Analysis of reactive oxygen species generating systems in rat epididymal spermatozoa

Epididymal sperm maturation culminates in the acquisition of functional competence by testicular spermatozoa. The expression of this functional state is dependent upon a redox-regulated, cAMP-mediated signal transduction cascade that controls the tyrosine phosphorylation status of the spermatozoa during capacitation. Analysis of superoxide anion (O2(-.)) generation by rat epididymal spermatozoa has revealed a two-component process involving electron leakage from the sperm mitochondria at complexes I and II and a plasma membrane NAD(P)H oxidoreductase. Following incubation in a glucose-, lactate-, and pyruvate-free medium (-GLP), O2(-.) generation was suppressed by 86% and 96% in caput and cauda spermatozoa, respectively. The addition of lactate, malate, or succinate to spermatozoa incubated in medium -GLP stimulated O2(-.) generation. This increase could be blocked by rotenone and oligomycin (R/O) in the presence of malate or lactate but not succinate. Stimulation with all three substrates, as well as spontaneous O2(-.) production in +GLP medium, was blocked by the flavoprotein inhibitor, diphenylene iodonium. Diphenylene iodonium, but not R/O, suppressed NAD(P)H-induced lucigenin-dependent chemiluminescence. This NAD(P)H-dependent enzyme resided in the sperm plasma membrane and its activity was regulated by zinc and uncharacterized cytosolic factors. Reverse transcription-polymerase chain reaction analysis indicated that the sperm NAD(P)H oxidoreductase complex is quite distinct from the equivalent leukocyte system.