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

• Published in: The Journal of biological chemistry Vol. 264; no. 36; pp. 21934 - 21942
• Main Authors: OONK, R. B, KRASNOW, J. S, BEATTIE, W. G, RICHARDS, J. A. S
• Format: Journal Article
• Language: English
• Published: Bethesda, MD American Society for Biochemistry and Molecular Biology 25-12-1989
• Subjects: Amino Acid Sequence; Analytical, structural and metabolic biochemistry; Animals; Base Sequence; Biological and medical sciences; Blotting, Northern; Cattle; Cells, Cultured; Cholesterol Side-Chain Cleavage Enzyme - genetics; Cholesterol Side-Chain Cleavage Enzyme - metabolism; Chorionic Gonadotropin - pharmacology; Cloning, Molecular; Corpus Luteum - drug effects; Corpus Luteum - enzymology; Cyclic AMP - physiology; DNA - genetics; Enzymes and enzyme inhibitors; Female; Fundamental and applied biological sciences. Psychology; Gene Expression Regulation, Enzymologic; Gene Library; Granulosa Cells - drug effects; Granulosa Cells - enzymology; Humans; Kinetics; Molecular Sequence Data; Oxidoreductases; Progesterone - biosynthesis; Prolactin - pharmacology; Protein Kinases - metabolism; Rats; Restriction Mapping; RNA - genetics; RNA - isolation & purification; RNA, Messenger - biosynthesis; RNA, Messenger - genetics; Sequence Homology, Nucleic Acid; Cell culture; Rat; Enzyme; Rodentia; Cyclic AMP; Vertebrata; Regulation(control); Mammalia; Messenger RNA; Gene; Flavoprotein-linked monooxygenase; Radioimmunoassay; Immunoblotting assay; Computer aid; Comparative study; Aminoacid sequence
• ISSN: 0021-9258; 1083-351X

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.