Estradiol regulation of follistatin and inhibin alpha- and beta(B)-subunit mRNA in avian granulosa cells

Estradiol modulation of granulosa cell growth and regulation of follistatin and inhibin alpha- and beta(B)-subunit mRNA were investigated in cultured chicken granulosa cells. Granulosa cells were isolated and pooled according to size from the F(4) + F(5), small yellow (SYF), and large white (LWF) follicles. Isolated and dispersed granulosa cells were then cultured in the absence or presence of 1 x 10(-5) M 17 beta-estradiol. In Experiment 1 (n = 4 replications) the effect of estradiol on the growth of granulosa cells from the different-sized follicles was examined at 24 and 48 h of culture. Untreated and treated granulosa cells from all three follicle sizes proliferated during culture, and cell viability for all cultures was over 95% throughout the experiment. After 48 h the untreated cultures for all follicle types had 1.6 to 2.2 times (P < 0.05) more cells than the estradiol-treated cultures. In Experiment 2 (n = 3 replications), the cultures were terminated at 4 and 24 h after plating. Follistatin mRNA levels were higher in estradiol-treated cells at 24 h in F(4) + F(5) follicles, at 4 and 24 h in the SYF, and at 4 h in the LWF. beta(B)-subunit mRNA levels were also increased by estradiol at 4 h in F(4) + F(5) cells and at 4 and 24 h in the LWF. Steady state mRNA levels for the alpha-subunit were higher (P < 0.05) in estradiol-treated cultures at 4 and 24 h in F(4) + F(5) follicles and at 24 h in the SYF. Immunoreactive alpha-subunit protein, however, was not increased by estradiol treatment. Thus, whereas estradiol inhibited granulosa cell growth, it exerted a generally stimulatory effect on the expression of FS and the inhibin alpha- and beta(B)-subunit mRNA.     

Photoperiod-dependent regulation of inhibin in Siberian hamsters: I. Ovarian inhibin production and secretion

Follicle-stimulating hormone (FSH) stimulates ovarian follicle development and the production of protein hormones including inhibin A and inhibin B. The inhibins are dimeric proteins (alpha-beta(A) or alpha-beta(B)) secreted by growing follicles that suppress FSH in a classical endocrine negative feedback loop. Siberian hamsters, Phodopus sungorus, exhibit seasonal variation in FSH levels. Given the role of inhibin in FSH regulation, we hypothesized that ovarian inhibin expression differs between animals reared in long (16 h light:8 h darkness) and short (6 h light:18 h darkness) photoperiods. To examine inhibin expression in animals housed under long or short photoperiods, hamster inhibin alpha-, beta(A)-, and beta(B)-subunits were cloned and used to detect and localize inhibin subunit mRNA in developing follicles. Ovarian inhibin alpha-subunit mRNA levels were significantly higher in long day-exposed (LD) than in short day-exposed (SD) hamsters. In addition, dimeric inhibin, as well as inhibin alpha-, beta(A)-, and beta(B)-subunit protein levels were higher in the LD than in the SD hamster ovaries.     

Regulation of inhibin subunit gene expression by FSH and estradiol in cultured rat granulosa cells

Roles of follicle-stimulating hormone (FSH) and sex steroids in regulating the expression of mRNA species encoding the alpha-, beta A- and beta B-subunits of inhibin were studied in cultured granulosa cells from immature rat ovaries. Inhibin subunit mRNAs were detected by Northern blot analysis of total RNA extracted from granulosa cell monolayers which had been incubated for 48 h in serum-free medium containing FSH (100 ng/ml) and/or a steroid (10(-6) M): estradiol (E), testosterone (T) or 5 alpha-dihydrotestosterone (DHT). Levels of mRNA encoding each inhibin subunit in untreated (control) cultures were low. In cultures treated with FSH alone, levels of inhibin alpha-, beta A- and beta B-subunit mRNA were approximately 60-fold, 70-fold and 66-fold greater than control, respectively. In cultures treated with E alone, levels of inhibin alpha- and beta B-subunit mRNA were elevated approximately 4-fold and 2-fold, respectively, but the level of inhibin beta A-subunit mRNA was not measurably affected. Treatment with T or DHT alone had no consistent effect on the levels of any inhibin subunit mRNA. The stimulatory effects of FSH were not consistently altered by the presence of either androgen or estrogen. These results confirm the role of FSH in regulating inhibin alpha-subunit gene expression and provide direct evidence that both inhibin beta-subunit genes are inducible by FSH in granulosa cells. All three inhibin subunit mRNAs followed the same pattern, suggesting that their expression is coordinately regulated by FSH during granulosa cell differentiation.     

