4 Local control of ovarian steroidogenesis

4 Local control of ovarian steroidogenesis H E L E N M A S O N ehD Senior Research Fellow

S T E P H E N F R A N K S MD, ~-~CP, Hon MD(Uppsala) Professor of Reproductive Endocrinology Department of Obstetrics and Gynaecolog); Imperial College School of Medicine at St Mary's, London W2 1PG, UK

A number of putative paracrine factors are now thought to interact with FSH in the control of ovarian steroidogenesis. The relative importance of these factors remains to be determined, but the presence of the insulin-like growth factors and their binding proteins and the mechanism of control of the latter through the local production of proteases suggests a role for this system in folliculogenesis. We have demonstrated over-production of steroid hormones in tissue from women with polycystic ovaries. Theca cells in monolayer culture produced excessive amounts of progesterone and androstenedione and granulosa cell oestradiol production was considerably enhanced in response to FSH. Recent evidence points to a genetic defect in the expression or translation of steroidogenic hormones as a cause of excess androgen production, but the gene or genes involved has not been established. Data from our group suggest that granulosa cells from anovulatory polycystic ovaries are prematurely matured and we hypothesize that this is due to the interaction of the raised circulating insulin levels with LH in these follicles, an interaction that results in arrested follicular growth.

Key words: granulosa; theca; steroidogenesis; polycystic ovaries; insulin; insulin-like growth factors. Ovarian steroidogenesis is primarily controlled by pituitary gonadotrophins, but a plethora of additional modulators of steroidogenesis have been identified in recent years. In addition to novel endocrine regulators, there are multiple factors that are produced within the ovary and are therefore, by definition, paracrine. Steroids are o f fundamental importance in reproductive signalling and skeletal growth stimulation, and it therefore seems logical that there are multiple systems available to modulate their production and release. Abnormalities in the steroidogenic capacity of the ovary have been found to be associated with disorders of ovulation, the most obvious Baillikre "s Clinical Obstetrics and Gynaecology-Vol. It, No. 2, June 1997 ISBN 0-7020-2263-2 0950-3552/97/020261 + 19 $12.00/00

261 Copyright © 1997, by Bailli~re Tindall All rights of reproduction in any form reserved

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example being polycystic ovary syndrome (PCOS). This is the most common cause of anovulation (Adams et al, 1986; Hull, 1987; Franks, 1996) and is associated with hyperandrogenism (Stein and Leventhal, 1935; Franks, 1989). Efforts to elucidate the mechanism of anovulation in women with polycystic ovary syndrome (PCOS) have revealed abnormalities in the steroid production of both compartments of the follicle in these ovaries. As a significant proportion of these women have normal luteinizing hormone (LH) secretion (Franks et al, 1989) and immuno- (Yen, 1980; Franks et al, 1989) or bio-active (Baird et al, 1988; Fauser et al, 1991) follicle-stimulating hormone (FSH) levels do not appear to be sufficiently abnormal to cause the characteristic cessation of follicular growth, a search for possible abnormalities in these secondary regulators has ensued. This chapter is comprised of a discussion of the interaction of these factors in the local control of steroid production by the ovary with special reference to the abnormalities found in PCOS. It has become a widely accepted concept that the steroidogenic enzymes are to a great extent compartmentalized according to the 'two-cell theory' (Short, 1962; Lieberman, 1996 for review) and so this review is arranged according to androgen production by theca and oestradiol production by granulosa cells. THE THECA CELL The pre-menopausal normal ovary contributes an equal amount of androgen to the circulation as the adrenal. In both glands the majority of this androgen is in the form of androstenedione with testosterone being secreted in lesser amounts, and approximately 50% of the circulating testosterone being derived from peripheral conversion of androstenedione (Kirshner and Bardin, 1972; Vermeulin, 1979). In the ovary, progesterone and androgens are produced by the theca cell from cholesterol, which are primarily under the control of LH. The major rate-limiting step in thecal androgen production is controlled by the enzyme P450c 17cq a single protein that catalyses both 17(xhydroxylase and 17,20 lyase (desmolase), the two reactions required to convert pregnenolone to dehydroepiandrosterone and progesterone to androstenedione (Miller, 1988; McAllister et al, 1989; Ehrmann et al, 1995) (Figure 1). There is evidence for differential regulation of these two processes despite the fact that P450c 17o~is a single protein (Miller, 1988). In addition to LH, the classic stimulator of theca cell steroidogenic enzyme activity, several other hormones and growth factors have been shown to possess this activity, at least in vitro. The actions of growth factors and other modulatory peptides on granulosa cells are better described than the actions on theca. This is mainly because granulosa cells are much more readily available, particularly from in vitro fertilization procedures, whereas it is difficult to obtain 'pure' preparations of theca without removal of the human ovary. In addition, in contrast to granulosa cells, obtaining monolayer cultures of theca cells requires enzymatic dispersion of the tissue.

