Na⁺,K⁺-ATPase α isoforms in sperm show a highly structured and distinct pattern of distribution

Abstract

Na⁺,K⁺-ATPase α4 is a unique Na⁺ and K⁺ transporter of the plasma membrane of spermatozoa, which is essential for male fertility. While previous studies have found Na⁺,K⁺-ATPase α4 to be mainly expressed in the sperm flagellum, less is known about its localization in the sperm head. Moreover, the spatial arrangement of Na⁺,K⁺-ATPase α4 at the subcellular level and its relationship to the functional state of the cells are unclear. We studied this here using stimulated emission depletion (STED) super-resolution microscopy. We show that, under noncapacitated conditions, Na⁺,K⁺-ATPase α4 is distributed in a trilinear pattern along the midpiece and as a scattered single line along the principal piece of the sperm flagellum. Under capacitated conditions, Na⁺,K⁺-ATPase α4 pattern undergoes remodelling and its distribution shifts to a single line along the flagellum. On the other hand, Na⁺,K⁺-ATPase α1, the somatic isoform of Na⁺,K⁺-ATPase, also present in sperm, exhibits a similar trilaminar localization at the flagellar midpiece but a bilinear pattern in the principal piece. This distribution, unlike that of Na⁺,K⁺-ATPase α4, does not change during sperm capacitation. We also found Na⁺,K⁺-ATPase α1 and α4 in the sperm head, where it presents a complex distribution both under non capacitated and capacitated conditions. These differences in the localization pattern and spatial dynamics of Na⁺,K⁺-ATPase isoform expression, along with their different functional properties, highlight the distinct roles that both isoforms play to support sperm function.

In Brief:

This manuscript shows that the Na⁺ and K⁺ transporter Na⁺,K⁺-ATPase α4, specific of sperm, is expressed on the surface of the sperm head and flagellum in a very structured manner. This is also true for Na⁺,K⁺-ATPase α1, the other Na⁺,K⁺-ATPase isoform present in sperm and also all cells. However, Na⁺,K⁺-ATPase α4 distribution changes when the cells are capacitated, an event necessary for fertilization. This dynamic remodeling, along with the distinct functional properties of Na⁺,K⁺-ATPase α4 and α1, provides evidence for the refined level of specialization that sperm have developed to achieve the amazing goal of fertilizing the oocyte.

Introduction

Spermatozoa maintain a tight regulation of their intracellular ion composition, which is essential for their function and fertilization capacity. To achieve this, sperm express a series of ion transport proteins at their cell plasma membrane, some of which are unique and not shared with those of somatic cells (Darszon, et al. 2006, Darszon, et al. 2001, Miller, et al. 2015, Navarro, et al. 2008, Nowicka-Bauer and Szymczak-Cendlak 2021, Puga Molina, et al. 2018). Among these transporters is Na⁺,K⁺-ATPase (EC/7.2.2.13), an integral plasma membrane protein complex that utilizes the energy from ATP hydrolysis to move 3 Na⁺ out of the cell in exchange for 2 K⁺ that are brought in (Blanco 2005, Dyla, et al. 2020, Jorgensen, et al. 2003). The asymmetrical Na⁺ and K⁺ distribution that Na⁺,K⁺-ATPase creates across the cell surface is crucial for various cellular and physiological processes including maintaining cell ion homeostasis, cell volume, resting membrane potential, and sodium-dependent secondary transport of different solutes and ions across the cell plasma membrane (Blaustein 2018, Contreras, et al. 2024).

The ion transport function and enzymatic hydrolysis of ATP catalyzed by Na⁺,K⁺-ATPase primarily depend on the α polypeptide or catalytic subunit of Na⁺,K⁺-ATPase. The other main subunit that constitutes Na⁺,K⁺-ATPase, the glycosylated β subunit, works as a chaperone molecule that facilitates the folding and delivery of the whole Na⁺,K⁺-ATPase transport complex to the plasma membrane (Blanco 2005, Geering 1991, Vagin, et al. 2007). Mammalian cells express multiple isoforms of the Na⁺,K⁺-ATPase α (α1, α2, α3, and α4) and β (β1, β2, and β3) subunits. The pairing of Na⁺,K⁺-ATPase α and β isoforms in different combinations generates distinct Na⁺,K⁺-ATPase isozymes, each of which exhibits a tissue-specific and developmentally regulated pattern of expression and have distinct functional properties (Blanco and Mercer 1998, Clausen, et al. 2017, Mobasheri, et al. 2000, Teixeira, et al. 2003).

