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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 THEHUMAN GRANULOSA-LUTEAL CELLSbyShideh KhorashehB.Sc., The University of Manitoba, 1986B.Ed., The University of Alberta, 1989A THESIS SUBMITT’ED IN PARTIAL FULFILLMENT OFTHE REQUIREMENTS FOR THE DEGREE OFMASTER OF SCIENCEinTHE FACULTY OF GRADUATE STUDIESDepartment of PhysiologyUniversity of British ColumbiaWe accept this thesis as conformingto the required standardTHE UNIVERSITY OF BRITISH COLUMBIADecember 1993© Shideh Khorasheh, 1993In presenting this thesis in partial fulfilment of the requirements for an advanceddegree at the University of British Columbia, I agree that the Library shall make itfreely available for reference and study. I further agree that permission for extensivecopying of this thesis for scholarly purposes may be granted by the head of mydepartment or by his or her representatives. It is understood that copying orpublication of this thesis for financial gain shall not be allowed without my writtenpermission.(Signature)Department of Phyio1o3)The University of British ColumbiaVancouver, CanadaDate Pecer?ber &, / i9q3DE-6 (2/88)ABSTRACTThe present study investigated the hypothesis that in addition to thegonadotropins, there is an autocrine mechanism controlling human ovarian steroidhormone production (estrogen and progesterone). The specific intra-ovariansubstances investigated were inhibin, activin, follistatin, and angiotensin II and III(Ang II and Ang III). These intraovarian substances may act either alone or inconcert to modulate the actions of gonadotropins, to influence the steroid hormoneproduction. In vitro culture of human granulosa cells, which are one of thesteroidogenic type of ovarian cells, were used to demonstrate the actions of thesesubstances on steroidogenesis. Granulosa cells were harvested from the follicularfluid contents of women undergoing oocyte retrieval in the In Vitro FertilizationProgram. The in vitro culture system was validated by confirming human chorionicgonadotropin (hCG)-mediated progesterone and estradiol production.Inhibin-related peptides have been isolated in follicular fluid. Inhibin-A did notaffect basal or hCG-stimulated progesterone and estradiol production in humangranulosa cells. Activin-A consistently stimulated basal progesterone production buteither inhibited or did not affect hCG-stimulated progesterone production. Activin-Aalso stimulated basal estradiol production, suggesting its role in maintaininggranulosa cells in a healthy (estrogenic) state of differentiation. Foffistatin is anactivin-binding protein. Foffistatin blocked the activin-induced increase in basalestradiol and progesterone production in the human granulosa cells, suggesting a role11as an activin-binding protein. However, follistatin may also have direct actions ongranulosa cells which are independent of its activin-binding activity. Follistatinstimulated basal progesterone and estradiol production; however, this stimulatoryeffect of follistatin on basal levels of steroid hormones was not present in all of theexperiments. Follistatin did not affect hCG-stimulated estradiol and progesteroneproduction by human granulosa cells.The biochemical pathways that lead to the formation ofthe Ang 111111 exists in thehuman ovary. The effects of Ang II and Ang III with regard to steroid production inthe ovary were investigated. Ang III but not Ang II inhibited hCG-stimulatedprogesterone production. On the other hand, Ang II stimulated basal estradiol levelswhile Ang III had no effect on basal or hCG-induced estradiol production. Takentogether, these results suggest that Ang II and Ang III may promote follicularmaturation in the human ovary by enhancing basal estradiol production fromgranulosa cells and preventing premature luteinization.In summary, the results from this study showed that local ovarian substancesplay a role in modulating the steroid hormone production by the granulosa cells. Itis anticipated that future studies may allow for a better understanding of thecoordinated action of many local substances involved in regulating steroid hormoneproduction in the human ovary.111TABLE OF CONTENTSABSTRACTLIST OF FIGURES viiACKNOWLEDGEMENTS xCHAPTER ONE: INTRODUCTION ANI) LITERATURE REVIEW 1I. Ovary 1A. Introduction 1B. Histology 2C. The life cycle of the ovarian follicle 4II. Steroid hormone production by the ovary 7A. Synthesis of sex steroid hormones 7B. Mechanism of action of steroid hormones 11III. Ovarian regulation 12A. Neuroendocrine regulation of the ovary 131. GnRH 132. Prolactin regulating factors 14B. Gonadotropic hormone regulation of the ovary 151. Chemistry of gonadotropic hormones 152. Role of gonadotropic hormones 16i) FSH actions 16ii) LH actions 19iii) Prolactin actions 21C. Steroid hormone regulation of the ovary 221. Role of estrogens 222. Role of progesterone 243. Role of androgens 25D. Role of local nonsteroidal regulators 261. Role of inhibin related peptides 27i) luhibin 27ii) Activin 31iii) Follistatin 352. Role of the ovarian renin-angiotensin system 39E. Role of other intragonadal factors 441. Intraovarian growth factors 44i) Insulin-like growth factors 44ii) Epidermal growth factor (EGF)/Transforming growthfactor-cc (TGF-cz) 46iviii) Transforming growth factor-B (TGF-13) 47IV. Signal transduction system in the ovary 48A. Introduction 481. Cyclic AMP-dependent protein kinase A 482. Calcium and protein kinase C pathway 513. Tyrosine Kinase 52CHAPTER TWO: OBJECTiVES 54I. Background and rationale 54II. Hypothesis and Objectives 56CHAPTER THREE: MATERIALS & METHODS 58I. Human granulosa cell culture system 58A. Human granulosa cell preparation 58B. Experimental designs 60C. Hormone analysis 641. Radioimmunoassay for steroids (progesterone and estradiol) . . 642. Statistical analysis 67II. Protein measurement of granulosa cells in culture 68A. Lowry Protein Assay 68B. Protein assay experiments 70CHAPTER FOUR: RESULTS 71I. Static culture experiments 71A. Effect of activin-A on basal and hCG-stimulated estradiolproduction 71B. Effect of activin-A on basal and hCG-stimulated progesteroneproduction 71C. Effect of inhibin-A on basal and hCG-stimulated progesterone andestradiol production 79D. Effect of follistatin on basal and hCG-stimulated estradiolproduction 79E. Effect of follistatin on basal and hCG-stimulated progesteroneproduction 82F. Interaction between activin and follistatin on basal and hCGstimulated estradiol production 82G. Interaction between activin and follistatin on basal and hCGstimulated progesterone production 89VH. Effects of Ang II and Ang III on basal and hCG-stimulated estradiolproduction 92I. Effects of Ang II and Ang III on basal and hCG-stimulatedprogesterone production 92II. Protein assay experiments 97A. Effect of inhibin, activin, or follistatin on protein content ofgranulosacells 97CHAPTER FiVE: DISCUSSION 99I. Regulation of steroid production by inhibin-A and activin-A 99II. Regulation of steroid production by follistatin-288 103III. Regulation of steroid production by angiotensins 107IV. Summary & conclusions 110A. Physiological roles of activin and follistatin 110B. Physiological roles of angiotensins 111REFERENCES 115viLIST OF FIGURESFig. 1 The biosynthetic pathway of steroid hormone production in theovary.Fig.2 Diagram of the “two-cell, two-gonadotropin theory” of folliclesteroidogenesis. . . . .18Fig.3 The biosynthetic pathways of the angiotensins. . . . .40Fig.4 Diagram of the main intracellular signalling pathways involvedin the mediation of hormone action in the ovary. ... .49Fig.5 Diagrammatic representation of protocol for human granulosacells processing. . .. .59Fig.6 Effect of activin on basal and hCG-stimulated estradiolproduction by the human granulosa cells.Fig.7 Dose-dependent effect of activin on basal estradliol productionby the human granulosa cells.Fig.8 Time-course effect of activin on basal estracliol production bythe human granulosa cells. . . . .74Fig.9 Effect of activin on basal and hCG-stimulated progesteroneproduction by the human granulosa cells.Fig. 10 Dose-dependent effect of activin on basal progesteroneproduction by the human granulosa cells. . .. .76Fig.11 Time-course effect of activin on basal progesterone productionby the human granulosa cells.Fig. 12 Dose-dependent effect of activin on hCG-stimulatedprogesterone production. . . . .78Fig. 13 Effect of inhibin on basal and hCG-stimulated estradiolproduction by the human granulosa cells. . . . .80Fig. 14 Effect of inhibin on basal and hCG-stimulated progesteroneproduction by the human granulosa cells.viiFig. 15 Effect of follistatin on basal and hCG-stimulated estradiolproduction by the human granulosa cells. . . . .83Fig. 16 Dose-dependent effect of follistatin on basal estradiolproduction by the human granulosa cells. . . . .84Fig.17 Time-course effect of follistatin on basal estradiol production bythe human granulosa cells. . .. .85Fig. 18 Effect of follistatin on basal and hCG-stimulated progesteroneproduction by the human granulosa cells. ... .86Fig. 19 Dose-dependent effect of follistatin on basal progesteroneproduction by the human granulosa cells. . . . .87Fig.20 Time-course effect of follistatin on basal progesteroneproduction by the human granulosa cells. . . . .88Fig.21 Interaction between activin and follistatin on basal and hCGstimulated estradliol production by the human granulosa cells 90Fig.22 Interaction between activin and follistatin on basal and hCGstimulated progesterone production by the human granulosacells. ....91Fig.23 Effect of angiotensin II & III on basal and hCG-stimulatedestradiol production by the human granulosa cells. . . . .93Fig.24 Dose-dependent effect of angiotensin II on basal estradiolproduction by the human granulosa cells. . . . .94Fig.25 Time-course effect of angiotensin II on basal estradiolproduction by the human granulosa cells. . . . .95Fig.26 Effect of angiotensin II & III on basal and hCG-stimulatedprogesterone production by the human granulosa cells. . .. .96Fig.27 Effect of imbibin, activin, or follistatin on protein content ofhuman granulosa cells. . . . .98viiiFig.28 Diagram summarizing the actions of activin and follistatin onhuman granulosa cells..... 112Fig.29 Diagram sinnmarizing the actions of angiotensin II & III onhuman granulosa cells. .... 114ixACKNOWLEDGEMENTSI would like to express my appreciation to my supervisor, Dr. Peter C.K. Leungfor providing advice and support throughout my study. I would also like to thank WeiLi 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 helpfulcomments.I would also like to thank all members of Dr. Leung laboratory for their supportthroughout my stay in the laboratory. Finally, I take this opportunity to express mydeepest gratitude to my parents, Mahin and Jamshid, for their love and continuingsupport throughout my academic endeavour.xCHAPTER ONE: I1’TRODUCTION AND LITERATURE REVIEWI. OvaryA. IntroductionThe ovaries are paired organs situated in the abdominal cavity. The function ofthe ovary is to produce oocytes and secrete ovarian hormones. The endocrine functionof the ovary in the female is a cyclic process. This cyclicity is referred to as theestrous cycle in subprimate species and the menstrual cycle in primates. The ovariancycle is designed to ensure that mature female oocytes are produced at ovulation sothat they are available for fertilization by sperm (Adashi, 1991).The ovarian cyclicity is controlled by a feedback system involving thehypothalamus, anterior pituitary and the ovaries. The gonadotropin-releasinghormone (GnRH) from the hypothalamus causes the release of follicle stimulatinghormone (FSH) and luteinizing hormone (LH) from the anterior pituitary. FSH andLH 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 offollicular development, not all follicles in a given ovary respond to pituitarygonadotropins during a given cycle. Only a limited number of selected follicles ovulateduring the life span of the female while most of the follicles undergo atresia. Thevariable fate of ovarian follicles subjected to comparable gonadotropic stimulationsuggests the existence of additional intraovarian modulatory mechanisms.1Intraovarian control of ovarian processes is likely exerted by means of local steroidalmodulation. Intraovarian peptides may have a potential for local modulation offollicular development by modulating steroid production. Inhibin (Hutchinson et al.,1987), activin and follistatin (Findlay et al., 1990; Depaolo et al., 1991), and/orangiotensins (Bumpus et al., 1988; Stirling et aL, 1990) are among potentialmodulators of ovarian steroid production.B. HistologyThe mammalian ovaries are paired organs, approximately equal in size situatedon either side of the uterus. The ovaries are enclosed in a peritoneal capsule (Beck,1972). The ovary contains the follicular apparatus, corpus luteum, stromal andconnective 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. Muchof the medullary region consists of connective, vascular, and nervous tissues. Thedense cortex is composed of a stroma of connective tissue, cells of epithelial origin,and numerous follicles (Franchi, 1962). Primordial follicles consist of a single layerof flattened epithelial cells surrounding each oocyte (Harrison and Weir, 1977). In agiven ovarian cycle, only one of these primordial follicles reaches maturity andruptures; the majority of follicles undergo atresia. As the follicle increase in size, thesingle layer offlattened cells enveloping the oocyte increases in thickness and its cellsbecome cuboidal or columnar to form a distinct layer referred to as the granulosa2layer. This layer rapidly becomes several cells thick (Harrison and Weir, 1977). Theenlargement of the granulosa layer is accompanied by the development of an outerlayer derived from the stroma. This layer which will then develop into a wellvascularized layer is referred to as the theca interna. The theca intema cells aresurrounded by another layer composed of connective tissue which is called the thecaexterna. Numerous blood vessels and lymphatics penetrate the theca externa tocommunicate with a fine plexus of vessels in the theca interna. A thick membranewhich is referred to as the basement membrane separates the granulosa cells fromthe theca interna. Prior to ovulation, the granulosa layer is avascular (Harrison andWeir, 1977). Both the theca interna and the granulosa layer are considered sites ofsteroid production.The maturing follicle enlarges as a result of proliferation of granulosa and thecallayers and a cavity soon forms in the granulosa. The enlarged cavity is referred to asthe antrum. The fluid-filled follicle becomes surrounded by a uniform layer ofgranulosa cells referred to as mural granulosa cells (Adashi, 1991). The oocyte issurrounded by an irregular cluster ofgranulosa cells referred to as the cumulus cells.Cumulus cells appear to be present in the majority ofmammals and they persist afterovulation until fertilization. The cumulus cells support and provide nutrients for thegrowth of the oocyte (Adashi, 1991). Early in follicular development the immatureoocytes are in contact with the granulosa cells. Later a jellylike substance containingpolysaccharides separates the membrane of the oocyte and the granulosa cells. Thislayer is referred to as the zona pellucida (Harrison and Weir, 1977).3The corpus luteum is the endocrine gland which normally develops from thecellular 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 thecorpus luteum, such as granulosa-luteal cells. The term luteinization is used to referto the enlargement and morphological changes associated with the post-ovulatoryfollicle.C. The life cycle of the ovarian follicleThe transformation of ovarian follicle into a corpus luteum was first describedin 1672 by Regner de Graaf (Jocelyn and Setchell, 1972). Ovarian follicles constitutethe fundamental functional unit of the ovary. Follicular maturation depends, exceptduring the earlier stages, on gonadotropic stimulation from the anterior pituitary andproceeds in cycles extending throughout the period of sexual maturity, unlessinterrupted by pregnancy and lactation (Eckstein, 1962). At each cycle a group offollicles enters a rapid phase of growth in which only a selected dominant folliclechanges 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 thanovulation (Adashi, 1991).The follicular development can be divided into three phases: 1. preantral growthphase, 2. tonic growth phase, and 3. gonadotropin-dependent growth phase. Thepreantral growth phase refers to conversion of primordial follicles into primary and4then secondary follicles. The conversion of a primordial follicle to a primary follicleis marked by little follicular growth and is independent ofgonadotropins. 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 asecondary follicle is formed, the granulosa cells develop FSH receptors and becomephysiologically coupled with gap junctions. Then the secondary follicle develops intoa class 1 follicle (Gougeon, 1986).The tonic phase of follicular development corresponds to the conversion of class1 (preantral) follicles (0.12-0.2 mm in diameter) to class 4 (antral) follicles with adiameter of up to 2 mm. The growth is characterized with increase in granulosa layerand overall follicular diameter. The growth of the follicle is accomplished throughgranulosa cell proliferation and enlargement of a central fluid-filled cavity, theantrum. 