Introduction. Reactive oxygen species are important regulators of sperm functions (hyperactivation, capacitation and acrosome reaction). However, an excessive ROS generation appears to be related to male infertility. Oxidative stress may have a negative impact on the activity of membrane-bound enzymes, such as Са2+, Мg2+-АТPase, which are involved in maintaining calcium homeostasis in spermatozoa.

The objective of the present work was to evaluate the H2O2 effect on the main kinetic parameters of ATP hydrolysis by thapsigargin-sensitive Са2+, Мg2+-АТPase of spermatozoa of fertile (normozoospermia) and infertile men (asthenozoospermia).

Material and Methods. 10 infertile men with asthenozoospermia and 8 healthy men with somatic fertility and confirmed parenthood (normozoospermia) were involved in this study. Са2+, Мg2+-АТPase activity was determined spectrophotometrically by production of Pi. Thapsigargin- sensitive ATPase activity was evaluated in presence of 10-6 M thapsigargin. The aliquots of sperm cells were subjected to exogenous reactive oxygen species stimulation with H2O2 as an oxidizing agent (5 min, 37 °C, 5 % CO2) at different concentrations (25, 50, 100 and 200 μM). Untreated cells were used for controlling the study.

Results. Since Са2+,Mg2+-АТPase is one of the targets for reactive oxygen species and is directly involved in oxidative stress, spermatozoa obtained from normo- and pathozoospermic samples were subjected to oxidative stress in the form of exogenous H2O2 and then ATP hydrolysis by thapsigargin-sensitive Ca2+, Mg2+-ATPase in media with different Ca2+ concentrations was measured. Enzyme activity of thapsigargin-sensitive Са2+, Mg2+-АТPase was reduced in the presence of H2O2 in the incubation medium. In order to elucidate the possible mechanisms of change in thapsigargin-sensitive Са2+, Mg2+-АТPase activity under H2O2-induced oxidative stress the concentration curves were linearized using Hanes–Woolf plot {[S]/V; [S]}. It is apparent from the kinetics of hydrolysis of ATP that the maximum rate of ATP hydrolysis in normozoospermic samples was decreased dramatically (more than in 2.5 fold). In patients with astnenozoospermia the decrease in Vmax was not sharp, but gradual. The apparent activation constant for Ca2+ (KCa2+) has not changed under H2O2-induced oxidative stress in the sperm cell obtained from both normo- and pathozoospermic samples. All these results indicate that in both normo- and pathozoospermic samples H2O2 implements its inhibitory action through the mechanism of uncompetitive inhibition of thapsigargin-sensitive Ca2+, Mg2+-ATPase activity (Vmax decreases with no appreciable change in KCa2+). Strategies for protecting against 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 oxidative stress.

Conclusion. Under H2O2-induced oxidative stress, the reduction in thapsigargin-sensitive Cа2+,Mg2+-ATPase activity in both normo- and pathozoospermic samples was associated with a decrease in maximum rate of ATP hydrolysis (Vmax) with no appreciable change in the affinity of the enzyme for Ca2+.

Recieved: 06.02.2018

Ключові слова: Са2+, Мg2+-АТPase, hydrogen peroxide, thapsigargin, inhibition, spermatozoa, male infertility, pathospermia

Повний текст: PDF (Eng)

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