Hedin L, Rodgers R J, Simpson E R, Richards J S
Department of Cell Biology, Baylor College of Medicine, Houston, Texas 77030.
Biol Reprod. 1987 Aug;37(1):211-23. doi: 10.1095/biolreprod37.1.211.
The following study was undertaken to determine which hormones (luteinizing hormone, LH, and prolactin, PRL) and enzymes (cytochrome P450(17)alpha, nicotinamide adenine dinucleotide phosphate [NADPH]-cytochrome P450 reductase, 3-hydroxy-3-methylglutaryl [HMG] CoA reductase, cholesterol side-chain cleavage cytochrome P450 [P450scc], and adrenodoxin) were associated with the regulation of androgen biosynthesis by developing rat follicles and corpora lutea in vivo as well as by thecal explants maintained in culture. Immunoblots of soluble cell extracts of small antral (SA), preovulatory (PO), and luteinizing (PO + human chorionic gonadotropin [hCG], 7 h) follicles, newly formed corpora lutea (PO + hCG, 24 h), and corpora luteal isolated on Day 15 of pregnancy, demonstrated that cytochrome P450(17)alpha was low in SA follicles, selectively increased 4-fold in PO follicles, and decreased to less than 10% within 7 h after hCG. Filter hybridization assays using a 32P-labeled cytochrome P450(17)alpha cDNA probe demonstrated that changes in the content of P450(17)alpha mRNA exhibited a pattern similar to that of the enzyme. Conversely, immunoblots for other microsomal enzymes either exhibited no change (NADPH cytochrome P450 reductase) or a transient increase after the hCG surge (HMG CoA reductase), whereas the mitochondrial enzymes either increased markedly in association with luteinization (cytochrome P450scc) or were increased in a more transient manner (adrenodoxin). The LH-induced loss of cytochrome P450(17)alpha in vivo was not associated with loss of androgen biosynthesis when luteinizing theca were placed in culture in medium containing either LH or LH and PRL, suggesting that other hormones, or the presence of other cell types, are required to maintain the decrease in cytochrome P450(17)alpha in vivo. Conversely, the LH-induced increase in cytochrome P450scc in vivo was associated with the maintenance of elevated progesterone production by theca in culture, suggesting that cytochrome P450scc may be constitutively expressed in luteinized theca. Thus, thecal cell cytochrome P450(17)alpha and the regulation of its content and mRNA by LH are pivotal to the biosynthesis of androgens, the obligatory precursors for estradiol biosynthesis and the consequent development of preovulatory follicles. The molecular basis for the different effects of low versus elevated concentrations of LH on cytochrome P450(17)alpha, as well as cytochrome P450scc, remain to be determined.
开展以下研究以确定哪些激素（促黄体生成素、LH，和催乳素、PRL）及酶（细胞色素P450(17)α、烟酰胺腺嘌呤二核苷酸磷酸[NADPH]-细胞色素P450还原酶、3-羟基-3-甲基戊二酰[HMG]辅酶A还原酶、胆固醇侧链裂解细胞色素P450[P450scc]，和肾上腺皮质铁氧化还原蛋白）与体内发育中的大鼠卵泡和黄体以及培养中的卵泡膜外植体雄激素生物合成的调节相关。对小腔卵泡（SA）、排卵前卵泡（PO）和促黄体化卵泡（PO + 人绒毛膜促性腺激素[hCG]，7小时）、新形成的黄体（PO + hCG，24小时）及妊娠第15天分离的黄体的可溶性细胞提取物进行免疫印迹分析，结果表明细胞色素P450(17)α在SA卵泡中含量较低，在PO卵泡中选择性增加4倍，并在hCG注射后7小时内降至不足10%。使用32P标记的细胞色素P450(17)α cDNA探针进行的滤膜杂交分析表明，P450(17)α mRNA含量的变化呈现出与该酶相似的模式。相反，其他微粒体酶的免疫印迹分析结果显示，要么没有变化（NADPH细胞色素P450还原酶），要么在hCG激增后短暂增加（HMG辅酶A还原酶），而线粒体酶要么在黄体化过程中显著增加（细胞色素P450scc），要么以更短暂的方式增加（肾上腺皮质铁氧化还原蛋白）。当将促黄体化的卵泡膜置于含有LH或LH与PRL的培养基中培养时，体内LH诱导的细胞色素P450(17)α缺失与雄激素生物合成的丧失无关，这表明在体内维持细胞色素P450(17)α的减少需要其他激素或其他细胞类型的存在。相反，体内LH诱导的细胞色素P450scc增加与培养中的卵泡膜孕酮产量升高的维持相关，这表明细胞色素P450scc可能在黄体化的卵泡膜中组成性表达。因此，卵泡膜细胞色素P450(17)α及其含量和mRNA受LH调节对于雄激素的生物合成至关重要，雄激素是雌二醇生物合成的必需前体，也是排卵前卵泡随后发育所必需的。低浓度与高浓度LH对细胞色素P450(17)α以及细胞色素P450scc产生不同影响的分子基础仍有待确定。
