Abstract
Production and secretion of steroid hormones throughout the ovarian cycle occurs in a highly episodic and coordinated fashion that requires precise and finely tuned regulatory mechanisms. The regulation of ovarian steroidogenesis by the gonadotropin follicle stimulating hormone (FSH) and luteinizing hormone (LH) as well as by other factors occurs, at least in part, at the level of expression of the genes encoding steroidogenic enzymes. The present study is aimed at the elucidation of regulatory mechansims by which cyclic adenosine monophosphate (cAMP) and protein kinase C regulate cytochrome P450scc ( CYP11A) gene expression in bovine granulosa cells in primary culture. As a first step we characterized the bovine granulosa cell cultures with regard to regulation of P450scc activity and mRNA levels upon treatment with forskolin and/or the phorbol ester TPA. Forskolin, a potent stimulator of cAMP generation, increased both progesterone secretion and P450scc mRNA levels. In contrast, treatment with TPA alone decreased both basal progesterone production and P450scc mRNA accumulation. Co-treatment with forskolin and TPA decreased progesterone and P450scc mRNA levels as compared to forskolin treatment alone. The possibility that TPA interfered with the forskolin-stimulated cAMP production could be excluded because simultaneous treatment of granulosa cells with TPA and forskolin potentiated the formation of cAMP. In order to identify regulatory sequences within the 5′ flanking region of the bovine CYP11A gene, chimeric DNA constructs comprizing regions of the CYP11A gene fused to a β-globin-derived reporter gene were transfected into granulosa cells in primary culture. The expression of reporter gene constructs containing −896 / − 32, −186/−32, −118/−83 and −118/100 bp of the 5′ upstream region of the CYP11A gene was markedly stimulated upon forskolin treatment. Expression in the absence of forskolin was essentially undetectable. Addition of TPA alone did not change the expression of the reporter gene constructs as compared to control, however, co-treatment with forskolin and TPA significantly decreased the stimulation observed with forskolin treatment alone. Examination of the −118/− 100 bp sequence revealed two regions exhibiting similarity to the consensus binding sites for AP1 and Sp1 transcription factors. It is likely therefore that differential regulation of bovine CYP11A by forskolin and phorbol esters is mediated by one or both of these sequences.
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