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IJMSInternational Journal of Molecular Sciences
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4 March 2026

16 Pages

The Sperm Acrosomal Reaction Depends on the Function of Both Na,K-ATPase α1 and α4 Isoforms

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1
Campus Centro-Oeste Dona Lindu, Universidade Federal de São João del Rei, Divinópolis 35501-296, Brazil
2
Department of Pathology and Laboratory Medicine, Kansas University Medical Center, Kansas City, KS 66160, USA
3
Department of Cell Biology and Physiology, Kansas University Medical Center, Kansas City, KS 66160, USA
*
Author to whom correspondence should be addressed.
Int. J. Mol. Sci.2026, 27(5), 2372;https://doi.org/10.3390/ijms27052372
This article belongs to the Special Issue The Na, K-ATPase in Health and Disease
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Abstract

Na,K-ATPase (NKA) is expressed as two isoforms in mouse sperm: the testis-specific Na,K-ATPase α4 (NKAα4) and the somatic Na,K-ATPase α1 (NKAα1). Currently, the role that NKAα4 and NKAα1 play in the sperm acrosome reaction (AR) is unknown, and is the subject of our investigation. We studied the following: (1) the differential sensitivity of NKAα4 and NKAα1 to inhibition by ouabain; (2) the effects of deleting NKAα4 in mice (using NKAα4-KO mouse). In sperm from wild type (WT) mice, inhibiting NKAα4 with a low concentration of ouabain reduced AR. Inhibiting NKAα1 with a higher ouabain concentration further reduced AR, indicating that both NKA isoforms are necessary for AR. Surprisingly, sperm from NKAα4-KO mice exhibited an abnormally high AR. This was not due to a lack of acrosome development during sperm differentiation, but rather from premature release of the acrosome after they were isolated from the epididymis. When WT and NKAα4-KO sperm were exposed to media with or without Na+, K+, and Ca2+, or with the ionophores nigericin, valinomycin, and A23187, they displayed abnormal AR, indicating that NKAα4-KO sperm are unable to control intracellular Na+, K+, and Ca2+ levels. NKAα4 directly maintains intracellular Na+ and K+ and indirectly influences Ca2+ levels. This provides the necessary ion environment for full development of sperm AR. Along with NKAα4, NKAα1 also contributes to sperm AR.

