Caspase 3 activation in human spermatozoa in response to hydrogen peroxide and progesterone

ObjectiveTo determine the role of calcium signaling on apoptosis evoked by the reactive oxygen species H2O2 and by the physiological agonist P in human ejaculated spermatozoa.DesignLaboratory study.SettingCenter for assisted human reproduction in a hospital in Spain.Patient(s)Forty-five healthy volunteers.Intervention(s)Spermatozoa were treated with increasing concentrations of hydrogen peroxide (H2O2; 10 μM, 100 μM, and 1 mM) or with 20 μM of P for 5–120 minutes.Main Outcome Measure(s)Activation of caspase-3 and -9 as well as phosphatidylserine externalization were examined in human ejaculated spermatozoa by fluorescence methods.Result(s)Hydrogen peroxide and P induced activation of caspase-3 and -9. In addition, the effect of H2O2 and P was time dependent. Dimethyl-1,2-bis (aminophenoxy) ethane-N,N,N′,N′-tetraacetic acid loading was able to inhibit H2O2- and P-induced caspase-3 activation and phosphatidylserine externalization. Pretreatment of spermatozoa with Ru360, to block the calcium uptake into mitochondria, also was able to decrease the activation of caspase-3 and phosphatidylserine exposure that was stimulated by either H2O2 or P.Conclusion(s)These findings suggest that H2O2- and P-induced mitochondrial apoptosis is dependent on calcium signaling.