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.     

Regulation of inhibin beta B-subunit mRNA expression in rat Sertoli cells: consequences for the production of bioactive and immunoreactive inhibin.

In Sertoli cells from 21-day-old rats, the expression of the mRNA encoding the alpha-subunit of inhibin, and the production of immunoreactive inhibin are stimulated by follicle-stimulating hormone (FSH). In contrast, the amount of beta B-subunit mRNA is not increased after FSH treatment of the cells, and the ratio between bioactive and immunoactive inhibin decreases after stimulation with FSH. These data suggest that the beta B-subunit is the limiting factor in the production of bioactive inhibin. The aim of the present experiments was to investigate the effect of changes in the amount of beta B-subunit mRNA on the production of bioactive and immunoreactive inhibin. During early postnatal testicular development, the relative amounts of the 4.2 kb and 3.5 kb mRNAs encoding the beta B-subunit of inhibin changed markedly. The meaning of this changing ratio between beta B-subunit mRNAs is not clear, since both mRNAs are actively translated, as demonstrated by polysomal analysis. The total amount of beta B-subunit mRNA correlated with the in vitro production of bioactive inhibin as published earlier. Prolonged stimulation of cultured Sertoli cells from 14-day-old rats with 4 beta-phorbol 12-myristate 13-acetate (PMA) caused a decreased expression of the beta B-subunit mRNAs, presumably by down-regulation of protein kinase C. A similar effect was obtained after addition of the calcium ionophore A23187. Concomitantly, a decreased production of bioactive inhibin was observed. Furthermore, Western blotting revealed that secretion of the 32 kDa inhibin alpha beta-dimer was decreased, whereas secretion of the combination of the C-terminal part with the pro-region of the alpha-subunit was increased. It is concluded that the level of the beta B-subunit of inhibin is rate-limiting for the production of bioactive inhibin in cultured Sertoli cells, and that its expression can be influenced by modulation of protein kinase C, and/or intracellular calcium levels.     

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.     

Effects of FSH and testosterone on highly purified rat Sertoli cells: inhibin alpha-subunit mRNA expression and inhibin secretion are enhanced by FSH but not by testosterone

The effects of FSH and testosterone on inhibin mRNA expression and inhibin production by highly purified Sertoli cell preparations were examined. Sertoli cells were isolated from testes of 22-day-old rats by sequential trypsin, collagenase and hyaluronidase treatments, with subsequent osmotic shock treatment on day 3 of culture. Contamination by peritubular and germ cells was less than 0.5 and 1-3% respectively. Intracellular and secreted inhibin levels were measured by radioimmunoassay, using Sertoli cells which were incubated for 24 h in the absence or presence of FSH and testosterone from days 4 to 5 of culture. FSH stimulated the cellular inhibin content and the secreted inhibin level by four- and sevenfold respectively, with a half-maximal effective dose of 5-50 ng/ml. Under the present incubation conditions, testosterone (1 mumol/l) had no effect on immunoreactive inhibin levels in either the presence or absence of FSH. Similarly, the expression of inhibin alpha-subunit mRNA was increased following FSH stimulation, whereas testosterone had no effect. The expression of inhibin beta B-subunit mRNAs was not influenced by FSH or testosterone. It is concluded that highly purified Sertoli cell preparations, with a very low number of peritubular or germ cells, are fully responsive to FSH with respect to inhibin mRNA expression and inhibin production.     

Follicle-stimulating hormone-stimulated secretion of an immunoreactive 29 kDa inhibin alpha-subunit complex, but not of 32 kDa bioactive inhibin, from cultured immature rat Sertoli cells

The medium of cultured Sertoli cells from immature rat testes contains 29 and 32 kDa proteins, which are recognized by an antiserum against the 22 N-terminal amino acids of the inhibin alpha-subunit. These proteins were detected by immunoprecipitation of labelled proteins after incubation of Sertoli cells with [35S]methionine, and by Western blotting. The amount of the 32 kDa protein was not affected by the addition of follicle-stimulating hormone (FSH) to the culture medium of the Sertoli cells, whereas FSH induced a large increase of the amount of the 29 kDa protein. Finally, the 29 and 32 kDa proteins in the medium from control and FSH-stimulated Sertoli cells were separated by sodium dodecyl sulfate-polyacrylamide electrophoresis, and inhibin bio- and immunoactivity were determined in eluates of the slices of the gel. Equal amounts of bioactivity were found in control and FSH-stimulated samples at 32 kDa, while the amount of immunoactivity at 29 kDa was increased; no bioactivity was detected in the eluates of these slices. It is concluded that FSH stimulates the secretion of a 29 kDa inhibin-like protein, which does not contain inhibin bioactivity. This indicates that results of experiments, in which antibodies against N-terminal peptides of the inhibin alpha-subunit are used to detect inhibin, do not necessarily reflect the amount of bioactive inhibin produced.     

