Rat thecal/interstitial cells produce a mitogenic activity that promotes the growth of granulosa cells

To test the hypothesis that the cells outside the basal lamina of the follicle secrete paracrine factors that influence the cells on the inside of the follicle, two ovarian cell populations were obtained from diethylstilbestrol-treated rats. Granulosa cells were obtained by extrusion from the follicles and an ovarian cell preparation, termed thecal/interstitial, was derived from the granulosa cell-depleted ovaries. Light microscopy showed that each cell population in culture had distinctive morphologies. Electrophoretic examination of the radiolabeled proteins secreted by the two ovarian cell preparations revealed that each secreted unique protein components into the culture medium. Rat thecal/interstitial cell-conditioned medium promoted [3H]thymidine incorporation into normal rat kidney cell line (NRK) DNA and into bovine granulosa cell DNA. The growth-promoting activity was stable to heating at 70 degrees C for 5 min whereas native fibroblast growth factor (FGF) lost its activity, showing that the factor was not characteristic of FGF. To further characterize the growth-promoting activity thecal/interstitial cell-conditioned medium was concentrated and the proteins separated by size exclusion high performance liquid chromatography. The growth-promoting activity eluted with an apparent molecular weight between 15,000 and 25,000. The finding that thecal/interstitial cells in culture secrete growth-promoting factors suggests that those cells that are in close proximity to the granulosa cells may secrete protein factors that diffuse into the follicular antrum and influence granulosa cell proliferation.     

17 beta-Estradiol biosynthesis in cultured granulosa and thecal cells of human ovarian follicles: stimulation by follicle-stimulating hormone.

Cellular sites and gonadotropic control of human follicular estrogen secretion have been assessed by culturing the theca and granulosa components separately under different hormonal conditions. Granulosa cells from human follicles were grown in chemically defined media containing gonadotropins and/or testosterone (T) for 24 h. The production of 17 beta-estradiol (E2) by cells cultivated in T-free media with or without FSH was very low during the culture period. There was a highly significant increase (P less than 0.001) in E2 production when T alone was added and a more marked increase was consistently noted in the presence of FSH and T. In all cases, hCG failed to exert any significant effect on E2 production by granulosa cells in the presence or absence of T. No treatments examined altered the E2 production of thecal cells during a 24-h culture period and the amounts of E2 released into media were negligible when compared with levels produced by granulosa cells from the same follicles. It is concluded that granulosa cells but not thecal cells are the prime site of follicular estrogen production and that FSH regulates estrogen secretion by nonluteinized granulosa cells of the human follicle.     

Ovarian nerve extracts influence androgen production by cultured ovarian thecal cells

There is growing evidence that ovarian steroidogenesis is controlled not only by pituitary gonadotropins but also by ovarian nerves. Nerves reach the ovary via the plexus nerve and via the superior ovarian nerve (SON), which runs in the suspensory ligament, and innervate theca cells of all sizes of follicles. To investigate the role of ovarian nerves in steroidogenesis we have examined the effects of adding an extract of SON from adult rats on androgen production by cultured porcine theca cells. Addition of SON extract to cultured theca interna from 3 to 6 mm diameter follicles of prepubertal gilts significantly inhibited (p less than 0.05) LH-stimulated androstenedione production in a dose-dependent manner; significant inhibition (10.8%) occurred with the addition of the extract of 2 mg of SON/ml culture medium, and near maximal inhibition (83%) resulted when the SON extract was increased to 60 mg SON/ml. Extracts of sciatic nerves, used as non-ovarian nerve controls, failed to inhibit, and in fact significantly increased (p less than 0.05) androstenedione production over the same concentration range of neural tissue extract. The inhibitory effect of the SON extract was unaffected by chymotrypsin digestion or by the presence of the beta-adrenergic antagonist propranolol (10(-6) M), but was removed by charcoal treatment. These results suggest that the nervous system has the potential for modulation of follicular steroid biosynthesis via direct innervation of the ovaries, in addition to the well-established indirect mechanism of neural control exerted via the hypothalamic-pituitary system.     

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.     