Immunohistochemical localization of inhibin/activin subunits in human ovarian follicles during the menstrual cycle.

The immunohistochemical localization of the alpha-, beta A-, and beta B-subunits of inhibin was examined in human follicles during follicular growth. Immunoreactive staining with antisera against the alpha-, beta B-subunits was observed in follicular granulosa cells, whereas no staining for each inhibin subunit was observed in thecal or interstitial cells. In the preantral and small antral follicles, the granulosa cells exhibited positive immunoreactive staining with antisera against beta A- and beta B-subunits and negative immunostaining with antiserum against alpha-subunit. In medium-sized healthy antral follicles obtained during the midfollicular phase, positive immunostaining with antisera against alpha-, beta B-subunits was detected in the granulosa cells. In contrast, immunostaining for alpha-, beta A-, and beta B-subunits was not detected in the granulosa cells of similarly sized atretic follicles. The granulosa cells of preovulatory follicles revealed enhanced positive staining for the three inhibin subunits. The present findings suggest that immunoreactive inhibin subunits are present in the follicular granulosa cells during the menstrual cycle, and that the localization and intensity of immunostaining for each inhibin subunit might change during follicular development and maturation.     

Activation of the bone morphogenetic protein signaling pathway induces inhibin beta(B)-subunit mRNA and secreted inhibin B levels in cultured human granulosa-luteal cells

During the human menstrual cycle the circulating levels of inhibin B, a dimer of inhibin alpha- and beta(B)-subunits, fluctuate in a fashion distinct from that of inhibin A, the alpha-beta(A)-subunit dimer. This suggests that human inhibin subunits are each regulated in a distinct manner in human ovarian granulosa cells by endocrine and local factors. We have previously shown using cultures of human granulosa-luteal (hGL) cells that gonadotropins stimulate the steady state mRNA levels of inhibin alpha- and beta(A)-subunits, but not those of the beta(B)-subunit, which, on the other hand, are up-regulated by, for instance, activin and TGF beta. We recently identified the TGF beta gene family member bone morphogenetic protein-3 (BMP-3) as a granulosa cell-derived growth factor, but whether BMP-3 or other structurally related BMPs regulate human granulosa cell inhibin production is not known. We show here that hGL cells express mRNAs for distinct serine/threonine kinase receptors (BMP-RIA and BMP-RII) and Smad signaling proteins (Smad1, Smad4, and Smad5) involved in the mediation of cellular effects of BMPs. Subsequently, we determined in hGL cell cultures the effects of distinct members of the BMP family previously found to be expressed in mammalian ovaries. Recombinant BMP-2 induces potently in a time- and concentration-dependent manner the expression of the inhibin beta(B)-subunit mRNAs in hGL cells without affecting the levels of alpha- or beta(A)-subunit mRNAs. BMP-6 has a similar, but weaker, effect than BMP-2, whereas BMP-3 and its close homolog, BMP-3b (also known as growth differentiation factor-10) had no effect on inhibin subunit mRNA expression. hCG treatment of hGL cells was previously shown to abolish the stimulatory effect of activin on beta(B)-subunit mRNA levels, and here hCG is also shown to suppress the effect of BMP-2. Furthermore, BMP-2 stimulates hGL cell secreted dimeric inhibin B levels in a concentration-dependent manner. Depending on the experiment, maximal increases in inhibin B levels of 6- to 28-fold above basal levels were detected during a 72-h culture period. We conclude that activation of the BMP-signaling pathway in hGL cells stimulates inhibin beta(B)-subunit mRNA levels and leads at the protein level to a dramatic stimulation of secreted inhibin B dimers. Our results are consistent with the suggestion that in addition to the distinct activin- and TGF beta-activated signaling pathways, the BMP-activated pathway is likely to be implicated in the complex regulation of inhibins in the human ovary.     

