Membrane receptor cross talk in gonadotropin-, IGF-I-, and insulin-mediated steroidogenesis in fish ovary: An overview.

Gonadal steroidogenesis is critical for survival and reproduction of all animals. The pathways that regulate gonadal steroidogenesis are therefore conserved among animals from the steroidogenic enzymes to the intracellular signaling molecules and G protein-coupled receptors (GPCRs) that mediate the activity of these enzymes. Regulation of fish ovarian steroidogenesis in vitro by gonadotropin (GtH) and GPCRs revealed interaction between adenylate cyclase and calcium/calmodulin-dependent protein kinases (CaMKs) and also MAP kinase pathway. Recent studies revealed another important pathway in GtH-induced fish ovarian steroidogenesis: cross talk between GPCRs and membrane receptor tyrosine kinases. Gonadotropin binding to Gαs-coupled membrane receptor in fish ovary leads to production of cAMP which in turn trans-activate the membrane-bound epidermal growth factor receptor (EGFR). This is followed by activation of ERK1/2 signaling that promotes steroid production. Interestingly, GtH-induced trans-activation of EGFR in the fish ovary uniquely requires matrix-metalloproteinase-mediated release of EGF. Inhibition of these proteases blocks GtH-induced steroidogenesis. Increased cAMP production in fish ovarian follicle upregulate follicular cyp19a1a mRNA expression and aromatase activity leading to increased biosynthesis of 17ß-estradiol (E2). Evidence for involvement of SF-1 protein in inducing cyp19a1a mRNA and aromatase activity has also been demonstrated. In addition to GtH, insulin-like growth factor (IGF-I) and bovine insulin can alone induced steroidogenesis in fish ovary. In intact follicles and isolated theca cells, IGF-I and insulin had no effect on GtH-induced testosterone and 17a,hydroxysprogeaterone production. GtH-stimulated E2 and 17,20bdihydroxy-4-pregnane 3-one production in granulosa cells however, was significantly increased by IGF-I and insulin. Both IGF-I and insulin mediates their signaling via receptor tyrosine kinases leading to activation of PI3 kinase/Akt and MAP kinase. These kinase signals then activates steroidogenic enzymes which promotes steroid production. PI3 kinase, therefore considered to be an initial component of the signal transduction pathways which precedes MAP kinase in IGF-1 and insulininduced steroidogenesis in fish ovary. Thus, investigation on the mechanism of signal transduction regulating fish ovarian steroidogenesis have shown that multiple, apparently independent signal transduction pathways are needed to convey the message of single hormone or growth factor.

Gonadotropin and sf-1 regulation of cyp19a1a gene and aromatase activity during oocyte development in the rohu, L. rohita.

Cytochrome P450 aromatase (P450arom), a product of cyp19a1 gene, plays pivotal roles in vertebrate steroidogenesis and reproduction. In this study, we isolated partial cDNA encoding the ovarian (cyp19a1a) and brain (cyp19a1b) P450arom genes from adult female rohu, Labeo rohita and investigated the regulation of cyp19a1a by gonadotropin and SF-1. The cyp19a1a and cyp19a1b were expressed predominantly in the ovary and brain respectively, with quantity of the former attuned to reproductive cycle. To elucidate gonadotropin regulation of cyp19a1a mRNA expression and P450 aromatase activity for 17ß-estradiol (E2) biosynthesis in vitro by the vitellogenic ovarian follicles, time- and dose-dependent studies were conducted with HCG and porcine FSH. Results demonstrated that HCG stimulated significantly higher expression of cyp19a1a mRNA and aromatase activity leading to increased biosynthesis of E2 than FSH. To understand the involvement of SF-1 to in the regulation of cyp19a1a and aromatase activity, ovarian follicles were incubated with increasing concentrations of HCG and expression of sf1gene and activation of SF-1 protein were measured. Results demonstrated that HCG significantly induced expression of sf-1 gene and activation of SF-1 protein suggesting a link between SF-1 and P450 aromatase activation in this fish ovary during gonadotropin-induced steroidogenesis.

Steroid-induced oocyte maturation in Indian shad Tenualosa ilisha (Hamilton, 1822) is dependent on phosphatidylinositol 3 kinase but not MAP kinase activation.

Fully grown fish and amphibian oocytes exposed to a maturation-inducing steroid (MIS) activates multiple signal transduction pathways, leading to formation and activation of maturation-promoting factor (MPF) and induction of germinal vesicle breakdown (GVBD). The present study was to investigate if phosphatidylinositol 3 kinase (PI3 kinase) and mitogen-activated protein kinase (MAP kinase) activation are required for naturally occurring MIS, 17α,20ß-dihydroxy-4-pregnen-3-one (17,20ß-P)-induced cdc2 activation and oocyte maturation (OM) in Tenualosa ilisha. We observed that 17,20ß-P-induced OM was significantly inhibited by PI3 kinase inhibitors Wortmannin and LY29400. 17,20 ß-P was shown to activate PI3 kinase maximally at 90 min and cdc2 kinase at 16 h of treatment. Relative involvement of PI3 kinase, MAP kinase and cdc2 kinase in 17,20ß-P-induced OM was examined. MAP kinase was rapidly phosphorylated and activated (60-120 min) after MIS treatment and this response preceded the activation of cdc2 kinase by several hours. A selective inhibitor of MAP kinase (MEK), PD98059, sufficiently blocked the phosphorylation and activation of MAP kinase. Inhibition of MAP kinase activity using PD98059 however, had no effect on MIS-induced cdc2 kinase activation and GVBD. These results demonstrate that activation of the PI3 kinase is required for 17,20ß-P-induced cdc2 kinase activation and OM in T. ilisha. MAP kinase although was activated in response to 17,20ß-P and PI3 kinase activation, it is not necessary for cdc2 activation and OM in this species.

Involvement of PI3 kinase and MAP kinase in IGF-I and insulin-induced ovarian steroidogenesis in common carp Cyprinus carpio.

Previously, we observed that in vitro steroidogenesis in intact ovarian follicles of common carp Cyprinus carpio can alone be induced by recombinant human insulin-like growth factor (IGF-I) and bovine insulin (b-insulin) and this induction was gonadotropin-independent. To investigate early signal transduction components involved in this process, the possible role of phosphatidylinositol 3-kinase (PI3 kinase) during ovarian steroidogenesis was examined. IGF-I and b-insulin induced testosterone and 17ß-estradiol production in carp ovarian theca and granulosa cells in short-term coincubation and this induction was significantly inhibited by Wortmannin and LY294002, two mechanistically different specific inhibitors of PI3 kinase. IGF-I and b-insulin were shown to activate PI3 kinase from 30 min onwards with a maximum at 90 min. In this study, we found the involvement of mitogen-activated protein kinase (MAP kinase) in the regulation of IGF-I- and b-insulin-induced steroidogenesis in carp ovary. An antagonist of mitogen-activated protein kinase kinase1/2 (MEK1/2) markedly attenuated IGF-I- and b-insulin-induced steroid production. Cells treated with IGF-I and b-insulin stimulated ERK1/2-dependent phosphorylation of extracellular signal regulated protein kinase1/2 (ERKs1/2) in a time-dependent manner, which was significantly attenuated in presence of MEK1/2 inhibitor. PI3 kinase inhibitors strongly attenuated phosphorylation and activation of MAP kinase, which was increased during IGF-I and b-insulin-induced steroidogenesis. Taken together, these results suggest that PI3 kinase is an initial component of the signal transduction pathway which precedes the MAP kinase during IGF-I- and b-insulin-induced steroidogenesis in C. carpio ovarian follicles.