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ECPB 2017, 80(4): 46–51
https://doi.org/10.25040/ecpb2017.04.046
Research articles

Effect of Hydrogen Peroxide on the Main Kinetic Parameters of ATP Hydrolysis by by Ca2 + ,Mg2 + -ATP-ase in Spermatozoa of Infertile Men

R.V. FAFULA, О.I. MESKALO, E.I. LYCHKOVSKYY, Z.D. VOROBETS
Abstract

Under the physiological conditions, Ca2+ ions regulate sperm motility, hyperactivation, chemotaxis, acrosomal response. [Сa2+]і level in cytoplasm is maintained by Са2+,Мg2+-АТPase which is one of the targets of reactive oxygen species. Hyperproduction of reactive oxygen species can damage sperm cells and is considered to be one of the mechanisms of male infertility. This paper is aimed at evaluation of the H2O2 effect on the main kinetic parameters of ATP hydrolysis by Са2+,Мg2+-АТPase of spermatozoa of fertile (normozoospermia) and infertility men (asthenozoospermia).

Са2+,Мg2+-АТPase activity has been determined spectrophotometrically by production of Pi. Concentration dependencies have been linearized in Lineweaver-Burk plot.

Effective inhibitory effect of H2O2 on thapsigargin-sensitive and thapsigargin-insensitive Са2+,Мg2+-АТPase activity of sperm cells in the fertile and infertile men has been demonstrated. The effects of H2O2 on the main kinetic parameters of the ATP hydrolysis with the involvement of Са2+,Мg2+-АТPase has been studied. In the whole range of studied concentrations of ATP the thapsigargin-sensitive and thapsigargin-insensitive Са2+,Мg2+-АТPase activity of spermatozoa in the fertile and infertile men has been reduced because of presence of H2O2 in the incubation medium. However, the optimal activity of the Са2+,Мg2+-АТPase activity of sperm cells in both normozoospermic and asthenozoospermic men has been observed taking into account 5 mM of ATP in the incubation medium. By linearization of concentration curves in Lineweaver-Burk plot the main kinetic parameters of Ca2+-activated, Mg2+-dependent ATP hydrolysis in the sperm cells of fertile and infertile men have been determined.

Under the influence of H2O2, the initial maximum rate of ATP hydrolysis by thapsigargin-sensitive Са2+,Мg2+-АТPase significantly decreases only in spermatozoa of men with normozoospermia. At the same time, the affinity constant of thapsigargin-sensitive Са2+,Мg2+-АТPase to ATP is significantly increased in spermatozoa of men with asthenozoospermia. A significant increase in the affinity constant of thapsigargin-insensitive Са2+,Мg2+-АТPase to ATP has been found in both normozoospermic and asthenozoospermic men. Moreover, in the pathospermic men the increase in the affinity constant to ATP is more expressed than in normozoospermic men. At the same time, there is a tendency of decreasing in the maximum rate of ATP hydrolysis by thapsigargin-insensitive Са2+,Мg2+-АТPase of spermatozoa of fertile men, but these changes are not significant.

Under conditions of H2O2-induced oxidative stress, inhibition of Са2+,Мg2+-АТPase activity in sperm cells of asthenozoospermic men occurs due to decreasing in the enzyme affinity to the substrate. In spermatozoa of normozoospermic men the inhibition of thapsigargin-sensitive Са2+,Мg2+-АТPase activity is caused by decreasing in the maximum reaction rate, and the inhibition of thapsigargin-insensitive Са2+,Мg2+-АТPase occurs by reducing the affinity to the substrate.

Added: 29.11.2017

Keywords: Са2+, Мg2+-АТPase; inhibition; kinetic analysis; spermatozoa; male infertility

Full text: PDF (Ukr) 1.45M

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