Mammalian male germ cells of the testis are the only cells that express the Na⁺,K⁺-ATPase α4 isoform. In addition, they also express the ubiquitous Na⁺,K⁺-ATPase α1 polypeptide of somatic cells and two of the β subunits (β1 and β3) (Jimenez, et al. 2011a). Na⁺,K⁺-ATPase α4 and α1 exhibit different affinities for Na⁺ and K⁺, with Na⁺,K⁺-ATPase α4 having a greater affinity for intracellular Na⁺ activation and a lower affinity for K⁺ compared to Na⁺,K⁺-ATPase α1 (Blanco 2005, Blanco and Mercer 1998). These particular biochemical properties better adapt Na⁺,K⁺-ATPase to the function of sperm. We have previously shown that genetic deletion of Na⁺,K⁺-ATPase α4 in mice results in complete infertility of male mice, with female mice being unaffected (Jimenez, et al. 2011a, McDermott, et al. 2021). Moreover, sperm from Na⁺,K⁺-ATPase α4 knock-out mice are unable to fertilize oocyte in-vitro (Jimenez, et al. 2011a). The main consequence of Na⁺,K⁺-ATPase α4 disruption is a severe reduction of sperm total and hyperactive motility, a characteristic pattern of sperm movement that sperm acquires after capacitation, which is essential for fertilization (Jimenez, et al. 2011a, Sanchez, et al. 2006, Suarez and Ho 2003). The mechanisms underlying this mouse model of asthenospermia are derived from the dissipation of the transmembrane Na⁺ gradient, depolarization of plasma membrane, cytoplasmic acidification, elevated Ca²⁺ levels, and reduced ATP production (Jimenez, et al. 2010, Numata, et al. 2022).

While previous studies have explored the overall expression and physiological relevance of Na⁺,K⁺-ATPase α4 in sperm function, there is little information regarding the subcellular organization of this protein at the plasma membrane. Na⁺,K⁺-ATPase α4 has been found to be localized mainly in the midpiece of the mouse sperm flagellum, with much lower presence in the sperm head. However, these studies were limited by the use of low-resolution microscopy (Jimenez, et al. 2011b, McDermott, et al. 2012). To determine the spatio-functional relationship of Na⁺,K⁺-ATPase α4, we here examined the 3D distribution of Na⁺,K⁺-ATPase α4 in sperm using stimulated emission depletion (STED) super-resolution microscopy. The nanometer-scale resolution that can be obtained with this equipment revealed that in noncapacitated sperm, Na⁺,K⁺-ATPase α4 shows a trilinear pattern along the flagellar midpiece and a single punctated line at the principal piece of the sperm flagellum. Interestingly, this distribution is subjected to remodeling when sperm undergoes capacitation, with Na⁺,K⁺-ATPase α4 being reorganized into a single column along the sperm flagellum. In contrast, Na⁺,K⁺-ATPase α1 has a trilinear distribution at the midpiece and bilinear localization at the principal piece, which is independent of the capacitated state of the cells. Moreover, we found that both Na⁺,K⁺-ATPase α4 and α1 are also present in the sperm head, something that was missed in our previous studies using regular fluorescence microscopy.

Results and Discussion

Na⁺,K⁺-ATPase α4 is organized in three columns in the midpiece of noncapacitated sperm

Na⁺,K⁺-ATPase α4 has been shown to be mainly expressed at the plasma membrane of the sperm flagellum, where it plays a critical role in sperm motility. However, previous studies were limited to the use of conventional fluorescence microscopy, which did not provide the resolution needed to distinguish the compartmentalization of proteins within different regions of the cell plasma membrane. This resulted in images that identified Na⁺,K⁺-ATPase α4 evenly present along the midpiece of the sperm flagellum, with lower expression in the principal piece and minimal or not appearance in the sperm head (Hlivko, et al. 2006, Jimenez, et al. 2011b, McDermott, et al. 2012, Wagoner, et al. 2005, Woo, et al. 2002). In contrast, STED microscopy provides a resolution of 40 nm surpassing the diffraction-limitation of regular fluorescent microscopes, thus enabling the visualization of proteins with enhanced resolution, and allowing to establish their subcellular organization at the nanometer level scale (Calovi, et al. 2021, Frolikova, et al. 2023). This is particularly important when considering cells like mouse spermatozoa, in which the flagellum has a width of ~1 µm and a total length of ∼120 µm (Albrechtová, et al. 2014, Cummins and Woodall 1985).

To identify Na⁺,K⁺-ATPase α4, we here used an antibody that we made against a N-terminal sequence (RPSTRSSTTNRQPKMKRR) of Na⁺,K⁺-ATPase α4, which shows excellent Na⁺,K⁺-ATPase α4 isoform selectivity when tested against tissues containing Na⁺,K⁺-ATPase α isoforms different from α4 (Fig. S1A). To further determine the specificity of this antibody, we tested its reactivity toward sperm from a mouse, which we had previously engineered, and lacks Na⁺,K⁺-ATPase α4 (Jimenez, et al. 2011a). Our results showed no label in the Na⁺,K⁺-ATPase α4 knock-out sperm, but nicely identified the protein in wild type sperm (Fig.S1B). This showed that the anti-Na⁺,K⁺-ATPase α4 antibody has the required specificity in identifying our protein of interest and therefore, appeared amenable for use in STED microscopy.

Interestingly, our analysis of wild type sperm with STED microscopy revealed that, rather than being evenly distributed over the entire surface of the cell plasma membrane, Na⁺,K⁺-ATPase α4 exhibits a highly organized cell surface pattern of distribution (movie S1). Thus, in sperm collected from the caudal portion of mouse epididymis and maintained under noncapacitated conditions, Na⁺,K⁺-ATPase α4 is organized in three lanes that run along the midpiece section of the sperm flagellum (Fig 1A,1B). This trilinear organization becomes discontinuous once entering the principal piece and appears at that point as a single line (Fig 1C). This pattern of distribution was the most commonly seen event and was observed in more than 80% of the cells present in each field of study.