263

OVARIAN STEROIDOGENESIS 1

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Androgen production by theca cells from PCO It has been recognized since the earliest definition that PCOS is associated with over-production of androgens (Stein and Leventhal, 1935). The advent of high-resolution ultrasound scanning has, however, revealed the presence of polycystic ovaries in a wider spectrum of women than was initially anticipated (Hull, 1987; Polson et al, 1988b; Clayton et al, 1992), but despite the fact that many of these women have regular ovulatory cycles, their androgen levels were found to be elevated (Conway et al, 1989; Franks, 1989). There has been considerable debate as to the origin of these androgens, but the weight of evidence points to the ovary being the primary site (Erickson et al, 1985; Poison et al, 1988a). Interest has focused on androgen production in these women, not only because symptoms related to excess androgen are one of the primary reasons for presentation (Yen, 1980; Franks, 1989), but also because androgens have been implicated in the mechanism of anovulation. This was suggested because administration of testosterone to oestrogenprimed, immature female rats resulted in atretic degeneration of pre-antral follicles (Payne et al, 1956; Hillier and Ross, 1979) and because androgens are highest in those women who are anovulatory (Franks, 1991). The in vitro experiments mentioned, however, were conducted in the absence of FSH and the weight of evidence suggests that androgens are beneficial to early follicle development when FSH is present (Goff et al, 1979; Hillier and Tetsuka, 1997). Hypersecretion of ovarian androgens by the polycystic ovary could be due to the increased production of steroid per cell, or might simply reflect the increased number of follicles in these ovaries. We therefore examined the production of androgens by theca cells isolated from normal or polycystic ovaries. Previous comparative studies of steroidogenesis, by normal

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and polycystic ovaries, have been almost exclusively performed on tissue slices or explants of theca tissue, or by a comparison of follicular fluid levels of hormones (Lanthier and Sandor, 1960; Short and London, 1961; Short, 1962; Mahesh and Greenblatt, 1964; Wilson et al, 1979; McNatty et al, 1980). The value of these studies has been limited by the wide variability in androgen production between tissue samples and the small numbers of samples obtained from both normal and polycystic ovaries. A method of primary theca-cell monolayer culture was therefore developed (Gilling-Smith et al, 1994), which had the added advantage of allowing the comparative effects of LH on these cells to be studied. Theca and granulosa cells (see below) were obtained from the ovaries of patients undergoing surgery for benign non-ovarian gynaecological disease. The ovaries were classified into three groups according to menstrual cycle history and macroscopic morphological features at the time of dissection. A polycystic ovary was diagnosed when an ovary had at least three of the following criteria; increased volume (>9ml), 10 or more follicles of 2-8 mm in diameter, an increase in the amount and density of stroma and thickening of the tunica (Adams et al, 1985). Patients with a history of anovulatory infertility and/or oligomenorrhoea or amenorrhoea and no evidence of recent corpora lutea were designated anovulatory PCO (anovPCO), and those ovaries that met the above morphological critera, but in which a dominant follicle and/or a recent corpus luteum was observed, were designated ovulatory (ovPCO). Normal morphology was assigned when the ovary was of normal size and contained no more than five follicles greater than 2 mm in diameter in a woman with regular menstrual cycles. The method of cell culture was, briefly, as follows. Follicles were dissected intact from the stroma, measured, the follicular fluid aspirated and then incised to reveal the granulosa cell layer. These cells were processed as outlined below. The theca layer, devoid of adhering granulosa cells was removed, minced and enzymatically dispersed into single cells. Cells were incubated at between 10-15 x 104 cells per well in 1 ml serumfree Medium 199 (Gibco, BRL, Paisley, Scotland) for 48 hours with or without the addition of purified human pituitary LH. Steroids were measured in conditioned medium by radioimmunoassay. The results revealed that, in quantitative terms, the major steroid produced was progesterone, followed by 17-hydroxyprogesterone (17OHP), dehydroepiandrosterone (DHEA) and androstenedione. These results have been summarized in Figure 2. Comparison of the results from nine pairs of PCO with five pairs of normal ovaries from age-matched women demonstrated that accumulation of androstenedione was approximately 20-fold higher per theca cell in the PCO than in the normal group (Figure 2B). Production of 17OHP was also significantly higher than normal (median 7-fold) suggesting increased activity of both 17-hydroxylase and 17,20 lyase. Interestingly, the enhancement of steroidogenesis was also observed with respect to progesterone production, although there was more overlap in values between cells from normal or polycystic ovaries (Figure 2C).

