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Regulation of steroid hormone production by the human granulosa-luteal cells Khorasheh, Shideh 1993

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REGULATION OF STEROID HORMONE PRODUCTION BY THE HUMAN GRANULOSA-LUTEAL CELLS  by  Shideh Khorasheh B.Sc., The University of Manitoba, 1986 B.Ed., The University of Alberta, 1989  A THESIS SUBMITT’ED IN PARTIAL FULFILLMENT OF  THE REQUIREMENTS FOR THE DEGREE OF MASTER OF SCIENCE in  THE FACULTY OF GRADUATE STUDIES Department of Physiology University of British Columbia  We accept this thesis as conforming to the required standard  THE UNIVERSITY OF BRITISH COLUMBIA  December 1993  © Shideh Khorasheh,  1993  In presenting this thesis in partial fulfilment of the requirements for degree at the University of British Columbia, I agree that the Library freely available for reference and study. I further agree that permission copying of this thesis for scholarly purposes may be granted by the department  or  by  his  or  her  representatives.  It  an advanced shall make it for extensive head of my  is understood that copying or publication of this thesis for financial gain shall not be allowed without my written permission.  (Signature)  Department of  3 Phy ) io1o  The University of British Columbia Vancouver, Canada  Date  DE-6 (2/88)  Pecer?ber &,  /  i9q3  ABSTRACT  The present study investigated the hypothesis that in addition to the gonadotropins, there is an autocrine mechanism controlling human ovarian steroid hormone production (estrogen and progesterone). The specific intra-ovarian substances investigated were inhibin, activin, follistatin, and angiotensin II and III (Ang II and Ang III). These intraovarian substances may act either alone or in concert to modulate the actions of gonadotropins, to influence the steroid hormone production. In vitro culture of human granulosa cells, which are one of the steroidogenic type of ovarian cells, were used to demonstrate the actions of these substances on steroidogenesis. Granulosa cells were harvested from the follicular fluid contents of women undergoing oocyte retrieval in the In Vitro Fertilization Program. The in vitro culture system was validated by confirming human chorionic gonadotropin (hCG)-mediated progesterone and estradiol production. Inhibin-related peptides have been isolated in follicular fluid. Inhibin-A did not affect basal or hCG-stimulated progesterone and estradiol production in human granulosa cells. Activin-A consistently stimulated basal progesterone production but  either inhibited or did not affect hCG-stimulated progesterone production. Activin-A also stimulated basal estradiol production, suggesting its role in maintaining granulosa cells in a healthy (estrogenic) state of differentiation. Foffistatin is an activin-binding protein. Foffistatin blocked the activin-induced increase in basal estradiol and progesterone production in the human granulosa cells, suggesting a role  11  as an activin-binding protein. However, follistatin may also have direct actions on granulosa cells which are independent of its activin-binding activity. Follistatin stimulated basal progesterone and estradiol production; however, this stimulatory effect of follistatin on basal levels of steroid hormones was not present in all of the experiments. Follistatin did not affect hCG-stimulated estradiol and progesterone production by human granulosa cells. The biochemical pathways that lead to the formation of the Ang 111111 exists in the human ovary. The effects of Ang II and Ang III with regard to steroid production in the ovary were investigated. Ang III but not Ang II inhibited hCG-stimulated progesterone production. On the other hand, Ang II stimulated basal estradiol levels while Ang III had no effect on basal or hCG-induced estradiol production. Taken together, these results suggest that Ang II and Ang III may promote follicular maturation in the human ovary by enhancing basal estradiol production from granulosa cells and preventing premature luteinization. In summary, the results from this study showed that local ovarian substances play a role in modulating the steroid hormone production by the granulosa cells. It is anticipated that future studies may allow for a better understanding of the coordinated action of many local substances involved in regulating steroid hormone production in the human ovary.  111  TABLE OF CONTENTS ABSTRACT LIST OF FIGURES  vii  ACKNOWLEDGEMENTS  x  CHAPTER ONE: INTRODUCTION ANI) LITERATURE REVIEW  1  I. Ovary A. Introduction B. Histology C. The life cycle of the ovarian follicle  1 1 2 4  II. Steroid hormone production by the ovary A. Synthesis of sex steroid hormones B. Mechanism of action of steroid hormones  7 7 11  III. Ovarian regulation A. Neuroendocrine regulation of the ovary 1. GnRH 2. Prolactin regulating factors B. Gonadotropic hormone regulation of the ovary 1. Chemistry of gonadotropic hormones 2. Role of gonadotropic hormones i) FSH actions ii) LH actions iii) Prolactin actions C. Steroid hormone regulation of the ovary 1. Role of estrogens 2. Role of progesterone 3. Role of androgens D. Role of local nonsteroidal regulators 1. Role of inhibin related peptides i) luhibin ii) Activin iii) Follistatin 2. Role of the ovarian renin-angiotensin system E. Role of other intragonadal factors 1. Intraovarian growth factors i) Insulin-like growth factors ii) Epidermal growth factor (EGF)/Transforming growth factor-cc (TGF-cz)  12 13 13 14 15 15 16 16 19 21 22 22 24 25 26 27 27 31 35 39 44 44 44  iv  46  iii) Transforming growth factor-B (TGF-13)  47  IV. Signal transduction system in the ovary A. Introduction 1. Cyclic AMP-dependent protein kinase A 2. Calcium and protein kinase C pathway 3. Tyrosine Kinase  48 48 48 51 52  CHAPTER TWO: OBJECTiVES  54  I. Background and rationale  54  II. Hypothesis and Objectives  56  CHAPTER THREE: MATERIALS & METHODS  58  I. Human granulosa cell culture system A. Human granulosa cell preparation B. Experimental designs C. Hormone analysis 1. Radioimmunoassay for steroids (progesterone and estradiol) 2. Statistical analysis  58 58 60 64 64 67  .  .  II. Protein measurement of granulosa cells in culture A. Lowry Protein Assay B. Protein assay experiments  68 68 70  CHAPTER FOUR: RESULTS  71  I. Static culture experiments A. Effect of activin-A on basal and hCG- stimulated estradiol production B. Effect of activin-A on basal and hCG-stimulated progesterone production C. Effect of inhibin-A on basal and hCG-stimulated progesterone and estradiol production D. Effect of follistatin on basal and hCG-stimulated estradiol production E. Effect of follistatin on basal and hCG-stimulated progesterone production F. Interaction between activin and follistatin on basal and hCG stimulated estradiol production G. Interaction between activin and follistatin on basal and hCG stimulated progesterone production  71  V  71 71 79 79 82 82 89  H. Effects of Ang II and Ang III on basal and hCG-stimulated estradiol production I. Effects of Ang II and Ang III on basal and hCG-stimulated progesterone production  92 92  II. Protein assay experiments A. Effect of inhibin, activin, or follistatin on protein content of granulosa cells  97  CHAPTER FiVE: DISCUSSION  99  I. Regulation of steroid production by inhibin-A and activin-A  99  97  II. Regulation of steroid production by follistatin-288  103  III. Regulation of steroid production by angiotensins  107  IV. Summary & conclusions A. Physiological roles of activin and follistatin B. Physiological roles of angiotensins  110 110 111  REFERENCES  115  vi  LIST OF FIGURES Fig. 1  The biosynthetic pathway of steroid hormone production in the ovary.  Fig.2  Diagram of the “two-cell, two-gonadotropin theory” of follicle steroidogenesis. .18 . . .  Fig.3  The biosynthetic pathways of the angiotensins.  Fig.4  Diagram of the main intracellular signalling pathways involved in the mediation of hormone action in the ovary. .49  . . .  .40  ...  Fig.5  Diagrammatic representation of protocol for human granulosa cells processing. .59 . ..  Fig.6  Effect of activin on basal and hCG-stimulated estradiol production by the human granulosa cells.  Fig.7  Dose-dependent effect of activin on basal estradliol production by the human granulosa cells.  Fig.8  Time-course effect of activin on basal estracliol production by the human granulosa cells. .74 . . .  Fig.9  Effect of activin on basal and hCG-stimulated progesterone production by the human granulosa cells.  Fig. 10  Dose-dependent effect of activin on production by the human granulosa cells.  basal  progesterone . ..  Fig.11  Time-course effect of activin on basal progesterone production by the human granulosa cells.  Fig. 12  Dose-dependent effect progesterone production.  Fig. 13  of  activin  on  hCG-stimulated . . .  .78  Effect of inhibin on basal and hCG-stimulated estradiol production by the human granulosa cells. .80 . . .  Fig. 14  .76  Effect of inhibin on basal and hCG-stimulated progesterone production by the human granulosa cells.  vii  Fig. 15  Effect of follistatin on basal and hCG-stimulated estradiol production by the human granulosa cells. .83 . . .  Fig. 16 Fig.17  Dose-dependent effect of follistatin on production by the human granulosa cells.  basal  estradiol . . .  .84  Time-course effect of follistatin on basal estradiol production by the human granulosa cells. .85 . . .  Fig. 18  Effect of follistatin on basal and hCG-stimulated progesterone production by the human granulosa cells. .86 ...  Fig. 19  Dose-dependent effect of follistatin on basal progesterone production by the human granulosa cells. .87 . . .  Fig.20 Fig.21  Time-course effect of follistatin on production by the human granulosa cells.  basal  progesterone . . .  Interaction between activin and follistatin on basal and hCG stimulated estradliol production by the human granulosa cells  .88 90  Fig.22  Interaction between activin and follistatin on basal and hCG stimulated progesterone production by the human granulosa cells. ....91  Fig.23  Effect of angiotensin II & III on basal and hCG-stimulated estradiol production by the human granulosa cells. .93 . . .  Fig.24  Dose-dependent effect of angiotensin II on basal estradiol production by the human granulosa cells. .94 . . .  Fig.25 Fig.26  Time-course effect of angiotensin II on production by the human granulosa cells.  basal  estradiol . . .  .95  Effect of angiotensin II & III on basal and hCG-stimulated progesterone production by the human granulosa cells. .96 . ..  Fig.27  Effect of imbibin, activin, or follistatin on protein content of human granulosa cells. .98 . . .  viii  Fig.28  Fig.29  Diagram summarizing the actions of activin and follistatin on human granulosa cells. Diagram sinnmarizing the actions of angiotensin II & III on human granulosa cells.  ix  ....  ....  112  114  ACKNOWLEDGEMENTS  I would like to express my appreciation to my supervisor, Dr. Peter C.K. Leung for providing advice and support throughout my study. I would also like to thank Wei Li for her advice, encouragement, and specially her friendship throughout my study. I would like to thank my supervisory committee, Dr. Pederson, Dr. Pearson, Dr. Kwok, and Dr. Rajamahendran for serving on my committee and their helpful comments. I would also like to thank all members of Dr. Leung laboratory for their support throughout my stay in the laboratory. Finally, I take this opportunity to express my deepest gratitude to my parents, Mahin and Jamshid, for their love and continuing support throughout my academic endeavour.  x  CHAPTER ONE: I1’TRODUCTION AND LITERATURE REVIEW I. Ovary A. Introduction The ovaries are paired organs situated in the abdominal cavity. The function of the ovary is to produce oocytes and secrete ovarian hormones. The endocrine function of the ovary in the female is a cyclic process. This cyclicity is referred to as the estrous cycle in subprimate species and the menstrual cycle in primates. The ovarian cycle is designed to ensure that mature female oocytes are produced at ovulation so that they are available for fertilization by sperm (Adashi, 1991). The ovarian cyclicity is controlled by a feedback system involving the hypothalamus, anterior pituitary and the ovaries. The gonadotropin-releasing hormone (GnRH) from the hypothalamus causes the release of follicle stimulating hormone (FSH) and luteinizing hormone (LH) from the anterior pituitary. FSH and LH act on the ovary to: 1. induce follicle maturation, 2. stimulate steroidogenesis (estradiol and progesterone production), and 3. induce ovulation of a fertilizable ovum (Adashi, 1991). Although pituitary gonadotropins (LH and FSH) are the major regulators of follicular development, not all follicles in a given ovary respond to pituitary gonadotropins during a given cycle. Only a limited number of selected follicles ovulate during the life span of the female while most of the follicles undergo atresia. The variable fate of ovarian follicles subjected to comparable gonadotropic stimulation suggests the existence of additional intraovarian modulatory mechanisms.  1  Intraovarian control of ovarian processes is likely exerted by means of local steroidal modulation. Intraovarian peptides may have a potential for local modulation of follicular development by modulating steroid production. Inhibin (Hutchinson et al., 1987), activin and follistatin (Findlay et al., 1990; Depaolo et al., 1991), and/or angiotensins (Bumpus et al., 1988; Stirling et aL, 1990) are among potential modulators of ovarian steroid production.  B. Histology The mammalian ovaries are paired organs, approximately equal in size situated on either side of the uterus. The ovaries are enclosed in a peritoneal capsule (Beck, 1972). The ovary contains the follicular apparatus, corpus luteum, stromal and connective tissue, interstitial tissue, vascular, nervous, and lymphatic tissues (Harrison and Weir, 1977). The ovary is made up of the cortex, surrounding a central core or medulla. Much of the medullary region consists of connective, vascular, and nervous tissues. The dense cortex is composed of a stroma of connective tissue, cells of epithelial origin, and numerous follicles (Franchi, 1962). Primordial follicles consist of a single layer of flattened epithelial cells surrounding each oocyte (Harrison and Weir, 1977). In a given ovarian cycle, only one of these primordial follicles reaches maturity and ruptures; the majority of follicles undergo atresia. As the follicle increase in size, the single layer of flattened cells enveloping the oocyte increases in thickness and its cells become cuboidal or columnar to form a distinct layer referred to as the granulosa  2  layer. This layer rapidly becomes several cells thick (Harrison and Weir, 1977). The enlargement of the granulosa layer is accompanied by the development of an outer layer derived from the stroma. This layer which will then develop into a well vascularized layer is referred to as the theca interna. The theca intema cells are surrounded by another layer composed of connective tissue which is called the theca externa. Numerous blood vessels and lymphatics penetrate the theca externa to communicate with a fine plexus of vessels in the theca interna. A thick membrane which is referred to as the basement membrane separates the granulosa cells from the theca interna. Prior to ovulation, the granulosa layer is avascular (Harrison and Weir, 1977). Both the theca interna and the granulosa layer are considered sites of steroid production. The maturing follicle enlarges as a result of proliferation of granulosa and thecal layers and a cavity soon forms in the granulosa. The enlarged cavity is referred to as the antrum. The fluid-filled follicle becomes surrounded by a uniform layer of granulosa cells referred to as mural granulosa cells (Adashi, 1991). The oocyte is surrounded by an irregular cluster of granulosa cells referred to as the cumulus cells. Cumulus cells appear to be present in the majority of mammals and they persist after ovulation until fertilization. The cumulus cells support and provide nutrients for the growth of the oocyte (Adashi, 1991). Early in follicular development the immature oocytes are in contact with the granulosa cells. Later a jellylike substance containing polysaccharides separates the membrane of the oocyte and the granulosa cells. This layer is referred to as the zona pellucida (Harrison and Weir, 1977).  3  The corpus luteum is the endocrine gland which normally develops from the cellular components of the ovarian follicle (granulosa and theca) after ovulation. Thus, the word luteal is referred to as any structure or cell which pertains to the corpus luteum, such as granulosa-luteal cells. The term luteinization is used to refer to the enlargement and morphological changes associated with the post-ovulatory follicle.  C. The life cycle of the ovarian follicle The transformation of ovarian follicle into a corpus luteum was first described in 1672 by Regner de Graaf (Jocelyn and Setchell, 1972). Ovarian follicles constitute the fundamental functional unit of the ovary. Follicular maturation depends, except during the earlier stages, on gonadotropic stimulation from the anterior pituitary and proceeds in cycles extending throughout the period of sexual maturity, unless interrupted by pregnancy and lactation (Eckstein, 1962). At each cycle a group of follicles enters a rapid phase of growth in which only a selected dominant follicle changes into a differentiated preovulatory stage characteristic of antral (graafian) follicles (Hisaw, 1947; Peters et al., 1975). The rest of the follicles will become atretic. Atresia is the process whereby oocytes are lost from the ovary by means other than ovulation (Adashi, 1991). The follicular development can be divided into three phases: 1. preantral growth phase, 2. tonic growth phase, and 3. gonadotropin-dependent growth phase. The preantral growth phase refers to conversion of primordial follicles into primary and  4  then secondary follicles. The conversion of a primordial follicle to a primary follicle is marked by little follicular growth and is independent of gonadotropins. Ultimately, proliferation of primary follicular granulosa cells give rise to multiple layers of cells, thereby enlarging the follicle to yield a secondary follicle (Gougeon, 1986). As a secondary follicle is formed, the granulosa cells develop FSH receptors and become physiologically coupled with gap junctions. Then the secondary follicle develops into a class 1 follicle (Gougeon, 1986). The tonic phase of follicular development corresponds to the conversion of class 1 (preantral) follicles (0.12-0.2 mm in diameter) to class 4 (antral) follicles with a diameter of up to 2 mm. The growth is characterized with increase in granulosa layer and overall follicular diameter. The growth of the follicle is accomplished through granulosa cell proliferation and enlargement of a central fluid-filled cavity, the antrum. The appearance of the antrum transforms the follicle into a small antral (tertiary) follicle which is also referred to as a graafian follicle. The final stages of the follicular development (class 5 to 8) are dependent on gonadotropins (Gougeon, 1986). During this time follicular dominance and selection are accomplished. FSH plays a crucial role in this selection process (McNatty and Baird, 1978). Large healthy antral follicles in women contain a large amount of FSH. Locally produced steroidal and non-steroidal factors also play a role in the selection of the dominant follicle by modulating the responsiveness of the ovarian cells to gonadotropins (Tonetta and DiZerega, 1989). Within this dominant follicle, the oocyte is surrounded by a layer of the granulosa cells. Ovulation occurs as a result of a  5  massive discharge of LH from the anterior pituitary. About 36 hour after the onset of LH surge, the follicular wall of the dominant follicle ruptures leading to the release of the oocyte. Ovulation marks the end of the first stage of the cycle which is referred to as the follicular phase. After ovulation, during the luteal phase of the cycle, the corpus luteum is formed from the cellular components of the ovarian follicle, the granulosa and theca cells (Rodgers et al., 1983). The word luteal cell is referred to as any structure or cell which pertains to the corpus lutei.un, such as granulosa luteal cells. The granulosa cells do not divide after ovulation, but they increase in size and undergo morphological changes (Auletta and Flint, 1988). These changes are referred to as luteinization. The corpus luteum consists of two types of steroidogenic luteal cells, the large luteal cells and the small luteal cells (Rodgers et al., 1985). The large luteal cells are derived from the granulosa cells while the small luteal cells are derived from the thecal cells (Rodgers et al., 1983). The corpus luteum is a large endocrine gland which secretes large amounts of steroid hormones especially progesterone, which prepares the uterine endometrium for implantation and maintains early pregnancy. If fertilization and implantation do not occur, the ovulatory cycle ends and the corpus luteum undergoes luteolysis (Auletta and Flint, 1988). However, if pregnancy is established, the continued secretion of progesterone by the corpus luteum is essential. Around the start of the implantation of the egg, about 7 days after ovulation, the embryo secretes hCG which rescues the corpus luteum from undergoing regression (Lenton and Woodward, 1988). Thus secretion ofprogesterone by the corpus luteum is maintained.  