Figure 1: Na⁺,K⁺-ATPase α4 organization along the flagellum of noncapacitated sperm.

(A) 3D reconstructed image of the sperm flagellum showing distribution of Na⁺,K⁺-ATPase α4 (NKAα4) along the whole length of the flagellum, including the midpiece (MP) and principal piece (PP). (B) Trilinear distribution of Na⁺,K⁺-ATPase α4 in the midpiece (MP). The scale bars represent 5 µm. (C) Sperm flagellar segment showing the mid-principal piece boundary demonstrates the three lanes merging into a single lane at the end of midpiece. (D) The depth color coded 3D image of Na⁺,K⁺-ATPase α4 is showed in the whole flagellum. (E) The y-z cross section of the image obtained from D, sectioned at the midpiece (at the site marked with an arrow in D) showing three tight clusters (1, 2 and 3) of Na⁺,K⁺-ATPase α4. (F) The y-z cross section of the image obtained from the principal piece (PP) (at the site marked with an arrow in D) showing one cluster. Colors in D, E and F represent the z positions (refer to the color scale bar G). (H) Distance between Na⁺,K⁺-ATPase α4 clusters measured from the cross section of 3D images using LAS X analysis software. D1 represents the distance between lane 1 and 3, D2 is the distance between lane 1 and 2 and D3 is the distance between lane 2 and 3. Bars represent the mean ±SEM for 8 different mice. ns indicates no significant statistical differences between values, with P < 0.05. (I) Schematic of the spatial distribution of three Na⁺,K⁺-ATPase α4 (NKAα4) clusters at the cross section of the midpiece shown in (E). The clusters or the lanes are equally distant from each other as derived from (H), forming an equilateral triangle connecting them as depicted in (I). The schematic was created with BioRender.com.

To determine the spatial position of the Na⁺,K⁺-ATPase α4 lanes, we obtained cross sections of the 3D STED images of the sperm mid- and principal piece. These images are shown as depth coded along the z axis direction (Fig 1D). The arrows in fig 1D mark the sites of the cross section from midpiece and principal piece resulting in fig 1E and 1F respectively. As shown, Na⁺,K⁺-ATPase α4 is arranged in three tight clusters in the midpiece that we designated as 1, 2 and 3 (Fig 1E), whereas it forms a one compact cluster at the flagellar principal piece (Fig 1F). From the color coded of depth, it can be estimated that cluster 1 and 3 lie parallel to each other being at the same level of z axis, whereas cluster 2 is on the top of the z axis (Fig 1E,1G). The distances between the three lanes were determined using the Leica Application Suite X (LAS X) analysis tool. This showed that the distance between lanes 1 and 3 (shown as D1) was 797 nm, between lanes 1 and 2 (designated D2) was 843 nm, and between lanes 2 and 3 (named D3) was 856 nm (Fig 1H). A one-way anova test showed that the mean distances were not statistically different. Therefore the Na⁺,K⁺-ATPase α4 arrangement shows that the lines are equally spaced. Figure 1I illustrates a schematic of the three clusters in the longitudinal cross-section, where the distances between the lanes (1, 2, and 3) are represented as D1, D2, and D3, respectively, forming an equilateral triangle. For further confirmation, we also determined the angle between the lanes from the cross section using the LAS X software and found that the angle between the lanes is ~60 ° suggesting the proteins are spatially organized in three equally spaced lanes.

The cross section of the mouse sperm flagellum is approximately circular (Phillips 1972). Based on the distribution of Na⁺,K⁺-ATPase α4, which is present at the sperm plasma membrane (Jimenez, et al. 2012), the diameter of the mouse sperm flagellar midpiece can be predicted using the formula:

$$\text{R}=\raisebox{1ex}{$a$}\!\left/ \!\raisebox{-1ex}{$\sqrt{3}$}\right.$$

Where, R is the radius of the circle and a is the side of the equilateral triangle inscribed within the circle. Taking the length of the side 832 (mean of D1, D2 and D3), we calculated a radius of 480 nm and hence the diameter of the midpiece stands at approximately 960 nm. This supports previous finding where the width of the mouse sperm tail was reported to go from 900 nm to 1 µm in mice (Cummins and Woodall 1985).

The distinct arrangement of Na⁺,K⁺-ATPase α4 shows the remarkable high organization that this ion transporter has in sperm. A particular arrangement for other ion transporters in sperm has also been reported by other authors. For instance, the sperm-specific Ca⁺ channel CatSper exhibits a quadrilateral organization along the principal piece of the sperm flagellum (Chung, et al. 2014). Likewise, the sperm H⁺ channel Hv1 also present in the flagellar principal piece, displays an asymmetric bilateral distribution. (Miller, et al. 2018).

