As spermatozoa progress along the epididymal duct, they are bathed in a specialized luminal fluid microenvironment which is crucial for their maturation and survival. These processes are dependent upon the presence of androgens and luminal factors of testicular origin. Gamma-glutamyl transpeptidase (GGT) is an enzyme that plays a critical role in the metabolism of the antioxidant glutathione (GSH). This study will examine how the expression of GGT mRNA (mRNA IV) is regulated by testicular factor(s). It will focus on the rat initial segment, the region which is influenced by testicular factors and is highly susceptible to oxidative damage. Our working hypothesis is that growth factors, of Sertoli cell origin, enter the epididymal duct and interact with their respective receptors located on the apical surface of epididymal cells. Here, second messenger pathways are activated which result in activation of transcription factors and transactivation of GGT mRNA-IV. GGT is synthesized and transported to the apical cell surface to protect the developing spermatozoa and/or epididymal cells from oxidative damage. Under normal physiological conditions this pathway is continuously stimulated. This contrasts with other cell types which only activate similar pathways during periods of growth and differentiation. Specifically, we propose: (1) To test the hypothesis that receptors for the testicular factor, fibroblast growth factor-2 (FGF-2; bFGF), are present in the cells of the rat initial segment, are localized to the apical surface and bind ligand. (2) To test the hypothesis that FGF-2 maintains the activity of second messenger pathways within the cells of the initial segment. (3) To test the hypothesis that FGF-2 regulates binding of PEA3 to GGT promoter IV via the ras-raf- MAPK pathway. (4) To test the hypothesis that FGF-2 maintains the activity of GGT promoter IV by activating the ras-raf-MAPK pathway. (5) To test the hypothesis that FGF-2 maintains GGT protein level and enzyme activity by activating the ras-raf-MAPK pathway. This laboratory is uniquely qualified to perform these studies because a combination of in vivo micropuncture with modern molecular biological techniques will be used. This proposal is part of a long term goal to understand the mechanisms by which the epididymis maintains an optimal luminal microenvironment for sperm maturation and survival. The findings from this proposal will provide fundamental information for the treatment of certain forms of male infertility and the development of a male contraceptive.
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