Hormonal regulation of thecal cell function during antral follicle development in bovine ovaries

The hormonal regulation of thecal cell function was investigated with cells isolated at various stages of antral follicle development. Bovine thecal cells were isolated from small antral, medium antral, and large Graffian follicles (small, medium, and large ovarian follicles). Serum-free cultures of thecal cells were established and viable for a minimum of 6-8 days of culture. The purity of the thecal cell population was characterized cytochemically and was found to contain less than 5% endothelial cell and/or granulosa cell contamination. The steroidogenic capacity of this purified population of thecal cells in serum-free culture was examined through an analysis of androgen and progesterone production. Androgen production was high during the first 3 days of culture, then declined to undetectable levels. Production of androstenedione was approximately 10-fold higher than production of testosterone. Progesterone production remained relatively constant throughout the 8-day culture period. hCG was found to stimulate androgen production during days 1-3 of culture, but had a negligible effect on progesterone production. In contrast, hCG stimulated progesterone production during days 3-6 of culture, but had a negligible effect on androgen production. Insulin stimulated progesterone production during days 3-6 of culture, but had no effect on androgen or progesterone production during days 1-3 of culture. The minimum effective concentrations of hCG and insulin required to stimulate steroidogenesis of the thecal cells ranged from approximately 1-10 ng/ml. Addition of serum to the cultures decreased androgen production and suppressed the hormone responsiveness of the cells. Thecal cells in culture appear to alter their steroidogenic capacity from an androgen-producing cell to a progesterone-producing cell. Analysis of the developmental regulation of thecal cell function revealed that androgen production and hormone responsiveness were relatively constant in small, medium, and large follicles. In contrast, progesterone production and hormone responsiveness were highest in small follicles, intermediate in medium follicles, and lowest in large follicles. A more general analysis of the developmental regulation of thecal cell function examined the secretion of radiolabeled proteins. A large number of radiolabeled proteins were secreted by thecal cells, ranging in molecular mass from 5-500 kDa. Interestingly, insulin and hCG had no major effect on secretion of proteins by cells isolated from any of the stages of development examined.(ABSTRACT TRUNCATED AT 400 WORDS)     

Differentiation of rat ovarian thecal cells: evidence for functional luteinization

To determine if thecal cells of rat preovulatory (PO) follicles become functionally luteinized, theca from small antral (SA) and PO follicles were isolated before and 8 h after iv injection of an ovulatory dose (10 IU) of hCG. Thecal explants were cultured for 30 days in Dulbecco's Modified Eagle's Medium-Ham's F-12 medium containing 1% fetal calf serum (FCS) with or without 5 ng/ml ovine LH or 10 microM forskolin. Whereas theca from SA, hCG-treated SA, and PO follicles were dependent on LH or forskolin to maintain progesterone (greater than 10 ng/ml) and androstenedione (greater than 10 ng/ml) accumulation, luteinizing theca (hCG-treated PO) accumulated more than 10 ng/ml progesterone and more than 2 ng/ml androstenedione with or without LH or forskolin for 30 days. Granulosa cells were isolated from these same follicles and cultured under similar conditions, including 10 ng/ml testosterone and 25 ng/ml ovine FSH. Only granulosa cells isolated from luteinizing follicles (hCG-treated PO) maintained progesterone (greater than 20 ng/ml) and estradiol (10 ng/ml) accumulation with or without FSH or forskolin for 30 days. Basal concentrations of cAMP were 5 to 10-fold higher in thecal and granulosa cells from luteinizing follicles than in these tissues isolated from SA or PO follicles. We conclude that thecal cells as well as granulosa cells of rat PO follicles respond to the LH/hCG surge by becoming functionally luteinized, less dependent on LH, and capable of maintaining an increased accumulation of basal cAMP. Furthermore, the data suggest that one luteinizing thecal explant produces a similar amount of progesterone as one follicle equivalent of luteinizing granulosa cells. Thus, luteinized theca have the potential of contributing significantly to progesterone secretion by the mature rat corpus luteum.     

