Equine P450 cholesterol side-chain cleavage and 3 beta-hydroxysteroid dehydrogenase/Delta(5)-Delta(4) isomerase: Molecular cloning and regulation of their messenger ribonucleic acids in equine follicles during the ovulatory process

The preovulatory LH rise is the physiological trigger of follicular luteini zation, a process during which the synthesis of progesterone is markedly in creased. To study the control of follicular progesterone biosynthesis in ma res, the objectives of this study were to clone and characterize the equine cholesterol side-chain cleavage cytochrome P450 (P450(scc)) and 3 beta -hy droxysteroid dehydrogenase/Delta (5)-Delta (4)-isomerase (3 beta -HSD), and describe the regulation and cellular localization of their transcripts in equine follicles during hCG-induced ovulation. Complementary DNA cloning an d primer extension analyses revealed that the equine P450(scc) transcript i s composed of a 5'-untranslated region (UTR) of 52 nucleotides, an open rea ding frame (ORF) of 1560 nucleotides, and a 3'-UTR of 225 nucleotides, wher eas the equine 3 beta -HSD mRNA consists of a 5'-UTR of 61 nucleotides, an ORF of 1119 nucleotides, and a 3'-UTR of 432 nucleotides. The equine P450(s cc) and 3 beta -HSD ORF encode 520 and 373 amino acid proteins, respectivel y, that are highly conserved (68-79% identity) when compared to homologs of other mammalian species. Northern blot analyses were performed with preovu latory follicles isolated 0, 12, 24, 30, 33, 36, and 39 h post-hCG, and cor pora lutea obtained on day 8 of the cycle. Results showed that levels of P4 50(scc) mRNA in follicular wall (theca interna with attached granulosa cell s) decreased after hCG treatment (30-39 h versus 0 h post-hCG, P < 0.05), a nd increased again after ovulation to reach their highest levels in corpora lutea (P < 0.05). Northern blots on isolated cellular preparations reveale d that theca interna was the predominant site of P450(scc) expression in fo llicles prior to hCG (P < 0.05). However, transcript levels decreased in th eca interna between 30-39 h (P < 0.05) and increased in granulosa cells at 39 h (P < 0.05), making the granulosa cell layer the predominant site of P4 50(scc) expression at the end of the ovulatory process. A different pattern of regulation was observed for 3<beta>-HSD, as transcript levels remained constant throughout the luteinization process (P > 0.05). Also, in contrast to other species, expression of 3 beta -HSD mRNA in equine preovulatory fo llicles was localized only in granulosa cells and not in theca interna. Thu s, this study characterizes for the first time the complete structure of eq uine P450(scc) and 3 beta -HSD mRNA and identifies novel patterns of expres sion and regulation of these transcripts in equine follicles prior to ovula tion.