Signal transduction pathways regulating steroidogenesis in the ovary of Atlantic croaker (Micropogonias undulatus)
Abstract
The overall aim of this research was to study signal transduction pathways regulating ovarian steroid production in a teleost model, Atlantic croaker (Micropogonias undulatus). The potential roles of calcium-, adenylyl cyclase- and/or mitogen-activated protein kinase (MAP kinase)-dependent signaling pathways in mediating steroidogenesis induced by gonadotropin, the steroid hormone 20β-S or the polychlorinated biphenyl mixture Aroclor 1254 were investigated. Two experimental incubation systems were utilized for the measurement of steroidogenesis in vitro: static ovarian tissue incubation and a primary co-culture system of theca and granulosa cells developed specifically to investigate signaling pathways in steroidogenic cells. Multiple calcium-dependent sites of regulation of steroidogenesis were identified, including voltage-sensitive calcium channels (VSCCs), inositol-1,4,5- triphosphate receptors, calmodulin, calcium/calmodulin-dependent protein kinase II (CaMK II) and aromatase. Gonadotropin-induced testosterone synthesis involves the rapid synthesis of cAMP and the activity of the cAMP-dependent protein kinase (PKA). Furthermore, evidence was obtained for an involvement of mitogen-activated protein kinase (MAP kinase) in gonadal steroidogenesis in a lower vertebrate model. Treatment with hCG induced MEK-dependent phosphorylation of ERK1/2 in a concentration- and time-dependent manner in co-cultured croaker theca and granulosa cells. Few interactions among the signaling pathways were observed; however, there is evident cross-talk between the adenylyl cyclase and MAP kinase pathways mediated by cAMP. Furthermore, inhibitors of VSCCs, calmodulin, CaMK II and MEK reduced forskolin- and dbcAMP-induced testosterone synthesis. Acute in vitro exposure to lead cause moderate decreases in basal testosterone and estradiol synthesis by whole ovarian follicles, while o,p’-DDT increased both basal and hCG-stimulated steroid production. Aroclor 1254 induced a dramatic increase in gonadotropin-stimulated estradiol synthesis; this stimulatory effect was attenuated by inhibitors of VSCCs and calmodulin, thus providing preliminary evidence for a novel mechanism of endocrine disruption in vertebrates. Finally, a stimulatory effect of the maturation-inducing steroid 17α,20β,21- trihydroxy-4-pregnen-3-one (20β-S) on testosterone synthesis by co-cultured theca and granulosa cells was characterized. Physiological concentrations of 20β-S augmented both basal and gonadotropin-stimulated testosterone synthesis by cells from mature ovarian follicles. The stimulatory action of 20β-S is independent of calcium-dependent signaling, and 20β-S does not alter adenylyl cyclase activity. However, 20β-S stimulation of testosterone synthesis may be mediated by the MAP kinase pathway.