1. Introduction

Na,K-ATPase (NKA) is an active ion transport system expressed at the surface of most animal cells. NKA exchanges intracellular Na+ for extracellular K+ across the cell plasma membrane using ATP as the energy source [1]. The resulting transmembrane Na+ and K+ gradients are essential to maintain cell osmotic balance, resting membrane potential, and the secondary movement of other ions and water in and out of the cell [2]. NKA is an oligomer composed of two main subunits: a catalytic α-subunit, and a glycosylated β-subunit [1,3]. The α-subunit is responsible for the binding, occlusion, and translocation of ions between the cell and its environment. The α-subunit contains the binding sites for Na+ and K+ and the substrate ATP, which is critical to provide the energy for ion transport [4]. The α-subunit is also the target of the glycoside ouabain, an inhibitor of NKA function [5,6]. The β-subunit is not directly involved in Na+ and K+ transport but assists in the folding of the α-subunit during its synthesis and translocation to the cell plasma membrane [7,8].
Na,K-ATPase α4 (NKAα4) is one of four isoforms of the NKA α-subunit of mammalian cells [9,10]. While the other three isoforms, named NKAα1, NKAα2, and NKAα3, are expressed in a cell- and tissue-specific manner throughout the body, NKAα4 is only present in male germ cells of the testis and in mature sperm [11,12]. Within sperm, NKAα4 is predominantly found in the mid-piece segment of the flagellum. However, recent data from our laboratory and others have shown that NKAα4 is also present in the sperm head [13,14]. NKAα4 expression varies during testis development, increasing at puberty and after male germ cells undergo meiosis [11,15]. Moreover, NKAα4 activity is upregulated during sperm capacitation [16], an event that is critical for acquisition of sperm fertilizing capacity [17,18]. In addition to NKAα4, sperm also expresses the ubiquitous somatic isoform of NKA, NKAα1. This isoform is evenly distributed throughout the sperm flagellum and in the sperm head [11,13].
Current data suggest that NKAα1 maintains the basal Na+ and K+ transmembrane gradient of sperm, and NKAα4 regulates ion transport to the unique requirements of sperm [19,20]. This model derives from studies that selectively inhibited NKAα4 with ouabain [21]. In rodents, NKAα isoforms have distinct responses to ouabain: NKAα4 has the highest affinity for ouabain (IC50 of ~1 nM), and NKAα1 has the lowest (IC50 of ~1 µM). This difference has been exploited to distinguish the function of NKAα4 from NKAα1 [21]. Thus, when NKAα4 is inhibited with relatively low ouabain concentrations, there is a significant reduction in total sperm motility, progressive motility, straight and curvilinear velocities, linearity, and amplitude of head displacement [15,21]. Importantly, inhibition of NKAα4 and NKAα1 with higher ouabain concentrations does not have additional effects on sperm kinematics, indicating that NKAα4 is the NKA isoform specifically suited to serve sperm flagellar movement [19]. Later studies used a genetic approach to validate this model by deleting NKAα4 in mice [22,23,24]. In these NKAα4-KO mice, there is a drastic reduction in sperm motility and hypermotility [22], the pattern of movement that sperm acquire during capacitation [25,26]. As a result, male (but not female) mice are completely infertile, and NKAα1 is not sufficient to revert this infertility phenotype.
Deletion of NKAα4 in vivo or inhibition of NKAα4 with ouabain in vitro alters the concentrations of several ions inside sperm, increasing intracellular Na+ and Ca2+ and reducing cell pH. This demonstrates that NKAα4 maintains the transmembrane Na+ gradient, which is used by the Na+/Ca2+ and Na+/H+ exchangers as the energy source to regulate sperm Ca2+ and H+ levels, respectively [27]. Moreover, ablation or inhibition of NKAα4 depolarizes the sperm plasma membrane, which may be attributed to an imbalance in the transmembrane K+ gradient [22]. The appropriate levels of Na+, K+, and Ca2+ are necessary for normal sperm motility, and their imbalance may explain the altered movement of NKAα4-KO sperm [28,29,30]. Additional support for the role of NKAα4 in sperm motility arose from experiments in which NKAα4 was blocked with synthetic ouabain derivatives, which preferentially inhibit NKAα4. These compounds reduced sperm motility and interfered with sperm fertility in in vitro fertilization assays [31]. Thus, NKAα4 plays an essential role in sperm kinematics and male fertility.
While the function of NKAα4 on sperm motility has been well documented, its role in other aspects of sperm function remains poorly understood. Previous reports showed that NKA activity is associated with the sperm acrosome reaction (AR) [32,33,34]. AR is an exocytotic process during which the sperm acrosome and plasma membrane fuse, and then hydrolytic enzymes are released from the sperm acrosome [17,35,36]. This event is key to fertilization since it helps sperm make their way through the cumulus cells and zona pellucida that cover the oocyte [37,38,39]. While NKA activity in general is known to play a role in sperm AR, it is unknown how NKAα4 and NKAα1 in particular contribute to this process. Moreover, while AR is highly dependent on intracellular Ca2+ levels [40,41,42], the effect of Na+ and K+ in this process remains poorly characterized [30,43]. Here, we investigated the role of NKAα4 in the sperm AR. We hypothesized that NKAα4 directly controls Na+ and K+, and it indirectly contributes to sperm intracellular Ca2+ levels. Our recent finding, that NKAα4 is expressed in the sperm head [13], suggests that its function could also extend to AR. Indeed, we found that NKAα4 is important for sperm AR by maintaining the intracellular concentrations of Na+, K+, and Ca2+ in the cells. We also found that NKAα1 also participates in sperm AR.

