LaPolt P S, Piquette G N, Soto D, Sincich C, Hsueh A J
Department of Reproductive Medicine, University of California, San Diego, La Jolla 92093.
Endocrinology. 1990 Aug;127(2):823-31. doi: 10.1210/endo-127-2-823.
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.
培养的大鼠颗粒细胞为研究抑制素分泌的激素调节提供了一个有用的模型。在本研究中，我们使用克隆的大鼠抑制素α亚基和βA亚基cDNA来表征促性腺激素、生长因子和GnRH对从未成熟雌激素处理大鼠获得的颗粒细胞中抑制素亚基mRNA水平的影响。细胞在添加或不添加激素的培养基中培养。从培养细胞中提取总RNA，并与32P标记的抑制素α亚基和βA亚基cRNA或β-肌动蛋白cDNA探针杂交，抑制素亚基mRNA水平用β-肌动蛋白mRNA水平进行标准化。用FSH处理颗粒细胞以剂量依赖的方式增加了抑制素α亚基和βA亚基mRNA水平。同样，LH（而非PRL）增加了用FSH预处理以诱导功能性LH和PRL受体的颗粒细胞中的α亚基和βA亚基mRNA水平。FSH和LH对抑制素亚基mRNA水平的影响被福斯可林模拟，福斯可林以剂量和时间依赖的方式增加了α亚基和βA亚基转录本，提示cAMP依赖性蛋白激酶-A途径的参与。由于已显示几种生长因子会影响抑制素分泌，因此也研究了它们对抑制素亚基mRNA水平的影响。用转化生长因子-β1处理细胞增加了基础和FSH刺激的抑制素α亚基和βA亚基mRNA含量，而胰岛素样生长因子-I没有显著影响。相反，表皮生长因子（EGF）和碱性成纤维细胞生长因子（FGF）均显著抑制基础和FSH刺激的抑制素亚基转录水平。EGF和碱性FGF的抑制作用是剂量依赖性的，并且在孵育12 - 72小时后持续存在。调节肽GnRH可降低抑制素分泌，也被发现以剂量依赖的方式抑制FSH刺激的抑制素α亚基和βA亚基mRNA水平。此外，GnRH的作用可通过与GnRH拮抗剂共同孵育而被抵消，提示特异性GnRH结合位点参与GnRH作用。这些研究表明，除了胰岛素样生长因子-I外，促性腺激素、生长因子（EGF、碱性FGF和转化生长因子-β1）和GnRH对抑制素分泌的影响与其对抑制素α亚基和βA亚基mRNA水平的调节有关。
