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.     

Control of immunoactive inhibin production by human granulosa cells

OBJECTIVE: The aim was to determine the relation between stage of antral follicular development and granulosa cell production of immunoactive inhibin. DESIGN: Primary granulosa cell cultures in serum-free Medium 199 were incubated at 37 degrees C for 96 hours with a change of medium at 48 hours. Inhibin and steroid levels in culture medium were determined by radioimmunoassay. The inhibin assay was based on the N-terminal 1-26 amino acid sequence of the alpha-chain of porcine 32 kDa inhibin using pl alpha 1-26-GLY27-TYR28 as the immunogen, tracer and standard. PATIENTS: Granulosa cells were obtained from the ovaries of women with regular menstrual cycles undergoing hysterectomy with unilateral or bilateral oophorectomy to treat non-malignant gynaecological disease. RESULTS: Basal production of immunoactive inhibin by granulosa cells from presumptive preovulatory follicles (greater than 15 mm diameter) was 5-13 times higher than that by granulosa cells from immature (less than 10 mm diameter) or intermediately mature (10-15 mm diameter) follicles. Basal production of progesterone and oestradiol followed a qualitatively similar pattern, establishing a positive relation between functional granulosa cell maturity and inhibin production. Treatment of granulosa cell cultures from immature follicles with follicle-stimulating hormone (FSH), but not luteinizing hormone (LH), increased inhibin production, time and dose dependently. FSH, but not LH, also brought about similar increases in steroid hormone synthesis by granulosa cells from immature follicles. The stimulatory effect of FSH on granulosa cell inhibin production was augmented at least twofold by the presence of testosterone or 5 alpha-dihydrotestosterone (1.0 mumol/l) but was unaffected by oestradiol. Granulosa cells from intermediately mature follicles undertook variable degrees of both FSH and LH-responsive inhibin production which generally corresponded with gonadotrophin-responsive steroid production. Granulosa cells from presumptive preovulatory follicles showed inconsistent inhibin responses to FSH. However, LH caused marked (at least twofold) increases in inhibin production, paralleling LH-responsive steroid production. CONCLUSION: These results show that for human beings, granulosa cell capacity to produce immunoactive inhibin in vitro increases with follicular maturity. FSH, but not LH, stimulates inhibin production by immature granulosa cells and this response to FSH is subject to modulation by androgen. During preovulatory follicular development, production of inhibin, like steroids, becomes increasingly responsive to LH. Such a development-related pattern of granulosa cell inhibin production helps explain how, post-ovulation, the corpus luteum is able to secrete inhibin as well as steroids. It is also compatible with the concept that locally produced inhibin could participate in the paracrine control of follicular development during the human menstrual cycle.     

Increase in catecholamine-stimulated cyclic AMP and progesterone synthesis in rat granulosa cells during culture

Catecholamines stimulated cyclic AMP and progesterone synthesis in rat corpora lutea but not in the pre-ovulatory follicles. We collected granulosa cells from follicles of immature rats treated with pregnant mare's serum gonadotropin (PMSG) and cultured them either in monolayer or in suspension. Freshly collected granulosa cells responded to isoproterenol and epinephrine with 2-fold increases in cyclic AMP accumulation and progesterone synthesis. However, granulosa cells cultured in monolayer for 2 days responded to isoproterenol and epinepherine with a 90-fold and a 6-fold increase in cyclic AMP accumulation and progesterone synthesis, respectively. The accumulation of cyclic AMP in response to Catecholamines gradually increased in cells cultured in suspension, from 2-fold over control after 5 h, to 8-fold after 24 h. Granulosa cells isolated from hypophysectomized and diethylstilbestrol (Hx-DES) treated immature rats (containing pre-antral follicles) also showed an increase in cyclic AMP accumulation in response to Catecholamines during culture. Because these cells are devoid of an LH-responsive adenylate cyclase system, we conclude that luteinization of granulosa cells in culture is not necessarily the process responsible for the increased response to Catecholamines.During culture, the number of β-adrenergic receptors in granulosa cells rose from 6020 ± 400 per 10⁶ cells shortly after isolation to 26400 ± 1800 after 2 days in culture. This increase in receptor density during culture may be responsible for the change in the responsiveness to Catecholamines, although other factors, such as changes in coupling efficiency between the hormone-receptor complex and the adenylate cyclase moiety and/or supersensitivity to Catecholamines, should also be considered.     

