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All mouse procedures were reviewed and approved by the Institutional Animal Care and Use Committee of Washington University in St. Louis (St. Louis, MO, United States) (protocol # 20-0126) and were performed according to the National Institutes of Health Guiding Principles for the care and use of laboratory animals. CatSper knock-out mice (in which the second exon encoding the first putative transmembrane domain was replaced with an IRESLacZ sequence followed by a neomycin resistance gene in 129S4/SvJae-derived J1 ES cells, Ren et al. (2001) were obtained from Jackson Laboratory. C57BL/6 males were used as wild-type controls. Soluble adenylyl cyclase (sAC) knock-out mice were generously provided by Lonni Levin and J Buck (Buck et al., 1999; Hess et al., 2005).

This study conformed with the Declaration of Helsinki (except for registration in a database) and was approved by the Washington University in St. Louis Institutional Review Board (protocol #201706077). All subjects signed written informed consent forms approved by the Washington University in St. Louis Institutional Review Board.

Adult (60–90 days old) males were euthanized by cervical dislocation, and the cauda epididymis was removed and incubated in non-capacitated or capacitated HS media (defined below), pH 7.4, for 15–20 min or 90 min, respectively, at 37^oC. After incubation, the motile sperm were removed for use in experiments.

Mouse ejaculated spermatozoa were isolated as described previously (Li et al., 2015). Briefly, each adult male (60–90 days old) was placed in a cage with a female in estrus before sunrise (3–5 am). Mice were watched under a red light to determine when coitus occurred. Five to ten minutes after coitus was complete, the females were euthanized, and their uteri dissected out. Sperm were retrieved by washing the uterus with 500 μl of non-capacitated HS media, yielding an average of 10.64 ± 6.85 (SD) million sperm per milliliter. Ejaculated mouse sperm did not appear to be capacitated, as the percentages of hyperactivated and acrosome-reacted sperm were similar to the percentages in non-capacitated epididymal sperm (data not shown).

Small (1 cm) vas deferens pieces were obtained from healthy donors undergoing elective vasectomy procedures under local anesthesia. Specimens were kept in non-capacitated HS media at room temperature and flushed within an hour after surgery with non-capacitated recording solution (described below) within 1 h of obtaining the sample.

Freshly ejaculated semen samples were obtained by masturbation after two days of abstinence from healthy donors with normal semen parameters as defined by the World Health Organization (≥32% progressive motility; ≥40% total motility; ≥15 × 10⁶ cells/ml). Within two hours, sperm were purified by the swim-up technique at 37°C in a VWR Borosilicate Glass tube with 2 ml of non-capacitated recording solution and 1 ml of sample at the bottom. Sperm were allowed to swim out of the semen for an hour, then collected in recording solution. Human sperm were capacitated by adding 25 mM HCO₃^– (Sigma-Aldrich) and 5 mg/ml BSA to the non-capacitating recording solution and incubating the cells for 18–24 h at 37^oC and 5% CO₂.

After swim up, motile sperm were incubated with 2–4 μM Fluo-4 AM (catalog number F-4201, Life Technologies) and 0.05% Pluronic F-127 (catalog number P3000MP, Life Technologies) in HS media at 37°C for 45 min (human sperm) or 90 min (mouse sperm). Then, sperm were centrifuged at 325 RCF for 5–10 min and resuspended in the corresponding media. Sperm were allowed to attach to the recording chamber floor for 5 min. Mouse sperm were then overlaid with a coverslip coated with1 mg/ml Laminin Mouse Protein, Natural (catalog number 23017015, Gibco^®), 0.1% (w/v) Poly-L-lysine in water (catalog number P8920-100ml, Sigma-Aldrich), or Cell-Tak (catalog number 354240, Corning Labware, Bedford, MA, United States). Laminin-coated cover slips were incubated at 4^oC for at least 12 hrs before the experiment. Human sperm were overlaid with a coverslip coated with 0.1% Poly-L-lysine or 1 mg/ml Cell-Tak. Non-capacitated sperm were not exposed to HCO₃^– at any time before the recordings. A perfusion device with an estimated exchange time of 10 s was used to apply test solutions. Sperm were pre-incubated with 15 mM (mouse) or 25 mM (human) HCO₃^– for 20 min before recording. Then, HCO₃^– was washed out for 2–4 min so that, at time “0” when the recording was started, no HCO₃^– was present in the recording solution. PKA inhibitors and sAC inhibitor were added only during the pre-incubation period and were not present at the time of stimulation with KCl. Ionomycin (5 μM) and HS plus 2 mM CaCl₂ were added at the end of the recordings as references of maximum fluorescence (F_Iono).