Pachytene spermatocytes in co-culture inhibit rat Sertoli cell synthesis of inhibin beta B-subunit and inhibin B but not the inhibin alpha-subunit

This study investigates the effects of spermatogenic germ cells on inhibin alpha-subunit and beta B-subunit expression, and inhibin alpha-subunit and inhibin B production by rat Sertoli cells in vitro. Sertoli cells isolated from 19-day-old rats were cultured for 48 h at 32 degrees C, in the presence or absence of FSH (2.3-2350 mIU/ml), and in the presence of pachytene spermatocytes, round spermatids or cytoplasts of elongated spermatids purified from adult rat testis by elutriation and density gradient separation. Sertoli cell secretion of inhibin alpha-subunit and inhibin B, as measured by immunoassay, was dose-dependently stimulated by FSH (maximal stimulation 13- and 2-fold, respectively). Round spermatids or cytoplasts co-cultured with Sertoli cells had no effect on basal or FSH-induced secretion of inhibin alpha-subunit or inhibin B. When Sertoli cells were co-cultured with pachytene spermatocytes, inhibin alpha-subunit secretion was unaltered, while inhibin B secretion was suppressed in a cell concentration-dependent manner to reach a maximal suppression of 45% compared with Sertoli cells alone (P<0.01). A similar suppression in inhibin B was still observed (64% of Sertoli cells alone) when the pachytene spermatocytes were separated from Sertoli cells by a 0.45 microm pore membrane barrier in bicameral chambers. Pachytene spermatocytes also suppressed FSH-induced inhibin B levels in Sertoli cell co-cultures and this suppression was attributed to a decrease in basal inhibin B production rather than a change in FSH responsiveness. Quantitation of Sertoli cell inhibin alpha- and beta B-subunit mRNA by quantitative (real-time) PCR demonstrated that pachytene spermatocytes did not alter Sertoli cell alpha-subunit mRNA expression, but significantly (P<0.01) suppressed basal and FSH-induced beta B-subunit mRNA expression to a similar degree to that seen with inhibin B protein levels. It is concluded that pachytene spermatocytes in vitro suppress Sertoli cell inhibin B secretion via factor-mediated suppression of inhibin beta B-subunit expression. These findings support the hypothesis that specific germ cell types can influence inhibin B secretion by the testis independent of FSH regulation.     

Possible role of luteinizing hormone and follicle-stimulating hormone in modulating inhibin secretion and expression during the estrous cycle of the rat

In the female rat, plasma immunoreactive inhibin alpha (irl alpha) levels show marked changes during proestrus and estrus. We investigated the modulating effect of LH and FSH on these changes by injecting the GnRH antagonist DNal-DCpa-DPal-Dpr-(Ac)Dal-Leu-Arg-Pro-Asn-NH2, with or without exogenous LH replacement. Administration of the antagonist at noon on proestrus abolished the primary (proestrus) LH and FSH surge and markedly reduced the secondary (estrus) FSH surge. This treatment also reduced the release of irl alpha normally measured during proestrus afternoon, and partially prevented the decrease in irI alpha secretion on proestrus evening. Exogenous LH injected at 1545 h on proestrus had no measurable effect on irI alpha or FSH levels in control rats; however, in antagonist-treated animals, it restored the secondary FSH surge to control values while augmenting the late proestrus fall in irI alpha. This suggests that the decrease in inhibin secretion measured after exogenous LH treatment represents the mechanism through which LH induced the secondary FSH surge in antagonist-blocked rats. We also used in situ hybridization techniques to examine the changes in the expression of inhibin subunits in the ovary at 0200 h on estrus. The antagonist reduced expression of the alpha-, beta A-, and beta B-subunits in all follicle and tissue types, with the exception of the granulosa cells of large tertiary (possibly preovulatory) follicles where the signal appeared greatly enhanced. These changes were reversed by LH. The alteration in inhibin subunit messages caused by blockade of the primary gonadotropin surge suggests the presence of a cross-regulation between LH and inhibin/activin secretion, so that a decline in circulating LH levels might stimulate inhibin/activin secretion in the granulosa cells of preovulatory follicles, while reducing the production of these proteins in less mature follicles and in other ovarian cell types.     

