Testosterone selectively increases primary follicles in ovarian cortex grafted onto embryonic chick membranes: relevance to polycystic ovaries.

Histological studies have demonstrated that polycystic ovaries (PCO) contain increased numbers of preantral follicles with a specific increase in primary follicles. Polycystic ovary syndrome is associated with hyperandrogenism and pre- and postnatal androgenization of primates increases the pool of growing follicles producing changes resembling PCO. In vitro studies could test the hypothesis that androgens alter early folliculogenesis, but conventional culture techniques for small follicles are generally unsuitable in non-rodent species. Our objective was to develop and use a method to investigate the effects of testosterone on early folliculogenesis. We adapted an in ovo technique in which lamb cortical ovarian fragments were grafted onto the chorioallantoic membrane of fertilised chick eggs. Optimal experimental conditions for vascularisation and survival of tissue were determined and the model then used to investigate the effects of testosterone on follicle growth. Eggs were inoculated with testosterone at the time of implantation of the ovarian tissue, which was retrieved 5 days later. Tissue was sectioned and follicles staged and counted. There was no wholesale initiation of primordial follicle growth over the 5-day in ovo culture. Importantly, the proportion of primordial, primary and secondary follicles remained similar to those in unimplanted tissue. Testosterone increased the number of primary follicles by 50% compared with controls, an effect that was largely due to a reduction in atresia. In conclusion, incubation of ovarian cortex with testosterone reproduces the changes in early folliculogenesis reported in histological studies of PCO.

Dual effect of metformin on growth inhibition and oestradiol production in breast cancer cells.

Evidence has been accumulating for a role for metformin in reducing breast cancer risk in post-menopausal women. It inhibits growth of breast cancer cells via several mechanisms, primarily the AMPK/mTOR signalling pathway. Another possible protective mechanism may be the ability of metformin to inhibit aromatase activity. In the present study, we investigated the effects of metformin on the basal growth of MCF-7 cells, after oestradiol (E2) stimulation and after the inhibition of mTOR by rapamycin. Secondly, we investigated the effects of metformin on the activity of a number of steroidogenic enzymes and the mRNA expression of aromatase and steroid sulphatase (STS). High doses of metformin significantly inhibited both basal and oestrogen-stimulated cell division. Low-dose rapamycin (10-10 M) did not inhibit growth, but the addition of metformin induced a significant reduction in growth. High-dose rapamycin (10-8 M) inhibited growth, and this was further attenuated by the addition of metformin. Exposure to low (10-7 M) and high (10-4 M) doses of metformin for 7-10 days significantly reduced the conversion of androstenedione (ANDRO) and testosterone (TESTO) (both requiring aromatase), but not the conversion of oestrone or oestrone sulphate (ES) via 17ß-hydroxysteroid dehydrogenase/sulphatase to E2. This attenuation was via a downregulation in the expression of total aromatase mRNA and promoter II, whilst the expression of sulphatase was unaffected by metformin. In conclusion, plasma levels of metformin have a dual therapeutic action, first by directly inhibiting cell proliferation which can be augmented by rapamycin analogues, and secondly, by inhibiting aromatase activity and reducing the local conversion of androgens to E2.

Developmentally regulated responses of human granulosa cells to insulin-like growth factors (IGFs): IGF-I and IGF-II action mediated via the type-I IGF receptor.

In experimental animal models, insulin-like growth factors (IGFs) have been found to be more potent stimulators of ovarian function than insulin. In human theca cells, however, insulin, IGF-I, and IGF-II have similar effects on androgen production. The relative effects of insulin and IGFs on human granulosa cell steroidogenesis is unknown. Furthermore, it is unclear whether effects of IGF-II on steroidogenesis are mediated by the type-I or type-II IGF receptor. The effects of insulin, IGF-I, and IGF-II on human granulosa cell steroidogenesis were compared in vitro. As expected, insulin, IGF-I, and IGF-II enhanced steroidogenesis. Previously, IGF-II has been shown to enhance granulosa cell steroid production after insulin preincubation. In this study, an effect of IGF-II, independent of insulin priming, also was observed. In granulosa cell cultures from small antral follicles (< or = 13 mm), insulin and IGF-I stimulated steroid production to a similar degree, whereas IGF-II was less effective. In contrast, IGFs were more effective than insulin (IGF-I > IGF-II > insulin) in granulosa cells isolated from preovulatory follicles. IGF-I and IGF-II actions were mediated via the type-1 IGF receptor. The increased responsiveness of mature granulosa cells to IGFs may be an important mechanism by which granulosa cells increase their steroidogenic output in the preovulatory follicle.