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Figure 2. (A) Steroid accumulation by theca from normal or polycysfic ovaries under basal conditions. Bars represent the median steroid accumulated (pmol) per 1000 cells per 48 hours with the dots representing the range. N= 5 and 9 for normal and polycystic ovaries respectively, except for DHEA where N = 4 and 7. a = P < 0 . 0 1 ; b = P < 0 . 0 0 5 (Mann-Whitney U-test). Comparison of; (B) adione; (C) progesterone accumulation in theca from five normal ([3) and nine polycystic ovaries ( I ) . Each data point is the average of duplicate or triplicate experiments, Note logarithmic scale for adione. Reproduced from Gilling-Smith et al (1994, Journal of Clinical Endocrinology and Metabolism 79:1158-1165) with permission, © The Endocrine Society.

Of fundamental importance was the finding that steroidogenesis was enhanced in theca cells from polycystic ovaries irrespective of the menstrual history of the patient. Thus, increased 17-hydroxylase/17,20 lyase activity appears to be an intrinsic characteristic of theca cells from polycystic ovaries.

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The results of stimulation of the cultured cells by LH showed that the magnitude of response in relative terms was similar in each case showing that increased sensitivity of the theca cell to LH cannot be responsible for the increased secretion of steroid. We have also demonstrated that the proportion of atretic follicles in PCO is not significantly higher than in normal ovaries and so the data cannot be explained by these follicles being atretic (Mason et al, 1994b). In further studies the response of androstenedione secretion to insulin and insulin-like growth factors -I and -II was compared between theca from normal or PCO. Equimolar concentrations of these three peptides produced a similar enhancement of basal or LHstimulated androstenedione accumulation within each group and there were no significant differences in the responsiveness between cells from normal or PCO (Gilling-Smith, 1993). These results are consistent with those obtained in vivo (Rosenfield et al 1990; White et al, 1995) and support the hypothesis that there is abnormal regulation of key steroidogenic enzymes in the androgen pathway in the PCO. This concept is explored in more detail in the chapter in this volume by Rosenfield. The presence of a genetic defect in steroid biosynthesis would be in accordance with the results of studies showing that PCOS is a familial disorder (Cooper et al, 1968; Ferriman and Purdie, 1979; Hague et al, 1988). In family studies carried out at St Mary's in which assignment of affected status was made on the basis of a positive ultrasound scan and in men by the presence of premature balding, the segregation ratio (the ratio of affected to non-affected individuals) was calculated to be 51% (Carey et al, 1993). In a subsequent study, a single base change was found in the promoter region of CYP17, the gene encoding P450c 17o~ and this variant form of CYP17 was associated with the presence of PCO (Carey et al, 1994). Although this may be a significant factor in the expression of hyperandrogenism in PCO the results also revealed that this could be ruled out as the primary defect. This study has since been extended and the data now reveal that CYP17 is not involved (Gharani et al, 1996). Further candidate genes will need to be investigated, using large well-characterized families, if the genetics of this condition are to be unravelled, but it appears that the final expression of the syndrome may be the result of a combination of steroidogenic enzyme gene defects and external influences.