Stage-dependent regulation of ovarian pituitary adenylate cyclase-activating polypeptide mRNA levels by GnRH in cultured rat granulosa cells

The present study was designed to test whether GnRH regulates pituitary adenylate cyclase-activating polypeptide mRNA levels in a stage-dependent manner during follicle development in the rat ovary. The granulosa cells of preovulatory and immature follicles obtained from PMSG- and estrogen-treated rats, respectively, were cultured in serum-free conditions in the presence of various hormones. GnRH receptor mRNA expression was detected in both preovulatory and immature granulosa cells and was down-regulated by gonadotropins. Treatment of preovulatory granulosa cells with GnRH agonist stimulated pituitary adenylate cyclase-activating polypeptide mRNA levels in a dose-dependent manner. In situ hybridization analysis of cultured preovulatory follicles revealed that GnRH-induced pituitary adenylate cyclase- activating polypeptide signals were detected in granulosa cells, but not thecal cells. In immature granulosa cells, cotreatment with GnRH agonist suppressed FSH-stimulated pituitary adenylate cyclase-activating polypeptide mRNA levels in a dose-dependent manner, whereas treatment with GnRH alone had no effect. Furthermore, treatment with GnRH antagonist inhibited LH-induced pituitary adenylate cyclase-activating polypeptide gene expression in preovulatory granulosa cells, whereas it stimulated FSH-induced pituitary adenylate cyclase-activating polypeptide gene expression in immature granulosa cells. Interestingly, GnRH-stimulated pituitary adenylate cyclase-activating polypeptide mRNA levels in preovulatory granulosa cells was inhibited by arachidonyltri fluoromethyl ketone, an inhibitor of phospholipase A(2), but not by an inhibitor of protein kinase A or C. Lastly, treatment of preovulatory follicles with pituitary adenylate cyclase-activating polypeptide antagonist suppressed GnRH-stimulated progesterone production during 6--9 h of culture. Taken together, these results demonstrate the stage-dependent regulation of pituitary adenylate cyclase-activating polypeptide mRNA levels by GnRH, the stimulatory and inhibitory effect in granulosa cells of preovulatory and immature follicles, respectively.     

Ovarian thecal cells produce transforming growth factor-beta which can regulate granulosa cell growth

Ovarian thecal cells in culture were found to synthesize and secrete transforming growth factor-beta (TGF beta). A component in thecal cell-conditioned medium was immunologically similar to TGF beta, as assessed with a RIA, and inhibited specific binding of TGF beta to its cell surface receptors. Thecal cell-secreted proteins also contained TGF beta biological activity, which was determined by stimulation of soft agar colony formation by AKR-2B indicator cells. Specific TGF beta antibodies precipitated a 25 K protein from radiolabeled thecal cell-secreted protein that comigrated with purified platelet-derived TGF beta. Both bovine thecal cell and rat thecal/interstitial cell preparations produced TGF beta, which required acid treatment to obtain fully active samples. The physiological significance of TGF beta production by thecal cells was addressed through an analysis of the effects of TGF beta on bovine granulosa cell growth. TGF beta inhibited epidermal growth factor stimulation of granulosa cell growth, but alone it had no apparent influence. Observations indicate that ovarian thecal cells produce TGF beta, which can regulate granulosa cell growth and differentiation. Discussion of thecal cell-granulosa cell interactions and the possible functions of TGF beta in the ovary is presented.     

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.     

Inhibin/activin and ovarian cancer

Inhibin and activin are members of the transforming growth factor beta (TGFbeta) family of cytokines produced by the gonads, with a recognised role in regulating pituitary FSH secretion. Inhibin consists of two homologous subunits, alpha and either betaA or betaB (inhibin A and B). Activins are hetero- or homodimers of the beta-subunits. Inhibin and free alpha subunit are known products of two ovarian tumours (granulosa cell tumours and mucinous carcinomas). This observation has provided the basis for the development of a serum diagnostic test to monitor the occurrence and treatment of these cancers. Transgenic mice with an inhibin alpha subunit gene deletion develop stromal/granulosa cell tumours suggesting that the alpha subunit is a tumour suppressor gene. The role of inhibin and activin is reviewed in ovarian cancer both as a measure of proven clinical utility in diagnosis and management and also as a factor in the pathogenesis of these tumours. In order to place these findings into perspective the biology of inhibin/activin and of other members of the TGFbeta superfamily is also discussed.     