Hormonal regulation of the growth and steroidogenic function of human granulosa cells.

The aim of this study was to assess development-related interactions between gonadotropins and insulin-like growth factor (IGF-I) on DNA synthesis and steroidogenesis in human granulosa cells. "Immature" granulosa cells were obtained from follicles during the late luteal phase or first half of the follicular phase; "mature" granulosa cells came from follicles during the second half of the follicular phase but before the midcycle LH surge; and granulosa-lutein cells were obtained as a by-product of in vitro fertilization. Granulosa cells were cultured for 96 h in serum-free medium 199 with and without LH or FSH, and in the presence and absence of IGF-I. The cell monolayers were then incubated with [3H]methyl thymidine to assess DNA synthesis. Spent culture medium was assayed for progesterone and estradiol content. Immature granulosa cells: Tritiated thymidine uptake in granulosa cell cultures from immature follicles were significantly increased by IGF-I. FSH was able to maintain or increase basal and IGF-I stimulated growth whereas LH had no effect. Basal progesterone production was low and not increased by either FSH or LH. However, treatment with FSH, but not LH, increased aromatase activity. Mature granulosa cells: IGF-I also stimulated thymidine uptake. However, whereas FSH either maintained or increased thymidine uptake by these cells, LH dose dependently suppressed thymidine uptake. This inhibitory action of LH was accentuated by the presence of IGF-I. Despite the inhibitory effect of LH on thymidine uptake, the gonadotropin markedly stimulated steroid production and the maximal steroidogenic response to LH was equivalent to 3-fold greater than that to FSH. Granulosa-lutein cells: Patterns of basal and IGF-I- and gonadotropin-stimulated steroid synthesis were similar to those observed for mature granulosa cells but steroid production rates were higher. Suppression of basal and IGF-I-stimulated thymidine uptake by LH was even more pronounced. These results suggest that the granulosa cell LH receptor, once expressed, negatively regulates cell growth and, simultaneously, positively regulates steroid synthesis. This development related event could be crucial to the mechanism whereby granulosa cells cease to divide and commence maximal rates of steroid synthesis in response to the LH surge.     

Relative effects of activin and inhibin on steroid hormone synthesis in primate granulosa cells.

Ovarian granulosa cells produce inhibin and activin, structurally related proteins with potentials to directly modulate follicular steroidogenesis. The aim of the present study was to compare development-related effects of inhibin-A and activin-A on steroidogenesis in marmoset monkey (Callithrix jacchus) granulosa cells. Granulosa cells from "immature" (< 1.0 mm diameter) and "mature" (> 2 mm diameter) follicles were incubated in serum-free culture medium for 96 h with and without peptide (1-100 ng/mL), in the presence and absence of gonadotropins [human (h) FSH or hLH] (10 ng/mL). Spent medium was collected and stored frozen for progesterone assay. Aromatase activity was determined by incubating cells for a further 6 h in the presence of 1 mumol testosterone and assaying accumulation of oestradiol. Granulosa cells from immature follicles showed characteristically low basal rates of steroid synthesis that were unaffected by treatment alone with either inhibin or activin. Treatment with hFSH stimulated both progesterone production and aromatase activity. Cotreatment with activin and hFSH further enhanced aromatase activity by up to 4-fold. The progesterone response to activin plus hFSH was related to the effect of hFSH in the absence of activin: high-level responsiveness to hFSH was suppressed by activin while low-level responsiveness was enhanced. Inhibin had no significant effect on FSH-responsive progesterone production, but at high concentrations (> 10 ng/mL) it caused slight (up to 30%) reduction in FSH-induced aromatase activity. Granulosa cells from mature follicles showed relatively high basal rates of steroidogenesis, and treatment with inhibin did not influence either basal or gonadotropin responsive steroidogenesis. Treatment with activin had divergent effects on aromatase activity and progesterone synthesis in that it increased both basal and hLH-responsive aromatase activity (up to 11-fold), had no effect on basal progesterone production, and markedly suppressed (by more than 50%) the progesterone response to hLH. These data reveal development-dependent effects of inhibin and activin on granulosa cell steroidogenesis that are likely to have physiological relevance to ovarian function in vivo.     

