Aromatase cytochrome P450 in rat ovarian granulosa cells before and after luteinization: adenosine 3',5'-monophosphate-dependent and independent regulation. Cloning and sequencing of rat aromatase cDNA and 5' genomic DNA

• Published in: Molecular endocrinology (Baltimore, Md.) Vol. 4; no. 1; pp. 3 - 12
• Main Authors: HICKEY, G. J, KRASNOW, J. S, BEATTIE, W. G, RICHARDS, J. A
• Format: Journal Article
• Language: English
• Published: Bethesda, MD Endocrine Society 1990
• Subjects: Amino Acid Sequence; Animals; Aromatase - biosynthesis; Aromatase - genetics; Base Sequence; Biological and medical sciences; Cloning, Molecular; Cyclic AMP - pharmacology; DNA - genetics; Female; Fundamental and applied biological sciences. Psychology; Genes. Genome; Gonadotropins, Pituitary - pharmacology; Granulosa Cells - drug effects; Granulosa Cells - enzymology; Granulosa Cells - metabolism; Humans; Molecular and cellular biology; Molecular genetics; Molecular Sequence Data; Promoter Regions, Genetic; Rats; Rats, Inbred Strains; Restriction Mapping; Rat; Nucleotide sequence; Enzyme; Transcription promoter; Rodentia; Cyclic AMP; Homology; Gene expression; Northern blotting; Vertebrata; Regulation(control); Mammalia; Messenger RNA; DNA; AMP-3',5'; Oestrogen synthase; Granulosa; Molecular cloning; Ovarian follicle; Aminoacid sequence
• ISSN: 0888-8809; 1944-9917
• DOI: 10.1210/mend-4-1-3

The complete nucleotide sequence for rat ovarian aromatase cytochrome P450 (P450arom) has been derived from four cDNA clones isolated from three rat granulosa/luteal cell lambda gt11 cDNA expression libraries. The composite P450arom cDNA extends 1597 basepairs, encodes a protein of 508 amino acids (calculated mol wt = 58,263), and hybridizes to three mRNA transcripts (3.3, 2.6, and 1.9 kilobases in size) in rat ovarian tissues. A 5' genomic fragment was isolated from a rat genomic library and shown to contain exon I and 538 basepairs of 5' flanking sequences, including putative promoter elements. Further, we document that P450arom mRNA and estradiol (E) biosynthesis are regulated by cAMP-dependent mechanisms in granulosa cells of preovulatory (PO) follicles, but are maintained by cAMP-independent mechanisms after LH/hCG-induced luteinization. The transition of the PO granulosa cell to the luteal cell (PO + hCG) phenotype requires 5 h of exposure to hCG in vivo. Once the luteal cell phenotype is programed, P450arom mRNA and E biosynthesis are maintained in the luteinized cells for up to 10 days in a constitutive manner in the absence of hormones or agents that increase intracellular cAMP. Furthermore, when PO + hCG (7 h) follicles were isolated and incubated for 1-3 h with reversible inhibitors of transcription (actinomycin-D) or translation (cycloheximide) before harvesting the granulosa cells, neither morphological nor functional luteinization of granulosa cells in culture was impaired. Thus, rapid cellular and molecular events occur in granulosa cells within 5-7 h after an ovulatory LH/hCG surge that alter the hormonal regulation of the aromatase gene.