Na⁺,K⁺-ATPase α4 is redistributed upon capacitation

We also determined the spatial organization of Na⁺,K⁺-ATPase α4 after incubating mouse sperm in medium which supports capacitation. To achieve capacitation, sperm was placed in Card FertiUp Preincubation Medium, which is a medium successfully used for in vitro fertilization purposes. This preincubation medium is optimized to maximize sperm capacitation and is a more efficient inducer of sperm capacitation than BSA. It contains, among other components, methyl-β-cyclodextrin which efficiently and rapidly extracts cholesterol from the sperm plasma membrane. Using Card FertiUp medium, we observed a twofold increase in sperm hyperactive motility compared to sperm capacitated under BSA and bicarbonate containing media. Moreover, sperm capacitation occurs faster in Card FertiUp media as opposed to BSA and bicarbonate containing media (60 vs 90 minutes respectively; data not shown).

Interestingly, under capacitated conditions, we found an important change in the pattern of Na⁺,K⁺-ATPase α4 distribution. The trilinear arrangement observed under the non-capacitation state, now reorganized as a single line with predominant presence in the midpiece and continuing, although with a more punctate configuration, along the principal piece and rest of the flagellum (Fig 2A,2B). The cross section at the midpiece from the depth coded 3D reconstructed image (Fig 2B) showed one compact cluster (Fig 2C, 2D). Approximately 26 ± 5% of the cells exhibited the reorganization of Na⁺,K⁺-ATPase α4. The remaining of the cells show the trilinear pattern of distribution found in noncapacitated cells. To confirm the capacitated status of the cells, we evaluated their ability to develop hyperactive motility, a characteristic high amplitude-low frequency movement that sperm acquires at capacitation (Jimenez, et al. 2010, Kay and Robertson 1998, Suarez and Ho 2003). Figure 2E shows that 41 ± 4% of sperm acquired hyperactivated motility as measured using computer assisted sperm analysis (CASA). This confirmed the capacitated state of the cells under our study conditions. We also established that there is a relatively good correlation between the amount of cells that achieve hyperactivation and the redistribution of Na⁺,K⁺-ATPase α4 in the cells (Fig 2G).

Figure 2: Na⁺,K⁺-ATPase α4 is redistributed upon sperm capacitation.

(A) 3D reconstructed image of Na⁺,K⁺-ATPase α4 along the entire flagellum in capacitated sperm. (B) Depth coded 3D image of Na⁺,K⁺-ATPase α4. (C) The y-z cross section of the flagellum showing a compact Na⁺,K⁺-ATPase α4 cluster. The site of cross section is marked with an arrow in B. The scale bars are 5 µm. Colors in B and C represent the z positions (see color scale bar D). (E) Hyperactive motility in noncapacitated (Noncap) and capacitated (Cap) sperm evaluated with CASA. (F) A schematic showing the three lanes to be remodeled into one. The schematic was created with BioRender.com. Experiments were conducted three times, and fifteen to twenty cells were analyzed in each of the experiments. (G) The scattered plot showing a positive trend between the hyperactive motility and the redistribution of Na⁺,K⁺-ATPase α4 (NKA α4) in sperm (n=3). (H) Percentage of cells with redistributed NKAα4 at different incubation time of capacitation were determined (Pearson’s coefficient, r = 0.97; p value <0.05). Sperm collected from cauda epididymis are incubated with FertiUp medium for 20 minutes, 40 minutes and 60 minutes. The percentage of hyperactive motile cells was also determined at those time points (Pearson’s coefficient, r = 0.95; p value <0.05).

To determine whether the relocalization of protein is dependent on the time of capacitation, we studied the development of the changes in Na⁺,K⁺-ATPase α4 distribution and sperm hyperactivated motility at different incubation times in the capacitation medium. This showed that there is a direct relationship and parallel increase of the change in Na⁺,K⁺-ATPase α4 localization and sperm capacitation with the incubation time in the capacitated medium (r= 0.97) (Fig 2H).

A capacitation dependent redistribution of other proteins, such as surface antigens has been reported in boar, pig, hamster, mouse, and rat sperm (Myles and Primakoff 1984, Rochwerger and Cuasnicu 1992, Saxena, et al. 1986, Young, et al. 2009). For instance, PT-1, a protein restricted to the last segment of the sperm tail appears in the entire flagellum after capacitation (Myles, et al. 1987). Also, cytoskeletal proteins, such as actin undergo dynamic remodeling during capacitation (Brener, et al. 2003). The capacitation associated changes that we observe in Na⁺,K⁺-ATPase α4 distribution may be linked to the upregulation of activity that we have previously reported for this isoform at capacitation (Jimenez, et al. 2012). This rearrangement of Na⁺,K⁺-ATPase α4 to specific areas of the plasma membrane (schematically shown in Fig 2F). may be advantageous to support the functional changes that sperm undergoes during capacitation. For example, enhancing the transmembrane Na⁺ gradient at specific microdomains of the sperm cytosol, will provide the chemical force that fuels the activity of other sperm ion transport systems, such as the Na⁺/H⁺ and Na⁺/Ca²⁺ exchanger, which are essential in increasing the sperm cytosol pH and Ca²⁺ levels required during sperm hyperactivation (Garcia and Meizel 1999, Peralta-Arias, et al. 2015, Yeste, et al. 2024). In addition, the K⁺ gradient that Na⁺,K⁺-ATPase α4 generates assists in the function of K⁺-channels, such as Slo3, which is critical for the hyperpolarization that cells acquire at capacitation (Chávez, et al. 2013, Chávez, et al. 2014). Interestingly, the capacitation dependent changes in the distribution pattern of Na⁺,K⁺-ATPase α4 have not been reported for CatSper or Hv1 (Chung, et al. 2014, Miller, et al. 2018). Therefore, it appears that relocalization upon capacitation is a phenomenon limited to selected sperm proteins like Na⁺,K⁺-ATPase α4.

