Maitra A, LaVoie H A, Day R N, Garmey J C, Veldhuis J D
Department of Internal Medicine, University of Virginia Health Science Center, Charlottesville 22908, USA.
Endocrinology. 1995 Nov;136(11):5111-7. doi: 10.1210/endo.136.11.7588248.
In species such as the pig and human, gonadal steroidogenesis is believed to be dependent upon the availability of low density lipoprotein (LDL) cholesterol. However, before ovulation, Graafian follicles are impermeant to lipoproteins in the LDL class. Thus, de novo cholesterol biosynthesis via the rate-determining enzyme 3-hydroxy-3-methylglutaryl coenzyme A (HMG-CoA) reductase is likely to provide a significant mechanism for generating sterol substrate for steroidogenesis by granulosa cells before follicular rupture. As serum-free monolayer culture of (swine) granulosa cells offers an in vitro model of hormonally responsive HMG-CoA reductase, we generated a (porcine) complementary DNA and homologous complementary RNA to investigate by sensitive and specific ribonuclease protection assay the hormonal regulation of HMG-CoA reductase gene expression in ovarian cells from immature Graafian follicles. Using reverse transcriptase-polymerase chain reaction, we cloned and sequenced a 238-base pair complementary DNA from porcine luteal tissue that encodes the catalytic region of HMG-CoA reductase. GenBank analysis of the DNA sequence homology between the pig and other species showed the greatest concordance with human (88%) and hamster (90%). Solution hybridization/ribonuclease protection analysis of total RNA isolated from serum-free monolayer cultures of porcine granulosa cells revealed that insulin (3 micrograms/ml) increased HMG-CoA messenger RNA (mRNA) concentrations corrected for constitutive 18S ribosomal RNA expression in a time-dependent fashion, with significant effects observed at 12 h and a 6-fold increase by 48 h. Recombinant human insulin-like growth factor I (IGF-I) peptide was able to mimic the action of insulin alone. Neither FSH (100 ng/ml) nor 8-bromo-cAMP (1 mM) had observable effects on HMG-CoA message accumulation at any time point studied. However, the combined action of either FSH and insulin or 8-bromo-cAMP and insulin resulted in synergistic increases in reductase mRNA by 31- and 17-fold, respectively. To assess the possible feedback effects of sterol on HMG-CoA gene expression, granulosa cells were treated with LDL. At physiological concentrations, LDL suppressed basal expression of HMG-CoA mRNA to levels below the control value. In addition, LDL inhibited insulin-stimulated HMG-CoA mRNA accumulation by 84% as well as the synergistic effects of insulin and FSH (by 94%) and of insulin and 8-bromo-cAMP (by 93%). We conclude that insulin alone or in combination with FSH or cAMP augments the accumulation of HMG-CoA reductase mRNA in ovarian (granulosa) cells.(ABSTRACT TRUNCATED AT 400 WORDS)
在猪和人类等物种中，性腺类固醇生成被认为依赖于低密度脂蛋白（LDL）胆固醇的可用性。然而，在排卵前，格拉夫卵泡对LDL类脂蛋白是不可渗透的。因此，通过限速酶3-羟基-3-甲基戊二酰辅酶A（HMG-CoA）还原酶进行的从头胆固醇生物合成可能是卵泡破裂前颗粒细胞生成类固醇生成所需固醇底物的重要机制。由于（猪）颗粒细胞的无血清单层培养提供了一种对激素有反应的HMG-CoA还原酶的体外模型，我们生成了一个（猪）互补DNA和同源互补RNA，通过灵敏且特异的核糖核酸酶保护试验来研究未成熟格拉夫卵泡卵巢细胞中HMG-CoA还原酶基因表达的激素调节。使用逆转录聚合酶链反应，我们从猪黄体组织中克隆并测序了一个238碱基对的互补DNA，其编码HMG-CoA还原酶的催化区域。对猪与其他物种之间DNA序列同源性的GenBank分析表明，与人类（88%）和仓鼠（90%）的一致性最高。对从猪颗粒细胞无血清单层培养物中分离的总RNA进行溶液杂交/核糖核酸酶保护分析显示，胰岛素（3微克/毫升）以时间依赖性方式增加了经组成型18S核糖体RNA表达校正后的HMG-CoA信使RNA（mRNA）浓度，在12小时时观察到显著影响，到48小时时增加了6倍。重组人胰岛素样生长因子I（IGF-I）肽能够模拟胰岛素单独的作用。在任何研究的时间点，促卵泡激素（FSH，100纳克/毫升）和8-溴环磷酸腺苷（8-bromo-cAMP，1毫摩尔）对HMG-CoA信息积累均无明显影响。然而，FSH与胰岛素或8-溴环磷酸腺苷与胰岛素的联合作用分别使还原酶mRNA协同增加了31倍和17倍。为了评估固醇对HMG-CoA基因表达的可能反馈作用，用LDL处理颗粒细胞。在生理浓度下，LDL将HMG-CoA mRNA的基础表达抑制到低于对照值的水平。此外，LDL抑制胰岛素刺激的HMG-CoA mRNA积累达84%，以及胰岛素与FSH的协同作用（94%）和胰岛素与8-溴环磷酸腺苷的协同作用（93%）。我们得出结论，单独的胰岛素或与FSH或cAMP联合使用可增强卵巢（颗粒）细胞中HMG-CoA还原酶mRNA的积累。（摘要截断于400字）
