Voutilainen R, Tapanainen J, Chung B C, Matteson K J, Miller W L
J Clin Endocrinol Metab. 1986 Jul;63(1):202-7. doi: 10.1210/jcem-63-1-202.
Conversion of cholesterol to pregnenolone in man is mediated by a single enzyme, P450scc. To study possible regulation of the single P450scc gene in ovarian steroid synthesis, we incubated human granulosa cells with potential hormonal stimulators, measured P450scc mRNA accumulation by hybridization to 32P-labeled human P450scc cDNA, and compared the results to secretion of progesterone into the culture medium. Primary cultures of human granulosa cells were optimally responsive after 8-14 days of culture. Incubation with hCG (1.0-100 ng/ml), FSH (1.0-50 ng/ml), and (Bu)2cAMP (0.02-2.0 mM) increased P450scc mRNA accumulation and progesterone secretion in dose-dependent fashions. Maximal stimulation increased P450scc mRNA accumulation and progesterone secretion to 490% and 240% of control values, respectively, with hCG, to 166% and 168% with FSH, and to 495% and 380% with (Bu)2cAMP. PRL (to 100 ng/ml), ACTH (10(-6) M), and butyric acid (2 mM) had no significant effect on progesterone secretion or P450scc mRNA accumulation. These data indicate gonadotropin-specific stimulation of cAMP-mediated regulation of P450scc mRNA accumulation in human granulosa cells, presumably mediated by increased P450scc gene transcription. Ovarian estrogen synthesis may require both thecal and granulosa cells, although this two-cell theory of estrogen synthesis is unproven in man. To examine this theory, we probed the same blots used in the experiments described above with 32P-labeled human P450c17 cDNA (P450c17 is the single enzyme mediating both 17 alpha-hydroxylase and 17,20-lyase activities). Only miniscule amounts of P450c17 mRNA were found in the human granulosa cells, and the amounts did not increase in response to any of the above stimuli. These data strongly support the two-cell theory of human ovarian estrogen synthesis.
人体内胆固醇向孕烯醇酮的转化由单一酶——细胞色素P450侧链裂解酶（P450scc）介导。为研究卵巢甾体激素合成过程中单一的P450scc基因可能的调控机制，我们将人颗粒细胞与潜在的激素刺激物一起孵育，通过与32P标记的人P450scc cDNA杂交来测定P450scc mRNA的积累，并将结果与培养基中孕酮的分泌情况进行比较。人颗粒细胞的原代培养物在培养8 - 14天后反应最为理想。用hCG（1.0 - 100 ng/ml）、FSH（1.0 - 50 ng/ml）和双丁酰环磷腺苷（(Bu)2cAMP，0.02 - 2.0 mM）孵育，可使P450scc mRNA的积累和孕酮的分泌呈剂量依赖性增加。最大刺激时，hCG使P450scc mRNA的积累和孕酮的分泌分别增加至对照值的490%和240%，FSH使其增加至166%和168%，(Bu)2cAMP使其增加至495%和380%。催乳素（至100 ng/ml）、促肾上腺皮质激素（ACTH，10(-6) M）和丁酸（2 mM）对孕酮分泌或P450scc mRNA的积累无显著影响。这些数据表明，促性腺激素特异性刺激人颗粒细胞中cAMP介导的P450scc mRNA积累的调控，推测是由P450scc基因转录增加介导的。卵巢雌激素的合成可能需要卵泡膜细胞和颗粒细胞两者参与，尽管雌激素合成的这种双细胞理论在人体中尚未得到证实。为验证这一理论，我们用32P标记的人P450c17 cDNA（P450c17是介导17α-羟化酶和17,20-裂解酶活性的单一酶）探测上述实验中使用的相同印迹。在人颗粒细胞中仅发现极少量的P450c17 mRNA，并且其含量在上述任何刺激下均未增加。这些数据有力地支持了人类卵巢雌激素合成的双细胞理论。