Inhibin A is a follicle stimulating hormone-responsive marker of granulosa cell differentiation, which has both autocrine and paracrine actions in sheep

The aim of these studies was to examine the origin, control and local actions of inhibin A in monovular species, using the sheep as a model. Experiment 1 examined the pattern of mRNA expression for the inhibin subunits in relation to follicular size and pattern of expression to other differentiative markers in granulosa (P450 aromatase) and thecal cells (P450 17alpha-hydroxylase). Experiment 2 examined the pattern of inhibin A production, in relation to oestradiol, by granulosa cells induced to differentiate in vitro with follicle-stimulating hormone (FSH). Experiment 3 examined possible paracrine and autocrine actions of inhibin A by determining the effect of addition of human recombinant inhibin A and/or antiserum to inhibin on gonadotrophin-stimulated cellular differentiation. The results of Experiment 1 showed that expression of mRNA encoding inhibin subnuits alpha, beta(A) and beta(B) is greater (P<0.05) in large oestrogenic follicles than in small follicles but that only expression of the inhibin beta(A) subunit differs (P<0.05) between large oestrogenic and non-oestrogenic follicles. Expression of 17alpha-hydroxylase, but not of the luteinising hormone (LH) receptor, in thecal cells was related to both the size and the oestrogenicity of antral follicles, in a manner similar to that of the inhibin subunits. Experiment 2 demonstrated that the production of inhibin A by sheep granulosa cells is FSH-responsive after prolonged exposure (P<0.001) and precedes the production of oestradiol by around 48 h in the differentiative cascade induced in granulosa cells by FSH. The results of Experiment 3 showed that inhibin A can augment gonadotrophin-stimulated steroid production by both granulosa and theca cells (P<0.01), and that the addition of antiserum to inhibin can inhibit FSH-stimulated oestradiol production by granulosa cells from both small and large follicles (P<0.001). We conclude that inhibin A is an FSH-responsive marker of granulosa cell differentiation which has both autocrine and paracrine actions in sheep.     

Comparison of the effects of cycloheximide and inhibin on the gonadotropin subunit messenger ribonucleic acids.

Cycloheximide (CHX) has been shown to mimic the action of inhibin on gonadotropin secretion by pituitary cell cultures. We showed previously that suppression of FSH secretion by inhibin is associated with a rapid and profound suppression of FSH beta mRNA levels. The present study was designed to examine the mechanism of action of CHX and to determine whether inhibin's actions involve new proteins synthesis. Pituitary cell cultures were treated with control medium or medium containing inhibin, CHX, or inhibin plus CHX for 2 or 6 h. At 6 h, secretion of FSH was decreased by inhibin (72% of control), CHX (58% of control), and the combined inhibitors (56% of control). LH secretion was not significantly changed, while that of free alpha-subunit was reduced only by CHX (68% of control). Levels of FSH beta, LH beta, and alpha-subunit mRNAs were measured by Northern analysis. At 2 h inhibin decreased FSH beta mRNA to 49% of the control value. CHX alone had no effect, while CHX plus inhibin produced intermediate levels (77% of control). By 6 h, however, inhibin and CHX each decreased FSH beta mRNA to very low levels (12% and 15% of control, respectively), and in cultures treated with both inhibin and CHX, this RNA was barely detectable. To determine the reversibility of the effects of these inhibitors, cells were incubated with fresh control medium after 6 h. Secretion of FSH and free alpha-subunit remained suppressed 4 h later; recovery was complete by 16 h in inhibin treated cultures. FSH beta mRNA returned to control levels by 4 h in inhibin-treated and by 16 h in CHX-treated cultures. Levels of LH beta and alpha-subunit mRNA were comparable to control values at all times. In conclusion, 1) CHX, like inhibin, suppresses FSH beta mRNA levels, although its actions are less rapid and less rapidly reversible; 2) inhibin requires ongoing protein synthesis for full expression of its inhibitory effects; 3) the synthesis and secretion of LH are much less sensitive to inhibition by either inhibin or CHX than are the synthesis and secretion of FSH; and 4) secretion of free alpha-subunit involves a labile protein(s).     

Reciprocal regulation of activin A and inhibin B by interleukin-1 (IL-1) and follicle-stimulating hormone (FSH) in rat Sertoli cells in vitro

