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.


METHODS
Experimental study.


METHODS
Andrology Center Leipzig (European Academy of Andrology).


METHODS
Ten healthy volunteers, 40 semen samples.


METHODS
Sperm populations were separated by annexin V magnetic activated cell sorting (MACS) according to externalized phosphatidylserine.


METHODS
Active caspases 8, 9, 1, and 3 were examined in human ejaculated spermatozoa before and after cryopreservation.


RESULTS
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.


CONCLUSIONS
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.
Paasch
Uwe
U
Department of Dermatology/Andrology Unit, University of Leipzig, Leipzig, Germany. paau@medizin.uni-leipzig.de
Grunewald
Sonja
S
Agarwal
Ashok
A
Glandera
Hans-Jürgen
HJ
D017304:Annexin A5; D020170:Caspase 1 / Q000378:metabolism; D053148:Caspase 3; D053181:Caspase 8; D053453:Caspase 9; D020169:Caspases / Q000378:metabolism*; D002462:Cell Membrane / Q000473:pathology; D002469:Cell Separation; D015925:Cryopreservation; D004789:Enzyme Activation; D006801:Humans; D008280:Magnetics; D008297:Male; D008954:Models, Biological; D010718:Phosphatidylserines / Q000378:metabolism; D012016:Reference Values; D013094:Spermatozoa / Q000201:enzymology* / Q000473:pathology; D014018:Tissue Distribution
D017304:Annexin A5; D010718:Phosphatidylserines; C505407:CASP3 protein, human; C505530:CASP8 protein, human; C506433:CASP9 protein, human; D053148:Caspase 3; D053181:Caspase 8; D053453:Caspase 9; D020169:Caspases; D020170:Caspase 1