Cryopreservation alters membrane sulfhydryl status of bull spermatozoa: Protection by oxidized glutathione

Cryopreservation induces extensive biophysical and biochemical changes in t he membrane of spermatozoa that ultimately decrease the fertility potential of the cells. Sulfhydryl groups of sperm proteins regulate a number of act ivities of the cells. Qualitative and quantitative analyses of sulfhydryl g roups in the sperm membrane were performed by fluorescence microscopy, fluo rimetry and electrophoresis. Fluorimetric analysis using 5-iodoacetamidoflu oresceine indicated a two-fold increase in the content of sulfhydryl groups in sperm membrane after a freezing/thawing cycle. Electrophoresis of Trito n-soluble sperm proteins after labeling with 3-(N-maleimidylpropionyl) bioc ytin indicated that proteins of 40-65 and 34 kDa range expose more sulfhydr yl groups after cooling at 4 degreesC and freezing/thawing. Cryopreservatio n of spermatozoa changed the distribution pattern of sulfhydryl groups on s perm surface measured with fluorescence microscopy using 5-iodoacetamidoflu oresceine. The percentage of spermatozoa labeled at the level of the mid-pi ece decreased by 50 and 90% after cooling and freezing/thawing, respectivel y. Spin labeling studies showed a 15% faster rotational diffusion (mobility ) of sulfhydryl containing proteins in the membrane of frozen/thawed sperma tozoa as compared to that of fresh spermatozoa. Addition of glutathione, re duced (GSH) or oxidized (GSSG), to the cryoprotectant partially prevented t he effects of freezing/thawing, such as higher exposure of sulfhydryl group s, changes in the cellular distribution, and enhanced rotational diffusion of sulfhydryl containing proteins of sperm membrane. Addition of GSSG to th e cryoprotectant reduced by 35% the loss of motility of spermatozoa undergo ing a freezing/thawing cycle. We concluded that, cryopreservation perturbs sperm membrane sulfhydryl containing proteins and that these modifications could be partially prevented by the addition of GSSG to the cryopreservatio n medium. Mol. Reprod, Dev. 60: 498-506, 2001. (C) 2001 Wiley-Liss, Inc.