6  II. Steroid hormone production by the ovary A. Synthesis of sex steroid hormones The principal ovarian hormones are steroids, estradiol and progesterone, secreted by the preovulatory follicle and corpus luteum (Strauss and Miller, 1991). The common precursor of steroids is cholesterol. Major pathways of steroid hormone synthesis in the ovaries are illustrated in figure 1. The ovaries are capable of de novo cholesterol synthesis (Schuler et al., 1979). Steroidogenic cells in the ovaries synthesize cholesterol from acetyl coenzyme A. 3-Hydroxy-3-methylglutaryl coenzyme A reductase (HMG-CoA reductase) is the rate-determining enzyme in de novo cholesterol synthesis (Strauss et al., 1981). Ovarian cells are also capable of taking up cholesterol from circulating lipoproteins by receptor-mediated mechanisms (Gwynne and Strauss, 1982). Cholesterol used for steroid hormone production is derived primarily from circulating serum lipoproteins (Brown and Goldstein, 1976) rather than from de novo cellular synthesis from acetate (Anderson and Dietschy, 1978). The lipoproteins are classified as low-density lipoproteins (LDL) and high-density lipoproteins (HDL). The human granulosa cells express many LDL receptors which indicates that cholesterol from LDL is the major precursor in human ovarian cells (Tureck and Strauss, 1982). The importance of LDL cholesterol for ovarian progesterone production is demonstrated by the observation that the presence of LDL is required for maximal progesterone  7  12  :cERL<  Cholesterol  5J7 Pregnenolone  -  O  20 cx -OH-Progesterone  Progesterone  12  17 cx  -  5 17 cx hydroxyprogesterOfle hydroxypregnenolone -  0  56 Dehydroepiandrostened-iOfle  —  Androstenedione  Testosterone  —  Estrone  Estradiol-17 3  Figure 1. The biosynthetic pathway of steroid hormone production in the ovary. The enzymes regulating this pathway are as follows: 1. 2. 3. 4. 5. 6. 7.  Cholesterol side-chain cleavage p450 17-cx-hydroxylase -lyase 17 20 C , 17-13-hydroxysteroid dehydrogenase 3-B-hydroxysteroid dehydrogenase/A , zV isomerase 5 Aromatase 20-.cx-hydroxysteroid dehydrogenase 8  production by cultured human granulosa cells (Tureck and Strauss, 1982). HDL does not support human ovarian progesterone biosynthesis (Gwynne and Strauss, 1982). LDL particles bind to specific membrane receptors, the LDL-receptor complexes enters the cell by receptor-mediated endocytosis (Anderson et al., 1977; Goldstein et al., 1979). Then the endocytotic vesicles are known to fuse to lysosomes, where LDL cholesterol esters are hydrolysed to yield free cholesterol (Brown et al., 1975). The free cholesterol is then re-esterified and is stored in the cytoplasm in lipid droplets. Upon steroidogenic demands, the cholesterol ester is hydrolysed and the free cholesterol is transported to mitochondria for steroidogenic processing. The human granulosa cells are capable of synthesizing both steroids, progesterone and estradiol. Cholesterol side-chain cleavage (cytochrome p450scc) cleaves the cholesterol side chain which results in C 21 compound, pregnenolone (Dimino and Campbell, 1976; lVliller, 1988). This is the first step in steroid hormone synthesis. This reaction takes place in the inner mitochondrial membrane. The rate of formation of pregnenolone in steroidogenic cells is determined by: 1. the availability of cholesterol to the mitochondria (Toaff et al., 1979; Nakamura et aL, 1980; Privalle et al., 1987), 2. the quantities of cholesterol side-chain cleavage enzyme, and 3. the degree of enzyme activity expressed (Simpson et al., 1987). Acute alterations in steroidogenesis result from changes in the availability of cholesterol to the enzyme and its expressed activity. Pregnenolone is the intermediate steroidogenic compound common to all classes of steroid hormones. Both the human granulosa and theca cells are capable of  9  21 steroid. The conversion of converting pregnenolone to progesterone, another C pregnenolone to progesterone occurs readily by the relative abundance of the cytoplasmic enzymes 3B-hydroxysteroid dehydrogenase and A 5,4 isomerase (Sulimovici and Boyd, 1969). The secretion of progesterone by ovarian cells is modulated by the conversion of progesterone to its metabolites. The main route of progesterone breakdown is mediated by 20-o-hydroxysteroid dehydrogenase which converts progesterone to its inactive metabolite, 20-a-hydroxyprogesterone (Rodway and Kuhn, 1975). The rate-limiting step in the biosynthesis of androgens in the follicle is that 10 C 2 , 7 enzyme (Sasano et al. 1989). This catalyzed by the 17--hydroxylase/ -lyase 19 reaction is capable of converting the C 21 pregnenolone and progesterone into C androgen, dehydroepiandrosterone and androsteneclione respectively. Studies of isolated human theca cells have revealed that the thecal layer is the major source of follicular androgen (Tsang et al., 1980). The granulosa cells contain low amounts of 17-cz-hydroxylase/ -lyase 10 C 2 , 7 enzyme, the enzyme which mediates the conversion of progestins to androgens. Thus, the granulosa cells are incapable of producing significant amounts of androgens (Tsang et al., 1979). The fact that the biosynthesis of estradiol from androgens requires the cooperation of the granulosa and their thecal neighbours was discovered by Falck in 1959. In all species estrone and estradiol are derived from the androgen precursors androstenedione and testosterone. Thus, the C 19 precursor steroids are produced by the thecal cells and are transferred across the basement membrane of the follicle to  10  the granulosa cells (Ryan and Petro, 1966). Ryan and colleagues in 1968 were also able to show that the conversion of acetate to estradiol is enhanced by the co incubation of granulosa and theca cells. In the granulosa cells, the conversion of androsteneclione and testosterone to estrone and estradiol takes place by the enzyme aromatase. Aromatase is the granulosa cell enzyme crucial to follicular estradiol synthesis (Ryan and Petro, 1966; Bjersing, 1967; Miller, 1988).  B. Mechanism of action of steroid hormones The classical ovarian steroid hormones are estrogens, progesterone, and androgens. Estrogens are important in regulating the developmental and physiologic functions of the female phenotype. On the other hand, progesterone is a vital hormone during pregnancy. To accomplish these tasks, the steroid hormones must bind and activate a group of specific-regulatory molecules called receptors. The steroids are secreted from their endocrine gland into the blood stream. The steroids diffuse into the cells and combine with specific receptors present in the target cells in which they will exert their functions. Free steroid enters the cell and binds to inactive receptors in either the cytoplasmic or nuclear compartments. After binding to their specific receptors, the steroid hormones cause the receptors to undergo a conformational change, which converts the receptors from an inactive to an active form (for review see Katzenellenbogan, 1980). The steroid receptor in its inactive form is bound to a heat shock protein. While complexed to the heat shock protein, steroid receptors cannot bind to the gene. However, once the steroid hormone enters the cell  11  and binds to its receptor, the heat shock protein comes off and as the result, the receptor hormone is then activated. These heat shock proteins are thought to help the receptor fold into an appropriate conformation that permits subsequent biological activity and tends to protect them from degradation by cellular proteases (for review see Katzenellenbogan, 1980). Once the receptor becomes activated, it has the capacity to bind to and activate the regulatory elements of genes. If the gene is activated, the enzyme RNA polymerase transcribes the information in the gene into messenger ribonucleic acid (mRNA), a molecule that carries the information to the cytoplasmic compartment of cells. The information is then decoded on structures termed ribosomes, which produce the appropriate protein specified by that certain gene (for review see Walters, 1985).  III. Ovarian regulation During the follicular phase of the human menstrual cycle, the follicle destined to ovulate increases in size from a diameter of 2-5 mm to over 20 mm and becomes the major ovarian source of secreted estradiol. This estracliol-secretory stage in its development encompasses a programmed sequence of cell growth and differentiation in the follicle wall which terminates with ovulation and transformation of the follicle into a corpus luteum. The entire sequence of events depends upon primary (endocrine) stimulation of the ovaries by the gonadotropins FSH and LH. There are also local (paracrine and autocrine) levels of control from within the follicle itself.  12  A. Neuroendocrine regulation of the ovary 1. GnRH  The secretion of both FSH and LH from the anterior pituitary gonadotrophs is stimulated by the hypothalamic decapeptide GnRH, also termed luteinizing hormonereleasing hormone (LHRH) (pyro-Glu-His-Trp-Ser-Tyr-Gly-Leu-Arg-Pro-Gly-Nh ). The 2 responsiveness of pituitary gonadotrophs to GnRH varies markedly during the menstrual cycle (Wang et al., 1976). In primates, including man, GnRH neurons are located mainly in the arcuate nucleus of the medial basal hypothalamus (MBH) and the preoptic area of the anterior hypothalamus. Axons from GnRH neurons project to many sites within the brain. One of the distinct projections is from arcuate nucleus of the MBH to the median eminence (Silverman et al., 1987). The median eminence is made up of extensive capillaries referred to as the primary plexus. These capillaries that penetrate the median eminence drain into the short portal vessels that pass down the pituitary stalk and branch into capillaries surrounding the anterior pituitary. In this way the GnRH is delivered to its target cells  -  the gonadotrophs of the anterior  pituitary (Schwanzel-Fukuda and Pfaff, 1989). GrLRH is released in a pulsatile manner from the median eminence into the portal system and rapidly binds to specific, high-affinity receptors on the surface of the pituitary gonadotrophs (Clayton and Catt, 1981). The binding of GnRH is followed rapidly by aggregation of the receptor-bound peptide resulting in the release of gonadotropins from the secretory granules. Much of the stored gonadotropins within  13  the secretory granules can be released during the secretory response to GnRH. In addition to the release of pre-formed gonadotropins from the secretory granules, there is evidence for increased synthesis of gonadotropins as well (Landefeld and Kepa, 1984). Prolonged or continuous exposure to GnRH or its agonists results in profound suppression of gonadotropin release (Hazum and Conn, 1988). This is due to a decrease in the number of GnRH receptors on the plasma membrane, a phenomena known as the down-regulation of the receptors. An optimal frequency of GnRH pulsatile stimulation of the pituitary is essential to maintain appropriate plasma levels of LH and FSH. The pulsatile pattern of gonadotropin release from the pituitary gland is the result of episodic secretory discharges of GnRH, a process that is governed by a pulse generator located in the arcuate region of the medial basal hypothalamus (Wilson et al., 1984). GnRH pulse generator activities are subject to neuromodulation. Both a-adrenergic and dopaminergic inputs have stimulatory effect on the GrLRH pulse generator. On the other hand, morphine and endogenous opioids have an inhibitory effect (Knobil, 1980). In the rhesus monkey, deviations from the physiologic frequency of GmRH pulses impairs gonadotropin secretion and ovarian function (Pohl et aL, 1983) suggesting that GnRH has a permissive role in the control of follicular maturation and ovulation.  2. Prolactin regulating factors  Prolactin-containing cells which are referred to as lactotrophs have been identified  14  in the human anterior pituitary gland. Lactotrophs are sparse in the human pituitary, except in the fetus and during pregnancy and lactation. The posterior and the intermediate lobe of the pituitary contain a prolactin-releasing factor (PRF), a small (5000 mol wt) peptide which is distinct from known prolactin secretagogues (Hyde and Ben-Jonathan, 1989; Laudon et al., 1990). The release of PRF may be subjected to short loop negative feedback regulation by prolactin (Laudon et al., 1990). In addition, thyroid releasing hormone (TRH), 13-endorphin, and vasopressin also stimulate prolactin release (Leong et al., 1983). The inhibitory control of prolactin is largely mediated by the action of dopamine (Ben-Jonathan, 1980). However, a prolactin-inhibiting factor (PIP) also exists in the neurointermedliate lobe. The PIP was active in suppressing prolactin release from the in vitro culture of bovine anterior pituitary cells during dopamine blockade (Samson et al., 1990).  B. Gonadotropic hormone regulation of the ovary Normal follicular development and steroid hormone secretion depend upon appropriate stimulation of the ovaries by adequate amounts of FSH and LH.  1. Chemistry of gonadotropic hormones LH and PSH are climers composed of two glycosylated polypeptide subunits (cx and 13) joined together by a noncovalent bond. The cx-subunit of LH, FSH, and that of human chorionic gonadotropin (hCG), is common to the glycoprotein hormones (Parsons and Pierce, 1981). Within different species, the ex-subunits of the  15  glycoprotein hormones possess the same amino acid sequence. In contrast, the Bsubunit of each hormone has a distinctive amino acid sequence that determines its specific hormonal activity expressed upon association with the cc-subunit. The molecular weight of LH is 28,000 and that of FSH is 33,000; the molecular weight of the common a-subunit is about 14,000. Prolactin has been characterized in many vertebrates. However, the human prolactin was not discovered until 1970. Human prolactin has a molecular weight of 22,000 and consists of a single polypeptide chain of 199 amino acids. Human prolactin has sequence homologies with animal prolactin, human growth hormone (GH), and human placental lactogen. These polypeptide hormones are believed to have evolved  from a common ancestral protein (Niall et al., 1973). In the human, the GH and placental lactogen are more closely related to each other than to prolactin. Whereas placental lactogen is produced in the placenta, prolactin and GH are produced in different pituitary cells.  2. Role of gonadotropic hormones i) FSH actions FSH induces ovarian follicle maturation and stimulates the granulosa cells to secrete steroids, estradiol and progesterone (Channing, 1970; McNatty, 1981). FSH regulates granulosa cell progesterone synthesis by modulating the activity of various steroidogenic enzymes (Moon et al., 1978). The major site of FSH action on progesterone synthesis is at the cholesterol side-chain cleavage activity (Toaff et al.  16  1983). FSH also increases 313-hydroxysteroid dehydrogenase activity which is responsible for the conversion of pregnenolone to progesterone in the granulosa cells (Zeleznik et al., 1974). F’SH also stimulates estradiol secretion by the rat ovaries in organ culture (Moon et al., 1975). FSH acts on the aromatase enzyme to enhance estradiol production in the rat granulosa cells (Dorrington et al., 1975). Luteinized granulosa cells have minimal ability to convert progesterone to androgens, and are deficient in l7czhydroxylase which converts progesterone to androgens. Fevold in 1941 and Greep et al. in 1942 were first to demonstrate that both FSH and LH are required for estradiol biosynthesis by immature, hypophysectomized rats. Faick in 1959 was first to discover that the biosynthesis of estradiol from androgens requires the cooperation between the granulosa and their thecal neighbours. Short in 1962 proposed a two-cell type theory suggesting the participation of theca cells for the conversion of progesterone to estradiol. Ryan et al. in 1968 demonstrated that combined incubation of both granulosa and theca cells in human tissues produces higher estradiol from acetate in vitro as compared to incubation of the individual cell types alone. The two cell, two-gonadotropin hypothesis for ovarian estracliol biosynthesis is summarized in figure 2. According to this model, LH stimulates the biosynthesis of androgen from cholesterol in the theca interna compartment (Markis and Ryan, 1975; Fortune and Armstrong, 1977; Hamberger et al., 1978; Tsang et al., 1980). Androgens diffuse across the basement membrane to the granulosa cells and are acted upon by the aromatase enzyme which converts androgens to estrogens.  17  THECA CELLS  LH  4  Cholesterol  cAMP Progesterone  4,  Androstenedione  FSH  LDL  Li (D Androstenedione  Cholesterol cAMP  ®c-Aromatase  Pregnenolone Estradiol  CD Progesterone  GRANLJLOSA CELLS  Figure 2. Diagram steroidogenesis.  of the  “two-cell,  18  two-gonadotropin theory”  of follicle  FSH also stimulates the granulosa cells to secrete various nonsteroidal substances such as inhibin (Lee et al., 1982), prostaglandins (LeMaire et al., 1973), and plasminogen activator (Martinat and Combarnous, 1983). These nonsteroidal secretions ensure optimal folliculogenesis and oocyte maturation by altering steroid hormone production by granulosa cells. FSH also induces an increase in LH (Nimrod et al., 1977), prolactin (Navickis et al., 1982), and epidermal growth factor (EGF) (Jones et al., 1982; St-Arnaud et al., 1983) receptor number in granulosa cells. During follicular development, granulosa cells undergo many cell divisions. FSH stimulates granulosa cell division which is reflected by an increase in the amount of DNA (Ryle, 1969). FSH also stimulates granulosa cell protein synthesis. During follicular development, granulosa cells undergo morphological changes (Albertini, 1980). The avascular nature of granulosa cells requires intercellular contacts between neighbouring cells. During follicular development, extensive gap junctions are found among granulosa cells (Albertini and Anderson, 1974). FSH treatment increases the number of gap junctions in granulosa cells of hypophysectomized rats (Burghardt and Matheson, 1982).  