Imaging was performed with a Leica DMi8 microscope equipped with a Zyla Andor sCMOS camera, and images were collected for 1.5 ms every 5–10 s over 10–30 min. Images were analyzed with Image J software. Fluorescence (F) of Fluo-4 changes was normalized to F_Iono(F/F_iono) after background subtraction. Calcium imaging data are presented as the average ± the standard deviation. The regions of interest were selected automatically and corroborated with ImageJ software. Pclamp 10 and Sigmaplot 12 were used to analyze data. Experiments were done at room temperature. Sperm in which the peak [Ca²⁺]_i reached 10% or more of the [Ca²⁺]_i in the presence of Ionomycin were considered to have responded to the stimuli.

Sperm samples were incubated for 60 min in the presence or absence of the soluble adenylyl cyclase inhibitor TDI-10229 (donated by Lonnie Levin and Johen Buck’s labs) (Balbach et al., 2021) in capacitating TYH media or in non-capacitating HS media. After incubation, sperm pH was evaluated as previously described (Gunderson et al., 2021). Briefly, samples were incubated for 10 min with 0.5 μM BCECF-AM, washed, and resuspended in non-capacitating TYH media, media plus 15 mM bicarbonate, or media plus 15 mM HCO₃^– and 5 μM TDI-10229. For each condition, high potassium-buffered solutions were used to calibrate the pH by adding 5 μM nigericin to equilibrate intracellular and extracellular pH and create a pH calibration curve. Fluorescence of BCECF was recorded as individual cellular events on a FACSCanto II TM cytometer (Becton Dickinson) (excitation 505 nm, emission 530 nm). Sperm intracellular pH was calculated by linearly interpolating the median of the histogram of BCECF fluorescence of the unknown sample to the calibration curve.

Non-capacitating HS medium (in mM): 135 NaCl, 5 KCl, 2 CaCl₂, 1 MgSO₄, 20 HEPES, 5 Glucose, 10 lactic acid (catalog number L13242, Alfa Aesar, Lancashire, United Kingdom), 1 sodium pyruvate (catalog number P2256, Sigma-Aldrich, Saint Louis, MO) at pH7.4 or as indicated. Capacitated HS medium: Same as non-capacitating HS medium plus 5 mg/ml bovine serum albumin (BSA, catalog number A2153, Sigma-Aldrich, Saint Louis, MO) and 15 mM NaHCO₃ (catalog number S5761, Sigma-Aldrich) (Chavez et al., 2014). 50 mM KCl HS (in mM): 90 NaCl, 50 KCl, 2 CaCl₂, 1 MgSO₄, 20 HEPES, 5 glucose, 10 lactic acid, 1 sodium pyruvate in pH as indicated. Recording solution (in mM): 110 NaCl, 4 KCl, 2 CaCl₂, 1 MgCl₂, 20 HEPES, 5 glucose, 10 lactic acid, 1 sodium pyruvate at pH as indicated. KCl Recording solution (in mM): 65 NaCl, 50 KCl, 2 CaCl₂, 1 MgCl₂, 20 HEPES, 5 glucose, 10 lactic acid, 1 sodium pyruvate at pH as indicated. TYH medium (in mM):135 NaCl, 4.7 KCl, 1.7 CaCl2, 1.2 KH2PO4, 1.2 MgSO4, 5.6 glucose, 0.56 pyruvate, 10 HEPES, pH 7.4 adjusted at 37°C with NaOH.

pH of the solutions after addition of HCO₃^– was measured with a Sartorious PB-10 basic bench top pH meter, with automatic temperature compensation.

Dimethyl sulfoxide (DMSO) (catalog number D8418, Sigma-Aldrich); CatSper inhibitor Mibefradil (catalog number 2198, Tocris Bioscience, Bristol, United Kingdom); PKI (Protein Kinase A inhibitor fragment 14-22, myristoylated trifluoroacetate salt, catalog number P9115; Sigma Aldrich); KT5720 (CAS number 108068-98-0, Cayman Chemicals).

