The modulation of apoptosis in testicular germ cells following toxicant-induced cellular stress

Di-(2-ethylhexyl) phthalate (DEHP) is a ubiquitous environmental toxicant. The active metabolite of DEHP, mono-(2-ethylhexyl) phthalate (MEHP), is ultimately responsible for disrupting the process of spermatogenesis and promoting germ cell death. In addition, this toxicant has been positively correlated with developmental problems such as cryptorchidism, a derangement of the seminiferous tubules, and a syndrome called testicular dysgenesis, leading to reduced sperm number. The potential impact of MEHP on human fertility justifies a detailed investigation into the mechanisms by which this agent causes germ cell death. MEHP is known to directly target and damage the Sertoli cell, a testicular cell whose main function is to support the development of the principle germ cell types from the earliest stem cell to the most mature spermatozoa. This dissertation examines the downstream effect of Sertoli cell damage on germ cell homeostasis and the proteins that modulate the sensitivity of germ cells to undergo apoptotic elimination. Specifically, the stabilization of the p53 protein is proposed to be an important upstream determinant of Fas-mediated apoptosis in germ cells following MEHP exposure. Furthermore, that the resulting cell death is the result of increased death receptor expression and c-FLIPL ubiquitinylation. The mechanism is speculated to reside in the spermatocyte sub-population of germ cells, which appears to be most responsive to changes in apoptosis. Exposures of wild type mice to MEHP caused an increased p53 stability and elevated protein levels of the membrane-bound death receptors Fas and DR5 in testicular spermatocytes. The expression of these proteins occur coincident with increases in spermatocyte apoptosis and are driven by p53 activity. To further assess the mechanisms responsible for the sensitivity of germ cells to undergo p53-mediated apoptosis, we used the germ cell line GC-2spd(ts) (a p53 temperature sensitive spermatocyte-like cell line that allows for p53 nuclear localization at 32°C but not 37°C). Induction of the p53 protein led to higher levels of the death receptors DR5 and Fas, activation of caspase-8, and decreases in c-FLIPL. Addition of TRAIL (the cognate ligand for DR5) and the agonistic DR5 agonistic antibody MD5-1, triggered a robust synergistic increase of apoptosis in GC-2 cells maintained at the p53 permissive temperature (32°C). DR5 levels on the germ cell plasma membrane were considerably enhanced following these treatments. Immunoprecipitation of c-FLIPL suggests that the protein is ubiquitinylated after cellular stress and concomitant with p53 activity. Experiments also reveal that c-FLIPL levels may be influenced by Itch, a regulatory protein able to label targets for the proteasomal degradation using a ubiquitinylating E3 ligase. Immunohistochemical detection in adult wild type mouse testis show robust increases in Itch protein levels upon MEHP treatment (1g/kg) and subsequently localization to the cytoplasm of meiotic spermatocyte germ cells. Western blot analysis of testis from MEHP treated mice also show a correlation between the reduction of c-FLIPL and an increase in Itch threonine-222 phosphorylation, a necessary modification for its E3 ligase function. These results provide a possible model in which the removal of Sertoli cell support promotes germ cell death through the extrinsic pathway, ultimately leading to disruption of spermatogenesis and testicular dysgenesis in mammals. However, removal of Itch also show increases in apoptosis and Itch protein deficient mice demonstrate defects in meiosis. Thus, Itch may also play a novel role in the cell cycle.