Cellular stress to ejaculated spermatozoa such as cryopreservation is known to induce caspase-derived, apoptotic signaling. Therefore, the proenzymes and active forms of caspases 1, 3, 8 and 9 were examined by western blot technique in unfrozen and frozen human spermatozoa of infertility patients and of healthy donors. Twenty-two semen samples derived from healthy donors and 26 semen samples of unselected infertility patients were divided into 3 parts, two of them were cryostored at -196 degrees C with 7% or 14% (v/v, final concentration) of glycerol. The caspases were detected by immunoblots with polyclonal rabbit-anti-caspases-antibodies after 15% sodium dodecyl sulfate-polyacrylgel electrophoresis (SDS-PAGE) under reducing conditions. For evaluation of the differences between amounts of caspase protein the luminol/H(2)O(2) method was applied. A significant increase of activated caspase-1 in donors, of caspase-8 in patients and caspase-9 in patients and donors after cryopreservation were found, whereas, the application of 14% glycerol resulted in higher amounts of activated caspase than did 7% glycerol. Possibly, glycerol may also contribute to activation of caspases via direct toxic effects to mitochondria during cryopreservation of spermatozoa. This finding strongly supports an hypothesis of a higher mitochondria-derived apoptosis-sensitivity of spermatozoa in patients than in healthy donors during cryopreservation. Inactive caspase-3 was reduced subsequent to cryopreservation in patients (p<0.05) and non-significant in donors (p<0.05). Active caspase-3 was detectable in all samples but without significant differences between the three assays. It is concluded that mechanisms associated with apoptotic processes deserve attention in cryopreservation of spermatozoa in order to conserve vital sperm functions after thawing.