Oxidative stress impairs function and increases redox protein modifications in human spermatozoa

Abstract

Oxidative stress, generated by excessive reactive oxygen species (ROS) or decreased antioxidant defenses (and possibly both), is associated with male infertility. Oxidative stress results in redox-dependent protein modifications, such as tyrosine nitration andS-glutathionylation. Normozoospermic sperm samples from healthy individuals were included in this study. Samples were incubated with increasing concentrations (0–5 mM) of exogenous hydrogen peroxide, tert-butyl hydroperoxide, or diethylamine NONOate (DA-NONOate, a nitric oxide (NO∙) donor) added to the medium. Spermatozoa treated with or without ROS were incubated under capacitating conditions and then levels of tyrosine phosphorylation and percentage of acrosome reaction (AR) induced by lysophosphatidylcholine were determined. Modified sperm proteins from cytosolic, triton-soluble, and triton-insoluble fractions were analyzed by SDS–PAGE immunoblotting and immunocytochemistry with anti-glutathione and anti-nitrotyrosine antibodies. Levels ofS-glutathionylation increased dose dependently after exposure to hydroperoxides (P<0.05) and were localized mainly to the cytosolic and triton-soluble fractions of the spermatozoa. Levels of tyrosine-nitrated proteins increased dose dependently after exposure to DA-NONOate (P<0.05) and were mainly localized to the triton-insoluble fraction. ROS-treated spermatozoa showed impaired motility without affecting viability (hypo-osmotic swelling test). These treated spermatozoa had tyrosine phosphorylation and AR levels similar to that of non-capacitated spermatozoa following incubation under capacitating conditions, suggesting an impairment of sperm capacitation by oxidative stress. In conclusion, oxidative stress promotes a dose-dependent increase in tyrosine nitration andS-glutathionylation and alters motility and the ability of spermatozoa to undergo capacitation.Free Spanish abstractA Spanish translation of this abstract is freely available athttp://www.reproduction-online.org/content/149/1/113/suppl/DC1.