Actions of Cyclic Adenosine Monophosphate on the Cytodifferentiation of Ovarian Cells: Studies in Cultured Swine Granulosa Cells Using a Novel Exogenous Adenylate Cyclase from Bordetella Pertussis

The regulation by cAMP of cholesterol side-chain cleavage activity and the synthesis of immunoiso-lated cytochrome P-450_scc and adrenodoxin proteins was investigated in primary cultures of swine ovarian (granulosa) cells. Administration of a novel adenylate cyclase toxin isolated from Bordetella pertussis increased granulosa-cell cAMP accumulation up to 200-fold over basal. These effects were additive with those of FSH, forskolin, and cholera toxin. In contrast, bacterial extracts BP 347 and BP 348 from mutant strains of B. pertussis that lack either all virulent factors or the adenylate cyclase toxin and hemolysin were devoid of effect. Granulosa-cell cAMP accumulation supported by active bacterial adenylate cyclase was accompanied by 2- to 11fold, time-dependent increases in [³⁵S]methionine incorporation into immunospecific cytochrome P-450_scc and adrenodoxin. These increases in the synthesis of cholesterol side-chain cleavage proteins were associated with enhanced pregnenolone production in response to exogenous sterol substrate, 25-hydroxycholesterol, and augmented progesterone secretion both in the absence and presence of exogenous lipoprotein. Moreover, the effects of Bordetella adenylate cyclase toxin on granulosa cell steroidogenesis were functionally integrated with other regulatory responses, since the non-cAMP dependent effector, estradiol 17β, interacted syner-gistically with bacterial adenylate cyclase in stimulating progesterone production. We conclude that exogenous adenylate cyclase isolated from B. pertussis can be functionally integrated into the cAMP-dependent effector pathway of granulosa cells with a resulting increase in intracellular cAMP concentrations, augmented biosynthesis of progesterone and pregnenolone, enhanced synthesis of immunospecific cytochrome P-450_scc and adrenodoxin, and synergistic interactions with a non-cAMP-dependent ovarian effector hormone (estradiol). These observations provide new evidence of a crucial role for cAMP in the differentiation of granulosa cell steroidogenesis.