PAUL F.TERRANOVA AND KARL K.ROZMAN

term studies, atrazine and simazine, each by itself, were given daily for 2 weeks at 100 or 300mg/kg BW to SD and Fischer 344 female rats (Eldridge et al., 1994). Treatments reduced ovarian weights and serum estradiol levels. SD rats exhibited a lengthening of the estrous cycle with an increase in the number of days in vaginal estrous and a decrease in the number of days in diestrus. Interestingly, Fischer 344 rats also exhibited an increase in the length of the estrous cycle, but there was an increase in the number of day s in diestrus with a reduction in vaginal estrus. It was concluded that prolonged blockage of ovulation associated with continuous secretion of estradiol in the SD rat led to increased secretion of prolactin, which in turn led to mammary gland tumors. SD rats have a high rate of spontaneous mammary tumors. Fischer 344 rats exhibiting prolonged diestrus mimicked pseudopregnancy resulting in activated corpora lutea with prolonged progesterone secretion; however, these rats are not prone to develop mammary tumors (Cutts and Noble, 1964). In fact, differences in reproductive aging of these two strains of rats were noted. SD rats enter constant estrus as they age whereas Fischer 344 rats exhibit repeated pseudopregnancies. Chronic administration of atrazine in the diet also resulted in a lengthening of the estrous cycle and the number of days in estrus in SD rats (Wetzel et al, 1994). In Fischer rats, however, although estrous cycles were slightly prolonged by atrazine there was no effect on serum concentrations of progesterone and estradiol (Wetzel et al., 1994). Exposure of the SD rat to maximally tolerated doses (or higher) of atrazine and simazine (both chloro-S-triazines) for 2 years altered the neuroendocrine axis, prolonged cycles and induced pathology in the mammary gland. The changes in hormones and the prolonged estrous cycles were similar to natural aging in the SD rat except they were more pronounced in the simazine-treated groups. Thus those studies revealed that chloro-S-triazines had detrimental effects on the female reproductive system in various strains of rats, and disrupted neuroendocrine control of ovarian function. In 1996, Cooper et al. found that high daily doses of atrazine (150mg/kg BW by gavage) over 21 consecutive days altered estrous cycles in Long Evans (LE)hooded and SD rats in favor of sustained diestrus, i.e., pseudopregnancy. A higher dose (300 mg/kg) of atrazine led to ovarian regression and an anestrous smear. This study confirmed the endocrine-disrupting effects of atrazine on ovarian function and extended the observations to an additional strain of rat. Later studies targeted the central nervous system, specifically the hypothalamicpituitary axis, as a potential site for endocrine disruption. Thus, it was proposed that atrazine increased dopamine and reduced norepinephrine concentrations in the hypothalamus (Cooper et al., 1998), because atrazine was shown to have direct effects on catecholamine neurons in vitro leading to alteration in catecholamine synthesis (Das et al, 2000, 2001). These alterations are likely to reduce the ability of estrogen to induce an LH surge. This was investigated using ovariectomized estrogen-treated SD and LE-hooded rats with spontaneous surges of LH and prolactin (Cooper et al., 2000). In this study, atrazine (50–300 mg/kg/