Independence of steroidogenic capacity and luteinizing hormone receptor induction in developing granulosa cells.

The relationship between FSH-induced acquisition of LH/hCG receptors and the steroidogenic capacity of granulosa cells from estrogen-primed hypophysectomized rat ovaries has been examined. Granulosa cells harvested from the immature preantral follicles of animals not treated with FSH (controls) displayed negligible specific human [125I]iodo-hCG binding and produced only minimal amounts of progesterone during 48 h of culture in vitro. Addition of highly purified hFSH or prostaglandin-E2 (PGE2) to the culture medium elicited substantial increases in progesterone production which were not accompanied by measurable increases in [125I]iodo-hCG binding. Treatment with oFSH in vivo for 24 h led to the initiation of antrum formation in many follicles and was accompanied by an 8-10-fold increase in hCG binding by freshly isolated granulosa cells. Basal, hFSH-, and PGE2-stimulated progesterone production during culture was also greater than controls. In contrast, cells from animals receiving oFSH in vivo for only 12 h showed no increase in hCG binding either before or after culture, yet basal and stimulated progesterone production in vitro was significantly greater than controls, indicating that the initiation of steroidogenesis was antecedent to LH/hCG receptor induction. Only those cells obtained after the 24-h in vivo treatment with oFSH produced elevated amounts of progesterone when incubated in the presence of hCG, thereby showing that the observed increases in [125I]iodo-hCG binding reflected the induction of functionally active LH/hCG receptors. Pharmacological stimulation of steroidogenesis by cell suspensions with N,O'-dibutyryl cAMP resulted in consistently high levels of progesterone production irrespective of previous treatment with FSH in vivo. This uniform expression of in vitro steroidogenic capacity occurred in the complete absence of measurable increases in LH/hCG receptors, suggesting that these two fundamental developmental processes are independent phenomena which may be under separate regulation in vivo.     

Development-related effects of recombinant activin on steroid synthesis in rat granulosa cells.

Activin is structurally related to polypeptide growth factors such as transforming-growth factor-beta, which may have paracrine and/or autocrine functions in the ovaries. We have investigated the action of activin on granulosa cell steroidogenesis in vitro in relation to preovulatory follicular development in vivo. Estrogen-primed immature female rats received no other treatment (nondifferentiated granulosa cells), treatment with ovine (o) FSH (differentiated granulosa cells), or treatment with oFSH followed by human (h) CG (preovulatory granulosa cells) to stimulate preovulatory follicular development. Granulosa cells were isolated and cultured in the presence and absence of recombinant human activin-A using serum-free medium supplemented with 1.0 microM testosterone as an aromatase substrate and hFSH, hLH, forskolin, or 8-bromo-cAMP to stimulate steroid synthesis in vitro. After 48 h, medium was collected for measurement of estradiol (aromatase activity), progesterone, and cAMP. Basal steroid synthesis in nondifferentiated granulosa cells was unaffected by activin, but both aromatase activity and progesterone production induced by treatment with FSH in vitro were dosedependently enhanced up to 10-fold by the presence of activin. FSH-stimulated cAMP production was not measurably altered by activin; however, steroidogenesis induced by forskolin or 8-bromo-cAMP was significantly enhanced by the factor. Thus the effect of activin on steroidogenesis includes action at a subcellular level(s) distal to the production of cAMP. After gonadotropin treatment in vivo, granulosa cell aromatase activity and progesterone production showed divergent responses to activin in vitro. Basal-, FSH-, and LH-stimulated aromatase activity were all enhanced by activin in cultures of differentiated and preovulatory granulosa cells. However, whereas basal progesterone production was stimulated by activin in cultures of differentiated granulosa cells, in preovulatory granulosa cells it was inhibited. Moreover, in vitro stimulation of progesterone production by treatment of both differentiated and preovulatory granulosa cells with FSH or LH was suppressed by the presence of activin. Thus rat granulosa cells display development-related steroidogenic responses to activin, aromatase production becoming enhanced and progesterone production suppressed as follicular maturation progresses. These results further implicate activin as a local modulator of granulosa cell steroid synthesis in the ovaries, although its functional significance has yet to be established.     