ii) LH actions LH stimulates preovulatory follicle growth, induces ovulation, and regulates corpus luteum function. After the FSH induces LH receptors in cultured rat granulosa cells, these cells are capable of responding to LH. Both gonadotropins can then act directly to stimulate granulosa cell steroidogenesis in the preovulatory  19  follicle (Channing and Tsafriri, 1977). LH receptors are located on theca interna cells and steroidogenesis is under direct LH control throughout the menstrual cycle (Erickson et al., 1985). LH acts on the thecal cells via its receptor to stimulate progesterone and androgen synthesis. As mentioned before, LH stimulation of androgens is required for maintenance of estracliol production by the granulosa cells. The midcycle surge of LH is essential for ovum maturation and ovulation. LH induces the resumption of oocyte maturation in preovulatory follicles (Tsafriri, 1978). LH also causes the granulosa and luteal cells to secrete several nonsteroidal substances such as prostaglandins (Marsh et al., 1974; Clark et al., 1978), plasminogen activator, and proteoglycan (Beers et al., 1975). These nonsteroidal substances, as well as progesterone (Rondell, 1974), may be important in the LH induction of follicle rupture which leads to ovulation. After ovulation, the granulosa and theca cells of the ovulatory follicle will luteinize and form the corpus luteum. LH stimulation sustains the steroid-secretory function of the corpus luteum (Filicori et al., 1984). Luteal tissue mainly produces progesterone, which prepares the uterine endometrium for implantation and maintains early pregnancy. The LH stimulation of progesterone production is accompanied by alterations in cholesterol metabolism and changes in the activities of steroidogenic enzymes. Brief exposure to LH activates cholesterol esterase activity, resulting in the mobilization of free cholesterol from pools of fatty acid esters (Behrman and Armstrong, 1969). LH may also increase the number of LDL receptors in the rat corpus luteum (Hwang and Menon, 1983). With prolonged exposure to LH,  20  the increase of progesterone synthesis occurs via stimulation of cholesterol side-chain cleavage enzyme (Armstrong et al., 1970) and 313-hydroxysteroid enzymes (Madej, 1980). If pregnancy occurs, the functional lifespan of the corpus luteum is extended by the direct action of hCG secreted by the trophoblastic tissue of placenta. If fertilization and implantation do not occur, the final stage of the ovarian cycle begins as the corpus luteum undergoes luteolysis (Auletta and Flint, 1988).  iii) Prolactm actions Prolactin is an important regulator of lactation. The actions of prolactin on the  mammary gland include promotion of mimmary growth and initiation of milk secretion. In addition to its action on the mammary gland, prolactin acts as a luteotropic hormone in rodents and several other species (Richards and Williams, 1976). The luteotropic action of prolactin varies among species. In rats, prolactin acts as a luteotropic agent by stimulating progesterone secretion (Smith, 1980) as well as maintaining the LH receptors during follicular luteinization (Holt et al., 1976). In the rat corpus luteum, prolactin contributes to the rise in progesterone by inhibiting progesterone catabolism. Prolactin inhibits the activity of 2Occ-hydroxysteroid dehydrogenase enzyme which converts progesterone to its inactive form (Zmigrod et al., 1972). In contrast to its luteotropic role, prolactin also exerts a luteolytic action on the rat and may be responsible for the luteal regression during the estrous cycle in the rat (McNeilly et al., 1982). In contrast to its ability to stimulate progesterone production in rats, in vitro  21  treatment with prolactin inhibits progesterone production by human granulosa cells (McNatty et al., 1974). Thus, the action of prolactin varies in different species.  C. Steroid hormone regulation of the ovary 1. Role of estrogens Estrogens induce and maintain secondary sexual characteristics and exert a feedback action on the hypothalamo-pituitary axis and consequently affect the release of gonadotropins. More importantly, estrogens play a vital role at the site of their production, the granulosa cells. Estrogen is considered an intrafollicular autocrine regulator (i.e., having an action within the cell which produces it). Estrogen stimulates granulosa cell mitosis (Rao et al., 1978), and exerts a antiatretic effect (Harman et al., 1975). Estrogen also raises gonadotropin receptor levels and amplifies follicular responsiveness to  exogenously administered  gonadotropin (Richards, 1980). Estrogen synergizes with FSH to promote follicular growth (Simpson et al., 1941; Reiter et aT., 1972), and enhances FSH-stimulated granulosa cell aromatase activity (Adashi and Hsueh, 1982; Zhuang et al., 1982). The ability of estrogens to augment the ability of the enzyme responsible for their formation accounts for the preovulatory rise in the circulating estradiol levels. This form of self amplification may play a central role in the follicular selection as well as in the establishment of follicular dominance. It has been reported that induction of atresia may be associated with the loss of estrogen receptors (Harman et al., 1975). The large healthy follicles contain detectable amount ofestradiol (McNatty and Baird,  22  1978). Once selected, the dominant follicle ensures that development of other follicles is suppressed. Current evidence suggests that this dominance is secured by the suppression of FSH secretion due to the rise in levels of estradiol secreted by the dominant follicle (Baird, 1987). The estrogen in the dominant follicle will exert negative feedback control to the pituitary leading to reduction in systemic (Ross et al., 1970) and intrafollicular FSH levels (McNatty et al., 1975). The reduced levels of FSH will not allow for the further growth of the other follicles. Once a chosen follicle produces larger amount of estrogens compared to the other follicles, the estrogen acts through a local positive feedback loop to stimulate granulosa cell proliferation and augments follicular responsiveness to gonadotropins, thus causing further increase in estrogen formation (Hillier, 1981; McNatty, 1981). In this way, the selection of the dominant follicle is secured and it will continue to mature even though the FSH levels are declining. On the other hand, the follicles that are unable to produce enough follicular estrogens will undergo atresia. During the late follicular phase, estrogen causes the preovulatory discharge of LH through positive feedback on the pituitary and hypothalamus. This surge in LH results in the ovulation of the dominant follicle. On the other hand, estrogen inhibits production of its precursor, androgen through negative feedback on the theca cells (Leung and Armstrong, 1979). Such an intraovarian negative feedback mechanism may be significant in limiting the estrogen and provide adequate time for oocyte maturation before ovulation.  23  2. Role of progesterone  Granulosa cells secrete large amounts of progesterone. The role of progesterone in modulating follicular growth and granulosa cell function is not clear. Specific progesterone receptors have been demonstrated in the rat ovary which supports a direct ovarian action of progesterone (Schreiber and Hseuh, 1979). Direct effects of progesterone on granulosa cells have been studied in vitro. Treatment with synthetic progesterone and FSH showed that progesterone causes an increase in FSH stimulated progesterone production (F’anjul et al., 1983). Also, progesterone augments the LH-stimulated progesterone production in FSH-primed granulosa cells in vitro (Fanjul et al., 1983). Thus luteal cell progesterone production is an autocrine control mechanism in which progesterone regulates its own production. The increase in progesterone secretion during the preovulatory stage may exert a local action at the ovarian level to induce ovulation. Progesterone treatment enhances LH-stimulated ovulation in hypophysectomized rats in vivo (Fanjul et al., 1983). High levels of progesterone secreted by the luteal cells of the corpus luteum during the luteal phase also prepare the endometrium for the implantation of the oocyte. Progesterone secreted by the corpus luteum also exerts negative feedback control on the synchronized discharge of GnRH. Thus, the frequency of LH pulses falls in response to rising levels of progesterone (Knobil, 1980). As mentioned earlier, the estradiol also inhibits the FSH secretion. Thus, during the luteal stage of the cycle, follicular development is suppressed due to low circulating levels of gonadotropins. But, when the corpus luteum regresses, the concentration of FSH and LH rise and a new  24  ovarian cycle recommences (Baird et al. 1984). However, the effect of progesterone on follicular growth is not clear. Progesterone inhibits FSH-stimulated estrogen production in cultured rat granulosa cells (Schreiber et aL, 1980). This finding suggests a role of progesterone as inhibitor of follicular growth. In monkeys, ovarian implants of progesterone directly inhibit follicular growth (Goodman and Hodgen, 1979). In contrast to the in vivo studies in the monkeys, in prepubertal rats exogenous administration of progesterone facilitates hCG-stimulated growth of small antral follicles (Richards and Bogovich, 1982). These discrepancies between rat and monkey studies may be related to species-specifity.  3. Role of androgens Androgens are produced by the theca cells and play a role in follicular development. As mentioned before, androgens serve as substrate for aromatase enzyme to form estradiol. Androgens also exert a direct action on the granulosa cells through interaction with androgen receptor. In vitro studies demonstrate that androgens augment gonadotropin-stimulated steroidogenesis. Androgens directly augment aromatase activity in cultured rat granulosa cells (Daniel and Armstrong, 1980; Hillier and DeZwart, 1981). Androgens also stimulate progesterone biosynthesis by granulosa cells. In vivo administration of androgens to intact rats enhances the ability of FSH and LH to increase progesterone production by rat granulosa cells (Leung et al., 1979). Androgens also act synergistically with FSH to stimulate progesterone production in cultured rat granulosa cells (Nimrod et al. 1980; Welsh  25  et al., 1982). The stimulatory effect of androgens on progesterone synthesis appears to be on the cholesterol side-chain cleavage and 313-hydroxysteroid dehydrogenase enzymes (Welsh et al., 1982). However, in the absence of gonadotropins, androgens stimulate follicular atresia and antagonize estradiol-induced follicular growth in hypophysectomized immature rats. The counteracting action of androgens on estrogens may be related to the fact that androgen treatment decrease ovarian estrogen receptor content (Saiduddin and Zassenhaus, 1978). Androgen and estrogen levels vary in the follicular fluid, depending upon the stages of follicular development. Elevation of androgen to estrogen ratio is invariably associated with signs of atresia (McNatty et aL, 1975).  D. Role of local nonsteroidal regulators The concept that regulation of ovarian function involves the actions of local regulators has gained increasing acceptance in recent years. It has been suggested that these local factors might regulate ovarian functions such as steroidogenesis, oocyte maturation, and ovulation. These locally produced factors act through paracrine or autocrine control mechanisms. In recent years, measurable amounts of a number of these nonsteroidal substances have been found and isolated in the ovary, including the inhibin family (inhibin, activin, and follistatin), angiotensins, growth factors, insulin and insulin-like growth factors, GnRH, prostaglandins, and many other factors. The study of paracrine and autocrine functions of these local hormones has become important to the further understanding of ovarian function.  26  1. Role of inhibin related peptides i) Inhibin 1. Structure Inhibin, a 32,000 kDa protein, was first isolated from bovine and porcine follicular fluid in 1985 and was shown to be a product of granulosa cells (Robertson et at, 1985; Rivier et at, 1985; Fukuda et al., 1985; Robertson et al., 1986). Inhibin is a heterodimeric glycoprotein consisting of an x subunit joined together to a B subunit by two disulfide bonds. Two related forms of the 13-subunit termed  A 13  and  B 13  have  been described (Ling et at, 1985), and both complex with the CL-subunit to form biologically active inhibin termed inhibin A and inhibin B. The complete amino acid sequences of inhibin for porcine, bovine, human, and rat sources have been deduced by molecular cloning of the corresponding cDNAs (Forage et al., 1986; Mayo et al., 1986; Woodruff et al., 1987; Esch et al., 1987a). Human, porcine, bovine, and murine inhibin are closely related in structure and highly conserved (Esch et al., 1987a). In addition, the B-subunits of inhibin share a substantial sequence identity with an emerging family of proteins with growth-regulating properties referred to as transforming growth factor-B (TGF-B) (Mason et al., 1985). 2. Localization Histochemical techniques have demonstrated that granulosa cells of the ovary are the primary producers of inhibin. The CL-subunit of inhibin has been localized in both granulosa and luteal cells of the ovary by immunohistochemical techniques (Cuevas et al., 1987; Merchanthaler et al., 1987). The localization and intensity of  27  inm-imiostaining for each inhibin subunit changes during the follicular development and maturation (Yamoto et al., 1992). The inhibin a subunit is mainly expressed in healthy, maturing follicles and in corpora lutea, but not in small antral follicles of the primate ovary (Schwall et al., 1990) the sheep, (Mann et al., 1989) and the rat (Woodruff et al. 1988). The presence of inhibin mRNA in luteal cells remains controversial in rats (Meunier et al., 1988a; Woodruff et al., 1988) and sheep (Rodgers  et al., 1989; Tsonis et al., 1988). However, evidence exists that human granulosa cells that were luteinized in culture produced inhibin (Tsonis et al. 1987; Eramaa et al., 1993) and niRNA for the a and BA subunits of inhibin has been identified in the human corpora lutea (Davis et al., 1987). Extragonadal sources of inhibin include the placenta (Merchanthaler et al., 1987), the adrenal, and the pituitary (Meunier et al., 1988b). 3. Regulation FSH has been shown to be a major regulator of inhibin. F’SH induced inhibin bioactivity from the rat cultured granulosa cells (Anderson and Hoover, 1982). FSH induced accumulation of inhibin a-subunit mRNA has been observed both in intact animals and in cultured granulosa cells (Davis et al., 1986). In addition to FSH, a variety of other hormones appear to affect either basal or FSH-induced inhibin production. Insulin-like growth factor I (IGF-I), insulin (Woodruff and Mayo, 1990), activin (LaPolt et al., 1989), and TGF-13 (Zwiwen et al., 1988) have a stimulatory effect on inhibin production. On the other hand, epidermal growth factor (EGF) (Zhiwen et al., 1987b) and GnRH (Rivier and Vale, 1989) inhibit inhibin secretion by  28  granulosa cells. 4. Actions Inhibin was originally identified as an inhibitor of FSH secretion from pituitary gonadotrophs and followed from experiments showing the inadequacy of estrogen as a feedback regulator of FSH following unilateral or bilateral ovariectomy (Welschon, et al. 1978). A substantial amount of evidence supports a role for inhibin in the regulation of FSH secretion (for review see De Jong, 1988; Ling et al. 1988; Woodruff and Mayo, 1990). Treatment of rat pituitary cell cultures with inhibin significantly suppressed a and 13 FSH mRNA levels with parallel changes in FSH secretion (Carroll et al., 1989). No change in LH mRNA levels was observed. There is an inverse relationship between the plasma levels of FSH and inhibin during both the follicular and luteal phase of the human menstrual cycle (McLachlan et al., 1987). During the human menstrual cycle, inhibin levels increase late in the follicular phase, reaching a peak coincident with the LH surge. The inhibin levels then decrease during the LH surge with a subsequent second and higher peak of immunoreactive inhibin during the luteal phase parallel to the luteal phase progesterone concentrations (McLachlan et al., 1987). The levels of inhibin correlate positively with both progesterone and estradiol levels in the luteal phase, providing evidence that the corpus luteum secretes inhibin. Late in the luteal phase, as the corpus luteum regresses, the levels of inhibin also decline before the next menstrual cycle. The declining levels of inhibin before the next menstrual cycle allows for the rise in the circulating levels of FSH which is required for the stimulation of follicular growth for  29  the next cycle (McLachlan et aL, 1987). This inverse relationship between FSH and inhibin supports the concept that inhibin is a negative feedback regulator of FSH. Besides its effect on pituitary FSH secretion, inhibin exerts actions within the ovary. Inhibin acts directly on the gonadal cells via paracrine and possibly autocrine mechanisms to influence steroidogenesis. Two paracrine actions of inhibin have been reported. Firstly, after diffusing from its site of production in granulosa cells to the theca cells inhibin enhances LH-stimulated androgen biosynthesis in thecal cells in rats (Hsueh et al., 1987) and in humans (Hillier et al., 1991a). In humans, inhibin alone at doses between 10 and 100 ng/ml caused an increase in production of androgens by theca cells (Hillier et al., 1991a). Secondly, there is evidence of a paracrine interaction between the inhibin produced in the cumulus granulosa cells  and the oocyte. Bovine inhibin caused inhibition of spontaneous maturation division of cumulus-enclosed and denuded oocytes obtained from immature rats (0 et al., 1989). These paracrine actions of inhibin are yet to be confirmed using tissues from other species. There are conflicting reports of an autocrine influence of inhibin on granulosa cell steroidogenesis. Ying et at in 1986 reported that porcine inhibin inhibited FSH induced estracliol production by rat granulosa cells in vitro. However, the inhibitory actions of inhibin on estradiol production was not confirmed using bovine inhibin (Hutchinson et al., 1987; Sugino et al., 1988a). Hutchinson et al. (1987) reported that bovine inhibin has no effect on either basal or FSH-stimulated estradiol or progesterone production in cultured rat granulosa cells. Autocrine regulation of  30  human ovarian steroidogenesis by inhibin has not been elucidated. Using cultured human granulosa cells from women undergoing in vitro fertilization, inhibin-A showed no effects on basal or gonadotropin-stimulated progesterone (Li et al., 1992; Rabinovici et al., 1992) and estradiol secretion (Rabinovici et al., 1992). Such experiments are complicated by the endogenous production of inhibin by granulosa luteal cells in culture (Zhiwen et al., 1987a, 1988).  ii) Activm  1. Structure During the purification of inhibin from porcine folliciilar fluid, a substance with FSH-stimulating activity was discovered. This material was characterized as a 28,000 KDa protein that was disulfide-linked dimers of the inhibin 13-subunits (Vale et al., 1986; Ling et al., 1986). This hormone which is known for its FSH-stimulating ability is referred to as activin. Three different forms of activins have been identified: activin A  (A/A),  activin B  13 ( B) 13 B’  and activin AB (BAIBB).  2. Localization  Considering that activin is composed of inhibin B-subunits, it is expected that any site of inhibin subunit production may be one of activin production (Meunier et al., 1988b). The RNA encoding the inbibin-B subunits is found in a multitude of diverse tissues, including the gonads, pituitary, placenta and bone marrow (Meunier et al., 1988b). Antibodies to activin are not widely available so evidence for the localization of activin in the gonads have been deduced from the presence of B-chain iURNA. In  31  rats, both B-subunit mRNAs are expressed in the ovary (Esch et al., 1987a; Meunier et al., 1988a). The 13-mRNAs are exclusively localized in the granulosa cells of both the adult ovary (Woodruff et aL, 1988) and the immature rat ovary (Meunier et al., 1989). Activin can also be isolated from the follicular fluid of the pig (Ling et al., 1986; Vale et al., 1986) and cow (McLachlan et al., 1987). Schwall et al. (1990) presented evidence for the expression of the B subunits (without a-subunit expression) in granulosa cells of small antral follicles in the primate ovary. Using immunohistochemical localization techniques, Yamoto et al. (1992) showed that the granulosa cells of human preantral and small antral follicles exhibited positive immunoreactive staining with antisera against BA and BB subunits and negative immunostaining with antiserum against a-subunit. Thus, in contrast to inhibin subunits (a, BA, and  UB)  which are located in the human granulosa cells of mature,  preovulatory follicles, activin subunits (BA and BB) are found in small antral follicles (Yamoto et al., 1992). 