OVARIAN TOXICITY CAUSED BY ENDOCRINE DISRUPTORS 73

day) was administered by gavage for 1, 3 or 21 days. A single dose of atrazine (300 mg/kg) suppressed the LH and prolactin surges in ovariectomized LE rats but not in SD rats. Three hundred mg/kg given on proestrus did not block ovulation but it did induce pseudopregnancy in the majority of LE rats. SD and LE rats responded to atrazine in a dose-dependent manner after 21 days of treatment with reduced LH and prolactin surges. The results of these studies indicated clearly that neuro-endocrine control of LH and prolactin was altered and continued treatment led to altered cycles in both strains of rats. These data are consistent with the hypothesis that atrazine reduces GnRH pulse frequency similar to that observed in aging rats and that this is caused by continuous exposure to estrogen (Cooper et al, 2000). However, atrazine does not bind to the estrogen receptor (Tennant et al, 1994; Connor et al, 1996), requiring other mechanisms to account for this action. Atrazine appears to alter regulation of the CNS component of the hypothalamic-pituitary axis and not the pituitary, since atrazine did not affect prolactin secretion when a pituitary graft was placed under the kidney capsule (Cooper et al, 2000). In addition, exogenous GnRH restored LH secretion in atrazine-treated rats to a level comparable to normal surge levels indicating that atrazine likely reduced GnRH secretion without altering pituitary gonadotrophs (Cooper et al., 2000). Lastly, secretion of LH and prolactin was unaffected by perifusion of pituitaries with atrazine in vitro (Cooper et al., 2000). Pre-pubertal female rats given atrazine on days 22–41 post-natally exhibited a delay in vaginal opening associated with the first ovulation (Laws et al., 2000). However, when treatment was stopped, vaginal opening occurred within 3–4 days but vaginal estrous cyclicity was irregular during the following 15 days. Thereafter, estrous cycles resumed as normal. In fact, high doses of atrazine prevented vaginal opening as long as it was administered. When the high-dose treatment was withdrawn, normal vaginal cycles eventually resumed. These data (Laws et al., 2000) are consistent with the inhibition of LH and prolactin secretion in adult rats in which cycles were prolonged (Cooper et al., 2000). In summary, atrazine appears to increase dopamine and reduce norepinephrine concentrations in the hypothalamus leading to a decrease in GnRH release and as a result to reduced LH secretion (Figure 4.6). The lack of LH secretion provides a satisfactory explanation for the disrupted cycles, delayed vaginal opening, and a blockage of ovulation (Das et al, 2000, 2001), although secretion of FSH with low levels of LH allows follicular development and estradiol secretion to continue. 2-Bromopropane (2BP), an ovarian axis ED It is clear that 2BP is an ED based on its ability to disrupt the menstrual cycles in humans, rat vaginal cycles and gonadotropin-induced ovulation in mice; all are endocrine-controlled events. Thus, the effects of 2BP are reviewed here to elucidate the mechanisms of endocrine disruption. 2BP is very volatile, permeable to the skin, and is used as a substitute for Freon 113 (trichlorotrifluoroethane),

74 PAUL F.TERRANOVA AND KARL K.ROZMAN

Figure 4.6 Hypothetical mechanism by which atrazine blocks ovulation in rats.

which contributes to the reduction of the stratospheric ozone levels. The use of Freon 113 has been curtailed because of this detrimental effect on the environment. 2BP has also been used as a substitute for trichloroethylene for the extraction of asphalt mixtures. Detrimental effects of 2BP on humans were first reported in 1996 (Kim et al., 1996a,b). The incidents occurred in 1995 in South Korea. Industrial workers who were exposed to 2BP at an electronic factory in Korea exhibited signs of reproductive and hematopoietic toxicity (Kim et al., 1996a,b; Maeng and Yu, 1997; Park et al., 1997). Exposure occurred in the cleansing portion of the tactile switch assembly area where levels of 2BP in the air were ~12 ppm as estimated by simulation studies. However, short-term exposure to much higher levels likely occurred inside the hoods of the cleaning baths with concentrations reaching ~4,100 ppm in the air during simulation studies. Often the heads of the workers were put inside the hood of the cleaning baths and they also dipped bare hands into the cleaning solution containing 2BP. No type of protective equipment (masks and gloves) was used by the exposed workers. Of twenty-five women exposed to 2BP, sixteen exhibited signs of ovarian dysfunction as evidenced by amenorrhea and serum concentrations of FSH greater than 40 mIU/ml (range: 28–137 mIU; normal serum FSH is 10mIU/ml for the follicular phase and premature ovarian failure is considered at >20 mIU/ ml). Only sera from three women were analyzed for estradiol and the values (