Release of 3H2O from 1 beta,2 beta[3H]androstenedione by equine granulosa cells

Granulosa cells were harvested from mares at various stages of the oestrous cycle and incubated in Krebs-Ringer bicarbonate buffer with 1 beta,2 beta[3H]androstenedione as substrate. The release of 3H2O expressed as CPM/h/mg protein varied from 44000 to 768000 in follicles from 7 mares. The release of 3H2O was not significantly altered by luteinizing hormone, follicle stimulating hormone or pregnant mare's serum gonadotrophin. There was a significant negative correlation between the release of 3H2O and the concentration of progesterone in the follicular fluid. Based on the assumption that the release of 3H2O represent total aromatization, these data suggest that the equine granulosa cells have a very active aromatizing enzyme system.     

Characterization of dissimilar steroid productions by granulosa, theca interna and theca externa cells during follicular maturation in the turkey (Meleagris gallopavo).

Unlike the established models for steroidogenesis in the rat and human, we have previously demonstrated that in the turkey progesterone (P), androgen (A), and estrodiol (E) are primarily produced by the granulosa, theca interna, and theca externa cells, respectively. In the present study, experiments were conducted to further characterize steroid productions by these cell types during follicular maturation. In Experiments 1 and 2, granulosa cells and theca internal cells, respectively, from the larger (F1) and fifth largest (F5) preovulatory follicles were incubated (1 x 10(5) cells/ml) with ovine luteinizing hormone (oLH) or porcine follicle stimulating hormone (pFSH) for 5 hr. Granulosa production of P from both follicles was stimulated in response to oLH, with both basal and LH-stimulated P production greater in the larger F1 than in the smaller F5 follicle. No A or E production was detected in granulosa cells from either follicle size tested. Theca interna cell productions of P and A were stimulated by oLH in the smaller F5, but not in the larger F1, with both basal and stimulated levels of A greater in F5 than in F1. Medium content of E was non-detectable in cultures of theca interna cells from all follicles tested. In Experiment 3, theca externa cells were incubated (1 x 10(6) cells/ml) from F1 and F5 follicles. The theca interna cells from F5 and the seventh largest follicle (F7) were pooled (1 x 10(5) cells/ml) to provide substrate(s) for theca externa steroidogenesis. Theca externa cells were incubated alone and in combination with the pooled theca interna cells and with or without oLH or pFSH.(ABSTRACT TRUNCATED AT 250 WORDS)     

The association between granulosa cell aromatase activity and oocyte-corona-cumulus-complex maturity from individual human follicles