While our data show that Na⁺,K⁺-ATPase α4 localization changes with sperm capacitation, other possibilities can be considered. Thus, capacitation might alter the accessibility of the Na⁺,K⁺-ATPase α4 epitope to the recognizing antibody. This masking of the epitope could be due to capacitation induced changes in Na⁺,K⁺-ATPase α4-protein association, which could prevent us from detecting the Na⁺,K⁺-ATPase α4 signal, even if the protein is still in its original trilaminar location. Alternatively, it is possible that the N-terminus of the Na⁺,K⁺-ATPase α4, recognized by our antibody, is cleaved during capacitation, rendering the remaining portion of the protein undetectable. While protein cleavage has been reported for the Hv1 cation transporter (Berger, et al. 2017), this is still unknown for Na⁺,K⁺-ATPase α4. In any case, the mechanisms involved in masking Na⁺,K⁺-ATPase α4 epitope would have to specifically occur for the Na⁺,K⁺-ATPase α4 present in only two of the three lines in which it is arranged; the ones for which the signal is lost with capacitation. All these different alternatives create exciting new avenues to be explored in future experiments.

Na⁺,K⁺-ATPase α1 also has a distinct pattern of expression in the sperm flagellum independent of the capacitated status of the cells

To assess the pattern of expression of the other Na⁺,K⁺-ATPase α isoform expressed in sperm, we investigated the distribution of Na⁺,K⁺-ATPase α1 along the sperm flagellum under noncapacitated and capacitated conditions. Our results show that Na⁺,K⁺-ATPase α1 is present along the entire length of the sperm flagellum as has been previously described (Wagoner, et al. 2005). Like Na⁺,K⁺-ATPase α4, Na⁺,K⁺-ATPase α1 is also distributed (in ~80% of the cells) as three lines in the midpiece of the sperm (Fig 3A,3B). The cross section of the depth coded images of the flagellar midpiece revealed three compact dots (denoted by 1, 2 and 3) showing the trilinear organization (Fig 3C,3D). From the depth coded z axis, it is found that lanes 1 and 3 are at the same depth level whereas Lane 2 is on the top of the axis (Fig 3D and 3F). Interestingly, beyond the flagellar midpiece and in the principal piece, Na⁺,K⁺-ATPase α1 follows a bilinear distribution which appears to be spatially at the same level (Fig 3E,3F).

Figure 3: Na⁺,K⁺-ATPase α1 organization in the sperm flagellum under noncapacitated conditions.

(A) 3D distribution of Na⁺,K⁺-ATPase α1 along the entire flagellum. (B) Na⁺,K⁺-ATPase α1 localization in the midpiece (MP) showing a trilinear pattern in the midpiece (MP) and a bilinear pattern at the principal piece (PP). Scale bar represents 5 µm (A) and 2 µm (B) respectively. (C) Color coded 3D image showing the position of Na⁺,K⁺-ATPase α1 along the z direction (see color bar in F for reference). (D) y-z cross section obtained from (C) shows three domains (1,2 and 3) at the midpiece (MP). (E) Cross section showing Na⁺,K⁺-ATPase α1 distributed as two lines at the principal piece of the flagellum. Arrows in C denote the sites of cross sections that result in D and E. (G) Distance between the lanes or clusters measured from the cross section of 3D images using LAS X analysis software. D1 represents the distance between lane 1 and 3, D2 is the distance between lane 1 and 2 and D3 is the distance between lane 2 and 3. Values are expressed as mean ±SEM for n=7 different mice. ns shows no significant statistical differences, with P < 0.05.

Like Na⁺,K⁺-ATPase α4, the distances between the lanes for Na⁺,K⁺-ATPase α1, measured with LASX software, were found to be statistically similar, indicating that the three clusters are equally spaced from each other (Fig 3G). However, unlike Na⁺,K⁺-ATPase α4, the localization pattern of Na⁺,K⁺-ATPase α1 was preserved upon sperm capacitation (Fig 4A–E). In addition, we found that Na⁺,K⁺-ATPase α1 distribution remains unchanged when we repeated the experiments in sperm from the Na⁺,K⁺-ATPase α4 knock out mouse (Fig S1C–E). This indicates that the distribution of Na⁺,K⁺-ATPase α1 and α4 is not interdependent.

Figure 4: The pattern of distribution of Na⁺,K⁺-ATPase α1 remains unchanged upon sperm capacitation.