3. Actions Activin is mainly known for its ability to stimulate FSH synthesis and release from the pituitary (Ling et al., 1986; Carroll et al., 1989). Infusion of activin into immature rats (Schwall et al., 1989) and monkeys (McLachlan et al., 1989) led to increased gonadotropin secretion. In addition to stimulating FSH synthesis and release, activin has a broad range of activities that includes effects on cell growth, differentiation, and maturation. These varied actions on growth and differentiation reflect the partial sequence homology of the inhibin-B subunits to TGF-B family  32  (Mason et al., 1985) and euthyroid differentiation factor (EDF). Subsequent to the identification of ovarian activins, an erythroid differentiation factor was also isolated and was found to be identical to activin-A (Eto et aL, 1987; Murata et aL, 1988). Rabinovici et al., (1990) reported that activin-A is able to modulate growth and induced proliferation of human luteinized granulosa cells in culture. Stimulation of oocyte maturation in the rat was induced by activin-A (Itoh et al.,1990); however, Tsafriri et al. (1989) failed to observe the effect of activin-A on the spontaneous maturation of oocytes. Besides its effect on the pituitary FSH and cellular growth, activin can affect granulosa cell function in vitro (Findlay et al., 1990) and in vivo (Woodruff et al., 1990). Binding sites for activinfEDF have been demonstrated in granulosa cells, suggesting an autocrine action and possibly a paracrine function (Lapolt et al., 1989; Sugino et al., 1988b). Activin exerts its paracrine effect by diffusing from its site of production, granulosa cells to the theca cells. Activin and inhibin have opposing paracrine actions on LH-induced secretion of androgen by rat theca cells (Hsueh et al., 1987) and human thecal cells (Hillier et al., 1991b). Activin suppressed LH stimulated androgen biosynthesis in rats and in humans (Hsueh et al., 1987; Hillier et al, 1991b). There are conflicting reports of an autocrine influence of activin on granulosa cell steroid production. The effect of activin on steroidogenesis differs in different species. Activin has a stimulatory action on FSH-induced estradiol production by rat granulosa cells in vitro (Ying et al., 1987b; Hutchinson et al., 1987). However, the  33  effect of activin on progesterone production is unclear in rats. It appears that the effect of activin on progesterone production is related to follicular maturity (Miro et al., 1991). Hutchinson et al. (1987) reported that activin caused a dose-dependent decrease in FSH-induced progesterone production by undifferentiated rat granulosa cells. In contrast to the findings of Hutchinson et al. (1987), Xiao et al. (1990) reported a time and dose-dependent increase in FSH-stimulated progesterone production by activin in differentiated rat granulosa cells. Miro et al. (1991) reported that activin alone enhanced basal levels of progesterone whereas it inhibited FSH stimulated progesterone production in differentiated rat granulosa cells. In contrast to the controversial reports on the effect of activin on the rat granulosa cell progesterone production, activin inhibited progesterone production in the bovine ovary (Shukovski and Findlay, 1990). In human granulosa cells, activin-A inhibited basal as well as FSH-stimulated estradiol (Rabinovici et al., 1992) and progesterone production (Rabinovici et al., 1992; Li et al., 1992). In addition to its effect on steroidogenesis, activin also stimulates inhibin production (Xiao et al., 1992a; LaPolt et al., 1989), causes an increase in the number of FSH receptors on granulosa cells (Xiao et al., 1992b), and stimulates the induction of LH receptors by FSH (Sugino et al., 1988a). Overall, the data from the rat, bovine, and human are consistent with an autocrine action of activin on the regulation of granulosa cell ontogeny, differentiation, and the variable fate of the follicles by modulating local steroid production.  34  iii) Follistatin 1. Structure During the isolation of inhibin from the porcine follicular fluid, a side fraction was found that also suppressed FSH secretion from the pituitary cell cultures. This novel FSH-release inhibitor, named follistatin or FSH-suppressing protein (FSP), has now been isolated from porcine and bovine follicular fluid (Robertson et al., 1987; Ueno et al., 1987). Follistatin, is a monomeric glycosylated protein with at least three isoforms with molecular weights of 32, 35, and 39 Kda, and is structurally different from the inhibins (Robertson et al., 1987; Ueno et al., 1987). Follistatin was first isolated from porcine and bovine follicular fluid and found to suppress FSH release in vitro with 5-30% of the potency of inhibin (Ying et al. 1987a; Robertson et at,  1990a). Genes encoding porcine (Esch et al., 1987b; Shimasaki et al., 1988b), human (Shimasaki et al., 1988a), and rat (Shimasaki et al., 1989) follistatin have been cloned. The amino acid sequences are highly homologous, and the gene structure is conserved among the three species. 2. Localization Follistatin is expressed in the ovary, and extragonadal tissues such as brain and the kidney (Shimasaki et al. 1989). Follistatin, like inhibins and activins, is primarily a product of the granulosa cells (DePaolo et at, 1991; Nakatani et al., 1991; Saito et al., 1991; Shimasaki et at, 1989). The intensity of follistatin expression changes during granulosa cell differentiation and the rat estrous cycle. In rats, in situ hybridization studies revealed that the mRNA levels of follistatin is low in primordial  35  follicles but increased in growing secondary and tertiary follicles (Nakatani et aL, 1991). In rat granulosa cells, immunohistochemistry studies showed that the follistatin protein is localized to a subpopulation of early tertiary follicles and the dominant follicles that are selected to ovulate (Nakatani et al., 1991). After ovulation, the hybridization and immunohistochemical signals continued to be strong in the newly formed corpus luteum. Follistatin mRNA was not detected in theca, stroma, or interstitial cells of rats (Nakatani et al. 1991). Follistatin production was shown to be stimulated by FSH, but not LH, in differentiated bovine granulosa cells harvested from preovulatory follicles (Klein et at, 1991). In the immature rat ovary, the follistatin mRNA level was stimulated by pregnant mare serum gonadotropin (PMSG) (Shimasaki et al., 1989). Follistatin is present in the human follicular fluid (Robertson et al., 1990b; Schneyer et al., 1992). Human ovarian hyperstimulation with human menopause gonadotropins (HMG) caused an increase in the circulating levels of serum follistatin and estradiol (Sugawara et al., 1990). There is a direct correlation between increased serum follistatin and estradiol after ovarian stimulation. The increased serum follistatin level may likely reflect follicular or oocyte maturation (Sugawara et al., 1990). 3. Actions Follistatin may also act locally to regulate steroid hormone production in the granulosa cells. Follistatin augmented FSH-stimulated progesterone production by rat granulosa cells (Xiao et al., 1990) and enhanced LH-stimulated progesterone production in undifferentiated bovine granulosa cells (Shukovski et al., 1991).  36  Follistatin also augmented progesterone production at a concentration of 0.3 nM in human granulosa-luteal cells (Li et al., 1993). Follistatin decreased FSH-stimulated estradiol production in the rat granulosa cells (Xiao et al., 1990); however, an effect of follistatin on estradiol production by human granulosa cells has not yet been reported. Together with the above findings, the presence of follistatin mRNA within the ovary (Shimasaki et al., 1989; Natakani et al., 1991) suggests local regulation of steroid production by follistatin. Recent studies indicate that follistatin binds to activins in pituitary and ovarian extracts (Nakamura et al., 1990; Saito et al., 1991; Shimonaka et al., 1991; Kogawa et al., 1991), suggesting that follistatin antagonism to activin actions may occur by follistatin binding activin and then blocking its action. As mentioned before, activins have multiple biological effects. In addition to FSH stimulation, activin A promotes differentiation and growth, modulates gonadal steroid production, induces inhibin production in the granulosa cells, causes an increase in the FSH receptor numbers, and induces mesoderm formation. It is possible that follistatin may also participate in the regulation of these effects by binding activin-A, thus blocking its action. The high affinity activin-bincling protein, follistatin, has recently been shown to block activin-stimulated activities in several in vitro systems. Carroll et al. (1989) reported that follistatin suppressed activin’s stimulation of FSH mRNA levels of rat pituitary cells in vitro. Follistatin also blocked activin’s action on steroidogenesis. Follistatin reversed the inhibitory effects of activin-A on LH-stimulated progesterone production in bovine granulosa cells in culture (Shukovski et al., 1991). Follistatin also reversed  37  the stimulatory action of activin on FSH-induced aromatase activity and progesterone production in the undifferentiated rat granulosa cells (Xiao and Findlay, 1991). Xiao et al. (1992a) also reported that follistatin suppressed activin induced inhibin production in cultured rat granulosa cells. In the chicken embryo, follistatin inhibited the mesoderm-inducing activity of recombinant human activin-A (Asashima et al., 1991). The effects of follistatin on the activity of activin in stimulating the re aggregation of sertoli cell monolayer and proliferation of testicular germ cells were examined (Mather et al., 1993). Follistatin blocked the ability of activin-A to stimulate reaggregation of sertoli cell monolayer. However, in these same cultures, follistatin had no effect on the ability of activin-A to stimulate proliferation of testicular germ cells (Mather et al., 1993). In all of the above studies, the concentration of follistatin required for effective suppression of activin was two-three fold that of activin (Asashima et al., 1991; Mather et al., 1993; Carroll et al., 1989; Xiao et al., 1992a). These results suggest that perhaps in the ovary follistatin may be able to regulate steroidogenesis by modulating activin’s action and regulating the bioavailability of activin. However, follistatin has a direct action on the granulosa cells, which is independent of binding to activin, since both follistatin and activin enhanced FSH induced progesterone production in undifferentiated rat granulosa cells (Xiao and Findlay, 1991).  38  2. Role of the ovarian renin-angiotensin system  1. Synthesis of angiotensins The principal function of the renin-angiotensin system (RAS) involves regulation of cardiovascular homeostasis. Renin is the primary, rate-limiting enzyme of the renin-angiotensin system. Renin is formed as a result of a cleavage of prorenin and is stored in the kidney. Renin is indirectly responsible for the formation of the active agents of the RAS which are angiotensin II (Ang II) and possibly angiotensin III (Ang III), which are formed as a result of a degradative cascade reaction, and angiotensin (1-7)[Ang (1-7)], which is formed directly from angiotensin I, and from Ang II (Fig.3). Once in the blood, renin cleaves its substrate, angiotensinogen, to form a decapeptide, angiotensin I. Angiotensin I is then converted to Ang II and other peptidases by angiotensin-converting enzyme (ACE). Ang II is the main active peptide of the renin-angiotensin system. Ang II has diverse physiological effects including vasoconstriction, aldosterone secretion, angiogenesis, and the induction of drinking behaviour and salt intake (Peart, 1969; Catt, 1970; Regoli et al., 1974). In the blood,  Ang II has a short half-life and is cleaved by anginotensinase to form Ang III. Although the Ang II is the main bioactive product of the RAS, Ang III and angiotensin I can exert their own action in the regulation of the cardiovascular homeostasis. Ang III is as potent a vasoconstrictor as Ang II in the adrenal glomerulosa in stimulating aldosterone secretion (Goodfriend and Peach, 1975). Ang III may also be the active angiotensin in the brain (Wright at al., 1990). Also recent studies have shown that Ang (1-7) which is a newly defined hormone of the RAS  39  Angiotensinogen Renin  -  Prorenin  Angiotensin I endopeptidase  Ang (1-7)  Angiotensin II angiotensinase Angiotensin III  Figure 3. The biosynthetic pathway of angiotensins (ACE= angiotensin converting enzyme). 40  mimicks some of the known actions of Ang II (Santos et al., 1989; Kohara et aL, 1991; Goodfriend, 1991). 2. Localization During the last two decades, recombinant DNA technology and molecular cloning have shown the presence of both renin and angiotensinogen genes in a variety of tissues (Campbell and Habener, 1986; Dzau et al., 1987; Burnham et al., 1987; Dzau et al., 1988). The classical concept that the RAS is only a regulator of blood pressure has been changed due to the identification of local endogenous renin-angiotensin systems in several different tissues. In addition to its well known target organs, the rat and human ovary may be an important site of angiotensins’ actions. Prorenin (Glorioso et al., 1986; Itskovitz et al., 1988), renin-like activity (Fernandez et al., 1985; Lightman et al., 1987; Do et al., 1988), and Ang 111111 (Culler et al., 1986, Lightman et al., 1987; Jarry et al., 1988) have been found in the follicular fluid of preovulatory gonadotrophin-stimulated and normally cycling women, suggesting that the ovary is producing these substances. Renin niRNA is expressed in the rat (Kim et al., 1987) and monkey ovaries (Itzkovitz et al., 1992), suggesting that prorenin is produced locally in the ovary. Using immunohistochemical techniques it has been shown that prorenin, renin, and Ang II are found within the stromal, thecal and luteal cells of human (Palumbo et al., 1989) and rat ovaries (Lightman et al., 1988).  Only granulosa cells of preovulatory follicles and atretic follicles stain for both renin and Ang II (Palumbo et al., 1989). This supports the idea that the ovarian RAS is under gonadotropin control. Also, renin-like activity and Ang 11)111 immunoreactivity  41  in follicular fluids from women stimulated with HMG and hCG were much higher than the follicular fluid from women not stimulated with exogenous gonadotropins (Lightman et al., 1987). Also, immunostaining of both granulosa and thecal cells for renin and angiotensin occurs during preovulatory period when the LH levels are high. This suggests that the ovarian RAS is regulated by LH. In addition to expression of Ang Il/Ill and renin in the ovary, Ang II receptors are expressed on granulosa and theca cells and luteal cells of different species (Pucell et al., 1987; Speth and Husain, 1988; Husain et al., 1987; Miyazaki et at, 1988; Pucell et al., 1991). 3. Actions Because the various proteins composing the cascade of biochemical pathways that leads to the formation of the peptide have been found in the ovary, modulation of ovarian functions by the angiotensins seems likely (Bumpus et al., 1988). Angiotensins may have an autocrine function in regulating steroidogenesis. Several reports have shown that Ang II affects steroidogenesis in the ovarian tissues. In vitro studies on rat ovarian fragments from PMSG-stimulated rats showed a stimulatory effect of Ang II on estradiol production (Pucell et al., 1987), but studies with granulosa cells from diethyistilbestrol-treated rats reported no effect of Ang II on estradiol production (Pucell et al., 1988). The results of in vitro experiments with gonadotropin-stimulated human granulosa-luteal cells showed that Ang II caused an increase in progesterone, testosterone and estradiol (Palumbo et al.,1988). In cultured bovine luteal cells, Ang II had no effect on basal progesterone secretion but inhibited LH-stimulated progesterone production by a mechanism that involves inhibition of  42  cholesterol-side chain cleavage enzyme activity (Stirling et aL, 1990). Thus, it appears that there may be differences in the effects of Ang II on ovarian steroidogenesis, depending upon species and/or differentiation stage of granulosa cells. Therefore, Ang II may play a role in follicular development, possibly through the modulation of follicular steroidogenesis. There is no report on the effect of Ang III on steroidogenesis in the ovary from different species. The angiotensin-converting enzyme which converts Ang II to Ang III and the angiotensinase which converts Ang II to Ang III have been reported to exist in the rat ovarian follicles (Speth and Husain, 1988; Duad at al., 1990). This supports the idea that the ovary might produce its own Ang III. Also Ang Ill and Ang II bind to similar receptors (Goodfriend, 1991). The functions of Ang II and Ang III with regards to steroid production in the ovary is unclear and requires further investigation. Ang II may also have a direct role in ovulation and oocyte maturation (Pellicer et al., 1988; Kuo et al., 1991; Yoshimura et al., 1992). Although others have not shown a direct role for Ang II in ovulation (Naftolin et al., 1989), a recent study by Peterson et al. (1993) has confirmed that Ang II is a regulator of ovulation. Peterson and colleagues showed that saralasin (Ang II blocker) reduced LH-stimulated ovulation rate in the perfused rat ovary. Ang II may also play a role in oocyte maturation. Prorenin concentrations correlate with follicular development, oocyte-cumulus complex maturity and oocyte viability (Cornwallis et al., 1990). In the human, there is a high level of Ang II in the preovulatory follicular fluid of dominant follicles that could facilitate the regulation of oocyte maturation and ovulation.  43  E. Role of other intragonadal factors 1. Intraovarian growth factors Numerous growth factors have been identified by their ability to stimulate proliferation and growth of different cell types. Many of these growth factors have been also isolated from the gonads. In the ovary, these growth factors may function in an autocrine or paracrine fashion to modulate steroidogenesis. i) Insulin-like growth factors The family of insulin-like growth factors (IGFs) are composed of: 1. IGF-I (also called somatomedin C), and 2. IGF-II. Both IGFs exert endocrine effects on tissue growth throughout the body, being secreted by the liver under the control of growth hormone (Slack, 1989). These compounds have been extensively studied in the ovary (for review see Adashi et al., 1985b). Among modulators of granulosa cells, IGFs have a potential role for local modulation of steroid production and follicular development. Granulosa cells are the main site of IGF-I production and synthesis (Hammond et al., 1985; Hernandez et al., 1989). Human follicular fluid also contains IGF-II (Ramasharma et al., 1986). IGF-I production is regulated by gonadotropins, and estradiol (Hsu and Hammond, 1987; Hammond et al., 1988). Insulin, IGF-I, and IGF-II each have a separate receptor. However, IGFs can cross-react with each other and insulin for cell surface receptor (Czech et al., 1983; King et al., 1982). Both IGF-I and IGE-Il receptors have been found on granulosa cells of rats (Adashi et al., 1988), pigs (Baranao and Hammond, 1984), ewes (Monget et al., 1989), and humans (Poretsky et al., 1985). Localization of IGFs’ receptors on  44  the granulosa cells suggests that these cells are a site of IGF action. Autocrine regulatory actions of IGF-I in isolated granulosa cells have been observed in vitro. The IGFs have mitogenic effects on the granulosa cells (Hammond  and English, 1987; May et al., 1988). Although the ability of PSH to induce granulosa cell progesterone and estrogen biosynthetic capacity is well established (Richards, 1979), the modulation of this important process remains under investigation. IGF-I has been shown to stimulate progesterone secretion from the granulosa and luteal cells (Adashi et aL, 1985c; Veldhuis and Furlanetto, 1985; Veldhuis et al., 1985). The stimulatory effect of FSH or estradiol on progesterone production from ovarian cells was partially blocked by using IGF antibodies (Mondschein et aL, 1989). Thus, IGF-I is capable of synergizing with FSH to stimulate progesterone secretion (Baranao and Hammond, 1984). IGF-I can also stimulate basal and FSH-stimulated estradiol production in both human and rat granulosa cells (Steinkampf et al., 1988; Erickson et al., 1989; Adashi et al., 1985a). In addition, IGF can also influence androgen biosynthesis. Both basal and LH-stimulated synthesis of androgen in cultured theca cells can be enhanced by treatment with insulin or IGF-I (Cara and Rosenfeld, 1988; Magoffin et aL, 1990). In summary, IGF-I is synthesized in the ovary and effects ovarian functions. In the ovary, IGF-I has a variety of actions, including affects on cell proliferation and steroidogenesis, and it may also modulate gonadotropin actions.  