Insulin preincubation enhances insulin-like growth factor-II (IGF-II) action on steroidogenesis in human granulosa cells.

Although abundant mRNA for IGF-II has been detected in the human ovary, a role for IGF-II in steroidogenesis has not yet been established. In rat adipocytes, incubation with insulin greatly increases cell-surface IGF-II receptor (5-fold) and the receptor is rapidly internalised in the absence of insulin. We have therefore investigated the effects of insulin preincubation on the response of granulosa cells from unstimulated ovaries to a range of doses of IGF-II. In the absence of insulin, IGF-II stimulated steroidogenesis in only one of three experiments. After incubation with 10 ng/ml insulin, there was a dose-dependent response to IGF-II in all experiments. Cells incubated with insulin produced 5-10 fold more estradiol in response to IGF-II than those incubated without. In contrast, insulin produced only a small increase of estradiol in response to IGF-I. These results demonstrate a synergistic interaction of insulin with IGF-II in human granulosa cells and suggest that there is an important role for IGF-II in human ovarian steroidogenesis.

Production of transforming growth factor-alpha by normal and polycystic ovaries.

The mechanism of anovulation in polycystic ovary (PCO) syndrome remains unknown. As circulating concentrations of FSH are apparently normal, and in vivo, granulosa cells from anovulatory PCO are hyperresponsive to FSH, it has been suggested that the lack of follicular development in anovulatory PCO is caused by overexpression of a paracrine growth factor that inhibits steroidogenesis. Epidermal growth factor and the structurally homologous transforming growth factor-alpha (TGF alpha) are suitable candidates for this role, but although the production of the latter has been demonstrated in the ovary, no comparison has been performed between the levels in normal ovaries and PCO. We compared the levels of TGF alpha in follicular fluid and in granulosa cell- and theca- and stroma-conditioned media from normal ovaries and PCO. TGF alpha was present in the range of 0.2-200 ng/mL in follicular fluid. There was a significant inverse correlation of TGF alpha with follicle size, with no differences between follicles from normal ovaries and PCO. Granulosa cell-conditioned medium contained concentrations of TGF alpha ranging from 0.1-200 ng/1000 cells. There was a wide range of concentrations in theca- and stroma-conditioned media, with levels varying from 0.2-100 ng/mg tissue and no consistent effect of LH. There were no significant differences between the levels from normal ovaries or PCO in medium conditioned by any compartment of the ovary. We conclude that the failure of folliculogenesis in PCO syndrome is not likely to be due to overproduction of TGF alpha by the ovary.

Expression of insulin-like growth factor (IGF), IGF-binding protein, and IGF receptor messenger ribonucleic acids in normal and polycystic ovaries.

Expression of the messenger ribonucleic acids (mRNAs) for insulin-like growth factors (IGFs), their binding proteins (IGFBP1 through IGFBP-6), and receptors was examined in normal and polycystic human ovaries (PCO). Northern and dot blots and RT-PCR were used to evaluate mRNA levels. The IGF system was studied in fresh granulosa, stromal, and thecal samples and in thecal tissue after incubation with LH and GH. IGF-II expression was high in granulosa and thecal compartments, whereas IGF-I was only weakly detectable, suggesting the importance of IGF-II in the human ovarian IGF system. Northern blot analysis revealed IGFBP-2 and -4 mRNA in all ovarian compartments and IGFBP-5 mRNA in stroma and theca. IGFBP-2 mRNA was the most abundant IGFBP mRNA in the human ovary. No IGFBP-1 or -3 mRNA was detected in fresh ovarian tissues. IGFBP-6 and type 1 and 2 IGF receptor mRNA expression was detectable in all ovarian compartments only by RT-PCR. In cultured theca, the expression of IG-FBP-1, -3, and 4 was induced. Only IGFBP-5 expression showed some dependence on trophic hormone (LH) stimulation during theca incubation. Otherwise, GH and LH had no effect on IGF or IGFBP expression in thecal tissue. This study indicates that thecal tissue is an essential part of the IGF system in the human ovary. However, no differences were found between normal ovaries and PCO in IGF, IGFBP, or IGF receptor expression. Thus, our results (from a limited number of patients) together with recent in situ hybridization and immunohistochemistry data of others provide no evidence for a role for IGFs in the functional disturbances related to PCO. Interestingly, our data show that in cultured thecal samples, the IGF and IGFBP expression pattern is different from that in fresh tissue.