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 ongonadotropins (Gougeon, 1986). During this time follicular dominance and selectionare accomplished. FSH plays a crucial role in this selection process (McNatty andBaird, 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 selectionof the dominant follicle by modulating the responsiveness of the ovarian cells togonadotropins (Tonetta and DiZerega, 1989). Within this dominant follicle, the oocyteis surrounded by a layer of the granulosa cells. Ovulation occurs as a result of a5massive discharge of LH from the anterior pituitary.About 36 hour after the onset of LH surge, the follicular wall of the dominantfollicle ruptures leading to the release of the oocyte. Ovulation marks the end of thefirst 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 cellularcomponents 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 thecorpus lutei.un, such as granulosa luteal cells. The granulosa cells do not divide afterovulation, but they increase in size and undergo morphological changes (Auletta andFlint, 1988). These changes are referred to as luteinization. The corpus luteumconsists of two types of steroidogenic luteal cells, the large luteal cells and the smallluteal cells (Rodgers et al., 1985). The large luteal cells are derived from thegranulosa cells while the small luteal cells are derived from the thecal cells (Rodgerset al., 1983). The corpus luteum is a large endocrine gland which secretes largeamounts of steroid hormones especially progesterone, which prepares the uterineendometrium for implantation and maintains early pregnancy. If fertilization andimplantation do not occur, the ovulatory cycle ends and the corpus luteum undergoesluteolysis (Auletta and Flint, 1988). However, if pregnancy is established, thecontinued secretion of progesterone by the corpus luteum is essential. Around thestart of the implantation of the egg, about 7 days after ovulation, the embryo secreteshCG which rescues the corpus luteum from undergoing regression (Lenton andWoodward, 1988). Thus secretion ofprogesterone by the corpus luteum is maintained.6II. Steroid hormone production by the ovaryA. Synthesis of sex steroid hormonesThe principal ovarian hormones are steroids, estradiol and progesterone, secretedby the preovulatory follicle and corpus luteum (Strauss and Miller, 1991). Thecommon precursor of steroids is cholesterol. Major pathways of steroid hormonesynthesis in the ovaries are illustrated in figure 1. The ovaries are capable of de novocholesterol synthesis (Schuler et al., 1979). Steroidogenic cells in the ovariessynthesize cholesterol from acetyl coenzyme A. 3-Hydroxy-3-methylglutaryl coenzymeA reductase (HMG-CoA reductase) is the rate-determining enzyme in de novocholesterol synthesis (Strauss et al., 1981).Ovarian cells are also capable of taking up cholesterol from circulatinglipoproteins by receptor-mediated mechanisms (Gwynne and Strauss, 1982).Cholesterol used for steroid hormone production is derived primarily from circulatingserum lipoproteins (Brown and Goldstein, 1976) rather than from de novo cellularsynthesis from acetate (Anderson and Dietschy, 1978). The lipoproteins are classifiedas low-density lipoproteins (LDL) and high-density lipoproteins (HDL). The humangranulosa cells express many LDL receptors which indicates that cholesterol fromLDL is the major precursor in human ovarian cells (Tureck and Strauss, 1982). Theimportance of LDL cholesterol for ovarian progesterone production is demonstratedby the observation that the presence of LDL is required for maximal progesterone712:cERL< Cholesterol5J7 OPregnenolone - Progesterone 20 cx -OH-Progesterone12517 cx - hydroxypregnenolone 17 cx - hydroxyprogesterOfle056Dehydroepiandrostened-iOfle— Androstenedione — EstroneTestosterone Estradiol-17 3Figure 1. The biosynthetic pathway of steroid hormone production in the ovary. Theenzymes regulating this pathway are as follows:1. Cholesterol side-chain cleavage p4502. 17-cx-hydroxylase3. C17,20-lyase4. 17-13-hydroxysteroid dehydrogenase5. 3-B-hydroxysteroid dehydrogenase/A5,zV isomerase6. Aromatase7. 20-.cx-hydroxysteroid dehydrogenase8production by cultured human granulosa cells (Tureck and Strauss, 1982). HDL doesnot support human ovarian progesterone biosynthesis (Gwynne and Strauss, 1982).LDL particles bind to specific membrane receptors, the LDL-receptor complexesenters the cell by receptor-mediated endocytosis (Anderson et al., 1977; Goldstein etal., 1979). Then the endocytotic vesicles are known to fuse to lysosomes, where LDLcholesterol esters are hydrolysed to yield free cholesterol (Brown et al., 1975). Thefree cholesterol is then re-esterified and is stored in the cytoplasm in lipid droplets.Upon steroidogenic demands, the cholesterol ester is hydrolysed and the freecholesterol is transported to mitochondria for steroidogenic processing.The human granulosa cells are capable of synthesizing both steroids, progesteroneand estradiol. Cholesterol side-chain cleavage (cytochrome p450scc) cleaves thecholesterol side chain which results in C21 compound, pregnenolone (Dimino andCampbell, 1976; lVliller, 1988). This is the first step in steroid hormone synthesis.This reaction takes place in the inner mitochondrial membrane. The rate of formationof pregnenolone in steroidogenic cells is determined by: 1. the availability ofcholesterol to the mitochondria (Toaff et al., 1979; Nakamura et aL, 1980; Privalle etal., 1987), 2. the quantities of cholesterol side-chain cleavage enzyme, and 3. thedegree of enzyme activity expressed (Simpson et al., 1987). Acute alterations insteroidogenesis result from changes in the availability of cholesterol to the enzymeand its expressed activity.Pregnenolone is the intermediate steroidogenic compound common to all classesof steroid hormones. Both the human granulosa and theca cells are capable of9converting pregnenolone to progesterone, another C21 steroid. The conversion ofpregnenolone to progesterone occurs readily by the relative abundance of thecytoplasmic enzymes 3B-hydroxysteroid dehydrogenase and A 5,4 isomerase(Sulimovici and Boyd, 1969). The secretion of progesterone by ovarian cells ismodulated by the conversion of progesterone to its metabolites. The main route ofprogesterone breakdown is mediated by 20-o-hydroxysteroid dehydrogenase whichconverts progesterone to its inactive metabolite, 20-a-hydroxyprogesterone (Rodwayand Kuhn, 1975).The rate-limiting step in the biosynthesis of androgens in the follicle is thatcatalyzed by the 17--hydroxylase/C17,20-lyase enzyme (Sasano et al. 1989). Thisreaction is capable of converting the C21 pregnenolone and progesterone into C19androgen, dehydroepiandrosterone and androsteneclione respectively. Studies ofisolated human theca cells have revealed that the thecal layer is the major source offollicular androgen (Tsang et al., 1980). The granulosa cells contain low amounts of17-cz-hydroxylase/C17,20-lyase enzyme, the enzyme which mediates the conversion ofprogestins to androgens. Thus, the granulosa cells are incapable of producingsignificant amounts of androgens (Tsang et al., 1979). The fact that the biosynthesisof estradiol from androgens requires the cooperation of the granulosa and their thecalneighbours was discovered by Falck in 1959.In all species estrone and estradiol are derived from the androgen precursorsandrostenedione and testosterone. Thus, the C19 precursor steroids are produced bythe thecal cells and are transferred across the basement membrane of the follicle to10the granulosa cells (Ryan and Petro, 1966). Ryan and colleagues in 1968 were alsoable to show that the conversion of acetate to estradiol is enhanced by the coincubation of granulosa and theca cells. In the granulosa cells, the conversion ofandrosteneclione and testosterone to estrone and estradiol takes place by the enzymearomatase. Aromatase is the granulosa cell enzyme crucial to follicular estradiolsynthesis (Ryan and Petro, 1966; Bjersing, 1967; Miller, 1988).B. Mechanism of action of steroid hormonesThe classical ovarian steroid hormones are estrogens, progesterone, andandrogens. Estrogens are important in regulating the developmental and physiologicfunctions of the female phenotype. On the other hand, progesterone is a vitalhormone during pregnancy. To accomplish these tasks, the steroid hormones mustbind and activate a group of specific-regulatory molecules called receptors. Thesteroids are secreted from their endocrine gland into the blood stream. The steroidsdiffuse into the cells and combine with specific receptors present in the target cellsin which they will exert their functions. Free steroid enters the cell and binds toinactive receptors in either the cytoplasmic or nuclear compartments. After bindingto their specific receptors, the steroid hormones cause the receptors to undergo aconformational change, which converts the receptors from an inactive to an activeform (for review see Katzenellenbogan, 1980). The steroid receptor in its inactive formis bound to a heat shock protein. While complexed to the heat shock protein, steroidreceptors cannot bind to the gene. However, once the steroid hormone enters the cell11and binds to its receptor, the heat shock protein comes off and as the result, thereceptor hormone is then activated. These heat shock proteins are thought to help thereceptor fold into an appropriate conformation that permits subsequent biologicalactivity and tends to protect them from degradation by cellular proteases (for reviewsee Katzenellenbogan, 1980). Once the receptor becomes activated, it has the capacityto bind to and activate the regulatory elements of genes. If the gene is activated, theenzyme RNA polymerase transcribes the information in the gene into messengerribonucleic acid (mRNA), a molecule that carries the information to the cytoplasmiccompartment of cells. The information is then decoded on structures termedribosomes, which produce the appropriate protein specified by that certain gene (forreview see Walters, 1985).III. Ovarian regulationDuring the follicular phase of the human menstrual cycle, the follicle destined toovulate increases in size from a diameter of 2-5 mm to over 20 mm and becomes themajor ovarian source of secreted estradiol. This estracliol-secretory stage in itsdevelopment encompasses a programmed sequence of cell growth and differentiationin the follicle wall which terminates with ovulation and transformation of the follicleinto a corpus luteum. The entire sequence of events depends upon primary(endocrine) stimulation of the ovaries by the gonadotropins FSH and LH. There arealso local (paracrine and autocrine) levels of control from within the follicle itself.12A. Neuroendocrine regulation of the ovary1. GnRHThe secretion of both FSH and LH from the anterior pituitary gonadotrophs isstimulated by the hypothalamic decapeptide GnRH, also termed luteinizing hormone-releasing hormone (LHRH) (pyro-Glu-His-Trp-Ser-Tyr-Gly-Leu-Arg-Pro-Gly-Nh2.Theresponsiveness of pituitary gonadotrophs to GnRH varies markedly during themenstrual cycle (Wang et al., 1976).In primates, including man, GnRH neurons are located mainly in the arcuatenucleus of the medial basal hypothalamus (MBH) and the preoptic area of theanterior hypothalamus. Axons from GnRH neurons project to many sites within thebrain. One of the distinct projections is from arcuate nucleus of the MBH to themedian eminence (Silverman et al., 1987). The median eminence is made up ofextensive capillaries referred to as the primary plexus. These capillaries thatpenetrate the median eminence drain into the short portal vessels that pass down thepituitary stalk and branch into capillaries surrounding the anterior pituitary. In thisway the GnRH is delivered to its target cells - the gonadotrophs of the anteriorpituitary (Schwanzel-Fukuda and Pfaff, 1989).GrLRH is released in a pulsatile manner from the median eminence into the portalsystem and rapidly binds to specific, high-affinity receptors on the surface of thepituitary gonadotrophs (Clayton and Catt, 1981). The binding of GnRH is followedrapidly by aggregation of the receptor-bound peptide resulting in the release ofgonadotropins from the secretory granules. Much of the stored gonadotropins within13the secretory granules can be released during the secretory response to GnRH. Inaddition to the release ofpre-formed gonadotropins from the secretory granules, thereis evidence for increased synthesis of gonadotropins as well (Landefeld and Kepa,1984). Prolonged or continuous exposure to GnRH or its agonists results in profoundsuppression of gonadotropin release (Hazum and Conn, 1988). This is due to adecrease in the number of GnRH receptors on the plasma membrane, a phenomenaknown as the down-regulation of the receptors.An optimal frequency of GnRH pulsatile stimulation of the pituitary is essentialto maintain appropriate plasma levels of LH and FSH. The pulsatile pattern ofgonadotropin release from the pituitary gland is the result of episodic secretorydischarges of GnRH, a process that is governed by a pulse generator located in thearcuate region of the medial basal hypothalamus (Wilson et al., 1984). GnRH pulsegenerator activities are subject to neuromodulation. Both a-adrenergic anddopaminergic inputs have stimulatory effect on the GrLRH pulse generator. On theother hand, morphine and endogenous opioids have an inhibitory effect (Knobil,1980). In the rhesus monkey, deviations from the physiologic frequency of GmRHpulses impairs gonadotropin secretion and ovarian function (Pohl et aL, 1983)suggesting that GnRH has a permissive role in the control of follicular maturationand ovulation.2. Prolactin regulating factorsProlactin-containing cells which are referred to as lactotrophs have been identified14in the human anterior pituitary gland. Lactotrophs are sparse in the humanpituitary, except in the fetus and during pregnancy and lactation. The posterior andthe intermediate lobe of the pituitary contain a prolactin-releasing factor (PRF), asmall (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 besubjected to short loop negative feedback regulation by prolactin (Laudon et al.,1990). In addition, thyroid releasing hormone (TRH), 13-endorphin, and vasopressinalso stimulate prolactin release (Leong et al., 1983). The inhibitory control ofprolactin 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 ofbovine anterior pituitary cells during dopamine blockade (Samson et al., 1990).B. Gonadotropic hormone regulation of the ovaryNormal follicular development and steroid hormone secretion depend uponappropriate stimulation of the ovaries by adequate amounts of FSH and LH.1. Chemistry of gonadotropic hormonesLH and PSH are climers composed of two glycosylated polypeptide subunits (cx and13) joined together by a noncovalent bond. The cx-subunit of LH, FSH, and that ofhuman chorionic gonadotropin (hCG), is common to the glycoprotein hormones(Parsons and Pierce, 1981). Within different species, the ex-subunits of the15glycoprotein hormones possess the same amino acid sequence. In contrast, the B-subunit of each hormone has a distinctive amino acid sequence that determines itsspecific hormonal activity expressed upon association with the cc-subunit. Themolecular weight of LH is 28,000 and that of FSH is 33,000; the molecular weight ofthe common a-subunit is about 14,000.Prolactin has been characterized in many vertebrates. However, the humanprolactin was not discovered until 1970. Human prolactin has a molecular weight of22,000 and consists of a single polypeptide chain of 199 amino acids. Human prolactinhas sequence homologies with animal prolactin, human growth hormone (GH), andhuman placental lactogen. These polypeptide hormones are believed to have evolvedfrom a common ancestral protein (Niall et al., 1973). In the human, the GH andplacental lactogen are more closely related to each other than to prolactin. Whereasplacental lactogen is produced in the placenta, prolactin and GH are produced indifferent pituitary cells.2. Role of gonadotropic hormonesi) FSH actionsFSH induces ovarian follicle maturation and stimulates the granulosa cells tosecrete steroids, estradiol and progesterone (Channing, 1970; McNatty, 1981). FSHregulates granulosa cell progesterone synthesis by modulating the activity of varioussteroidogenic enzymes (Moon et al., 1978). The major site of FSH action onprogesterone synthesis is at the cholesterol side-chain cleavage activity (Toaff et al.161983). FSH also increases 313-hydroxysteroid dehydrogenase activity which isresponsible 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 (Moonet al., 1975). FSH acts on the aromatase enzyme to enhance estradiol production inthe rat granulosa cells (Dorrington et al., 1975). Luteinized granulosa cells haveminimal ability to convert progesterone to androgens, and are deficient in l7cz-hydroxylase which converts progesterone to androgens. Fevold in 1941 and Greep etal. in 1942 were first to demonstrate that both FSH and LH are required for estradiolbiosynthesis by immature, hypophysectomized rats. Faick in 1959 was first todiscover that the biosynthesis of estradiol from androgens requires the cooperationbetween the granulosa and their thecal neighbours. Short in 1962 proposed a two-celltype theory suggesting the participation of theca cells for the conversion ofprogesterone to estradiol. Ryan et al. in 1968 demonstrated that combined incubationof both granulosa and theca cells in human tissues produces higher estradiol fromacetate in vitro as compared to incubation of the individual cell types alone. The twocell, two-gonadotropin hypothesis for ovarian estracliol biosynthesis is summarizedin figure 2. According to this model, LH stimulates the biosynthesis of androgen fromcholesterol in the theca interna compartment (Markis and Ryan, 1975; Fortune andArmstrong, 1977; Hamberger et al., 1978; Tsang et al., 1980). Androgens diffuseacross the basement membrane to the granulosa cells and are acted upon by thearomatase enzyme which converts androgens to estrogens.17THECA