Regulation of inhibin secretion by Sertoli cell-enriched cultures

Sertoli cells secrete a factor which has the same bioactivity as ovine testicular lymph inhibin: it selectively suppresses the secretion of FSH by cultured pituitary cells. We investigated the factors that acutely modulate the secretion of this inhibin by cultured Sertoli cells derived from immature rats. The secretion of inhibin was studied on day 7 of culture after a 24 h period of incubation in the presence or absence of steroids, gonadotrophins and foetal bovine serum, added alone or in various combinations. It could be demonstrated that aromatisable as well as non-aromatisable natural and synthetic androgens promote the secretion of inhibin in a dose-dependent way. FSH and pregnant mare serum gonadotrophin--at concentrations that clearly stimulate Sertoli cell aromatase activity--did not affect basal or androgen-stimulated production of inhibin. hCG was equally uneffective. The effect of androgens was not modified by the addition of an aromatase inhibitor but it was neutralized by the antiandrogen cyproterone acetate. Oestradiol-17 beta did not influence the secretion of inhibin whereas progesterone decreased it. Serum enhanced basal as well as androgen stimulated secretion of inhibin. It is concluded that androgens are the major factor which acutely stimulates the production of Sertoli cell inhibin.     

Effects of orchidectomy on gonadotropin and inhibin subunit messenger ribonucleic acids in the pituitary of the rhesus monkey (Macaca mulatta).

Our research programs required the preparation of hypophysectomized and orchidectomized rhesus monkeys. This afforded us the possibility to characterize and compare levels of the gonadotropin and inhibin subunit mRNAs in pituitaries from intact and castrate monkeys. Eighteen adult male monkeys, four of which had been bilaterally orchidectomized 5-9 months previously, were used in this study. Plasma concentrations of LH and FSH were, respectively, 188.5 +/- 5.3 and 246.8 +/- 25.2 ng/ml in the castrate monkeys and 25.8 +/- 4.5 and 4.1 +/- 1.1 ng/ml (mean +/- SEM) in the intact animals. Total pituitary RNA was hybridized to cDNA probes for cynomolgus monkey gonadotropin subunits (FSH beta, LH beta, and the common alpha-subunit) and for human inhibin subunits (alpha, beta B, and beta A) by Northern blot analysis, and mRNA levels were normalized by subsequent hybridization to cyclophilin. Each of the gonadotropin subunit probes hybridized to a single RNA species with the approximate sizes of 1.6 kilobases (kb; FSH beta), 0.7 kb (LH beta), and 0.8 kb (alpha). Levels of LH beta and alpha-subunit mRNAs in pituitaries from castrate monkeys were about 5- and 2-fold higher, respectively, than those in pituitaries from intact monkeys. FSH beta mRNA, on the other hand, was elevated about 27-fold in castrate monkeys [mean +/- SEM, 3176 +/- 408 cpm bound (n = 4 castrate) and 116 +/- 30 cpm bound (n = 8 intact]). Inhibin beta B-subunit mRNA was present in the monkey pituitary as a doublet of about 5 kb, and it was approximately twice as abundant in intact pituitaries as in castrate pituitaries. Hybridizations involving inhibin beta A cDNA revealed a faint band in the region expected for monkey beta A mRNA (6.5 kb) in three of six RNA samples from intact monkeys and a 0.3- to 0.4-kb mRNA species. mRNA encoding the inhibin alpha-subunit was undetectable by Northern blot hybridization. These results indicate that the postpubertal testis imposes an inhibition on the expression of the genes encoding FSH beta, LH beta, and glycoprotein hormone alpha-subunit and that this suppression of the FSH beta gene in the monkey is much greater than that in the rat. In addition, the monkey pituitary may be a source of activin, which may act locally to modulate FSH gene expression and secretion.     

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.     

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.     

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).     

