Hormonal Regulation of P450scc (20,22-desmolase) and P450cl7 (17α-hydroxylase/17,20-lyase) in Cultured Human Granulosa Cells*

Abstract

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.

Human cholesterol side-chain cleavage enzyme, P450scc: cDNA cloning, assignment of the gene to chromosome 15, and expression in the placenta.

“…Placental RNA was prepared by guanidine thiocyanate/LiCl extraction (23), and RNA from primary cultures was prepared as described (16). One 63-mer and three 72-mers corresponding to various regions of the bovine P450scc cDNA sequence (11) were produced using a noncommercial synthesizer (24); the sequences and hybridization characteristics of these oligonucleotides have been described (25).…”

“…P450scc mRNA accumulation is regulated hormonally (13)(14)(15)(16) and in development (17), presumably due to increased transcription of the single P450scc gene (18). Similarly, gonadotropins and cyclic AMP stimulate P450scc mRNA accumulation in cultured human ovarian granulosa cells (16).…”

“…Similarly, gonadotropins and cyclic AMP stimulate P450scc mRNA accumulation in cultured human ovarian granulosa cells (16). Pregnenolone is also produced from cholesterol in the placenta (19,20), but the nature of the enzyme(s) mediating placental progesterone synthesis is unclear (21).…”

Deletion of the Mouse P450c17 Gene Causes Early Embryonic Lethality

“…The conversion of pregnenolone and progesterone to their 17␣-hydroxylated products and then to either dehydroepiandrosterone (DHEA) or androstenedione, respectively, is mediated by a single microsomal enzyme, P450c17 (33,34,48), encoded by a single gene (22,39). The pattern of P450c17 expression in steroidogenic tissues is species specific: it is expressed in the human adrenal and gonad but not placenta (9,11,15,44), and it is expressed in the rodent gonad and placenta but not adrenal (19,23,25).…”

Follicle stimulating hormone effects on immature human oocytes: In vitro maturation and hormone production

“…The higher production of P by the oocyte-cumulus complex cultured in FSH was probably achieved by an FSH increase in cytochrome P-450 scc (25). That there was no increase in Ez when the medium without the oocytecumulus complex was compared to that with oocytecumulus complex may be due to the limited number of cells available to aromatize androgens to estrogens, the amount of FSH added to the control medium, or to the fact that the decreased aromatase activity observed in the cumulus cells of other species compared to membrana granulosa ceils may hold true in the human as well (26).…”