45  ii) Epidermal growth factor (EGF)/Transforming growth factor-x (TGF-c) EGF and TGF-a are able to modulate development of epidermis, breast and gut (Gill et al., 1987; Waterfield, 1989). Both TGF-a and EGF bind to the same receptors and have similar actions in many cell types, but they also have distinct effects (for review see Derynck, 1986). In the ovary, TGF-ocfEGF may play a physiological role by affecting granulosa cell mitogenesis. Granulosa cells express gonadotropin regulated receptors for EGFJTGF-cz (St-Arnaud et al., 1983; Feng et aL, 1987; Kudlow et al., 1987; Roy and Greenwald, 1990). EGF binding sites have been located on granulosa (Hopkins et al., 1981; Jones et al., 1982), luteal, and thecal cells (Chabot et aL, 1986). Treatment with EGF/TGF-a promotes granulosa cell growth (Gospodarowicz and Bialecki, 1978; Gospodarowicz and Bialecki, 1979; Hammond and English, 1987). EGF/TGF-c have also been shown to have effects on steroidogenesis. EGF)TGF-a inhibited FSH-stimulated estradiol production in rats (Hsueh et al., 1981; Adashi and Resnick, 1986 ; Adashi et al., 1987) and human granulosa cell cultures  (Steinkampf et al., 1988). In humans, EGF’ stimulated basal and hCG-stimulated progesterone production from luteinized granulosa cells (Richardson et al., 1989). In summary, EGFIPGF-oL modulated steroidogenesis and granulosa cell differentiation. This is supported by the fact that EGF receptors are found in the ovary. Also, the ovary is a site of EGF synthesis since both immunoreactive EGF and EGF mRNA have been localized in the ovary.  46  iii) Transforming growth factor-B (TGF-B) The TGF-13 belongs to a family of peptides that includes inhibin, activin, and mullerian inhibiting substance (for review see Knecht et al., 1989). The protein is highly conserved among species. The cDNA sequence of TGF-13 shows complete homology for the human, porcine and bovine polypeptides (Sporn et al., 1987). Since its isolation in 1981 by Roberts and colleagues, TGF-B has been found to have many effects in tissues and cells. TGF-B is considered to be an intraovarian regulator of ovarian function. Ovarian thecallinterstitial cells (Skinner et al., 1987) and granulosa cells (Kim and Schomberg, 1989; Mulheron and Schomberg, 1990) have been identified as sites of TGF-B synthesis, and steroid synthesis in both cell types is influenced by treatment with TGF-13 in vitro. In rat granulosa cell cultures, treatment with TGF-13 stimulated FSH-induced estradiol and progesterone secretion (Adashi and Resnick, 1986; Hutchinson et aL, 1987; Ying et al., 1986; Dodson and Schomberg, 1987). TGF-13 also acts on the thecal cells to increase progesterone production but inhibits androgen secretion (Magoffin et al., 1989). TGF-13 also affects other aspects of cellular differentiation. It has a stimulatory effect on ESH-induced LH receptor induction in rat granulosa cell cultures (Dodson and Schomberg, 1987). TGF-13 can act as a positive or negative regulator of growth in many different cells (Sporn et al., 1986). Likewise, the effects of TGF-B on the replication of granulosa cells are also varied and dependent on the species in question, growth conditions and presence of other growth factors (Knecht et al., 1987; Roberts et al., 1988). In vitro,  47  TGF-13 promotes rat granulosa cell proliferation (Dorrington et al., 1988). In contrast, TGF-13 suppressed the proliferation of bovine granulosa cells stimulated by EGF (Skinner et al., 1987). In summary, TGF-13 has a variety of actions, including effects on granulosa cell proliferation and steroidogenesis, and it can also modulate gonadotropin actions.  W. Signa’ transduction system in the ovary  A. Introduction Ovarian cellular functions are regulated by peptide hormones, neurotransniitters, and nonsteroidal factors. The hormone must bind to its specific receptor in order for a given cell to respond to that hormone. When the hormone receptors are occupied by their specific hormone, a second messenger system is stimulated. The hormones regulate ovarian functions via second messenger systems which enable the hormonal signal to spread rapidly throughout the cell. The three major intracellular signalling pathways that are involved in the mediation of hormone action in the ovary are: 1. cAMP-dependent protein kinase A, 2. protein kinase C, and 3. tyrosine kinase. These pathways are illustrated in figure 4.  1. Cyclic AMP-dependent protein kinase A  The role of cAMP in mediating gonadotropin (FSH and LH) action has been recognized for many years. The steroidogenic effects of FSH on immature granulosa  48  cAMP  1P3  DAG  PKA  Ca2+  PKC  +  4  +  +  Tyrosine 1P3  4k  Phosphorylation I dephosphorylation of numerous target-protein substrates. Tyrosine phosphorylation of target proteins  Figure 4. The three main intracellular signalling pathways involved in the mediation of hormone action in the ovary (AC=adenylate cyclase, PKA=protein kinase A, PLC= phospholipase C, DAG= diacyiglycerol, PKC= Protein kinase C, 1P = inositol 3 triphosphate). 49  cells and LH on thecal cells and mature cells are mediated through intracellular production of cAMP (for review see Leung and Steele, 1992). hCG also mediates its steroidogenic actions via the stimulation of cAMP. Both LH and hCG share the same receptors (Ascoli and Segaloff, 1989). The mechanism of action of inhibin family of peptides is not elucidated yet. It has been speculated that stimulation of granulosa cell steroidogenesis by activin may involve the enhancement of cAMP action (Miro et al., 1991). The mechanism of action of follistatin is not known yet, and the follistatin receptor has not yet been cloned. Hormonal activation of cA1VIP formation involves the action of three proteins: 1. the receptor; 2. a guanine nucleotide coupling protein (G protein) comprising three subunits a, 13 and 6 subunits; and 3. adenylate cyclase (Johnson and Dhanasekaran, 1989; Neer et al., 1990). Receptors which lead to the activation cAMP are transmembrane glycoproteins with the hormone-binding site on the outer membrane surface and a signalling domain at the cytoplasmic face of the membrane. Membrane receptors are associated with two classes of G proteins; G is responsible for the activation of adenylate cyclase, while G 1 is involved in the inhibition of this enzyme. Binding of hormone to its receptor triggers a conformational change in the G. In its inactivated form the G nucleotide binds guanine diphosphate (GDP). Activation of G proteins occurs when hormone occupies the receptor, releasing GDP from the G protein and allowing guanine triphosphate (GTP) to bind in its place. Once GTP binds to the a-subunit of G, the a-subunit dissociates from its 13-6 subunits and the receptor itself. The free a-subunit will then activate the adenylate cyclase (Michell,  50  1989). The activation of adenylate cyclase leads into the hydrolysis of ATP to cyclic AMP which then activates the cAMP-dependent protein kinase A in the cytoplasm. Binding of cAMP to the protein kinase A allows for the phosphorylation of protein substrates which leads to the stimulation of steroidogenesis (Kurten and Richards, 1989). Alternatively, receptor-mediated inhibition of adenylate cyclase activity involves the activation of G 1 proteins. Deactivation of adenylate cyclase results from GTP hydrolysis to GDP, which terminates G 8 protein activation. The final step in adenylate cyclase deactivation comes with the reassociation of a-subunit to the J3complex (Birnbaumer et al., 1990).  2. Calcium and protein kinase C pathway Autocrine and paracrine information affecting steroid production in the ovarian cells also involves inositol phospholipid hydrolysis in the plasma membrane (Leung and Wang, 1989). Among the nonsteroidal products that may regulate steroid hormone productions via the inositol phospholipid hydrolysis are Ang II. Ang II has been shown to stimulate polyphosphoinositide turnover in several tissues including vascular smooth muscle (Dostal et al., 1990), adrenal glomerulosa (Kojima et al., 1985; Spat, 1988), and heart (Baker et al., 1989). However, there is conflicting evidence regarding the role of Ang II in calcium mobilization. Ang II was found to stimulate a rapid, transient increase in intracellular calcium levels in rat granulosa cells (Wang et al., 1989), but not in human granulosa cells (Currie et al., 1992). This response was completely blocked by an Ang II antagonist, saralasin, suggesting a  51  receptor-mediated mechanism. In contrast, Pucell et al. (1991) failed to show any effects of Ang II on intracellular calcium levels, phosphoinositide turnover or cAMP production in rat granulosa cells. Post-receptor mechanism of Ang II requires further investigation. Some growth factors such as EGF also utilize inositol lipid hydrolysis (For review see Catt et aL, 1991). The role of second messengers derived from inositol lipids was recently reviewed by Catt et al. (1991). Receptors using inositol lipid hydrolysis pathway transmit information by a G protein which activates the enzyme phospholipase C (PLC). PLC then metabolizes inositol phospholipids to inositol triphosphate and dliacylglycerol (DAG) (Berridge, 1987b; Taylor et al., 1986; Berridge and Irvine, 1989). Inositol triphosphate in turn induces an increase in intracellular calcium levels by causing the release of calcium from the endoplasmic reticulum (Burgess et al., 1984), while DAG activates calcium-dependent protein kinase C (Nishizuka, 1988). Protein kinase C causes the phosphorylation of cellular proteins. This signal pathway based on inositol triphosphate/calcium and DAG/protein kinase C is used for a variety of actions in the ovarian tissue, and ovarian follicles and corpora lutea are sites of protein kinase C activity (Noland and Dimino, 1986).  3. Tyrosine Kinase  The third postreceptor signalling pathway in the ovary involves tyrosine kinases. Granulosa cells possess other receptors which regulate intracellular kinase activity. Receptors in this group include those of EGF/TGFx (Feng et al., 1987; Waterfield,  52  1989) and IGF-I (Adashi et al., 1985b, Gates et al., 1987). Factors which stimulate tyrosine kinases promote cell proliferation and positively or negatively regulate gonadotropin-stimulated steroidogenesis. Growth factors have also been shown to induce tyrosine phosphorylation of PLC which will lead into inositol phosphate hydrolysis (Berridge, 1987a; Wahl et aL, 1988). Very little is known yet about the mechanism of action of TGF-13. Unlike other growth factors, TGF-B does not activate a tyrosine kinase (Sporn et al., 1987) and has negligible effects on cAMP production (Knecht et al., 1986; Dodson and Schomberg, 1987). The tyrosine kinase postreceptor pathway differs from the cAMP and protein kinase C pathway in that the binding of ligand to its receptor does not involve a G protein. The ligand-binding and tyrosine kinase domains of the receptor are on different portions of a single polypeptide chain spannlng the plasma membrane: the ligand binding site is outside coupled to tyrosine kinase inside. Binding of ligand to the extracelluar binding sites activates intracellular tyrosine kinase, promoting the phosphorylation of tyrosine residues in protein involved in cell growth and/or differentiation (Michell, 1989).  53  CHAPTER TWO: OBJECTWES  L Background and rationale The concept that regulation of ovarian function involves the actions of local regulators has gained increasing acceptance in recent years. This stems from two main lines of evidence. Firstly, it is not possible to explain all of the processes of ovarian differentiation and function simply by changes in the patterns of secretion of pituitary gonadotropins, FSH and LH. For instance, not all follicles in a given ovary respond to pituitary gonadotropins during a given cycle. The hormonal profiles of follicular fluid differ among the follicles. Only a limited number of selected follicles ovulate during the life span of the female while most of the follicles become atretic. The variable fate of ovarian follicles subjected to comparable gonadotropic stimulation suggests the existence of additional intraovarian mechanisms. Intraovarian control is likely exerted by means of local steroidal modulation. Secondly, there is increasing evidence for the existence of substances in ovarian tissues and fluids which are able to act locally, either alone or by modulating the actions of gonadotropins, to modulate steroid production. In recent years, measurable amounts of a number of other elements besides the ovarian steroids have been found and isolated in the ovary, including imhibin (Robertson et al., 1985; Rivier et al., 1985), activin (Vale et al., 1986; Ling et al., 1986), follistatin (Ueno et al., 1987), and angiotensins (Culler et aL, 1987; Lightman et at, 1988). Study of the local actions of these hormones has become important to the further understanding of ovarian  54  functions. Local actions of these putative intraovarian regulators can be demonstrated by the following method. In vitro culture of ovarian cells can be used to demonstrate an action of these local regulators on basal and gonadotropin-stimulated production of steroids. Granulosa cells which are one of the steroidogenic type of ovarian cells are used in this study to demonstrate the actions of these substances on steroidogenesis. The two main types of steroids produced by the ovary are estrogen and progesterone. The process of steroidogenesis as it occurs in granulosa cells is regulated by enzymes. First to be synthesized is the rate limiting enzyme cytochrome P450 scc, which converts cholesterol to pregnenolone and further to progesterone (Miller, 1988). The second enzyme is P450-aromatase which converts androgens to estrogens. The granulosa cells can synthesize progesterone but cannot produce ancirogens. In vivo, androgens diffuse into the granulosa cells from the theca cells where they are acted upon by P450-aromatase enzyme, which converts androgens to estrogens (Ryan and Petro, 1966). In vitro granulosa cells lack the thecal contribution of androgens found in vivo. Thus, androstenedione (a form of androgen which exists in theca cells) at low concentrations of 5x10 M was added as substrate for estradiol 7 formation to the in vitro cultured granulosa cells in this study (Benoit et al., 1988; Rabinovici et al., 1992). Local regulators can also affect gonadotropin-stimulated steroid production. The gonadotropin used in these experiments was hCG. The use of hCG as a gonadotropin was preferred over LH or FSH for several reasons: 1. It is difficult to obtain purified  55  FSH or LH, 2. hCG has a long half-life and it occupies the same receptors as LH, 3. hCG causes an increase in both estradiol and progesterone production. 4. hCG is easily obtained.  II. Hypothesis and Objectives  The hypothesis to be tested is that in addition to the gonadotropins, there is an autocrine mechanism controlling human ovarian steroid hormone production. This intra-ovarian regulatory mechanism involves inhibin, activin, follistatin, and/or angiotensins. The principal objective of this thesis project was to examine whether human  granulosa cell steroid (progesterone & estradiol) production is under the control of locally produced regulators such as activin, inhibin, follistatin, and angiotensins within the ovary. Study of the role of these local regulators on granulosa cell differentiation and steroid production has mainly been limited to small laboratory mammals. Few studies of ovarian regulation have utilized human granulosa cells. In vitro culture of granulosa cells was used to demonstrate the action of these regulators on basal and gonadotropin-stimulated progesterone and estradiol production. Using this system, the following specific objectives were addressed:  1.  To investigate the effect ofinhibin on basal and gonadotropin-stimulated progesterone and estradiol production in cultured human granulosa cells.  56  2.  To investigate the effect of activin on basal and gonadotropin-stimulated progesterone and estradiol production in cultured human granulosa cells.  3.  To investigate the effect of follistatin on basal and gonadotropin stimulated progesterone and estradiol production in cultured human granulosa cells.  4.  To investigate the interaction between follistatin (an activin binding protein) and activin on basal and gonadotropin-stimulated progesterone and estradiol production in cultured human granulosa cells.  5.  To investigate the effect of angiotensins (Ang II and Ang III) on basal and gonadotropin-stimulated progesterone and estradiol production in cultured human granulosa cells.  57  CHAPTER THREE: MATERIALS & METHODS  L Human granulosa cell culture system A. Human granulosa cell preparation The use of human granulosa cells was approved by the Clinical Screening Committee for Research and Other Studies Involving Subjects of the University of British Columbia. Human granulosa cells were harvested during oocyte collection in the University of British Columbia In Vitro Fertilization Program (IVF). Follicular development had been stimulated by using two main protocols: 1. a combination of human menopausal gonadotropins (Serono) and a GnRH analogue (Serono), 2. a combination of human menopausal gonadotropins and clomiphene citrate (Serono) until adequate response was achieved. The criteria for adequate response included three follicles >16 mm in diameter. Final maturation of the oocytes was effected with human chorionic gonadotropin (hCG, 10,000 IU, Serono). Retrieval of the oocytes was accomplished 32 h after the injection with hCG. Granulosa cells were then harvested from the follicular fluid contents after the oocyte was identified for in vitro fertilization. The method for the cell culture of granulosa cells is outlined in figure 5. The freshly harvested granulosa cells were layered onto 40% percoll (Sigma, St. Louis, MO) and 60% Hank’s balance salt solution (Gibco, Burlington, ON) and centrifuged at 1700 x g for 20 mm at 20°C. The cells were then washed and resuspended in  58  Layer cells on single density 40% percoll gradient Cells Percoll gradient  Centrifuge (20 mm, 1700 g, 25 C)  Resuspend cells in 25 ml M 199 containing 10% FBS  Red blood cells Culture in 48-well culture dishes for 48 hr. (0.5 mI/well) 1. Replace media with M199 containing 2% FBS and incubate cells for 48 hr.  iII  2. Replace media with M199 containing 0.5% BSA and various treatments and incubate cells for 24 hr.  0 00 0  ) 0 L  LI  ‘-1  Q  0000  C  Figure 5. Diagrammatic representation of protocol for human granulosa cells processing and plating for culture. Cells were centrifuged on a Percoll gradient to exclude hematocytes. The cells were then plated at a density of 0.2-1 x iO cells/mi . 