The steroidogenic capability of granulosa cells isolated from 12 preovulatory human follicles was correlated with the stage of maturation of the corresponding oocyte-corona-cumulus-complex ( OCCC ). Individual follicles from human menopausal gonadotropin (hMG) stimulated cycles were aspirated 36 h after administration of hCG. Granulosa cells were cultured for 150 min and corresponding OCCC were evaluated for maturity before fertilization with human sperm. Granulosa cell aromatase activity was measured using 1 beta-3H-testosterone as substrate by quantitating the amount of 3H2O produced. Progesterone production by the granulosa cells was measured as was follicular fluid levels of combined hCG and LH activity and FSH and PRL. Follicular fluid concentrations of combined hCG plus LH activity decreased somewhat while FSH levels increased as OCCC matured. PRL levels did not vary. Granulosa cell progesterone production did not change with maturity of OCCC . However, aromatase activity decreased as OCCC matured with levels from granulosa cells with immature OCCC vs. intermediate and mature OCCC of 260 +/- 148 vs. 129 +/- 53 (SE) pg E2/10(5) cells, respectively (P less than 0.07). Although granulosa cells responded variably to hMG stimulation from individual to individual, and the response was not predictable from peripheral serum estradiol levels, follicles isolated from the same patient had a definite diminution in aromatase activity with OCCC maturation. From these preliminary results, aromatase activity in immediately preovulatory granulosa cells declined as OCCC matured in hMG/hCG stimulated cycles.     

Developmental changes in luteinizing hormone/human chorionic gonadotropin steroidogenic responsiveness in marmoset granulosa cells: effects of follicle-stimulating hormone and androgens

Factors regulating LH/hCG responsiveness in primate granulosa cells were examined in the marmoset monkey (Callithrix jacchus). Granulosa cells were isolated and pooled from small antral (0.5-1.0 mm) and large preovulatory (greater than or equal to 2 mm) follicles from mid- to late follicular phase ovaries of cyclic marmosets. The cells from small and large follicles were cultured in serum-free medium for 48 h in the absence or presence of increasing concentrations of hCG (0.1-100 ng/ml) with or without 0.1 microM androgen [testosterone or 5 alpha-dihydrotestosterone (DHT]). Granulosa cells from small follicles were also cultured in the absence or presence of a constant concentration of human FSH (30 ng/ml) with or without androgen for 48 h before exposure to hCG for an additional 48 h. Steroidogenic responsiveness was assessed by measuring progesterone accumulation in culture medium and aromatase activity in washed monolayers. Granulosa cells from large follicles showed dose-dependent increases in both progesterone accumulation and aromatase activity in response to treatment with hCG. In contrast, granulosa cells from small follicles were unresponsive to hCG. However, pretreatment of granulosa cells from small follicles for 48 h with FSH stimulated hCG responsiveness. The effects of both testosterone and DHT on hCG-stimulated aromatase activity and progesterone accumulation by granulosa cells from large preovulatory follicles were inhibitory. Testosterone and DHT also suppressed basal (no hCG) progesterone accumulation in these cells, but had no effect on basal aromatase activity. The effects of androgens on FSH-induced hCG responsiveness in immature granulosa cells were variable. The results show a development-related increase in marmoset granulosa cell responsiveness to LH/hCG and provide evidence that FSH and androgens interact to regulate the onset and expression of this critical event during preovulatory follicular development in the primate ovary.     

