MECHANISM OF SUPEROXIDE-DISMUTASE LOSS FROM HUMAN SPERM CELLS DURING CRYOPRESERVATION

Earlier studies on human sperm cryodamage have shown that plasma membr ane stress is the primary process and that phospholipid peroxidation i n cryopreserved samples is not inhibited by addition of antioxidants. One consistent effect of cryopreservation is loss of enzymatic activit y of the peroxidation defense enzyme, superoxide dismutase (SOD). To c larify this aspect of the freeze-thaw process and to develop a more co mplete resolution of the reactions leading to cryodamage, we sought to identify which of the two most probable mechanisms, loss of enzyme pr otein from the cells or denaturation of the protein, operates. If the first operates, cellular enzymatic activity and enzyme protein as iden tified by immunocytochemistry should give a linear correlation. If the second operates, there should be no correlation. In this study, five individual samples were analyzed before and after cryopreservation for immunoreactive Cu/Zn SOD and cell intactness by flow cytometry, for S OD enzymatic activity by a highly sensitive fluorimetric method, and f or motility characteristics by Hamilton-Thorn motility analyzer. Fresh samples were obtained by the ''swim-up'' method and had >95% intact c ells with >78% motile cells. After freeze-thaw, about half the cells w ere intact. SOD enzymatic activity was determined on Triton X-100 cell extracts, a method that removes all enzymatic activity from the cell structure, and compared with immunoreactive SOD in the cells as determ ined by indirect immunofluorescence mean intensities. Residual immunof luorescence was observed in the cells after Triton X-100 treatment; if this was taken into account, a close linear correlation between SOD e nzyme activity and SOD immunoreactivity was obtained (r = 0.90; P = 0. 00014). There was no correlation between SOD enzyme activity ratios fo r crypreserved and fresh cells and fraction of intact cells after free ze-thaw. We conclude that loss of SOD protein from the subset of cells undergoing acute membrane damage is the most probable primary mechani sm of SOD enzymatic activity loss from the sample and that resistance to cryodamage and SOD activity in any given cell are quite independent of one another.