2 in 48-well cell culture plates and cultured at 37°C in humidified air with 5% CO  59  media 199 (M199, Gibco) supplemented with 10% fetal bovine serum (FBS,Gibco), sodium penicillin (100 lU/mi, Gibco) and streptomycin (100 uglml, Gibco). The cells were counted using a hemocytometer (Improved Newbauer hemocytometer, Ingram & Bell, London, ON). The cells were then plated at a density of 0.2-1 x iO cells/mi and cultured in 48-well culture plates (Celiwell, Corning), 0.5 ml per well in replicates of 8 wells/treatment. The incubation media were replaced with M199 containing 2% FBS at 48 h and subsequently with M199 containing 0.5% bovine serum albumin (BSA, Sigma) and the various treatments at 96 h after plating. The culture media was collected 24 hour after treatment and concentrations of progesterone and estradiol in media were determined by radioimmunoassay (RIA). Granulosa cells were incubated at 37°C in humidified air with 5% CO . All procedures 2 were carried out under sterile conditions in a biosafety hazard hood (Biological Safety Cabinet Model 1128, Forma Scientific).  B. Experimental designs 1. Effects of activin-A or inhibin-A on basal and hCG-stimulated steroidogenesis The effects of inhibin-A or activin-A on basal and hCG-stimulated estradiol and progesterone production were investigated by adding recombinant human activin-A or inhibin-A (3.6 nM, and 3 nM respectively, Genentech, San Francisco, CA) in M199 with 0.5% BSA, with or without hCG (1 lU/mi, Sigma). Androstenedione (5 x 10 M, 7 Sigma) was added to culture medium as substrate for estradiol formation. Estradiol  60  and progesterone concentrations were measured in medium collected after 24 h incubation. This experiment was performed 3 times with granulosa cells from 3 different 1VF patients (n=8 wells per treatment in each experiment).  2.  Dose-dependency  and  time-course  study  of activin-A on basal  progesterone and estradiol production Increasing doses of activin-A (0.1 to 3.6 nM) with androstenedione (5 x 10 M) 7 were added to the cultured cells and incubated for 24 h. Estradiol and progesterone concentrations were measured in medium collected after 24 h incubation. This experiment was performed 3 times with granulosa cells from 3 different IVF patients (n=8 wells per treatment in each experiment). For time-course analysis, androstenedione (5 X 10 M) with and without activin-A (3.6 nM) was added to the 7 cultured cells and incubation media was collected after 6, 12, 24, and 48 h for estradiol and progesterone analysis.  3. Dose-dependency study of activin-A on hCG- stimulated progesterone production Increasing doses of activin-A (0.1 to 3.6 nM) were added with hCG (1 lU/mI) to the cultured cells and incubated for 24 h. Progesterone concentrations were measured in medium collected after 24 h incubation. This experiment was performed 2 times with granulosa cells from 2 different 1VF’ patients (n=8 wells per treatment in each experiment).  61  4. Effects of foffistatin-288 on basal and hCG-stimulated steroidogenesis The effects of follistatin (recombinant human follistatin with 288 amino acids was expressed in Chinese hamster ovary cells under the control of the simian virus-40 promoter, as detailed in a previous report by Inouye et al., 1991 was used in this study. Follistatin-288 is a more potent FSH suppressor than the native porcine follistatin.) on basal and hCG-stimulated estradiol and progesterone production were investigated by adding recombinant human follistatin (3 nM, NIH) in M199 with 0.5% BSA, with or without hCG (1 lU/rid). Androstenedione (5 x 10 M) was added to 7 culture medium as substrate for estradiol formation. Estracliol and progesterone concentrations were measured in medium collected after 24 h incubation. This experiment was performed 5 times with granulosa cells from 5 different IVF patients (n=8 wells per treatment in each experiment).  5.  Dose-dependency and time-course study  of foffistatin on basal  progesterone and estradlol production Increasing doses of follistatin (0.1 to 3 nM) with androstenedione (5 x 10 M) were 7 added to the cultured cells and incubated for 24 h. Estradiol and progesterone concentrations were measured in medium collected after 24 h incubation. This experiment was performed 2 times with granulosa cells from 2 different 1VF patients (n=8 wells per treatment in each experiment). For time-course analysis, androstenedione (5 X 10 M) with and without follistatin (3 nM) was added to the 7 cultured cells and incubation media was collected after 6, 12, 18, and 24 h for  62  estradiol and progesterone analysis.  6. Combined effects of activin-A and foffistatin on steroidogenesis The effects of activin-A and follistatin on basal and hCG-stimulated estradiol and progesterone production were investigated by adding recombinant human activin-A (3.6 nM) or follistatin (3 nM) in M199 with 0.5% BSA, with or without hCG (1 lU/mi). M) was added to culture medium as substrate for estradiol 7 Androstenedione (5 x 10 formation. The interaction between activin and follistatin on basal and hCG stimulated estradiol and progesterone production was investigated by adding recombinant human follistatin (12 nM) together with activin-A (3.6 nM) in M199 with 0.5% BSA, with or without hCG (1 lU/mi). Cells were incubated for 24 h. Estradiol and progesterone concentrations were measured in medium collected after 24 h incubation. This experiment was repeated 5 times with granulosa cells obtained from 5 different lyE patients (n=6 wells per treatment in each experiment).  7. Effects of Ang II and Ang IH on basal and hCG-stimulated steroidogenesis The effects of Ang II and Ang III on basal and hCG-stimulated estradiol and progesterone production were investigated by adding Ang II or Ang III (10 M, Sigma) 5 in M199 with 0.5% BSA, with or without hCG (1 lU/mi, Sigma). Androstenedione (5 x 10 M) was added to culture medium cultured as substrate for estradiol formation. 7 Estradiol and progesterone concentrations were measured in medium collected after 24 h incubation. This experiment was repeated 4 times with granulosa cells obtained  63  from 4 different IVF’ patients (n=8 wells per treatment in each experiment).  8. Dose-dependency and time-course study of Ang II on basal estradiol production Increasing doses of Ang II (10MM to 5 10 ) with androstenedione (5 x 10 M M) were 7 added to the cultured cells and incubated for 24 h. Estradiol production was measured in medium collected after 24 h incubation. This experiment was repeated 3 times with granulosa cells obtained from 3 different IVE patients (n=8 wells per treatment in each experiment). For time-course analysis, androstenedione (5 X 10 M) 7 with and without Ang II (10 M) was added to the cultured cells and incubation media 5 was collected after 6, 12, 24, and 48 h for estradiol analysis.  C. Hormone analysis Concentrations of progesterone and estradiol in media were determined by validated RIA with specific antisera provided by Dr. D. T. Armstrong of the University of Western Ontario (Katz and Armstrong, 1976; Leung and Armstrong, 1979).  1. Radloimmunoassay for steroids (progesterone and estradiol) Reagents and buffer: 1)  The assay buffer used was 0.1 M phosphate buffered saline (PBS, pH 7.4) supplemented with 0.1% gelatin (PBSG). The 0.1 M PBS buffer consisted of 80  64  gIL of NaCl (BDH chemicals, Vancouver, B.C.), 2 g/L of KC1 (BDH chemicals), HPO (BDH chemicals), 2 g/L of 4 2 Na 11.5 g/L of 4 PO (BDH chemicals), and 2 KH 0.1% thimerosal (BDH chemicals). The buffer was stored at 4°C. 2)  The progesterone (Sigma P0 130) standards used were serially diluted in PBS buffer from an initial 0.32 mM stock solution which was reconstituted in redistilled absolute ethanol. The stock solution was kept at -20°C. A standard curve was set up with 8 reference concentrations ranging from 0.5 to 64 nM.  3)  The estradiol standards (Sigma E8875) used were serially diluted in PBS buffer from an initial 1 miVi stock solution which was diluted in redistilled absolute ethanol. The stock solution was stored at -20°C. A standard curve was set up with 8 reference concentrations ranging from 0.25 to 32 nM.  4)  The progesterone antiserum used was rabbit anti-progesterone provided by D .T.Armstrong, raised against 4-pregnen-613-ol-3,20-dione hemisuccinate:bovine serum albumin conjugate (Steraloids, Wilton, NH). This was used at a final dilution of 1:12500 w/v in PBSG and gave approximately 50% binding of tracer.  5)  Estradiol antiserum was rabbit #3, bled 26/10/82, raised against 1,3,5(10)estratriene-3 17B-diol-6-one-6-carboxymethyl-oxime:BSA conjugate (Steraloids). ,  This was used at a final dilution of 1:200,000 w/v in PBSG and gave approximately 60% binding of tracer. 6)  The progesterone labelled hormone used was 3 H-progesterone (Amersham, Oakville, ON). The labelled hormone had a specific activity of approximately 90-130 CiJmmol. The initial stock was 250 pCiJul in toluene:ethanol (9:1, v:v).  65  Solvents were evaporated and initial stock was further diluted in 10 ml ethanol  and stored at -20°C. The stock was further diluted in PBS such that 80 p1 in 10 ml of PBS gave 10000 cpm. 7)  Labelled hormone for estradiol was 2,4,6,7,16,17H-E2 (Amersham) with a 3 specific activity of 140-170 Ci/mmol. The initial stock was 250 jiCi/ul in toluene:ethanol. Solvents were evaporated and initial stock was diluted in 10 ml ethanol and stored at -20°C. The stock was further diluted in PBS such that 80 p1 in 10 ml of PBS gave 10000 cpm.  5)  The separation reagent was charcoal/dextran solution with 0.025% dextran (Sigma) and 0.25% charcoal (BDH chemicals) dissolved in PBSG.  6)  The scintillation cocktail used was Scintiverse (Fisher Scientific).  Protocol 1)  100 p,l of steroid (progesterone or estracliol) standard or sample solution was added to 10 x 75 mm assay tubes (Fisher scientific). Each standard was assayed in triplicate and each sample was assayed in duplicate.  2)  100 p1 of diluted antibody was added to each tube except the tubes containing the tracer only which is referred to as the total binding tube.  3)  100 p1 3 H-progesterone or 3 H-estradiol (10,000 cpm) was added to each tube.  4)  All tubes were then vortexed, and incubated at 4°C for a period of 20-24 h.  5)  500 p1 of charcoalldextran solution was added to each tube, followed by vortexing.  66  6)  Tubes were then incubated for 10 mm at 4°C.  7)  Tubes were then centrifuged at 2500 x g for 10 mm at 4°C.  9)  The supernatant was then immediately transferred into polyethylene scintillation vials (Ingram & Bell Scientific, Vancouver, B.C.)  10)  3 ml scintillation cocktail was added to each tube. The tubes were then vortexed and were counted by LKB 13-counter for one minute.  The sensitivity of progesterone assay was 0.5 pmollml of estradiol assay was 0.25 pmollml  =  =  160 pg/nil. The sensitivity  60 pg/mI. The intra-assay and inter-assay  coefficients of variation for the progesterone RIA were 7.1% and 8.8%, respectively, and for estradiol RIA were 3.8% and 6.6% respectively.  2. Statistical analysis Statistical significance of the data were determined by one way analysis of variance followed by Scheffe’s multiple comparisons test (pc0.05). In some cases, estradiol and progesterone values are reported as the mean±SEM. Mean estradiol and progesterone levels for control and treatment groups were compared between groups by one-way analysis of variance. Scheffe’s test was used to differentiate between means after a significant F test. In other cases, the data were pooled from all replicates in separate cell culture experiments and expressed as percentages of control values.  67  II. Protein measurement of granulosa cells in culture The protein assays were performed in order to assess the changes in the protein content of granulosa cells after treatment with inhibin-related peptides. The design of the protein measurement experiments was as follows: 1. The granulosa cells were cultured in the same way as described previously. 2. Cells were collected on day 1, 3, and 5 of the culture and 24 h after treatment with activin, inhibin or follistatin. 3. The cells were detached from the 48 well cell culture plates by the use of a sonicator (Fisher Sonic Dismembrator Model 300). The media was removed from the culture wells and was replaced with 0.5 ml of PBS buffer. The cells were then sonicated at 10 W for 20 seconds. The sonicator not only removed the cells from the bottom of the wells but also broke down the cell membrane. 4. The 0.5 ml PBS buffer containing the cells was then transferred to the microcentrifuge tubes. The cells were then centrifuged at 10,000 cpm for 15 minutes. 5. The supernatant was then carefully removed and transferred to another microcentrifuge tube and it was stored at -20°C until the time of protein assay.  A. Lowry Protein Assay Reagents All of the reagents were prepared in distilled water (dH O). 2 1.  Na-K tartarate  (2.5 g/40m1, BDH Chemicals)  2.  4 CuSO  (1.0 g/40m1, BDH Chemicals)  68  3.  3 solution: a. NaOH (4 gfL, BDH Chemicals) NaHCO b. 3 CO (20 gIL, BDH Chemicals) 2 Na  4.  The Lowry reagent which was prepared fresh every time. The Lowry reagent consists of: a. 0.8 ml Na-K tartarate b. 0.2 ml CuSO 4 3 c. 50.0 ml NaHCO  5.  iN Folin-Phenol reagent (BDH Chemicals)  Standards 1.  The stock solution was 500 mg bovine serum albumin in 10 ml of CIH O. 2 For the working solution, 200 p1 of the stock solution was diluted in lOmi of distilled water to give a concentration of 1 pg/j.il. A standard curve was set up with 8 reference concentrations ranging from 0 to 35 pg.  Protocol Samples and standards were assayed in duplicates. Assay tubes used were 16 x 100 mm glass tubes (Fisher Scientific). 1.  300 p1 of standards (100 ul of standard  +  200 p1 of distilled water) and  300 p1 of samples were added to the assay tubes. 2.  2 ml of freshly prepared Lowry reagent was added to all the tubes, followed by vortexing.  3.  0.2 ml of iN Folin-Phenol reagent was then added to all the tubes,  69  followed by vortexing. 4.  The samples were then heated in a water bath at 60°C for 10 minutes. The samples were allowed to cool to room temperature.  5.  The amount of protein in each sample was then measured by reading the absorbance at 660 nm in the spectrophotometer (SP8-400 UV/VTS).  6.  In order to find out the amount of protein in each sample the absorbance of each sample recorded by the spectrophotometer, the linear regression program was used in order to determine the amount of protein in the samples. The sensitivity of the protein assay was 5 jig/jil.  Statistical Analysis Statistical significance of the data were determined by one-way analysis of variance followed by Scheffe’s test (p<0.05).  B. Protein assay experiments 1. Effect of inhibin, activin, and foffistatin on protein content of human granulosa cells In order to assess whether or not the protein concentration changed throughout the cell culture period, the protein concentrations were measured on day 1, 3, 5 of culture and 24 h after the application of treatments with inhibin (3 nM), activin (3.6 nM), or follistatin (3 nM). This experiment was performed 4 times with granulosa cells from 4 different 1VF patients (n=8 wells per treatment in each experiment).  70  CHAPTER FOUR: RESULTS  I. Static culture experiments A. Effect of activin-A on basal and hCG-stimulated estradiol production  Activin-A (3.6 nM) exhibited a stimulatory effect on basal estradiol levels (Fig.6, p<0.Ol). By comparison, estradiol concentrations were increased to approximately the same extend by 1 lU/mi hCG (p<O.Ol). The effect of activin-A on estradiol production was dose dependent and became significant at a concentration of 0.4 nM (Fig.7, p<O.Ol). Time-course experiment of action of activin-A (3.6 nM) on basal estradiol levels revealed that estradiol production was stimulated in a time-dependent manner. Estradiol production was stimulated significantly at 24 and 48 h of treatment (Fig.8,  p<0.Ol). Estradiol production was increased by hCG (1 lU/rn]., Fig.6, p<0.Ol). Activin A (3.6 nM) did not affect hCG-stimulated estradiol production.  B. Effect of activin-A on basal and hCG-stimulated progesterone production Activin-A (3.6 nM) exhibited a stimulatory effect on basal progesterone levels (Fig.9, p<O.0l). As shown in Fig.10, dose response studies revealed that the stimulatory effect of activin-A on basal progesterone production was significant at a concentration of 3.6 nM (p<0.05). Time-course experiment of action of activin-A (3.6 nM) on basal progesterone levels revealed that progesterone production was stimulated in a time-dependent manner. Progesterone production was stimulated  71  b  1200  b  I I I  —  0  i... I—  1000  O C—) 0  I  I  b I I  ‘  8O0  .‘  ‘  ‘  ‘  ‘  .‘  ‘  ‘  o C  o  600  C.)  .D 0  D—  400.  —  O  .  CU  .%%%\%  200  C,)  a ______  W  I,,•,,,-,•,-i  0  I,‘‘‘‘I ‘‘‘‘‘  control  ‘\S%\’  __..__, _________  —  hCG  —  activin  hCG+  activin  Figure 6. The in vitro stimulation of basal estracliol production by activin-A in human granulosa-luteal cells. Cells were cultured for 24 h in medium (control), hCG (1 lU/mi), activin-A (3.6 nM), hCG+activin-A in the presence of androstenedione (5 x 10 M). The data were pooled from 3 separate experiments (n=8 wells per treatment 7 in each experiment) and expressed as a percentage of control±SEM. Different letters above the SEM bars denote statistical significance (b differs from a and is significant at p<O.Ol by one-way analysis of variance followed by Scheffe’s test). 72  12 (I) —  **  11  **  .  Lf)  0  o 1  10.  control  .1  .4  1.2  3.6  Activin (nM)  Figure 7. Dose-dependent effects of activin-A on basal estradiol production by human granulosa-luteal cells. Cells were cultured for 24 h in medium (control) or medium supplemented with different doses of activin (0.1-3.6 nM) in the presence of androstenedione (5 X 10 M). Values are meam±SEM of eight replicate wells of one 7 experiment. Similar results were obtained in two other separate experiments. (** p<O.Ol by one-way analysis of variance followed by Scheffe’s test). 73  **  0.7  control Activin  G) C.)  **  0.6  LC)  0  E  C  0.5  0.4  C  30  20  40  50  Time (hr)  Figure 8. Time-course effect of activin-A on basal estradiol production by human granulosa-luteal cells. Cells were cultured for 6, 12,24, and 48 h in medium (control), M). Values are 7 or activin (3.6 nM) in the presence of androstenedione (5 x 10 mean±SEM of eight replicate wells of one experiment ( ** p<O.Oi by one-way analysis of variance followed by Scheffe’s test). 74  b  500  -ö C  0 C.) 0 C  C  300  C  I-  0  /  0 0 G) C  a  0 1  /<‘7  C) Cl) C)  0 0 0  control  activi n  hCG  hCG+ activin  Figure 9. Effect of activin-A on basal and hCG-stimulated progesterone production by human granulosa-luteal cells. Different letters above the SEM bars denote statistical significance (b differs from a, c differs from a and b and is significant at pO.Ol by one-way analysis of variance followed by Scheffe’s test). (See legend to figure 6 for details). 75  3. *  0 L() 0  E  2  C C  0 0  I  1•  II  C 0 C) Cl) C)  0) 0  L.  0• Control  .1  .4  1.2  3.6  Activin (nM)  Figure 10. Dose-dependent effects of activin-A on basal progesterone production by human granulosa-luteal cells (See legend to figure 7 for details). * p<0.05.  76  t2  —ci——  Control Activin  *  *  **  C.) If)  o  E 0.8  0 o  0.6  0 C  0 0 0)  0  0.2  0  I  I  10  20 Time  30  40  50  (hr)  Figure 11. Time-course effect of activin-A on basal progesterone production by human granulosa-luteal cells (See legend to figure 8 for details). ** p<O.Ol. 77  0.4 C) C.)  0  E  03  C.)  Activin (nM)  Figure 12. Dose-dependent effects of activin-A on hCG-stimulated progesterone production by human granulosa-luteal cells. Cells were treated with hCG (1 lU/mi), and different doses of activin (0.1-3.6 nM) in the presence of hCG for 24 h. Values are mean±SEM of eight replicate wells of one experiment. Similar results were obtained in one other separate experiments (* p<O.O5 by one-way analysis of variance followed by Scheffe’s test). 78  signfficantly at 24 and 48 h of treatment (Fig.11,  p<O.Ol). Progesterone production  was increased by hCG (1 IU/rril, F’ig.9, p<O.Ol). Activin-A (3.6 nM) significantly inhibited hCG-stimulated progesterone production (Fig.9, p<O.Ol). The inhibitory effect of activin-A on progesterone production in response to hCG was dose-dependent and became significant at a concentration of 1.2 nM (Fig.12, p<O.05).  