Potent inhibition of human ovarian steroidogenesis by insulin-like growth factor binding protein-4 (IGFBP-4).

Several studies have now documented the existence of IGFBPs in follicular fluid and their correlation with the health of the follicle. In particular, increased levels of IGFBP-4 have been reported in androgen-dominant atretic follicles and those from polycystic ovaries. The aim of this study was to elucidate the role of IGFBP-4 in ovarian steroidogenesis. Granulosa cells and theca tissue were incubated with or without LH or FSH in the presence or absence of IGFBP-4 (0.5-50 ng/ml). Inhibition by IGFBP-4 of estradiol production in the presence of testosterone alone was seen in three of four experiments. IGFBP-4 completely inhibited FSH-stimulated estradiol production in three experiments and caused 67% inhibition in a fourth. Similar results were obtained for theca, in which concurrent incubation with IGFBP-4 completely negated the stimulatory effects of LH on androstenedione production. The mechanism by which IGFBP-4 exerts these potent effects and the possibility that this may by IGF-independent are currently being investigated.

Insulin-like growth factor-I (IGF-I) inhibits production of IGF-binding protein-1 while stimulating estradiol secretion in granulosa cells from normal and polycystic human ovaries.

We have investigated the actions of FSH and insulin-like growth factor-I (IGF-I) on the production of IGF-binding protein-1 (IGFBP-1) by granulosa cells from unstimulated normal and polycystic (PCO) human ovaries and related these effects to those on estradiol (E2). IGFBP-1 concentrations were measured in granulosa cell-conditioned medium (48-h culture with 10(-7) M testosterone) and follicular fluid. IGF-I (50 ng/mL), in the absence of FSH, stimulated E2 production by granulosa cells from both normal and polycystic ovaries, and there was a synergistic action between IGF-I and FSH. Granulosa cells secreted IGFBP-1 in concentrations ranging from 20-500 pg/1000 cells.48 h, with cells from two of four normal and three of four polycystic ovaries showing a dose-related increase in IGFBP-1 production in response to FSH. In contrast, the addition of as little as 100 pg/mL IGF-I to cells incubated with testosterone or testosterone plus FSH, caused complete inhibition of IGFBP-1 production. FSH treatment produced the expected dose-related increase in E2 accumulation. IGFBP-1 was detectable in fluid from all sizes of follicle tested, but there was no correlation of IGFBP-1 concentrations with follicle size and no difference between normal and polycystic ovaries. These data indicate that IGFBP-1 and E2 are differentially regulated by IGF-1 in the human ovary.

Premature response to luteinizing hormone of granulosa cells from anovulatory women with polycystic ovary syndrome: relevance to mechanism of anovulation.

Polycystic ovary syndrome is the most common cause of anovulatory infertility. Anovulation in polycystic ovary syndrome is characterized by the failure of selection of a dominant follicle with arrest of follicle development at the 5-10 mm stage. In an attempt to elucidate the mechanism of anovulation associated with this disorder we have investigated at what follicle size human granulosa cells from normal and polycystic ovaries respond to LH. Granulosa cells were isolated from individual follicles from unstimulated human ovaries and cultured in vitro in serum-free medium 199 in the presence of LH or FSH. At the end of a 48-h incubation period, estradiol (E2) and progesterone (P) were determined in the granulosa cell-conditioned medium by RIA. In ovulatory subjects (with either normal ovaries or polycystic ovaries), granulosa cells responded to LH once follicles reached 9.5/10 mm. In contrast, granulosa cells from anovulatory women with polycystic ovaries responded to LH in smaller follicles of 4 mm. Granulosa cells from anovulatory women with polycystic ovaries were significantly more responsive to LH than granulosa cells from ovulatory women with normal ovaries or polycystic ovaries (E2, P < 0.0003; P, P < 0.03). The median (and range) fold increase in estradiol and progesterone production in response to LH in granulosa cell cultures from size-matched follicles 8 mm or smaller were E2, 1.0 (0.5-3.9) and P, 1.0 (0.3-2.5) in ovulatory women and E2, 1.4 (0.7-25.4) and P, 1.3 (0.3-7.0) in anovulatory women. Granulosa cells from anovulatory (but not ovulatory) women with polycystic ovaries prematurely respond to LH; this may be important in the mechanism of anovulation in this common endocrinopathy.

