engleski

Oxidation of human spermatozoa: the action of gangliosides

Introduction: Different gangliosides (sialic acid containing glycosphingolipids) have been shown to protect some human cells from the oxidation-induced damages. The aim of this study was to examine the ability of gangliosides from the b-series (GD1b and GT1b) to reduce changes in human sperm membrane fluidity triggered by the external oxidative stimulus. In addition, the scavenger and chelating properties of gangliosides were addresed. Material and methods: Spermatozoa from 40 normozoospermic infertile men were investigated. Membrane fluidity was monitored by electron paramagnetic resonance (EPR) spectroscopy using 5-doxylstearic acid nitroxide (5-NS) as spin label. Lipid peroxidation was estimated by quantifying malondialdehyde (MDA) concentration (Bioxytech LPO-586 test) following the iron/ascorbate induced LPO in the presence/absence of GD1b and GT1b. The dismutase-inhibitable reduction of ferricytochrome c was applied to determine the phorbol myristate acetate (PMA)-induced superoxide anion formation. Results: The EPR spectra revealed that membrane rigidity increased during induced lipid peroxidation (2Amax =5.76 +/- 0.06 mT vs. 2Amax =5.56 +/- 0.04 mT in the oxidized vs. native state, respectively ; p<0.0001, n=9). In the presence of 100 uM GT1b the oxidation-induced spectral changes were suppressed (different spectral peak proportions as compared to native state). GT1b simultaneously inhibited MDA formation (18.5 +/- 1.2 at 50 uM GT1b and 14.5 +/- 2.4 at 100 uM GT1b vs. 22.9 +/- 3.1 uM MDA/108 spermatozoa in untreated samples, n=6). GD1b exhibit similar effect (21.6 +/- 2.5 and 16.9 +/- 0.8 at 50 and 100 uM GD1b respectively, in comparison with 24.6 +/- 0.6 uM MDA/108 spermatozoa in controls). Metal chelator EDTA also suppressed MDA formation and 10 uM (24.3 +/- 4.4 at 5 uM and 17.4 +/- 2.6 at 10 uM EDTA compared with 35.4 +/- 5.4 uM MDA/108 spermatozoa in controls) Furthermore, preincubation of spermatozoa (15 min, 37 °C) with GD1b and GT1b (50 and 100 uM) affected PMA-induced superoxide anion generation about 30% and 54%, respectively (n=9). Spermatozoa of normospermic infertile men released significantly less superoxide anionsm in the presence of 100 uM GT1b (0.45 +/- 0.1 vs. 0.71 +/- 0.1 nM O2-/106 spermatozoa ; p<0.0001 ; n=16). Conclusion: So far no study has examined the mechanisms of the protective action of exogenous gangliosides on sperm membrane fluidity changes upon external oxidative stimulus. This study demonstrated that gangliosides of GD1b and GT1b effectively reduced oxidation-induced changes in sperm membrane fluidity. The obtained data suggest that this protective action could be related to both scavenging of free radicals and/or transition metal chelating properties of the examined ganglisides.

human spermatozoa; gangliosides; membrane fluidity; oxidation-induced damages; EPR

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