Inhibin: studies of stored and secreted forms by biosynthetic labeling and immunodetection in cultured rat granulosa cells

The biosynthesis of inhibin in rat granulosa cells was studied by biosynthetic labeling, immunoblotting, and immunocytochemical techniques. Granulosa cells from immature hypophysectomized estrogen-treated rats were cultured in the presence of [35S]cysteine. Both conditioned media and cell extracts were subjected to immunoprecipitation with an antibody directed against the N-terminal 26 amino acids of the alpha-chain of porcine inhibin (pI alpha 1-26), followed by sodium dodecyl sulfate-polyacrylamide gel electrophoresis (SDS-PAGE) and fluorography. Treatment with FSH (100 ng/ml) and delta 4-androstenedione (10(-7) M) increased the secretion of 35S-labeled inhibin immunoreactivity by 2.6-fold over that in control cultures treated with androstenedione alone. The radiolabeled inhibin had mol wt (Mr) values of 45,000 and 30,000. Upon reduction, the 45,000 Mr polypeptide remained (with increased apparent Mr of 49,000), but the 30,000 Mr species disappeared with the concomitant appearance of two bands with 18,000 and 11,000 Mr. Competition studies with pI alpha 1-26 confirmed that these polypeptides were all related to inhibin. Furthermore, immunoblotting with an antibody directed against the porcine inhibin beta-A chain (pI beta A81-113) indicated that the 11,000 Mr peptide was the inhibin beta-A chain. Extracts of cells treated with FSH contained only a high Mr alpha-related species (Mr, 41,000 nonreduced; 49,200 reduced). The inhibin alpha antibody was also used to immunocytochemically stain cultured granulosa cells. Cells that had been treated with FSH or the adenyl cyclase activator forskolin (3 x 10(-5) M), but not untreated cells, exhibited positive staining. These results indicate that granulosa cells synthesize and store inhibin alpha-chain precursor with 49,000 Mr. Although some of the high Mr alpha-form was secreted, the majority of the alpha-subunit was processed to the 18,000 Mr form and dimerized with the 11,000 Mr beta-chain to form the mature inhibin dimer immediately before secretion. The cultured granulosa cells may provide a model for future studies on the hormonal regulation of inhibin alpha- and beta-gene expression as well as subunit dimerization and secretion.     

Tumor necrosis factor-α (TNF-α) inhibits steroidogenesis of bovine ovarian granulosa and thecal cells in vitro

The effect of recombinant bovine tumor necrosis factor-α (TNF-α) on steroidogenesis and numbers of bovine ovarian granulosa and thecal cells has been studied, and specific binding sites for ¹²⁵I-TNF-α on ovarian cells have been determined. Granulosa cells have been examined from small (surface diameter 1–5 mm) follicles, whereas thecal cells from large (≥ 8 mm) follicles were utilized. Increasing doses of TNF-α significantly attenuated insulin- and IGF-I-induced estradiol production by granulosa cells from small follicles, but had no effect on basal estradiol production. Moreover, TNF-α significantly attenuated insulin- and LH-induced androstenedione production by thecal cells from large follicles. TNF-α had little or no effect on the numbers of granulosa and thecal cells in these same studies. Specific high-affinity, low-capacity binding of ¹²⁵I-TNF-α was also demonstrable in granulosa and thecal cells. Thus, it appears that TNF-α inhibits insulin-and IGF-I-induced estradiol production by granulosa cells and androstenedione production by thecal cells via TNF-α binding to its own receptor.     

Estradiol-17β production in amago salmon (Oncorhynchus rhodurus) ovarian follicles: Role of the thecal and granulosa cells

