The focus of this research project is on the mechanisms responsible for initiating testicular germ cells to undergo apoptosis after toxicant-induced Sertoli cell injury. Recent evidence suggests that exposure to environmental toxicants (organic chemicals, metals, and heat) is responsible for a decline in semen quality in men over the Last 50 years. However, despite this association of toxicant exposure and male infertility, Little is known of the mechanisms by which these agents cause a loss of germ cells. In this research project, the investigators will use mono-(2-ethyLhexyL) phthalate (MEHP), a widely characterized Sertoli cell toxicant, as our primary model agent to study the mechanisms initiating germ cells to undergo apoptosis. In the previous grant-funding period the investigators established that the Fas (CD95)- signaling pathway participates in the initiation of testicular germ cell apoptosis after MEHP-induced Sertoli cell injury. Recently, they made two exciting and critical observations: 1) The re-distribution of Fas in germ cell changes from a cytosolic to a membrane localization after MEHP exposure and, 2) young gld mice, that lack a functional form of FasL, are not sensitive to MEHP-induced apoptosis whereas adult gld mice are sensitive. These fundamental observations have led to the development of two working hypotheses. 1) Fas plays the dominant role in the initiation of MEHP- induced germ cell apoptosis, and, 2) p53 activation leads to the expression of Fas on the germ cell membrane and confers its sensitivity to apoptosis. The first specific aim of this proposal utilizes mutant mice that express either dysfunctional Fas (lpr mice) or FasL (gld mice) to directly examine the dependence of MEHP-mediated germ cell apoptosis on the cellular expression of Fas. In the second aim, the involvement of p53 in mediating the membrane distribution of Fas is tested both in vitro, using GC-2spd cells that express a temperature sensitive mutant of p53, or in vivo, using p53 null mice. In the Last two aims, the two hypotheses are further challenged by evaluating both the importance of the soluble form of FasL and the participation of alternative Fas-independent signaling mechanisms in the pathogenesis of MEHP-induced increases in germ cell apoptosis. This investigation of MEHP-stimulated germ cell apoptosis will provide fundamental insights into mechanisms regulating the sensitivity of germ cell to undergo apoptosis after chemical-induced testicular injury.
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