(A) 3D organization of Na⁺,K⁺-ATPase α1 along the sperm flagellum, showing a trilinear pattern in the midpiece which is discontinued into two lanes in the principal piece. Scale bar represents 5 µm. (B) Expression of Na⁺,K⁺-ATPase α1 color-coded along the z axis (refer to color bar E for depth of field). (C) y-z cross section at the midpiece showing three domains (1, 2 and 3) (D) y-z cross section at the principal piece showing only two Na⁺,K⁺-ATPase α1 clusters. Arrows in B shows the sites of cross section from midpiece (C) and principal piece (D) respectively. Experiments were conducted thrice and in total 40 cells per sample were analyzed.

Na⁺,K⁺-ATPase α4 and α1 isoforms are also expressed in the sperm head

Previous studies performed by us and others using conventional fluorescence microscopy and antibodies different than the ones used in this study (Wagoner, et al. 2005, Woo, et al. 2000) showed that Na⁺,K⁺-ATPase α4 was primarily expressed in the sperm flagellar midpiece. Here, we found that Na⁺,K⁺-ATPase α4 is present in the sperm head (Fig 5A). Similarly, Na⁺,K⁺-ATPase α1 is also found in the sperm head (Fig 5B). Moreover, it appears that the localization of both Na⁺,K⁺-ATPase α1 and α4 isoforms is different and that this even changes when one compares cells incubated under noncapacitated and capacitated media (Fig 5A, 5B). While the distribution of both Na⁺,K⁺-ATPase isoforms appear to change with capacitation, after analyzing multiple cells, we found it was difficult to establish a specific capacitation dependent pattern in the arrangement of these proteins. In any case, the presence of Na⁺,K⁺-ATPase isoforms in the sperm head is interesting. Similar to our results in mice, the localization of Na⁺,K⁺-ATPase in the sperm head has been also reported in the bull and rat (Hickey and Buhr 2012, Milewski and James 2025). The finding of Na⁺,K⁺-ATPase in the sperm head poses the question for its functional relevance in this region of the sperm. As a primary generator of the Na⁺ and K⁺ gradients across the plasma membrane, it is possible to speculate that Na⁺,K⁺-ATPase contributes to the cell membrane hyperpolarization and Ca²⁺ increase that are necessary to support the sperm acrosomal reaction (Gardner and James 2023). Further experiments regarding the role of Na⁺,K⁺-ATPase in the sperm head are currently on going in our lab.

Figure 5: Na⁺,K⁺-ATPase α4 and Na⁺,K⁺-ATPase α1 are also expressed in the sperm head under both noncapacitated and capacitated conditions.

(A) 3D organization of Na⁺,K⁺-ATPase α4 in sperm head under both noncapacitated and capacitated condition. (B) Expression of Na⁺,K⁺-ATPase α1 in sperm under both capacitated and noncapacitated condition. Scale bar represents 5 µm.

Colocalization of Na⁺,K⁺-ATPase α4 and α1 in sperm

Next, we investigated the mutual localization of Na⁺,K⁺-ATPase α4 and α1 isoforms in noncapacitated (Fig 6A) and capacitated sperm (Fig 6B). For this we captured 3D images of the cells using STED. Then the images were run with Fiji to obtain the sum of the intensity of all the slices along the z direction of the sample. Equal number of slices (n=25) were taken to get the final images for colocalization. We have observed weak to moderate colocalization of these proteins in noncapacitated (Fig 6C) and capacitated sperm (Fig 6D). From the colocalization analysis of these proteins, we have found the Pearson’s correlation coefficient (r) to be 0.45 ± 0.05 and 0.535 ± 0.04 under noncapacitated and capacitated conditions respectively. These r values indicate that there is partial but not full colocalization of Na⁺,K⁺-ATPase α4 and α1, suggesting that both isoforms maybe spacially separated.

Figure 6: Mutual distribution of Na⁺,K⁺-ATPase α4 and Na⁺,K⁺-ATPase α1 in sperm captured by 3D STED.

(A) The 2D projection of 3D images was obtained by summing the intensity of all slices along z-axis showing the localization of Na⁺,K⁺-ATPase α4 (NKAα4), Na⁺,K⁺-ATPase α1 (NKAα1) under noncapacitated condition. Each z stack is 0.16 µm and in total 25 slices were included. (B) shows the colocalization under capacitated condition. Scale bar represents 5 µm. (C) y-z cross section of the 3D images (obtained with 3D viewer) at the midpiece showing three domains representing localization NKAα4 (red), NKAα1 (green) and both under noncapacitated condition. (D) y-z cross section at the midpiece showing one cluster representing localization NKAα4 (red), three domains for NKAα1 (green) and both under capacitated condition.

The lack of complete overlap, as well as the capacitation dependent changes in distribution in the sperm flagellum between Na⁺,K⁺-ATPase α1 and α4 is an interesting phenomenon. This may be related to the different roles that these isoforms have in sperm function. Thus, earlier findings from our laboratory have shown that, while Na⁺,K⁺-ATPase α1 maintains the basal ion concentrations in the cell, Na⁺,K⁺-ATPase α4, which can generate a steeper Na⁺ gradient across the cell membrane, is dedicated to sperm motility, hypermotility and capacitation (McDermott, et al. 2021). Therefore, the repositioning of Na⁺,K⁺-ATPase α4 in the sperm flagellum during capacitation, may be related to the need to maintain a steeper Na⁺ gradient in specific intracellular sperm domains at capacitation, required to prepare sperm for fertilization. These capacitation dependent changes in Na⁺,K⁺-ATPase α4 distribution also raise new questions regarding the mechanisms that are operating in sperm to shift the distribution of this protein. The cellular localization of the Na⁺,K⁺-ATPase α2 has been shown to change from intracellular stores to the plasma membrane in skeletal muscle. This is triggered by different stimuli, which cause posttranslational modifications of the α2 isoform (Pirkmajer and Chibalin 2016). At this point, we can speculate that similar mechanisms maybe operating for the Na⁺,K⁺-ATPase α4 isoform in sperm.

