BMP15 regulates the inhibin/activin system independently of ovulation rate control in sheep

Polymorphisms in the gene encoding bone morphogenetic protein 15 (BMP15) have been associated with multiple ovulations in sheep. As BMP15 regulates inhibin expression in rodents, we assumed that the ovarian inhibin/activin system could mediate part of the effect of BMP15 mutations in the regulation of ovulation rate in sheep. To answer this question, we have studied the effects of two natural loss-of-function mutations of BMP15 on the expression of components of this system. The FecXR and the FecXGr mutations, when present respectively in Rasa Aragonesa ewes at the heterozygous state and in Grivette ewes at the homozygous state, were associated with a twofold increase in ovulation rate. There were only small differences between mutant and wild-type ewes for mRNA expression of INHA, INHBA, ACVR1B, ACVR2A, FST or TGFBR3 in granulosa cells and inhibin A or activin A concentrations in follicular fluid. Moreover, the effects of mutations differed between breeds. In cultures of granulosa cells from wild-type ewes, BMP15, acting alone or in synergy with GDF9, stimulated INHA, INHBA and FST expression, but inhibited the expression of TGFBR3. Activin A did not affect INHBA expression, but inhibited the expression of ACVR2A also. The complexity of the inhibin/activin system, including positive and antagonistic elements, and the differential regulation of these elements by BMP15 and activin can explain that the effects of BMP15 mutations differ when present in different genetic backgrounds. In conclusion, the ovarian inhibin/activin system is unlikely to participate in the increase of ovulation rate associated with BMP15 mutations in sheep.

Growth Differentiation Factor-9 Stimulates Inhibin Production and Activates Smad2 in Cultured Rat Granulosa Cells

Ovarian inhibin production is stimulated by FSH and several TGFβ family ligands including activins and bone morphogenetic proteins. Growth differentiation factor-9 (GDF-9) derived by the oocyte is a member of the TGFβ/activin family, and we have previously shown that GDF-9 treatment stimulates ovarian inhibin-α content in explants of neonatal ovaries. However, little is known about GDF-9 regulation of inhibin production in granulosa cells and downstream signaling proteins activated by GDF-9. Here, we used cultured rat granulosa cells to examine the influence of GDF-9 on basal and FSH-stimulated inhibin production, expression of inhibin subunit transcripts, and the GDF-9 activation of Smad phosphorylation. Granulosa cells from small antral follicles of diethylstilbestrol-primed immature rats were cultured with FSH in the presence or absence of increasing concentrations of GDF-9. Secreted dimeric inhibin A and inhibin B were quantified using specific ELISAs, whereas inhibin subunit RNAs were analyzed by Northern blotting using 32P-labeled inhibin subunit cDNA probes. Similar to FSH, treatment with GDF-9 stimulated dose- and time-dependent increases of both inhibin A and inhibin B production. Furthermore, coincubation of cells with GDF-9 and FSH led to a synergistic stimulation of both inhibin A and inhibin B production. GDF-9 treatment also increased mRNA expression for inhibin-α and inhibin-β subunits. To investigate Smad activation, granulosa cell lysates were analyzed in immunoblots using antiphosphoSmad1 and antiphosphoSmad2 antibodies. GDF-9 treatment increased Smad2, but not Smad1, phosphorylation with increasing doses of GDF-9 leading to a dose-dependent increase in phosphoSmad2 levels. To further investigate inhibin-α gene promoter activation by GDF-9, granulosa cells were transiently transfected with an inhibin-α promoter-luciferase reporter construct and cultured with different hormones before assaying for luciferase activity. Treatment with FSH or GDF-9 resulted in increased inhibin-α gene promoter activity, and combined treatment with both led to synergistic increases. The present data demonstrate that oocyte-derived GDF-9, alone or together with pituitary-derived FSH, stimulates inhibin production, inhibin subunit mRNA expression, and inhibin-α promoter activity by rat granulosa cells. The synergistic stimulation of inhibin secretion by the paracrine hormone GDF-9 and the endocrine hormone FSH could play an important role in the feedback regulation of FSH release, thus leading to the modulation of follicle maturation and ovulation.

Photoperiod-dependent regulation of inhibin in Siberian hamsters: II. Regulation of inhibin production and secretion by pregnant mare serum gonadotropin

Inhibin production differs in ovaries of Siberian hamsters (Phodopus sungorus) exposed to long days (LD) or short days (SD). We believe that seasonal differences in serum follicle-stimulating hormone contribute to this difference. However, given the profound photoperiodic differences in follicle maturation, serum gonadotropins alone may not account for all of the observed differences in inhibin processing. To test this hypothesis, we challenged LD and SD female hamsters with exogenous gonadotropins. While both groups responded with increased inhibin expression, the effects were muted in ovaries of SD females and there was no evidence of ovulation in these animals. These data indicate that the ovaries of SD females are not immediately equipped to respond to gonadotropin stimulation. More generally, these data suggest that photoperiodic history affects ovarian inhibin production and secretion in response to gonadotropins.

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.