The Rat Cholesterol Side-Chain Cleavage Cytochrome P450 (P450scc) Gene: cAMP-Dependent and -Independent Regulation in Ovarian Cells

Summary

Using a 5′ human P450scc cDNA probe and a human 57-mer oligonucleotide probe, 2 rat P450scc cDNA clones were identified and isolated from combined granulosa cell and luteal cell λgt11 cDNA expression libraries. One 350 bp clone contained the start codon. The other 750 bp clone overlapped on the 5′ end with the 350 bp clone and on the 3’ end with a previously isolated 1.2 kb cDNA clone (1), which contains a poly (A) tail, suggesting that the complete cDNA sequence for rat P450scc is encoded in these 3 cDNA clones. Using the same 5′ human P450scc probes, we have isolated a rat genomic P450scc clone from a rat liver genomic library. The genomic clone contained 3 distinct Bam HI fragments, 3.7, 4.0 and approximately 14 kb in size. The 3.7 and 4.0 kb Bam HI fragments hybridized to the 1.2 kb cDNA and appear to contain 3′ located exons. After subcloning, the 14 kb fragment yielded several transformants with inserts ranging from 8 to 0.5 kb in size. Hybridization with the rat cDNA clones revealed that the 8 kb fragment may represent 5′ upstream or intron sequences. A 6 kb fragment contained the first and probably the next 2 exons. Northern blot hybridization with the analysis, using the 750 bp cDNA probe, demonstrated that P450scc mRNA was low in granulosa cells of preovulatory (PO) follicles but could be induced by an ovulatory dose of LH/hCG in vivo or by forskolin added in vitro. Once induced by LH/hCG in vivo, P450scc mRNA and progesterone biosynthesis were constitutively maintained by luteinized cells in vivo or in vitro. Thus, the induction of P450scc mRNA in granulosa cells from developing follicles is hormone and cAMP dependent. In contrast, the expression of P450scc mRNA in luteinized granulosa cells has become cAMP independent.