Estradiol production by granulosa cells of normal and polycystic ovaries: relationship to menstrual cycle history and concentrations of gonadotropins and sex steroids in follicular fluid.

The underlying cause of anovulation in polycystic ovary syndrome is unknown. Circulating levels of immuno- and bioactive FSH are within the normal range, and the follicles contain measurable levels of bioactive FSH. The aim of this study was to compare estradiol (E2) production in response to FSH by granulosa cells from normal ovaries with those from polycystic ovaries derived from both anovulatory (anovPCO) and ovulatory subjects (ovPCO). Intrafollicular levels of immunoactive FSH, E2, and androstenedione in follicles of less than 12 mm were also measured. Follicular fluid steroid concentrations were obtained from 41 pairs of normal ovaries and 23 pairs of polycystic ovaries (8 anovPCO and 15 ovPCO). In size-matched follicles from each group there were no significant differences in follicular fluid FSH or E2 concentrations, but androstenedione levels were significantly higher in 5- to 11-mm follicles from ovPCO than in corresponding follicles from normal ovaries. Dose responses to FSH were determined in granulosa cells derived from 9 pairs of normal ovaries, 7 anovPCO, and 8 ovPCO. Cells from anovPCO produced 6- to 10-fold more E2 in response to FSH than normal cells, although there was no significant difference in the ED50 values. The response in cells from ovPCO was reduced compared to normal, but this difference did not reach significance. In summary, as judged by their FSH and E2 contents, polycystic ovaries do not have a higher proportion of atretic follicles than normal. Indeed, cells from anovPCO are hyperesponsive to FSH in vitro. This could be explained by stimulation of aromatase in vivo by either paracrine or, more probably, by endocrine factors, of which insulin is an arguable candidate.

Insulin-like growth factor (IGF) I and II, IGF-binding proteins, and IGF-binding protein proteases are produced by theca and stroma of normal and polycystic human ovaries.

There is increasing evidence for an important regulatory role for the insulin-like growth factor (IGF) system in the human ovary. IGF-I and -II and IGF-binding proteins (IGFBPs)-1 to -4 have been identified by analysis of follicular fluid and granulosa cell-conditioned medium and by in situ hybridization and Northern and dot blot analyses of ovarian tissues. It has been suggested that abnormalities of intraovarian IGF-I or IGFBPs may play a part in the pathogenesis of polycystic ovary syndrome. The aim of this study was to identify production of IGF-I and -II and IGFBP-1 to -4 by unstimulated normal and polycystic ovaries. IGF-I and -II were measured by RIA after acid-gel exclusion chromatography in medium conditioned by incubation for 48 h with granulosa cells or explants of theca or stroma. Both IGF-I and -II were present in the low nanograms per mL range in theca- and stroma-conditioned medium (T+SCM). IGFBPs in T+SCM were initially analyzed by Western ligand blotting, which revealed that low mol wt IGFBPs were predominant, especially IGFBP-2 (35 kDa). There was a band corresponding to 26 kDa with smaller amounts of a 31-kDa band, but only a trace of IGFBP-3 (44 and 40 kDa, confirmed by immunoblot). We found no consistent differences between normal and polycystic ovary syndrome ovaries, and although there was a trend toward increased IGFBP accumulation in response to LH, this was not consistent. We were unable to detect IGFs or IGFBPs by Western ligand blotting in granulosa cell-conditioned medium. In further studies we attempted to measure IGFBP-3 by RIA using two different antisera (alpha-BP-3gl and 1287-2-14) that detect different epitopes of IGFBP-3 and allow the presence of proteolytic activity to be demonstrated. Results obtained using alpha-BP-3gl were lower than those using 1287-2-14, suggesting proteolysis of IGFBP-3 in the medium. There was no evidence of proteolysis of serum IGFBP-3 after incubation with conditioned medium, but in contrast, radiolabeled [125I]IGFBP-3 was cleaved after incubation with T+SCM. Immunoblotting revealed intact IGFBP-2 (35 kDa) and bands of various sizes between 16-33 kDa. Immunoreactive fragments of IGFBP-3 between 13-40 kDa were seen. In conclusion, T+SCM contained IGF-I and -II. IGFBP-2 and -4 were the predominant species of IGFBP in T+SCM. T+SCM also contained significant protease activity directed toward IGFBP-2 and -3. Proteolytic activity may be an important mechanism by which bioactive IGFs are made available to these tissues.

