In this research project, activation of an apoptotic signal transduction process in germ cell is proposed as a ~final common pathway~ of germ cell death in the testis after toxicant-induced testicular Sertoli cell injury. To test this idea, the Sertoli cell toxicant, mono- (2-ethylhexyl) phthalate (MEHP), will be used to stimulate germ cell apoptosis in young (28 day old) rat testis. Apoptosis of germ cells in the testis occurs in rats routinely as a physiologic mechanism to limit the clonal expansion of germ cells in the testis. In preliminary experiments, exposure of young rats to MEHP resulted in an unexpected initial (3 h) inhibition of germ cell apoptosis followed later by a stimulation of germ cell apoptosis (12 h). These findings indicate that MEHP exposure alters the regulation of germ cell apoptosis in the testis. In addition, the expression of three components of the Fas-mediated pathway in testis: Fas receptor (Fas), Fas ligand (FasL) and FAP-1, was induced after MEHP exposure. The Fas-mediated pathway is a well characterized apoptotic signal transduction system where Fas acts as a receptor protein to trigger an intrinsic suicide program in the cell. These fundamental observations have led to the development of the following working hypothesis: MEHP disrupts Sertoli cell-germ cell interactions resulting in a stimulation of the Fas-mediated signal transduction process between these cells altering the physiologic mechanism of Sertoli cell-directed germ cell apoptosis. This hypothesis will be tested by specific aims designed 1) to characterize MEHP-induced alterations in the expression of components of the Fas-mediated pathway in testis and determine the cell-specific localization of the Fas-associated protein components and, 2) to delineate the role of Fas-mediated apoptosis in MEHP- induced germ cell loss by inhibiting the expression of FasL both in vitro and in vivo. Recent evidence suggests that environmental exposures to toxicants play a role in male infertility. However, despite the association of exposure to environmental agents and infertility, little is known of the mechanisms by which these agents cause decreased numbers of sperm. Phthalates, the model toxicants used in this project, are found widespread in the environment due to their use as plasticizers in food packaging and biomedical devices. The investigation of phthalate-induced testicular injury in the rat will provide clues into the mechanisms of both physiologic and toxicant-induced germ cell death in the testis and give insight into mechanisms of infertility resulting from environmental toxicant exposures.
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