Suggested modulators of androgen production in PCO IGF-I and -H actions in theca

IGF-I has been shown to stimulate steroidogenesis (Cara and Rosenfield, 1988; Hernandez et al, 1988a; Erickson et al, 1989) and side chain-cleavage mRNA expression (Magoffin et al, 1990) in many species. In monolayer cultures of human theca insulin-like growth factor (IGF)-I and -II were seen to enhance both basal and LH-stimulated androstenedione production (Bergh et al, 1993; Gilling-Smith, 1993; Nahum et al, 1995). IGF-I and -II were detectable by radioimmunoassay in medium conditioned by explants

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of human theca, albeit at low levels, following rigorous acid-gel exclusion chromatography to remove interfering IGF-binding proteins (IGFBP) (Mason et al, 1996). Using this method, IGF-II levels were found to be several fold higher than those of IGF-I and this agrees with the results of further studies in which Northern and dot blots, and reverse transcription (RT)-potymerase chain reaction (PCR) were used to evaluate mRNAs for the IGFs (Voutilainen et al, 1996). These results suggest the likelihood of a modulatory role for the IGFs in theca cell function, with IGF-II being the more likely candidate for a paracrine effector in the human ovary. The IGFs have also been implicated in the mechanism of hyperandrogenaemia in PCO, but serum IGF-I levels appear essentially normal in these women. Furthermore, we have been unable to demonstrate any consistent differences in production of either IGF-I or -II between tissue from normal or polycystic ovaries. IGFBPs and IGFBP proteases The actions of the IGFs are dependent on their interactions with locally produced IGFBPs, of which six are well characterized and a further two putative. We demonstrated the presence of IGFBP-1 by immunoradiometric assay in medium conditioned by explants of human theca (Mason et al, 1996). Other studies have revealed the presence of a range of IGFBPs in the ovary by Western ligand blotting (WLB) of media (San Roman and Magoffin, 1992), in situ hybridization of cells and tissue (El Roiey et al, 1994) and by measurements in follicular fluid (Hamori et al, 1991; Cataldo and Giudice, 1992). We and others have demonstrated the ability of the IGFBPs to inhibit granulosa cell steroidogenesis (Ui et al, 1989; Bicsak et al, 1990; Adashi et al, 199l; Angervo et al, 1991; Mason et al, 1992) and such an effect may have important implications in the context of a disorder such as PCO in which the arrest of folliculogenesis occurs. For example, IGFBP-4 has been shown to be associated with atretic follicles and is present in higher concentrations in follicular fluid from PCO follicles than from normal oestrogen-dominant follicles (Cataldo and Giudice, 1992; San Roman and Magoffin, 1992). The IGFBPs themselves are also subject to proteotytic cleavage, and proteolytic enzymes capable of performing this function have been reported to be present in various biological fluids (Giudice et al, 1990; Cohen et al, 1992; Cwyfan-Hughes et al, 1992) and in the ovary (Grimes and Hammond, 1994; Cwyfan-Hughes et al, 1997). We therefore examined the theca cell-conditioned medium and follicular fluid from these follicles for the presence of IGFBP protease activity using Western immunoblotting (WIB) and by incubation of the media or fluid with recombinant radiolabelled IGFBPs, using a method originally described by Lamson et al (1991). Our earliest experiments in this field using a WLB and immunoblotting revealed considerable variability in the profile of IGFBPs according to the size and health of the follicle (Mason et al, 1996). We therefore began a systematic analysis of well characterized individual follicles to enable valid

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comparisons between follicles to be made. To examine the theca production of these proteins we incubated explants of theca (in quadruplicate) from individual follicles in which the health of the follicle had been established by estimation of oestradiol in granulosa cell-conditioned medium and in follicular fluid. In most follicles the predominant binding proteins in medium conditioned by theca were IGFBP-2 and -4. One surprising finding, in view of the fact that IGFBP-4 is regarded as an 'atretogenic' IGFBP when found in follicular fluid, was that theca from dominant follicles appeared to produce increased amounts of IGFBP-4 per milligram of tissue (as visualized by WLB) compared with theca from small atretic follicles from the same ovary. IGFBP-4 was not present in the follicular fluid in these follicles. This was due to the presence of a protease that was able to fragment radiolabeiled recombinant IGFBP-4 as well as IGFBP-4 from serum. We are currently investigating the effects of IGFBP-4 on theca steroidogenesis in the light of the paradoxical increase in production by dominant follicles. We were also able to demonstrate variability in the effect of LH on the IGFBP profile according to follicle size. Whereas LH had no effect on IGFBPs in theca cultured from small follicles, it invoked a marked dosedependent decrease in all binding proteins produced by theca from dominant follicles. This appeared to be due to direct inhibition of production of IGFBP-3 and -2 rather than an increase in protease activity. These results, taken together, demonstrate that the changes in the IGF system within the maturing follicle appear to be due to changes in local production of IGFBPs as well as to the induction of specific proteases activated within different compartments. The flexibility of such a system would enable the dominant follicle to alter the surrounding IGF milieu to its advantage by proteolytically cleaving IGFBPs and allowing free IGFs to accelerate steroidogenesis. Other follicles, in which the IGFBP levels remained high, would undergo accelerated atresia. The exact role of the IGF system in normal folliculogenesis remains to be elucidated, however. Only then will we be able to determine its significance in terms of disordered folliculogenesis.

