Aim: To estimate the dissipation of mitochondrial transmembrane potential (mTMP, m) and activation of sperm caspases (aCP) as signs of apoptosis in human spermatozoa during cryopreservation and to evaluate the efficiency of immunomagnetic cell separation (MACS) of these spermatozoa via annexin V-binding.

Methods: The mTMP and aCP in fresh and cryopreserved spermatozoa were detected by fluorescence microscopy and by Western blots. The sperm suspensions were divided into two sperm fractions (with intact and deteriorated membranes) by magnetic cell separation (MiniMACS, Miltenyi Biotec, Bergisch Gladbach, Germany) in dependence on their binding to superparamagnetic annexin V-microbeads (AN-MB).

Results: The cryopreservation decreased the portion of spermatozoa with intact mTMP from 80.1 %±7.2 % to 53.5 %±13.1 % and increased the spermatozoa with activated pancaspases (aCP) from 21.8 %±2.6 % to 47.7 %±5.8 % (n = 10; mean SEM; P < 0.01). The activation of caspases 1, 3, 8, and 9 in the cryopreserved spermatozoa was confirmed by Western blots (n = 22). MACS reduced significantly the percentage of cryopreserved spermatozoa with dissipated mTMP to 8.1± 3.9 (P < 0.01) and also those with aCP to 9.3 %±2.2 %. Western blot analyses confirmed the increase of the activated caspase3, 9, and 8 in the AN-MB-positive fraction (P < 0.05) compared with the AN-MB-negative fraction. The MACS separation effect was confirmed by anti-annexin V-antibodies. There was no significant influence of the separation column and the magnetic field on the sperm functions.

Conclusion: The cryopreservation impaired the mTMP and enhanced the activation status of caspases in human spermatozoa. The immunomagnetic sperm separation via binding of AN-MB could deplete low quality spermatozoa from cryopreserved semen samples.

Keywords: spermatozoa, cryopreservation, immunomagnetic separation, plasma membrane, annexin V, apoptosis, caspases, mitochondria

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