We have investigated the possibility that the generation of hydrogen peroxide (H2O2) by spermatozoa plays a physiological role during capacitation. Capacitation is defined as the incubation period required for fertilization in mammals. Capacitation culminates in an exocytotic event, the acrosome reaction (AR). Mammalian sperm generate H2O2 during aerobic incubation and do not contain catalase, the enzyme that promotes scavenging of H2O2. In the present work we show that added catalase inhibited the AR, while glucose oxidase (GO), an enzyme that generates H2O2, accelerated the onset of the AR. Direct addition of H2O2 also stimulated the AR; catalase inhibited both the stimulation by GO and by H2O2. The onset of the AR was always preceded by the appearance of hyperactivated motility. The stimulation of the AR by H2O2 was manifest 1-2 h after the addition of H2O2. Catalase added at 3 h of incubation was less effective in inhibiting the AR than catalase added at the beginning. Incubation of sperm with catalase prevented the induction of the AR by the membrane-perturbing lipid, lysophosphatidyl choline. Taken together, these results suggest that H2O2 produced by hamster sperm plays a significant role during capacitation, possibly in membrane reorganization to facilitate the fusion that takes place during exocytosis of the acrosomal contents.
K-Cl cotransport can participate in volume regulation in a number of cell types. Swelling activation of K-Cl cotransport in sheep erythrocytes proceeds by a two-step process, A<-->B<-->C (Dunham et al., J. Gen. Physiol. 101: 733-765, 1993). The A state, with a low flux, predominates at physiological volume. A-->B is rate limiting and can be activated by reducing cell Mg concentration ([Mg]c); complete activation (B-->C) requires cell swelling. Inhibitors of protein kinases and phosphatases were employed in an attempt to identify enzymatic reactions in the activation process. Staurosporine, a kinase inhibitor, activated K-Cl cotransport by approximately sixfold. Swelling of staurosporine-treated cells caused further activation that proceeded without delay. The effects of staurosporine and reducing [Mg]c were not additive. These two results indicate that staurosporine, like reducing [Mg]c, promotes the rate-limiting A-->B conversion. Unlike swelling, staurosporine activated cotransport without delay. Therefore staurosporine activates by promoting the forward reaction in the A<-->B conversions, in contrast to swelling, which activates by inhibiting the reverse reaction. Calyculin A, a phosphatase inhibitor, inhibited K-Cl cotransport but did not inhibit after activation by reducing [Mg]c, confirming earlier proposals that A-->B is promoted by a phosphatase. Calyculin A, added before or after staurosporine, abolished activation by staurosporine, confirming that staurosporine promotes A-->B. It is proposed that the phosphatase promoting this reaction is regulated by an inhibitory kinase, the staurosporine target.
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