The signaling events leading to apoptosis can be divided into two pathways, involving either mitochondria (intrinsic) or death receptors (extrinsic). In earlier studies, we have shown the involvement of the intrinsic pathway signaling for induction of male germ cell apoptosis after hormone deprivation or by mild testicular hyperthermia. In additional studies, using the gld and lpr cg mice, which harbor loss-of-function mutations in Fas L and Fas, respectively, we further demonstrated that the Fas signaling system had little, if any, role in male germ cell death triggered by mild testicular hyperthermia. To gain further insight into molecular mechanisms that regulate germ cell apoptosis, here we characterized the up-stream modulators of the intrinsic pathway signaling after suppression of intratesticular testosterone (T). Groups of adult male rats were given a daily injection of vehicle for 14 days or GnRH antagonist (GnRH-A) for 2, 5, and 14 days. Within 2 days of GnRH-A treatment testicular T concentrations declined markedly to 10.7% of control values and plasma T levels fell below detectable limits. The mean incidence of apoptotic germ cells (expressed as numbers per 100 Sertoli cells) exclusively at stages VII-VIII increased significantly (108.1 ± 7.4; P ≤ 0.001) by Day 5, and increased another 1.9-fold (over the 5-day treatment value) on Day 14 after GnRH-A treatment when compared to controls, where no apoptosis was detected. Activation of p38 mitogen-activated protein kinase (p38 MAPK), a critical mediator of a variety of environmental stresses, and induction of inducible nitric oxide (NO) synthase (iNOS), which is critical for NO-mediated cell death, were clearly evident in the testicular lysates, coincident with the fall in testicular T concentrations as early as 2 days after GnRH-A treatment. These events were followed by a marked alteration in the mitochondrial Bax and Bcl-2 ratio, cytosolic translocation of mitochondrial cytochrome c, caspase activation, and PARP cleavage. Taken together, these results indicate that withdrawal of gonadotropins and consequently intratesticular T induces germ cell apoptosis in the testis by stimulating p38 MAPK and NO-mediated intrinsic pathway signaling. Supported by NIH grants HD 39293 and GM 08683.
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