Differential production and regulation of inhibin subunits in rat testicular cell types

The distributions of the alpha-, beta A-, and beta B subunits of inhibin/activin polypeptides were studied in the testis of adult (60-day-old) and immature (12-day-old) rats. Immunohistochemical techniques using antisera selective for each subunit were used to localize the polypeptide chains. In situ hybridization using radiolabeled complementary RNA probes enabled localization of the messenger RNAs (mRNAs) encoding these subunits. In 12-day-old rats, immunostaining and mRNA signal for the alpha-subunit was found in Leydig cell clusters. The beta A- and beta B-subunit staining and beta A-subunit message were detectable in isolated interstitial cells, but the clusters appeared to lack these subunits. Positive immunostaining for each subunit was localized in a Sertoli cell-like pattern in seminiferous tubules, as was a positive mRNA signal for the alpha- and beta B-subunit over regions containing these cell types. Treatment with human CG (hCG) and PMSG greatly enhanced the production of the alpha-subunit in the Leydig cell clusters, but not within the tubules, of these young rats. In adult rats, alpha- and beta B-subunit staining, and alpha-subunit mRNA signal, was observed in the interstitial cells. As in the immature animals, all three subunits were localized in a Sertoli cell-like pattern in the tubules, and a positive mRNA signal for the alpha- and beta B-subunits was found over these cells. There was, however, no obvious change in the expression of the subunits in the testis of adult rats after gonadotropin treatment. The present findings suggest that: 1) in the rat testis, both Sertoli and interstitial cells produce inhibin/activin subunits; 2) the alpha- and beta-subunits are produced by different types of interstitial cells in immature rats; and 3) the production of the alpha-subunit in the Leydig cells of immature rats is regulated by LH-like hormones.     

Stimulation of Sertoli cell inhibin secretion by the testicular paracrine factor PModS

The testicular paracrine factor PModS is produced by peritubular myoid cells under androgen control and modulates Sertoli cell function and differentiation. The observation that luteinizing hormone (LH) stimulates inhibin production in vivo, but has no effect on isolated Sertoli cells in vitro, suggested an indirect mode of LH action, potentially mediated by PModS. The effects of the testicular paracrine factor PModS and hormones on inhibin secretion by Sertoli cells were investigated to provide insight into the endocrine control of inhibin expression. An inhibin radioimmunoassay was utilized which showed essentially parallel displacement curves with purified bovine follicular fluid inhibin, Sertoli cell conditioned medium and concentrated Sertoli cell secreted proteins. An immunoblot analysis of Sertoli cell secreted proteins with the inhibin antisera consistently detected a 32 kDa protein which is the expected size of the mature of inhibin (alpha beta) and periodically detected a 57 kDa protein which is speculated to be an incomplete processed form of the inhibin precursor (alpha 43 beta). Follicle-stimulating hormone (FSH) was found to stimulate inhibin secretion initially between days 2 and 5 of Sertoli cell culture. Insulin and retinol alone had no significant effect on inhibin secretion; however, together they appeared to enhance the ability of FSH to stimulate inhibin secretion. Testosterone had no effect on inhibin production alone or in combination with other regulatory agents. PModS was found to stimulate inhibin secretion approximately 3-fold, but with a delayed time course of stimulation which did not occur until days 5-7 of Sertoli cell culture. Treatment with a combination of PModS and FSH resulted in an apparent maximal stimulation of inhibin secretion. Both forms of PModS, PModS (A) and PModS (B), were found to have equivalent biological activities in their ability to stimulate inhibin production with an apparent half-maximal effective concentration between 10 and 15 ng/ml. The current study provides evidence for the local testicular control of inhibin production and adds to the complexity of the endocrine control of inhibin expression. The cellular interaction is proposed in which LH acts on Leydig cells to stimulate androgen production which in turn acts on peritubular cells to regulate PModS production which subsequently can act on Sertoli cells to control inhibin production. Testicular control of inhibin production provides a potential short feedback loop for the local regulation of androgen production and an additional regulatory element for the pituitary-gonadal axis.     

Hormonal regulation of inhibin production by cultured Sertoli cells

The hormonal regulation of inhibin production by cultured rat Sertoli cells was examined using a specific radioimmunoassay (RIA) which detects the N-terminal portion of the porcine inhibin alpha chain. FSH, but not hCG or prolactin caused a dose-dependent increase in inhibin production (EC50 for FSH = 2.4 ng/ml); both secreted and intracellular levels of inhibin were increased, but the secreted form represented one-half to two-thirds of the total. The FSH-stimulated production of inhibin was augmented by addition of a phosphodiesterase inhibitor, and could be mimicked by cholera toxin, forskolin, or dibutyryl cAMP, all of which are known to increase intracellular cAMP levels. Inclusion of either dihydrotestosterone or estradiol in the cultures had no effect on inhibin production, both in the presence and absence of FSH. Examination of the conditioned media from forskolin-treated Sertoli cells by gel filtration chromatography revealed a single peak of bioactive and immunoreactive inhibin, at a molecular weight of approximately 32,000, similar to that observed for the porcine and bovine follicular fluid inhibins. Thus, FSH activated the cAMP pathway to stimulate Sertoli cell production of inhibin which in turn suppresses pituitary FSH release to form a closed-loop feedback system.     