The insulin-like growth factors (IGFs) in follicular fluid are predominantly bound in the ternary complex.

The presence of IGFs and their associated binding proteins (IGFBPs) in human follicular fluid is well documented. Furthermore, most of the constituents of the IGF system in follicular fluid have been found to vary, either in total amount or by proteolytic cleavage, depending on the health status of the follicle. In this study we have examined the acid-labile subunit (ALS) and found that levels in follicular fluid (mean 146 nmol/l) were almost 50% of those in the circulation. This amount of ALS was considerably greater than that found in other extracirculatory fluids (20.9 for synovial fluid and 31.4 nmol/l for skin blister fluid). As in the circulation, ALS levels were in molar excess and did not vary between atretic and dominant follicles. Although the source of ALS is probably from blood (conditioned medium from ovarian cell cultures had no measurable ALS) it would appear that this glycoprotein is not merely diffusing from the circulation as the capillary endothelium becomes more permeable in dominant follicles and this is not reflected in the level of ALS. Analysis of the distribution of IGF-I, IGF-II and IGFBP-3 in fluid from healthy and atretic follicles revealed that the majority of these growth factors (> 80% of total IGF-II) were in the 150KDa complex, indicating that the ALS present was functional, in that it formed the ternary complex with a molecule of IGFBP-3 and IGF. No free IGF-II was found in any of the follicular fluids analysed nor was there any increase in the amount of unsaturated IGFBP-3 in atretic follicles. In summary, we have shown that the majority of IGF measured in follicular fluid, whether from healthy or atretic follicles, is bound in the ternary complex.

Stimulation of prolactin secretion by oestradiol in the rat is associated with increased hypothalamic release of thyrotrophin-releasing hormone.

We have studied the effect of oestradiol (OE2) on secretion of prolactin and TSH by rat pituitary glands and correlated this with changes in hypothalamic content and release of thyrotrophin-releasing hormone (TRH). Ovariectomized Wistar rats received s.c. silicone elastomer implants of OE2 at a dose known to give pro-oestrous OE2 levels. After 1 week rats were decapitated, blood was collected for assay of prolactin and TSH, blocks of hypothalamus were dissected out and pituitary glands were removed and bisected. Medium bathing hemipituitary glands was collected for measurement of prolactin and TSH after a 30-min incubation. Immunoreactive TRH was measured in medium removed from hypothalami and in extracts of homogenized hypothalami. Serum prolactin was higher in OE2-treated than in control animals (59.3 +/- 19.5 (S.E.M.) vs 9.4 +/- 1.5 micrograms/l; P less than 0.05) and this was associated with a threefold increase in pituitary prolactin in the medium. By contrast, TSH concentrations in serum and pituitary incubation medium were not significantly different in the two groups. There was no difference between the groups in hypothalamic content of TRH but TRH release in the incubation medium was increased by OE2 (30.2 +/- 6.5 vs 10.0 +/- 1.3 pg/mg protein per 30 min; P less than 0.01). In summary, physiological levels of OE2 stimulated prolactin secretion without change in TSH and this was associated with a threefold increase in hypothalamic release of TRH. These findings suggest that the stimulating effect of OE2 on prolactin secretion may, in part, be mediated by hypothalamic TRH.

