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407 REGULATION OF HUMAN MALE GERM CELL DEATH BY MODULATORS OF ATP PRODUCTION

Purpose ATP production regulates the death process of many cells, including testicular cells. However, it is not known whether it is the ATP (or its level) or the ATP producing machinery, or some component, such as the mitochondrial respiratory chain or its complexes, or the ATP synthetase, or other mediators, which may be crucial. Based on this uncertainty, we investigated the role of ATP production in human male germ cell death. Results and Methods We found, that incubation of human seminiferous tubule segments under serum and hormone free culture conditions induced germ cell apoptosis, detected by Southern blot analysis of DNA fragmentation, TUNEL and electron microscopy. Inhibitors of different complexes (I to IV) of mitochondrial respiration and of the ATP synthetase (oligomycin) depleted the ATP levels (analyzed by HPLC), and suppressed apoptosis at 4h. An uncoupler (DNP) and oligomycin, the combination that allows activity of the respiratory chain while ATP synthetase is blocked, also suppressed death at 4h, as did the DNP by itself. Inhibition of glycolysis by 2-DG neither suppressed apoptosis nor altered the results obtained with the mitochondrial modulators. Furthermore, at 4h, apoptosis was not induced by Fas activation in the presence of inhibitors of ATP production. After 24h, many of the cells underwent delayed apoptosis despite ATP-depletion mediated by any of the mitochondrial modulators (± 2-DG). In addition, some of the spermatocytes and spermatids showed signs of necrosis or toxicity. Conclusion The mitochondrial ATP production plays an important role in regulating Fas associated primary pathways of human male germ apoptosis, triggered by the culture conditions. It appears that it is not any of the studied distinct mitochondrial components, but rather 1) the whole functional mitochondrial ATP production machinery, 2) some yet undefined part, or 3) possibly the ATP level as such, which is crucial for these primary pathways. We also conclude that there seem to secondary pathways of human testicular apoptosis that do not require mitochondrial ATP production and perhaps not ATP either.