Transforming growth factor-alpha and -beta differentially regulate growth and steroidogenesis of bovine thecal cells during antral follicle development

The actions and interactions of transforming growth factor-alpha (TGF alpha) and TGF beta on growth and differentiation of bovine thecal cells were investigated. Bovine thecal interna cells were isolated from small (less than 5 mm), medium (5-10 mm), and large (greater than 10 mm) antral follicles and cultured in the presence or absence of TGF alpha and/or TGF beta. Both [3H]thymidine incorporation and changes in cell number (i.e. DNA levels) were evaluated to determine effects on thecal cell growth. Short term treatment of cells with TGF alpha (18-24 h) stimulated thymidine incorporation, and longer term treatments (4 days) increased cell number. TGF beta suppressed thymidine incorporation below that observed in untreated cultures, but had no effect on cell number. When combined with TGF alpha, TGF beta suppressed the ability of TGF alpha to stimulate thymidine incorporation and increase cell number. The response to these growth factors was similar for cells isolated from the different stages of antral follicle development. The effects of TGF alpha and TGF beta on thecal cell differentiation were evaluated by quantitating changes in androstenedione and progesterone accumulation in cultures treated with TGFs in the absence (basal) or presence of hCG, estradiol (E2), or a combination of hCG and E2. E2 and hCG were included in this study because previous research has demonstrated that these hormones alter thecal cell steroidogenesis. Treatment with TGF alpha resulted in a suppression of basal and hormonally stimulated accumulation of androstenedione during days 0-3 of culture, whereas TGF beta did not significantly alter androstenedione accumulation. TGF alpha also suppressed progesterone accumulation during days 0-3 of culture in the absence or presence of hormones. In contrast, TGF beta stimulated accumulation of progesterone in cultures that did not contain E2, which suppressed progesterone during this period. Therefore, during days 0-3 of culture, TGF alpha appears to have suppressive effects on androstenedione and progesterone production, whereas TGF beta can stimulate progesterone production in the absence of E2. During days 3-6 of culture, thecal cell differentiation changes, and the capacity to produce androstenedione dramatically declines, while the capacity to produce progesterone increases. During this period, either TGF alpha or TGF beta slightly increased basal progesterone accumulation and partially suppressed the ability of hCG to stimulate progesterone. The effects of TGFs on thecal cell steroidogenesis were similar with cells isolated from the different stages of antral follicle development. Results from these studies provide evidence that THF alpha and TGF beta can modulate thecal cell growth and differentiation (i.e. steroidogenesis).(ABSTRACT TRUNCATED AT 400 WORDS)     

Short term androgen production by rat ovarian follicles and long term steroidogenesis by thecal explants in culture

To characterize the aromatizable and 5 alpha-reduced androgens produced by developing ovarian follicles, small antral (SA) and preovulatory (PO) follicles, theca and granulosa cells were incubated for 4 h with or without 8-bromo-cAMP and androstenedione. In addition, thecal explants were cultured for 10 days with or without ovine LH (oLH) to determine if the hormone-induced changes in androgen synthesis by developing follicles could be mimicked in vitro. Short term incubations of SA and PO follicles, theca and granulosa cells in medium alone resulted in limited accumulation of androgen [testosterone, 5 alpha-androstan-17 beta-ol-3-one (DHT), 5 alpha-androstan-3 alpha, 17 beta-diol (3 alpha diol), and androsterone], as determined by RIA. In the presence of 8-bromo-cAMP, PO follicles produced large quantities of testosterone (3 ng), DHT (1 ng), 3 alpha diol (15 ng), and androsterone (14 ng), while SA follicles accumulated much less androgen (0.69, 0.05, 1.23, and 1.3 ng, respectively). In the presence of androstenedione and 8-bromo-cAMP, both SA and PO follicles and theca produced large amounts of aromatizable and 5 alpha-reduced androgens. SA and PO granulosa cells required the presence of the substrate androstenedione to produce androgens, primarily testosterone and 3 alpha diol. Therefore, progesterone, androstenedione, and 5 alpha-reduced androgens were used to monitor LH action on thecal cell function in culture. Small antral theca cultured in basic culture medium alone (containing 10% fetal calf serum) displayed an increased ability to accumulate androstenedione by day 6, approximately 3 times that observed on day 2. However, a 5-fold further increase in androstenedione accumulation was observed by day 6 for SA theca cultured in the presence of oLH. Maintenance of progesterone accumulation by SA theca throughout the culture period also was dependent on the presence of LH. In contrast, androstenedione accumulation by PO theca required the presence of LH in the culture medium, while progesterone accumulation in these cultures did not. Little or no 5 alpha-reduced androgen accumulated in the media of SA and PO theca cultured in basic culture medium alone. However, SA and PO theca cultured with oLH accumulated approximately 1 ng androsterone by day 10. We conclude that 1) SA and PO follicles, theca and granulosa cells possess the enzymes required to produce large amounts of 3 alpha diol and androsterone; 2) low concentrations of oLH are required to stimulate SA thecal steroidogenesis and to maintain PO thecal androstenedione accumulation in culture.(ABSTRACT TRUNCATED AT 400 WORDS)     

Inhibition by estradiol-17 beta of porcine thecal androgen production in vitro.

