Activation pattern of caspases in human spermatozoa.

OBJECTIVE We investigated the activation of caspases 8, 9, 1, and 3 in human ejaculated spermatozoa to study main pathways of apoptosis. Potential functional impact of this phenomenon and possible activation mechanisms were examined by [1] subjecting cells to freezing and thawing, and [2] testing the dependence of caspase activity on membrane integrity. DESIGN Experimental study. SETTING Andrology Center Leipzig (European Academy of Andrology). PATIENT(S) Ten healthy volunteers, 40 semen samples. INTERVENTION(S) Sperm populations were separated by annexin V magnetic activated cell sorting (MACS) according to externalized phosphatidylserine. MAIN OUTCOME MEASURE(S) Active caspases 8, 9, 1, and 3 were examined in human ejaculated spermatozoa before and after cryopreservation. RESULT(S) The active caspases were localized in the postacrosomal region (caspases 8, 1, and 3) or in the midpiece (caspase 9). Cryopreservation led to a significant increase in the number of cells showing activation of all caspases. The MACS separation resulted in a significant depletion of sperm positive for activated caspases within the externalized phosphatidylserine-negative fraction. CONCLUSION(S) Caspases 8, 9, 1, and 3, representing the main pathways of apoptosis, are present in human spermatozoa and can become activated, particularly after freezing and thawing, and are associated with changes in the outer cell membrane. Spermatozoa showing these changes and caspase activation can be efficiently removed from semen samples by the annexin V MACS technique.