CELLS LH(DCholesterolCholesterol 4cAMPProgesterone4,AndrostenedioneFSHLDLLiAndrostenedionecAMP®c--AromatasePregnenoloneEstradiol CDProgesteroneGRANLJLOSA CELLSFigure 2. Diagram of the “two-cell, two-gonadotropin theory” of folliclesteroidogenesis.18FSH also stimulates the granulosa cells to secrete various nonsteroidal substancessuch as inhibin (Lee et al., 1982), prostaglandins (LeMaire et al., 1973), andplasminogen activator (Martinat and Combarnous, 1983). These nonsteroidalsecretions ensure optimal folliculogenesis and oocyte maturation by altering steroidhormone production by granulosa cells. FSH also induces an increase in LH (Nimrodet 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. FSHstimulates granulosa cell division which is reflected by an increase in the amount ofDNA (Ryle, 1969). FSH also stimulates granulosa cell protein synthesis. Duringfollicular development, granulosa cells undergo morphological changes (Albertini,1980). The avascular nature ofgranulosa cells requires intercellular contacts betweenneighbouring cells. During follicular development, extensive gap junctions are foundamong granulosa cells (Albertini and Anderson, 1974). FSH treatment increases thenumber ofgap junctions in granulosa cells ofhypophysectomized rats (Burghardt andMatheson, 1982).ii) LH actionsLH stimulates preovulatory follicle growth, induces ovulation, and regulatescorpus luteum function. After the FSH induces LH receptors in cultured ratgranulosa cells, these cells are capable of responding to LH. Both gonadotropins canthen act directly to stimulate granulosa cell steroidogenesis in the preovulatory19follicle (Channing and Tsafriri, 1977). LH receptors are located on theca interna cellsand steroidogenesis is under direct LH control throughout the menstrual cycle(Erickson et al., 1985). LH acts on the thecal cells via its receptor to stimulateprogesterone and androgen synthesis. As mentioned before, LH stimulation ofandrogens is required for maintenance of estracliol production by the granulosa cells.The midcycle surge of LH is essential for ovum maturation and ovulation. LHinduces the resumption of oocyte maturation in preovulatory follicles (Tsafriri, 1978).LH also causes the granulosa and luteal cells to secrete several nonsteroidalsubstances such as prostaglandins (Marsh et al., 1974; Clark et al., 1978),plasminogen activator, and proteoglycan (Beers et al., 1975). These nonsteroidalsubstances, as well as progesterone (Rondell, 1974), may be important in the LHinduction of follicle rupture which leads to ovulation.After ovulation, the granulosa and theca cells of the ovulatory follicle willluteinize and form the corpus luteum. LH stimulation sustains the steroid-secretoryfunction of the corpus luteum (Filicori et al., 1984). Luteal tissue mainly producesprogesterone, which prepares the uterine endometrium for implantation andmaintains early pregnancy. The LH stimulation of progesterone production isaccompanied by alterations in cholesterol metabolism and changes in the activitiesof steroidogenic enzymes. Briefexposure 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 receptorsin the rat corpus luteum (Hwang and Menon, 1983). With prolonged exposure to LH,20the increase ofprogesterone synthesis occurs via stimulation ofcholesterol side-chaincleavage enzyme (Armstrong et al., 1970) and 313-hydroxysteroid enzymes (Madej,1980). If pregnancy occurs, the functional lifespan of the corpus luteum is extendedby the direct action of hCG secreted by the trophoblastic tissue of placenta. Iffertilization and implantation do not occur, the final stage of the ovarian cycle beginsas the corpus luteum undergoes luteolysis (Auletta and Flint, 1988).iii) Prolactm actionsProlactin is an important regulator of lactation. The actions of prolactin on themammary gland include promotion of mimmary growth and initiation of milksecretion. In addition to its action on the mammary gland, prolactin acts as aluteotropic hormone in rodents and several other species (Richards and Williams,1976). The luteotropic action of prolactin varies among species. In rats, prolactin actsas a luteotropic agent by stimulating progesterone secretion (Smith, 1980) as well asmaintaining the LH receptors during follicular luteinization (Holt et al., 1976). In therat corpus luteum, prolactin contributes to the rise in progesterone by inhibitingprogesterone catabolism. Prolactin inhibits the activity of 2Occ-hydroxysteroiddehydrogenase enzyme which converts progesterone to its inactive form (Zmigrod etal., 1972). In contrast to its luteotropic role, prolactin also exerts a luteolytic actionon the rat and may be responsible for the luteal regression during the estrous cyclein the rat (McNeilly et al., 1982).In contrast to its ability to stimulate progesterone production in rats, in vitro21treatment 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 ovary1. Role of estrogensEstrogens induce and maintain secondary sexual characteristics and exert afeedback action on the hypothalamo-pituitary axis and consequently affect the releaseof gonadotropins. More importantly, estrogens play a vital role at the site of theirproduction, the granulosa cells. Estrogen is considered an intrafollicular autocrineregulator (i.e., having an action within the cell which produces it).Estrogen stimulates granulosa cell mitosis (Rao et al., 1978), and exerts aantiatretic effect (Harman et al., 1975). Estrogen also raises gonadotropin receptorlevels and amplifies follicular responsiveness to exogenously administeredgonadotropin (Richards, 1980). Estrogen synergizes with FSH to promote folliculargrowth (Simpson et al., 1941; Reiter et aT., 1972), and enhances FSH-stimulatedgranulosa cell aromatase activity (Adashi and Hsueh, 1982; Zhuang et al., 1982). Theability of estrogens to augment the ability of the enzyme responsible for theirformation accounts for the preovulatory rise in the circulating estradiol levels. Thisform of self amplification may play a central role in the follicular selection as well asin the establishment of follicular dominance. It has been reported that induction ofatresia may be associated with the loss of estrogen receptors (Harman et al., 1975).The large healthy follicles contain detectable amount ofestradiol (McNatty and Baird,221978). Once selected, the dominant follicle ensures that development of other folliclesis suppressed. Current evidence suggests that this dominance is secured by thesuppression of FSH secretion due to the rise in levels of estradiol secreted by thedominant follicle (Baird, 1987). The estrogen in the dominant follicle will exertnegative feedback control to the pituitary leading to reduction in systemic (Ross etal., 1970) and intrafollicular FSH levels (McNatty et al., 1975). The reduced levels ofFSH will not allow for the further growth of the other follicles. Once a chosen follicleproduces larger amount of estrogens compared to the other follicles, the estrogen actsthrough a local positive feedback loop to stimulate granulosa cell proliferation andaugments follicular responsiveness to gonadotropins, thus causing further increasein estrogen formation (Hillier, 1981; McNatty, 1981). In this way, the selection of thedominant follicle is secured and it will continue to mature even though the FSHlevels are declining. On the other hand, the follicles that are unable to produceenough follicular estrogens will undergo atresia. During the late follicular phase,estrogen causes the preovulatory discharge of LH through positive feedback on thepituitary and hypothalamus. This surge in LH results in the ovulation of thedominant follicle.On the other hand, estrogen inhibits production of its precursor, androgenthrough negative feedback on the theca cells (Leung and Armstrong, 1979). Such anintraovarian negative feedback mechanism may be significant in limiting the estrogenand provide adequate time for oocyte maturation before ovulation.232. Role of progesteroneGranulosa cells secrete large amounts of progesterone. The role of progesteronein modulating follicular growth and granulosa cell function is not clear. Specificprogesterone receptors have been demonstrated in the rat ovary which supports adirect ovarian action of progesterone (Schreiber and Hseuh, 1979). Direct effects ofprogesterone on granulosa cells have been studied in vitro. Treatment with syntheticprogesterone and FSH showed that progesterone causes an increase in FSHstimulated progesterone production (F’anjul et al., 1983). Also, progesterone augmentsthe LH-stimulated progesterone production in FSH-primed granulosa cells in vitro(Fanjul et al., 1983). Thus luteal cell progesterone production is an autocrine controlmechanism in which progesterone regulates its own production. The increase inprogesterone secretion during the preovulatory stage may exert a local action at theovarian level to induce ovulation. Progesterone treatment enhances LH-stimulatedovulation in hypophysectomized rats in vivo (Fanjul et al., 1983). High levels ofprogesterone secreted by the luteal cells of the corpus luteum during the luteal phasealso prepare the endometrium for the implantation of the oocyte. Progesteronesecreted by the corpus luteum also exerts negative feedback control on thesynchronized discharge of GnRH. Thus, the frequency of LH pulses falls in responseto rising levels ofprogesterone (Knobil, 1980). As mentioned earlier, the estradiol alsoinhibits the FSH secretion. Thus, during the luteal stage of the cycle, folliculardevelopment is suppressed due to low circulating levels of gonadotropins. But, whenthe corpus luteum regresses, the concentration of FSH and LH rise and a new24ovarian cycle recommences (Baird et al. 1984).However, the effect of progesterone on follicular growth is not clear. Progesteroneinhibits FSH-stimulated estrogen production in cultured rat granulosa cells(Schreiber et aL, 1980). This finding suggests a role of progesterone as inhibitor offollicular growth. In monkeys, ovarian implants of progesterone directly inhibitfollicular growth (Goodman and Hodgen, 1979). In contrast to the in vivo studies inthe monkeys, in prepubertal rats exogenous administration ofprogesterone facilitateshCG-stimulated growth of small antral follicles (Richards and Bogovich, 1982). Thesediscrepancies between rat and monkey studies may be related to species-specifity.3. Role of androgensAndrogens are produced by the theca cells and play a role in folliculardevelopment. As mentioned before, androgens serve as substrate for aromataseenzyme to form estradiol. Androgens also exert a direct action on the granulosa cellsthrough interaction with androgen receptor. In vitro studies demonstrate thatandrogens augment gonadotropin-stimulated steroidogenesis. Androgens directlyaugment aromatase activity in cultured rat granulosa cells (Daniel and Armstrong,1980; Hillier and DeZwart, 1981). Androgens also stimulate progesterone biosynthesisby granulosa cells. In vivo administration of androgens to intact rats enhances theability of FSH and LH to increase progesterone production by rat granulosa cells(Leung et al., 1979). Androgens also act synergistically with FSH to stimulateprogesterone production in cultured rat granulosa cells (Nimrod et al. 1980; Welsh25et al., 1982). The stimulatory effect of androgens on progesterone synthesis appearsto be on the cholesterol side-chain cleavage and 313-hydroxysteroid dehydrogenaseenzymes (Welsh et al., 1982).However, in the absence of gonadotropins, androgens stimulate follicular atresiaand antagonize estradiol-induced follicular growth in hypophysectomized immaturerats. The counteracting action of androgens on estrogens may be related to the factthat androgen treatment decrease ovarian estrogen receptor content (Saiduddin andZassenhaus, 1978). Androgen and estrogen levels vary in the follicular fluid,depending upon the stages of follicular development. Elevation of androgen toestrogen ratio is invariably associated with signs of atresia (McNatty et aL, 1975).D. Role of local nonsteroidal regulatorsThe concept that regulation of ovarian function involves the actions of localregulators has gained increasing acceptance in recent years. It has been suggestedthat these local factors might regulate ovarian functions such as steroidogenesis,oocyte maturation, and ovulation. These locally produced factors act throughparacrine or autocrine control mechanisms. In recent years, measurable amounts ofa number of these nonsteroidal substances have been found and isolated in the ovary,including the inhibin family (inhibin, activin, and follistatin), angiotensins, growthfactors, insulin and insulin-like growth factors, GnRH, prostaglandins, and manyother factors. The study of paracrine and autocrine functions of these local hormoneshas become important to the further understanding of ovarian function.261. Role of inhibin related peptidesi) Inhibin1. StructureInhibin, a 32,000 kDa protein, was first isolated from bovine and porcine follicularfluid 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 aheterodimeric glycoprotein consisting of an x subunit joined together to a B subunitby two disulfide bonds. Two related forms of the 13-subunit termed 13A and 13B havebeen described (Ling et at, 1985), and both complex with the CL-subunit to formbiologically active inhibin termed inhibin A and inhibin B. The complete amino acidsequences of inhibin for porcine, bovine, human, and rat sources have been deducedby 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 murineinhibin are closely related in structure and highly conserved (Esch et al., 1987a). Inaddition, the B-subunits of inhibin share a substantial sequence identity with anemerging family of proteins with growth-regulating properties referred to astransforming growth factor-B (TGF-B) (Mason et al., 1985).2. LocalizationHistochemical techniques have demonstrated that granulosa cells of the ovary arethe primary producers of inhibin. The CL-subunit of inhibin has been localized in bothgranulosa and luteal cells of the ovary by immunohistochemical techniques (Cuevaset al., 1987; Merchanthaler et al., 1987). The localization and intensity of27inm-imiostaining for each inhibin subunit changes during the follicular developmentand maturation (Yamoto et al., 1992). The inhibin a subunit is mainly expressed inhealthy, maturing follicles and in corpora lutea, but not in small antral follicles of theprimate 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 remainscontroversial in rats (Meunier et al., 1988a; Woodruff et al., 1988) and sheep (Rodgerset al., 1989; Tsonis et al., 1988). However, evidence exists that human granulosa cellsthat 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 thehuman corpora lutea (Davis et al., 1987). Extragonadal sources of inhibin include theplacenta (Merchanthaler et al., 1987), the adrenal, and the pituitary (Meunier et al.,1988b).3. RegulationFSH has been shown to be a major regulator of inhibin. F’SH induced inhibinbioactivity from the rat cultured granulosa cells (Anderson and Hoover, 1982). FSHinduced accumulation of inhibin a-subunit mRNA has been observed both in intactanimals and in cultured granulosa cells (Davis et al., 1986). In addition to FSH, avariety of other hormones appear to affect either basal or FSH-induced inhibinproduction. 