DIFFERENTIAL REGULATION OF CHOLESTEROL SIDE-CHAIN CLEAVAGE (P450SCC) AND AROMATASE (P450AROM) ENZYME MESSENGER-RNA EXPRESSION BY GONADOTROPINS AND CYCLIC-AMP IN HUMAN GRANULOSA-CELLS

The co-ordinated biosynthesis of progesterone and oestradiol in the hu man ovary is critical for reproductive cyclicity and eventual pregnanc y. The crucial regulatory enzymes for progesterone and oestradiol bios ynthesis in granulosa cells are the cholesterol side-chain cleavage (P 450scc) and aromatase (P450arom) enzymes respectively. We utilized the cDNA sequences encoding P450arom and P450scc to examine the roles of FSH and LH, and their intracellular second messenger, cyclic AMP (cAMP ), in regulating steroidogenic gene expression. Mature granulosa cells (aspirated before the onset of the endogenous LW surge) and granulosa lutein cells (obtained after an ovulatory dose of human chorionic gon adotrophin) were cultured for 4 days with FSH, LH or dibutyryl cAMP (d bcAMP). After the period of culture, total RNA was extracted from gran ulosa cells and Northern analyses were performed utilizing P-32-labell ed cDNAs encoding P450arom and P450scc. Spent culture media were analy sed for steroid and cAMP content. Both FSH and LH strongly stimulated P450arom mRNA expression and oestradiol production in mature granulosa cells. On the other hand, P450scc mRNA expression and progesterone bi osynthesis were weakly induced by FSH; maximal synthesis occurred only in the presence of LH. With both gonadotrophins at equivalent concent rations, LH generated a 30-fold higher level of cAMP than FSH. Further more, the differential effects of FSH and LH on P450 mRNA expression w ere reproduced by the presence of low and high concentrations of dbcAM P respectively. LH (and high levels of dbcAMP) increased P450arom mRNA expression in mature granulosa cells but inhibited its accumulation i n granulosa lutein cells. In contrast, it stimulated P450scc mRNA expr ession and progesterone synthesis in both mature granulosa and granulo sa lutein cells. Therefore, FSH/low cAMP levels stimulated P450arom ge ne expression and oestradiol production, while LH/high cAMP levels max imally induced P450scc gene expression and function, in a development- related manner consistent with steroid production in vivo. These findi ngs support the hypothesis that one set of genes (like P450arom) in hu man granulosa cells is regulated by FSH/low cAMP levels and another (l ike P450scc) by LH/high cAMP levels.