engleski

Do exogenous gangliosides protect from cryopreservation-induced DNA damage of human spermatozoa?

Introduction: Recent studies have reported that exogenous gangliosides, the sialic acid containing glycosphingolipids modulate many cellular functions and properties. We have previously demonstrated the protective effect of exogenous trisialoganglioside GT1b against hydrogen peroxide (H2O2)-induced DNA fragmentation and apoptotic changes of human spermatozoa. The aim of this study was to determine whether gangliosides added to cryoprotective media prior to cryostorage could protect human spermatozoa from DNA fragmentation induced by the freezing/thawing process. Materials & methods: Samples from 16 infertile normozoospermic men (aged 38-41 ; non-smokers) undergoing routine fertility evaluation were collected after three days of sexual abstinence and classified according to the 2010 WHO laboratory manual. All patients gave written informed consent. Spermatozoa were obtained by a two-step discontinuous Percoll density gradient centrifugation. The mixture of sperm suspension and cryoprotective media was divided into aliquots with and without gangliosides, transferred to 0.5 ml straws and stored in liquid nitrogen at -196°C for two weeks. Single-cell gel electrophoresis (Comet assay) was used to assess sperm DNA integrity in fresh and cryostorage-exposed spermatozoa. Sperm cell viability was measured by means of flow cytometry using dual fluorescent staining with 6-carboxyfluorescein diacetate (6-CFDA) in combination with propidium iodide labelling. Results were analysed by ANOVA and post-hoc Scheffe test. Results: Untreated human spermatozoa exhibited significantly increased DNA Comet test parameters after freezing/thawing as compared with unfrozen spermatozoa (1.97±0.05 vs. 1.26±0.05 and 0.36±0.01 vs 0.23±0.01 for tail intensity and tail moment, respectively, p<0.05). In vitro supplemented mono- and trisialoganglioside GM1 and GT1b at a concentration of 100 μM, which was above their respective critical micellar concentrations (cmc), completely abolished DNA damage induced by cryopreservation. Comet parameters in the presence of gangliosides corresponded to the values before cryostorage. Conclusion: Cold shock is known to induce changes in the structure, content and lipid composition of the membrane bilayer. One of the proposed mechanisms of cell protection against cryostorage-induced stress is the sperm cell capability to shed off hydrophilic lipids leading to enhanced hydrophobic properties of the sperm membrane and better tolerance of sperm to cryopreservation. The interaction between the exogenous gangliosides and the membrane lipid bilayer has been shown to render the membrane more hydrophobic. This is in accordance with our previous findings on the influence of gangliosides on molecular ordering and changes in sperm membrane fluidity, suggesting that pre-treatment of spermatozoa with gangliosides modifies membrane properties. The phenomena observed in this study suggest that ganglioside micelles attached at the sperm surface modulate membrane hydrophobicity, thus protecting human spermatozoa from DNA fragmentation induced by cryostorage.

human spermatozoa; cryopreservation; DNA damage; gangliosides

nije evidentirano