Effect of hydrogen peroxide on Na+,K+-ATPase activity in spermatozoa of infertile men
AbstractNa+,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.
Barwe, S. P., Anilkumar, G., Moon, S. Y., Zheng, Y., Whitelegge, J. P., Rajasekaran, S. A., & Rajasekaran, A. K. (2005). Novel role for Na,K-ATPase in phosphatidylinositol 3-kinase signaling and suppression of cell motility. Molecular Biology of the Cell, 16(3), 1082–1094.
Bogdanova, A., Petrushanko, I., Boldyrev, А., & Gassmann, M. (2006). Oxygen- and redox-induced regulaton of the Na/K ATPase. Current Enzyme Inhibition, 2(1), 37–59.
Derham, B. K., Ellory, J. C., Bron, A. J., & Harding, J. J. (2003). The molecular chaperone a-crystallin incorporated into red cell ghosts protects membrane Na/K-ATPase against glycation and oxidative stress. European Journal of Biochemistry, 270, 2605–2611.
Fafula, R. V., Onofrovych, O. K., Iefremova, U. P., Nakonechnyi, I. A., & Vorobets, Z. D. (2017). Intensity of processes of lipid peroxidation in sperm of men with fertility breach. Bulletin of Problems Biology and Medicine, 135, 199–204.
Groeger, G., Mackey, A. M., Pettigrew, C. A., Bhatt, L., & Cotter, T. G. (2009). Stress-induced activation of Nox contributes to cell survival signalling via production of hydrogen peroxide. Journal of Neurochemistry, 109, 1544–1554.
Januszewski, A. S., Alderson, N. L., Jenkins, A. J., Thorpe, S. R., & Baynes, J. W. (2005). Chemical modification of proteins during peroxidation of phospholipids. Journal of Lipid Research, 56, 1440–1449.
Khundmiri, S. J., Metzler, M. A., Ameen, M., Amin, V., Rane, M. J., & Delamere, N. A. (2006). Ouabain induces cell proliferation through calcium-dependent phosphorylation of Akt (protein kinase B) in opossum kidney proximal tubule cells. American Journal of Physiology, Cell Physiology, 291(6), 1247–1257.
Koçak-Toker, N., Aktan, G., & Aykaç-Toker, G. (2002). The role of Na,K-ATPase in human sperm motility. International Journal of Andrology, 25(3), 180–185.
Koppers, A. J., De Iuliis, G. N., Finnie, J. M., McLaughli, E. A., & Aitken, R. J. (2008). Significance of mitochondrial reactive oxygen species in the generation of oxidative stress in spermatozoa. The Journal of Clinical Endocrinology and Metabolism, 93(8), 3199–3207.
Liu, C.-C.,Garcia, A., Mahmmoud, Y. A., Hamilton, E. J., Galougahi, K. K., Fry, N. A. S., Figtree, G. A., Cornelius, F., Clarke, R. J., & Rasmussen, H. H. (2012). Susceptibility of β1 Na+-K+ pump subunit to glutathionylation and oxidative inhibition depends on conformational state of pump. Journal of Biological Chemistry, 287, 12353–12364.
Liu, J.., Kesiry, R., Periyasamy, S. M., Malhotra, D., Xie, Z., & Shapiro, J. I. (2004). Ouabain induces endocytosis of plasmalemmal Na/K-ATPase in LLC-PK1 cells by a clathrin-dependent mechanism. Kidney International, 66, 227–241.
Liu, L., Li, J., Liu, J., Yuan, Z., Pierre, S. V., Qu, W., Zhao, X., & Xie, Z. (2006). Involvement of Na+/K+-ATPase in hydrogen peroxide-induced hypertrophy in cardiac myocytes. Free Radical Biology and Medicine, 41(10), 1548–1556.
Mahfouz, R. Z., Aziz, N., Sharma, R., Bykova, M., Sabanegh, E., & Agarwal, A. (2008). Assessment of intracelular human sperm reactive oxygen species after hydrogen peroxide exposure using four different probes. Fertility and Sterility, 90(Suppl 1), 320–321.
Mahfouz, R. Z., du Plessis, S. S., Aziz, N., Sharma, R., Sabanegh, E., & Agarwal, A. (2010). Sperm viability, apoptosis, and intracellular reactive oxygen species levels in human spermatozoa before and after induction of oxidative stress. Fertility and Sterility, 93(3), 814–821.
