title: Cyclic AMP-dependent and -independent regulation of cholesterol side chain cleavage cytochrome P-450 (P-450scc) in rat ovarian granulosa cells and corpora lutea. cDNA and deduced amino acid sequence of rat P-450scc.
abstract: We report the isolating and sequencing of three cDNA clones encoding rat P-450scc, the nucleotide and protein sequences of which are highly homologous to those of bovine and human P-450scc, especially in the putative heme and steroid binding domains. We document that different molecular mechanisms regulate P-450scc in granulosa cells of preovulatory (PO) follicles prior to and after luteinization. Luteinizing hormone/human chorionic gonadotropin (LH/hCG) and cAMP are obligatory to induce P-450scc mRNA in PO granulosa cells in vivo and in vitro. Once P-450scc mRNA is induced as a consequence of the LH/hCG surge it is constitutively maintained by luteinized cells in vivo (0-4 days) and in vitro (0-9 days) in the absence of gonadotropins, is susceptible to modulation by prolactin and is no longer regulated by cAMP. Exposure to elevated concentrations of hCG in vivo for 5-7 h was required for PO granulosa cells to undergo a functional transition establishing the stable luteal cell phenotype. Transient exposure of PO + hCG (7 h) follicles in vitro to the RNA synthesis inhibitor actinomycin D (1 microgram/ml) or the protein synthesis inhibitor cycloheximide (10 micrograms/ml), for 1-5 h prior to culturing the granulosa cells failed to disrupt the induction of P-450scc mRNA, progesterone biosynthesis, and appearance of the luteal cell morphology. Inhibitors of protein kinase A (Rp-cAMPS; 1-500 microM and N-[2-(methylamino)ethyl]-5-isoquinolinesulfonamide dihydrochloride (H8); 1-200 microM) added directly to the luteinized cell cultures also failed to alter P-450scc mRNA in these cells, although the cells contain in vivo amounts of mRNA for RII beta, RI alpha, and C alpha, the primary subunits of protein kinase A found in the rat ovary. These data suggest that expression of the P-450scc gene in rat ovarian follicular cells is regulated in a sequential manner by cAMP-dependent and cAMP-independent mechanisms associated with granulosa cells and luteal cells, respectively.
authors: Department of Cell Biology, Baylor College of Medicine, Houston, Texas 77030.
authors: R B
authors: RB
authors: Oonk
authors: J S
authors: JS
authors: Krasnow
authors: W G
authors: WG
authors: Beattie
authors: J S
authors: JS
authors: Richards
mesh_terms: D000595:Amino Acid Sequence; D000818:Animals; D001483:Base Sequence; D015152:Blotting, Northern; D002417:Cattle; D002478:Cells, Cultured; D002786:Cholesterol Side-Chain Cleavage Enzyme / Q000235:genetics* / Q000378:metabolism; D006063:Chorionic Gonadotropin / Q000494:pharmacology; D003001:Cloning, Molecular; D003338:Corpus Luteum / Q000187:drug effects / Q000201:enzymology*; D000242:Cyclic AMP / Q000502:physiology*; D004247:DNA / Q000235:genetics*; D005260:Female; D015971:Gene Expression Regulation, Enzymologic; D015723:Gene Library; D006107:Granulosa Cells / Q000187:drug effects / Q000201:enzymology*; D006801:Humans; D007700:Kinetics; D008969:Molecular Sequence Data; D011374:Progesterone / Q000096:biosynthesis; D011388:Prolactin / Q000494:pharmacology; D011494:Protein Kinases / Q000378:metabolism; D012313:RNA / Q000235:genetics / Q000302:isolation & purification; D012333:RNA, Messenger / Q000096:biosynthesis / Q000235:genetics; D051381:Rats; D015183:Restriction Mapping; D012689:Sequence Homology, Nucleic Acid
chemical_list: D006063:Chorionic Gonadotropin; D012333:RNA, Messenger; D011374:Progesterone; D012313:RNA; D011388:Prolactin; D004247:DNA; D000242:Cyclic AMP; D002786:Cholesterol Side-Chain Cleavage Enzyme; D011494:Protein Kinases