Effect of hydrogen peroxide on Na+,K+-ATPase activity in spermatozoa of infertile men

Na+,K+-ATPase plays an essential role in sperm motility, hyperactivation, chemotaxis, acrosome reaction etc. Na+,K+-ATPase is sensitive to ROS insult. Apart from production of highly reactive molecules, H2O2 can exert a number of direct effects on cells, their metabolism and enzymes. In the present study, exposure to exogenous H2O2 was used to characterize the effects of H2O2 on Na+,K+-ATPase activity in spermatozoa of infertile men with different forms of pathospermia. It was shown that Na+,K+-ATPase activities in spermatozoa of infertile men with different forms of pathospermia were inhibited by exposure to H2O2 (50−500 μM). H2O2, one of the most toxic oxygen species, has the ability to depress Na+,K+-ATPase activity in a dose-dependent manner. Severe inhibition of the hydrolytic activity was observed when higher H2O2 were used. The time course of incubation with 100 μM H2O2 showed a sharp decrease in the enzyme activity during the first 5 min of incubation for both normozoospermic and pathozoospermic men. The enzymatic activity of Na+,K+-ATPase in the sperm was completely destroyed at 20 min for asthenozoospermic men and 30 min for normozoospermic men. We show that an administation of H2O2 inhibited Na+,K+-ATPase activity in normozoospermic samples with IC50 of 106.6 ± 7.9 μM. IC50 for patients with asthenozoospermia was two times less than for healthy men with preserved fertility. For other studied groups, the differences in IC50 were not significant. These observations suggest that Na+,K+-ATPase in pathozoospermic samples is more vulnerable to H2O2-induced damage than in normozoospermic men. The Hill coefficient was significantly increased only for patients with asthenozoospermia, indicating increased positively cooperative binding. The decreases in Na+,K+-ATPase hydrolase activity in H2O2-treated sperm cells in men with normozoospermia were largely attenuated by exogenous GSH at 5 mM. This suggests that GSH partially protects the Na+,K+-ATPase from inhibition under experimental oxidative stress. However, treatment of oligo-, astheno- and oligoasthenozoospermic samples with 100 μM H2O2 and 5 mM GSH did not result in protection of Na+,K+-ATPase against induced oxidation, suggesting that the impaired Na+,K+-ATPase in pathozoospermic samples appears to be an irreversible event. In contrast, presence of GSH only after H2O2 treatment does not reverse Na+,K+-ATPase inhibition. This study has provided a deeper insight into the role Na+,K+-ATPase plays in sperm cells,it also could offer clues to the clinical application of antioxidant therapy in male infertility therapy

Kinetics of inhibitory effect of hydrogen peroxide on activity of plasma membrane transporting Cа2+, Mg2+-ATPase of sperm cells

Decreased potential of spermatozoa fertility is closely associated with the development of oxidative stress and dysfunction of ion-transporting ATPases. Oxidative stress may have negative impact on the activity of membrane-bound enzymes, such as Са2+,Мg2+-АТPase that is involved in maintaining calcium homeostasis in sperm cells. The aim of present work was to evaluate the exogenous H2O2 effect on the main kinetic parameters of ATP hydrolysis by plasma membrane Са2+,Мg2+-АТPase of spermatozoa of fertile (normozoospermia) and infertility (asthenozoospermia) men. Since Са2+,Mg2+-АТPase is one of the targets for the reactive oxygen species and is directly involved in oxidative stress, spermatozoa obtained from normo- and asthenozoospermic samples were subjected to oxidative stress in the form of exogenous H2O2. Then ATP hydrolysis by thapsigargin-resistant Ca2+,Mg2+-ATPase in media with different Ca2+ concentrations was measured. An effective inhibitory effect of H2O2 on the activity of the thapsigargin-resistant component of Са2+,Мg2+-АТPase of sperm cells was demonstrated. In order to elucidate possible mechanisms of change in Ca2+,Mg2+-ATPase activity under H2O2-induced oxidative stress, the concentration curves were linearized using Hanes–Woolf plot {[S]/V; [S]}. The apparent activation constant for Ca2+ (KCa2+) in sperm cell obtained from men with proven fertility was not changed, whereas in the asthenozoospermic samples, it was decreased almost twice under H2O2-induced oxidative stress. These results indicate that in normozoospermic samples H2O2 implements its inhibitory action through the mechanism of uncompetitive inhibition of plasma membrane Ca2+,Mg2+-ATPase activity. According to formal features of kinetics in the asthenozoospermic samples a mixed type of enzyme inhibition occurs under the oxidative stress induced by H2O2. Strategies to protect against a loss in Cа2+,Mg2+-ATPase activity may be useful to prevent the harmful biochemical cascades leading to Ca2+ overload and dysfunction of spermatozoa as a result of the oxidative stress