Separation of the follicular thecal cell and granulosa cell layers of oocytes of the amago salmon (Oncorhynchus rhodurus) facilitated assessment of their roles in estradiol-17β production by an in vitro incubation method. Four different follicular preparations, intact follicles (oocytes with complete follicle layers), thecal layers contaminated with less than 10% granulosa cells, pure granulosa layers and zona radiata, and thecal layer-granulosa layer co-cultures, were incubated in the presence or absence of partially purified chinook salmon gonadotropin (SG-G100). Estradiol-17β and testosterone levels in the medium were measured by specific radioimmunoassay. SG-G100 (0.1, 1 μg/ml) stimulated estradiol-17β production by intact follicles and co-culture preparations, but not by the isolated thecal or granulosa layers, indicating that both layers are necessary for gonadotropin-stimulated estradiol-17β production. In contrast, SG-G100 greatly stimulated testosterone production by thecal layers (up to 80 times basal control values) but only slightly stimulated testosterone production by the other follicular preparations. Incubation of granulosa layers with exogenous testosterone (0.01, 1 μg/ml) or with testosterone and SG-G100 (1 μg/ml) together resulted in elevated testosterone levels. No significant difference was found between the two treatments, suggesting that gonadotropin did not stimulate the conversion of testosterone to estradiol-17β. Isolated thecal layers incubated with testosterone produced relatively small amounts of estradiol-17β, 7–8 times less than granulosa layers under the same conditions. These results suggest a two-cell-type model for the production of follicular estrogens, the thecal layer possibly contributing to estradiol-17β production by synthesizing androgens which are transferred to the granulosa layer and aromatized to estradiol-17β.     

The production of progesterone, androgens, and estrogens by granulosa cells, thecal tissue, and stromal tissue from human ovaries in vitro.

The concentrations of steroids in antral fluid, the number of granulosa cells, the status of the oocyte, and the diameter of each follicle were determined in human ovaries so that follicles at each stage of the menstrual cycle could be classified as large (greater than or equal to 8 mm diameter) or small (less than 8 mm diameter) and healthy or atretic. The granulosa cells and thecal-enriched tissue from each follicle and the stromal tissue from each ovary were cultured for 6 days in vitro. The amounts of progesterone (P), androstenedione (delta 4), testosterone, dihydrotestosterone, estrone, and estradiol (E2) generated by the different tissues were measured on days 0, 2, 4, and 6 of culture. It was found that granulosa cells, thecal tissue, and stromal tissue all have the biosynthetic capacity to produce P, delta 4, testosterone, dihydrotestosterone, estrone, and E2. No individual steroid-secreting compartment of the ovaries studied, whether part of the follicle or of the stroma, had the exclusive capability of producing any of the above-named steroids at any stage of the menstrual cycle or at any stage of antral follicle growth or atresia. Although the steroids produced by the human follicle appear not to be unique to any one cell type, the patterns of steroidogenesis by the granulosa and thecal compartments differ from one another and from the stroma throughout follicular maturation and atresia. During follicular development, granulosa cells produce large amounts of E2 and small amounts of delta 4. During the preovulatory phase, cells from large follicles (greater than or equal to 8 mm diameter) differentiate from an estrogen-secreting state into a P- and, to a lesser extent, an delta 4-secreting one. By contrast, during follicular atresia, granulosa cells continue to synthesize delta 4, but their capacity to synthesize estrogen is substantially reduced. Furthermore, granulosa cells from atretic follicles are incapable of transforming from an androgen-secreting state into a P-secreting one in tissue culture. During follicular growth, thecal tissue secretes about 2--3 times more delta 4 than E2. By contrast, during follicular atresia, thecal tissue retains its capacity to synthesize delta 4 but loses much of its capacity to synthesize E2. The in vitro capacity of thecal tissue to produce steroids exceeds that of the stroma (on a per weight basis) from 2- to 500-fold. Thecal tissue from healthy but not from atretic follicles is capable of differentiating from an androgen- and estrogen-secreting state to a predominantly P-secreting one in tissue culture. It is postulated that although steroid synthesis may not be rigidly compartmentalized during follicular development, appreciable amounts of the steroids secreted by the granulosa and theca may enter different compartments before leaving the ovary...     