Thecal preparations from medium-sized procine ovarian follicles (3.5-5 mm diameter) were incubated for 4 h in a chemically defined medium in the presence or absence of highly purified luteinizing hormone (LH) and/or estradiol-17 beta (estradiol). LH (1 microgram/ml) stimulated the thecal production of testosterone (T) and dihydrotestosterone (DHT) by 2- to 3-fold. Although estradiol (10 microgram/ml) alone had only a slight but non-significant inhibitory effect on basal testosterone production, it significantly inhibited the production of both T and DHT as well as decreasing the DHT/T ratio in a dose-related manner in the presence of LH. Production of cyclic adenosine 3',5'-monophosphate (cAMP) and progesterone by the thecal cells was stimulated 50-to 200-fold and 2.5-fold, respectively, by LH. Estradiol had no significant effect on thecal cAMP and progesterone production in the presence or absence of the gonadotropin. These findings are consistent with the concept that estradiol produced by granulosa cells following hormonal stimulation may serve as a local negative feedback mechanism to control thecal androgen production.     

Interleukin-2 affects steroidogenesis and numbers of bovine ovarian granulosa cells but not thecal cellsin vitro

The effect of recombinant bovine interleukin-2 (IL-2) on steroidogenesis and numbers of bovine ovarian granulosa and thecal cells has been studied. Granulosa cells have been examined from both small (surface diameter ≤5 mm) and large (≥8 mm) follicles, whereas thecal cells from only large follicles were utilized. Estradiol and progesterone production per cell by granulosa cells from large follicles was 2- to 3-times greater than those from small follicles. Increasing doses of IL-2 significantly attenuated FSH-induced estradiol production by cells from small follicles but not large follicles. In general, progesterone production per cell by granulosa cells was almost double that of thecal cells. Moreover, IL-2 significantly attenuated FSH-induced progesterone production by granulosa cells from small and large follicles but had no effect on LH-induced progesterone or and-rostenedione production by thecal cells. Co-treatment of TNFα with IL-2 enhanced the responsiveness of granulosa cells to IL-2. The effect of IL-2 on the numbers of granulosa and thecal cells were studied independently under serum-free conditions and media enriched with 10% fetal calf serum. In serum-free medium containing insulin, IL-2 dosage significantly increased numbers of granulosa cells from large follicles, whereas IL-2 had no effect on numbers of granulosa cells from small follicles or thecal cells from large follicles. When cells were grown in medium enriched with serum, increasing doses of IL-2 significantly inhibited numbers without affecting viability of granulosa cells from small follicles, but had no effect on numbers of thecal cells. Thus, it appears that granulosa cells are more sensitive to IL-2 than are thecal cells. Approved for publication by the Director, Oklahoma Agriculture Experiment Station. This research was supported in part under project H-2088.     

Regulation of steroidogenesis in cultured porcine theca cells by growth factors

Growth factors [insulin-like growth factors (IGF-I, IGF-II), transforming growth factor-beta (TGF beta), epidermal growth factors (EGF)], found in the ovary and known to alter granulosal function, were assessed for their ability to modulate porcine thecal steroidogenesis. Theca cells from large porcine follicles (8-10 mm) were plated (5 x 10(5) cells/ml.well) in serum-free M199, treated with increasing doses of growth factors: IGF-1 (0.1-50 ng/ml), IGF-II (0.5-200 ng/ml), EGF (0.021-100 ng/ml), TGF beta (0.001-40 ng/ml), or insulin (0.01-50 micrograms/ml), with or without human CG [(hCG); 20 ng/ml], and incubated for 72 h. Levels of steroids in media were determined by RIA. Insulin increased (P less than 0.05) basal and gonadotropin-induced secretion of androstenedione, progesterone, estradiol, and testosterone. IGF-I increased (P less than 0.05) the basal and hCG-induced secretion of progesterone and androstenedione at the highest doses, but did not affect basal secretion of estradiol or testosterone. IGF-II, at the highest doses, increased (P less than 0.05) thecal steroidogenesis, but only after administration of hCG. In contrast, TGF beta increased (P less than 0.05) basal and gonadotrophin-induced secretion of estradiol but inhibited thecal secretion of progesterone, androstenedione, and testosterone. EGF did not alter thecal secretion of progesterone, androstenedione, or testosterone but significantly (P less than 0.05) inhibited basal and hCG-stimulated secretion of estradiol. In conclusion, insulin IGF-I, IGF-II, EGF, and TGF beta can modulate steroidogenesis in porcine theca cells.     

