Tilly J L, LaPolt P S, Hsueh A J
Department of Gynecology and Obstetrics, Stanford University School of Medicine, California 94305-5317.
Endocrinology. 1992 Mar;130(3):1296-302. doi: 10.1210/endo.130.3.1311235.
The maturation of ovarian granulosa cells is dependent upon the pituitary gonadotropin FSH, the actions of which are mediated via specific plasma membrane receptors. To study the regulation of ovarian FSH receptor expression at the mRNA level, we used a specific cRNA probe to evaluate changes in FSH receptor transcripts in cultured granulosa cells. Granulosa cells obtained from immature estrogen-treated rats contained two predominant FSH receptor mRNA transcripts (7.0 and 2.5 kilobases), the levels of which declined in a time-related manner during a 2-day culture period. However, inclusion of FSH (30 ng/ml) in the culture medium prevented the decline in FSH receptor mRNA levels. Compared to controls, treatment of granulosa cells for 48 h with FSH (1-100 ng/ml) increased FSH receptor mRNA levels in a dose-dependent manner (ED50, 4.5 ng/ml), with a maximal 5.9 +/- 0.7-fold increase observed in response to 30 ng/ml FSH. The stimulatory actions of FSH were mimicked by the adenyl cyclase activator forskolin (0.1-30 microM), suggesting the involvement of cAMP in FSH receptor gene transcription and/or mRNA stability. Incubation of granulosa cells for 48 h with epidermal growth factor (EGF; 0.3-10 ng/ml), basic fibroblast growth factor (bFGF; 1-30 ng/ml), or insulin-like growth factor-I (IGF-I; 1-30 ng/ml) did not affect basal FSH receptor mRNA levels, whereas the highest doses of EGF and bFGF, but not IGF-I, completely suppressed the stimulatory effects of FSH (30 ng/ml) on its own receptor mRNA levels. Similarly, GnRH (10-1000 nM) attenuated the actions of FSH on its receptor mRNA levels in a dose-dependent manner (ID50, 8 nM). The inhibitory effects of GnRH (100 nM) were reversed by cotreatment with a GnRH antagonist ([Ac-D-Phe1,D-pCl-Phe2,D-Trp3,6]GnRH; 100 nM), indicating that the actions of GnRH are mediated via specific GnRH receptors. These data indicate that treatment of granulosa cells with FSH increases the levels of two FSH receptor mRNA transcripts. However, this positive feedback system, which may lead to an amplification of FSH action, is tightly regulated by the inhibitory actions of EGF, bFGF, and GnRH. Thus, the use of cultured rat granulosa cells provides a model system to analyze the hormonal regulation of FSH receptor gene expression in the ovary.
卵巢颗粒细胞的成熟依赖于垂体促性腺激素促卵泡激素（FSH），其作用通过特定的质膜受体介导。为了在mRNA水平研究卵巢FSH受体表达的调控，我们使用特异性cRNA探针评估培养的颗粒细胞中FSH受体转录本的变化。从未成熟的经雌激素处理的大鼠获得的颗粒细胞含有两种主要的FSH受体mRNA转录本（7.0和2.5千碱基），在2天的培养期内，其水平以时间相关的方式下降。然而，在培养基中加入FSH（30 ng/ml）可防止FSH受体mRNA水平下降。与对照组相比，用FSH（1 - 100 ng/ml）处理颗粒细胞48小时，FSH受体mRNA水平呈剂量依赖性增加（半数有效剂量，4.5 ng/ml），在30 ng/ml FSH刺激下，最大增加5.9±0.7倍。FSH的刺激作用可被腺苷酸环化酶激活剂福斯可林（0.1 - 30 μM）模拟，提示cAMP参与FSH受体基因转录和/或mRNA稳定性。用表皮生长因子（EGF；0.3 - 10 ng/ml）、碱性成纤维细胞生长因子（bFGF；1 - 30 ng/ml）或胰岛素样生长因子-I（IGF-I；1 - 30 ng/ml）孵育颗粒细胞48小时，不影响基础FSH受体mRNA水平，而最高剂量的EGF和bFGF（而非IGF-I）完全抑制FSH（30 ng/ml）对其自身受体mRNA水平的刺激作用。同样，促性腺激素释放激素（GnRH；10 - 1000 nM）以剂量依赖性方式减弱FSH对其受体mRNA水平的作用（半数抑制剂量，8 nM）。GnRH（100 nM）的抑制作用可被与GnRH拮抗剂（[Ac-D-Phe1,D-pCl-Phe2,D-Trp3,6]GnRH；100 nM）共同处理所逆转，表明GnRH的作用通过特异性GnRH受体介导。这些数据表明，用FSH处理颗粒细胞可增加两种FSH受体mRNA转录本的水平。然而，这个可能导致FSH作用放大的正反馈系统受到EGF、bFGF和GnRH抑制作用的严格调控。因此，培养的大鼠颗粒细胞的使用提供了一个模型系统，用于分析卵巢中FSH受体基因表达的激素调控。
