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.

Agency
National Institute of Health (NIH)
Institute
Eunice Kennedy Shriver National Institute of Child Health & Human Development (NICHD)
Type
Research Project (R01)
Project #
5R01HD043143-02
Application #
6757869
Study Section
Biochemical Endocrinology Study Section (BCE)
Program Officer
Rankin, Tracy L
Project Start
2003-06-09
Project End
2008-05-31
Budget Start
2004-06-01
Budget End
2005-05-31
Support Year
2
Fiscal Year
2004
Total Cost
$233,953
Indirect Cost
Name
University of Pittsburgh
Department
Physiology
Type
Schools of Medicine
DUNS #
004514360
City
Pittsburgh
State
PA
Country
United States
Zip Code
15213
Toocheck, Corey; Clister, Terri; Shupe, John et al. (2016) Mouse Spermatogenesis Requires Classical and Nonclassical Testosterone Signaling. Biol Reprod 94:11
Smith, Lee B; Walker, William H (2014) The regulation of spermatogenesis by androgens. Semin Cell Dev Biol 30:2-13
Walker, William H (2011) Testosterone signaling and the regulation of spermatogenesis. Spermatogenesis 1:116-120
Shupe, John; Cheng, Jing; Puri, Pawan et al. (2011) Regulation of Sertoli-germ cell adhesion and sperm release by FSH and nonclassical testosterone signaling. Mol Endocrinol 25:238-52
Walker, William H (2010) Non-classical actions of testosterone and spermatogenesis. Philos Trans R Soc Lond B Biol Sci 365:1557-69
Walker, William H (2009) Molecular mechanisms of testosterone action in spermatogenesis. Steroids 74:602-7
Cheng, Jing; Watkins, Simon C; Walker, William H (2007) Testosterone activates mitogen-activated protein kinase via Src kinase and the epidermal growth factor receptor in sertoli cells. Endocrinology 148:2066-74
Walker, William H; Cheng, Jing (2005) FSH and testosterone signaling in Sertoli cells. Reproduction 130:15-28
Fix, Charity; Jordan, Cynthia; Cano, Patricia et al. (2004) Testosterone activates mitogen-activated protein kinase and the cAMP response element binding protein transcription factor in Sertoli cells. Proc Natl Acad Sci U S A 101:10919-24