C. Effect of inhibin-A on basal and hCG-stimulated progesterone and estradiol production Inhibin-A (3 nM) did not affect basal estradiol production (Fig.13). Estradiol production was increased by hCG (1 IU/ml, Fig.13, p<O.Ol). Inhibin-A (3 nM) did not affect hCG-stimulated estradiol production. Inhibin-A (3 nM) also did not affect basal progesterone production (Fig. 14). Progesterone production was increased by hCG (1 IU/ml, Fig.14, p<O.Ol). Inhibin-A (3 nM) did not affect hCG-stimulated progesterone production.  D. Effect of foffistatin on basal and hCG-stimulated estradiol production The effect of follistatin on estradiol production was examined in the presence or absence of hCG (Fig.15). Follistatin (3 nM) exhibited a stimulatory effect on basal estradiol levels (Fig.15, p<O.O5). The effect of follistatin on estradiol production was dose dependent and became significant at a concentration of 1 nM (Fig.16, p<O.Ol). Time-course experiment of action of follistatin (3 nM) on basal estradiol levels revealed that estradiol production was stimulated in a time-dependent manner.  79  1200b —  0  I I  1000  O C.) IS  b I  800  ‘ f_/f,,,, f_f,,,,, f/_f,,,,  o C  o  ,,,,/_,,  600  (.)  .D O L.. D.. — O .D CU  __,__,__  ,,,_,,__  ,,,_,,__  ,,,,,___ ‘.%%\‘\  •///  400  “  ,,,,,,,,  -  “ ‘ .,,_,_,,,  ‘  ‘f__f,,,,  if,,,,,, ,,,,,_,,,  200-  a  ‘‘‘‘‘‘‘‘ ‘‘‘‘‘‘‘‘  Cl,  a  ‘‘‘ii,,, ‘‘‘if,,,  W  ‘i_i,,,, ___________  ‘‘‘‘‘‘‘‘  0-  ________________  control  __________  hCG  L,,’,,’4 ‘‘‘‘‘“‘I Inhibin  if’’.,, ___________ __________  hCG÷ Inhibin  Figure 13. Effect of inhibin-A on basal and hCG-stimulated estradiol production by human granulosa-luteal cells. Cells were cultured for 24 h with hCG (1 lU/mi), inhibin-A (3 nM), hCG-i-inhibin in the presence of androstenedione (5 X 10 M). The 7 data were pooled from 3 separate experiments (n=8 wells per treatment in each experiment) and expressed as a percentage of control±SEM. Different letters above the SEM bars denote statistical significance (b differs from a and is significant at p<0.01 by one-way analysis of variance followed by Scheffe’s test). 80  600 0 C  0 C-)  b  b  500  0 400C  0 0  0 0 a) C 0 a) Cl) C) 0) 0 I  300-  200 a 100  a-  0  Li  Control  hCG  Inhibin  hCG+ Inhibin  Figure 14. Effect of inhibin-A on basal and hCG-stimulated progesterone production by human granulosa-luteal cells (See legend to figure 13 for details). 81  Estradiol production was stimulated significantly at 18 and 24 h of treatment (Fig.17, p<0.01). Follistatin did not affect hCG-stimulated estradiol production, i.e. in cells treated with follistatin (3 nM) and hCG (1 IU/ml) concomitantly, follistatin did not further augment hCG-stimulated estradiol secretion (Fig. 15, p>0.05).  E. Effect of follistatin on basal and hCG-stimulated progesterone production The effect of follistatin was examined in the presence and absence of hCG (Fig. 18). Follistatin (3 nM) stimulated basal progesterone production in the absence of hCG (Fig. 18,  p<O.Ol). The effect of follistatin on progesterone production was dose-  dependent and became significant at a concentration of 0.1 nM (Fig. 19, p<O.05). Time-course experiment of action of follistatin (3 nM) on basal progesterone levels revealed that progesterone production was stimulated in a time-dependent manner. Progesterone production was stimulated significantly at 18 and 24 h of treatment (Fig.20, p<0.Ol). Follistatin did not affect hCG-stimulated progesterone production, i.e. in cells treated with follistatin (3 nM) and hCG (1 111/mi) concomitantly, follistatin  did not further augment hCG-stimulated progesterone production (Fig.18, p>O.O5).  F. Interaction between activin and follistatin on basal and hCG-stimulated estradiol production The combined effect of follistatin (activin binding protein) and activin on estradiol production in human granulosa cells was examined in the presence and absence of hCG (Fig.21). Follistatin at a concentration of 3 nM did not affect basal or  82  b  1200 b —  I  o  I  I  C O  I  C.) 0 C  o  8O0  b  600  O .D 0 O—  4 00  —  O .  CU  200  a  I-  Co  ‘/,,——, ___________  W  0  I”  ‘A  i—i—’—  __,_,_,.1  ________  _____,_.1  _______F % ‘ ‘. %  ‘  ,,4 ‘ ‘  control  ____‘  tollistatin  hCG  —  %  hCG÷ follistatin  Figure 15. Effect of follistatin on basal and hCG-stimulated estradiol production by human granulosa-luteal cells. Cells were cultured in medium (control), hCG (1 RJ/ml), follistatin (3 riM), hCG+follistatin in the presence of androstenedione (5 X 10 M). The 7 data were pooled from 5 separate experiments (n=8 wells per treatment in each experiment) and expressed as a percentage of control±SEM. Different letters above the SEM bars denote statistical significance (b differs from a and is significant at p<O.O5 by one-way analysis of variance followed by Scheffe’s test). 83  **  400  300  **  200 0 0 L.  a 100 Cu L.  Cl)  Ui  0  control  I  I  I  I  .1  .3  1  3  Follistatin (nM)  Figure 16. Dose-dependent effects of follistatin on basal estracliol production by human granulosa-luteal cells. Cells were cultured for 24 h in medium (control) or medium supplemented with different doses of follistatin (0.1-3 nM) in the presence of androsteneclione (5 X 10 M). The data were pooled from 2 separate experiments 7 (n=8 wells per treatment in each experiment) and expressed as a percentage of the control±SEM ( ** p<0.01 by one-way analysis of variance followed by Scheffe’s test). 84  30  control Follistatin  °  0  **  25  If)  **  0 1  20  E C  o  15  0  o I  0  Cu Cl)  w  0 0  5  10  15  20  25  Time (hr)  Figure 17. Time-course effect of follistatin on basal estradiol production by human granulosa-luteal cells. Cells were cultured for 6, 12, 18, and 24 h in medium (control), or follistatin (3 nM) in the presence of androstenedione (5 x 10 M). Values are 7 mean±SEM of eight replicate wells of one experiment ( ** p<O.O1 by one-way analysis of variance followed by Scheffe’s test). 85  500  b b  400  b  4  //// / / :.•::.:‘.:.:•::..-::.:.><.<  300  0  /  /  •:z--’/ -//,Q..  0  0 0  200-  0  a  /  //  // ,//  /  i/  Ib0:  control  hCG  follistatin  hCG+ follistatin  Figure 18. Effect of follistatin on basal and hCG-stimulated progesterone production by human granulosa-luteal cells (See legend to figure 15 for details). 86  **  500 0 400 ‘4-  0 **  300  o  *  L..  0 C)  .S  Cl) C)  II  io  0 0  0 Control  I  I  I  .1  .3  1  3  Follistatin (nM)  Figure 19. Dose-dependent effects of follistatin on basal progesterone production by human granulosa-luteal cells (See legend to figure 16 for details). ** p<O.Ol. 87  120  •  *  Control Follistatin  T  0 LC)  100  .  **  I  110  1’5  2’O  2’5  Time (hr)  Figure 20. Time-course effect of follistatin on basal progesterone production by human granulosa-luteal cells (See legend to figure 17 for details). ** p<O.Ol. 88  hCG (1 IU/ml)-stimulated estradiol production. On the other hand, activin-A (3.6 nM) stimulated basal estradiol production (Fig.21,  p<O.O5)  without affecting hCG  stimulated estradiol production. By comparison, activin stimulated basal estradiol concentrations to approximately the same extent by 1 lU/mi hCG (p<O.O1). When follistatin (12 nM) was added to the cell culture along with activin (3.6 nM), the stimulatory action of activin on basal estradiol production was inhibited.  G. Interaction between activin and follistatin on basal and hCG-stimulated progesterone production The combined effect of follistatin and activin on progesterone production in human granulosa cells was examined in the presence and absence ofhCG (Fig.22). Follistatin at a concentration of 3 nM did not stimulate basal or hCG (1 IU/ml)-stimulated progesterone production. On the other hand, activin-A (3.6 nM) stimulated basal progesterone production (Fig.22, p.<O.05) but did not affect hCG (1 IU/ml)-stimulated progesterone production. When follistatin (12 nM) was added to the cell culture along with activin (3.6 nM), the stimulatory action of activin on basal progesterone production was inhibited.  89  b  1600-  1400 .  C  o  C..)  if,, f__f  1200-  f__i ‘f_f  b  II-  ‘i_i  ‘f_f  b  ‘f_f f_f,  1000-  f__i f_f’ ‘f_f \‘\\  f__f,  C  .2  I-  %\‘‘s  ___ff  800-  o  o  I—  Ll.I  __f_f  f_f_f  f__f,  ___f_ ‘\\\\ f_f_f ‘‘\\\ f__/f \\\%% ____f \%\‘\ f_ff_ \\\‘% f_f__ ‘\•% i__f,  f__f \\%‘.\ f__f ‘\%\\ f___ %\% f__f %%%‘•. f__f •.‘%‘\ /_f_ •.%\ fffi  %‘‘\  %‘.%%  \‘.%\  f_f/f  400  fffff  f__f,  Cu I  f__f,  _ff__ \\\\ f____ %%‘% f_f_f  ffff  .D Cl)  f_f_f  f_f_f  —  __f_f f_f_f f__if  200  ‘‘‘  a  s’\ ‘.‘.%  fffff  s’’’  r’ ‘fl 0 f_f_f ‘••%%  control  s\%\  f__f \\%S% f__f %\%%% f__f %%\\% f__f  f__f,  f_f_f ‘‘ ‘\\ f_f_f \\\ if,,,  [  ifff %%%‘‘.  a  %•.‘% f_i_f  a 1—  ifff  ffff ss’\ f__f %\%%  f_f_I ,fji  iffi ff %%%\% ifff ffff  f_f_f  f_ff %‘‘.% iffi ‘%\‘•.  ffif ‘‘i’’  _/_f_ ‘‘ f/ff %% f_f_f El  activin follistatinactivin+ follistatin  ‘.‘..‘.\  f_f_f %,‘%\ f_f_f \•% f_f_f •.\%\ __f__ %\%\ f_f_f %\\ f_f_f f_f_f f_f__ ‘\\ __f__ •%\‘ ___ff \\\ fff,f  fff f/ ‘%‘S f_f_f f_f_f ‘‘%  fffff \\%\  s%%  \\  fffff  fi \‘%•% fffi  ff/f  fff  ffff  %‘‘.%  ffff  ff-f ‘‘‘‘%  ‘f__f ‘f__f %%%%% ‘f__f ‘f__f %%% ‘f__f %%‘.% ‘f__f f__f ffff \\\‘ \‘\  f_f, \\‘‘.  f/_f f/_f %%%% fff  ‘f ,‘‘‘  f__if %\\ fffff ‘.\‘  f_f_f  f__f  _fff_  ,,_  f__i  __ff  hCG  ‘__f__ %‘.‘.%\ ‘f__f %%%% ‘f_f, \%‘ ‘f_f,  ‘\‘‘  ffff  %.%%‘  f_f,,  _f__f  \\•.\%  f_/f  f_f,  %%‘‘\  ‘‘  f_f_f  f_f_f  f__f, f_f_f  fffff  f_f_f  f__f_  ,I  f__f,  Q—  f__f  f_f_f  ___ff  600-  f__f \‘‘%. \\‘‘  f__f  \\s\’  C.)  .D  %\‘‘  Tb  ‘%\  ‘‘,‘‘  \\\\  /fff ffff \\‘% ‘f//f \%%%%  hCG+ hCG+ hCG+ activin follistatin actlvin+ follistatin  Figure 21. The in vitro effect of activin in combination with follistatin on basal and hCG-stimulated estradiol production by human granulosa-luteal cells. Cells were cultured for 24 h in medium (control), activin (3.6 nM), follistatin (3 nM), activin (3.6 nM) plus follistatin (12 nM), hCG (1 lU/nil), hCG (1 ITJIml) plus activin (3.6 nM), hCG (1 lU/nil) plus follistatin (3 nM), and hCG (1 ITJ/ml) plus activin (3.6 nM) plus M). The data were 7 follistatin (12 nM) in the presence of androstenedione (5 x 10 pooled from 5 separate experiments (n=6 wells per treatment in each experiment) and expressed as a percentage of the control±SEM (b differs from a and is significant at p<O.O5 by one-way analysis of variance followed by Scheffe’s test). 90  600, b  I  400  0  300-  500  0 0 C)  200-  0 C) Cl)  C)  100  0 0  0control  activn follistatln actlvln+ hCG follistatin  hCG+ hCG+ hCG+ activin follistatin actlvln÷ follistatin  Figure 22. The in vitro effect of activin in combination with follistatin on basal and hCG-stimulated progesterone production by human granulosa-luteal cells. (See legend to figure 21 for details). 91  H. Effects of Ang II and Ang III on basal and hCG-stimulated estradiol production Treatment of cells with Ang III (10 M) did not affect basal estradiol production 5 (Fig.23,  p>O.O5);  whereas, treatment of cells with Ang II (10 M) stimulated basal 5  estradiol production (Fig.23, p<O.Ol). The effect of Ang II on basal estradiol production was dose-dependent and became significant at a concentration of 10 M 6 (Fig.24, p<O.Ol). Time-course experiments revealed that basal estradiol production was stimulated significantly by Ang II (10 M) at 24 and 48 h of treatment (Fig.25, 5 p<O.O5). Estracliol concentrations were stimulated by hCG (I lU/mi, Fig.23, pcO.O1). Treatments of cells with Ang II (10 M) or Ang III (1O-5M) did not affect hCG 5 stimulated estradiol production (Fig.23, p>O.O5).  I. Effects of Ang II and Ang HI on basal and hCG-stimulated progesterone production Treatment of cells with Ang II or Ang III (10 M) did not affect basal progesterone 5 production (Fig.26, p>O.O5). Progesterone production was increased by hCG (1 lU/mi, p<O.O1). Ang III (10 M) suppressed hCG-stimulated progesterone production (Fig.26, 5 pcO.O1). On the other hand, Ang II (10 M) did not affect hCG-stimulated 5 progesterone production (p>O.05).  92  1000 b .  I  800  b  0  C) I  I  O 600  ___,,  _,_,_,  _,_,,  ,__,,_  __,_,  O — O  O— a  —  O  ,_,_ ___,_  __,_,_  ,,,,, \%‘\% ,,,__ /_,_,  a  Cl) W  ____  I%\\%%I %‘\%I  ,,,__1  %%‘%I  —,—,,I  _____  _,_,,, _,____  ___,_  ____, \S.%’ __,,, %%% _,_,_  __,,,_ ,,_,_  I,,,,  ,,,l,  ,_,__ ‘‘%%‘% ,__,, %%%\ _,___ \%%%\‘  __.,_  _,‘_,_  ,__,,  ,_,__, ‘s%%%  %‘\‘s%  _,,_,_ \%_\  ‘.‘%‘%  0  control  %%%%%‘ ___,_  ,_,__  %\%\%‘ ___,, %%%.‘‘ _,, %\%% __,,_  ,___,_  \%‘‘ ____  S ‘% _,,___  ,____  __,___ ‘%%% ,_,,,_  _,_,,  ____,_  ,,_,_  ,,_,,  ____.._  ____,  __,___  ,____  __,__  /,,,,,  ____,  ,___,  ___,,  _,__ %%‘%‘ ____ \%%‘  __,,,_ %%%%% ____,_  I’  ‘\‘‘s%’  %%%%S  %‘\‘, /,,,, %%‘%‘ ,__,_ ‘‘.‘‘-‘  Angil  %‘‘.‘%%  ___,_  %%%%•.  —  _,___  %%%% _____ %%%%‘ __,___ %%%% _____  —  Anglil  %\%%% _____ %%%%‘ ___,_ %‘%%% ,/__,  __,__  ______ %%%%  __,__  _____,  _____  __,,,_  __,__ %‘%‘.%%  ‘%\‘‘  ,,__,,  _.,,_/ %%%‘%%  ‘s’’%%  __,,.., —  hCG  ,____,  s%%  __,__ %.%%%%  __,__,  .\\\  ‘‘-‘  _,_,_ \%\‘% ,,,,,  _,_,_  ___,_ %%%%‘.% _____  200  __,,_  ,___  ____.,_ %\‘ __..,__ \‘.‘\ _,_,,_  _______  O  \%%%‘s  ‘\‘\  ___,_ ‘%%%% _____  400  _,___ _,,,_ \%\\‘%  _,,___ \\\ ___,,_  ____,  I  I _?,•_• %‘%‘.\  ______ \\\ _,,___ %•.\‘  %‘%%%% ___,_  I  I I  ‘‘\\  ___,_ %%%  b  Cu  b  7f  “‘-.“-  hCG+ AnglI  —  __,_,  hCG+ Anglil  Figure 23. The in vitro effects of angiotensins (II & III) on basal and hCG-stimulated estradiol production by human granulosa-luteal cells. Cells were cultured for 24 h in medium (control), Ang II (10 M), hCG (1 IU/ml), Ang II+hCG, and 5 M), Ang III (10 5 Ang III÷hCG in the presence of androstenedione (5 X 10 M). The data were pooled 7 from 4 separate experiments (n=8 wells per treatment in each experiment) and expressed as a percentage of the control±SEM (b differs from a and is significant at p<O.05 by one-way analysis of variance followed by Scheffe’s test). 93  400 **  0 **  o  300  C.) ‘4-  0  C  .2  200  0  z 0 0 L.  .  (U I  C’)  w  0 0  I  I  I  I  1  10  100  1000  10000 100000  Angiotensin II (nM)  Figure 24. Dose-dependent effect of Ang II on basal estracliol production by human granulosa-luteal cells. Cells were cultured for 24 h in medium (control) or medium supplemented with different doses of Ang II (1O M 10 9 M) in the presence of 5 androstenedione (5 X 10 M). The data were pooled from 3 separate experiments (n=8 7 wells per treatment in each experiment) and expressed as a percentage of the control±SEM ( ** P<O.O1 by one-way analysis of variance followed by Scheffe’s test). -  94  40  control Angil  In  *  *  C)  o  30-  E  03’O4’O5’O  Time (hr)  Figure 25. Time-course effect of Ang II on basal estradiol production by human granulosa-luteal cells. Cells were cultured for 6, 12, 24, and 48 h in medium (control), or Ang II (10 M). Values are 7 M) in the presence of androstenedione (5 x 10 5 * mean±SEM of eight replicate wells of one experiment ( P<O.05 by one-way analysis of variance followed by Scheffe’s test).  95  b  400-  b  300  0 0’  a C.)  200 a  0 0  a)  a  C  a) 0 a)  100  0) 0 0 control  Angli  Anglil  hCG  hCG+ Angil  hCG÷ Anglil  Figure 26. The in vitro effects of angiotensins (II & III) on basal and hCG-stimulated progesterone production by human granulosa cells (See legend to figure 23 for details).  96  II. Protein assay experiments A. Effect of inhibin, activin, or foffistatin on protein content of granulosa cells The protein content did not change throughout the cell culture period from day 1 to day 5 of culture period (Fig. 27). After treatment with inhibin (3 nM), activin (3.6 nM), or follistatin (3 nM) for a 24 h period, the protein content of the cultured granulosa cells did not change when compared to the cells cultured in medium alone (control) or to day 1 of culture (Fig.27,  p>O.O5).  97  30  I  a  Day 1  Day 3  Day 5  Control  7/’  7/  Activln  Inhibin  Follistatln  Figure 27. Effects of inhibin, activin, and follistatin on protein content of cultured granulosa cells. Protein content of granulosa cells were measured on day 1, 3, and 5 of the culture. On day 5 of the culture, cells were cultured for 24 h with medium (control), activin (3.6 nM), inhibin (3 nM), or foffistatin (3 nM) and the protein concentrations of granulosa cells were measured. Values are mean±SEM (n=8 wells per treatment) from a representative experiment. Similar results were obtained from 3 other separate experiments.  98  CHAPTER FWE: DISCUSSION  I. Regulation of steroid production by inhibin-A and activin-A Inhibin and activin were originally identified as inhibitors and stimulators, respectively, of FSH secretion from pituitary gonadotrophs. In this study the local effects of recombinant human inhibin-A and activin-A were examined on human granulosa-luteal cells. The present study demonstrates that recombinant human activin-A, but not recombinant human inhibin-A, can modulate steroidogenesis in cultured human granulosa-luteal cells. The role of inhibin as an autocrine regulator of granulosa cell function has remained controversial. While some investigators have reported inhibitory effects of inhibin on FSH-stimulated estrogen production in granulosa cells (Ying et al., 1986), others have not been able to confirm these results (Hutchinson et al., 1987; Sugino et al., 1988a). In this study, inhibin did not affect basal or hCG-stimulated progesterone and estracliol production. These observations were similar to those of Hutchinson et al. (1987) and Rabinovici et al. (1992) studies in which inhibin had no effect on either basal or gonadotropin-stimulated estradiol or progesterone production in cultured rat and human granulosa cells respectively. Thus, it appears that inhibin-A does not exert specific effects on human granulosa luteal cells under the present experimental conditions. However, granulosa-luteal cells secrete immunoreactive imbibin in response to gonadotropins (Tsonis et al., 1987). Therefore, cellular secretion of high levels of inhibin could lead to saturation of inhibin receptors so that addition of exogenous inhibin might not elicit any  99  additional response. In this study, activin-A is capable of modulating steroidogenesis. Activin-A stimulated basal progesterone levels. Dose-dependency experiments revealed that activin significantly stimulated basal progesterone levels with maximal effective concentration of 3.6 nM. In addition, activin-A attenuated hCG-stimulated progesterone production, although in a later set of experiments, activin-A did not affect hCG-stimulated progesterone production. The cause for such discrepancies are not entirely clear. However, differences in the maturation stages of granulosa cells obtained from the 1VF program and different combination of exogenous gonadotropins used to induce ovulation in the patients can contribute to such discrepancies. It is possible that the effect of recombinant activin on granulosa cell steroidogenesis is related to follicular maturity. Thus, activin-A may augment or inhibit steroidogenesis in follicular cells depending upon their states of differentiation and maturation. Activin augmented FSH- stimulated progesterone production in undifferentiated granulosa cells from ovaries of immature rats (Xiao et al., 1990; Miro et al., 1991). By contrast, activin-A inhibited hCG-stimulated progesterone secretion from differentiated bovine, human, and rat granulosa cells (Shukovski and findlay, 1990; Li et al., 1992; Rabinovici et al., 1992; Miro et al., 1991). The effect of activin-A on basal progesterone production is somewhat controversial. Rabinovici et al. (1992) reported that activin-A inhibited both basal and hCG-induced progesterone production in human granulosa cells. In the present studies, treatment with activin alone stimulated basal progesterone production in human granulosa cells, but  100  attenuated progesterone produätion when administered in the presence of hCG in most experiments. These results agreed with the study of activin on differentiated granulosa cells in the rat (Miro et al., 1991). Such a gonadotropin-dependent transition from stimulation to inhibition of progesterone