Reactive oxygen species generation by human spermatozoa is induced by exogenous NADPH and inhibited by the flavoprotein inhibitors diphenylene iodonium and quinacrine
Version of Record online: 7 DEC 1998
Copyright © 1997 Wiley-Liss, Inc.
Molecular Reproduction and Development
Volume 47, Issue 4, pages 468–482, August 1997
How to Cite
Aitken, R. J., Fisher, H. M., Fulton, N., Gomez, E., Knox, W., Lewis, B. and Irvine, S. (1997), Reactive oxygen species generation by human spermatozoa is induced by exogenous NADPH and inhibited by the flavoprotein inhibitors diphenylene iodonium and quinacrine. Mol. Reprod. Dev., 47: 468–482. doi: 10.1002/(SICI)1098-2795(199708)47:4<468::AID-MRD14>3.0.CO;2-S
- Issue online: 7 DEC 1998
- Version of Record online: 7 DEC 1998
- Manuscript Accepted: 6 FEB 1997
- Manuscript Received: 29 JUL 1996
- reactive oxygen species;
- diphenylene iodonium
Human spermatozoa possess a specialized capacity to generate reactive oxygen species (ROS) that is thought to be of significance in the redox regulation of sperm capacitation (De Lamirande and Gagnon, 1993; Aitken et al., 1995). However, the mechanisms by which ROS are generated by these cells are not understood. In this study we have examined the possible significance of NADPH as a substrate for ROS production by human spermatozoa. Addition of NADPH to viable populations of motile spermatozoa induced a sudden dose-dependent increase in the rate of superoxide generation via mechanisms that could not be disrupted by inhibitors of the mitochondrial electron transport chain (antimycin A, rotenone, carbonyl cyanide m-chlorophenylhydrazone [CCCP], and sodium azide), diaphorase (dicoumarol) xanthine oxidase (allopurinol), or lactic acid dehydrogenase (sodium oxamate). However, NADPH-induced ROS generation could be stimulated by permeabilization and was negatively correlated with sperm function. Both NADH and NADPH were active electron donors in this system, while NAD+ and NADP+ exhibited little activity. Stereospecificity was evident in the response in that only the β-isomer of NADPH supported superoxide production. The involvement of a flavoprotein in the electron transfer process was indicated by the high sensitivity of the oxidase to inhibition by diphenylene iodonium and quinacrine. These results indicate that NAD(P)H can serve as an electron donor for superoxide generation by human spermatozoa and present a simple strategy for the production of motile populations of free radical generating cells with which to study the significance of these molecules in the control of normal and pathological sperm function. Mol. Reprod. Dev. 47:468–482, 1997. © 1997 Wiley-Liss, Inc.