We first repeated previous experiments on mouse epidydimal sperm (Chavez et al., 2014) and confirmed that, at pH 7.4, KCl induced an increase in [Ca²⁺]_i in 12% of sperm in non-capacitating conditions and 61% of sperm in capacitating conditions (bicarbonate [HCO₃^–] plus bovine serum albumin). In this and all subsequent experiments, sperm in which the peak [Ca²⁺]_i reached 10% or more of the [Ca²⁺]_i in the presence of Ionomycin were considered to have responded. As expected, bypassing the increase in pH that occurs during capacitation by incubating the sperm at alkaline pH (pH 8.6) allowed 77% of the non-capacitated sperm to respond to KCl. The top row of Figure 1 shows representative traces of the most prevalent response in each condition.

We next performed similar experiments with human ejaculated sperm, which have been most commonly used to study sperm Ca²⁺ signaling and CatSper activation. For these experiments, we first tested pH 7.4 as in the mouse sperm experiments but found that it caused Ca²⁺ responses with varying kinetics (not shown). Thus, we instead used pH 6.8 and pH 8.0 because they yielded consistent and reproducible Ca²⁺ responses similar to those obtained with mouse sperm at pH 7.4 and pH 8.6. In ejaculated human sperm at pH 6.8, membrane depolarization with KCl triggered an increase in [Ca²⁺]_i in 67% of sperm in non-capacitating conditions and 60% of sperm in capacitating conditions. As with mouse epididymal sperm, bypassing the increase in pH that occurs during capacitation with alkaline pH (pH 8.0) allowed a high percentage (81%) of sperm to respond to KCl. The bottom row of Figure 1 shows representative traces of the most prevalent response in each condition. We conclude that, at low pH in non-capacitating conditions, epididymal mouse and ejaculated human sperm sharply differ in their abilities to increase [Ca²⁺]_i in response to KCl-mediated depolarization.

To determine whether sperm from the two species at similar stages of maturation would respond to KCl-induced membrane depolarization similarly, we compared epididymal mouse sperm to epididymal human sperm and compared ejaculated mouse sperm to ejaculated human sperm. In each case, we assessed response by measuring the percentage of sperm that responded to the KCl stimulus. Additionally, given that the slope of the [Ca²⁺]_i increase is a good indicator of the number of CatSper channels that are open in response to membrane depolarization (Wennemuth et al., 2003), we compared the rate of [Ca²⁺]_i increase in the responsive sperm during the first 20 s of the depolarization-evoked response.

In non-capacitating conditions, 12% of mouse epididymal sperm increased [Ca²⁺]_i in response to high external KCl at pH 7.4 (Figures 2A,G). Similarly, 8% of human epididymal sperm increased [Ca²⁺]_i in response to high external KCl pH 6.8 (Figures 2C,G). In contrast, 42% of mouse ejaculated sperm and 67% of human ejaculated sperm increased [Ca²⁺]_i in response to high external KCl (Figures 2B,D,G). In both mouse and human sperm, a significantly higher percentage of ejaculated sperm responded than did epididymal sperm (Figure 2G). When we examined only those sperm in each condition that responded, we found that the rate of [Ca²⁺]_i increase was significantly higher in ejaculated mouse and human sperm than in epididymal mouse and human sperm, respectively (Figures 2E,F).

In capacitating conditions, mouse epididymal sperm showed [Ca²⁺]_i increases in response to high external KCl at low pH, and both non-capacitated and capacitated sperm from human and mouse species showed [Ca²⁺]_i increases in response to high external KCl at high pH (Supplementary Figure 1; Wennemuth et al., 2000; Chavez et al., 2014). As expected (Xia et al., 2007; Chavez et al., 2014), capacitated epididymal sperm from CatSper knockout mice did not show [Ca²⁺]_i increases in response to high external KCl (Supplementary Figure 1C). Similarly, in ejaculated human sperm, the [Ca²⁺]_i increase in response to high external KCl was inhibited by the CatSper channel blocker Mibefradil (Supplementary Figure 1F). Together, these results indicate that human and mouse ejaculated sperm show comparable [Ca²⁺]_i increases in response to membrane depolarization by high extracellular KCl. This depolarization similarly activates CatSper channels in mouse and human sperm at similar maturational stages.