In several biological systems, the inhibin beta(A) homodimer activin A is stimulated by, and in turn, inhibits the action of interleukin (IL)-1 (both IL-1alpha and IL-1beta) and IL-6. The possibility that a similar regulatory relationship operates within the testis was investigated. Sertoli cells from immature (20-day-old) rats were cultured with human IL-1alpha or IL-1beta, human IL-6 and/or ovine FSH or dibutyryl cAMP. Activin A and the inhibin dimers, inhibin A and inhibin B, were measured by specific ELISA. Immunoreactive inhibin (ir-inhibin) was measured by RIA. Activin/inhibin subunit mRNA expression was measured by quantitative real-time PCR. Both IL-1 isoforms, but not IL-6, stimulated activin A secretion through increased synthesis of beta(A)-subunit mRNA. IL-1 also stimulated activin A secretion by testicular peritubular cells. In contrast to the effect on activin A, IL-1 suppressed inhibin beta(B)-subunit and, to a lesser extent, alpha-subunit mRNA expression, thereby reducing basal and FSH-stimulated inhibin B secretion by the Sertoli cells. Conversely, FSH inhibited basal activin A secretion and antagonised the stimulatory effects of IL-1. Dibutyryl cAMP partially inhibited the action of IL-1 on activin A secretion, but had no significant effect on basal activin A secretion. Secretion of inhibin A was low in all treatment groups. These data demonstrate that IL-1 and FSH/cAMP exert a reciprocal regulation of activin A and inhibin B synthesis and release by the Sertoli cell, and suggest a role for activin A as a potential feedback regulator of IL-1 and IL-6 activity in the testis during normal spermatogenesis and in inflammation.     

Developmental expression of activin/inhibin alpha- and beta(A)-subunit genes in the gonads of male and female chick embryos

The expression of inhibin alpha- and beta(A)-subunits was investigated in gonads of male and female chick embryos during the last week of their 21-day incubation period. Fertilized Hisex brown laying hen eggs were incubated at 37.8 +/- 0.2 degrees and 60% relative humidity in an automatic forced-draft incubator with constant lighting. Embryos were killed after 14, 18, and 21 days of incubation, sexed by macroscopical inspection of the gonadal phenotype, and further dissected to obtain the gonads. Total RNA was isolated using the ultraspec RNA method. The expression of alpha- and beta(A)-subunits was evaluated by competitive RT-PCR. Significant differences were found within and between sexes in the expression of the alpha- and beta(A)-subunits. The level of the alpha-subunit in the testis was about 23-fold higher than that in the ovary at all ages. Testicular content of inhibin alpha mRNA levels was similar at days 14 and 18 but declined significantly at day 21 of incubation, whereas no significant differences were observed between the three age groups in the ovary. Testicular and ovarian inhibin beta(A)-subunit increased significantly from day 14 to day 18 followed by a significant decline before hatch. However, inhibin beta(A) level at day 14 was significantly higher in the ovary than in the testis. At days 18 and 21, there were no differences in the levels of the inhibin beta(A) in the sexes. The expression of inhibin beta(A)-subunit in the ovary was significantly higher than that of the alpha-subunit at all ages. In the testis, however, the expression of the beta(A)-subunit was higher at days 18 and 21 than at day 14. The sex difference in gonadal inhibin subunits expression suggests differential roles of inhibin/activin in the development of the chicken gonads. The changing level of expression during incubation also suggests changing biological roles within sexes.     

Localization and secretion of inhibin/activin subunits in the human and subhuman primate fetal gonads

Little is known about the ability of the fetal primate gonads to produce inhibin/activin. We investigated the presence of the alpha-, beta A-, and beta B-subunits of inhibin/activin in fetal human (16-23 weeks gestational age) and rhesus monkey (days 150-157 of gestation; term = 165 days) testes and ovaries by immunocytochemistry. The regulation of alpha-inhibin secretion by gonadotropins was studied in fetal testicular cultures. In the human fetal testis, alpha-subunit immunostaining was found in interstitial and intratubular cells, while beta A- and beta B-subunit immunostaining occurred in clusters of Leydig cells that were clearly demarcated from groups of Leydig cells that were immunonegative. In the late gestational monkey testis, the alpha-subunit was localized in tubular cells, and the beta B-subunit was present in the tubules and interstitium. Testicular cells from midgestation human testes secreted detectable immunoreactive alpha-inhibin in response to FSH and hCG stimulation; alpha-inhibin levels were significantly higher after hCG than FSH. In contrast, levels of alpha-inhibin secreted by rhesus monkey testicular cells were significantly increased by FSH, but not hCG. In the ovary, only weak beta B-subunit immunoreactivity was detected in granulosa cells of a few primary follicles from midgestational human fetal ovaries. In contrast, all three subunits were found in granulosa cells of numerous primary and secondary follicles in the late gestation rhesus monkey ovary. In light of recent evidence that inhibins/activins have actions on gonadal differentiation and growth modulation in vitro, as well as endocrine effects on the fetal pituitary, we propose that these proteins may have intragonadal and endocrine roles in human and subhuman intrauterine gonadal development.     

