Regulation of Inhibin Subunit Messenger Ribonucleic Acid Levels by Gonadotropins, Growth Factors, and Gonadotropin-Releasing Hormone in Cultured Rat Granulosa Cells*

Abstract

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 alpha- and beta A-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 alpha- and beta A-subunit cRNA or beta-actin cDNA probes, and inhibin subunit mRNA levels were normalized with beta-actin mRNA levels. Treatment of granulosa cells with FSH increased inhibin alpha- and beta A-subunit mRNA levels in a dose-dependent manner. Similarly, LH, but not PRL, increased alpha- and beta A-subunit mRNA levels in granulosa cells pretreated 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 alpha- and beta A-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-beta 1 increased both basal and FSH-stimulated inhibin alpha- and beta A-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 alpha- and beta A-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-beta 1), and GnRH on inhibin secretion are related to their regulation of inhibin alpha- and beta A-subunit mRNA levels.

Epidermal growth factor (EGF) decreased endothelin‐2 (ET‐2) production in human renal adenocarcinoma cells

“…On the other hand, EGF decreases mRNA levels of inhibin in cultured rat granulosa cells [21]. Similar to the observation that the ET-2 secretion was inhibited by bFGF, the transcription of the inhibin gene was also repressed by bFGF [21]. Since the EGF and bFGF receptors have cytoplasmic tyrosine domains [22,23], signal transduetions triggered by tyrosine kinase may participate in the regulation of the transcriptions of ET-2 and inhibin genes by EGF, TGF-ct and bFGF.…”

Effects of Indomethacin on the Selective Release of Follicle-Stimulating Hormone during the Period of Ovulation in the Rat.

“…In the granulosa cells, inhibin-α expression is stimulated by LH, FSH, cAMP analogs, and activin [2,16,37,41,46,47], whereas treatment with GnRH, protein kinase C activator, estradiol and estradiol with progesterone suppress FSH-stimulated inhibin-α expression [2,15,37,41]. Transforming growth factor (TGF)-α/epidermal growth factor (EGF) is thought to be a primary mitogen of granulosa cells [21], and the arachidonic acid metabolites by PGS, not the lipoxygenase metabolites, mediate the mitogenic effects of TGF-α/EGF, as demonstrated in the hen granulosa cells [17].…”

Genomic Cloning and Sequence Analyses of the Bovine α‐, βA‐ and βB‐Inhibin/activin Genes

“…Miyanaga et al, 1993 and references therein). The adenylate cyclase activator forskolin and a number of CAMP analogues have been shown to stimulate expression of a-inhibin subunit mRNA (Klaij et al, 1990;LaPolt et al, 1990;Najmabadi et al, 1993a). Pei et al (1991) identified a CRE element in the rat a-inhibin promoter that binds CREB and is responsive to forskolin in rat primary granulosa cells.…”