Conclusion

Our findings reveal a distinct pattern of localization for Na⁺,K⁺-ATPase α4 and α1 in the sperm flagellum and head. This, along with the specific redistribution of Na⁺,K⁺-ATPase α4 in the flagellum during capacitation, emphasizes the complexity of sperm in general and of the Na⁺,K⁺-ATPase in particular. Considering that Na⁺,K⁺-ATPase α4 and α1 have different functional properties, our present data provides important evidence for the refined level of specialization that sperm have developed to function and achieve the amazing goal of fertilizing the oocyte.

Materials and Methods:

Sperm preparation and in vitro capacitation

Sperm from the caudal portion of the epididymis were isolated from adult wild type and Na⁺,K⁺-ATPase α4-KO male mice (12–14 weeks old, C57Bl/6 J) as previously reported(Numata, et al. 2024). Briefly, sperm were collected by swim-up in the modified Whitten’s Media containing 100mM NaCl, 4.7mM KCl, 1.2mM KH₂PO4, 1.2mM MgSO4, 5.5mM glucose, 0.8mM pyruvic acid, 4.8mM lactic acid, 20mM Hepes, and 1.7mM CaCl₂ (pH 7.20–7.40). To induce capacitation, cells were incubated in Card FertiUp Preincubation Medium: PM, containing modified Krebs-Ringer bicarbonate solution, without BSA, methyl-β-cyclodextrin and polyvinylalcohol (Cosmo Bio LTD, Koto-ku, Tokyo, Japan) at 37˚C for 10 mins for swimming up and then incubated at the same temperature in 5% CO₂ for an hour. In order to examine whether the relocalization of capacitation is a time dependent event, sperm were collected at different time points during capacitation including 0, 20, 40 and 60 minutes and then prepared for immunocytochemistry.

Immunocytochemistry

For Immunolabelling, sperm was washed in Phosphate Buffered Saline (PBS) (ThermoFisher Scientific, Waltham MA, USA) twice (120x g, 5 mins for each wash) at 4°C and fixed in 4% paraformaldehyde (PFA) for 15 min. The cells were then washed three times in PBS (380x g, 5mins for each wash) and then resuspended in PBS. The diluted cells in suspension were placed on a slide and air-dried. To minimize autofluorescence the slides were exposed to light emitting diode (LED) light for 48 hrs. Then, samples were permeabilized with 0.3% Triton‐X for 10 min in PBS, washed three times with PBS, and then blocked with 2% BSA in PBS for 1h at RT. The cells were incubated at 4°C overnight in a humid chamber with the primary antibodies. These antibodies included anti-ATP1A4 (in-house antibody raised in rabbits against epitope RPSTRSSTTNRQPKMKRR of Na⁺,K⁺-ATPase α4; used at a 1:1500 dilution); and anti-ATP1A1 (ProteinTech, Rosemont IL, USA; made against a peptide that spans amino acids 347–681 of the recombinant human ATP1A1 protein; used at a 1:1500 dilution). For the colocalization studies of ATP1A1 and ATP1A4, samples were incubated with the anti-ATP1A4 in-house antibody (1:1500 dilution) and anti-ATP1A1 antibody (Santa Cruz Biotechnology, Texas, USA; raised against purified rabbit renal outer medulla; used at a dilution of 1:500). The samples were then washed three times with PBS and incubated at RT for 1h with secondary antibodies. These include a goat anti‐rabbit Alexa Flour 594 conjugated secondary antibody (Invitrogen, Carlsbad CA, USA) and goat anti-mouse ATTO 647N (MIlipore Sigma, USA) at a 1:1000 and 1:500 dilution in PBS with 2% BSA respectively. Cells were then washed three times in PBS, mounted with Prolong Gold (ThermoFisher, Waltham MA, USA), and cured for 24 hours for imaging with STED microscopy. For Nikon Eclipse 80i Fluorescence Microscope, the sample was mounted with Dapi and kept at 4°C and imaging was done at 20x magnification.

Immunoblot

To determine the selectivity and specificity of in-house antibody, protein expression of Na⁺,K⁺-ATPase α4 in different tissue samples was evaluated by SDS-PAGE and immunoblotting as described previously(Numata, et al. 2024). Briefly, tissues from kidney, liver, heart, brain, testis and sperm from wild type mice are homogenized and treated with lysis buffer. Samples were run on 8% SDS-PAGE gel and transferred on a PVDF membrane. Blocking for an hour in 5% blotto with TBST followed by overnight incubation with primary anti-ATP1A4 antibody at 4°C were performed. Horseradish peroxide conjugated anti-goat secondary antibody (Jackson ImmunoResearch Europe Ltd, Cambridgeshire, UK) at 1:2000 dilution and chemiluminescence were used for detection.

