Detection of superoxide anion generation by equine spermatozoa

OBJECTIVE: To identify the generation of the superoxide anion by equine spermatozoa. SAMPLE POPULATION: Multiple ejaculates collected from 3 Thoroughbred stallions. PROCEDURES: Induced superoxide production by reduced nicotinamide adenine dinucleotides (NADP]H; ie, reduced nicotinamide adenine dinucleotide NADH] and reduced nicotinamide adenine dinucleotide phosphate NADPH]) was measured by use of a nitroblue tetrazolium (NBT) reduction assay on whole spermatozoa and a cytochrome c reduction assay on isolated membrane fractions of spermatozoa. Localization of superoxide generation was determined by use of NBT cytochemistry. RESULTS: A dose-dependent increase in NBT reduction was found in the presence of NADPH, which was inhibited by superoxide dismutase (SOD). The flavoprotein inhibitor, diphenyleneiodonium (DPI; 5 or 15 microM), significantly decreased NBT reduction. Cytochrome c reduction by plasma membranes of spermatozoa was significantly higher in the presence of NADPH than in its absence. Cytochemical staining of equine spermatozoa in the presence of NADPH and NADH revealed diaphorase labeling in the spermatozoon midpiece and head. This staining was inhibited by DPI and SOD. CONCLUSIONS AND CLINICAL RELEVANCE: Results of our study indicate that superoxide generation is associated with a membrane-associated NAD(P)H oxidase present in equine spermatozoa, although mitochondrial generation of superoxide is also detected. This oxidase may play a role in cell signaling or may also contribute to cytopathic effects associated with oxidative stress in equine spermatozoa.