Sperm output in humans has declined by 50 percent in the past 50 years. The cause for the decline is unknown, but exposure to very low levels of many toxicants may be responsible. To contend with this highly significant problem, a better understanding is needed of mechanisms by which toxicants affect spermatogenesis in mammals. The long-term goal of this project is to better understand the cellular/molecular mechanisms of reproductive toxicants. Many testicular toxicants induce the synthesis of stress or heat-shock proteins (i.e., the stress response). Inducers of the stress response cause disruption of actin microfilaments and loss of tight junctions in cells in vitro and in vivo. One stress protein, hsp27, has been shown to affect microfilament polymerization in vitro and microfilament-dependent cell activities in vivo. Hsp27 also co- localizes with microfilaments in Sertoli cells (SC) of the testis. We hypothesize that hsp27 regulates SC microfilaments, and that toxicants disrupt spermatogenesis by altering hsp27 regulation of SC microfilament dynamics. To gain insight into the mechanism of regulation of SC microfilaments, we will develop antibodies that exclusively recognize each phosphorylated isoform of hsp27 and localize each isoform in frozen sections of testis and in seminiferous tubules, using confocal microscopy. To learn how hsp27 expression is regulated in SC during spermatogenesis we will study, by northern blot, gel shift assay, and immunolocalization, expression and localization of heat-shock transcription factors HSF1 and HSF2 during the maturational stages of the cycle of the seminiferous epithelium. To better understand the mechanism by which hsp27 regulates SC microfilaments, we will discover and characterize SC protein(s) that bind hsp27 using the two-hybrid system to screen a differentiated SC cDNA library. Lastly, we will develop transgenic mice containing hsp27 cDNA in sense and antisense orientation. Transcription from hsp27 cDNA in the mice will be driven by a novel inducible promoter so that hsp27 protein expression can be increased or decreased in testes, without the need for systemic toxicant treatment which would have side effects and also affect expression of genes other than hsp27. These studies will provide significant new knowledge about how toxicants affect SC function at a cellular/molecular level, about hsp27 regulation of microfilaments, and about hsp27 function in pathological and normal conditions. This knowledge may lead to better recognition, prevention and treatment of toxin-induced infertility and to more targeted and novel approaches for development of safe, reliable and reversible male contraceptives.
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