THE NA,K-ATPASE α4 ISOFORM: MECHANISMS OF ACTION, REGULATION AND RELEVANCE TO MALE FERTILITY

Abstract

Ion movement across the plasma membrane is essential for the function of spermatozoa and defects in the mechanisms that drive ion transport between the environment and cell cytoplasm can affect male fertility. The active transmembrane exchange of Na+ and K+ in most cells is under the control of the Na,K-ATPase. Spermatozoa express a specific isoform of the catalytic subunit of the Na,K-ATPase, the α4 polypeptide, which exhibits a series of biochemical properties that are unique. In this thesis, we have studied the mechanisms of action and the role of the α4 isoform in the physiology and fertilizing capacity of sperm. We show that selective inhibition of α4 with ouabain affects many aspects of sperm motility. Inhibition of α4 also results in an increase in intracellular Na+ concentration and this alteration in the Na+ gradient secondarily affects essential parameters of sperm function. Among these is the depolarization of the sperm plasma membrane, increase in cytosolic calcium and acidification of the cells. Since all these changes are known to influence sperm motility, they represent important mechanisms through which α4 is involved in maintenance of normal sperm movement. In addition, we find that α4 activity is regulated with physiological changes in sperm function, increasing during sperm capacitation. This increase in α4 activity results in a decrease in intracellular sodium and in hyperpolarization of the sperm plasma membrane. Concomitantly, the cells show higher total, progressive and hyperactive motility. The capacitation-dependent up-regulation of α4 activity depends on increases of the isoform at the plasma membrane, which appear to be regulated by phosphorylation of serine and tyrosine residues in the α4 isoform. To study the role of α4 in vivo, we have generated mice in which we have expressed the rat α4 isoform in sperm. These mice show increased activity of the α4 isoform, exhibit normal testis and sperm morphology, and no differences in fertility. However, compared to wild type, sperm from transgenic mice displayed plasma membrane hyperpolarization and higher total, progressive and hyperactive motility. In contrast, augmented α4 activity did not produce changes in progesterone induced acrosome reaction, suggesting that the main function of α4 is to maintain sperm motility and not acrosomal exocytosis. As another approach to understanding α4 function, we blocked its expression in mice using knockout technology. Males from α4 null mice are completely sterile, while females are normal, confirming the gender specificity of α4 expression. Sperm from these mice are also unable to fertilize eggs in vitro, and show severe reduction in sperm motility. Absence of α4 causes a characteristic bend in the sperm flagellum, similar to that reported for abnormal sperm ion regulation. Altogether, our results demonstrate that α4 is a specific Na,K-ATPase isoform with a unique role, and that it is an absolute requirement for sperm motility and fertility. In addition, these findings make the α4 isoform an attractive target for male contraception and open the possibility for the potential use of this Na,K-ATPase isoform as a biomarker for male fertility.