Multiple steroidogenic cell populations in the thecal layer of preovulatory follicles of the chicken ovary

The thecal layer of the preovulatory follicle of the chicken ovary produces primarily androgens and estrogens. However, the precise cellular sites of androgen and estrogen synthesis in the thecal layer have not been identified. Therefore, our aims were 1) to identify steroidogenic cells in the thecal layer of the preovulatory follicles by localizing specific steroidogenic enzymes in these cells by immunocytochemistry, and 2) to confirm that these cells which contained the specific steroidogenic enzymes secreted the expected steroid in a short term cell incubation. Follicles were collected 2 h after oviposition and prepared for immunocytochemistry and short term cell incubation. Immunocytochemistry for cholesterol side-chain cleavage cytochrome P450 (P450SCC), 17 alpha-hydroxylase cytochrome P450 (P450C17), and aromatase cytochrome P450 (P450AROM) was performed to localize pregnenolone-, androgen-, and estrogen-producing cells, respectively, on frozen sections and paraffin sections of the five largest preovulatory follicles. Interstitial cells showed immunoreactivity for both P450SCC and P450C17, whereas a specific cell population of the theca externa, hereafter termed aromatase cells, showed immunoreactivity for P450AROM. Furthermore, fibroblasts in the theca externa indicated immunoreactivity for P450C17. The immunoreactivity of P450C17 and P450AROM enzymes in specific cells in the theca externa appeared to decrease with follicular maturation. The third largest, fourth largest, and fifth largest follicles were selected for short term cell incubations because the immunoreactivity for P450 enzymes in the thecal layer of these follicles was high. Isolated theca interna cells, theca externa cells, and a combination of interna and externa cells were incubated with/without ovine LH (oLH) for 3 h. The medium was assayed for progesterone, testosterone, and 17 beta-estradiol by RIAs. Incubation of theca interna cells with oLH increased progesterone and testosterone production in a dose-dependent manner. However, we did not observe any production of progesterone and testosterone by theca externa cells. Theca externa cells produced 17 beta-estradiol, and its production was increased significantly when theca interna and externa cells were coincubated in the present of oLH. Based on these data, we propose a multiple cell theory for steroidogenesis in the thecal layer of preovulatory follicles of the chicken ovary which states that interstitial cells in the theca interna produce progestins and androgens, fibroblasts in the theca externa may function as an additional site for the conversion of progestins to androgens, and aromatase cells in the theca externa require androgens as substrate to produce estrogens.     

Ovulation induction with human menopausal gonadotropin compared to human urinary follicle-stimulating hormone results in a significant shift in follicular fluid androgen levels without discernible differences in granulosa-luteal cell function

Follicular fluid estradiol, progesterone, testosterone, and androstenedione levels were compared in 2 groups of spontaneously ovulatory women undergoing ovulation induction with human menopausal gonadotropin (hMG; which contains equal amounts of LH and FSH) or human urinary FSH (huFSH). The results were correlated with the ratios of embryo cleavage and pregnancy. Although significantly more FSH [1268 +/- 38 (+/- SEM) vs. 953 +/- 38 IU; P less than 0.05] was required for equivalent hyperstimulation in hMG compared to huFSH cycles, the number of oocytes retrieved and fertilized and the number of embryos transferred were similar for the 2 ovulation induction protocols. Forty-two follicles from 21 women stimulated with hMG and 38 follicles from 15 women stimulated with huFSH were examined and found to be representative of the total cohort of aspirated follicles. Follicular fluid estradiol and progesterone levels were similar, but hMG-stimulated follicles contained significantly more testosterone [7.83 +/- 0.52 (+/- SEM) vs. 6.30 +/- 0.42 ng/ml; P less than 0.03] and less androstenedione (24.4 +/- 3.6 vs. 37.8 +/- 5.0 ng/ml; P less than 0.03) than did huFSH-stimulated follicles. Embryonic cleavage rates were similar for all fertilized oocytes from both hMG- and huFSH-stimulated cycles, although pregnancy rates were significantly higher in huFSH cycles (40% vs. 9.5%; P less than 0.05). In addition, aromatase activity, progesterone production, and [125I]hCG-binding activity were compared in granulosa-luteal cells isolated from some of these women. Cells from 21 follicles from 9 women stimulated with hMG and 24 follicles from 9 women stimulated with huFSH were studied. There were no significant differences in aromatase activity, progesterone production, or [125I]hCG binding. Thus, the presence or absence of exogenous LH during ovulation induction with FSH has little direct effect on granulosaluteal cell function. However, the presence of LH during ovulation induction with FSH does appear to alter thecal androgen metabolism, resulting in higher testosterone and lower androstenedione levels in follicular fluid. Such a shift in androgen milieu may impair oocyte development and successful implantation.     