Contrasting effects of prolactin on luteal and follicular steroidogenesis

To determine whether prolactin affects both luteal and follicular production of testosterone and oestradiol, pseudopregnant rats, either intact or hypophysectomized on day 8, were injected daily between days 8 and 9 with 1.5 i.u. human chorionic gonadotrophin (hCG), 250 micrograms prolactin or a combination of both. Control rats were given vehicle. On day 9, blood was obtained from the ovarian vein and corpora lutea and follicles were isolated and incubated in vitro for 2 h. Administration of hCG to intact rats increased ovarian secretion of testosterone and oestradiol dramatically, but did not affect progesterone secretion. Hypophysectomy on day 8 of pseudopregnancy was followed by a drop in ovarian steroid secretion. Prolactin treatment of hypophysectomized rats markedly enhanced progesterone production but had no stimulatory effect on either testosterone or oestradiol. In contrast, hCG dramatically enhanced ovarian secretion of both testosterone and oestradiol without affecting progesterone secretion. Prolactin administered together with hCG antagonized the stimulation of both testosterone and oestradiol secretion by hCG, yet increased progesterone production. When the specific effects of hCG and prolactin administration on follicles and corpora lutea were studied separately, it was found that hCG treatment in vivo greatly stimulated testosterone and oestradiol production by both tissues in vitro. Since hCG only marginally affected aromatase activity in the follicle, had no effect on aromatase activity in luteal cells and did not increase progesterone synthesis, it appears that hCG acts to increase the formation of androgen substrate for oestradiol biosynthesis. Prolactin, administered with or without hCG, inhibited both basal and hCG-stimulated testosterone and oestradiol synthesis by the follicle. In sharp contrast to its inhibitory effect on follicular production of steroids, prolactin appears to be essential for LH stimulation of testosterone and oestradiol by the corpus luteum. In the absence of prolactin, luteal cells gradually ceased to respond to LH and decreased their output of testosterone and oestradiol. Prolactin administration to hypophysectomized rats did not affect luteal cell production of either steroid. However, corpora lutea of rats treated with prolactin responded to the hCG challenge with an increase in testosterone and oestradiol synthesis. In summary, results of this investigation demonstrate that prolactin affects follicular and luteal production of testosterone and oestradiol in opposite ways.(ABSTRACT TRUNCATED AT 400 WORDS)     

LH stimulation of estrogen secretion by cultured rat granulosa cells

The direct effect of LH on estrogen secretion by rat granulosa cells was investigated. Ovarian granulosa cells from immature hypophysectomized diethylstilbestrol-treated rats were primed with FSH for 2 days in vitro to induce LH receptors. After the FSH priming, the granulosa cells were washed, and recultured for 4 additional days in media containing aromatase substrate (10(-7) M androstenedione) and purified FSH or LH. After the incubations, estrogen (E), progesterone (P) and 20 alpha-dihydroprogesterone (20 alpha-OH-P) in the media were measured by RIA. When granulosa cells from hypophysectomized DES-treated rats were cultured for 6 days with FSH and androstenedione, the production of E, P and 20 alpha-OH-P was stimulated to a maximum of 100-, 200- and 270-fold, respectively, above that of control levels. In contrast, LH did not increase steroidogenesis in these cells. Following 2 days of FSH priming in vitro, however, the cultured granulosa cells exhibited marked increases (400-600%) in E, P and 20 alpha-OH-P production in response to LH treatment over a 4-day incubation period. This stimulatory effect of LH on estrogen and progestin production was dose-related; the minimum and maximum effective doses of LH for steroid production were 3 and 30 ng/ml, respectively, and the ED50 was calculated to be 6 ng/ml of LH. As with LH, FSH also stimulated steroidogenesis in a dose-related manner and the apparent ED50 of FSH on steroidogenesis was 45 ng/ml. To investigate whether LH can also stimulate aromatase activity in granulosa cells primed with FSH in vivo, immature hypophysectomized DES-treated rats were injected for 2 days with FSH after which the granulosa cells were isolated and cultured for 4 days in medium containing 10(-7) M androstenedione and LH or FSH. Both LH and FSH stimulated E, P and 20 alpha-OH-P production, and the maximum steroidogenic responses of LH and FSH were similar to those observed in cultured granulosa cells primed with FSH in vitro. THese results have demonstrated that LH is effective in stimulating both estrogen and progestin secretion in rat granulosa cells pretreated with FSH. This suggests an important role of LH in the direct control of both aromatization and luteinization in the granulosa cell.     

Estradiol-17beta biosynthesis in cultured granulosa cells from hypophysectomized immature rats; stimulation by follicle-stimulating hormone.