2. Results

To assess the specific role of NKAα4 in sperm AR, we used a combined genetic and pharmacological approach. The genetic approach used sperm from both WT mice and a mouse model lacking NKAα4 (NKAα4-KO mice), which we previously engineered [22]. The pharmacological approach leveraged differences in ouabain affinity between NKAα1, which is relatively resistant to ouabain, and NKAα4, which is relatively sensitive to ouabain [44]. This allowed us to specifically block the activity of NKAα4 using a low dose of ouabain (10−6 M), or to block both NKAα4 and NKAα1 with a higher dose of ouabain (10−3 M) [21]. We found that, when sperm from WT mice are maintained in non-capacitated medium (NC), relatively few undergo acrosome loss (Figure 1A). This observation is consistent with previous reports, which demonstrated that capacitation is required for sperm to achieve AR [22]. Ouabain treatment had no significant effect on AR at either 10−3 M or 10−6 M (Figure 1A, white bars). In contrast, in medium that supports capacitation (capacitating medium, Cap), AR was increased, as expected (Figure 1A, grey bars). In this case, treatment with ouabain at 10−6 M inhibited AR, and treatment with 10−3 M further inhibited AR, reducing it to levels similar to those found in sperm in NC medium. These results show that both NKAα1 and NKAα4 participate in sperm AR, and that they contribute to this process in an approximately equal manner.
Figure 1. Acrosome reaction in epididymal sperm from (A) WT and (B) NKAα4-KO mice. AR was measured after incubating the cells in non-capacitating (NC) or capacitating (Cap) media and in the absence or presence of 10−6 M and 10−3 M ouabain. Data are presented as mean ± SEM (n = 3). Asterisks indicate statistical significance of ouabain treated samples with respect to the controls without ouabain for WT and NKAα4-KO sperm under both NC and Cap media, with p < 0.001 (A) and p < 0.05 (B) as determined by one-way ANOVA test followed by Tukey test.
To further ascertain the role of NKAα4 in AR, we studied sperm from NKAα4-KO mice. Surprisingly, NKAα4-KO sperm displayed unexpectedly abnormal behavior with respect to AR. In NC medium, NKAα4-KO sperm exhibited abnormally high AR levels, ~2-fold higher than that of WT sperm under the same conditions. This was not reduced by the addition of 10−6 M or 10−3 M ouabain (Figure 1B, white bars). The AR of NKAα4-KO sperm was increased by an additional ~10% when they were incubated in Cap medium. In this case, 10−6 M or 10−3 M ouabain did not significantly affect AR (Figure 1B, dark bars). These data show that, unexpectedly, lack of NKAα4 exacerbates AR under both non-capacitating and capacitating conditions.
Because deletion of NKAα4 appears to trigger AR in both NC medium and Cap medium, we wanted to confirm if sperm lacking NKAα4 developed the acrosomal vesicle or if their acrosomes were abnormally released early during sperm differentiation. To assess this, we used electron microscopy of sperm directly after their collection from the cauda epididymis, and without exposure to any added media. As shown in Figure 2A,B, sperm from NKAα4-KO mice had an intact acrosome, with a similar morphology as that of WT sperm. This confirmed the structural integrity of the acrosome of NKAα4 sperm.
Figure 2. Electron micrographs of sperm obtained from the cauda of the epididymis. (A) WT and (B) NKAα4-KO mouse sperm were smeared onto slides, fixed, processed, and subjected to electron microscopy. The intact acrosomes in the head of both WT and NKAα4-KO sperm are shown with a white arrow labelled with a letter “A”.
As a complementary experiment, we quantified and compared the acrosomes in WT and NKAα4-KO sperm, which were directly retrieved from the epididymis and labeled with the acrosomal marker PNA-Fluorescein. As shown in Figure 3A,B, without the addition of medium, WT and NKAα4-KO sperm have intact and morphologically normal acrosomes. In contrast, when sperm was collected in NC medium, WT sperm maintained their acrosomes, but NKAα4-KO sperm experienced acrosomal loss (Figure 3C,D). Figure 3E shows quantitative data for these experiments. When we compared sperm that were smeared versus those that were processed by swim up technique, we observed a significantly higher AR in both WT and NKAα4-KO. This suggests that, even under non-capacitating conditions, the addition of media to the cells promotes AR. Importantly, AR was significantly higher in NKAα4-KO than in WT sperm (Figure 3E). Together, these results show that, when exposed to the NC medium, NKAα4-KO sperm abnormally undergo AR.
Figure 3. Acrosome analysis by PNA-Fluorescein. (A,B) Sperm obtained from the epididymal cauda were directly smeared onto slides without the addition of any media, and acrosome presence was determined; (A) WT and (B) NKAα4-KO sperm. (C,D) Sperm were processed by swim-up technique in NC medium and acrosome occurrence was determined; (C) WT and (D) NKAα4-KO sperm. The presence of acrosomes is indicated with a white arrow. (E) Quantification of acrosome numbers in WT and NKAα4-KO in smeared and in swim-up processed samples. Data are presented as mean ± SEM (n = 3). **, p < 0.01; ns, non-significant; one-way ANOVA test followed by Tukey test.