Sperm-motility assay

Noncapacitated or capacitated sperm from wild type mice were analyzed for sperm motility using Computer Aided Sperm Analysis tool or CASA (version 3.9.8, Penetrating Innovations) as previously described (Jimenez, et al. 2011b). Briefly, 1X10⁶ sperm were labelled with 2 μl of a 75-μM stock of SITO 21, a nucleic acid staining fluorescence to track sperm movement. After incubating for 2 mins with the dye the sample was visualized under the microscope and sperm hyperactive motility was analyzed with the CASA. Different sperm motility parameters were obtained, including total motility, progressive motility, curvilinear, average path, and straight-line velocities, amplitude of lateral head displacement, straightness, and linearity. The analytical setup parameters used considered a cell identification or a cell size area between 05 and 900 μm², a cutoff velocity corresponding to a minimum straight-line velocity of 5 µm/s, a progressive motility threshold corresponding to a straight-line velocity of more than 20 µm/s. Linearity was calculated from the ratio between straight line velocity and curvilinear velocity during the measurement period. Amplitude of lateral head displacement (ALH) was obtained as the maximum distance of the sperm head from the average trajectory of the sperm during the analysis period. Curvilinear velocity of more than 100  µm/s, linearity less than 65% and ALH greater than 7.5  µm were the threshold measurements taken to determine hyperactive motility. An average of 60 cells/field was captured at a rate of 30 frames per field, and a total of ten fields in each sample were analyzed. Therefore, ∼600 cells were analyzed per condition for each experiment. Each field was taken randomly by scanning the slide following a pre-established path to ensure consistency in the method.

STED Microscopy and image analysis

The STED images were recorded with a Leica TCS SP8 STED 3X Super Resolution Microscope with an oil immersion objective (HC PL APO 100x/1.40 OIL CS2 objective, Oil immersion type F; Leica Wetzlar, Germany). The White Light Laser (WLL) was used for the excitation and a pulsed 775 nm Laser Beam (40% turned on) was used for emission depletion without gating. The WLL had an excitation wavelength of 594 nm and 647 nm for Alexa Flour 594 and Atto 647N respectively. The detection ranges for these fluorophores were 598–625 nm and 655–750 nm respectively. The bit depth was set to 12. To get sharp and more sensitive images for both one- and two-color STED, the HyD was turned on as the photodetector. The HyD (Hybrid Detector) provides high contrast image with low-dark noise and reduce photobleaching of the sample. A pixel size of 170 to 180 nm was used with dwell time of 3.16 µs and each line was scanned for 3 times (Line Accumulation). The pinhole was set to 1.0 AU. The images were obtained as z-stacked with z-step size 0.16 µm. XY format was optimized and set as suggested by STED. Speed was set to 400 Hz, meaning repeated scanning with depletion laser for 400 times per second which facilitates faster imaging. The image is then deconvoluted which reveals the fine structure of the image reducing the background noises which might be otherwise missed.

For one-color STED, 3D images were visualized with 3D viewer and depth coded images are generated using Leica Image Analysis software (Leica Application Suite X, RRID:SCR_013673). The images are then saved in tiff file which are adjusted in brightness and contrast with an open software, Fiji ImageJ (Schindelin, et al. 2012). For determining the distance, and angle between the three lines, LASX Leica Analysis Software (Leica Application Suite X, RRID:SCR_013673) was used.

Colocalization Analysis:

Two color STED images were recorded with the Leica TCS SP8 STED 3X Super Resolution Microscope as mentioned above. Each line was scanned thrice (line accumulation) and images were obtained line sequentially. To assess the colocalization of ATP1A4 and ATP1A1, STED images were processed using Fiji (ImageJ). First, 3D z-stacked images were converted into 2D projections by summing the intensity of equal number of slices along the z-axis. The channels were then split to generate separate images for ATP1A1 and ATP1A4 localization. These individual channel images were subsequently merged. Colocalization between ATP1A4 and ATP1A1 were analyzed using the plugin named “JaCoP” (Just Another Co-localization Plugin) which include assessing correlation between the two fluorescence signals (Bolte and Cordelières 2006, Schneider, et al. 2012). The rate of association between two signals (ATP1A4 and ATP1A4) is determined by Pearson’s coefficient which will assess the correlation between two signals by comparing the intensity values. The cross sections of sperm were obtained from the 3D STED images using the orthogonal slices tool in the 3D viewer of Leica LASX Analysis software.

Statistical Analysis

Experiments were repeated at least three times and the statistical significance of mean and differences between distances were determined by one-way anova test. Pearson’s correlation coefficient was used to assess the direction and strength of relationships between capacitation time and either protein remodeling or hyperactive motility as well as between signal intensities of Na⁺,K⁺-ATPase α isofroms. For determining the statistical significance of the differences between hyperactive motility, paired t-test using GraphPad Prism (San Diego, CA, USA, www.graphpad.com) was employed. Statistical significance was defined as p < 0.05.