Modulation of the insulin-like growth factor-binding proteins by follicle size in the human ovary.

The IGFs are believed to play an important role in the regulation of steroidogenesis and follicular maturation in the human ovary. The activities of the IGFs are regulated by a family of binding proteins (IGFBPs) which are subject to a number of potential post-translational modifications. The aim of this study was to investigate both the production and modification of the IGFBPs in follicular fluid and in medium conditioned by granulosa cells and theca from individual follicles at different stages of maturation. In follicular fluid from healthy, dominant follicles there was an increase in the amount of IGFBP-2, -3 and -4 present as lower molecular weight forms (23 kDa, 29 kDa and 16.5 kDa respectively) in comparison to that seen in atretic follicles from the same ovary. Furthermore for IGFBP-4, this fragmentation was confirmed to be attributable to the presence of a specific protease which could be inhibited not only by the addition of metal ion chelators or serine protease inhibitors, but also by the addition of other recombinant unsaturated IGFBPs, particularly IGFBP-3. IGF-I did not modulate the activity of the IGFBP-4 protease in solution but was able to prevent the inhibition seen with IGFBP-3. Analysis of granulosa cell conditioned medium from the same series of healthy and atretic follicles revealed that IGFBP-2 and -4 were the predominant IGFBPs with no fragments seen on immunoblotting. In contrast, IGFBP-3 in conditioned medium from theca of atretic follicles was always found as an intact doublet, but was found partially fragmented (30 and 32 kDa) in medium conditioned by theca from healthy, dominant follicles with the proportion of IGFBP-3 in this lower molecular weight or fragmented doublet increasing with follicular maturation. A similar situation was also found for IGFBP-4 with the progressive increase in the amount of the 15 and 16.5 kDa fragments. IGFBP-2 was always found to be intact. Finally, IGFBP production from stroma explants was also examined. This revealed a wide variation in IGFBP pattern between different ovaries, although there was a remarkable degree of consistency between different stroma explant cultures from the same ovary. Immunoblotting for IGFBP-3 revealed that, where present, it existed as both an intact and a lower molecular weight doublet and that IGFBP-2 was again always found to be intact. In conclusion we have demonstrated alterations in the proteolytic modification of the IGFBPs which differ in the various follicular compartments and are closely linked to the stage of follicular development.

Direct gonadotrophic effect of growth hormone on oestradiol production by human granulosa cells in vitro.

In-vitro studies in both rodents and man suggest that GH can stimulate ovarian steroidogenesis, but it is not clear whether this effect is mediated by changes in circulating concentrations of insulin-like growth factor-1 (IGF-1) or whether it is a direct action on the ovary (or, indeed, both). In this study the effects of biosynthetic human GH (hGH) on the production of oestradiol and IGF-1 by human granulosa cells in culture were examined using ovarian tissue (from both normal and polycystic ovaries) which had not previously been exposed to exogenous gonadotrophin therapy. Addition of hGH (1 or 10 ng/ml) to the incubation medium resulted in a significant (1.7 to 3.6 fold) increase in oestradiol accumulation after 48h in culture. Human GH also had a significant additive effect on the dose-related responsiveness of granulosa cell oestradiol production to hFSH. Concentrations of IGF-1 in the medium were undetectable in each of these experiments. These studies demonstrate that hGH has a potent, direct stimulatory effect on production of oestradiol by the human ovary which is independent of the effect of FSH. These findings have important implications for understanding the physiological role of hGH in human ovarian function as well as for therapeutic use of biosynthetic hGH for induction of ovulation.

Human granulosa cells are a site of sulphatase activity and are able to utilize dehydroepiandrosterone sulphate as a precursor for oestradiol production.