Purification and characterization of high molecular weight forms of inhibin from bovine follicular fluid.

High molecular mass forms [95 kilodaltons (kDa)] of bovine inhibin-A as well as the known forms of intermediate (55 kDa) and low (32 kDa) mass were purified from bovine follicular fluid by ion exchange chromatography on DEAE-Sepharose, immunoaffinity chromatography using a monoclonal antibody directed against bovine 32-kDa inhibin-A, gel permeation HPLC on TSK-gel, and reverse phase HPLC. The 95-kDa inhibin-A had similar suppressive activity on FSH secretion from cultured rat anterior pituitary cells as the 55- and 32-kDa inhibins. There is, however, a possibility that the inhibin activity detected with larger forms may be due to that of the 32-kDa form that results from proteolytic processing during incubation with rat pituitary cells. Both sodium dodecyl sulfate-polyacrylamide gel electrophoresis (SDS-PAGE) and immunoblotting analysis using monoclonal antibodies specific for 32-kDa inhibin alpha- or beta A-subunits revealed that the 95-kDa inhibin preparation contained two forms of inhibin (105 and 95 kDa), which were composed of either a 50- or a 40-kDa alpha-subunit linked by a disulfide bond(s) to a 55-kDa beta A-subunit. Amino-terminal sequence analysis showed that the 50-kDa alpha-subunit and the 55-kDa beta A-subunit were generated by removal of a signal peptide from each corresponding primary translation product [the first NH2-terminal 17 residues of the inhibin alpha-subunit (residues 1-360) and the first 20 residues of the inhibin beta A-subunit (residues 1-425)] and suggested that the 40-kDa alpha-subunit was formed by proteolytic processing of the 50-kDa alpha-subunit. On the basis of our findings, we propose that in bovine follicular fluid, the larger 105-kDa form of inhibin is processed successively to form the lowest molecular mass form, 32 kDa inhibin, through the smaller 95- and 55-kDa forms.     

In vitro bioactive and immunoactive inhibin concentrations in bovine fetal ovaries and testes throughout gestation.

This study investigates the concentrations of inhibin in the bovine fetal ovary and testis throughout gestation (days 40 to 270/term) as determined by inhibin in vitro bioassay and RIA techniques. In addition, the expression of the inhibin alpha- and beta-subunits (beta A and beta B) in these tissues was evaluated by Northern blot analysis and in situ hybridization. Testicular concentrations of inhibin bioactivity and immunoactivity increased 2.5-fold (103 +/- 24.5 to 256 +/- 11.7 U/g wet weight; mean +/- SE) and 10.4-fold (139 +/- 56 to 1430 +/- 172) respectively, between 90 days and term. The corresponding ratio of inhibin biological: immunological activities (B/I ratio) decreased 8.3-fold (1.5 +/- 0.7 to 0.18 +/- 0.01). The concentration of ovarian bioactive inhibin increased significantly (P less than 0.05) 4.6-fold between 90 days and term (73.6 +/- 14.7 to 340 +/- 11.1 U/g wet weight), whereas the immunoactive inhibin concentration increased 10.3-fold between 120 and 210 days of gestation (7.2 +/- 1.9 to 40.0 +/- 8.7). The corresponding B/I ratio remained unchanged throughout gestation (8.3 +/- 2.4 to 12.5 +/- 4.0). Although the levels of alpha subunit mRNA in the testis and ovary increased over gestation, the levels of testicular beta A subunit mRNA remained low and unchanged. Ovarian levels of beta A subunit mRNA were also low but variable. Furthermore, no beta B subunit mRNA could be detected in gonadal tissue throughout gestation. alpha-Subunit mRNA was detected by in situ hybridization in the sex cords of the fetal testis and the granulosa cells of the fetal ovary while beta A subunit mRNA was detected only in the granulosa cells of the fetal ovary. It is concluded that inhibin is produced by the fetal testis and ovary and these tissue levels increase throughout gestation. The location of alpha- and beta A-subunit mRNA to the sex cords of the testis and granulosa cells of the ovary indicate that these cells are the primary source of inhibin production. The rapid fall in inhibin B/I ratio in testicular extracts over gestation is attributed to the production of an inhibin-related protein with limited or negligible biological activity.