Inhibin-like activity in media from cultured rat granulosa cells collected throughout the oestrous cycle

Granulosa cells of antral ovarian follicles from adult 5-day cyclic rats were cultured on each day of the cycle. The rat granulosa cell conditioned medium (rGCCM) was harvested and renewed on each day of a 4-day culture period. Inhibin-like activity and progesterone were estimated in rGCCM using an in-vitro bioassay system with dispersed rat anterior pituitary cells and radioimmunoassay respectively. Removal of steroids from rGCCM with dextran-coated charcoal was effective and did not significantly change the inhibin-like activity of the treated samples. On day 1 of culture the inhibin-like activity of rGCCM for each day of the oestrous cycle was 20-90% higher than on days 2, 3 and 4 of culture when low and constant levels were observed. Media collected after culture on days 1 and 2 from pro-oestrous cells contained larger amounts of inhibin-like activity than media collected on the other days of the cycle. On day 1 of culture, rGCCM from pro-oestrous cells contained higher concentrations of progesterone than that from cells collected on the other days of the cycle. On days 2, 3 and 4 progesterone levels in rGCCM were undetectable (less than 320 pmol/l) except in media from pro-oestrous cultures on day 2. Addition of FSH (62 micrograms/l) to granulosa cell cultures in medium with or without 10% fetal calf serum (FCS) did not alter the inhibin-like activity of rGCCM from pro-oestrous cells. The presence of FCS maintained the production of inhibin-like activity since rGCCM from cells cultured without FCS was devoid of FSH-suppressing activity after 3 days of culture.(ABSTRACT TRUNCATED AT 250 WORDS)     

Biological properties of chicken luteinizing hormone-releasing hormone: gonadotropin release from rat and chicken cultured anterior pituitary cells and radioligand analysis

We have examined the biological properties of chicken LHRH, which has been isolated and characterized in our laboratory, using primary monolayer cultures of rat and chicken anterior pituitary cells. The radioligand receptor analysis using rat pituitary LHRH receptor was also performed. Biological activities of mammalian LHRH and a more potent analog, [D-Leu6]des-Gly10-LHRH N-ethylamide ([D-Leu6] LHRH analog), were compared with those of chicken LHRH. The ED50 values for [D-Leu6]LHRH analog, mammalian LHRH, and chicken LHRH were, respectively, 0.0166, 0.455, and 18.2 nM for LH secretion and 0.0089, 0.263, and 8.28 nM for FSH secretion from rat pituitary cells. Relative potencies of chicken LHRH were 2.5% that of mammalian LHRH for LH and 3.2% that for FSH. On the other hand, the ED50 for chicken LHRH for LH release from chicken anterior pituitary cells was 1.03 nM, indicating that the biological potency of chicken LHRH acting on chicken pituitary cells was higher than that of chicken LHRH acting on mammalian pituitary cells. The ID50 of chicken LHRH to inhibit the binding of 125I-labeled [D-Leu6]LHRH analog to rat anterior pituitary membrane was 708.5 nM, and was 200 times that of mammalian LHRH (3.39 nM). These results suggest that the substitution of arginine residue of mammalian LHRH for glutamine residue of chicken LHRH causes a decrease in the binding affinity of the hormone for the mammalian LHRH receptor and that the mammalian LHRH receptor has a binding site for the cationic center of the arginine residue of LHRH. On the other hand, chicken pituitary LHRH receptor seems to have more broad specificity than mammalian LHRH receptor.     

Inhibin and activin-like activity in fluids from male and female gonads: different molecular weight forms and bioactivity/immunoactivity ratios

The existence of various molecular weight forms of inhibin in ovarian follicular fluid has been reported earlier, while there is no information on the form of inhibin in testicular tissue. Inhibin bioactivity was therefore estimated in eluates of slices, obtained after SDS-PAGE of rat testicular and ovarian homogenates, rat Sertoli cell-conditioned medium (rSCCM) and bovine ovarian follicular fluid (bFF). The only form of inhibin detected in testes from 22-day-old rats and in rSCCM was a 30 kDa protein. In rat ovarian extracts, larger forms of inhibin were also found as well as the predominant 30 kDa form. An activin-like activity was found in the 25 kDa SDS-PAGE eluates of both rSCCM and ovarian homogenates, which caused a dose-dependent increase of FSH release from cultured pituitary cells. Activin-like activity and several forms of inhibin were found in bFF after SDS-PAGE. After purification of inhibin from bFF using dye affinity, anion-exchange, lentil lectin affinity chromatography and a subsequent reversed phase chromatography step, two pools of inhibin activity were obtained. These were separated by SDS-PAGE revealing 30 and 58 kDa inhibin forms. The immunoactivity of these forms of inhibin was then estimated using antibodies against the 22 N-terminal amino acids of the alpha subunit of 30 kDa bovine inhibin. It appeared that the two molecular weight forms of inhibin had bioactivity/immunoactivity ratios which differed more than five-fold. This indicates that results of radioimmunoassays of inhibin of ovarian origin, using peptide antisera, should be interpreted with caution.     