Dehydroepiandrosterone sulphate (DHEAS) is the most abundant androgen in the circulation and in ovarian follicular fluid. A steroid sulphatase accepting DHEAS as a substrate has been identified in the follicle, but the cellular location has not been determined. As DHEAS is also a potential source of oestrogen for endocrine-dependent tumours, a potent steroid sulphatase inhibitor oestrone-3-O-sulphamate (EMATE) has been developed which inhibits this activity in rat liver and mammary tumour. The aim of this study was to investigate human granulosa cells as a site of steroid sulphatase activity, to determine whether DHEAS can be utilized as a precursor for oestrogen synthesis and to investigate the inhibitory capacity of EMATE in these cells. Conversion of DHEAS to DHEA was assessed in luteinized granulosa cells by tritiated steroid assay following incubation with or without LH or insulin and steroid accumulation in the medium measured by RIA. The effects of EMATE were assessed by addition of a range of doses during the measurement of conversion of DHEAS to DHEA. Cells from three sizes of small follicles from an unstimulated ovary were also assessed for their ability to produce oestradiol from DHEAS. Sulphatase enzyme activity was present in all cells; the mean conversion of tritiated DHEAS to DHEA was 50% (range 4-65%). LH and EMATE inhibited and insulin stimulated this activity. Addition of DHEAS to granulosa cells caused a dose-dependent increase in oestradiol and androstenedione production with no change in progesterone concentration. LH increased the accumulation of oestradiol in the medium. DHEAS also stimulated oestradiol production by granulosa cells from small follicles. This is the first demonstration that granulosa cells are a site of sulphatase activity and that DHEAS can be utilized as a substrate for androstenedione and oestrogen production. This may be of physiological importance for both normal folliculogenesis and oestrogen-dependent tumour growth.

Inhibitory effects of insulin-like growth factor-binding proteins on steroidogenesis by human granulosa cells in culture.

The effects of insulin-like growth factor-binding proteins (IGFBPs) 1 and 3 on steroidogenesis by human granulosa cells has been examined. Both IGFBP-1 and IGFBP-3 produced a dose-related inhibition of IGF-I-stimulated oestradiol accumulation in granulosa cell-conditioned medium with complete reversal of the effects of IGF-I in the presence of a molar excess of binding protein. IGFBPs 1 and 3 also exerted a small (25-40%) but significant and consistent inhibition of oestradiol secretion in response to follicle-stimulating hormone (FSH) alone. The progesterone response to IGF-I was inhibited by IGFBPs 1 and 3 but there was no effect on FSH-stimulated progesterone production. These data support the concept of a physiologically important intraovarian IGF system in the human ovary and demonstrate an unequivocally inhibitory effect of IGFBPs 1 and 3 on IGF-I-stimulated granulosa cell steroidogenesis.

Polycystic ovary syndrome: interaction of follicle stimulating hormone and polypeptide growth factors in oestradiol production by human granulosa cells.

The mechanism of the ovarian dysfunction in polycystic ovary syndrome, the most common cause of anovulatory infertility, remains obscure. Clinical data suggest that follicle stimulating hormone (FSH) action may be inhibited at the ovarian level by paracrine factors derived, presumably, from interstitial cells. The greater responsiveness to FSH of granulosa cells isolated from polycystic ovaries (PCO) compared with that seen in cells derived from normal ovaries, provides some support for this hypothesis and we present data which suggests that epidermal growth factor, or more likely transforming growth factor alpha, could be a candidate for this inhibitor. It should be emphasized, however, that the cardinal biochemical feature of the PCO is hypersecretion of androgens by interstitial cells. Stromal tissue from the PCO will secrete significant quantities of androstenedione in response to LH, whereas there is a negligible response in stroma from normal ovaries. It remains to be determined whether androgens have a direct inhibitory effect on FSH-induced oestradiol production in the human follicle, or whether they might exert an indirect effect by activating inhibitory polypeptide growth factors.

Induction of ovulation with clomiphene citrate in women with polycystic ovary syndrome: the difference between responders and nonresponders.

To identify why some women with polycystic ovary syndrome (PCO) fail to respond to clomiphene citrate (CC), the authors have monitored the endocrine and ovarian response to CC 100 mg/day given for 5 days. Of 40 cycles studied in 27 women, 73% were ovulatory. In 8 of 9 anovulatory women, there was no follicular development despite a significant rise in serum gonadotrophin concentrations within 3 to 5 days of starting CC. There were no significant differences between the ovulatory and anovulatory groups in the peak response of either luteinizing hormone (LH) or follicle-stimulating hormone (FSH). The authors conclude that, in women with polycystic ovaries, the most common reason for the failure to ovulate is an absent ovarian response to an appropriate rise in serum FSH.