Effects of insulin on theca steroidogenesis An indication that insulin is a possible stimulator of theca activity came from early observations of the association between insulin resistance and ovarian hyperandrogenism (Khan, 1976; Burghen et al, 1980). It was postulated that insulin stimulation of the ovary could be directly responsible for this over-production of androgens despite the presence of insulin resistance. It has been clearly demonstrated that insulin is able to perform this stimulatory action in cultured slices of theca of rodent (Cara and Rosenfield, 1988; Hernandez et al, 1988b), porcine (Barbieri and Ryan, 1983) and human (Barbieri et al, 1986; Garzo and Dorrington, 1984) origin. How therefore is insulin able to exert its effects on the ovary in an insulin resistant state? The most likely explanation is that the insulin resistance is

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selective and is confined to the muscle and fat tissues 'sparing' individual organs such as the liver (Peiris et al, 1989; Willis, 1996a). Another possibility is that there is a cross-reaction of insulin with the type-I IGF receptor although this is more likely to occur when the levels of insulin are very high, i.e. in severely insulin resistant states. The fact that hyperinsutinaemia is primarily associated with anovulatory women with PCO and not with weight-matched ovulatory women with PCO (Robinson et al, 1993) indicates that insulin may be implicated in the mechanism of anovulation in these women. However, we hypothesize that this is due to insulin effects on the granulosa cell and not on the theca. THE GRANULOSA CELL The granulosa cell is separated from the circulation by the basal lamina, which precursors required for continuing steroidogenesis must traverse. In addition to the production of progesterone, the granulosa cell is the site of conversion of androgens from the theca into oestrogens, a process that is principally under the control of FSH. It is generally acknowledged that granulosa cells lack P450c 17 and although there is evidence that some androgen is released by these cells in culture (McNatty et al, 1980; Mason and Franks, 1992), it is unlikely that any P450c 17 activity present in granulosa cells is of physiological relevance. In addition to FSH, a large number of other endocrine and paracrine modulators of granulosa cell steroidogenesis, both inhibitory and stimulatory have been described. This section will concentrate on oestradiol production by granulosa cells in culture, in particular the hyper-responsiveness of cells from PCO, and on those modulators that may be of importance in normal steroidogenesis and therefore of potential importance in this abnormality.

Steroid production by granulosa cells from PCO Our initial interest in culturing human granulosa cells stemmed from a search for a potential inhibitor of steroidogenesis that could cause the arrested follicular development associated with PCOS. We demonstrated that EGF was a potent aromatase inhibitor in these cells (Mason et al, 1990). However, during the course of these experiments it became clear that, per cell, oestradiol production by granulosa cells from PCO was much greater in response to FSH than by cells from normal ovaries. We therefore extended this study specifically to compare responses by the two types of ovary. The source of ovarian tissue and its morphological classification were as stated in the section on theca cell culture. Initial experiments were performed by pooling cells from size-matched follicles from individual patients from each of the three groups. Because of the known variation of FSH responsiveness with age, the patient groups were also carefully matched for age. Cells were cultured as previously described (Mason et al, 1990) and oestradiol measured in the overlying medium after 48 hours. Dose responses to FSH were determined in

