Testosterone is absolutely essential for spermatogenesis and targets Sertoli cells in the mammalian testis through the androgen receptor (AR) to produce factors required for germ cell development and survival. We now have evidence for an alternative, rapid (< 5 min) mechanism by which testosterone stimulates the phosphorylation (activation) of the Erk MAP kinase and the CREB transcription factor in primary rat Sertoli cells. This novel mechanism of testosterone action will impact a broad number of processes in Sertoli cells because MAP kinase and CREB are focal points for the control of numerous signaling pathways. Furthermore, this proposal addresses a longstanding gap in the understanding of the mechanisms by which testosterone regulates spermatogenesis as few AR-regulated genes have been identified. We will test the overall hypothesis that testosterone binding to AR activates a Src kinase and/or Ca2+-mediated signaling cascade resulting in the phosphorylation and activation of MAP kinase and CREB.
Aim 1 is to test the hypothesis that AR is required to mediate the rapid phosphorylation of MAP kinase and CREB. Androgen-induced ERK and CREB phosphorylation will be assayed in normal and AR deficient rat primary Sertoli cells. AR domains required for androgen signaling will be identified. The hypothesis that populations of AR are associated with the Sertoli cell plasma membrane will be tested.
Aim 2 is to test the hypothesis that androgen activates MAP kinase and CREB by a Src kinase-regulated pathway and/or via a Ca2+-mediated pathway. Androgen-induced Src kinase activity will be determined and androgen-activation of Erk and CREB wilt be assayed after addition of pharmacological and gene-based inhibitors of Src activity. Specific blockers of Ca2+ channels and Ca2+ actions will be used to test the hypothesis that Ca2+-mediated signaling pathways propagate androgen signals.
Aim 3 is to test the hypothesis that androgen activates CREB and MAP kinase-regulated gene expression. Androgen regulation of MAP kinase and CREB-mediated gene expression will be tested using reporter plasmids in transient transfection assays. The regulation of specific endogenous Sertoli cell target genes via CREB will be assessed using RNAse protection assays. The information generated from this study will be directly applicable to divining the mechanisms by which testosterone supports spermatogenesis and maintains male fertility.