In a previous report, we showed that sperm lacking NKAα4 have several defects, including an imbalance in the concentrations of several ions inside the cells [20]. Since the primary role of NKA is to export Na+ from the cell, sperm from NKAα4-KO mice have higher than normal cytosolic Na+ [20]. Additionally, the dissipation of the transmembrane Na+ gradient in NKAα4-KO sperm increases cytoplasmic Ca2+, possibly via reduced activity of the Na+/Ca2+ exchanger, and it also lowers pH, via decreased activity of the Na+/H+ exchanger [20]. The ion composition of the media surrounding sperm and the intracellular ion levels in the sperm cytoplasm are critical for AR [45,46,47]. Therefore, to understand how AR is influenced by ions in general, and in NKAα4-KO sperm in particular, we investigated the influence of Na+, K+, and Ca2+ in sperm AR. Each of these ions are directly or indirectly related to NKA function.
We first studied the effect of Na+. To achieve this, sperm from WT and NKAα4-KO mice were collected in NC or Cap media with or without 100 mM Na+. As shown in Figure 4A, sperm from WT mice had low basal levels of AR in NC media with or without Na+. In Cap medium with Na+, WT sperm underwent AR, but it remained at the low basal levels seen in NC medium without Na+. In contrast, NKAα4-KO sperm had high levels of AR in both NC medium with Na+ and Cap medium with Na+. In NC medium without Na+ and Cap medium without Na+, NKAα4-KO sperm had AR reduced to levels similar to that of WT sperm in NC medium (Figure 4A). These results indicate that Na+ is necessary for the development of the sperm AR in both WT and in NKAα4-KO sperm. In addition, the data suggest that, in NKAα4-KO sperm, higher-than-normal intracellular Na+ [20] could trigger abnormally high AR, even in the absence of extracellular Na+.
Figure 4. (A,B) Acrosome reaction in sperm from WT and NKAα4-KO mice in NC or Cap media with or without 100 mM Na+ (A), or in the same media after pretreatment with 10 μM monensin (B). Data are presented as mean ± SEM (n = 4). Asterisks indicate statistical differences compared to the corresponding values in the absence of Na+, with * p < 0.1. The # sign shows differences with respect to WT in the presence of Na+, with ** p < 0.01, respectively. (C) Intracellular Na+ levels as measured with CoroNa Green in sperm from WT and NKAα4-KO mice with or without 100 mM NaCl, and with or without nigericin. Data represent the mean ± SEM of 10,000 events obtained from cytometric determinations and are expressed relative to WT samples in the absence of Na+. Asterisks show significant differences with respect to WT in medium without Na+. The number sign indicates differences with WT sperm in Na+ containing medium.
To further explore the impact of Na+ on AR of WT and NKAα4-KO sperm, we pretreated the cells with 10 µM of monensin, an Na+ ionophore, for 20 min in NC and Cap media, both with and without Na+ in the medium. As shown in Figure 4B, monensin treatment increased AR in WT sperm in NC medium with Na+ (compare WT in NC medium between Figure 4A,B). Monensin did not significantly change the AR of sperm in Cap medium with or without Na+. In NKAα4-KO sperm, monensin reduced the abnormally high AR observed in NC medium (compare NKAα4-KO in NC medium between Figure 4A,B). Monensin did not significantly affect the AR of NKAα4-KO sperm in Cap medium with or without Na+ (compare NKAα4-KO in Cap medium between Figure 4A,B). These data show that Na+ is important for AR, since its absence prevents AR from fully developing. Monensin promotes AR in WT sperm by allowing Na+ to enter the cell, following its transmembrane gradient. In addition, it appears that, when intracellular Na+ is higher (as is the case of NKAα4-KO sperm), the movement of additional Na+ into the cell by monensin does not further affect AR. This is also the case of WT sperm in which monensin has no significant effect in Cap medium with Na+.
To determine if changes in intracellular Na+ were induced by the conditions to which the cells were subject, we labeled WT and NKAα4-KO sperm with the Na+ indicator CoroNa Green in NC medium, with or without Na+, and with or without nigericin. As shown in Figure 4C, intracellular Na+ levels were relatively low in WT and NKAα4-KO sperm in medium without Na+, and this did not significantly change when nigericin was added. Instead, in medium with Na+, intracellular Na+ levels increased, with a greater increase in NKAα4-KO sperm compared to WT sperm (Figure 4C). Nigericin increased intracellular Na+ in WT and especially in NKAα4-KO sperm, reflecting the movement of this cation into the cells following its inward concentration gradient. These data show that this experimental manipulation changed Na+ levels in the cells.
To assess the involvement of K+ in AR, we subjected sperm from WT and NKAα4-KO mice to medium with or without K+. As shown in Figure 5A, in NC medium without K+, WT sperm had basal AR levels. In Cap medium without K+, WT sperm had lower levels (Figure 5A). Removing K+ from the medium reduced AR in NKAα4-KO sperm in both non-capacitated and capacitated medium (Figure 5A).
Figure 5. Acrosome reaction in sperm from WT and NKAα4-KO mice. Sperm were subjected to (A) NC or Cap media with or without K+ (4.7 mM), or to (B) the presence of K+ and pretreatment with 10 μM valinomycin. Data are presented as mean ± SEM (n = 4 for (A), and n = 3 for (B)). Statistical differences are indicated by asterisks, with * p < 0.05, ** p < 0.01 and *** p < 0.001. (C) Intracellular K+ levels as measured with PBFI AM in sperm from WT and NKAα4-KO mice with or without 4.7 mM KCl and with or without valinimicyn. Data represent the mean ± SEM of 10,000 events obtained from cytometric determinations and are expressed relative to WT samples in the absence of K+. Asterisks show differences with WT in medium without K+.
