The sodium-proton exchangers sNHE and NHE1 control plasma membrane hyperpolarization in mouse sperm

Abstract

Sperm capacitation is a complex process that takes place in the female reproductive tract and empowers mammalian sperm with the competence to fertilize an egg. It consists of an intricate cascade of events that can be mimicked in vitro through incubation in a medium containing essential components, such as bicarbonate, albumin, Ca²⁺, and energy substrates, among others. Genetic and pharmacological studies have underscored the unique significance of the K⁺ channel SLO3 in membrane potential hyperpolarization, as evidenced by the infertility of mice lacking its expression. Notably, two key molecular events, sperm hyperpolarization and intracellular alkalinization, are central to the capacitation process. SLO3 is activated by alkalinization. However, the molecular mechanisms responsible for intracellular alkalization and activation of SLO3 are not completely understood. In this study, we examined the impact of Na⁺/H⁺ exchangers (NHEs) on mouse sperm membrane hyperpolarization during capacitation. Pharmacological inhibition of the NHE1 blocked membrane hyperpolarization. A similar effect was observed in sperm deficient of the Ca²⁺ channel CatSper because of NHE1 not being activated by Ca²⁺. In addition, the sperm-specific NHE (sNHE) KO did not show membrane hyperpolarization upon capacitation or induction with cAMP analogs. Our results show that sNHE is dually modulated by cAMP and membrane hyperpolarization probably through its cyclic nucleotide-binding domain and the voltage-sensor motif, respectively. Together, sNHE and NHE1 provide the alkalinization need for SLO3 activation during capacitation.

Keywords: adenylate cyclase; cyclic AMP; fertilization; membrane hyperpolarization; potassium channel; sodium–proton exchange; sperm.

Figure 1

Mouse sperm Em hyperpolarization can dispense PKA catalytic activity.A, fluorescence traces showing the values of the sperm E_m obtained after sperm incubation in either noncapacitating (NC) or capacitating (cap) conditions containing or not 15 μM sPKI for 60 min. Each experiment displays its calibration curve and the estimated Em value. B, summary of E_m measurements of sperm incubated in conditions depicted in A (mean ± SD; n ≥ 5). One-way ANOVA with Tukey’s multiple comparisons test was performed; different letters indicate statistically significant differences (p < 0.001). C, sperms were incubated for 60 min in NC or cap medium containing or not 15 μM sPKI. Each condition was processed for Western blot analysis with a monoclonal anti-pPKAs antibody. Membrane was stripped and analyzed for the presence of tubulin using anti-β-tub (clone E7). D, sperm E_m measurements obtained after 60 min incubation of either Slo3 KO (gray boxes) or WT sperm (white boxes) in either NC or cap medium containing or not 15 μM sPKI. One-way ANOVA with Tukey’s multiple comparisons test was performed (mean ± SD; n = 4; ∗p < 0.001). Each colored dot represents the value for each independent sample. E, sperm E_m measurements obtained after 60 min incubation in NC or cap medium; as indicated, capacitating media were supplemented with either 30 μM H-89, 500 μM 8Br-cAMP (8Br), or 500 μM Rp-cAMPS (Rp). One-way ANOVA with Tukey’s multiple comparisons test was performed (mean ± SD; n = 3); different letters indicate statistically significant differences (p < 0.001). Each colored dot represents the value for each independent sample. F, sperms were incubated for 60 min in NC or cap medium; as indicated, cap media were supplemented with either 30 μM H-89 or 500 μM Rp. Each condition was processed for Western blot analysis with a monoclonal anti-pPKAs antibody. Membrane was stripped and analyzed for the presence of tubulin using anti-β-tub (clone E7).