Granulosa cells isolated from the ovaries of hypophysectomized immature rats synthesize and secrete estradiol-17beta and estrone when grown for 2 days in monolayer culture in a synthetic medium containing testosterone (0.5 muM) and a highly purified follicle-stimulating hormone (FSH) preparation (0.25 mug/ml). Secretion is negligible in the absence of either testosterone or FSH, and a highly purified luteinizing hormone (LH) preparation (0.25 mug/ml) was without significant stimulatory effect. It is concluded that FSH regulates estrogen biosynthesis in granulosa cells of hypophysectomized rats by a specific stimulation of the aromatizing enzyme system.     

Follicular fluid stimulation of steroidogenesis in immature granulosa cells in vitro.

Granulosa cells from small (1-2 mm) immature porcine follicles were cultured in monolayer in culture media composed of equal parts of culture medium 199 and either (a) fluid from small follicles, (b) fluid from large (6-12 mm) follicles or (c) adult female porcine serum for 6 days, with or without 100 ng LH and/or 2 microgram FSH/ml. Both basal and gonadotrophin-stimulated progesterone secretion were greater in the presence of fluid from large follicles than in serum, for all 6 days. After 4 days of culture, fluid from small follicles enhanced gonadotrophin-stimulated progesterone secretion over that occurring in serum, but to a lesser extent than fluid from large follicles. These studies suggest the presence of a maturation stimulating molecule(s) in follicular fluid which increases in activity or concentration as the follicles enlarge. This factor may be essential for normal granulosa cell maturation in vivo.     

Effect of gonadotrophins on progesterone secretion by cultured granulosa cells obtained from human preovulatory follicles

Granulosa cells were obtained from human preovulatory follicles in 31 women undergoing in vitro fertilization and embryo transfer due to tubal infertility. Follicular maturation was stimulated and synchronized by treatment with Clomiphene or human menopausal gonadotrophin (hMG), or both, plus human chorionic gonadotrophin (hCG). Follicles were aspirated by ultrasound guided puncture approximately 34-36 h after the hCG injection. The granulosa cells were washed and suspended in modified medium 199 containing 10% foetal bovine serum and cultured as monolayers for 6-8 days in the absence and presence of hormones and reactants. Progesterone formation was analyzed by RIA. In general, the cells underwent morphological luteinization and secreted high amount of progesterone. Under basal conditions the secretion of progesterone was highest during the first 2 days in culture and then gradually declined. Progesterone secretion was stimulated by human LH, hCG and the adenylate cyclase stimulator forskolin, with a maximal effect between days 2-6. The beta-adrenergic agonist isoproterenol in preliminary experiments potentiated the stimulatory effect of hCG but had no own stimulatory effect. No clear differences in progesterone secretion or responsiveness to in vitro stimulation relating to the various in vivo stimulation protocols were found.     

Steroidogenesis by cultured granulosa cells aspirated from human follicles using pregnenolone and androgens as precursors

Human granulosa cells from Graafian follicles aspirated 3-4 h before the expected time of ovulation were incubated with various steroid substrates, including pregnenolone, androstenedione, testosterone and dehydroepiandrosterone (DHA). Steroid production after 3 and 10 h of incubation was determined by radioimmunoassay. Progesterone and 17alpha-hydroxyprogesterone were the major products of granulosa cells in control short-term cultures with endogenous substrates. The addition of pregnenolone increased the synthesis of progesterone and 17alpha-hydroxyprogesterone compared with the controls, although the response varied considerably between paired short-term cultures. Little or no oestradiol-17beta was produced from endogenous precursors or short-term cultures to which pregnenolone had been added; one follicle, however, produced similar amounts of oestradiol-17beta in the control cultures and after incubation with pregnenolone. When granulosa cells were cultured with various amounts of androstenedione, DHA or testosterone, large amounts of oestradiol-17beta were produced, especially in short-term cultures in which larger amounts of substrate were added. Progesterone production continued and progesterone was synthesized more rapidly or in greater amounts in some short-term test cultures than in the controls. The results indicate that human granulosa cells are one source of oestradiol-17beta during the preovulatory phase. The data support the two-cell theory for oestradiol synthesis, for granulosa cells do not appear to undertake steroid conversion via the 5-unsaturated pathway, but aromatize androgens known to be produced by thecal cells. It is also suggested that either androgens or oestradiol-17beta stimulate progesterone production by granulosa cells, at least in vitro.     