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 stimulatoryeffect on inhibin production. On the other hand, epidermal growth factor (EGF)(Zhiwen et al., 1987b) and GnRH (Rivier and Vale, 1989) inhibit inhibin secretion by28granulosa cells.4. ActionsInhibin was originally identified as an inhibitor of FSH secretion from pituitarygonadotrophs and followed from experiments showing the inadequacy of estrogen asa feedback regulator of FSH following unilateral or bilateral ovariectomy (Welschon,et al. 1978). A substantial amount of evidence supports a role for inhibin in theregulation of FSH secretion (for review see De Jong, 1988; Ling et al. 1988; Woodruffand Mayo, 1990). Treatment of rat pituitary cell cultures with inhibin significantlysuppressed 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 aninverse relationship between the plasma levels of FSH and inhibin during both thefollicular 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 decreaseduring the LH surge with a subsequent second and higher peak of immunoreactiveinhibin during the luteal phase parallel to the luteal phase progesteroneconcentrations (McLachlan et al., 1987). The levels ofinhibin correlate positively withboth progesterone and estradiol levels in the luteal phase, providing evidence that thecorpus luteum secretes inhibin. Late in the luteal phase, as the corpus luteumregresses, the levels of inhibin also decline before the next menstrual cycle. Thedeclining levels of inhibin before the next menstrual cycle allows for the rise in thecirculating levels ofFSH which is required for the stimulation of follicular growth for29the next cycle (McLachlan et aL, 1987). This inverse relationship between FSH andinhibin supports the concept that inhibin is a negative feedback regulator of FSH.Besides its effect on pituitary FSH secretion, inhibin exerts actions within theovary. Inhibin acts directly on the gonadal cells via paracrine and possibly autocrinemechanisms to influence steroidogenesis. Two paracrine actions of inhibin have beenreported. Firstly, after diffusing from its site of production in granulosa cells to thetheca cells inhibin enhances LH-stimulated androgen biosynthesis in thecal cells inrats (Hsueh et al., 1987) and in humans (Hillier et al., 1991a). In humans, inhibinalone at doses between 10 and 100 ng/ml caused an increase in production ofandrogens by theca cells (Hillier et al., 1991a). Secondly, there is evidence of aparacrine interaction between the inhibin produced in the cumulus granulosa cellsand the oocyte. Bovine inhibin caused inhibition of spontaneous maturation divisionof 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 fromother species.There are conflicting reports of an autocrine influence of inhibin on granulosa cellsteroidogenesis. Ying et at in 1986 reported that porcine inhibin inhibited FSHinduced estracliol production by rat granulosa cells in vitro. However, the inhibitoryactions of inhibin on estradiol production was not confirmed using bovine inhibin(Hutchinson et al., 1987; Sugino et al., 1988a). Hutchinson et al. (1987) reported thatbovine inhibin has no effect on either basal or FSH-stimulated estradiol orprogesterone production in cultured rat granulosa cells. Autocrine regulation of30human ovarian steroidogenesis by inhibin has not been elucidated. Using culturedhuman granulosa cells from women undergoing in vitro fertilization, inhibin-Ashowed no effects on basal or gonadotropin-stimulated progesterone (Li et al., 1992;Rabinovici et al., 1992) and estradiol secretion (Rabinovici et al., 1992). Suchexperiments are complicated by the endogenous production of inhibin by granulosaluteal cells in culture (Zhiwen et al., 1987a, 1988).ii) Activm1. StructureDuring the purification of inhibin from porcine folliciilar fluid, a substance withFSH-stimulating activity was discovered. This material was characterized as a 28,000KDa 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 abilityis referred to as activin. Three different forms of activins have been identified: activinA (A/A), activin B(13B’) and activin AB (BAIBB).2. LocalizationConsidering that activin is composed ofinhibin B-subunits, it is expected that anysite 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 diversetissues, including the gonads, pituitary, placenta and bone marrow (Meunier et al.,1988b). Antibodies to activin are not widely available so evidence for the localizationof activin in the gonads have been deduced from the presence of B-chain iURNA. In31rats, both B-subunit mRNAs are expressed in the ovary (Esch et al., 1987a; Meunieret al., 1988a). The 13-mRNAs are exclusively localized in the granulosa cells of boththe 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-subunitexpression) in granulosa cells of small antral follicles in the primate ovary. Usingimmunohistochemical localization techniques, Yamoto et al. (1992) showed that thegranulosa cells of human preantral and small antral follicles exhibited positiveimmunoreactive staining with antisera against BA and BB subunits and negativeimmunostaining with antiserum against a-subunit. Thus, in contrast to inhibinsubunits (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. ActionsActivin is mainly known for its ability to stimulate FSH synthesis and releasefrom the pituitary (Ling et al., 1986; Carroll et al., 1989). Infusion of activin intoimmature rats (Schwall et al., 1989) and monkeys (McLachlan et al., 1989) led toincreased gonadotropin secretion. In addition to stimulating FSH synthesis andrelease, activin has a broad range of activities that includes effects on cell growth,differentiation, and maturation. These varied actions on growth and differentiationreflect the partial sequence homology of the inhibin-B subunits to TGF-B family32(Mason et al., 1985) and euthyroid differentiation factor (EDF). Subsequent to theidentification of ovarian activins, an erythroid differentiation factor was also isolatedand 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 andinduced proliferation of human luteinized granulosa cells in culture. Stimulation ofoocyte 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 spontaneousmaturation of oocytes.Besides its effect on the pituitary FSH and cellular growth, activin can affectgranulosa 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 ofproduction, granulosa cells to the theca cells. Activin and inhibin have opposingparacrine actions on LH-induced secretion of androgen by rat theca cells (Hsueh etal., 1987) and human thecal cells (Hillier et al., 1991b). Activin suppressed LHstimulated androgen biosynthesis in rats and in humans (Hsueh et al., 1987; Hillieret al, 1991b).There are conflicting reports of an autocrine influence of activin on granulosa cellsteroid production. The effect of activin on steroidogenesis differs in different species.Activin has a stimulatory action on FSH-induced estradiol production by ratgranulosa cells in vitro (Ying et al., 1987b; Hutchinson et al., 1987). However, the33effect of activin on progesterone production is unclear in rats. It appears that theeffect of activin on progesterone production is related to follicular maturity (Miro etal., 1991). Hutchinson et al. (1987) reported that activin caused a dose-dependentdecrease in FSH-induced progesterone production by undifferentiated rat granulosacells. In contrast to the findings of Hutchinson et al. (1987), Xiao et al. (1990)reported a time and dose-dependent increase in FSH-stimulated progesteroneproduction by activin in differentiated rat granulosa cells. Miro et al. (1991) reportedthat activin alone enhanced basal levels of progesterone whereas it inhibited FSHstimulated progesterone production in differentiated rat granulosa cells. In contrastto the controversial reports on the effect of activin on the rat granulosa cellprogesterone production, activin inhibited progesterone production in the bovine ovary(Shukovski and Findlay, 1990). In human granulosa cells, activin-A inhibited basalas well as FSH-stimulated estradiol (Rabinovici et al., 1992) and progesteroneproduction (Rabinovici et al., 1992; Li et al., 1992). In addition to its effect onsteroidogenesis, activin also stimulates inhibin production (Xiao et al., 1992a; LaPoltet 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 etal., 1988a).Overall, the data from the rat, bovine, and human are consistent with anautocrine action of activin on the regulation of granulosa cell ontogeny,differentiation, and the variable fate of the follicles by modulating local steroidproduction.34iii) Follistatin1. StructureDuring the isolation of inhibin from the porcine follicular fluid, a side fraction wasfound that also suppressed FSH secretion from the pituitary cell cultures. This novelFSH-release inhibitor, named follistatin or FSH-suppressing protein (FSP), has nowbeen isolated from porcine and bovine follicular fluid (Robertson et al., 1987; Uenoet al., 1987). Follistatin, is a monomeric glycosylated protein with at least threeisoforms with molecular weights of 32, 35, and 39 Kda, and is structurally differentfrom the inhibins (Robertson et al., 1987; Ueno et al., 1987). Follistatin was firstisolated from porcine and bovine follicular fluid and found to suppress FSH releasein 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 beencloned. The amino acid sequences are highly homologous, and the gene structure isconserved among the three species.2. LocalizationFollistatin is expressed in the ovary, and extragonadal tissues such as brain andthe kidney (Shimasaki et al. 1989). Follistatin, like inhibins and activins, is primarilya product of the granulosa cells (DePaolo et at, 1991; Nakatani et al., 1991; Saito etal., 1991; Shimasaki et at, 1989). The intensity of follistatin expression changesduring granulosa cell differentiation and the rat estrous cycle. In rats, in situhybridization studies revealed that the mRNA levels offollistatin is low in primordial35follicles but increased in growing secondary and tertiary follicles (Nakatani et aL,1991). In rat granulosa cells, immunohistochemistry studies showed that thefollistatin protein is localized to a subpopulation of early tertiary follicles and thedominant follicles that are selected to ovulate (Nakatani et al., 1991). After ovulation,the hybridization and immunohistochemical signals continued to be strong in thenewly formed corpus luteum. Follistatin mRNA was not detected in theca, stroma,or interstitial cells of rats (Nakatani et al. 1991). Follistatin production was shownto be stimulated by FSH, but not LH, in differentiated bovine granulosa cellsharvested 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 hyperstimulationwith human menopause gonadotropins (HMG) caused an increase in the circulatinglevels of serum follistatin and estradiol (Sugawara et al., 1990). There is a directcorrelation between increased serum follistatin and estradiol after ovarianstimulation. The increased serum follistatin level may likely reflect follicular oroocyte maturation (Sugawara et al., 1990).3. ActionsFollistatin may also act locally to regulate steroid hormone production in thegranulosa cells. Follistatin augmented FSH-stimulated progesterone production byrat granulosa cells (Xiao et al., 1990) and enhanced LH-stimulated progesteroneproduction in undifferentiated bovine granulosa cells (Shukovski et al., 1991).36Follistatin also augmented progesterone production at a concentration of 0.3 nM inhuman granulosa-luteal cells (Li et al., 1993). Follistatin decreased FSH-stimulatedestradiol production in the rat granulosa cells (Xiao et al., 1990); however, an effectof follistatin on estradiol production by human granulosa cells has not yet beenreported. Together with the above findings, the presence of follistatin mRNA withinthe ovary (Shimasaki et al., 1989; Natakani et al., 1991) suggests local regulation ofsteroid production by follistatin.Recent studies indicate that follistatin binds to activins in pituitary and ovarianextracts (Nakamura et al., 1990; Saito et al., 1991; Shimonaka et al., 1991; Kogawaet al., 1991), suggesting that follistatin antagonism to activin actions may occur byfollistatin binding activin and then blocking its action. As mentioned before, activinshave multiple biological effects. In addition to FSH stimulation, activin A promotesdifferentiation and growth, modulates gonadal steroid production, induces inhibinproduction in the granulosa cells, causes an increase in the FSH receptor numbers,and induces mesoderm formation. It is possible that follistatin may also participatein the regulation of these effects by binding activin-A, thus blocking its action. Thehigh affinity activin-bincling protein, follistatin, has recently been shown to blockactivin-stimulated activities in several in vitro systems. Carroll et al. (1989) reportedthat follistatin suppressed activin’s stimulation of FSH mRNA levels of rat pituitarycells in vitro. Follistatin also blocked activin’s action on steroidogenesis. Follistatinreversed the inhibitory effects of activin-A on LH-stimulated progesterone productionin bovine granulosa cells in culture (Shukovski et al., 1991). Follistatin also reversed37the stimulatory action ofactivin on FSH-induced aromatase activity and progesteroneproduction in the undifferentiated rat granulosa cells (Xiao and Findlay, 1991). Xiaoet al. (1992a) also reported that follistatin suppressed activin induced inhibinproduction in cultured rat granulosa cells. In the chicken embryo, follistatin inhibitedthe mesoderm-inducing activity of recombinant human activin-A (Asashima et al.,1991). The effects of follistatin on the activity of activin in stimulating the reaggregation of sertoli cell monolayer and proliferation of testicular germ cells wereexamined (Mather et al., 1993). Follistatin blocked the ability of activin-A tostimulate reaggregation of sertoli cell monolayer. However, in these same cultures,follistatin had no effect on the ability of activin-A to stimulate proliferation oftesticular germ cells (Mather et al., 1993). In all of the above studies, theconcentration of follistatin required for effective suppression of activin was two-threefold 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 regulatesteroidogenesis by modulating activin’s action and regulating the bioavailability ofactivin. However, follistatin has a direct action on the granulosa cells, which isindependent of binding to activin, since both follistatin and activin enhanced FSHinduced progesterone production in undifferentiated rat granulosa cells (Xiao andFindlay, 1991).382. Role of the ovarian renin-angiotensin system1. Synthesis of angiotensinsThe principal function of the renin-angiotensin system (RAS) involves regulationof cardiovascular homeostasis. Renin is the primary, rate-limiting enzyme of therenin-angiotensin system. Renin is formed as a result of a cleavage of prorenin andis stored in the kidney. Renin is indirectly responsible for the formation of the activeagents 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, andangiotensin (1-7)[Ang (1-7)], which is formed directly from angiotensin I, and fromAng II (Fig.3). Once in the blood, renin cleaves its substrate, angiotensinogen, to forma decapeptide, angiotensin I. Angiotensin I is then converted to Ang II and otherpeptidases by angiotensin-converting enzyme (ACE). Ang II is the main active peptideof the renin-angiotensin system. Ang II has diverse physiological effects includingvasoconstriction, aldosterone secretion, angiogenesis, and the induction of drinkingbehaviour 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 andangiotensin I can exert their own action in the regulation of the cardiovascularhomeostasis. Ang III is as potent a vasoconstrictor as Ang II in the adrenalglomerulosa in stimulating aldosterone secretion (Goodfriend and Peach, 1975). AngIII may also be the active angiotensin in the brain (Wright at al., 1990). Also recentstudies have shown that Ang (1-7) which is a newly defined hormone of the RAS39AngiotensinogenRenin - ProreninAngiotensin IendopeptidaseAngiotensin II Ang (1-7)angiotensinaseAngiotensin IIIFigure 3. The biosynthetic pathway of angiotensins (ACE= angiotensin convertingenzyme).40mimicks some of the known actions ofAng II (Santos et al., 1989; Kohara et aL, 1991;Goodfriend, 1991).2. LocalizationDuring the last two decades, recombinant DNA technology and molecular cloninghave shown the presence of both renin and angiotensinogen genes in a variety oftissues (Campbell and Habener, 1986; Dzau et al., 1987; Burnham et al., 1987; Dzauet al., 1988). The classical concept that the RAS is only a regulator of blood pressurehas been changed due to the identification of local endogenous renin-angiotensinsystems in several different tissues. In addition to its well known target organs, therat 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 ofpreovulatory gonadotrophin-stimulated and normally cycling women, suggesting thatthe ovary is producing these substances. Renin niRNA is expressed in the rat (Kimet al., 1987) and monkey ovaries (Itzkovitz et al., 1992), suggesting that prorenin isproduced locally in the ovary. Using immunohistochemical techniques it has beenshown that prorenin, renin, and Ang II are found within the stromal, thecal andluteal 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 reninand Ang II (Palumbo et al., 1989). This supports the idea that the ovarian RAS isunder gonadotropin control. Also, renin-like activity and Ang 11)111 immunoreactivity41in follicular fluids from women stimulated with HMG and hCG were much higherthan the follicular fluid from women not stimulated with exogenous gonadotropins(Lightman et al., 1987). Also, immunostaining of both granulosa and thecal cells forrenin 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 ofAng Il/Ill and renin in the ovary, Ang II receptors are expressed on granulosa andtheca 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. ActionsBecause the various proteins composing the cascade ofbiochemical pathways thatleads to the formation of the peptide have been found in the ovary, modulation ofovarian functions by the angiotensins seems likely (Bumpus et al., 1988).Angiotensins may have an autocrine function in regulating steroidogenesis. Severalreports have shown that Ang II affects steroidogenesis in the ovarian tissues. In vitrostudies on rat ovarian fragments from PMSG-stimulated rats showed a stimulatoryeffect of Ang II on estradiol production (Pucell et al., 1987), but studies withgranulosa cells from diethyistilbestrol-treated rats reported no effect of Ang II onestradiol production (Pucell et al., 1988). The results of in vitro experiments withgonadotropin-stimulated human granulosa-luteal cells showed that Ang II caused anincrease in progesterone, testosterone and estradiol (Palumbo et al.,1988). In culturedbovine luteal cells, Ang II had no effect on basal progesterone secretion but inhibitedLH-stimulated progesterone production by a mechanism that involves inhibition of42cholesterol-side chain cleavage enzyme activity (Stirling et aL, 1990). Thus, it appearsthat there may be differences in the effects of Ang II on ovarian steroidogenesis,depending upon species and/or differentiation stage ofgranulosa cells. Therefore, AngII may play a role in follicular development, possibly through the modulation offollicular steroidogenesis. There is no report on the effect of Ang III onsteroidogenesis in the ovary from different species. The angiotensin-convertingenzyme which converts Ang II to Ang III and the angiotensinase which converts AngII to Ang III have been reported to exist in the rat ovarian follicles (Speth andHusain, 1988; Duad at al., 1990). This supports the idea that the ovary might produceits 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 ovaryis unclear and requires further investigation.Ang II may also have a direct role in ovulation and oocyte maturation (Pellicer etal., 1988; Kuo et al., 1991; Yoshimura et al., 1992). Although others have not showna direct role for Ang II in ovulation (Naftolin et al., 1989), a recent study by Petersonet al. (1993) has confirmed that Ang II is a regulator of ovulation. Peterson