reinforces the likelihood that more than one second messenger system is involved (Miro et al., 1991). In this study, activin stimulated basal estradiol levels without affecting hCG stimulated estradiol production in human granulosa-luteal cells. Activin increased basal estradiol concentrations at doses as low as 0.4 and 1.2 nM. In contrast to these observations, Rabinovici et al. (1992) reported that activin-A inhibited basal and gonadotropin-stimulated estradiol production in human granulosa cells. The reason for this discrepancy is not known. On the other hand, the results obtained from this study are also similar to the action of activin-A on rat granulosa cells in which activin stimulates basal estradiol (Miro et al, 1991) and FSH-induced estradiol production by differentiated rat granulosa cells in vitro (Hutchinson et al., 1987; Xiao et al., 1990). It appears that stimulation of estracliol is enhanced by activin at all stages of follicular development, whereas FSH- stimulated progesterone production was initially enhanced (nondifferentiated cells) but became suppressed by activin in vitro after exposure to FSH or hCG in vivo (Miro et al., 1991). The existence of specific binding sites for activin on the granulosa cells suggest a role for activin in regulation of ovarian functions (LaPolt at al., 1989; Sugino et al., 1988a). Given the in vitro effects of activin in modulating steroidogenesis obtained from this study, as well as activin’s subunit gene expression in this human granulosa  101  cell culture system (Li and Leung, unpublished data), it is tempting to speculate that activin may be a local endogenous ovarian regulator. The specific inhibitory action of activin on hCG-stimulated progesterone production suggests that activin may prevent or delay premature luteinization of granulosa cells. The enhancement of basal progesterone production by activin may reflect an overall stimulation of the steroidogenic pathway leading to increased estradiol formation. The stimulation of estradiol production by activin suggests that activin could prevent the transition from healthy to atretic follicle by maintaining granulosa cells in a healthy (estrogenic) state of differentiation. The formation of estradiol and its actions on the granulosa cells are critical to continued follicular development through to ovulation (Gibori and Miller, 1982). It has been reported that activin-A increases proliferation of cultured human preovulatory luteinizing ovarian follicular cells in a dose- and time-dependent manner (Rabinovici et al., 1990). The hypothesis that activin’s action on granulosa cell steroid production may involve changes in cell number of granulosa cells in culture was examined. The protein content per well was not altered after treatment with either activin or inhibin for 24 h period. The protein content of the granulosa cells did not change throughout the cell culture period. Thus, activin does not exert its steroidogenic effect on the granulosa cells by altering the protein content per well. In conclusion, the present findings suggest that activin but not inhibin modulates steroidogenesis. Thus, it is plausible that the overall action of activin may be to promote folliculogenesis and delay the onset of luteinization.  102  II. Regulation of steroid production by follistatin-288 Follistatin was originally characterized as an FSH-releasing inhibitor (Robertson et al., 1987; Ueno et al., 1987; Ying et al., 1987a) and more recently, as a local factor having the ability to affect differentiation of rat granulosa cells (Ying, 1988; Xiao et al., 1990). Follistatin-288 is similar in potency to inhibin-A in suppressing FSH  activity in vitro and more potent and longer acting than inhibin-A in viva (Inouye et at, 1991). A number of studies have localized follistatin message and/or protein in the granulosa cells of ovaries from different species (Shimasaki et al., 1989; Ying, 1988; Findlay et al., 1990; Nakatani et al., 1991), however, the follistatin receptor has not been cloned yet. The physiological role of follistatin in the human ovary is unknown. In this study the local effects of recombinant follistatin on steroid production were examined on the human granulosa-luteal cells. In the first set of experiments, a stimulatory effect of follistatin (3 nM) on basal progesterone and estradiol production without any effect on hCG-stimulated progesterone or estracliol production in differentiated human granulosa-luteal cells in culture was observed. Follistatin increased basal estradiol and progesterone concentrations at dose of 1 nM. However, in a later set of experiments, follistatin did not affect basal progesterone and estradiol levels. The cause for this discrepancy is not entirely clear. However, differences in the maturation stages of granulosa cells obtained from the 1YF program and the different combination of exogenous gonadotropins used to induce ovulation in the patients could contribute to such discrepancies. It is possible that the effect of follistatin on progesterone production  103  is related to the follicular maturity. The 31, 35, and 39 Kda forms of follistatin had no detectable effects on progesterone production by fully differentiated bovine granulosa cells, which had presumably been exposed to an LH surge in vivo (Shukovski et al., 1990). However, follistatin augmented LH-stimulated progesterone production in the undifferentiated stage of bovine granulosa cells (Shukovski et al., 1991). In undifferentiated rat granulosa cells, follistatin augmented FSH-stimulated, but not basal progesterone production (Xiao et al., 1990). In contrast to the present study where follistatin stimulated basal estradiol production in human granulosa cells, an inhibitory effect of follistatin on FSH-stimulated estradiol production was reported in rat granulosa cells (Xiao et al., 1990). The cause of this apparent species difference in the granulosa cell response to the steroidogenic action of follistatin is unknown. The outcome of this study reveals a potential physiological role for follistatin as a modulator of luteinization in the human ovary. Follistatin is mainly located in the preovulatory follicles (Sugawara et al., 1990). Increased progesterone secretion from human preovulatory granulosa cells by follistatin supports the notion that follistatin could contribute in part to the rise in progesterone in large ovarian follicles before the preovulatory LH surge (McNatty et. al., 1979). Follistatin’s stimulation of basal estradiol production may perhaps contribute to the maintenance of estradiol production by the granulosa cells in the preovulatory follicle. Inimunohistochemistry studies have shown that the follistatin protein is localized to a subpopulation of early tertiary follicles and the dominant follicles that are selected to ovulate (Nakatani et  104  al., 1991). These observations, together with the stimulatory effect of follistatin on basal progesterone and estracliol production suggests a role for follistatin as a potential modulator of steroidogenesis in the human ovary. The hypothesis that the stimulatory effect offollistatin on progesterone production may involve changes in the protein content of granulosa cells per well in this culture system was examined. The protein concentrations of granulosa cells did not change throughout the cell culture period or after 24 h treatment with follistatin. Thus, follistatin does not exert its effect on steroidogenesis by altering changes in the granulosa cell protein content. Recent studies indicate that follistatin is a binding protein of activins in pituitary and ovarian extracts (Nakamura et al., 1990; Saito et al., 1991; Shimonaka et al., 1991; Kogawa et al., 1991), suggesting that by binding activin, follistatin can block activin’s action. In the present study, the interaction between these two peptides was examined. These findings provide evidence that follistatin can also serve as a binding protein for activin. It seems that follistatin formed a complex with activin-A which led to the neutralization of activin’s activity. Thus, it appears that follistatin can neutralize the folliculogenic actions of endogenous activin presumably by acting as an activin-binding protein. Follistatin protein is detected in the healthy growing follicles of the human ovary (Sugawara et al., 1990). However, the activin 13-subunits are located only in the small antral follicles of the human ovary and preovulatory follicles until the time of LH surge (Yamoto et al., 1992). The levels of niRNAs for activin and follistatin by granulosa cells are FSH dependent (Sugawara et al., 1990).  105  This provides a basis for a short loop feed-back system whereby PSH could stimulate production of both activin and follistatin, and follistatin then regulates the local actions of activin (Xiao et at, 1992b). However, the possibility that follistatin may also have direct actions on granulosa cells which is independent of its activin-binding activity cannot be ignored. In most experiments, both activin and follistatin stimulated basal progesterone and estradiol production independently. This implicates a direct action for follistatin on steroidogenesis. The present results agreed with that of Xiao and Findlay (1991) who also showed that follistatin has a direct action which is independent of binding to activin, since both follistatin and activin enhanced FSH-induced progesterone production in undifferentiated rat granulosa cells. The enhancement of basal progesterone production in human granulosa-luteal cells by follistatin independent of its activin-binding activity would be facilitated by its continued expression and production after the gonadotropin surge (Sugawara et aL, 1990) when the levels of inhibin 13-subunit mRNA and activin protein in granulosa would fall sharply (Yamoto et al., 1992). Thus, it is possible that follistatin may have a direct effect on the cells  and can also neutralize activin’s action. However, the model of this study predicts a higher foflistatinlactivin ratio in dominant follicles exposed to the preovulatory LH surge. In the present studies, the concentration of follistatin required for effective suppression of activin was four fold that of activin. Normally the concentration of follistatin used in our experiments was 3 nM. However, when the interaction between activin and follistatin were examined, the concentration of follistatin used was 12 nM.  106  In the interaction studies, follistatin at concentration of 3 nM was ineffective in suppressing the activin’s actions (data not shown). These results agreed with others who have reported that the concentration of follistatin required for effective suppression of activin was two-three fold that of activin (Carroll et al., 1989; Asashima et al., 1991; Xiao et al., 1992a; Mather et al., 1993). In summary, the present observations show that follistatin is a potential local regulator of steroid production in the human granulosa cells. These observations support the hypothesis that the action of follistatin on granulosa cells is either dependent on its function as an activin-binding protein or by a direct action of follistatin on the granulosa cells.  IlL Regulation of steroid production by angiotensins Follicular development is regulated by the interplay between cyclic pituitary gonadotropin secretion and ovarian responsiveness to these pituitary hormones. The observation that not all follicles respond to FSH demonstrates functional  heterogeneity of follicles within the human ovary. The role of intraovarian regulatory mechanisms in producing follicular heterogeneity is becoming an important area of study in research. Because the various proteins composing the cascade ofbiochemical pathways that leads to the formation of the Ang IT/Ill have been found in the ovary, modulation of ovarian functions by the angiotensins seems likely. The role of Ang II and Ang III with regards to steroid production in the ovary is unclear and requires further investigation.  107  Ang II (10 M) stimulated basal estradiol production but did not affect basal or 5 hCG-stimulated progesterone production. Dose-dependency experiments revealed that Ang II stimulated basal estradiol levels at 10 M and 10 6 M significantly. A 5 stimulatory effect of Ang II on estradiol secretion from PMSG-stimulated rat ovary and human granulosa cells agreed with the results of this study (Pucell et al., 1987; Bumpus et al., 1988; Palumbo et al., 1988). Ang II inhibited and stimulated gonadotropin-stimulated progesterone production in bovine and human granulosa cells respectively (Stirling et al., 1990; Palumbo et al., 1988). Thus, it appears that there may be differences in the effects of Ang II on ovarian steroidogenesis, depending upon species and/or granulosa cell differentiation. According to present study, Ang II effects on ovarian steroid production appear to be selective; Ang II stimulated basal estradliol production without affecting progesterone production. These observations, in conjunction with evidence for binding sites of Ang II on this culture system of human granulosa cells (Li and Leung, unpublished data) suggests a role for Ang II as a local modulator of steroidogenesis. Synthesis of estradiol by granulosa cells is an important component of follicular development (Dorrington, 1977; Gibori and Miller, 1982). The formation of estradiol and its actions on the granulosa cells are critical to continued follicular development through to ovulation (Gibori and Miller, 1982). If Ang II is an important stimulant of estradiol synthesis, Ang II may play a role in follicular development. Thus, the presence of Ang II receptors on a subpopulation of developing follicles (Husain et al., 1987), similar to a restricted distribution of FSH receptors in the ovary, suggests a  108  possible role for Ang II in maintaining intrafollicular estradiol levels during follicular maturation. There are no reports on the effect of Ang III on steroidogenesis in the ovary from different species. However, it is possible that Ang II is secreted into the follicle and is then converted to the Ang III by the angiotensinase enzyme. There are reports indicating that Ang III may be the active angiotensin in the brain (Wright et al., 1990), and that Ang III is at least as powerful as Ang II in the stimulation of aldosterone secretion. In this study, Ang III inhibited hCG-stimulated progesterone production but had no effect on basal or hCG-stimulated estradiol production, which suggests a specific role for Ang III as a potent inhibitor of progesterone production in the human ovary. Ang II and Ang III may act together to promote the final maturation of the dominant follicle by enhancing basal estradiol production and suppressing hCG-stimulated progesterone production, respectively. In summary, according to this study, Ang III inhibits hCG-stimulated progesterone production while Ang II stimulates basal estradiol production in human granulosa-luteal cells. These observations, in conjunction with evidence for binding sites of Ang 11/Ill on the granulosa-luteal cells (Li and Leung, unpublished data), suggests that ovarian angiotensins play a role in modulating steroidogenesis. Ang II may influence follicular maturation by its stimulatory effect on estracliol production. Ang III may contribute to folliculogenic action of Ang II by blocking hCG-stimulated progesterone production and thus preventing or delaying premature luteinization.  109  1V. Summary & conclusions A. Physiological roles of activrn and follistatin  The regulation of steroid production in the ovary through the endocrine limb of hypothalamus-pituitary-ovarian axis is well established. However, increasing evidence suggests that local regulators are also involved in the regulation of ovarian function. The specific aim of this study was to examine whether human granulosa steroid production is under the influence of several specific and the locally produced regulators such as inbibin, activin, and follistatin. The results from this study show that activin and foflistatin affect steroid  hormone production in the human granulosa cells. The overall findings from the studies show that treatment with activin stimulated basal progesterone and estradiol production consistently in differentiated human granulosa-luteal cells; whereas, treatment with activin in the presence of hCG either inhibited or did not affect progesterone production. The enhancement of basal progesterone production by activin may reflect an overall stimulation of the steroidogenic pathway leading to increased estradiol formation. The stimulation ofbasal estradiol production by activin suggests that activin could prevent the transition from healthy to atretic follicle by maintaining granulosa cells in a healthy (estrogenic) state of differentiation. The specific inhibitory action of activin on hCG-stimulated progesterone production in a significant number of cases examined suggests that activin may prevent or delay premature luteinization of granulosa cells until the time of preovulatory surge of LH. In this study, follistatin either stimulated basal progesterone and estradiol  110  production or did not affect basal progesterone and estradiol production by the human granulosa cells. The outcome of this study support a potential influence of follistatin as a modulator of luteinization in the human ovary. Follistatin and activin may also form a balanced network to regulate steroid production by the granulosa cells. The present findings provide evidence that follistatin can overcome the actions of activin in vitro. Follistatin blocked the activin-induced increase in basal estradiol and progesterone production in the human granulosa-luteal cells. Thus, it seems that follistatin can neutralize the actions of activin presumably by acting as an activin binding protein. The ratio of activin to follistatin may be critical to steroid production by the individual follicles. The model presented a higher follistatin/activin ratio in dominant follicles exposed to the preovulatory LH surge. In the present studies, the concentration of follistatin required for effective suppression of activin was four fold that of activin. Figure 28 summarizes the action of activin and follistatin on the human granulosa cells.  B. Physiological roles of angiotensins  The various proteins comprising the cascade of biochemical pathways that leads to the formation of the Ang IT/ITT have been found in the ovary, modulation of ovarian functions by the angiotensins seems likely. The results of these studies suggest a role for angiotensins in the regulation of steroid secretion from the granulosa cells. According to the present study, Ang II effects on ovarian steroid production appear to be distinct; Ang II stimulated basal estradiol secretion without affecting  111  I  I  gonadotropin  /\\  /  AROMATASE  Q /  ANDROGENS  cc  CHOLESTEROL  PROGESTWS  Q  I  activin  I  Figure 28. Diagram summarizing the actions of activin and follistatin on human granulosa-luteal cells (AC=adenylate cyclase, P45Oscc=side chain cleavage P450 enzyme).  112  basal or hCG-stimulated progesterone production. In this study, Ang III inhibited hCG-stimulated progesterone production but had no effect on basal or hCG stimulated estradiol production, which suggests a specific role for Ang III as a potent inhibitor of progesterone production in the human ovary. Ang II and Ang III may act in concert to enhance basal estradiol production and suppress hCG-stimulated progesterone production, thereby promote the final maturation and prevent premature luteinization of the dominant follicle. Figure 29 summarizes the action of Ang II and Ang III on the human granulosa cells.  113  I  I I gonadotro In  I  AC  1  //\[ €TASE  ANDR’)  CHOLES  :ctcDH ESTROGENS  PROGESTINS  Figure 29. Diagram summarizing the actions of angiotensins on human granulosa luteal cells (AC=adenylate cyclase, P45Oscc=side chain cleavage P450 enzyme).  114  REFERENCES Adashi, E.Y. 1991. The ovarian life cycle. In: Yen S.S.C., and Jaffe R.B. (eds). Reproductive Endocrinology. London, W.B. Saunders Company. pp. 181-238. 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