These results show the importance of K+ in the development of sperm AR. To further explore this, we studied the effect of the K+ ionophore valinomycin on AR in WT and NKAα4-KO sperm. We incubated sperm in NC and Cap media with K+ and with 10 µM valinomycin. Figure 5B shows that, in WT sperm in NC medium with K+, valinomycin had no effect on AR levels. In contrast, in Cap medium with K+, valinomycin decreased AR. In NKAα4-KO, valinomycin decreased AR in both NC and Cap medium, with a stronger effect in NC medium.
The changes in intracellular K+ in the cells were determined by labelling WT and NKAα4-KO sperm with the K+ indicator Potassium Binding Benzofuran Isophthalate Acetoxymethyl Ester (PBFI AM) in NC medium with or without K+ and with or without valinomycin. Figure 5C shows that intracellular K+ levels were high inside the cells, both in the absence and presenced of extracellular K+, and K+ levels were higher in WT than in NKAα4-KO sperm. This reflects the reduction in K+ uptake in NKAα4-KO sperm due to the absence of NKAα4. In the presence of valinomycin, intracellular K+ decreased, as expected by the raise in permeability caused by valinomicyn. Altogether, these results suggest that K+ plays a role in the development of AR during capacitation in both WT and NKAα4-KO sperm and in both non-capacitating and capacitating conditions.
Next, we assessed the combined effect of eliminating both Na+ and K+ from the medium (Figure 6). As expected, the absence of Na+ and K+ impaired the development of AR. However, the absence of Na+ and K+ did not further affect AR compared to the absence of either cation individually (Figure 4 and Figure 5). These results further support the relevance of both Na+ and K+ in sperm AR.
Figure 6. Effect of Na+ and K+ on AR from WT or NKAα4-KO mouse sperm. Cells were placed in NC or Cap medium in the absence and presence of 100 mM of Na+ and 4.7 mM of K+, respectively. Data are presented as mean ± SEM (n = 4), with ** p < 0.01.
The role of Ca2+ in sperm AR in WT mice has been well established [48], but the involvement of NKAα4 in the control of Ca2+ in sperm is poorly understood. We previously determined that NKAα4-KO sperm have abnormally high intracellular Ca2+ levels [20]. We further explored this in WT and NKAα4-KO sperm in media with or without Ca2+. In NC medium without Ca2+, the AR of WT sperm was unchanged, and remained at basal levels (Figure 7A). In contrast, in Cap medium without Ca2+, AR was significantly reduced in WT sperm. In NKAα4-KO sperm, medium without Ca2+ decreased the abnormally high AR displayed under non-capacitating and capacitating conditions. These findings show the important role of Ca2+ in sperm AR and demonstrate that the increase in Ca2+, which is characteristic of sperm devoid of NKAα4 [20], is at least one of the factors driving the aberrantly high AR in the cells.
Figure 7. (A) Effect of Ca2+ on acrosome reaction in sperm from WT and NKAα4-KO mice in NC or Cap media. Data are presented as mean ± SEM (n = 4). Statistical differences are indicated with asterisks, with * p < 0.05 and *** p < 0.001. (B) Effect of the Ca2+ ionophore A23187 on AR from WT and NKAα4-KO mice with or without Na+ and K+. Data are presented as mean ± SEM (n = 3), with * p < 0.01 respectively. ns, non-significant. (C) Intracellular Ca2+ levels as measured with Fluo-4 AM in sperm from WT and NKAα4-KO mice with or without CaCl2 and with or without A23187. Data represent the mean ± SEM of 10,000 events obtained from cytometric determinations and are expressed relative to WT samples in the absence of Ca2+. Asterisks show differences with WT in medium without Ca2+.
To further study the effect of Ca2+ and its relationship with Na+ and K+, we subjected sperm from WT and NKAα4-KO mice to the Ca2+ ionophore A23187 in media with or without Na+ and K+. Figure 7B shows that, independent of the presence of Na+ and K+ and with A23187, AR was not statistically different for WT sperm. For WT sperm in Cap medium, A23187 stimulated AR when Na+ or K+ were present in the medium. It also partially counteracted the lack of Na+ and K+, slightly increasing AR, albeit not to the levels reached in Na+ or K+ containing medium. In NKAα4-KO sperm in NC or Cap media, A23187 increased the low AR when Na+ or K+ were absent (Figure 7B).
To determine the effect that changes in Ca2+ in the medium and the action of A23187 on intracellular Ca2+, we labelled WT and NKAα4-KO sperm with the Ca2+ indicator Fluo-4 AM in NC medium. Figure 7C shows that intracellular Ca2+ levels are low inside the cells in the absence of extracellular Ca2+. When Ca2+ was present in the medium, its levels inside sperm increase, with slightly higher levels observed in NKAα4-KO sperm compared to WT sperm. This demonstrates the effect of NKAα4 on Ca2+ levels in the cells. A23187 increased intracellular Ca2+, with a more robust effect in NKAα4-KO sperm than in WT sperm. Our experimental manipulations thus produced intracellular Ca2+ changes in both WT and NKAα4-KO sperm. Altogether, these data show that promoting Ca2+ entrance into sperm facilitates AR, even when Na+ or K+ are absent, suggesting that Ca2+ plays a higher hierarchical role in the effect of ions on AR.

