Regulation of Germ Cell Apoptosis via the Activity of the Itch E3 Ligase

Abstract

Testicular germ cell numbers are continuously regulated by apoptosis. The balance of anti- and pro-apoptotic proteins determine whether the cell favors death or survival. We have shown that stress generated with the testicular toxicant MEHP sensitizes germ cells to death receptor-mediated apoptosis. Experiments with the spermatocyte-like germ cell line GC2-spd (which possesses a TS mutation that controls p53 activity at defined temperatures) suggests that cell death is achieved via ubiquitinylation of the anti-apoptotic protein c-FLIP. Furthermore, p53 expression appears to be an important contributor to this process as robust ubiquitinylation occurs only at the p53 permissive temperature. Initial experiments suggest that the modulatory protein Itch is a candidate for the post-transcriptional regulation of c-FLIP because it possesses a ubiquinylating E3 ligase. Itch is also present in testis and highly expressed in the germ cell subtypes most sensitive to apoptosis. Immunohistochemical detection of Itch in mouse testicular tissue reveals that Itch protein levels increase upon MEHP treatment and subsequently localize to the cytoplasm of meiotic spermatocyte germ cells. Western blotting of these tissues also show a correlation between c-FLIP reduction and the activated Itch protein, indicated by the phosphorylation of thr-222. These data provide the first evidence that the Itch protein may play an important role in the regulation of spermatocyte germ cell numbers and ensure apoptosis occurs following toxicant stress.