Figure 2

Mouse sperm Em hyperpolarization is cAMP regulated and soluble Na⁺/H⁺exchanger (sNHE) dependent.A, sperm E_m obtained after incubation in either noncapacitating (NC) or capacitating (cap) conditions containing 500 μM 8Br-cAMP alone or in addition to15 μM sPKI for 60 min. Results are expressed as a normalization of percentage of hyperpolarization considering mean NC and cap values as 0% and 100%, respectively (mean ± SD; n = 4). One-way ANOVA with Tukey’s multiple comparisons test was performed; different letters indicate statistically significant differences (p < 0.001). B, sperm E_m obtained after incubation in either cap (with either 10 μM TDI10229 alone or in combination with 500 μM 8Br-cAMP) or NC conditions containing or not 500 μM 8Br-cAMP for 60 min. Results are expressed as a normalized percentage of hyperpolarization considering NC mean and cap mean values as 0% and 100%, respectively (mean ± SD; n = 9). One-way ANOVA with Tukey’s multiple comparisons test was performed. Different letters indicate statistically significant differences (p < 0.001). Each colored dot represents the value for each independent sample. C, sperms were incubated for 60 min in NC media containing increasing concentrations of 6Bnz-cAMP, as indicated. Each condition was processed for Western blot analysis with a monoclonal anti-pPKAs antibody. The membrane was stripped and analyzed for the presence of tubulin using anti-β-tub (clone E7). D, summary of densitometry analysis of sperm cells incubated as in A. One-way ANOVA with Tukey’s multiple comparisons test was performed (mean ± SD; n = 3). Different letters indicate statistically significant differences (p < 0.05). E, E_m measurements of sperm incubated in NC conditions containing either 30 μM 8-pCPT-2′-O-Me-cAMP (8pCPT), 50 nM 6Bnz-cAMP, 500 μM 8Br-cAMP, or 500 μM dibutyryl-cAMP (db-cAMP) for 60 min (mean ± SD; n = 3). One-way ANOVA with Tukey’s multiple comparisons test was performed; different letters indicate statistically significant differences (p < 0.001). F, summary of E_m sperm measurements from sNhe KO (gray boxes) or WT mice (black boxes) obtained after incubation in either cap or NC conditions containing 500 μM 8Br-cAMP or 10 mM NH₄Cl for 60 min. One-way ANOVA with Tukey’s multiple comparisons test was performed (mean ± SD; n = 5; ∗p < 0.05).

Figure 3

Em hyperpolarization induces intracellular alkalinization.A, sperms were incubated in capacitating (cap) or noncapacitating (NC) media either in the absence (NC) or the presence of 1 μM valinomycin (NC + Val). Representative BCECF (pH-sensitive fluorophore) versus propidium iodide (PI) two-dimensional fluorescence dot plot analysis. Squares marked as Q1 (upper left squares) define sperm with high intracellular pH displaying membrane integrity (low PI staining). Numbers in each square indicate percentage of total population detected. B, sperms were incubated in cap or NC media either in the absence (NC) or the presence of 1 μM valinomycin (NC + Val). The two-dimensional dot plots shown in A were used to select sperm with low (live) PI staining. The histogram analysis depicts normalized frequency of sperm and BCECF fluorescence of live sperm populations. C, normalized median fluorescence intensity of BCECF compared to the control capacitating condition (mean ± SD, n = 4, ∗p < 0.05).

