engleski

Detection of spermatozoa membrane lipid peroxidation by end-products analysis and electron paramagnetic resonance assay

Lipid peroxidation-induced changes in sperm membrane physical properties may have deleterious consequences on spermatozoa functioning. Detection of membrane lipid peroxidation (LPO) has been based on spectrophotometric measurement of LPO-end products such as malondialdehyde (MDA) and 4-hydroxyalkenals (4HA), or by the evaluation of extracted membrane lipids. Although these methods are sensitive to peroxidative damage sustained by spermatozoa, they do not provide evidence of cellular membrane changes. There have been only a few reports on the application of physical methods to investigate changes in human sperm fluidity upon experimentally induced LPO by iron/ascorbate promoter. In this study, electron paramagnetic resonance (EPR) method was applied to study changes in human sperm fluidity after the iron/ascorbate treatment, as an alternative to detection of LPO-end products. Ferrous ion promotes the catalysis of pre-existing lipid peroxides to alkoxyl and peroxyl radicals, which is important in the propagation of the chain reaction of lipid peroxidation in the sperm membrane. The accumulation of MDA in human spermatozoa after exposure to iron/ascorbate system in Ca2+-and Mg2+-free HBSS (Hank's balanced salt solution, pH 7.4) was estimated spectrophotometrically using thiobarbituric acid reactive substances (TBARS) measurement and MDA+4HA assay (LPO-586 ; Bioxytech, Portland, OR, USA), respectively. Three different stearic acid nitroxides (NS) were used to label plasma membrane for EPR assay: 5-NS, whose paramagnetic center resides near the lipid-water interface, and 7-NS and 13-NS, in which the nitroxide moiety localizes deeper in the lipid bilayer. Spin labeling was performed after membrane incubation with iron/ascorbate promoter. The results showed that measurements of LPO-end products concentration obtained by the two biochemical methods were highly correlated (r=0.78 ; p<0.0001 ; n=25). In 5 sperm samples, simultaneous to biochemical assay of LPO, the EPR assay revealed that upon the iron/ascorbate treatment of the cells the oxidation induced perturbation of membrane structural organization could only be detected in the samples labeled with 5-NS. The experimental EPR spectra showed that the motion of the reporter group slowed down after the oxidative modification of the membrane. We conclude that EPR assay might be a useful method to monitor oxidation-induced sperm membrane changes, which could be applied in the research of human spermatozoa.

spermatozoa; lipid peroxidation; EPR; membrane fluidity

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