Aminoglutethimide augments follicle-stimulating hormone-induced aromatase activity in cultured porcine granulosa cells

The role of endogenous progestin synthesis in the modulation of FSH-induced aromatase activity was examined. Granulosa cells isolated from nonatretic medium-sized (3-5 mm) follicles of prepubertal pigs were cultured for an initial 48-h period, during which time aromatase activity was induced by FSH in the absence or presence of aminoglutethimide (AG). After induction, the cell monolayers were washed before being cultured for a further 6-h period in the presence of the substrate testosterone (0.5 microM). The aromatase activity was assessed by measuring the accumulation of estradiol during the test period. Basal aromatase activity was negligible and was unaffected by the presence of AG (0.1-100 microM) during the induction period. But when cells were cultured with FSH and AG (0.1-1000 microM) during the induction period, there was a dose-dependent, biphasic increase in the FSH-induced estradiol synthesis during the test period. Maximal enhancement was obtained with 10 microM AG (3.5-fold). Thereafter the aromatase activity declined and, at 1000 microM AG, was significantly (P less than 0.05) inhibited. At the same time, the FSH-stimulated progestin production during the induction period was inhibited in a dose-related fashion by AG. This AG-enhanced aromatase activity was dose and time dependent but was independent of the FSH concentration used. The apparent median effective dose of AG was 2.4 microM and a minimal time of 24 h or less was needed to potentiate the induction of aromatase activity by FSH. If AG was, however, added to the cell cultures during the test period, the FSH-induced aromatase activity was inhibited, showing that AG is an inhibitor of FSH-induced aromatase activity. This action of AG during the test period could be alleviated by the addition of testosterone during the induction period. The viability of the granulosa cells and the total cellular protein were not significantly (P greater than 0.05) altered by AG. These results show that the induction of aromatase activity by FSH could be enhanced by AG, which probably acts by inhibiting progestin production during the induction period, leading us to conclude that endogenous progestins might play an important role in modulating the induction of aromatase activity by FSH.     

Rat thecal/interstitial cells secrete a transforming growth factor-beta-like factor that promotes growth and differentiation in rat granulosa cells

Rat granulosa cells respond to transforming growth factor-beta (TGF beta) with increases in DNA synthesis and FSH-stimulated aromatase activity. To determine if the cells surrounding the granulosa cells generate TGF beta-like activity, which influences the growth and differentiation of granulosa cells, we cultured rat ovarian thecal/interstitial cells under serum-free conditions and collected their secretion products. Conditioned medium generated by primary cultures of thecal/interstitial cells over a 6-day collection period stimulated DNA synthesis, suggesting the presence of TGF beta-like bioactivity. At the onset of the collection period extracts of cultured cells contained undetectable levels of TGF beta-like activity, suggesting that the factor was produced and released by thecal/interstitial cells. FSH augmented the actions of both TGF beta and conditioned medium on DNA synthesis. Using an independent assay that relies on the response of the aromatase complex we showed that TGF beta and conditioned medium had parallel effects; TGF beta and conditioned medium alone did not influence the basal levels of aromatase activity, but both treatments augmented FSH-induced aromatase activity. Treatment of the conditioned medium with heat, acid, or alkali resulted in an increase in TGF beta-like bioactivity, suggesting that the TGF beta-like factor could be activated under these conditions. A commercially available antibody known to neutralize the bioactivity of TGF beta blocked the actions of TGF beta on DNA synthesis and blocked the growth-promoting activity of conditioned medium in a dose-dependent manner. Fractionation of the proteins in conditioned medium by elution through a Sephadex G-75 (superfine) column under acidic conditions showed that the growth-promoting activity and the aromatase activator eluted in fractions containing proteins with mol wt 25K. Standard TGF beta eluted in the same fractions. In summary, rat thecal/interstitial cells in culture secrete a TGF beta-like factor. The TGF beta-like factor may act together with FSH to promote growth and activate the aromatase complex of granulosa cells developing in the intrafollicular lumen in close association with the oocyte.