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granulosa cells derived from nine pairs of normal ovaries, seven anovPCO and eight ovPCO. We were surprised to find that cells from anovPCO produced 6- to 10fold more oestradiol in response to FSH than cells from normal ovaries (Figure 3). There was no significant difference in the EDs0 values between the groups, but the cells from anovPCO were more sensitive in terms of the lowest effective dose of FSH. In contrast, the response of cells from ovPCO was reduced compared with normal cells. In order to further investigate the status of the follicles in these ovaries we analysed the oestradiol concentration in a large series of follicular fluids and found that in the size group studied, which excluded pre-ovulatory follicles, there was no difference in oestradiol content. There were also no differences in

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androstenedione concentration between follicles less than 5 mm in the three groups. There was, however, significantly more androstenedione in follicles from 5- to 10-ram in diameter in the ovPCO group than in the other two. We attributed this to the fact that although the theca in both PCO groups produces excessive amounts of androgen, the granulosa cells in anovPCO had an increased capacity to convert this androgen to oestradiol.

Suggested modulators of steroid production by granulosa cells in PCO At the time, the results from the granulosa cells appeared to be something of a paradox, in that follicular growth was arrested despite the simultaneous stimulation of aromatase. The most logical explanation was that there was production of an inhibitor of steroidogenesis in the anovPCO follicles, the effect of which was negated when the cells were removed from the follicular environment and cultured in vitro.

Transforming growth factor-a One putative inhibitor that initially appeared interesting was the polypeptide transforming growth factor ~ (TGF~). This is a ligand of the epidermal growth factor (EGF) receptor (Marquart et al, 1984; Derynk, 1986), but unlike EGF, has been shown to be produced by ovarian tissues (Kudlow et al, 1987). Using a highly specific antiserum (Owens et al, 199 l), we measured the levels of TGF~ in follicular fluid and in medium conditioned by granulosa cells or theca and stroma tissue from normal and polycystic ovaries (Mason et al, 1995). Although the levels of TGF~ in follicular fluid were in excess of those required to inhibit completely steroidogenesis in follicular fluid, we were unable to find any differences in the levels or production rates by normal or PCO that could explain the inhibition of follicular maturation in the latter.

IGF-I and -H actions in granulosa cells Another possible cause of this inhibition of follicle growth could be reduced production of an amplifier of steroidogenesis, particularly a paracrine factor. As mentioned in the section on theca cells, various components of the IGF system had been implicated in this role and in contrast to theca, a large body of data has accumulated on the production and actions of IGFs in the granulosa cell. Although IGF-I was proposed as the most powerful of these amplifiers, in contrast to other species, its production by human granulosa cells has not been demonstrated (Geisthovel et al, 1989). In the human, IGF-II is the most likely candidate for this role (for review see Giudice, 1992). However, its presence in significant amounts, particularly in dominant follicles, appeared to be incongruous with its apparent lack of effect on granulosa cell steroidogenesis (Erickson et al, 1990). We have recently repeated these

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experiments and found stimulation of oestradiol and progesterone by incubation of granulosa cells with IGF-II, particularly following incubation with insulin (Figure 4) (Mason et al, 1994a). These results may also help to explain the inhibitory effects of addition of IGFBPs to granulosa cells in the absence of IGF-I (Mason et al, 1992a), in that they may be explained by neutralization of endogenous IGF-II, thereby removing its amplification of FSH action. It remains to be determined, however, if abnormal regulation of IGF-II action or production has a role to play in PCOS. 60

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IGFBPs and IGFBP proteases in granulosa cells As the granulosa cell is a site of production of IGF-II it became important to evaluate the possible modulatory effects of locally produced IGFBPs on IGF-II action. We have previously demonstrated the presence of IGFBP-I by immunoradiometric assay in granulosa cell-conditioned medium and its regulation by insulin or IGF-I (Mason et al, 1993). In more recent studies, however, we were unable to demonstrate the presence of any IGFBPs using WLB or immunoblotting (for IGFBP-2 and -3) even when cells were incubated at 10~cells per well. Our failure to detect an abnormality in the production of a paracrine effector of steroidogenesis in PCO caused us to reconsider the actions of LH in these women. LH is known to cause the differentiation of granulosa cells, directing the pathway of steroidogenesis towards progesterone production and acting as a stimulator of this process, whilst inhibiting

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granulosa cell mitosis and arresting follicle growth, for instance at the time of the LH surge (Yong et al, t992a, b). Although the majority of women with anovPCO have been shown to have elevated serum LH (and in this group this could therefore be proposed as the mechanism), there remains a significant number in whom LH levels are essentially normal and so this cannot be the entire explanation. We therefore approached the problem by looking for the differences between ovulatory and anovulatory women with PCO, and so the insulin resistance and associated compensatory hyperinsulinaemia (Dunaif et al 1987; Robinson et al, 1993) came under scrutiny. The following section reviews the results of our experiments in this area.

