Exposure of rats to 2,5-bexanedione (2,5-HD) a reactive metabolite of the environmental toxicant n-hexane, results in the development of a persistent testicular atrophy. The toxicity of 2,5-HD has been attributed to the formation of reactive pyrroles which cross-link Sertoli cell microtubules, thereby disrupting microtubule transport required for the support of germ cell proliferation and differentiation. The reason for the persistence of testicular atrophy long after 2,5-HD exposure is unclear, as spermatogonial stem cells proliferate yet fail to develop into more advanced germ cell populations due to enhanced apoptosis. Recently, studies from our laboratory and others have demonstrated that testicular atrophy can be reversed by therapies which produce a prolonged suppression of intratesticular testosterone, including therapy with the GnRH agonist leuprolide. In this proposal, we present preliminary data which indicates that GnRH therapy suppresses testicular interstitial testosterone and restores spermatogenesis, but not when administered with ethane dimethane sulphonate, an alkylating agent which selectively ablates testosterone-producing Leydig cells. Our proposed studies will explore the hypothesis that paracrine acting factors, produced by Leydig cells, are critical to the reversal of 2,5-HD-induced testicular atrophy by GnRH agonist therapy. Soluble paracrine-acting factors produced by Leydig cells and important to the reversal of testicular atrophy will be identified by 2D-PAGE followed by LC/MS, and changes in Leydig cell expression will be examined by cDNA array technology. Finally, candidate Leydig cell factors will be quantitated by RT-PCR analysis and correlated with the localization of Leydig cells to populated versus atrophic tubules using laser capture microdissection.