Regulation of androstenedione production by adenosine 3',5'-monophosphate and phorbol myristate acetate in ovarian thecal cells of the domestic hen

Although factors that regulate cAMP and steroid production in granulosa cells of hen preovulatory follicles have been well studied, much less is known of the mechanisms that control steroidogenesis in the adjacent thecal layer. These studies were conducted to examine the involvement and interaction of cAMP and protein kinase-C in modulating androstenedione output from isolated ovarian thecal cells collected from the second largest preovulatory follicle. Treatment of thecal cells with ovine LH (0.01-100 ng/tube) caused a dose-dependent increase in androstenedione secretion. Although coincubation of cells with the phosphodiesterase inhibitor 3-isobutyl-1-methylxanthine (0.1 mM) potentiated the effects of LH on steroid production, cAMP levels increased only in response to the higher doses of LH (10-100 ng/tube). Small but significant increases in cAMP accumulation and androstenedione production were observed in response to vasoactive intestinal peptide (0.1 and 1.0 microM), but not to 100 ng/tube chicken FSH, in the presence of 0.1 mM 3-isobutyl-1-methylxanthine. Treatment of thecal cells with cholera toxin (0.001-100 ng/tube) or forskolin (0.001-10 microM) resulted in a dose-dependent increase in cellular cAMP levels and androstenedione secretion. Thecal cell androstenedione production was also stimulated by the cAMP analog 8-bromo-cAMP (0.1-1.0 mM). Incubation of thecal cells with phorbol 12-myristate 13-acetate (PMA; 0.32-162 nM) or 1-oleoyl-2-acetylglycerol (OAG; 2.5-126 microM) increased basal steroidogenesis (progesterone and androstenedione production) in the absence of a rise in cAMP levels. By contrast, the stimulatory effects of 1 ng/tube LH on androstenedione, but not progesterone, production were attenuated by the presence of PMA (3.2-162 nM) or OAG (25-126 microM). Only a high concentration of OAG (126 microM) suppressed cAMP accumulation stimulated by LH (50 ng/tube). Phorbol ester treatment (32-162 nM PMA) also inhibited androstenedione production in thecal cells stimulated by the presence of 8-bromo-cAMP (1 mM), indicating a post-cAMP effect of protein kinase-C activity on steroidogenesis. In contrast to the effects of PMA, phorbol 13-monoacetate (162 nM), a nontumor-promoting analog of PMA which does not activate protein kinase-C, did not alter basal steroidogenesis, nor did it affect androstenedione secretion stimulated by LH or 8-bromo-cAMP. Data from the present studies indicate that the adenylyl cyclase-cAMP pathway can mediate the induction of thecal cell steroidogenesis by extracellular signals (i.e. LH and vasoactive intestinal peptide), whereas activated protein kinase-C can both stimulate and inhibit androstenedione production, depending upon the hormonal environment.(ABSTRACT TRUNCATED AT 400 WORDS)     

Granulosa and thecal cells from human ovarian follicles under growth: steroid formation in vitro and responsiveness to human chorionic gonadotropin

In order to characterize the patterns of steroid production and gonadotropin responsiveness in growing human follicles, follicular thecal and granulosa cells were incubated for two hours in the presence or absence of human chorionic gonadotropin (hCG). After incubation, tissue cyclic AMP (cAMP) levels and medium content of progesterone (P), androstenedione (A) and estradiol-17 beta (E) were determined. A was the dominant steroid formed by the thecal cells, regardless if these were derived from small (diameter: 4-7.5 mm) or from large (diameter: 8-15 mm) follicles. Granulosa cells from small follicles formed minimal amounts of all steroids measured, while granulosa cells from large follicles produced considerable amounts of E in vitro. Thecal cells from both small and large follicles increased their production of cAMP in the presence of hCG. Steroid formation was significantly increased by hCG in thecal cells from large follicles only. Granulosa cells from large follicles responded to hCG in vitro with increased cAMP and steroid formation, while granulosa cells from small follicles appeared insensitive to hCG in vitro.     

