EFFECTS OF HYDROGEN-PEROXIDE ON HUMAN SPERMATOZOA

Purpose: Reactive oxygen species (ROS) have been reported widely to cause deleterious effects on sperm viability and function due to peroxidation of membrane lipids. However, their action appears more selective at low concentrations; recent evidence indicates that the superoxide anion can promote capacitation and induce hyperactivated motility (HA) in human spermatozoa and that hydrogen peroxide (H2O2) may participate in capacitation of hamster spermatozoa. The objective of these studies was to investigate the direct effects of H2O2 on functions crucial to fertilization in human spermatozoa. Methods: In these prospective studies, we examined the dose- and time-dependent effects of H2O2 on sperm membrane-mediated events (binding to the zona pellucida and changes in intracellular calcium concentration [Ca2+](i), motility patterns, and acrosome reaction). Sperm from fertile donors were used in the experiments under capacitating conditions after separation of the motile fraction by wash/swim-up. [Ca2+](i) was measured by the fluorescent fura-2 indicator, and sperm-zona pellucida binding was assessed with the hemizona assay (HZA). Hyperactivated motility was evaluated by computerized analysis, and the percentage of acrosome reacted sperm was detected by FITC-Pisum sativum lectin and indirect immunofluorescence. Results: In the HZA, H2O2 did not influence sperm-zona pellucida binding at low concentrations (0.05 mM and 0.1 mM), but significantly reduced binding at 0.2 mM (P < 0.004 vs controls). H2O2 significantly decreased HA in a dose-dependent manner (P < 0.0001) and had a significant effect (P < 0.01) on acrosome reaction (stimulatory effect at 0.01 mM). H2O2 did not affect basal [Ca2+](i); however, H2O2 (0.1 mM through 10 mM) decreased the initial phase of progesterone-induced (P-4: 1 mu M) enhancement of [Ca2+](i) in a dose- and time-dependent fashion. Preincubation of sperm with catalase (20 mu g/ml) potentiated the P-4-induced increase of[Ca2+](i). H2O2 did not significantly modify [Ca2+](i) increase in response to inomycin (10 mu M). Conclusions: These experiments show that H2O2 directly affects sperm functions crucial to fertilization in a dose-and time-dependent fashion. Low concentrations maintain capacitation, whereas higher concentrations have deleterious effects, as determined by the end points of the capacitation process. The latter effects are probably dependent on modifications of plasma membrane and intracellular homeostasis by the oxidative process.