Submitted on March 30, 2005
Accepted on January 4, 2006
Regulation of Human Male Germ Cell Death by Modulators of ATP production
Krista Erkkila1*, Sauli Kyttanen2, Marten Wikstrom3, Kimmo Taari4, Amiya P Sinha Hikim5, Ronald S Swerdloff5, and Leo Dunkel6
1 Program for Developmental and Reproductive Biology, Biomedicum Helsinki, and Hospital for Children and Adolescents, University of Helsinki, Helsinki, Finland; Division of Endocrinology, Department of Medicine, Harbor-UCLA Medical Center and Los Angeles Biomedical Research Institute, Torrance / Los Angeles, California, USA
2 Program for Developmental and Reproductive Biology, Biomedicum Helsinki, and Hospital for Children and Adolescents, University of Helsinki, Helsinki, Finland
3 Helsinki Bioenergetics Group, Institute of Biotechnology, University of Helsinki, Helsinki, Finland
4 Department of Urology, Helsinki University Hospital, Helsinki, Finland
5 Division of Endocrinology, Department of Medicine, Harbor-UCLA Medical Center and Los Angeles Biomedical Research Institute, Torrance / Los Angeles, California, USA
6 Program for Developmental and Reproductive Biology, Biomedicum Helsinki, and Hospital for Children and Adolescents, University of Helsinki, Helsinki, Finland; Department of Pediatrics, Kuopio University Hospital, University of Kuopio, Kuopio, Finland
* To whom correspondence should be addressed. E-mail: kerkkila{at}labiomed.org.
The understanding of testicular physiology, pathology and male fertility issues requires knowledge of male germ cell death and energy production. Here, we induced human male germ cell apoptosis (detected by Southern blot analysis of DNA fragmentation, TUNEL, activation of caspases 3 and 9, and electron microscopy) by incubating seminiferous tubule segments under hormone- and serum-free conditions. Inhibitors of complexes I to IV of mitochondrial respiration, exposure to anoxia, and inhibition of F0F1-ATPase (with oligomycin) decreased the ATP levels (analyzed by HPLC) and suppressed apoptosis at 4h. Uncoupler 2-4-dinitrophenol (DNP) and oligomycin -combination also suppressed death at 4h, as did the DNP alone. Inhibition of glycolysis by 2-deoxyglucose neither suppressed nor further induced apoptosis, nor altered the antiapoptotic effects of the mitochondrial inhibitors. Furthermore, Fas system activation did not modify the effects of mitochondrial modulators. After 24h, delayed male germ cell apoptosis was observed despite the presence of the mitochondrial inhibitors. We conclude that the mitochondrial ATP production machinery plays an important role in regulating in vitro induced primary pathways of human male germ apoptosis. The ATP synthesized by the F0F1-ATPase seems to be the crucial death regulator, rather than any of the complexes (I-IV) alone, or the functional electron transport chain, or the membrane potential. We also conclude that there seem to be secondary pathways of human testicular cell apoptosis that do not require mitochondrial ATP production. The present study emphasizes the role of the main catabolic pathways in the complex network of regulating events of male germ cell life and death.
