Secretory pattern of inhibin A, inhibin B and inhibin pro-alpha C during induced follicular atresia and subsequent follicular development in the golden hamster (Mesocricetus auratus).

The changes in plasma concentrations of inhibins A, B and pro-alpha C were determined in the cyclic golden hamster during follicular atresia induced with antiserum against luteinizing hormone releasing hormone (LHRH-AS) at 1100 h on day 4 (day 1=day of ovulation). Follicular status in the ovary was also studied by determining the number of follicles ovulating in response to human chorionic gonadotrophin (hCG) injection. The time-courses of changes in plasma concentrations of inhibins A, B and pro-alpha C were different from each other during induced follicular atresia and subsequent follicular development. Plasma concentrations of inhibin A decreased to 58.6% of initial values by 24 h after LHRH-AS treatment, and then remained relatively low until at least 60 h later. Plasma concentrations of inhibin B decreased to 64.2% of the initial values by 18 h after LHRH-AS treatment and remained at basal values for 36 h, but increased abruptly to greater than initial values at 42 h after the treatment. Plasma concentrations of inhibin pro-alpha C increased at 6 and 12 h, decreased suddenly to 21.9% of the initial values by 24 h after LHRH-AS treatment, and then gradually increased until 60 h after LHRH-AS. The number of follicles responding to hCG decreased gradually between 0 and 30 h after LHRH-AS, when no ovulations were observed, and then gradually increased until 60 h. The changes in follicular ovulatory responses to hCG correlated with the plasma profile of inhibin A throughout the experiment. These results suggest that inhibin A is mainly secreted by large antral follicles. In contrast, during the subsequent follicular development, the plasma concentration of inhibin B increased earlier than that of inhibin A. These results suggest that inhibin B is secreted by small and large antral follicles. Plasma concentrations of inhibin pro-alpha C were high at a time when plasma concentrations of oestradiol-17 beta had already decreased, indicating that inhibin pro-alpha C is secreted not only from healthy follicles but also from early atretic antral follicles.

Potential role of activin A in follicular development during the second half of pregnancy in the golden hamster: utero-placental source of activin A.

Numerous antral follicles develop during the second half of pregnancy in the golden hamster. However, mechanisms regulating follicular development during this period are unknown. Because inhibin and activin are related to follicular development, these hormones were studied to gain insight into any potential roles in follicular development. Plasma inhibin A and B suddenly increased from day 8 of pregnancy, reached peak levels on day 10 and gradually declined to term. Plasma activin A gradually increased from day 8 to day 15 of pregnancy, and this was followed by an abrupt decrease at day one of lactation. Ovariectomy on day 12 of pregnancy rapidly reduced plasma inhibin A and B, but not activin A levels. Hysterectomy or placentectomy on day 12 of pregnancy caused an abrupt decrease in the levels of plasma activin A and FSH, but not inhibin A and B at 6 h after surgery. Hysterectomy also induced atresia of large antral follicles at 24 h after surgery. These results indicate that antral follicles are the main source of circulating inhibin A and B, whereas uteri and placentae are the main source of circulating activin A. These results suggest that increased levels of activin A may be involved in folliculogenesis in the ovary during the second half of pregnancy in the golden hamster.

Inhibins in the male Göttingen miniature pig: Leydig cells are the predominant source of inhibin B.

The expression of inhibin subunits in the testes of the Göttingen miniature pig was examined by in situ hybridization and immunohistochemistry. In addition, the major forms were determined by enzyme-linked immunosorbent assay (ELISA). Strong positive immunostaining for the inhibin alpha subunit was observed in Sertoli and late-stage germ cells, but it was weak in Leydig cells. However, Leydig cells showed strong positive staining for the betaA subunit, but Sertoli cells and spermatogonia showed a weak reaction. Strong positive immunostaining for the betaB subunit was observed in Leydig cells but spermatogonia showed weak staining for it. In contrast to the staining specificity of inhibin alpha and betaA subunits, the betaB subunit did not exhibit positive staining in Sertoli cells. In situ hybridization revealed that although the a subunit mRNA signal was highly expressed in all cell types, the reaction appeared to be stronger in Sertoli cells and spermatogonia than in Leydig cells. betaA subunit mRNA expression was somewhat identical to that of the alpha subunit, however, germ cells showed a weak stain for it. A strong, positive mRNA signal for the betaB subunit was confined to Leydig cells and late-stage germ cells. ELISA results showed that concentrations of inhibin B and inhibin pro-alphaC were high in the circulation and testes. In contrast, inhibin A levels in both plasma and testes were undetectable. The present results strongly suggest that inhibin B is the major form of circulating inhibin and that Leydig cells are the predominant source of this dimeric inhibin in male Göttingen miniature pigs. Furthermore, the germ cells also appear to be an important source of circulating inhibins.

Contribution of endogenous inhibin to the decline of the secondary surge of follicle-stimulating hormone in the rat.