Stimulation of androstenedione and progesterone release by LHRH and LHRH agonist from isolated rat preovulatory follicles

Incubation of preovulatory rat follicles isolated from PMSG-treated immature rats for periods of less than 24 h in the presence of LHRH or LHRH agonist resulted in stimulation of basal androstenedione and progesterone release. The stimulatory effects seen were time-dependent, occurring from 2-3 h for androstenedione and from 8-20 h incubation for progesterone. Dose-dependent stimulation occurred with both LHRH agonist (in the range 5 X 10(-10) to 10(-8) M) and native LHRH (from 10(-9) to 10(-6) M), both being in the range of the reported Ka for ovarian LHRH receptors. LHRH did not enhance the stimulatory effect of 1-100 mIU hCG on androstenedione and progesterone release. These results show for the first time that LHRH and LHRH agonist can exert a stimulatory action on androgen release from isolated preovulatory follicles. This suggests that LHRH may be acting either directly at the thecal cell level or indirectly via decreased granulosa cell aromatization.     

Site at which ovarian nerve extracts inhibit thecal androgen production

We have recently reported that extracts of the superior ovarian nerve (SON) of rats inhibit porcine theca cell androstenedione production. Theca cells obtained from prepubertal gilts were cultured under serum-free conditions for 48 h. The inhibitory effect of SON extracts occurred within 6 h of treatment, was irreversible and independent of the dose of luteinizing hormone (LH) employed. The SON extracts' actions were exerted at a site(s) distal to the generation of adenosine 3',5'-monophosphate (cyclic AMP), since they did not affect extracellular cyclic AMP accumulation, while causing a significant inhibition of androstenedione production. The SON extract decreased 17 alpha-hydroxyprogesterone, androstenedione, testosterone, estradiol and estrone production while increasing progesterone and pregnenolone production, suggesting that the SON extract causes an inhibition of the 17 alpha-hydroxylase:C17-20 lyase complex. These results indicate that a factor(s) in the SON may play an important role in the regulation of follicular development, since thecal androgens are substrates for granulosa cell estrogen biosynthesis and are also involved in follicular atresia.     

Human chorionic gonadotropin stimulation of estradiol production and androgen antagonism of gonadotropin-stimulated responses in cultured human granulosa-luteal cells

Baseline and gonadotropin-stimulated estradiol production were examined in long term cultures of human granulosa-luteal cells isolated from women undergoing in vitro fertilization. Estradiol production declined by 70% during the first 48 h in culture and was minimally stimulated by the addition of hCG to the culture medium. During subsequent culture from 48-120 h estradiol production was significantly increased over control levels by hCG concentrations greater than 0.1 IU/ml. Incubation with testosterone stimulated estradiol production 100-fold in the presence and absence of gonadotropin. hCG (0.01-10 IU/ml) stimulated a 3- to 13-fold increase in progesterone production. However, at hCG concentrations greater than 1 IU/ml, coincubation with testosterone (10(-7) M) significantly inhibited progesterone production. Dihydrotestosterone also inhibited progesterone production, but to a lesser extent than testosterone. Freshly isolated granulosa-luteal cells specifically bound small amounts of [125I]hCG (less than 1,000 cpm/10(5) cells). Glycine buffer wash was shown to reversibly remove more than 88% of bound hCG and, in freshly isolated cells, increased [125I]hCG binding by 100%. In 5-day cultures, specific [125I] hCG binding nearly doubled from 52,000 cpm/10(5) cells in control cultures to 87,000 cpm/10(5) cells in cultures treated with hCG (0-5 IU/ml). At the highest concentration of hCG (5 IU/ml), testosterone (10(-7) M) significantly inhibited the amount of [125I]hCG specifically bound. In summary, estradiol production in long term cultures of granulosa-luteal cells appears to be gonadotropin dependent. In addition, the presence of testosterone (10(-7) M) antagonizes hCG-stimulated progesterone and LH receptor production by these cells.     