Effects of oestradiol-17 beta on FSH-stimulated steroidogenesis in cultured marmoset granulosa cells.

A role for oestradiol in ovarian follicular development is well recognized. However, a number of disparate effects have been reported for the action of oestradiol in the primate ovary. To investigate this further, we have examined the effects of oestradiol on the differentiation of granulosa cells isolated from small (0.5-1 mm) antral follicles obtained from the ovaries of prepubertal marmoset monkeys (Callithrix jacchus). Granulosa cells were co-cultured with oestradiol and human FSH (hFSH) for 48 h, or were pretreated with oestradiol for 48 h before addition of gonadotrophin for a further 48 h. Oestradiol (0.01-100 nmol/l) had no effect on basal or hFSH-stimulated progesterone accumulation and aromatase activity when the hormones were added concurrently. Furthermore, oestradiol did not influence the ability of hFSH to induce LH/human chorionic gonadotrophin (hCG) responsiveness in immature granulosa cells. The absence of synergism between oestradiol and hFSH in the induction of marmoset granulosa cell differentiation was independent of the presence of phenol red in culture medium. However, gonadotrophin-stimulated steroidogenesis was attenuated when cells were cultured in the presence of phenol red compared with in its absence; this effect was more pronounced for gonadotrophin-stimulated aromatase activity but was evident for LH/hCG-stimulated progesterone accumulation at higher doses of hCG (10 and 100 ng/ml). An effect of phenol red on basal steroidogenesis was less obvious.(ABSTRACT TRUNCATED AT 250 WORDS)     

Androgens augment FSH-induced progesterone secretion by cultured rat granulosa cells.

Granulosa cells have been isolated from ovaries of estrogen-treated immature intact and hypophysectomized rats, and have been maintained in culture in a chemically-defined medium. Progesterone secretion by these cells was testosterone or 17beta-OH-5alpha-androstan-3-one (DHT), progesterone secretion was low or undetectable. However, the addition of testosterone or DHT together with FSH caused a dramatic 8- to 19-fold increase over that caused by FSH alone. On the other hand, luteinizing hormone (LH) alone had no effect on progesterone secretion, but produced a small stimulation when added together with testosterone. These results demonstrate synergism between androgens and FSH in the control of progesterone secretion by granulosa cells in culture.     

Steroidogenesis in isolated rat granulosa cells--changes during follicular maturation

Steroid release was investigated in granulosa cells isolated from rat ovarian follicles at different maturation stages. Immature rats were treated with 10 IU of pregnant mare's serum gonadotrophin (PMSG) to induce follicular growth and maturation. Granulosa cells were isolated and subsequently incubated for 4 h in the absence or presence of follicle stimulating hormone (FSH), luteinizing hormone (LH) or forskolin. The concentrations of progesterone (P), 20 alpha-dihydroprogesterone (20 alpha DHP), testosterone (T) and oestradiol (E2) in the media were determined by radioimmunoassay. During maturation, basal release of T increased markedly at late dioestrus. At pro-oestrus, concomitant with a sharp rise in E2 and P release, T release returned to previously low levels. Addition of FSH or LH stimulated P release at late dioestrus and pro-oestrus but had no effect on E2 or T levels. Basal 20 alpha DHP levels remained low until a sharp rise at mid pro-oestrus. A stimulatory effect of gonadotrophins on 20 alpha DHP was first seen at this stage. The adenylate cyclase stimulator forskolin resembled FSH in all its effects. In summary, the present study demonstrates that maturing, but not preovulatory, granulosa cells have an increased capacity to release T during a relatively short period. It is suggested that this T peak could be of great importance for the further progress of follicular maturation.