The involvement of inhibin in the decline of the secondary surge of follicle-stimulating hormone (FSH) was investigated in the rat. After ovariectomy or treatment with inhibin antiserum conducted at 2300 hours during pro-oestrus, plasma concentrations of FSH were maintained at high levels compared with control rats. However, plasma FSH started to decline at 0500 hours during oestrus in both the groups. The same treatments conducted during metoestrus markedly increased plasma FSH after 24 h (twofold compared with the treatments during pro-oestrus), suggesting that the treatments sufficiently depleted circulating inhibin. To examine whether the decline of plasma FSH occurred through a transcriptional mechanism or through a translational mechanism, FSH-beta mRNA expression and the pituitary concentration of FSH were measured. Neither ovariectomy nor inhibin immunization conducted during the night of pro-oestrus, affected the pituitary concentration of FSH after 24 h, whereas a noticeable increase was observed after the treatments conducted during metoestrus. In both stages, both ovariectomy and inhibin immunization significantly increased FSH-beta mRNA expression compared with control rats. In contrast with the pituitary concentration of FSH, the effect of inhibin immunization on FSH-beta mRNA expression was not different between the stages. The present data demonstrate the involvement of inhibin in the decline of the secondary surge of FSH, and suggest that a factor or factors other than inhibin may also be responsible for the fall in FSH. Changes in the pituitary concentration of FSH and FSH-beta mRNA expression suggest that post-transcriptional mechanisms may be involved in the suppression of FSH secretion during oestrus.

Testicular secretion of inhibin in the male golden hamster (Mesocricetus auratus).

To identify the cellular source of inhibin in the male golden hamster, we have used complementary approaches, immunohistochemistry and enzyme-linked immunosorbent assay (ELISA). Strong positive staining of the inhibin alpha subunit was observed in both the Sertoli and Leydig cells of the testes. No specific staining was observed for the inhibin betaA subunit, whereas specific staining for the inhibin betaB subunit was strongly positive in the Leydig cells. Inhibin pro-alphaC and inhibin B were detected in peripheral plasma, and testicular homogenate also contained large amounts of inhibin pro-alphaC and inhibin B. However, inhibin A was not detected either in peripheral plasma or in testicular homogenate. Plasma concentrations of inhibin pro-alphaC and inhibin B were significantly (P < .001) decreased 24 hours after orchidectomy. These results strongly suggest that the Leydig cells are the main source of dimeric inhibin B in the male golden hamster.

Preovulatory follicles in the ovary as the source of circulating inhibin in the duck.

Inhibin secretion in the adult female duck was investigated. The bovine inhibin radioimmunoassay (RIA) system and human enzyme-linked immunosorbent assay (ELISA) of inhibin A and inhibin B were first validated for use in the duck. In both RIA and ELISA, the dilution curves of plasma and homogenate of the first largest follicle (F1) were parallel to each standard curve, indicating that plasma and the F1 follicle contained immunoreactive (ir) and dimeric inhibins. Short-term food deprivation caused follicular atresia in the ovary and significantly depressed the plasma concentration of ir-inhibin. Positive immunostaining for inhibin alpha-, betaA-, and betaB-subunits was clearly detected in the granulosa cells of the four largest preovulatory follicles. Immunolocalization of these three inhibin subunits was also weakly seen in the interna theca cells of these follicles. These results demonstrate that inhibin alpha-, betaA-, and betaB-subunit proteins are colocalized in the granulosa cell and theca cell of the four largest preovulatory follicles in the duck ovary. The present results, therefore, indicate that the four largest follicles in the ovary are the main source of circulating inhibin in the female duck.

Identification and characterization of a novel follistatin-like protein as a binding protein for the TGF-beta family.

Follistatin is an activin-binding protein that prevents activin from binding to its receptors and neutralizes its activity. Follistatin also binds bone morphogenetic proteins (BMPs). In this study, we report the identification of a novel follistatin-like protein from mouse. The mouse cDNA encodes a 256-residue precursor and most likely a mouse homologue of human FLRG, which was found at the breakpoint of the chromosomal rearrangement in a B-cell line. Whereas follistatin has three follistatin domains, which are presumed to be growth factor binding motifs, FLRG possesses only two follistatin domains. Northern blotting revealed that mRNAs for FLRG were abundantly expressed in heart, lung, kidney, and testis in mouse. The recombinant mouse FLRG proteins were found to have binding activity for both activin and bone morphogenetic protein-2. Like follistatin, FLRG has higher affinity for activin than for BMP-2. The FLRG protein inhibited activin-induced and BMP-2-induced transcriptional responses in a dose-dependent manner. Glutathione S-transferase fusion proteins encoding various regions of FLRG were produced and studied. Ligand blotting using (125)I-activin revealed that the COOH-terminal region containing the second follistatin domain was able to bind activin. Our finding implies that cellular signaling by activin and BMPs is tightly regulated by multiple members of the follistatin family.

Involvement of inhibin in the regulation of follicle-stimulating hormone secretion in the young adult male Shiba goat.

The roles of inhibin and testosterone in the regulation of gonadotropin secretion were investigated in young adult male Shiba goats (8-12 months of age). Plasma levels of inhibin but not testosterone abruptly decreased after hemicastration (75% of the initial level), concomitant with a progressive rise in plasma levels of FSH. Removal of the remaining testis at 33 days after the hemicastration quickly decreased plasma levels of both inhibin and testosterone and induced a progressive increase in plasma FSH and LH. Implantation of testosterone sheets immediately after castration suppressed the increase in plasma FSH in part only, whereas the increase in LH secretion was almost completely suppressed. An i.v. injection of antiserum against [Tyr30] porcine inhibin alpha(1-30) resulted in a significant increase in plasma FSH in a dose-dependent manner, without altering plasma concentrations of LH. These findings clearly indicate that both inhibin and testosterone physiologically regulate FSH secretion and that testosterone is the principal gonadal factor regulating LH secretion in the adult male goat.