The main difference between epididymal and ejaculated sperm is that ejaculated sperm are exposed to semen. Thus, we aimed to identify the component in semen that was responsible for the differences in [Ca²⁺]_i increases in response to membrane depolarization between epididymal and ejaculated sperm. We focused on bicarbonate (HCO₃^–) because it is a major component of semen (Okamura et al., 1985) and influences sperm function and intracellular Ca²⁺ regulation (Austin, 1952; Austin and Bishop, 1958; Wennemuth et al., 2003; Chavez et al., 2014). For example, Wennemuth et al. showed that a pulse of HCO₃^– facilitated Ca²⁺ increase in mouse sperm by enhancing the rate of depolarization-evoked [Ca²⁺]_i increase (Wennemuth et al., 2003). Additionally, de la Vega-Beltran et al. (2012) and Chavez et al. (2014) showed that exposure to HCO₃^– and bovine serum albumin triggered Ca²⁺ responses in mouse sperm, and Orta et al. (2018) showed that CatSper activity was modulated by HCO₃^– in mouse sperm.

To assess the effect of HCO₃^–, we pre-incubated mouse epididymal sperm without or with 15 mM HCO₃^– (a concentration similar to that in seminal fluid) (Okamura et al., 1985) for 20 min. Whereas 12% of sperm not exposed to HCO₃^– showed increased [Ca²⁺]_i in response to high extracellular KCl at pH 7.4 (Figures 3A,G), 48% of sperm exposed to HCO₃^– showed increased [Ca²⁺]_i in response to high extracellular KCl at pH 7.4 (Figures 3B,G). Exposure to HCO₃^– also increased the percentage of cells showing increased [Ca²⁺]_i in response to high extracellular KCl at pH 8.6 (Figures 3C,D,G). When we only examined the responding sperm in each condition, we found that pre-incubation with HCO₃^– enhanced the rate of depolarization-evoked [Ca²⁺]_i increase in mouse epididymal sperm (Figures 3E,F), indicating an increase in the number of open CatSper channels in response to KCl depolarization. These [Ca²⁺]_i increases were not observed in sperm from CatSper knock-out mice (Figure 3G). Importantly, 15 mM HCO₃^– did not significantly increase the pH of the HS media or the recording solution (Supplementary Figure 2). Moreover, HCO₃^– was not present in the chamber during the recordings.

To assess the effect of HCO₃^– on [Ca²⁺]_i responses in non-capacitated human epididymal sperm, we incubated them in the presence or absence of HCO₃^– for 20–30 min and then exposed them to high external KCl. High external KCl at pH 6.8 induced [Ca²⁺]_i increases in 9% of the human epididymal sperm in control conditions (Figures 4A,E). In contrast, high external KCl at pH 6.8 induced [Ca²⁺]_i increases in 37% of sperm pre-incubated with HCO₃^– (Figures 4B,E). Pre-incubation with HCO₃^– also significantly increased the rate of depolarization-evoked [Ca²⁺]_i increase in human epididymal sperm (Figures 4C,D), indicating an increase in the number of open CatSper channels in response to KCl depolarization. These results indicate that [Ca²⁺]_i responses to membrane depolarization depend on HCO₃^– in both mouse and human sperm.

One of the early events of sperm capacitation, which can occur as soon as sperm are ejaculated, is HCO₃^–-induced activation of soluble adenylyl cyclase (sAC), leading to increased intracellular cyclic adenosine monophosphate (cAMP) and activation of protein kinase A (PKA) (Chen et al., 2000; Hess et al., 2005; Xie et al., 2006; Visconti, 2009). Given a report suggesting that CatSper channels are modulated by PKA (Orta et al., 2018), we wondered whether the effect of HCO₃^– on the Ca²⁺ responses in epididymal sperm depended on PKA activation. Recent work from Orta et al. showed that the PKA inhibitor PKI reduced HCO₃^–-induced [Ca²⁺]_i increases in mouse epididymal sperm without directly blocking mouse CatSper currents (Orta et al., 2018). Thus, we incubated mouse epididymal sperm in HCO₃^– in the absence or presence of 10 μM PKI for 20 min before recording, then stimulated them with KCl in the absence of HCO₃^– or PKI. Compared to sperm not pre-treated with PKI, significantly fewer sperm pre-treated with PKI showed an increase in [Ca²⁺]_i in response to high external KCl at both pH 7.4 and pH 8.6 (Figure 3G). The rate of [Ca²⁺]_i increase of the responsive cells was also significantly lower in cells pre-incubated with HCO₃^– in the presence than in the absence of PKI (Figures 3E,F).

