REACTIVE OXYGEN SPECIES GENERATION BY HUMAN SPERMATOZOA IS INDUCED BYEXOGENOUS NADPH AND INHIBITED BY THE FLAVOPROTEIN INHIBITORS DIPHENYLENE IODONIUM AND QUINACRINE

Human spermatozoa possess a specialized capacity to generate reactive oxygen species (ROS) that is thought to be of significance in the redo x regulation of sperm capacitation (De Lamirande and Gagnon, 1993; Ait ken 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 hu man spermatozoa. Addition of NADPH to viable populations of motile spe rmatozoa induced a sudden dose-dependent increase in the rate of super oxide generation via mechanisms that could not be disrupted by inhibit ors of the mitochondrial electron transport chain (antimycin A, roteno ne, carbonyl cyanide m-chlorophenylhydrazone [CCCP], and sodium azide) , diaphorase (dicoumarol) xanthine oxidase (allopurinol), or lactic ac id dehydrogenase (sodium oxamate). However, NADPH-induced ROS generati on could be stimulated by permeabilization and was negatively correlat ed with sperm function. Both NADH and NADPH were active electron donor s in this system, while NAD(+) and NADP(+) exhibited little activity. Stereospecificity was evident in the response in that only the beta-is omer of NADPH supported superoxide production. The involvement of a fl avoprotein in the electron transfer process was indicated by the high sensitivity of the oxidase to inhibition by diphenylene iodonium and q uinacrine. These results indicate that NAD(P)H can serve as an electro n donor for superoxide generation by human spermatozoa and present a s imple strategy for the production of motile populations of free radica l generating cells with which to study the significance of these molec ules in the control of normal and pathological sperm function. (C) 199 7 Wiley-Liss, Inc.