Rabbit spermatozoa from the cauda epididymis produced 0.7–0.8nmol of H2O2/min per 108 cells at cell concentrations below 107 cells/ml with linear dependence on cell concentration. Above 2 × 107 cells/ml, the rate again became linear with cell concentration but decreased to 0.1–0.2nmol/min per 108 cells. Spermatozoa treated with amphotericin B, which makes the plasma membrane highly permeable to low-molecular-weight compounds, showed a similar dependence of H2O2 production rate on cell concentration; below 107 cells/ml the rate was 0.3–0.4nmol/min per 108 cells; above 2 × 107 cells/ml, the rate was 0.1–0.2nmol/min per 108 cells. Hypo-osmotically treated rabbit epididymal spermatozoa, a preparation useful for studying mitochondrial function in sperm [Keyhani & Storey (1973) Biochim. Biophys. Acta305, 557–565] produced 0.1–0.2nmol/min per 108 cells in the absence of added substrates. The dependence of rate on cell concentration was linear from 107 to 2.2 × 108 cells/ml. This endogenous rate was unaffected by rotenone, but stimulated 4-fold by antimycin A. Addition of the mitochondrial substrates lactate plus malate increased the rate of H2O2 production to 0.3nmol/min per 108 cells. The decreased rate of H2O2 production observed with intact sperm at high cell concentrations is attributed to reaction of H2O2 with the cells, possibly with the plasma membrane, which is lost after hypo-osmotic treatment. Rabbit spermatozoa have glutathione peroxidase and glutathione reductase activities, but these seem to play little role in removal of H2O2 generated. The rate at low cell concentration is taken to be the unperturbed rate. The sources of H2O2 production in rabbit spermatozoa have been tentatively resolved into a low-molecular-weight component, lost after amphotericin treatment, a mitochondrial component and a rotenone-insensitive component that has not been identified.

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