Analysis of reactive oxygen species generating systems in rat epididymal spermatozoal

Epididymal sperm maturation culminates in the acquisition of functional com petence by testicular spermatozoa. The expression of this functional state is dependent upon a redox-regulated, cAMP-mediated signal transduction casc ade that controls the tyrosine phosphorylation status of the spermatozoa du ring capacitation. Analysis of superoxide anion (O-2(-.)) generation by rat epididymal spermatozoa has revealed a two-component process involving elec tron leakage from the sperm mitochondria at complexes I and II and a plasma membrane NAD(P)H oxidoreductase. Following incubation in a glucose-, lacta te-, and pyruvate-free medium (-GLP), O-2(-.) generation was suppressed by 86% and 96% in caput and cauda spermatozoa, respectively. The addition of l actate, malate, or succinate to spermatozoa incubated in medium -GLP stimul ated O-2(-) generation. This increase could be blocked by rotenone and olig omycin (R/O) in the presence of malate or lactate but not succinate. Stimul ation with all three substrates, as well as spontaneous O-2(-.) production in +GLP medium, was blocked by the flavoprotein inhibitor, diphenylene iodo nium. Diphenylene iodonium, but not R/O, suppressed NAD(P)H-induced lucigen in-dependent chemiluminescence. This NAD(P)H-dependent enzyme resided in th e sperm plasma membrane and its activity was regulated by zinc and uncharac terized cytosolic factors. Reverse transcription-polymerase chain reaction analysis indicated that the sperm NAD(P)H oxidoreductase complex is quite d istinct from the equivalent leukocyte system.