While the function of the ubiquitous Na,K-ATPase a1 subunit has been well documented, the role of the sperm-specific a4 isoform of this ion transporter is less known. We have explored the importance of a4 in rat sperm physiology by taking advantage of the high sensitivity of this isoform for the inhibitor ouabain. Using concentrations that selectively block a4 activity, we found ouabain to reduce not only sperm total motility, but also multiple parameters of sperm movement, including progressive motility, straight line, curvilinear, and average path velocities, lateral head displacement, beat cross frequency, and linearity. According to a direct role of a4 in Na C transport, ouabain inhibition of a4 increased [Na C ] i in the male gametes. In addition, interference of a4 activity with ouabain produced cell membrane depolarization, diminished pH, and increased [Ca 2C ] i in spermatozoa. Inhibition of a4 was sufficient to cause all these effects and additional blockage of a1, the other Na,K-ATPase a isoform expressed in sperm, and higher doses of ouabain did not result in further changes in the cell parameters studied. These results show that a4 is the Na,K-ATPase isoform primarily involved in controlling the transmembrane Na C gradient in sperm, and that a4 activity is necessary for maintaining membrane potential, [Ca ] i , and acid-base balance suggests that their regulation is the mechanism by which a4 maintains motility of the male gametes.

Regulation of ion balance in spermatozoa has been shown to be essential for sperm motility and fertility. Control of intracellular ion levels requires the function of distinct ion-transport mechanisms at the cell plasma membrane. Active Na + and K + exchange in sperm is under the control of the Na,K-ATPase. Two molecular variants of the catalytic subunit of the Na,K-ATPase, α1 and α4, coexist in sperm. These isoforms exhibit different biochemical properties; however, their function in sperm fertility is unknown. In this work, we show that Na,K-ATPase α4 is essential for sperm fertility. Knockout male mice lacking α4 are completely sterile and spermatozoa from these mice are unable of fertilizing eggs in vitro. Furthermore, α4 deletion results in severe reduction in sperm motility and hyperactivation typical of sperm capacitation. In addition, absence of α4 causes a characteristic bend in the sperm flagellum, indicative of abnormal sperm ion regulation. Accordingly, α4-null sperm present increased intracellular Na + and cell plasma membrane depolarization. These results are unique in demonstrating the absolute requirement of α4 for sperm fertility. Moreover, the inability of α1 to compensate for α4 suggests that α4 is the Na,K-ATPase-α isoform directly involved in sperm fertility. Our findings show α4 as 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.

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The Na,K-ATPase alpha4 (ATP1A4) isoform is specifically expressed in male germ cells and is highly prevalent in spermatozoa. Although selective inhibition of alpha4 activity with ouabain has been shown to affect sperm motility, a more direct analysis of the role of this isoform in sperm movement has not yet been demonstrated. To establish this, we engineered transgenic mice that express the rat alpha4 isoform fused to green fluorescent protein in male germ cells, under the control of the mouse protamine 1 promoter. We showed that the rat Atp1a4 transgene is expressed in mouse spermatozoa and that it is localized to the sperm flagellum. In agreement with increased expression of the alpha4 isoform, sperm from transgenic mice displayed higher alpha4-specific Na,K-ATPase activity and binding of fluorescently labeled ouabain than wild-type mice. In contrast, expression and activity of ATP1A1 (alpha1), the other Na,K-ATPase alpha isoform present in sperm, remained unchanged. Similar to wild-type mice, mice expressing the alpha4 transgene exhibited normal testis and sperm morphology and no differences in fertility. However, compared to wild-type mice, sperm from transgenic mice displayed plasma membrane hyperpolarization and higher total and progressive motility. Other parameters of motility also increased, including straight-line, curvilinear, and average path velocities and amplitude of lateral head displacement. In addition, sperm from the transgenic mice showed enhanced sperm hyperactive motility, but no changes in progesterone-induced acrosome reaction. Altogether, these results provide new genetic evidence for the role of the ATP1A4 isoform in sperm motility, under both noncapacitating and capacitating conditions.

ABSTRACT:The a4 polypeptide is a testis-specific isoform of the catalytic subunit of the Na,K-ATPase, which is essential for sperm motility and fertility. In the present study, we have investigated the regulation of activity of the a4 isoform and the relevance of this event for sperm capacitation. We have performed this by taking advantage of the selective high affinity of a4 for the inhibitor ouabain. Our results show that ouabain-sensitive hydrolysis of ATP and uptake of 86 Rb, corresponding to the enzymatic and ion transport activities of a4, respectively, increased during sperm capacitation in a time-dependent manner. Specific labeling of a4 with the fluorescent indicator bodipy-ouabain and immunoblot analysis of biotinylated and streptavidin-precipitated sperm plasma membrane proteins indicated a capacitation-and time-dependent rise in levels of active a4 isoform at the sperm surface. Ouabain inhibition of a4 blocked the increase in total sperm motility and the hyperactive motility pattern characteristic of sperm capacitation. Moreover, interference of a4 activity with ouabain partially prevented the intracellular decrease in Na + and the plasma membrane hyperpolarization that typically accompany sperm capacitation. In contrast, ouabain inhibition of a4 did not affect the spontaneous sperm acrosomal reaction following capacitation. Together, these results demonstrate that Na,K-ATPase a4 activity is up-regulated during sperm capacitation through mechanisms that involve both increases in molecular activity and levels of a4 at the sperm plasma membrane. This increase in a4 activity helps maintain the changes in motility that are associated with sperm capacitation, emphasizing the biologic relevance of the Na,K-ATPase a4 isoform in sustaining sperm function.