andcolleagues showed that saralasin (Ang II blocker) reduced LH-stimulated ovulationrate in the perfused rat ovary. Ang II may also play a role in oocyte maturation.Prorenin concentrations correlate with follicular development, oocyte-cumuluscomplex maturity and oocyte viability (Cornwallis et al., 1990). In the human, thereis a high level of Ang II in the preovulatory follicular fluid of dominant follicles thatcould facilitate the regulation of oocyte maturation and ovulation.43E. Role of other intragonadal factors1. Intraovarian growth factorsNumerous growth factors have been identified by their ability to stimulateproliferation and growth of different cell types. Many of these growth factors havebeen also isolated from the gonads. In the ovary, these growth factors may functionin an autocrine or paracrine fashion to modulate steroidogenesis.i) Insulin-like growth factorsThe family of insulin-like growth factors (IGFs) are composed of: 1. IGF-I (alsocalled somatomedin C), and 2. IGF-II. Both IGFs exert endocrine effects on tissuegrowth throughout the body, being secreted by the liver under the control of growthhormone (Slack, 1989). These compounds have been extensively studied in the ovary(for review see Adashi et al., 1985b). Among modulators of granulosa cells, IGFs havea 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, andestradiol (Hsu and Hammond, 1987; Hammond et al., 1988).Insulin, IGF-I, and IGF-II each have a separate receptor. However, IGFs cancross-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 granulosacells of rats (Adashi et al., 1988), pigs (Baranao and Hammond, 1984), ewes (Mongetet al., 1989), and humans (Poretsky et al., 1985). Localization of IGFs’ receptors on44the granulosa cells suggests that these cells are a site of IGF action.Autocrine regulatory actions of IGF-I in isolated granulosa cells have beenobserved in vitro. The IGFs have mitogenic effects on the granulosa cells (Hammondand English, 1987; May et al., 1988). Although the ability of PSH to induce granulosacell progesterone and estrogen biosynthetic capacity is well established (Richards,1979), the modulation of this important process remains under investigation. IGF-Ihas been shown to stimulate progesterone secretion from the granulosa and lutealcells (Adashi et aL, 1985c; Veldhuis and Furlanetto, 1985; Veldhuis et al., 1985). Thestimulatory effect of FSH or estradiol on progesterone production from ovarian cellswas partially blocked by using IGF antibodies (Mondschein et aL, 1989). Thus, IGF-Iis capable of synergizing with FSH to stimulate progesterone secretion (Baranao andHammond, 1984). IGF-I can also stimulate basal and FSH-stimulated estradiolproduction in both human and rat granulosa cells (Steinkampf et al., 1988; Ericksonet al., 1989; Adashi et al., 1985a). In addition, IGF can also influence androgenbiosynthesis. Both basal and LH-stimulated synthesis of androgen in cultured thecacells 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 theovary, IGF-I has a variety of actions, including affects on cell proliferation andsteroidogenesis, and it may also modulate gonadotropin actions.45ii) 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 receptorsand have similar actions in many cell types, but they also have distinct effects (forreview see Derynck, 1986). In the ovary, TGF-ocfEGF may play a physiological roleby affecting granulosa cell mitogenesis. Granulosa cells express gonadotropinregulated receptors for EGFJTGF-cz (St-Arnaud et al., 1983; Feng et aL, 1987; Kudlowet al., 1987; Roy and Greenwald, 1990). EGF binding sites have been located ongranulosa (Hopkins et al., 1981; Jones et al., 1982), luteal, and thecal cells (Chabotet aL, 1986). Treatment with EGF/TGF-a promotes granulosa cell growth(Gospodarowicz and Bialecki, 1978; Gospodarowicz and Bialecki, 1979; Hammond andEnglish, 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-stimulatedprogesterone production from luteinized granulosa cells (Richardson et al., 1989).In summary, EGFIPGF-oL modulated steroidogenesis and granulosa celldifferentiation. This is supported by the fact that EGF receptors are found in theovary. Also, the ovary is a site ofEGF synthesis since both immunoreactive EGF andEGF mRNA have been localized in the ovary.46iii) Transforming growth factor-B (TGF-B)The TGF-13 belongs to a family of peptides that includes inhibin, activin, andmullerian inhibiting substance (for review see Knecht et al., 1989). The protein ishighly conserved among species. The cDNA sequence of TGF-13 shows completehomology for the human, porcine and bovine polypeptides (Sporn et al., 1987). Sinceits isolation in 1981 by Roberts and colleagues, TGF-B has been found to have manyeffects in tissues and cells.TGF-B is considered to be an intraovarian regulator of ovarian function. Ovarianthecallinterstitial cells (Skinner et al., 1987) and granulosa cells (Kim andSchomberg, 1989; Mulheron and Schomberg, 1990) have been identified as sites ofTGF-B synthesis, and steroid synthesis in both cell types is influenced by treatmentwith TGF-13 in vitro. In rat granulosa cell cultures, treatment with TGF-13 stimulatedFSH-induced estradiol and progesterone secretion (Adashi and Resnick, 1986;Hutchinson et aL, 1987; Ying et al., 1986; Dodson and Schomberg, 1987). TGF-13 alsoacts on the thecal cells to increase progesterone production but inhibits androgensecretion (Magoffin et al., 1989). TGF-13 also affects other aspects of cellulardifferentiation. It has a stimulatory effect on ESH-induced LH receptor induction inrat granulosa cell cultures (Dodson and Schomberg, 1987).TGF-13 can act as a positive or negative regulator ofgrowth in many different cells(Sporn et al., 1986). Likewise, the effects of TGF-B on the replication of granulosacells are also varied and dependent on the species in question, growth conditions andpresence of other growth factors (Knecht et al., 1987; Roberts et al., 1988). In vitro,47TGF-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 effectson granulosa cell proliferation and steroidogenesis, and it can also modulategonadotropin actions.W. Signa’ transduction system in the ovaryA. IntroductionOvarian cellular functions are regulated by peptide hormones, neurotransniitters,and nonsteroidal factors. The hormone must bind to its specific receptor in order fora given cell to respond to that hormone. When the hormone receptors are occupiedby their specific hormone, a second messenger system is stimulated. The hormonesregulate ovarian functions via second messenger systems which enable the hormonalsignal to spread rapidly throughout the cell. The three major intracellular signallingpathways 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 AThe role of cAMP in mediating gonadotropin (FSH and LH) action has beenrecognized for many years. The steroidogenic effects of FSH on immature granulosa48cAMP 1P3 DAG+ + + TyrosinePKA Ca2+ PKC 1P34 4kPhosphorylation I dephosphorylationof numerous target-protein substrates.Tyrosine phosphorylation oftarget proteinsFigure 4. The three main intracellular signalling pathways involved in the mediationof hormone action in the ovary (AC=adenylate cyclase, PKA=protein kinase A, PLC=phospholipase C, DAG= diacyiglycerol, PKC= Protein kinase C, 1P3= inositoltriphosphate).49cells and LH on thecal cells and mature cells are mediated through intracellularproduction of cAMP (for review see Leung and Steele, 1992). hCG also mediates itssteroidogenic actions via the stimulation of cAMP. Both LH and hCG share the samereceptors (Ascoli and Segaloff, 1989). The mechanism of action of inhibin family ofpeptides is not elucidated yet. It has been speculated that stimulation of granulosacell steroidogenesis by activin may involve the enhancement of cAMP action (Miro etal., 1991). The mechanism of action of follistatin is not known yet, and the follistatinreceptor 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 threesubunits a, 13 and 6 subunits; and 3. adenylate cyclase (Johnson and Dhanasekaran,1989; Neer et al., 1990). Receptors which lead to the activation cAMP aretransmembrane glycoproteins with the hormone-binding site on the outer membranesurface and a signalling domain at the cytoplasmic face of the membrane. Membranereceptors are associated with two classes of G proteins; G is responsible for theactivation of adenylate cyclase, while G1 is involved in the inhibition of this enzyme.Binding of hormone to its receptor triggers a conformational change in the G. In itsinactivated form the G nucleotide binds guanine diphosphate (GDP). Activation ofG proteins occurs when hormone occupies the receptor, releasing GDP from the Gprotein and allowing guanine triphosphate (GTP) to bind in its place. Once GTP bindsto the a-subunit of G, the a-subunit dissociates from its 13-6 subunits and thereceptor itself. The free a-subunit will then activate the adenylate cyclase (Michell,501989). The activation of adenylate cyclase leads into the hydrolysis of ATP to cyclicAMP 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 proteinsubstrates which leads to the stimulation of steroidogenesis (Kurten and Richards,1989). Alternatively, receptor-mediated inhibition of adenylate cyclase activityinvolves the activation of G1 proteins. Deactivation of adenylate cyclase results fromGTP hydrolysis to GDP, which terminates G8 protein activation. The final step inadenylate cyclase deactivation comes with the reassociation of a-subunit to the J3-complex (Birnbaumer et al., 1990).2. Calcium and protein kinase C pathwayAutocrine and paracrine information affecting steroid production in the ovariancells also involves inositol phospholipid hydrolysis in the plasma membrane (Leungand Wang, 1989). Among the nonsteroidal products that may regulate steroidhormone productions via the inositol phospholipid hydrolysis are Ang II. Ang II hasbeen shown to stimulate polyphosphoinositide turnover in several tissues includingvascular smooth muscle (Dostal et al., 1990), adrenal glomerulosa (Kojima et al.,1985; Spat, 1988), and heart (Baker et al., 1989). However, there is conflictingevidence regarding the role of Ang II in calcium mobilization. Ang II was found tostimulate a rapid, transient increase in intracellular calcium levels in rat granulosacells (Wang et al., 1989), but not in human granulosa cells (Currie et al., 1992). Thisresponse was completely blocked by an Ang II antagonist, saralasin, suggesting a51receptor-mediated mechanism. In contrast, Pucell et al. (1991) failed to show anyeffects of Ang II on intracellular calcium levels, phosphoinositide turnover or cAMPproduction in rat granulosa cells. Post-receptor mechanism ofAng II requires furtherinvestigation. 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 reviewedby Catt et al. (1991). Receptors using inositol lipid hydrolysis pathway transmitinformation by a G protein which activates the enzyme phospholipase C (PLC). PLCthen metabolizes inositol phospholipids to inositol triphosphate and dliacylglycerol(DAG) (Berridge, 1987b; Taylor et al., 1986; Berridge and Irvine, 1989). Inositoltriphosphate in turn induces an increase in intracellular calcium levels by causingthe release of calcium from the endoplasmic reticulum (Burgess et al., 1984), whileDAG activates calcium-dependent protein kinase C (Nishizuka, 1988). Protein kinaseC causes the phosphorylation of cellular proteins. This signal pathway based oninositol triphosphate/calcium and DAG/protein kinase C is used for a variety ofactions in the ovarian tissue, and ovarian follicles and corpora lutea are sites ofprotein kinase C activity (Noland and Dimino, 1986).3. Tyrosine KinaseThe 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,521989) and IGF-I (Adashi et al., 1985b, Gates et al., 1987). Factors which stimulatetyrosine kinases promote cell proliferation and positively or negatively regulategonadotropin-stimulated steroidogenesis. Growth factors have also been shown toinduce tyrosine phosphorylation of PLC which will lead into inositol phosphatehydrolysis (Berridge, 1987a; Wahl et aL, 1988). Very little is known yet about themechanism of action of TGF-13. Unlike other growth factors, TGF-B does not activatea 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 proteinkinase C pathway in that the binding of ligand to its receptor does not involve a Gprotein. The ligand-binding and tyrosine kinase domains of the receptor are ondifferent portions of a single polypeptide chain spannlng the plasma membrane: theligand binding site is outside coupled to tyrosine kinase inside. Binding of ligand tothe extracelluar binding sites activates intracellular tyrosine kinase, promoting thephosphorylation of tyrosine residues in protein involved in cell growth and/ordifferentiation (Michell, 1989).53CHAPTER TWO: OBJECTWESL Background and rationaleThe concept that regulation of ovarian function involves the actions of localregulators has gained increasing acceptance in recent years. This stems from twomain lines of evidence. Firstly, it is not possible to explain all of the processes ofovarian differentiation and function simply by changes in the patterns of secretionof pituitary gonadotropins, FSH and LH. For instance, not all follicles in a givenovary respond to pituitary gonadotropins during a given cycle. The hormonal profilesof follicular fluid differ among the follicles. Only a limited number of selected folliclesovulate during the life span of the female while most of the follicles become atretic.The variable fate ofovarian follicles subjected to comparable gonadotropic stimulationsuggests the existence of additional intraovarian mechanisms. Intraovarian controlis likely exerted by means of local steroidal modulation.Secondly, there is increasing evidence for the existence of substances in ovariantissues and fluids which are able to act locally, either alone or by modulating theactions ofgonadotropins, to modulate steroid production. In recent years, measurableamounts of a number of other elements besides the ovarian steroids have been foundand 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), andangiotensins (Culler et aL, 1987; Lightman et at, 1988). Study of the local actions ofthese hormones has become important to the further understanding of ovarian54functions.Local actions of these putative intraovarian regulators can be demonstrated bythe following method. In vitro culture of ovarian cells can be used to demonstrate anaction of these local regulators on basal and gonadotropin-stimulated production ofsteroids. Granulosa cells which are one of the steroidogenic type of ovarian cells areused in this study to demonstrate the actions of these substances on steroidogenesis.The two main types of steroids produced by the ovary are estrogen andprogesterone. The process of steroidogenesis as it occurs in granulosa cells isregulated by enzymes. First to be synthesized is the rate limiting enzyme cytochromeP450 scc, which converts cholesterol to pregnenolone and further to progesterone(Miller, 1988). The second enzyme is P450-aromatase which converts androgens toestrogens. The granulosa cells can synthesize progesterone but cannot produceancirogens. In vivo, androgens diffuse into the granulosa cells from the theca cellswhere they are acted upon by P450-aromatase enzyme, which converts androgens toestrogens (Ryan and Petro, 1966). In vitro granulosa cells lack the thecal contributionof androgens found in vivo. Thus, androstenedione (a form of androgen which existsin theca cells) at low concentrations of 5x107M was added as substrate for estradiolformation 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. Thegonadotropin used in these experiments was hCG. The use of hCG as a gonadotropinwas preferred over LH or FSH for several reasons: 1. It is difficult to obtain purified55FSH 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 iseasily obtained.II. Hypothesis and ObjectivesThe hypothesis to be tested is that in addition to the gonadotropins, there is anautocrine mechanism controlling human ovarian steroid hormone production. Thisintra-ovarian regulatory mechanism involves inhibin, activin, follistatin, and/orangiotensins.The principal objective of this thesis project was to examine whether humangranulosa cell steroid (progesterone & estradiol) production is under the control oflocally produced regulators such as activin, inhibin, follistatin, and angiotensinswithin the ovary. Study of the role of these local regulators on granulosa celldifferentiation and steroid production has mainly been limited to small laboratorymammals. Few studies of ovarian regulation have utilized human granulosa cells. Invitro culture ofgranulosa cells was used to demonstrate the action of these regulatorson basal and gonadotropin-stimulated progesterone and estradiol production. Usingthis system, the following specific objectives were addressed:1. To investigate the effect ofinhibin on basal and gonadotropin-stimulatedprogesterone and estradiol production in cultured human granulosacells.562. To investigate the effect of activin on basal and gonadotropin-stimulatedprogesterone and estradiol production in cultured human granulosacells.3. To investigate the effect of follistatin on basal and gonadotropinstimulated progesterone and estradiol production in cultured humangranulosa cells.4. To investigate the interaction between follistatin (an activin binding protein)and activin on basal and gonadotropin-stimulated progesterone and estradiolproduction in cultured human granulosa cells.5. To investigate the effect of angiotensins (Ang II and Ang III) on basal andgonadotropin-stimulated progesterone and estradiol production in culturedhuman granulosa cells.57CHAPTER THREE: MATERIALS & METHODSL Human granulosa cell culture systemA. Human granulosa cell preparationThe use of human granulosa cells was approved by the Clinical ScreeningCommittee for Research and Other Studies Involving Subjects of the University ofBritish Columbia. Human granulosa cells were harvested during oocyte collection inthe University of British Columbia In Vitro Fertilization Program (IVF). Folliculardevelopment had been stimulated by using two main protocols: 1. a combination ofhuman menopausal gonadotropins (Serono) and a GnRH analogue (Serono), 2. acombination of human menopausal gonadotropins and clomiphene citrate (Serono)until adequate response was achieved. The criteria for adequate response includedthree follicles >16 mm in diameter. Final maturation of the oocytes was effected withhuman chorionic gonadotropin (hCG, 10,000 IU, Serono). Retrieval of the oocytes wasaccomplished 32 h after the injection with hCG. Granulosa cells were then harvestedfrom the follicular fluid contents after the oocyte was identified for in vitrofertilization.The method for the cell culture of granulosa cells is outlined in figure 5. Thefreshly harvested granulosa cells were layered onto 40% percoll (Sigma, St. Louis,MO) and 60% Hank’s balance salt solution (Gibco, Burlington, ON) and centrifugedat 1700 x g for 20 mm at 20°C. The cells were then washed and resuspended in58PercollgradientLayer cells on single density40% percoll gradientCells1. Replace media with M199containing 2% FBS andincubate cells for 48 hr. 0002. Replace media with M199containing 0.5% BSA and 0 0L) Qvarious treatments and LI ‘-10000incubate cells for 24 hr. CFigure 5. Diagrammatic representation of protocol for human granulosa cellsprocessing and plating for culture. Cells were centrifuged on a Percoll gradient toexclude hematocytes. The cells were then plated at a density of 0.2-1 x iO cells/miin 48-well cell culture plates and cultured at 37°C in humidified air with 5% CO2.Centrifuge(20 mm, 1700 g, 25 C)Resuspend cells in 25 mlM 199 containing 10% FBSRedbloodcellsCulture in48-well culturedishes for 48 hr.