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

Characteristics of Ca2+, Mg2+-dependent ATP hydrolysis in sperm cells of infertile men

Disturbances of fertilizing potential of spermatozoa are closely associated with dysfunction of ion-transporting ATPases, in particular Ca2+, Mg2+-АТРase. Reduced activity of tapsigargin-resistant and tapsigargin-sensitive Ca2+, Mg2+-АТРase leads to disruption of Ca2+-homeostasis and is characteristic for abnormal spermatozoa (pathoospermia). In order to study the peculiarities of action of Ca2+, Mg2+-АТРase, we determined the initial reaction rate, the maximum (plateau) amount of the reaction product and the cha­­racteristic reaction time. To determine these kinetic parameters of Ca2+, Mg2+-dependent hydrolysis of ATP catalyzed by Ca2+, Mg2+-ATPase, the dynamics of product accumulation of the ATP-hydrolases reaction was studied. The obtained curves were linearized in the coordinates {P/t; P}. Analyzing the changes in the activity of Ca2+, Mg2+-ATPase, the kinetics of primary-active transport of calcium ions through the plasma membrane and membranes of intracellular Ca2+-stores in saponin-permeabilized spermatozoa of infertile men was studied. It was shown that in normozoospermic samples, the transport of Ca2+ ions through the plasma membrane is characterized by a higher capacity than through the membranes of intracellular Ca2+-stores, but it occurs with practically the same initial velocity and characteristic reaction time. It was found that in pathospermic samples, transport of Ca2+ ions with the participation of both components of Ca2+, Mg2+-ATPase occurs less intensively and is characterized by a lower capacity compared to spermatozoa of men with preserved fertility. Specific changes in the kinetic parameters of Ca2+, Mg2+-dependent hydrolysis of ATP lead to inhibition of tapsigargin-resistant and tapsigargin-sensitive Ca2+, Mg2+-ATPase activity and cause a decrease in fertilizing potential of spermatozoa

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

Na+, K+-ATPase and Ca2+, Mg2+-ATPase activity in spermatozoa of infertile men with different forms of pathospermia

Ion-exchanging ATPases play an essential role in biology of spermatozoa, inclu­ding their motility, hyperactivation, chemotaxis, acrosome reaction etc. The aim of present study was to analyze Na+, K+-ATPase and Ca2+, Mg2+-ATPase activities in spermatozoa of the infertile men with different forms of pathospermia and to explore a possible role that they may play in male infertility. A significant reduction in ouabain-sensitive Na+, K+-ATPase activity in sperm cells of infertile men with oligozoo-, asthenozoo-, oligoasthenozoo- and leucocytospermia was shown. The results show that asthenozoo-, oligoasthenozoo- and leucocytospermic patients have significantly impaired thapsigargin-sensitive and thapsigargin-insensitive Ca2+, Mg2+-ATPase activity compared to healthy men. However, Ca2+, Mg2+-ATPase activity has a tendency to increase in patients with oligozoospermia. The depressed ATPase activity in the infertile men could be due to a reduction in intracellular adenosine triphosphate level and damage of spermal membranes caused by lipid peroxidation products. The most significant decrease in Ca2+, Mg2+-ATPase activity was observed in patients with leucocytospermia which could be explained by an excessive formation of the reactive oxygen species by the leucocytes. It is suggested that a decrease in the ion-exchanging ATPase activity may damage sperm functions and be one of possible causes of male infertility