Activity of the Na,K‐ATPase α4 Isoform Is Regulated During Sperm Capacitation to Support Sperm Motility

Abstract

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.

Role of human Na,K‐ATPase alpha 4 in sperm function, derived from studies in transgenic mice

“…Sperm hyperactivation is also accompanied by hyperpolarization of the sperm plasma membrane (Hernandez-Gonzalez et al, 2006;Chavez et al, 2013;Lopez-Gonzalez et al, 2014). In previous reports, we observed that a4 activity helps maintain the membrane potential (Jimenez et al, 2012). Using the fluorescent indicator DiSC3(5), as previously described (Hernandez-Gonzalez et al, 2006), we were able to investigate the effect of human a4 expression on membrane potential in sperm from wild-type and transgenic mice.…”

“…Na,K‐ATPase α4 exhibits affinities for Na + ,K + , ATP, and the inhibitor ouabain, which is distinct from the other Na,K‐ATPases; The high affinity of α4 for ouabain has been used as a tool to distinguish the function of α4 from the other Na,K‐ATPase α isoforms (Blanco et al, ; Woo et al, ). This distinct pharmacology has linked Na,K‐ATPase α4 activity to sperm motility, particularly contributing to sperm flagellar beat (Woo et al, ; Woo et al, ; Wagoner et al, ; Jimenez et al, ; Jimenez et al, ). Recent studies in which the α4 isoform was either over‐expressed or knocked‐out in genetically modified mice further established a role for α4 in sperm motility, and showed that this isoform is important for male fertility (Jimenez et al, ,).…”

The Structure and Function of the Na,K-ATPase Isoforms in Health and Disease

“…Interestingly it has been demonstrated that as spermatozoa capacitate in the female genital tract, the catalytic activity of alpha4 is up-regulated due to an increased availability of pumps in the membrane (Jimenez et al, 2012), suggesting that alpha4 supports the hyperpolarization and decreased intracellular sodium concentration characteristic of capacitated spermatozoa. An alternative, and not necessarily conflicting, hypothesis is that since pumps in the membrane of spermatozoa experience dramatic changes in e.g., extracellular sodium levels and membrane potential as the cell passes from testis to oviduct, it could be advantageous with a pump that, like alpha4, responds less to environmental alterations (Clausen et al, 2016).…”

Ouabain Modulates the Distribution of Connexin 43 in Epithelial Cells

“…Binding of ouabain to Na+/K+-ATPase unleash a cascade of interrelated signal transduction pathways that involves inositol (3, 4, 5)-tris-phosphate receptor, Src kinase, tyrosine phosphorylation, epidermal growth factor receptor, Ras and the Ras/Raf/MEK/MAPK signaling pathway [13][14][15]. Following this scheme, ouabain has shown to influence a wide variety of physiological processes, including proliferation [16,17], growth [18], apoptosis [19][20][21] and cell mobility [22] in different tissues and organs. It also has been shown to influence the regulation of salt-sensitive blood pressure [23], salt handling in the kidney [24], vascular tone, Na + homeostasis [25] and embryonic kidney cell´s survival during malnutrition [26].…”