Ovarian follicle growth is a dynamic process that requires exquisite regulation. Follicles are restrained at the preantral stage until they are stimulated by follicle stimulating hormone (FSH). FSH signals through its cell-surface receptor via cAMP and cAMP-dependent protein kinase (PKA). The requirement for PKA is demonstrated by the ability of the specific PKA inhibitor peptide, PKI, to block acute signaling events, such as histone H3 phosphorylation and activation of the extracellular regulated kinase ERK, as well as to prevent induction of genes/proteins required for development of the preovulatory phenotype. However, FSH-stimulated granulosa cell maturation is also blocked by chemical inhibitors of the phosphoinositide-3 (PI-3) kinase pathway. PI-3 kinase and its downstream kinase target AKT are classically activated by the insulin or insulin-like growth factor 1 receptors upon binding the adaptor protein insulin receptor substrates (IRS) 1/2. FSH-stimulated AKT phosphorylation is also blocked by PKI. We hypothesize that PKA uniquely orchestrates all of the FSH-regulated signaling pathways to direct granulosa cell differentiation. We also hypothesize that FSH signals through the PI-3 kinase pathway by inducing the transcription factor hypoxia inducible factor-1alpha (HIF-1alpha) and inhibiting signals from the transcription factor FKHR that repress granulosa cell maturation. In this proposal we will test the hypotheses that FSH signals via PKA to activate PI-3 kinase via the adapters IRS1 and/or IRS2, that FSH via PI-3 kinase induces HIF-1alpha and that HIF-1alpha is a key positive signal leading to induction of FSH-responsive genes, and that signaling from FKHR which maintains granulosa cells in an undifferentiated state is neutralized by its phosphorylation via the PI-3 kinase pathway. Induction of key regulatory genes that characterize mature granulosa cells therefore requires regulated signaling not only through CREB, histone H3, and ERK but also through PI-3 kinase targets HIF-1alpha and FKHR. Understanding how follicular maturation is regulated can translate into safer and more effective treatments for fertility.
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