REGULATION OF INHIBIN SUBUNIT MESSENGER-RIBONUCLEIC-ACID LEVELS BY GONADOTROPINS, GROWTH-FACTORS, AND GONADOTROPIN-RELEASING-HORMONE IN CULTURED RAT GRANULOSA-CELLS

Cultured rat granulosa cells have provided a useful model to examine the hormonal regulation of inhibin secretion. In the present study we have used the cloned rat inhibin α- and βA-subunit cDNAs to characterize the influences of gonadotropins, growth factors, and GnRH on inhibin subunit mRNA levels in granulosa cells obtained from immature estrogen-treated rats. Cells were cultured in medium with or without added hormones. Total RNA from cultured cells was extracted and hybridized with 32P-labeled inhibin α- and βA-subunit cRNA or β-actin cDNA probes, and inhibin subunit mRNA levels were normalized with β-actin mRNA levels. Treatment of granulosa cells with FSH increased inhibin α- and βA-subunit mRNA levels in a dose-dependent manner. Similarly, LH, but not PRL, increased a- and βA-subunit mRNA levels in granulosa cells pre-treated with FSH to induce functional LH and PRL receptors. The effects of FSH and LH on inhibin subunit mRNA levels were mimicked by forskolin, which increased a- and βA-subunit transcripts in a dose- and time-dependent manner, suggesting involvement of the cAMP-dependent protein kinase-A pathway. Since several growth factors have been shown to influence inhibin secretion, their effects on inhibin subunit mRNA levels were also studied. Treatment of cells with transforming growth factor-β1 increased both basal and FSH-stimulated inhibin α-and βA-subunit mRNA content, whereas insulin-like growth factor-I had no significant effect. In contrast, both epidermal growth factor (EGF) and basic fibroblast growth factor (FGF) markedly suppressed both basal and FSH-stimulated inhibin subunit transcript levels. The inhibitory effects of EGF and basic FGF were dose dependent and persisted from 12-72 h of incubation. The regulatory peptide GnRH, which decreases inhibin secretion, was also found to suppress FSH-stimulated inhibin α- and βA-subunit mRNA levels in a dose-dependent manner. Furthermore, the effects of GnRH could be counteracted by coincubation with a GnRH antagonist, suggesting the involvement of specific GnRH-binding sites in GnRH action. These studies indicate that, except for insulin-like growth factor-I, the effects of gonadotropins, growth factors (EGF, basic FGF, and transforming growth factor-β1), and GnRH on inhibin secretion are related to their regulation of inhibin a- and βA-subunit mRNA levels. © 1990 by The Endocrine Society.