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 microM, 100 microM, and 1 mM) or with 20 microM 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.