3. Discussion

Previous studies on NKAα4 focused primarily on sperm motility [12,19,20,22]; we thus had a poor understanding of how it controls sperm AR, and how this control was impacted by Na+, K+, and Ca2+. In this study, we showed that NKA plays an important role in sperm AR. Previous studies showed that NKAα4 is expressed predominantly in the sperm flagellum, but more recent studies have identified it in the sperm head as well [13]; this supports its involvement in AR. We found that both the testis specific NKAα4 and the somatic NKAα1 regulate AR, with both isoforms contributing approximately equally to the total sperm AR. This is different from what we previously found for sperm motility, where NKAα4 is the only isoform responsible for maintaining sperm kinematics [22,49]. NKAα4 has a higher affinity for Na+ than NKAα1 [44]; therefore, NKAα4 can maintain the intracellular concentration of this cation at lower levels than NKAα1. It is possible, then, that sperm motility requires the steeper transmembrane Na+ gradient created by NKAα4, while AR can still proceed at the lower transmembrane Na+ gradient generated by NKAα1.
Surprisingly, sperm from NKAα4-KO mice exhibited abnormally high AR levels, even under non-capacitating conditions. This observation appeared at first counterintuitive, considering that ouabain inhibition of NKAα4 in WT sperm prevents full AR development. The dissimilar response between pharmacological inhibition and genetic ablation of NKAα4 may be due to the quantitative and temporal differences inherent to each model. Therefore, NKAα4 deletion has a more prolonged effect than acute inhibition with ouabain, resulting in a more drastic impairment of the capacity of the cells to regulate their ion levels. The chronic ionic disturbance in NKAα4-KO sperm therefore causes a permanent ion imbalance that prevents the cells from controlling AR, which then proceeds spontaneously and in a faster manner. The high spontaneous rate of AR in NKAα4-KO sperm may be another factor, in addition to the motility defects, which makes male NKAα4-KO mice infertile. The adequate spatial-temporal occurrence of AR in the female reproductive tract is important for proper fertilization of the egg [50,51,52]. Therefore, an early AR will render sperm without the acrosome contents, at the moment they meet the egg, preventing fertilization. In support of this, our previous experiments show that NKAα4-KO sperm are incapable of fertilizing eggs in vitro, an approach that highly favors sperm contact with the egg [22].
In NKAα4-KO sperm, the aberrant high spontaneous AR does not depend on the lack of acrosome formation or on the early release of the acrosomes during sperm differentiation, but it is observed upon exposure to the NC medium. It is important to note that the ion composition of the NC medium, like the fluid in the female genital tract, has higher Na+ and lower K+ concentrations than the epididymal environment from which the sperm were obtained [53]. This suggests that, when exposed to the NC medium used for sperm collection, NKAα4-KO sperm, but not WT sperm, are unable to cope with the ionic changes of the surrounding medium and cannot properly maintain intracellular ion balance.
NKAα4 directly controls intracellular Na+ and K+ levels and indirectly regulates Ca2+ and H+ levels, through the Na+/Ca2+ and Na+/H+ exchangers, respectively. Additionally, NKAα4 together with K+ channels maintain sperm membrane potential [19]. NKAα4-KO sperm have increased intracellular Na+ and Ca2+ levels and exhibit cell plasma membrane depolarization [20,22]. Our data suggest that these ion imbalances trigger the abnormal AR of NKAα4-KO sperm. For example, when considering Na+, its retrieval from the medium significantly reduces the high AR of NKAα4-KO sperm. This occurs in both NC and Cap medium. The relevance of Na+ in AR is also observed in WT sperm, where its absence partially prevents AR. Furthermore, treatment with monensin, which increases Na+ permeability [54], has no effect on the acrosome reaction in NKAα4-KO sperm, regardless of the capacitated status of the cells. This agrees with the idea that these cells already have high intracellular Na+ levels, and thus the additional influx of this cation does not modify the already high AR of NKAα4-KO sperm. In contrast, monensin enhances AR in WT sperm, even in NC medium, indicating that Na+ influx alters the behavior of WT sperm to mimic that of NKAα4-KO sperm. Together, these results show that the development of sperm AR requires the appropriate concentration of intracellular Na+.