(0.5 mI/well)iII59media 199 (M199, Gibco) supplemented with 10% fetal bovine serum (FBS,Gibco),sodium penicillin (100 lU/mi, Gibco) and streptomycin (100 uglml, Gibco). The cellswere 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/miand cultured in 48-well culture plates (Celiwell, Corning), 0.5 ml per well inreplicates of 8 wells/treatment. The incubation media were replaced with M199containing 2% FBS at 48 h and subsequently with M199 containing 0.5% bovineserum albumin (BSA, Sigma) and the various treatments at 96 h after plating. Theculture media was collected 24 hour after treatment and concentrations ofprogesterone and estradiol in media were determined by radioimmunoassay (RIA).Granulosa cells were incubated at 37°C in humidified air with 5% CO2.All procedureswere carried out under sterile conditions in a biosafety hazard hood (Biological SafetyCabinet Model 1128, Forma Scientific).B. Experimental designs1. Effects of activin-A or inhibin-A on basal and hCG-stimulatedsteroidogenesisThe effects of inhibin-A or activin-A on basal and hCG-stimulated estradiol andprogesterone production were investigated by adding recombinant human activin-Aor inhibin-A (3.6 nM, and 3 nM respectively, Genentech, San Francisco, CA) in M199with 0.5% BSA, with or without hCG (1 lU/mi, Sigma). Androstenedione (5 x 107M,Sigma) was added to culture medium as substrate for estradiol formation. Estradiol60and progesterone concentrations were measured in medium collected after 24 hincubation. This experiment was performed 3 times with granulosa cells from 3different 1VF patients (n=8 wells per treatment in each experiment).2. Dose-dependency and time-course study of activin-A on basalprogesterone and estradiol productionIncreasing doses of activin-A (0.1 to 3.6 nM) with androstenedione (5 x 107M)were added to the cultured cells and incubated for 24 h. Estradiol and progesteroneconcentrations were measured in medium collected after 24 h incubation. Thisexperiment 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 107M) with and without activin-A (3.6 nM) was added to thecultured cells and incubation media was collected after 6, 12, 24, and 48 h forestradiol and progesterone analysis.3. Dose-dependency study of activin-A on hCG- stimulated progesteroneproductionIncreasing doses of activin-A (0.1 to 3.6 nM) were added with hCG (1 lU/mI) tothe cultured cells and incubated for 24 h. Progesterone concentrations were measuredin medium collected after 24 h incubation. This experiment was performed 2 timeswith granulosa cells from 2 different 1VF’ patients (n=8 wells per treatment in eachexperiment).614. Effects of foffistatin-288 on basal and hCG-stimulated steroidogenesisThe effects of follistatin (recombinant human follistatin with 288 amino acids wasexpressed in Chinese hamster ovary cells under the control of the simian virus-40promoter, as detailed in a previous report by Inouye et al., 1991 was used in thisstudy. Follistatin-288 is a more potent FSH suppressor than the native porcinefollistatin.) on basal and hCG-stimulated estradiol and progesterone production wereinvestigated by adding recombinant human follistatin (3 nM, NIH) in M199 with0.5% BSA, with or without hCG (1 lU/rid). Androstenedione (5 x 107M) was added toculture medium as substrate for estradiol formation. Estracliol and progesteroneconcentrations were measured in medium collected after 24 h incubation. Thisexperiment 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 basalprogesterone and estradlol productionIncreasing doses of follistatin (0.1 to 3 nM) with androstenedione (5 x 107M) wereadded to the cultured cells and incubated for 24 h. Estradiol and progesteroneconcentrations were measured in medium collected after 24 h incubation. Thisexperiment 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 107M) with and without follistatin (3 nM) was added to thecultured cells and incubation media was collected after 6, 12, 18, and 24 h for62estradiol and progesterone analysis.6. Combined effects of activin-A and foffistatin on steroidogenesisThe effects of activin-A and follistatin on basal and hCG-stimulated estradiol andprogesterone 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).Androstenedione (5 x 107M) was added to culture medium as substrate for estradiolformation. The interaction between activin and follistatin on basal and hCGstimulated estradiol and progesterone production was investigated by addingrecombinant human follistatin (12 nM) together with activin-A (3.6 nM) in M199 with0.5% BSA, with or without hCG (1 lU/mi). Cells were incubated for 24 h. Estradioland progesterone concentrations were measured in medium collected after 24 hincubation. This experiment was repeated 5 times with granulosa cells obtained from5 different lyE patients (n=6 wells per treatment in each experiment).7. Effects of Ang II and Ang IH on basal and hCG-stimulated steroidogenesisThe effects of Ang II and Ang III on basal and hCG-stimulated estradiol andprogesterone production were investigated by adding Ang II or Ang III (105M, Sigma)in M199 with 0.5% BSA, with or without hCG (1 lU/mi, Sigma). Androstenedione (5x 107M) was added to culture medium cultured as substrate for estradiol formation.Estradiol and progesterone concentrations were measured in medium collected after24 h incubation. This experiment was repeated 4 times with granulosa cells obtained63from 4 different IVF’ patients (n=8 wells per treatment in each experiment).8. Dose-dependency and time-course study of Ang II on basal estradiolproductionIncreasing doses ofAng II (10MM to 105M ) with androstenedione (5 x 107M) wereadded to the cultured cells and incubated for 24 h. Estradiol production wasmeasured in medium collected after 24 h incubation. This experiment was repeated3 times with granulosa cells obtained from 3 different IVE patients (n=8 wells pertreatment in each experiment). For time-course analysis, androstenedione (5 X 107M)with and without Ang II (105M) was added to the cultured cells and incubation mediawas collected after 6, 12, 24, and 48 h for estradiol analysis.C. Hormone analysisConcentrations of progesterone and estradiol in media were determined byvalidated RIA with specific antisera provided by Dr. D. T. Armstrong of theUniversity 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 8064gIL of NaCl (BDH chemicals, Vancouver, B.C.), 2 g/L of KC1 (BDH chemicals),11.5 g/L ofNa2HPO4(BDH chemicals), 2 g/L ofKH2PO4(BDH chemicals), and0.1% thimerosal (BDH chemicals). The buffer was stored at 4°C.2) The progesterone (Sigma P0 130) standards used were serially diluted in PBSbuffer from an initial 0.32 mM stock solution which was reconstituted inredistilled absolute ethanol. The stock solution was kept at -20°C. A standardcurve 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 PBSbuffer from an initial 1 miVi stock solution which was diluted in redistilledabsolute ethanol. The stock solution was stored at -20°C. A standard curve wasset up with 8 reference concentrations ranging from 0.25 to 32 nM.4) The progesterone antiserum used was rabbit anti-progesterone provided byD .T.Armstrong, raised against 4-pregnen-613-ol-3,20-dione hemisuccinate:bovineserum albumin conjugate (Steraloids, Wilton, NH). This was used at a finaldilution 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 gaveapproximately 60% binding of tracer.6) The progesterone labelled hormone used was3H-progesterone (Amersham,Oakville, ON). The labelled hormone had a specific activity of approximately90-130 CiJmmol. The initial stock was 250 pCiJul in toluene:ethanol (9:1, v:v).65Solvents were evaporated and initial stock was further diluted in 10 ml ethanoland stored at -20°C. The stock was further diluted in PBS such that 80 p1 in10 ml of PBS gave 10000 cpm.7) Labelled hormone for estradiol was 2,4,6,7,16,17-3H-E2 (Amersham) with aspecific activity of 140-170 Ci/mmol. The initial stock was 250 jiCi/ul intoluene:ethanol. Solvents were evaporated and initial stock was diluted in 10ml ethanol and stored at -20°C. The stock was further diluted in PBS such that80 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).Protocol1) 100 p,l of steroid (progesterone or estracliol) standard or sample solution wasadded to 10 x 75 mm assay tubes (Fisher scientific). Each standard wasassayed in triplicate and each sample was assayed in duplicate.2) 100 p1 of diluted antibody was added to each tube except the tubes containingthe tracer only which is referred to as the total binding tube.3) 100 p13H-progesterone or3H-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 byvortexing.666) 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 polyethylenescintillation vials (Ingram & Bell Scientific, Vancouver, B.C.)10) 3 ml scintillation cocktail was added to each tube. The tubes were thenvortexed and were counted by LKB 13-counter for one minute.The sensitivity of progesterone assay was 0.5 pmollml = 160 pg/nil. The sensitivityof estradiol assay was 0.25 pmollml = 60 pg/mI. The intra-assay and inter-assaycoefficients 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 analysisStatistical significance of the data were determined by one way analysis ofvariance followed by Scheffe’s multiple comparisons test (pc0.05). In some cases,estradiol and progesterone values are reported as the mean±SEM. Mean estradioland progesterone levels for control and treatment groups were compared betweengroups by one-way analysis of variance. Scheffe’s test was used to differentiatebetween means after a significant F test. In other cases, the data were pooled fromall replicates in separate cell culture experiments and expressed as percentages ofcontrol values.67II. Protein measurement of granulosa cells in cultureThe protein assays were performed in order to assess the changes in the proteincontent of granulosa cells after treatment with inhibin-related peptides. The designof 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 withactivin, inhibin or follistatin.3. The cells were detached from the 48 well cell culture plates by the use of asonicator (Fisher Sonic Dismembrator Model 300). The media was removed from theculture wells and was replaced with 0.5 ml of PBS buffer. The cells were thensonicated at 10 W for 20 seconds. The sonicator not only removed the cells from thebottom of the wells but also broke down the cell membrane.4. The 0.5 ml PBS buffer containing the cells was then transferred to themicrocentrifuge tubes. The cells were then centrifuged at 10,000 cpm for 15 minutes.5. The supernatant was then carefully removed and transferred to anothermicrocentrifuge tube and it was stored at -20°C until the time of protein assay.A. Lowry Protein AssayReagentsAll of the reagents were prepared in distilled water (dH2O).1. Na-K tartarate (2.5 g/40m1, BDH Chemicals)2. CuSO4 (1.0 g/40m1, BDH Chemicals)683. NaHCO3 solution: a. NaOH (4 gfL, BDH Chemicals)b. Na2CO3(20 gIL, BDH Chemicals)4. The Lowry reagent which was prepared fresh every time. The Lowryreagent consists of:a. 0.8 ml Na-K tartarateb. 0.2 ml CuSO4c. 50.0 ml NaHCO35. iN Folin-Phenol reagent (BDH Chemicals)Standards1. The stock solution was 500 mg bovine serum albumin in 10 ml of CIH2O.For the working solution, 200 p1 of the stock solution was diluted inlOmi of distilled water to give a concentration of 1 pg/j.il. A standardcurve was set up with 8 reference concentrations ranging from 0 to 35pg.ProtocolSamples and standards were assayed in duplicates. Assay tubes used were 16 x 100mm glass tubes (Fisher Scientific).1. 300 p1 of standards (100 ul of standard + 200 p1 of distilled water) and300 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,69followed 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 readingthe absorbance at 660 nm in the spectrophotometer (SP8-400 UV/VTS).6. In order to find out the amount of protein in each sample theabsorbance ofeach sample recorded by the spectrophotometer, the linearregression program was used in order to determine the amount ofprotein in the samples. The sensitivity of the protein assay was 5 jig/jil.Statistical AnalysisStatistical significance of the data were determined by one-way analysis ofvariance followed by Scheffe’s test (p<0.05).B. Protein assay experiments1. Effect of inhibin, activin, and foffistatin on protein content of humangranulosa cellsIn order to assess whether or not the protein concentration changed throughoutthe cell culture period, the protein concentrations were measured on day 1, 3, 5 ofculture and 24 h after the application of treatments with inhibin (3 nM), activin (3.6nM), or follistatin (3 nM). This experiment was performed 4 times with granulosacells from 4 different 1VF patients (n=8 wells per treatment in each experiment).70CHAPTER FOUR: RESULTSI. Static culture experimentsA. Effect of activin-A on basal and hCG-stimulated estradiol productionActivin-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 thesame extend by 1 lU/mi hCG (p<O.Ol). The effect of activin-A on estradiol productionwas 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 estradiollevels 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). ActivinA (3.6 nM) did not affect hCG-stimulated estradiol production.B. Effect of activin-A on basal and hCG-stimulated progesterone productionActivin-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 thestimulatory effect of activin-A on basal progesterone production was significant at aconcentration of 3.6 nM (p<0.05). Time-course experiment of action of activin-A (3.6nM) on basal progesterone levels revealed that progesterone production wasstimulated in a time-dependent manner. Progesterone production was stimulated71b1200b_____I I— b I0_ _i... 1000 II— IO IC—) I8O0 ‘ .‘ ‘ ‘ ‘ ‘ .‘ ‘ ‘0oCo 600C.).D0 400.D——O..%%%\%CU 200 aC,)______W I,,•,,,-,•,-i‘\S%\’,‘‘‘‘I_________I‘‘‘ ‘0 .. , — —_____control hCG activin hCG+activinFigure 6. The in vitro stimulation of basal estracliol production by activin-A inhuman 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 (5x 107M). The data were pooled from 3 separate experiments (n=8 wells per treatmentin each experiment) and expressed as a percentage of control±SEM. Different lettersabove the SEM bars denote statistical significance (b differs from a and is significantat p<O.Ol by one-way analysis of variance followed by Scheffe’s test).7212(I)— 11 **. **0Lf)o 10.1control .1 .4 1.2 3.6Activin (nM)Figure 7. Dose-dependent effects of activin-A on basal estradiol production by humangranulosa-luteal cells. Cells were cultured for 24 h in medium (control) or mediumsupplemented with different doses of activin (0.1-3.6 nM) in the presence ofandrostenedione (5 X 107M). Values are meam±SEM of eight replicate wells of oneexperiment. 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____controlActivin0.6**G)C.)LC)0 0.5EC 0.4C20 30 40 50Time (hr)Figure 8. Time-course effect of activin-A on basal estradiol production by humangranulosa-luteal cells. Cells were cultured for 6, 12,24, and 48 h in medium (control),or activin (3.6 nM) in the presence of androstenedione (5 x 107M). Values aremean±SEM of eight replicate wells of one experiment ( ** p<O.Oi by one-way analysisof variance followed by Scheffe’s test).74500 b-öC0C.)0C 300000G)C a01___C)Cl)C)000control hCGCCI-//<‘7hCG+activi nactivinFigure 9. Effect of activin-A on basal and hCG-stimulated progesterone productionby human granulosa-luteal cells. Different letters above the SEM bars denotestatistical significance (b differs from a, c differs from a and b and is significant atpO.Ol by one-way analysis of variance followed by Scheffe’s test). (See legend tofigure 6 for details).753.