Urban R J, Garmey J C, Shupnik M A, Veldhuis J D
Department of Internal Medicine, University of Virginia Health Sciences Center, Charlottesville 22908.
Endocrinology. 1991 Apr;128(4):2000-7. doi: 10.1210/endo-128-4-2000.
FSH is the primary hormonal inducer of ovarian follicle maturation and a critically important regulator of steroidogenesis in granulosa cells. We examined possible molecular mechanisms subserving FSH action by assessing concentrations of cytochrome P450 cholesterol side-chain cleavage (P450scc) mRNA in porcine granulosa cells maintained in serum-free culture. Cellular concentrations of specific P450scc mRNA were measured by Northern blot hybridization using a 32P-labeled 1-kilobase porcine cDNA clone. Specificity was tested by estimating the granulosa cell mRNA content of the constitutively expressed enzyme, glyceraldehyde-3-phosphate dehydrogenase. Steroidogenesis was evaluated by measuring concomitant progesterone accumulation in the culture medium. Treatment with ovine FSH (100 ng/ml) increased P450scc mRNA concentrations in a time-dependent fashion, with significant effects on both P450scc mRNA concentrations and progesterone accumulation by 4 h and a maximal increase (8- to 10-fold) at 48 h. FSH dose-response studies at 48 h revealed a significant stimulatory effect of 30 ng/ml FSH on P450scc mRNA accumulation and progesterone production, with a maximal effect at 100 ng/ml FSH. To examine the role of cAMP in mediating granulosa cell P450scc mRNA accumulation, granulosa cells were treated with forskolin, cholera toxin, 8-bromo-cAMP, 8-bromo-cGMP, 5'AMP, or cAMP analogs that differentially stimulate the two isoenzymes of protein kinase-A. Increased specific P450scc mRNA accumulation and progesterone production occurred in response to each agent except 5'AMP and 8-bromo-cGMP. No effects of these agents were observed on glyceraldehyde-3-phosphate dehydrogenase mRNA. To assess possible feedback effects of steroid or sterol on FSH-stimulated P450scc mRNA concentrations, granulosa cells were treated with aminoglutethimide to block or with low density lipoprotein to stimulate steroid production. Inhibition of sterol utilization by the cholesterol side-chain cleavage enzyme had no effect on basal or FSH-stimulated concentrations of P450scc mRNA, but markedly suppressed progesterone production. Low density lipoprotein, which increases intracellular sterol, also did not alter basal or FSH-stimulated P450scc mRNA accumulation, suggesting that neither the utilization nor the availability of sterol regulates specific P450scc mRNA levels. Estradiol alone did not increase P450scc mRNA accumulation, but did augment progesterone production. Treatment of granulosa cells with estradiol and FSH produced a synergistic increase in progesterone concentrations, but did not affect FSH-stimulated P450scc mRNA accumulation.(ABSTRACT TRUNCATED AT 400 WORDS)
促卵泡激素（FSH）是卵巢卵泡成熟的主要激素诱导剂，也是颗粒细胞中类固醇生成的关键调节因子。我们通过评估无血清培养的猪颗粒细胞中细胞色素P450胆固醇侧链裂解酶（P450scc）mRNA的浓度，研究了FSH作用的可能分子机制。使用32P标记的1千碱基猪cDNA克隆，通过Northern印迹杂交法测量特定P450scc mRNA的细胞浓度。通过估计组成型表达酶甘油醛-3-磷酸脱氢酶的颗粒细胞mRNA含量来测试特异性。通过测量培养基中同时积累的孕酮来评估类固醇生成。用羊FSH（100 ng/ml）处理以时间依赖性方式增加P450scc mRNA浓度，在4小时时对P450scc mRNA浓度和孕酮积累均有显著影响，在48小时时最大增加（8至10倍）。48小时的FSH剂量反应研究表明，30 ng/ml FSH对P450scc mRNA积累和孕酮产生有显著刺激作用，在100 ng/ml FSH时达到最大效应。为了研究cAMP在介导颗粒细胞P450scc mRNA积累中的作用，用福司可林、霍乱毒素、8-溴-cAMP、8-溴-cGMP、5'AMP或差异刺激蛋白激酶A两种同工酶的cAMP类似物处理颗粒细胞。除了'AMP和8-溴-cGMP外，每种试剂处理后均出现特异性P450scc mRNA积累和孕酮产生增加。未观察到这些试剂对甘油醛-3-磷酸脱氢酶mRNA有影响。为了评估类固醇或甾醇对FSH刺激的P450scc mRNA浓度的可能反馈作用，用氨鲁米特阻断或用低密度脂蛋白刺激类固醇产生来处理颗粒细胞。胆固醇侧链裂解酶对甾醇利用的抑制对P450scc mRNA的基础浓度或FSH刺激浓度没有影响，但显著抑制了孕酮产生。增加细胞内甾醇的低密度脂蛋白也未改变基础或FSH刺激的P450scc mRNA积累，表明甾醇的利用和可用性均不调节特定P450scc mRNA水平。单独的雌二醇不会增加P450scc mRNA积累，但会增加孕酮产生。用雌二醇和FSH处理颗粒细胞会使孕酮浓度协同增加，但不影响FSH刺激的P450scc mRNA积累。（摘要截短至400字）