We next asked whether the cAMP/PKA pathway was also involved in [Ca²⁺]_i increases induced by external KCl in ejaculated mouse sperm. At pH 7.4 (Figures 5A,B), treatment with PKI reduced the percentage of ejaculated mouse sperm that showed increased [Ca²⁺]_i in response to high extracellular KCl from 42 to 16% (Figure 5G, top panel). PKI also significantly reduced the rate of [Ca²⁺]_i increase in the responsive sperm (Figures 5E,F). Likewise, at pH 8.6 (Figures 5C,D), treatment with PKI reduced the percentage of ejaculated sperm that showed increased [Ca²⁺]_i in response to high extracellular KCl from 71 to 32% (Figure 5G, bottom panel). PKI also significantly reduced the rate of [Ca²⁺]_i increase in the responsive sperm (Figures 5E,F). These results indicated that PKI reduced the number of CatSper channels that opened in response to membrane depolarization in mouse ejaculated sperm.

We wanted to determine whether PKA was required for the [Ca²⁺]_i increase in ejaculated human sperm. However, because PKI has been reported to directly inhibit human CatSper currents (47), we instead used KT5720, a PKA inhibitor that does not directly inhibit human CatSper currents (Wang et al., 2020). Pre-incubation of ejaculated human sperm with HCO₃^– in the presence of 50 μM KT5720 reduced the percentage of sperm responding from 67 to 32% (Figures 6A–D,G). Additionally, KT5720 significantly decreased the rate of [Ca²⁺]_i increase in responsive sperm (Figures 6E,F). These results indicate that the cAMP/PKA pathway is involved in the [Ca²⁺]_i responses to membrane depolarization in ejaculated sperm from both mice and humans.

As mentioned above, HCO₃^– regulates the production of cAMP and PKA activity through stimulation of a soluble adenylyl cyclase (sAC) (Chen et al., 2000; Hess et al., 2005; Xie et al., 2006; Visconti, 2009). Moreover, KCl-induced Ca²⁺ entry is impaired in sAC (Adcy10) knock-out mice (Xie et al., 2006). To determine whether sAC was required for the depolarization-induced [Ca²⁺]_i increase in sperm, we took two approaches. First, in a genetic approach, we isolated epididymal sperm from sAC knockout mice. HCO₃^– had no effect on the percentages of sperm from sAC knockout mice that increased [Ca²⁺]_i in response to extracellular KCl (Figure 7H). Likewise, HCO₃^– had no effect on the rate of [Ca²⁺]_i increase in responding sperm from sAC knockout mice (Figures 7F,G).

Second, in a pharmacologic approach, we treated epididymal sperm from wild-type mice with the sAC-specific inhibitor TDI-10229 (Balbach et al., 2021) and found that it prevented an increase in the percentage of sperm responding to high extracellular KCl (Figures 7C,H). Treatment with TDI-10229 also reduced the rate of [Ca²⁺]_i increase in responsive sperm (Figures 7A,B,D,E). Likewise, TDI-10229 prevented the increase in the percentage of ejaculated human sperm responding to high extracellular KCl (Figure 8G) and reduced the rate of [Ca²⁺]_i increase in the responsive sperm (Figures 8A–F). Together, these data indicate that activation of CatSper channels by membrane depolarization is facilitated by activation of the HCO₃^–-sAC-cAMP/PKA pathway in both mouse and human sperm.

Several studies have revealed that HCO₃^– raises sperm intracellular pH (pH_i) (Demarco et al., 2003; Chavez et al., 2019; Matamoros-Volante and Trevino, 2020) and that HCO₃^– can activate CatSper channels in mouse sperm (Orta et al., 2018). Therefore, we investigated whether the [Ca²⁺]_i changes induced by HCO₃^– were due to a change in pH_i. We found that addition of HCO₃^– to the media (which only raised the media pH by 0.1 unit; Supplementary Figure 2) caused a 0.4–0.5 unit increase in non-capacitated mouse epididymal sperm pH_i within 1–2 min (Figure 9A). This increase in pH_i was similar to the increase in pH_i seen in capacitated mouse sperm and was completely inhibited by incubation with the sAC inhibitor TDI-10229 (Figure 9B). Finally, we asked whether we could bypass the need for sAC by treating sperm with 20 mM NH₄Cl, which increases pH_i. This treatment increased the percent of sperm from sAC knock-out mice that increased [Ca²⁺]_i in response to high extracellular KCl from 33.8 to 61.5% (Figure 7H). These data indicate that the change in pH_i induced by sAC/PKA activation is responsible for sensitizing CatSper channels to membrane depolarization.