Figure 4

NHE1 activity through Ca²⁺stimulation is conducive to Em hyperpolarization.A, E_m obtained after sperm incubation in either noncapacitating (NC) or capacitating (cap) conditions containing different concentrations of 5-(N,N-dimethyl)-amiloride (DMA) as indicated, for 60 min. One-way ANOVA with Tukey’s multiple comparisons test was performed (mean ± SD; n = 4; ∗p < 0.001). B, sperms were incubated for 60 min in NC or in cap medium in the presence or the absence of 10 μM DMA. Each condition was processed for Western blot analysis with a monoclonal anti-pPKAs antibody. C, sperms were incubated for 60 min in NC or cap medium containing or not 10 μM TDI-10229 (TDI) or 1 μM DMA. One-way ANOVA with Dunnett’s multiple comparisons test was performed; different letters indicate statistically significant differences (p < 0.001). D, E_m obtained after sperm incubation in either NC or cap conditions containing different concentrations of cariporide, as indicated, for 60 min. One-way ANOVA with Tukey’s multiple comparisons test was performed; different letters indicate statistically significant differences (mean ± SD; n = 4; ∗p < 0.001). E, E_m obtained after sperm incubation in NC conditions in the presence or not of 500 μM 8Br-cAMP (8Br) or in cap conditions. As specified, cap conditions were supplemented with either 1 μM DMA or 10 μM cariporide for 60 min. As indicated, these conditions were also supplemented with 500 μM 8Br-cAMP or 10 mM NH4Cl. One-way ANOVA with Tukey’s multiple comparisons test was performed (mean ± SD; n = 4; ∗p < 0.01). F, sperm E_m from either CatSper1 KO (left panel) or WT mice (right panel) were obtained after sperm incubation in either NC (with boxes) or cap conditions (gray boxes) containing or not either 500 μM 8Br-cAMP or 10 mM NH₄Cl for 60 min. One-way ANOVA with Tukey’s multiple comparisons test was performed (mean ± SD; n = 5; ∗p < 0.005).

Figure 5

Intracellular Ca²⁺increase promotes cytoplasmic alkalinization in sperm cells. Noncapacitated (NC) sperm cells were loaded with 0.5 μM BCECF-AM for 30 min before smearing onto laminin-precoated coverslips to record fluorescence. A, representative fluorescence images of WT (upper panels) and sNhe KO (lower panels) sperm exposed to 10 μM of the ionophore A23187, followed by 10 mM NH₄Cl. Reference bar for fluorescence intensity is depicted. Scale bar represents 10 μm. B, summary average traces of experiments performed in A, including a mock treatment on WT sperm performed with dimethyl sulfoxide instead of A23187. About 122 of 140 cells and 77 of 99 cells analyzed responded in WT and sNHE KO, respectively; n = 5. C, summary average traces of either WT or sNhe KO sperm were exposed to 10 μM ionomycin followed by 20 mM NH₄Cl. About 117 of 147 cells and 92 of 110 cells analyzed responded in WT and sNHE KO, respectively; n = 4. D, summary average traces of either WT or sNhe KO sperm incubated in nominal zero Ca²⁺ (no added Ca²⁺ salts) challenged with 1.7 mM CaCl₂ and followed by 20 mM NH₄Cl. About 106 of 139 cells (n = 4) and 69 of 94 cells (n = 3) analyzed responded in WT and sNHE KO, respectively. E, summary average traces of sNhe KO sperm incubated in nominal zero Ca²⁺ (no added Ca²⁺ salts) in the presence or not of 10 μM cariporide, and challenged with 1.7 mM CaCl_2, followed by 20 mM NH₄Cl. About 78 of 101 cells (n = 3) and 20 of 83 cells (n = 3) analyzed responded in the absence or the presence of cariporide, respectively. sNHE, soluble Na⁺/H⁺ exchanger.

Figure 6

Working model proposing a dual action of sNHE and NHE1 on sperm Em hyperpolarization. (1) Synthesis of cAMP induces alkalinization via sNHE, which, together with the action of NHE1 driven by intracellular Ca²⁺ increase (1′), promote the necessary alkalinization (2 and 2′) to increase the conductance of SLO3 (3). In turn, Em hyperpolarization stimulates a positive feedback loop that further activates sNHE (4). It is worth noting that the sole action of sNHE (inhibition of NHE1) or of NHE1 (in the case of sNhe KO) is not sufficient, under physiological conditions, to promote SLO3 opening. sNHE, soluble Na⁺/H⁺ exchanger.

Supplemental Fig. S1

Ba²⁺ blocks Em hyperpolarization even in the presence of sPKI. Sperm E_m measurements obtained after incubation for 60 min in capacitating conditions containing either 1 mM BaCl₂ or 15 μM sPKI or a combination of both. Results are expressed as a normalization of percentage of hyperpolarization considering mean NC and cap values as 0% and 100%, respectively (mean ± SD; n = 4); one-way ANOVA with Turkey’s multiple comparisons test was performed; different letters indicate statistically significant differences (p < 0.05).