We also found that K+ is important for sperm AR. We observed the following: (1) K+ is necessary to trigger AR in WT sperm when in Cap medium, (2) removal of K+ from the medium prevents AR in NKAα4-KO sperm, in both NC and Cap medium, and (3) the addition of valinomycin reduces AR in both WT and NKAα4-KO sperm. The effect of K+ on sperm AR agrees with previous results showing that hamster sperm in low K+ medium fails to undergo AR [32]. Since the intracellular K+ concentration is normally higher inside than outside the cell, its efflux from sperm caused by valinomycin reduces its intracellular levels. Therefore, the effect of valinomycin supports a role for K+ in AR. In addition, K+ movement through K+ channels is important to maintain the voltage difference across the cell plasma membrane and cell membrane potential. Thus, K+ efflux from sperm reduces their membrane potential to more negative values [55]. Membrane hyperpolarization is important for sperm capacitation, but acrosome-reacted sperm have a depolarized membrane potential [56]. It is possible that the membrane depolarization of NKAα4-KO sperm [19] reflects the acrosomal-reacted status of these cells. Future experiments will be needed to directly measure and correlate the changes in K+, Na+, and Ca2+ in relation to sperm membrane potential and AR.
The reduction in AR that occurs when either Na+ or K+ are separately eliminated from the medium also occurs when both Na+ and K+ are eliminated from the medium simultaneously. Moreover, in WT mice, the absence of both Na+ and K+ reduces AR even after the addition of the calcium ionophore A23187 under capacitated conditions. This shows that both Na+ and K+ are required for full development of sperm AR. However, the effect of the simultaneous retrieval of the ions is not synergistic, and the reduction of only one of them is sufficient to bring AR down to almost non-capacitated levels.
The influx of Ca2+ is essential for sperm capacitation and AR [56]. The increase in Ca2+ in sperm is highly regulated during sperm capacitation through different active and passive Ca2+ transport mechanisms, which operate in a coordinated manner at the cell plasma membrane and intracellular stores [57,58]. Among these transporters, the sperm-specific Ca2+ channel CatSper plays a prominent role [42,59]. Alkalinization of sperm and cell hyperpolarization during capacitation activate CatSper, and further stimulation of CatSper by progesterone and other agonists continues to activate CatSper-dependent Ca2+ influx to ultimately trigger AR [41,60]. The importance of Ca2+ in AR was confirmed by our findings here. Ca2+ in the medium is required for WT sperm to fully undergo AR. On the other hand, the absence of Ca2+ prevents the abnormal AR in NKAα4-KO sperm, and A23187 has no significant effect on the AR of NKAα4-KO sperm in either NC or Cap media. We previously showed that NKAα4-KO sperm has high intracellular Ca2+ levels [20]; therefore, it appears that the uncontrolled high intracellular Ca2+ of NKAα4-KO sperm may be triggering the premature AR in the cells. It is also known that the sperm plasma membrane potential influences dynamic changes in intracellular Ca2+. Thus, the presence of Ca2+ oscillations in non-capacitated cells prevents the premature occurrence of AR [61]. It is possible that the sustained increase in Ca2+ of NKAα4-KO sperm may also abolish the oscillatory pattern and promote AR.
The relevance of Ca2+ for sperm AR is also highlighted by the ability of A23187 to counteract the low AR obtained when Na+ and K+ are missing in the medium. This shows that, while Na+ and K+ are important to support AR, Ca2+ exerts a predominant role. This agrees with data published by others, which show that Ca2+ ionophores can bypass several key signaling pathways in sperm that are typically required for sperm capacitation and AR [44]. In addition, A23187 effectively rescues the fertility of sperm from sterile mouse models which lack Catsper, the K+ channel Slo3, and the protein kinase activator soluble adenylyl cyclase [62]. Moreover, A23187 also improves fertilization of cryopreserved bovine sperm [63]. In this manner, our data confirm that Ca2+ is a critical factor that induces sperm AR. Proper intracellular Na+ and K+ levels maintained by NKAα4 are important not only by themselves, but also by their indirect effects on Ca2+ intracellular concentrations, changes in membrane potential, and pH. It is clear, then, that the effects of Na+, K+, and Ca2+ on sperm AR are intertwined and that regulation of one can influence the levels of the others.