The effects of insulin on granulosa cell steroidogenesis There appeared to be something of a paradox in proposing that insulin could have an effect on the ovary in women who were often profoundly insulin resistant. Our initial efforts, therefore, were directed towards determining the status of the ovary in terms of insulin resistance in these women. We were able to demonstrate a clear dose-response to physiological levels of insulin, in terms of steroidogenesis, in a woman with PCO and profound insulin resistance as evidenced by the presence of acanthosis nigricans (Willis et al, 1996a) (Figure 5A). Although this could provide an explanation for the increased aromatase level in these women this would not explain why the follicle ceases to grow. We therefore wondered if there was a possible interaction between insulin and LH in these ovaries that could be responsible for these combined phenomena. To address this question a series of experiments were performed in which granulosa cells were pre-incubated with insulin to mimic the hyperinsulinaemic conditions in vivo, the insulin was then removed and replaced with medium, with or without the addition of 5 ng/ml, of either LH or FSH. Insulin pre-incubation specifically enhanced the subsequent steroidogenic response to LH, but not to FSH (Figure 5B) (Willis et al, 1996a). These data are consistent with the hypothesis that hyperinsulinaemia in women with PCOS may be responsible for the increased granulosa cell steroidogenesis. At the same time, the enhanced response to what are in many cases already elevated LH levels, results in the premature activation of those events that are normally occurring at the time of the LH surge. That is, cessation of mitosis of the granulosa cell and increased steroid production (Willis et al, 1996b). In support of this we now have preliminary data to show that granulosa cells from anovPCO acquire LH responsiveness in smaller follicles than cells from normal ovaries (Willis et al, 1996c). In an earlier study obese women with anovulation and PCO were placed on a calorie-restricted diet and achieved a marked improvement in ovarian function (Kiddy et al, 1992). The weight loss was accompanied by a significant reduction in serum insulin concentrations, which our latest results indicate may have been the mechanism of the improved reproductive function.

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LH

Gonadotrophin Figure 5. (A) ~ production in response to insulin in granulosa cells from follicles from a patient with anovulatory PCO, peripheral insulin resistance and acanthosis nigricans. C represents basal E2 production in the ab~nce of testosterone. (B) Progesterone response to FSH (5 ng/ml) and LH in granulosa cells from an ovulatory woman with PCO, after pre-incubation with medium alone (control), FSH, insulin (INS) or insulin plus FSH. FSH was used in the pre-incubation stage to mimic the in vivo phenomenon of FSH-mediated LH receptor induction in the late follicular phase of the cycle. Pre-incubation with insulin alone and with FSH specifically increased LH induced P and E~ (data not shown) production. Reproduced from Willis et al (1996a, Journal of Clinical Endocrinology and Metabolism 81: 302-309) with permission, © The Endocrine Society.

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SUMMARY A large number of paracrine regulators of ovarian function have been identified and investigated in the last decade. Although clearly able to influence steroidogenesis and presumably also ovarian function, the relative importance of these factors in terms of normal folliculogenesis remains to be established. The presence and compartmentalization of all of the components of the IGF/IGFBP system and their ability to be regulated would indicate the likelihood that this system is of importance. Investigation of folliculogenesis in polycystic ovary syndrome (PCOS) has revealed over-production of steroidogenic hormones by both theca and granulosa cells in vitro. There is appreciable evidence to suggest that the increase in steroid production by theca is associated with a genetic defect in the expression and/or translation of steroidogenic enzymes in these women, but as yet the gene or genes involved have not been identified. We hypothesize that increased steroidogenesis by the granulosa cells in those women with anovulation, however, appears to be due to the interaction of LH and insulin, which, possibly via interaction with IGF-II, causes premature arrest of follicular growth.

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