McKenna, M. J., Medved, I., Goodman, C. A., Brown, M. J., Bjorksten, A. R., Murphy, K. T., Petersen, A. C., Sostaric, S., & Gong, X. (2006). N-acetylcysteine attenuates the decline in muscle Na+,K+-pump activity and delays fatigue during prolonged exercise in humans. Journal of Physiology, 576(1), 279–288.
Meharg, J. V., McGowan-Jordan, J., Charles, A., Parmelee, J. T., Cutaia, M. V., & Rounds, S. (1993). Hydrogen peroxide stimulates sodium-potassium pump activity in cultured pulmonary arterial endothelial cells. American Journal of Physiology, 265(6), 613–621.
Meskalo, О., Fafula, R. V., Lychkovskyj, E. I., & Vorobets, Z. D. (2017). Na+,K+-ATPase and Ca2+,Mg2+-ATPase activity in spermatozoa of infertile men with different forms of pathospermia. Studia Biologica, 11(2), 5–12.
Nguyen, N. T., Wallace, D. P., & Blanco, G. (2007). Ouabain binds with high affinity to the Na,K-ATPase in human polycystic kidney cells and induces extracellular signal-regulated kinase activation and cell proliferation. Journal of the American Society of Nephrology, 18(1), 46–57.
Petrushanko, I. Y., Yakushev, S., Mitkevich, V. A., Kamanina, Y. V., Ziganshin, R. H., Meng, X., Anashkina, A. A., Makhro, A., Lopina, O. D., Gassmann, M., Makarov, A. A., & Bogdanova, A. (2012). S-glutathionylation of the Na,K-ATPase catalytic alpha subunit is a determinant of the enzyme redox sensitivity. Journal of Biological Chemistry, 287, 32195–32205.
Petrushanko, N., Bogdanov, E., Bulygina B., Grenacher, T., Leinsoo, A., Boldyrev, M., Gassmann, M., & Bogdanova, A. (2006). Na-K-ATPase in rat cerebellar granule cells is redox sensitive. American Journal of Physiology, Regulatory Integrative and Comparative Physiology, 290(4), 916–925.
Reifenberger, M. S., Arnett, K. L., Gatto, C., & Milanick, M. A. (2008). The reactive nitrogen species peroxynitrite is a potent inhibitor of renal Na-K-ATPase activity. American Journal of Physiology, 295(4), 1191–1198.
Rocco-Machado, N., Cosentino-Gomes, D., & Meyer-Fernandes, J. R. (2015). Modulation of Na+/K+ ATPase activity by hydrogen peroxide generated through heme in L. amazonensis. PLoS One, 10(6), e0129604.
Sanchez, G., Nguyen, A. T., Timmerberg, B., Tash, J. S., & Blanco, G. (2006). The Na,K-ATPase a4 isoform from humans has distinct enzymatic properties and is important for sperm motility. Molecular Human Reproduction, 12, 565–576.
Schulpis, K. H., Papassotiriou, I., Parthimos, T., Tsakiris, T., & Tsakiris, S. (2006). The effect of L-cysteine and glutathione on inhibition of Na+,K+-ATPase activity by aspartame metabolites in human erythrocyte membrane. European Journal of Clinical Nutrition, 60(5), 593–597.
Shi, L. G., Yang, R. J., Yue, W. B., Xun, W. J., Zhang, C. X., Ren, Y. S., Shi, L., & Lei, F. L. (2010). Effect of elemental nano-selenium on semen quality, glutathione peroxidase activity, and testis ultrastructure in male Boer goats. Animal Reproduction Science, 118 (2–4), 248–254.
Thundathil, J. C., Anzar, M., & Buhr, M. M. (2006). Na+/K+-ATPase as a signaling molecule during bovine sperm capacitation. Biology of Reproduction, 75, 308–317.
Tsai-Turton, M., & Luderer, U. (2006). Opposing effects of glutathione depletion and follicle-stimulating hormone on reactive oxygen species and apoptosis in cultured preovulatory rat follicles. Endocrinology, 147, 1224–1236.
Vignini, A., Buldreghini, E., Nanetti, L., Amoroso, S., Boscaro, M., Ricciardo-Lamonica, G., Mazzanti, L., & Balercia, G. (2009). Free thiols in human spermatozoa: are Na+/K+-ATPase, Ca2+-ATPase activities involved in sperm motility through peroxynitrite formation? Reproductive BioMedicine Online, 18(1), 132–140.
WHO Laboratory Manual for the examination and processing of human semen, 5th ed. (2010). WHO.
Authors retain copyright and grant the journal right of first publication with the work simultaneously licensed under a Creative Commons «Attribution» 4.0 License that allows others to share the work with an acknowledgement of the work's authorship and initial publication in this journal.