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

Objective: To 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. Design: Laboratory study. Setting: Center 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 mu M, 100 mu M, and 1 mM) or with 20 mu M 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.