4. Materials and Methods

Design and Generation of Mouse Models. All experimental protocols involving animals were approved and monitored by the University of Kansas Medical Center Institutional Animal Care and Use Committee. The design and characterization of NKAα4-KO were previously published [22].
Sperm Isolation. Sperm from 12-week-old wild-type (WT), and NKAα4-KO mice were obtained from the cauda of adult mice epididymis after swim-up of the cells in Whitten’s non-capacitated (NC) medium, containing: 100 mM NaCl, 4.7 mM KCl, 1.2 mM KH2PO4, 1.2 mM MgSO4, 5.5 mM glucose, 0.8 mM pyruvic acid, 4.8 mM lactic acid, 20 mM HEPES (pH 7.4), and 1.7 mM CaCl2 (used for a number of assays). For some experiments, sperm was capacitated in Whitten’s capacitated medium (Cap), which contained all the components of NC medium but was supplemented with 25 mM sodium bicarbonate and 0.5% bovine serum albumin (BSA). Cells were counted, and the concentration was corrected to 106 cells/mL. Treatment with ouabain was performed at concentrations of 10−6 M and 10−3 M for two hours. For some experiments, sperm were incubated in NC or Cap medium without either NaCl, CaCl2, or KCl, but with the addition of the corresponding amount of choline chloride to maintain osmolality of the media. In some experiments, ionophores were added to increase the permeability for Na+ (nigericin), K+ (valinomycin), and Ca2+ (A23187). To ensure that the observed effects were due to the changes in ion levels and composition, the osmolarity of all media were adjusted with choline chloride and the pH was set at 7.4. The osmolarity of all media was determined using a Vapro 5520 vapor pressure osmometer (Wescor Inc., Logan, UT, USA) and was 294 ± 8 mOsm/kg.
Determination of the acrosome reaction. After subjecting to different media, sperm were washed with phosphate-buffered saline (PBS, pH 7.4) by centrifugation three times at 1000 rpm for 5 min. Sperm was then resuspended in 1 mL of 4% PFA (4% final concentration of formaldehyde in PBS), and centrifuged at 4 °C for 5 min at 5000× g. Samples were washed 3 times and resuspended in 1 mL of PBS. Aliquots of the suspension (12 μL each) were spread onto glass slides and air-dried at room temperature. Then, cells were hydrated with PBS for 10 s and fixed with 99% ethanol for 10 min. PNA-Fluorescein (Vector laboratories, Burlingame, CA, USA) was added, and the slides were incubated for 30 min at 37 °C in the dark. The slides were then washed with PBS, and DAPI (SlowFade™ Gold Antifade Mountant with DAPI, Invitrogen, Carlsbad, CA, USA) was added. After covering the samples with a coverslip, the slides were photographed under an immunofluorescence microscope (80i upright manual fluorescence microscope). To calculate the percentage of cells that underwent AR, at least 300 cells were assayed per experimental condition, and the images were analyzed using ImageJ software 1.54p (NIH, Bethesda, MA, USA). The percentage of acrosome-reacted sperm was calculated for each experimental condition by dividing the number of acrosome-reacted sperm by the total number of sperm scored and multiplying this ratio by 100.
Determination of intracellular ion concentrations. Sperm intracellular ion concentrations were assessed using the fluorescent indicators CoroNa Green, Fluo-4 AM, and PBFI AM (Life Technologies, Carlsbad, CA, USA), to detect Na+, Ca2+, and K+, respectively. Samples were processed as mentioned previously, using the appropriate NC or Cap media, and with or without the specific salt being studied. For intracellular Na+ detection, samples were labelled with 10 μM CoroNa Green for 20 min at 37 °C. For intracellular Ca2+ detection, 2 μM Fluo-4 AM was used for 20 min at 37 °C. To determine intracellular K+ concentrations, cells were loaded with 5 μM PBFI AM for 30 min at 37 °C. In all cases, cells were centrifuged at 500× g for 5 min at room temperature and washed twice with the corresponding medium in which the experiment was performed. For the experiments involving Na+, the samples were further treated with or without 2 μg/mL of the Na+ ionophore nigericin for 20 min at 37 °C. For the experiments involving Ca2+, cells were treated with or without 2 μM of the Ca2+ ionophore A23187 for 20 min at 37 °C. Finally, for the experiments involving K+, the cells were loaded with 5 μM valinomycin for 30 min at 37 °C. Once cells were treated with ionophores, they were centrifuged and washed once, resuspended in the media corresponding to each experiment, and 200 nM propidium iodide (PI) was added. Data were recorded as individual cellular events using an Attune NXT (Life Technologies, Carlsbad, CA, USA) cytometer. Debris and cell aggregates were excluded from analysis using two-dimensional sideways (SSC)–forward (FSC) scatter dot plots. FSC and SSC fluorescence data were collected from 10,000 events per sample. Cells negative for PI, using the filter for PI (585/40 nm), and positive for the various ion indicators were gated in logarithmic mode and collected as data. CoroNa Green and Fluo-4 AM were detected with a fluorescein isothiocyanate (FITC; 530/30 nm) filter, and PBFI was detected using a 450/50 nm filter.

5. Conclusions

The NKAα4 isoform is important not only for sperm motility, as we previously demonstrated, but also for sperm AR. We found that NKAα4 directly controls intracellular Na+ and K+ and indirectly influences intracellular Ca2+. In addition, we showed that NKAα1 is also required for the full development of AR, but that it cannot compensate if NKAα4 is missing. The complexity and coordinated interplay between Na+, K+, and Ca2+ requires further investigation of how their temporal and spatial changes modulate sperm function and male fertility.

Author Contributions

Conceptualization, G.B., J.M., L.A.B. and V.F.C. Sperm isolation, acrosome reaction experiments and preparation of data, M.M.T. and A.M. Writing, reviewing and editing, G.B., J.M., L.A.B. and V.F.C. All authors have read and agreed to the published version of the manuscript.

Funding

This work was supported with funds from the National Institutes of Health, grant R01 HD102623.

Institutional Review Board Statement

Maintenance and handling of mice, as well as all experimental protocols involving animals were approved and monitored by the University of Kansas Medical Center Institutional Animal Care and Use Committee (ACUP #22-09-261, approved date: 20 January 2026).

Data Availability Statement

All data are presented in this manuscript, and the raw data generated in this study will be provided by the authors upon request.

Acknowledgments

We thank the KUMC Imaging Core for their help with the images used to determine the sperm acrosome reaction.

Conflicts of Interest

The authors declare no conflicts of interest with the work reported in this article.

Abbreviations

The following abbreviations are used in this manuscript:
ARAcrosome reaction
CapCapacitation
NCNon-capacitated
NKANa,K-ATPase
NKAα4Na,K-ATPase α4
NKAα1Na,K-ATPase α1

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