Inhibin removes the inhibitory effects of activin on steroid enzyme expression and androgen production by normal ovarian thecal cells

Activin and inhibin are important local modulators of theca cell steroidogenesis in the ovary. Using a serum-free primary theca cell culture system, this study investigated the effects of inhibin on theca cell androgen production and expression of steroidogenic enzymes. Androstenedione secretion from theca cells cultured in media containing activin, inhibin and follistatin was assessed by RIA over 144?h. Activin (1-100?ng/ml) suppressed androstenedione production. Inhibin (1-100?ng/ml) blocked the suppressive effects of added activin, but increased androstenedione production when added alone, suggesting it was blocking endogenous activin produced by theca cells. Addition of SB-431542 (activin receptor inhibitor) and follistatin (500?ng/ml) increased androstenedione production, supporting this concept. Infection of theca cells with adenoviruses expressing inhibitory Smad6 or 7 increased androstenedione secretion, confirming that the suppressive effects of activin required activation of the Smad2/3 pathway. Activin decreased the expression levels of steroidogenic acute regulatory protein (STAR), whereas STAR expression was increased by inhibin and SB-431542, alone and in combination. CYP11A was unaffected. The expression of CYP17 encoding 17α-hydroxylase was unaffected by activin but increased by inhibin and SB-431542, and when added in combination the effect was further enhanced. The expression of 3β-hydroxysteroid dehydrogenase (3β-HSD) was significantly decreased by activin, while inhibin alone and in combination with SB-431542 both potently increased the expression of 3β-HSD. In conclusion, activin suppressed theca cell androstenedione production by decreasing the expression of STAR and 3β-HSD. Inhibin and other blockers of activin action reversed this effect, supporting the concept that endogenous thecal activin modulates androgen production in theca cells.     

Influence of cortisol on insulin- and insulin-like growth factor 1 (IGF-1)-induced steroid production and on IGF-1 receptors in cultured bovine granulosa cells and thecal cells

During stress, hyperactivity of the adrenal gland can directly and indirectly inhibit ovarian function. However, little evidence existed to support the notion that glucocorticoids could influence insulin-like growth factor 1 (IGF-1) action within the ovary. Therefore, the effect of cortisol on IGF-1-induced granulosa and thecal cell function was evaluated. Granulosa and thecal cells from bovine ovarian follicles were cultured for 2 d in the presence of 10% fetal calf serum and then cultured for an additional 2 d in serum-free medium with added hormones. Cortisol had little or no effect (p>0.05) on IGF-1-induced progesterone production by granulosa cells from both small (1–5 mm) or large (≥8 mm) follicles. Also, cortisol had little or no effect (p>0.05) on basal, insulin-, or IGF-1-induced estradiol production by granulosa cells from small or large follicles, or on the number of IGF-1 receptors in granulosa cells from small follicles. Cortisol had no effect (p>0.10) on insulin-induced granulosa cell numbers, but increased IGF-1-induced granulosa cell numbers. In thecal cells, doses of 1–100 ng/mL of cortisol increased (p<0.05) insulin- and IGF-1-induced thecal cell numbers by 10–20%, progesterone production by 18–36%, and androstenedione production by two- to fourfold. The estimated dose of cortisol necessary to stimulate 50% of the maximum androstenedione production in the presence of IGF-1 was 7 ng/mL. In contrast, cortisol decreased (p<0.05) the number of IGF-1 receptors in thecal cells by 45%. In conclusion, cortisol at physiological levels can directly influence ovarian follicular function in cattle, especially thecal androstenedione production.     

Effect of recombinant activin on androgen synthesis in cultured human thecal cells

The effect of activin-A on ovarian androgen synthesis was tested in vitro using serum-free monolayer cultures of human thecal cells. Maximal rates of androgen (androstenedione and dehydroepiandrosterone) production were induced by treating the cells for 4 days with LH (10 ng/mL) in the presence of insulin-like growth factor-I (greater than or equal to 30 ng/mL). The additional presence of recombinant activin-A (1-100 ng/mL) in culture medium caused dose-dependent suppression of thecal cell androgen production, with 50% maximal inhibition occurring at an activin-A concentration of about 10 ng/mL. Progesterone production was only suppressed by high dose (100 ng/mL) activin-A, and inhibition of steroid production occurred without inhibition of DNA synthesis (tritiated thymidine uptake). These results reveal a potent and selective inhibitory action of activin-A on thecal cell androgen synthesis, consistent with a paracrine function for activin(s) in modulating follicular androgen biosynthesis in the human ovary.     