*0L()0E 2CC00I1• IIC0C)Cl)C)0)0L.0•Control .1 .4 1.2 3.6Activin (nM)Figure 10. Dose-dependent effects of activin-A on basal progesterone production byhuman granulosa-luteal cells (See legend to figure 7 for details). * p<0.05.76Control * *t2—ci—— Activin**C.)If)oE0.80o 0.60C000)00.2 I I0 10 20 30 40 50Time (hr)Figure 11. Time-course effect ofactivin-A on basal progesterone production by humangranulosa-luteal cells (See legend to figure 8 for details). ** p<O.Ol.77__0.4C)C.)0E 03C.)Activin (nM)Figure 12. Dose-dependent effects of activin-A on hCG-stimulated progesteroneproduction 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 aremean±SEM of eight replicate wells of one experiment. Similar results were obtainedin one other separate experiments (* p<O.O5 by one-way analysis of variance followedby Scheffe’s test).78signfficantly at 24 and 48 h of treatment (Fig.11, p<O.Ol). Progesterone productionwas increased by hCG (1 IU/rril, F’ig.9, p<O.Ol). Activin-A (3.6 nM) significantlyinhibited hCG-stimulated progesterone production (Fig.9, p<O.Ol). The inhibitoryeffect of activin-A on progesterone production in response to hCG was dose-dependentand 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 andestradiol productionInhibin-A (3 nM) did not affect basal estradiol production (Fig.13). Estradiolproduction was increased by hCG (1 IU/ml, Fig.13, p<O.Ol). Inhibin-A (3 nM) did notaffect 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 productionThe effect of follistatin on estradiol production was examined in the presence orabsence of hCG (Fig.15). Follistatin (3 nM) exhibited a stimulatory effect on basalestradiol levels (Fig.15, p<O.O5). The effect of follistatin on estradiol production wasdose 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 levelsrevealed that estradiol production was stimulated in a time-dependent manner.791200-b—0 1000 I bI_OC.) IIS 800 ‘________________f_/f,,,,f_f,,,,,of/_f,,,,C ,,,,/_,,o 600(.)__,__,__.D ,,,_,,__ ,,,_,,__,,,,,___O ‘.%%\‘\•///400 “ ‘ ‘ “L.. - ,,,,,,,,D.. .,,_,_,,,— ‘f__f,,,,Oif,,,,,,.D ,,,,,_,,,CU 200- ‘‘‘‘‘‘‘‘__a ‘‘‘‘‘‘‘‘ a‘‘‘ii,,,Cl, ‘‘‘if,,,W___________‘i_i,,,,‘‘‘‘‘‘‘‘L,,’,,’4_____________________ _________________if’’.,,__________________________‘‘‘‘‘“‘I___0-___ ___ __ __ __ __ ___control hCG Inhibin hCG÷InhibinFigure 13. Effect of inhibin-A on basal and hCG-stimulated estradiol production byhuman 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 107M). Thedata were pooled from 3 separate experiments (n=8 wells per treatment in eachexperiment) and expressed as a percentage of control±SEM. Different letters abovethe SEM bars denote statistical significance (b differs from a and is significant atp<0.01 by one-way analysis of variance followed by Scheffe’s test).800C0C-)0C0000a)C0a)Cl)C)0)0Ia-Inhibinb ba600500400-300-2001000Figure 14. Effect of inhibin-A on basal and hCG-stimulated progesterone productionby human granulosa-luteal cells (See legend to figure 13 for details).LiControl hCG Inhibin hCG+81Estradiol 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 cellstreated with follistatin (3 nM) and hCG (1 IU/ml) concomitantly, follistatin did notfurther augment hCG-stimulated estradiol secretion (Fig. 15, p>0.05).E. Effect of follistatin on basal and hCG-stimulated progesterone productionThe effect of follistatin was examined in the presence and absence of hCG(Fig. 18). Follistatin (3 nM) stimulated basal progesterone production in the absenceof 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 levelsrevealed 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, follistatindid not further augment hCG-stimulated progesterone production (Fig.18, p>O.O5).F. Interaction between activin and follistatin on basal and hCG-stimulatedestradiol productionThe combined effect of follistatin (activin binding protein) and activin on estradiolproduction in human granulosa cells was examined in the presence and absence ofhCG (Fig.21). Follistatin at a concentration of 3 nM did not affect basal or82b1200b—__o I______I ICIOC.)8O00 bCo 600O.D0 400O——O.CU 200 aI-Co ___________ ‘/,,——,W ‘A i—i—’—__,_,_,.1 _____________,_.1 _______FI” ‘ ‘ ‘ ‘. % % ‘ %0 ,,4 ____‘—control hCG tollistatin hCG÷follistatinFigure 15. Effect of follistatin on basal and hCG-stimulated estradiol production byhuman granulosa-luteal cells. Cells were cultured in medium (control), hCG (1 RJ/ml),follistatin (3 riM), hCG+follistatin in the presence ofandrostenedione (5 X 107M). Thedata were pooled from 5 separate experiments (n=8 wells per treatment in eachexperiment) and expressed as a percentage of control±SEM. Different letters abovethe SEM bars denote statistical significance (b differs from a and is significant atp<O.O5 by one-way analysis of variance followed by Scheffe’s test).83**400300**20000L.a100CuL.Cl)Ui0 I I IIcontrol .1 .3 1 3Follistatin (nM)Figure 16. Dose-dependent effects of follistatin on basal estracliol production byhuman granulosa-luteal cells. Cells were cultured for 24 h in medium (control) ormedium supplemented with different doses of follistatin (0.1-3 nM) in the presenceof androsteneclione (5 X 107M). The data were pooled from 2 separate experiments(n=8 wells per treatment in each experiment) and expressed as a percentage of thecontrol±SEM ( ** p<0.01 by one-way analysis of variance followed by Scheffe’s test).8430_____control **___° Follistatin250If) **0120ECo 150oI0CuCl)w00 5 10 15 20 25Time (hr)Figure 17. Time-course effect of follistatin on basal estradiol production by humangranulosa-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 107M). Values aremean±SEM of eight replicate wells of one experiment ( ** p<O.O1 by one-way analysisof variance followed by Scheffe’s test).85500 b400b30000200-000Ib0:control hCGba// 4 ////:.•::.:‘.:.:•::..-::.:.><.<//•:z--’/ -//,Q..///,/////i/follistatin hCG+follistatinFigure 18. Effect of follistatin on basal and hCG-stimulated progesterone productionby human granulosa-luteal cells (See legend to figure 15 for details).86500 **0400‘4-0300 **o *L..0C) II.S ioCl)C)000 I I IControl .1 .3 1 3Follistatin (nM)Figure 19. Dose-dependent effects of follistatin on basal progesterone production byhuman granulosa-luteal cells (See legend to figure 16 for details). ** p<O.Ol.87Control * *120• Follistatin T0LC)100.I110 1’5**2’O 2’5Time (hr)Figure 20. Time-course effect offollistatin on basal progesterone production by humangranulosa-luteal cells (See legend to figure 17 for details). ** p<O.Ol.88hCG (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 hCGstimulated estradiol production. By comparison, activin stimulated basal estradiolconcentrations to approximately the same extent by 1 lU/mi hCG (p<O.O1). Whenfollistatin (12 nM) was added to the cell culture along with activin (3.6 nM), thestimulatory action of activin on basal estradiol production was inhibited.G. Interaction between activin and follistatin on basal and hCG-stimulatedprogesterone productionThe combined effect offollistatin and activin on progesterone production in humangranulosa cells was examined in the presence and absence ofhCG (Fig.22). Follistatinat a concentration of 3 nM did not stimulate basal or hCG (1 IU/ml)-stimulatedprogesterone production. On the other hand, activin-A (3.6 nM) stimulated basalprogesterone production (Fig.22, p.<O.05) but did not affect hCG (1 IU/ml)-stimulatedprogesterone production. When follistatin (12 nM) was added to the cell culture alongwith activin (3.6 nM), the stimulatory action of activin on basal progesteroneproduction was inhibited.89b1600-1400.___if,,C f__ff__io 1200-‘f_fC..) b ‘i_ibII-‘f_f‘f_ff_f,f__i1000-_ _Tb,I\‘\\ ‘%\f_f’_____‘f_ff__f, f__f f_f_fC %\‘‘s \‘‘%.___ff f__f f_f_f_____%\‘‘ \\‘‘ s\%\f__f, f__f f__f_.2 800-______f_f_f f_f_f f__f f_f_f\\s\’ \\%S% ‘.‘..‘.\I- f_f_f _ff__ f__f f_f_f ‘__f__C.) \\\\ %\%%% %,‘%\ %‘.‘.%\___ff f____ f__f f_f_f ‘f__f%%‘% %%\\% \•% %%%%f__f, f_f_f f__f f_f_f ‘f_f,\‘.%\ •.\%\ \%‘__f_f f_f_f f_f, __f__ ‘f_f,.D %\%\f__f, ___f_ f__f f_f_f ‘f__f‘\\\\ \\%‘.\ %\\f__f, f_f_f f__f f_f_f ‘f__fo 600- ‘‘\\\ ‘\%\\ %%%%%fffff f__/f f___ f_f_f ‘f__f\\\%% %\%Q— f__f, ____f f__f f_f__ ‘f__f\%\‘\ %%%‘•. ‘\\ %%%f_f_f f_ff_ f__f __f__ ‘f__f\\\‘% •.‘%‘\ •%\‘ %%‘.%f_f/f f_f__ /_f_ ___ff ‘f__f—‘\•% •.%\ \\\ffff i__f, fffi fff,f f__f%‘‘\ %‘.%% ‘\‘‘o 400 fffff ifff iffi ffff ffff%%%‘‘. %.%%‘ \\\‘I—a ff.D %•.‘% %%%\% \‘\f__f, f_i_f ifff fff f/ f_f,%%‘‘\ ‘%‘S \\‘‘.Cu __f_f f_f,, ffff f_f_f f/_ff_f_f f_f_f f_ff f_f_f f/_f%‘‘.% ‘‘% %%%%I f__if ffif iffi fffff fffs’\ %‘‘.% s%% \\Cl) 200 ‘‘‘ ‘‘ ‘‘i’’Ll.I a___‘.‘.% \\•.\% 1— \‘%•% ,‘‘‘ \\\\fffff ifff____ _fffff fffi ff/f /fff[ a ‘%\‘•. \\%\_______f_f_f f_/f _/_f_ fff ffff f__if fffff_f_I_f__f fi ‘fffff f/ff ffff ff-f fffffs’’’El‘fl ‘‘ ‘‘ %\\f_f_f f_f_f f__f f_f_f f_f_f f__f _fff_ ‘f//fr’ ‘\\ ss’\ %% ‘‘‘‘% ‘.\‘ \\‘%‘••%% \\\ %\%% ‘‘,‘‘ \%%%%0 if,,, ,fji ,,_ f__i __ff___control activin follistatinactivin+ hCG hCG+ hCG+ hCG+follistatin activin follistatin actlvin+follistatinFigure 21. The in vitro effect of activin in combination with follistatin on basal andhCG-stimulated estradiol production by human granulosa-luteal cells. Cells werecultured for 24 h in medium (control), activin (3.6 nM), follistatin (3 nM), activin (3.6nM) 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) plusfollistatin (12 nM) in the presence of androstenedione (5 x 107M). The data werepooled from 5 separate experiments (n=6 wells per treatment in each experiment) andexpressed as a percentage of the control±SEM (b differs from a and is significant atp<O.O5 by one-way analysis of variance followed by Scheffe’s test).90600,hCG hCG+ hCG+ hCG+activin follistatin actlvln÷follistatinFigure 22. The in vitro effect of activin in combination with follistatin on basal andhCG-stimulated progesterone production by human granulosa-luteal cells. (See legendto figure 21 for details).bI000C)0C)Cl)C)00500400300-200-1000-control activn follistatln actlvln+follistatin91H. Effects of Ang II and Ang III on basal and hCG-stimulated estradiolproductionTreatment of cells with Ang III (105M) did not affect basal estradiol production(Fig.23, p>O.O5); whereas, treatment of cells with Ang II (105M) stimulated basalestradiol production (Fig.23, p<O.Ol). The effect of Ang II on basal estradiolproduction was dose-dependent and became significant at a concentration of 106M(Fig.24, p<O.Ol). Time-course experiments revealed that basal estradiol productionwas stimulated significantly by Ang II (105M) at 24 and 48 h of treatment (Fig.25,p<O.O5).Estracliol concentrations were stimulated by hCG (I lU/mi, Fig.23, pcO.O1).Treatments of cells with Ang II (105M) or Ang III (1O-5M) did not affect hCGstimulated estradiol production (Fig.23, p>O.O5).I. Effects of Ang II and Ang HI on basal and hCG-stimulated progesteroneproductionTreatment of cells with Ang II or Ang III (105M) did not affect basal progesteroneproduction (Fig.26, p>O.O5). Progesterone production was increased by hCG (1 lU/mi,p<O.O1). Ang III (105M) suppressed hCG-stimulated progesterone production (Fig.26,pcO.O1). On the other hand, Ang II (105M) did not affect hCG-stimulatedprogesterone production (p>O.05).921000__b.__I800 b0______C) b7fI IO II___,, _,_,_, I_,_,,,__,,__?,•_•‘‘\\ %‘%‘.\600 __,_, ______ _,___\\\___,__,,____,,,_%%% %•.\‘ \%\\‘%b %‘%%%% ‘\‘\ \%%%‘s___,__,,_____,,_\\\O ____, ___,,_ ,__________,_____.,__,_,_I ‘%%%% %\‘ \%\‘%_______..,__ ,,,,,— I \‘.‘\O _______ ,_,_ _,_,,_ ,,,,,\%‘\%___,___,_,_ ,,,________,_,,, /_,_,400 _,_,_ _,_______,_O ____, __,,,_ ,_,__\S.%’ %‘\‘s% ‘‘%%‘%__,,,,,_,_ ,__,,%%% %%%\_,_,__,,_,__,___O— \%_\ \%%%\‘I,,,, ,,,l,%%%%%‘__.,__,‘_,____,_a— ,__,, ,_,__, ,_,__O ‘.‘%‘% ‘s%%% %\%\%‘___,_ ,___,_ ___,,%%%%‘.% %%%.‘‘_______,____,,I’ ‘%%% %\%%,____ ,_,,,_ __,,_200 \%‘‘ S ‘%Cu ____ _,,___ _,_,, ____,_ ,,_,_,,_,,____.._____,__,___ ,____a__,__ /,,,,,____, ,___,___,,‘\‘‘s%’ %%%%S %‘‘.‘%%Cl) ____ _,__ __,,,_ ___,_ ,____, __,______%%‘%‘ %%%%% %%%%•. s%%________,___,__________,__W I%\\%%I \%%‘ %.%%%% %%%%__,__, _,___ _____, _____%‘\%I %‘\‘, %%%% %\%%%,,,__1 /,,,,____________,,,___,__%%‘%I %%‘%‘ %%%%‘ %%%%‘ ‘%\‘‘ %‘%‘.%%—,—,,I __,___ ___,_ ,,__,, _.,,_/.\\\ %%%% %‘%%% ‘s’’%% %%%‘%%,__,_ _____ ,/__, __,,.., __,_,0 ‘‘-‘ ‘‘.‘‘-‘ — — — “‘-.“- —control Angil Anglil hCG hCG+ hCG+AnglI AnglilFigure 23. The in vitro effects of angiotensins (II & III) on basal and hCG-stimulatedestradiol production by human granulosa-luteal cells. Cells were cultured for 24 h inmedium (control), Ang II (105M), Ang III (105M), hCG (1 IU/ml), Ang II+hCG, andAng III÷hCG in the presence of androstenedione (5 X 107M). The data were pooledfrom 4 separate experiments (n=8 wells per treatment in each experiment) andexpressed as a percentage of the control±SEM (b differs from a and is significant atp<O.05 by one-way analysis of variance followed by Scheffe’s test).93400**0**o 300C.)‘4-0C.2 2000z0L.0.(UIC’)w0 I I I I0 1 10 100 1000 10000 100000Angiotensin II (nM)Figure 24. Dose-dependent effect of Ang II on basal estracliol production by humangranulosa-luteal cells. Cells were cultured for 24 h in medium (control) or mediumsupplemented with different doses of Ang II (1O9M- 105M) in the presence ofandrostenedione (5 X 107M). The data were pooled from 3 separate experiments (n=8wells per treatment in each experiment) and expressed as a percentage of thecontrol±SEM ( ** P<O.O1 by one-way analysis of variance followed by Scheffe’s test).9440_____controlAngil **C)Ino 30-E03’O4’O5’OTime (hr)Figure 25. Time-course effect of Ang II on basal estradiol production by humangranulosa-luteal cells. Cells were cultured for 6, 12, 24, and 48 h in medium (control),or Ang II (105M) in the presence of androstenedione (5 x 107M). Values aremean±SEM of eight replicate wells of one experiment ( * P<O.05 by one-way analysisof variance followed by Scheffe’s test).95b400-30000’C.) 20000a)C100a)0a)0)00Figure 26. The in vitro effects of angiotensins (II & III) on basal and hCG-stimulatedprogesterone production by human granulosa cells (See legend to figure 23 fordetails).baaacontrol Angli Anglil hCG hCG+ hCG÷Angil Anglil96II. Protein assay experimentsA. Effect of inhibin, activin, or foffistatin on protein content of granulosacellsThe protein content did not change throughout the cell culture period from day1 to day 5 of culture period (Fig. 27). After treatment with inhibin (3 nM), activin (3.6nM), or follistatin (3 nM) for a 24 h period, the protein content of the culturedgranulosa 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).9730aI_7/’ 7/_Day 1 Day 3 Day 5 Control Activln Inhibin FollistatlnFigure 27. Effects of inhibin, activin, and follistatin on protein content of culturedgranulosa cells. Protein content of granulosa cells were measured on day 1, 3, and 5of 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 proteinconcentrations of granulosa cells were measured. Values are mean±SEM (n=8 wellsper treatment) from a representative experiment. Similar results were obtained from3 other separate experiments.98CHAPTER FWE: DISCUSSIONI. Regulation of steroid production by inhibin-A and activin-AInhibin and activin were originally identified as inhibitors and stimulators,respectively, of FSH secretion from pituitary gonadotrophs. In this study the localeffects of recombinant human inhibin-A and activin-A were examined on humangranulosa-luteal cells. The present study demonstrates that recombinant humanactivin-A, but not recombinant human inhibin-A, can modulate steroidogenesis incultured human granulosa-luteal cells. The role of inhibin as an autocrine regulatorof granulosa cell function has remained controversial. While some investigators havereported inhibitory effects of inhibin on FSH-stimulated estrogen production ingranulosa 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 affectbasal or hCG-stimulated progesterone and estracliol production. These observationswere similar to those of Hutchinson et al. (1987) and Rabinovici et al. (1992) studiesin which inhibin had no effect on either basal or gonadotropin-stimulated estradiolor progesterone production in cultured rat and human granulosa cells respectively.Thus, it appears that inhibin-A does not exert specific effects on human granulosaluteal cells under the present experimental conditions. However, granulosa-lutealcells secrete immunoreactive imbibin in response to gonadotropins (Tsonis et al.,1987). Therefore, cellular secretion of high levels of inhibin could lead to saturationof inhibin receptors so that addition of exogenous inhibin might not elicit any99additional response.In this study, activin-A is capable of modulating steroidogenesis. Activin-Astimulated basal progesterone levels. Dose-dependency experiments revealed thatactivin significantly stimulated basal progesterone levels with maximal effectiveconcentration of 3.6 nM. In addition, activin-A attenuated hCG-stimulatedprogesterone production, although in a later set of experiments, activin-A did notaffect hCG-stimulated progesterone production. The cause for such discrepancies arenot entirely clear. However, differences in the maturation stages of granulosa cellsobtained from the 1VF program and different combination ofexogenous gonadotropinsused to induce ovulation in the patients can contribute to such discrepancies. It ispossible that the effect of recombinant activin on granulosa cell steroidogenesis isrelated to follicular maturity. Thus, activin-A may augment or inhibit steroidogenesisin follicular cells depending upon their states of differentiation and maturation.Activin augmented FSH-stimulated progesterone production in undifferentiatedgranulosa cells from ovaries of immature rats (Xiao et al., 1990; Miro et al., 1991).By contrast, activin-A inhibited hCG-stimulated progesterone secretion fromdifferentiated 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 onbasal progesterone production is somewhat controversial. Rabinovici et