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.     

Reproductive deficiencies in transgenic mice expressing the rat inhibin alpha-subunit gene

Inhibin is an important modulator of reproductive function at both the endocrine level, through its regulation of pituitary FSH biosynthesis, and at the paracrine and autocrine levels, as an intragonadal regulatory factor. To investigate the in vivo actions of inhibin in FSH regulation and gonadal function, transgenic mice that overexpress the rat inhibin alpha-subunit gene were generated. A transgene that includes the mouse metallothionein-I gene promoter (MT-alpha) fused to the rat inhibin alpha-subunit precursor coding sequences was used to produce three lines of transgenic mice. Transgene mRNA is expressed in numerous tissues, including the pituitary, liver, testis, ovary, and kidney. Inhibin alpha-subunit protein was also increased in transgenic pituitary and ovary. Serum inhibin alpha-subunit levels are highly increased compared with control mice. Inhibin beta(A)- and beta(B)-subunit protein amounts are lower in transgenic ovaries compared with wild type, although serum levels of activin A are not significantly reduced in transgenic female mice. FSH levels are reduced in both male and female transgenic mice, whereas LH levels are increased in MT-alpha female mice. MT-alpha transgenic females are subfertile and exhibit a 52% reduction in litter size compared with wild-type females. The smaller litter size of MT-alpha female mice was correlated with a reduction in the number of oocytes ovulated during a normal cycle. Treatment of the transgenic females with exogenous gonadotropins resulted in an ovulation rate similar to that of stimulated wild-type animals, suggesting that altered gonadotropin levels may be responsible for the decreased ovulation rates. MT-alpha transgenic male mice are fertile and sire litters of equivalent size to those sired by wild-type males, despite an approximately 50% reduction in sperm numbers. These results indicate that overexpression of the rat inhibin alpha-subunit gene in mice leads to a disruption of the normal inhibin-to-activin ratio and to reproductive deficiencies, and they support the hypothesis that inhibin and activin act to regulate FSH secretion in vivo and are essential for normal gonadal function.     

Regulation of Sertoli cell inhibin production and of inhibin alpha-subunit mRNA levels by specific germ cell types

To elucidate the endocrine and paracrine regulation of testicular inhibin production, the effects of follicle-stimulating hormone (FSH), (Bu)2cAMP, germ cells (either crude or enriched preparations) and germ cell-conditioned media on inhibin production (immuno- and bio-activities) and the levels of alpha- and beta B-subunit mRNAs were assessed in cultured Sertoli cells isolated from 20-day-old rats. FSH and (Bu)2-cAMP stimulated both secreted and intracellular inhibin levels in a dose-dependent manner. Using cDNA probes corresponding to the alpha-subunit and the beta B-subunit of rat inhibin it was also shown that both FSH and (Bu)2cAMP markedly increased the level of alpha-subunit mRNA but had no effect on the beta B-subunit mRNA. Addition of a crude mixture of germ cells to Sertoli cell monolayers was found to enhance inhibin secretion. Of the different germ cell fractions tested in co-culture, early spermatids reproducibly stimulated both basal and (Bu)2cAMP-induced production of inhibin whereas pachytene spermatocytes only increased the latter; cytoplasts from elongated spermatids (CES) had no effect. Co-culture of Sertoli cells with liver epithelial cells (LEC) significantly enhanced (Bu)2cAMP-induced inhibin levels. Media conditioned by early spermatids consistently and dramatically stimulated the secretion of both bioactive and immunoactive inhibin by Sertoli cells while spent media from pachytene spermatocytes displayed less activity. CES-conditioned media had only minor stimulatory effects, which may have resulted from the contamination of this fraction by spermatids. Media conditioned by LEC had no effect on inhibin production, confirming that the activity of this cell line is not mediated via a diffusible factor. Early spermatids were found to increase levels of the alpha-subunit mRNA. The current study provides evidence for the involvement of germ cells, in particular of early spermatids, in the local testicular regulation of inhibin gene expression and production in the rat. This may be of crucial importance for the ontogeny of this parameter of Sertoli cell function, and has important implications with regard to the postulated endocrine and paracrine roles of inhibin.     

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.