Gonadotropin regulation of testosterone production by primary cultured theca and granulosa cells of Atlantic croaker: I. Novel role of CaMKs and interactions between calcium- and adenylyl cyclase-dependent pathways

Theca and granulosa cells for in vitro primary culture were obtained by enzymatic digestion of mature ovarian tissue from Atlantic croaker ( Micropogonias undulatus ) and separation from the other cell types by Percoll density-gradient centrifugation. Histochemical staining and treatment with pregnenolone confirmed the presence in the cultured cells of enzymes involved in synthesizing the major sex steroids in croaker ovaries: testosterone, estradiol, and 17α,20β,21-trihydroxy-4-pregnen-3-one (20β-S). Croaker theca and granulosa cells maintained their steroidogenic response to gonadotropin when cultured with serum-supplemented media and produced high levels of testosterone for up to 5 days, although estradiol production was low. Multiple signal transduction pathways mediating gonadotropin stimulation of androgen production were identified in Atlantic croaker ovarian theca and granulosa cells in primary co-culture. Inhibitors of voltage-sensitive calcium channels (VSCCs) and calmodulin decreased the steroidogenic response to gonadotropin, whereas activators of adenylyl cyclase and protein kinase A (PKA) increased testosterone production, indicating that both calcium and PKA-dependent signaling pathways are involved in the regulation of follicular steroid production. In addition, the first evidence in vertebrates for an involvement of calcium/calmodulin-dependent protein kinases (CaMKs) in gonadal steroidogenesis was obtained, since the stimulatory effects of gonadotropin on testosterone media accumulation were attenuated by specific inhibitors of CaMKs. Some interactions among the signaling pathways were observed as demonstrated by the positive effect of elevated intracellular calcium on adenylyl cyclase activity and the reduction of forskolin- and dbcAMP-induced testosterone production by inhibitors of VSCCs, calmodulin, and CaMKs.