al. (1992)reported that activin-A inhibited both basal and hCG-induced progesteroneproduction in human granulosa cells. In the present studies, treatment with activinalone stimulated basal progesterone production in human granulosa cells, but100attenuated progesterone produätion when administered in the presence of hCG inmost experiments. These results agreed with the study of activin on differentiatedgranulosa cells in the rat (Miro et al., 1991). Such a gonadotropin-dependenttransition from stimulation to inhibition ofprogesterone reinforces the likelihood thatmore than one second messenger system is involved (Miro et al., 1991).In this study, activin stimulated basal estradiol levels without affecting hCGstimulated estradiol production in human granulosa-luteal cells. Activin increasedbasal estradiol concentrations at doses as low as 0.4 and 1.2 nM. In contrast to theseobservations, Rabinovici et al. (1992) reported that activin-A inhibited basal andgonadotropin-stimulated estradiol production in human granulosa cells. The reasonfor this discrepancy is not known. On the other hand, the results obtained from thisstudy are also similar to the action of activin-A on rat granulosa cells in which activinstimulates basal estradiol (Miro et al, 1991) and FSH-induced estradiol productionby 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 offollicular development, whereas FSH-stimulated progesterone production was initiallyenhanced (nondifferentiated cells) but became suppressed by activin in vitro afterexposure to FSH or hCG in vivo (Miro et al., 1991).The existence of specific binding sites for activin on the granulosa cells suggesta 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 obtainedfrom this study, as well as activin’s subunit gene expression in this human granulosa101cell culture system (Li and Leung, unpublished data), it is tempting to speculate thatactivin may be a local endogenous ovarian regulator. The specific inhibitory action ofactivin on hCG-stimulated progesterone production suggests that activin may preventor delay premature luteinization of granulosa cells. The enhancement of basalprogesterone production by activin may reflect an overall stimulation of thesteroidogenic pathway leading to increased estradiol formation. The stimulation ofestradiol production by activin suggests that activin could prevent the transition fromhealthy to atretic follicle by maintaining granulosa cells in a healthy (estrogenic)state of differentiation. The formation of estradiol and its actions on the granulosacells are critical to continued follicular development through to ovulation (Gibori andMiller, 1982).It has been reported that activin-A increases proliferation of cultured humanpreovulatory luteinizing ovarian follicular cells in a dose- and time-dependent manner(Rabinovici et al., 1990). The hypothesis that activin’s action on granulosa cell steroidproduction may involve changes in cell number of granulosa cells in culture wasexamined. The protein content per well was not altered after treatment with eitheractivin or inhibin for 24 h period. The protein content of the granulosa cells did notchange throughout the cell culture period. Thus, activin does not exert itssteroidogenic effect on the granulosa cells by altering the protein content per well.In conclusion, the present findings suggest that activin but not inhibin modulatessteroidogenesis. Thus, it is plausible that the overall action of activin may be topromote folliculogenesis and delay the onset of luteinization.102II. Regulation of steroid production by follistatin-288Follistatin was originally characterized as an FSH-releasing inhibitor (Robertsonet al., 1987; Ueno et al., 1987; Ying et al., 1987a) and more recently, as a local factorhaving the ability to affect differentiation of rat granulosa cells (Ying, 1988; Xiao etal., 1990). Follistatin-288 is similar in potency to inhibin-A in suppressing FSHactivity in vitro and more potent and longer acting than inhibin-A in viva (Inouye etat, 1991). A number of studies have localized follistatin message and/or protein in thegranulosa 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 notbeen 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 wereexamined on the human granulosa-luteal cells.In the first set of experiments, a stimulatory effect of follistatin (3 nM) on basalprogesterone and estradiol production without any effect on hCG-stimulatedprogesterone or estracliol production in differentiated human granulosa-luteal cellsin culture was observed. Follistatin increased basal estradiol and progesteroneconcentrations at dose of 1 nM. However, in a later set of experiments, follistatin didnot affect basal progesterone and estradiol levels. The cause for this discrepancy isnot entirely clear. However, differences in the maturation stages of granulosa cellsobtained from the 1YF program and the different combination of exogenousgonadotropins used to induce ovulation in the patients could contribute to suchdiscrepancies. It is possible that the effect of follistatin on progesterone production103is related to the follicular maturity. The 31, 35, and 39 Kda forms of follistatin hadno detectable effects on progesterone production by fully differentiated bovinegranulosa cells, which had presumably been exposed to an LH surge in vivo(Shukovski et al., 1990). However, follistatin augmented LH-stimulated progesteroneproduction 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 presentstudy where follistatin stimulated basal estradiol production in human granulosacells, an inhibitory effect of follistatin on FSH-stimulated estradiol production wasreported in rat granulosa cells (Xiao et al., 1990). The cause of this apparent speciesdifference in the granulosa cell response to the steroidogenic action of follistatin isunknown.The outcome of this study reveals a potential physiological role for follistatin asa modulator of luteinization in the human ovary. Follistatin is mainly located in thepreovulatory follicles (Sugawara et al., 1990). Increased progesterone secretion fromhuman preovulatory granulosa cells by follistatin supports the notion that follistatincould contribute in part to the rise in progesterone in large ovarian follicles before thepreovulatory LH surge (McNatty et. al., 1979). Follistatin’s stimulation of basalestradiol production may perhaps contribute to the maintenance of estradiolproduction by the granulosa cells in the preovulatory follicle. Inimunohistochemistrystudies have shown that the follistatin protein is localized to a subpopulation of earlytertiary follicles and the dominant follicles that are selected to ovulate (Nakatani et104al., 1991). These observations, together with the stimulatory effect of follistatin onbasal progesterone and estracliol production suggests a role for follistatin as apotential modulator of steroidogenesis in the human ovary.The hypothesis that the stimulatory effect offollistatin on progesterone productionmay involve changes in the protein content of granulosa cells per well in this culturesystem was examined. The protein concentrations of granulosa cells did not changethroughout the cell culture period or after 24 h treatment with follistatin. Thus,follistatin does not exert its effect on steroidogenesis by altering changes in thegranulosa cell protein content.Recent studies indicate that follistatin is a binding protein of activins in pituitaryand 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 blockactivin’s action. In the present study, the interaction between these two peptides wasexamined. These findings provide evidence that follistatin can also serve as a bindingprotein for activin. It seems that follistatin formed a complex with activin-A whichled to the neutralization of activin’s activity. Thus, it appears that follistatin canneutralize the folliculogenic actions of endogenous activin presumably by acting asan activin-binding protein. Follistatin protein is detected in the healthy growingfollicles of the human ovary (Sugawara et al., 1990). However, the activin 13-subunitsare located only in the small antral follicles of the human ovary and preovulatoryfollicles until the time of LH surge (Yamoto et al., 1992). The levels of niRNAs foractivin and follistatin by granulosa cells are FSH dependent (Sugawara et al., 1990).105This provides a basis for a short loop feed-back system whereby PSH could stimulateproduction of both activin and follistatin, and follistatin then regulates the localactions of activin (Xiao et at, 1992b).However, the possibility that follistatin may also have direct actions on granulosacells which is independent of its activin-binding activity cannot be ignored. In mostexperiments, both activin and follistatin stimulated basal progesterone and estradiolproduction independently. This implicates a direct action for follistatin onsteroidogenesis. The present results agreed with that ofXiao and Findlay (1991) whoalso showed that follistatin has a direct action which is independent of binding toactivin, since both follistatin and activin enhanced FSH-induced progesteroneproduction in undifferentiated rat granulosa cells. The enhancement of basalprogesterone production in human granulosa-luteal cells by follistatin independentof its activin-binding activity would be facilitated by its continued expression andproduction after the gonadotropin surge (Sugawara et aL, 1990) when the levels ofinhibin 13-subunit mRNA and activin protein in granulosa would fall sharply (Yamotoet al., 1992). Thus, it is possible that follistatin may have a direct effect on the cellsand can also neutralize activin’s action. However, the model of this study predicts ahigher foflistatinlactivin ratio in dominant follicles exposed to the preovulatory LHsurge. In the present studies, the concentration of follistatin required for effectivesuppression of activin was four fold that of activin. Normally the concentration offollistatin used in our experiments was 3 nM. However, when the interaction betweenactivin and follistatin were examined, the concentration offollistatin used was 12 nM.106In the interaction studies, follistatin at concentration of 3 nM was ineffective insuppressing the activin’s actions (data not shown). These results agreed with otherswho have reported that the concentration of follistatin required for effectivesuppression 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 localregulator of steroid production in the human granulosa cells. These observationssupport the hypothesis that the action of follistatin on granulosa cells is eitherdependent on its function as an activin-binding protein or by a direct action offollistatin on the granulosa cells.IlL Regulation of steroid production by angiotensinsFollicular development is regulated by the interplay between cyclic pituitarygonadotropin secretion and ovarian responsiveness to these pituitary hormones. Theobservation that not all follicles respond to FSH demonstrates functionalheterogeneity of follicles within the human ovary. The role of intraovarian regulatorymechanisms in producing follicular heterogeneity is becoming an important area ofstudy in research. Because the various proteins composing the cascade ofbiochemicalpathways 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 IIand Ang III with regards to steroid production in the ovary is unclear and requiresfurther investigation.107Ang II (105M) stimulated basal estradiol production but did not affect basal orhCG-stimulated progesterone production. Dose-dependency experiments revealed thatAng II stimulated basal estradiol levels at 106M and 105M significantly. Astimulatory effect of Ang II on estradiol secretion from PMSG-stimulated rat ovaryand 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 stimulatedgonadotropin-stimulated progesterone production in bovine and human granulosacells respectively (Stirling et al., 1990; Palumbo et al., 1988). Thus, it appears thatthere 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 appearto be selective; Ang II stimulated basal estradliol production without affectingprogesterone production. These observations, in conjunction with evidence for bindingsites 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 folliculardevelopment (Dorrington, 1977; Gibori and Miller, 1982). The formation of estradioland its actions on the granulosa cells are critical to continued follicular developmentthrough to ovulation (Gibori and Miller, 1982). If Ang II is an important stimulantof estradiol synthesis, Ang II may play a role in follicular development. Thus, thepresence 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 a108possible role for Ang II in maintaining intrafollicular estradiol levels during follicularmaturation.There are no reports on the effect of Ang III on steroidogenesis in the ovary fromdifferent species. However, it is possible that Ang II is secreted into the follicle andis then converted to the Ang III by the angiotensinase enzyme. There are reportsindicating 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 ofaldosterone secretion. In this study, Ang III inhibited hCG-stimulated progesteroneproduction but had no effect on basal or hCG-stimulated estradiol production, whichsuggests a specific role for Ang III as a potent inhibitor of progesterone productionin the human ovary. Ang II and Ang III may act together to promote the finalmaturation of the dominant follicle by enhancing basal estradiol production andsuppressing hCG-stimulated progesterone production, respectively.In summary, according to this study, Ang III inhibits hCG-stimulatedprogesterone production while Ang II stimulates basal estradiol production in humangranulosa-luteal cells. These observations, in conjunction with evidence for bindingsites 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 IImay influence follicular maturation by its stimulatory effect on estracliol production.Ang III may contribute to folliculogenic action of Ang II by blocking hCG-stimulatedprogesterone production and thus preventing or delaying premature luteinization.1091V. Summary & conclusionsA. Physiological roles of activrn and follistatinThe regulation of steroid production in the ovary through the endocrine limb ofhypothalamus-pituitary-ovarian axis is well established. However, increasing evidencesuggests that local regulators are also involved in the regulation of ovarian function.The specific aim of this study was to examine whether human granulosa steroidproduction is under the influence of several specific and the locally producedregulators such as inbibin, activin, and follistatin.The results from this study show that activin and foflistatin affect steroidhormone production in the human granulosa cells. The overall findings from thestudies show that treatment with activin stimulated basal progesterone and estradiolproduction consistently in differentiated human granulosa-luteal cells; whereas,treatment with activin in the presence of hCG either inhibited or did not affectprogesterone production. The enhancement of basal progesterone production byactivin may reflect an overall stimulation of the steroidogenic pathway leading toincreased estradiol formation. The stimulation ofbasal estradiol production by activinsuggests that activin could prevent the transition from healthy to atretic follicle bymaintaining granulosa cells in a healthy (estrogenic) state of differentiation. Thespecific inhibitory action of activin on hCG-stimulated progesterone production in asignificant number of cases examined suggests that activin may prevent or delaypremature luteinization ofgranulosa cells until the time ofpreovulatory surge of LH.In this study, follistatin either stimulated basal progesterone and estradiol110production or did not affect basal progesterone and estradiol production by the humangranulosa cells. The outcome of this study support a potential influence of follistatinas a modulator of luteinization in the human ovary. Follistatin and activin may alsoform a balanced network to regulate steroid production by the granulosa cells. Thepresent findings provide evidence that follistatin can overcome the actions of activinin vitro. Follistatin blocked the activin-induced increase in basal estradiol andprogesterone production in the human granulosa-luteal cells. Thus, it seems thatfollistatin can neutralize the actions of activin presumably by acting as an activinbinding protein. The ratio of activin to follistatin may be critical to steroid productionby the individual follicles. The model presented a higher follistatin/activin ratio indominant follicles exposed to the preovulatory LH surge. In the present studies, theconcentration of follistatin required for effective suppression of activin was four foldthat of activin. Figure 28 summarizes the action of activin and follistatin on thehuman granulosa cells.B. Physiological roles of angiotensinsThe various proteins comprising the cascade of biochemical pathways that leadsto the formation of the Ang IT/ITT have been found in the ovary, modulation of ovarianfunctions by the angiotensins seems likely. The results of these studies suggest a rolefor angiotensins in the regulation of steroid secretion from the granulosa cells.According to the present study, Ang II effects on ovarian steroid productionappear to be distinct; Ang II stimulated basal estradiol secretion without affecting111I Igonadotropin/\\AROMATASE Q/ cc/ANDROGENS CHOLESTEROL___PROGESTWSQ I activin IFigure 28. Diagram summarizing the actions of activin and follistatin on humangranulosa-luteal cells (AC=adenylate cyclase, P45Oscc=side chain cleavage P450enzyme).112basal or hCG-stimulated progesterone production. 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