Role of Hydrogen Peroxide in Sperm Capacitation and Acrosome Reaction1

The generation of reactive oxygen species (ROS) has been implicated in the regulation of sperm capacitation and acrosome reaction; however, the mechanisms underlying this regulation remain unclear. To examine the cellular processes involved, we studied the effect of different concentrations of hydrogen peroxide (H2O2) on protein tyrosine phosphorylation under various conditions. Treatment of spermatozoa with H2O2 in medium without heparin caused a time- and dose-dependent increase in protein tyrosine phosphorylation of at least six proteins in which maximal effect was seen after 2 h of incubation with 50 μM H2O2. At much higher concentrations of H2O2 (0.5 mM), there is significant reduction in the phosphorylation level, and no protein tyrosine phosphorylation is observed at 5 mM H2O2 after 4 h of incubation. Exogenous NADPH enhanced protein tyrosine phosphorylation similarly to H2O2. These two agents, but not heparin, induced Ca2+-dependent tyrosine phosphorylation of an 80-kDa protein. Treatment with H2O2 (50 μM) caused approximately a twofold increase in cAMP, which is comparable to the effect of bicarbonate, a known activator of soluble adenylyl cyclase in sperm. This report suggests that relatively low concentrations of H2O2 are beneficial for sperm capacitation, but that too high a concentration inhibits this process. We also conclude that H2O2 activates adenylyl cyclase to produce cAMP, leading to protein kinase A-dependent protein tyrosine phosphorylation.