Immunomagnetic removal of cryo-damaged human spermatozoa.
OBJECTIVE
To estimate the dissipation of mitochondrial transmembrane potential (mTMP, DeltaPsim) 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 pan-caspases (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.


CONCLUSIONS
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.
Paasch
Uwe
U
Division of Andrology, Department of Dermatology, University of Leipzig, Stephanstrasse 11, D-04103 Leipzig, Germany.
Grunewald
Sonja
S
Wuendrich
Katja
K
Jope
Torsten
T
Glander
Hans-Jurgen
HJ
D017209:Apoptosis; D015153:Blotting, Western; D020169:Caspases / Q000378:metabolism; D003080:Cold Temperature / Q000009:adverse effects*; D015925:Cryopreservation; D006801:Humans; D018189:Immunomagnetic Separation; D007425:Intracellular Membranes / Q000201:enzymology / Q000502:physiology; D008297:Male; D008856:Microscopy, Fluorescence; D013094:Spermatozoa / Q000201:enzymology / Q000502:physiology
D020169:Caspases