Signal transduction pathways regulating steroidogenesis in the ovary of Atlantic croaker (Micropogonias undulatus)
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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.