Calcium-dependent stimulation of estrogen secretion by FSH from theca cells of the domestic hen (Gallus domesticus)

There is little information about the stimulation of estrogen secretion from theca cells of the domestic hen by follicle-stimulating hormone (FSH), and the mechanism of action of FSH through calcium has not been considered previously. The theca interna and externa cells from the third (F3) and fourth (F4) largest ovarian follicles of hens were separated, dispersed, and incubated in M199 with FSH (0.5 micrograms ml-1) or A23187 (0.1 or 1 microgram ml-1) with or without ethylene glycol bis(beta-aminoethyl ether)N,N'-tetraacetic acid (EGTA) (2 mM) in calcium-replete or in calcium-free M199. Alternatively, androstenedione (10(-6) M) was added to the cells as aromatizable substrate, with or without FSH and/or EGTA. Estradiol and estrone secreted into the media during a 4-hr incubation period were measured by RIA. FSH stimulated estradiol and estrone secretion from all the cell preparations. The effect of FSH was abolished by the addition of EGTA or in calcium-free medium. A23187 stimulated estradiol and estrone secretion by the same extent as FSH, but did not do so in calcium-free medium. Androstenedione enhanced estradiol and estrone secretion, but estrogen secretion was further raised by the simultaneous addition of FSH. This action of FSH on aromatization was also abolished by EGTA. This evidence supports the hypothesis that calcium, possibly of extracellular origin, is an important mediator in the stimulation of aromatase systems in thecal cells of chickens.     

Effect of recombinant inhibin on androgen synthesis in cultured human thecal cells

Effects of inhibin (recombinant human inhibin-A) on ovarian androgen synthesis were tested in vitro using serum-free monolayer cultures of human thecal cells. Treatment for 4 days with inhibin alone at doses between 10 and 100 ng/ml caused modest (approximately 2-fold) increases in production of androgen (androstenedione and dehydroepiandrosterone): similar to the maximal level of stimulation caused by luteinizing hormone (LH) (10 ng/ml) alone but only about one-third of that caused by insulin-like growth factor I (IGF-I) (30 ng/ml) alone. Combined treatment with LH and inhibin elicited additive effects on androgen production whereas LH and IGF-I were synergistic, giving rise to androgen production rates at least 40 times greater than control. Additional presence of inhibin caused up to 10-fold augmentation of the response to LH + IGF-I. Activin (recombinant human activin-A) was previously shown to inhibit LH + IGF-I-induced androgen synthesis in this human thecal cell culture system. In the present study we found that the additional presence of inhibin (greater than 1 ng/ml) completely neutralized this inhibitory action of activin (10 ng/ml). These effects of inhibin were dose-dependent (ED50 1-10 ng/ml) and maximal at approximately 100 ng/ml. Inhibin stimulation of androgen synthesis occurred in the absence of measurable effects on progesterone production, and cell numbers in cultured cell monolayers were unaltered by the protein. It is concluded that inhibin exerts potent and selective stimulation of human thecal cell androgen synthesis in vitro. These results a paracrine role for inhibin(s) in modulating follicular androgen biosynthesis in the human ovary.     

Estradiol and luteinizing hormone regulation of insulin-like growth factor binding protein production by bovine granulosa and thecal cells

To determine the effects of estradiol and luteinizing hormone (LH) on insulin-like growth factor-binding protein (IGFBP) production by bovine granulosa and thecal cells, both cell types were collected and cultured in serum-free medium with various hormone treatments, arranged in three experiments. In thecal cells, insulin stimulated (p < 0.05) production of IGFBP-2 and IGFBP-5, but had no effect (p > 0.10) on IGFBP-3 and IGFBP-4 production; LH stimulated (p < 0.05) production of IGFBP-2 and IGFBP-3 but had no effect (p > 0.05) on IGFBP-4 and IGFBP-5. Estradiol had no effect (p > 0.10) on IGFBP-2, IGFBP-3, IGFBP-4, and IGFBP-5 production by thecal cells. Production of IGFBP-2/-5 by granulosa cells from small follicles was inhibited (p < 0.05) by insulin, but estradiol and LH did not influence (p > 0.10) insulin's inhibitory effect on basal IGFBP-2/-5 production. Insulin, LH, and estradiol each inhibited IGFBP-4 production by small-follicle granulosa cells, but their effects were not additive. IGFBP-3 was not produced by small-follicle granulosa cells. In large-follicle granulosa cells, insulin and LH inhibited (p < 0.05) production of IGFBP-2/-5 and IGFBP-3, whereas estradiol had no effect. Insulin alone had no effect (p > 0.10) on production of IGFBP-4, but estradiol and LH inhibited (p < 0.05) production by large-follicle granulosa cells, and their effects were not additive. These results suggest that production of IGFBP-2, IGFBP-3, IGFBP-4, and IGFBP-5 by granulosa and thecal cells is differentially affected by hormonal stimuli.