Percoll was obtained from GE Healthcare BioSciences (Little Chalfont, UK). Lysis buffer and other reagents for sodium dodecyl sulfate-polyacrylamide gel electrophoresis (SDS-PAGE) were purchased from the Beyotime Institute of Biotechnology (Shanghai, China). Dimethyl sulfoxide (DMSO) was acquired from Merck (Darmstadt, Germany). Enhanced chemiluminescence (ECL) Plus Chemiluminescence Kit, protein loading buffer, and pre-dyed protein markers were acquired from Thermo Fisher Scientific (Burlington, NC, United States). Polyvinylidene fluoride (PVDF) membranes were obtained from Millipore Corporation (Bedford, MA, United States). Fluorescein isothiocyanate-conjugated Pisum sativum agglutinin (PSA-FITC), KCNQ1 inhibitor chromanol 293B, and carbonyl cyanide m-chlorophenylhydrazone (CCCP) were obtained from Sigma-Aldrich (St. Louis, MO, United States). Complete mini EDTA-free protease inhibitor cocktail and phosphatase inhibitor cocktail (broad-spectrum phosphatase inhibitor, including Ser/Thr and Tyr phosphatase inhibitors) were obtained from Roche (Mannheim, Germany). The antibodies used in this study were as follows: KCNQ1 (ab84819), KCNE1 (ab65795), rabbit anti-β-tubulin (ab6046), Alexa Fluor 555-conjugated goat anti-mouse antibody (ab150118), Alexa Fluor 488-conjugated goat anti-rabbit antibody (ab150077), and Alexa Fluor 488-conjugated goat anti-mouse antibody (ab150113) were purchased from Abcam (Cambridge, UK); p-Tyr (sc-7,020) and KCNQ1 (sc-365,764) were obtained from Santa Cruz Biotechnology Inc (Dallas, Texas, United States). KCNE1 (31195A31) and horseradish peroxidase-conjugated goat antibodies were purchased from Invitrogen (Carlsbad, CA, United States). Fluo3-AM and MQAE were obtained from Beyotime Institute of Biotechnology (Shanghai, China). BCECF-AM, DisC3(5), and PBFI-AM were purchased from Invitrogen (Carlsbad, CA, United States).

Human tubal fluid (HTF) medium was prepared as previously described (Li et al., 2021; Sun et al., 2021). The HTF medium comprised 5.06mM KCl, 90mM NaCl, 25.3mM NaHCO₃, 1.17mM KH₂PO₄, 1.8mM CaCl₂, 1.01mM MgSO₄, 0.27mM sodium pyruvate, 5.56mM glucose, 21.6mM sodium lactate, 20mM HEPES, 4g/L bovine serum album, 5mg/L phenol red and 60mg/L penicillin. The pH was adjusted to 7.4. All chemicals were obtained from Sigma-Aldrich.

This study was approved by the Medical Ethics Committee of Hangzhou Medical College (no. 2018004). Written informed consent was obtained from 15 healthy male donors (aged 25–35years). Sperm preparation was performed as previously described (Sun et al., 2021). The donors abstained from sexual intercourse for 3days before sample collection. Fresh semen were obtained via masturbation, collected in sterile containers, and subsequently liquefied at 37°C for 1h. According to the World Health Organization (WHO) requirements, semen samples in this study met the following criteria: sperm viability ≥85%, sperm motility ≥50%, morphologically normal sperm ≥15%, and sperm concentration≥20×10⁶ sperm/mL. To remove dead sperm and cell debris, semen samples were centrifuged with 40 and 80% discontinuous Percoll gradients at 750×g for 15min and the precipitate was resuspended in HTF medium. Sperm collected from at least three donors were mixed, washed, adjusted to a density of approximately 20×10⁶ sperm/mL, and analyzed in the following experiments. The prepared samples were incubated in a 5% CO₂ incubator at 37°C.

According to our previously reported method (Sun et al., 2021), sperm samples were washed with phosphate-buffered saline (PBS) and resuspended in lysis buffer (P0013G, Beyotime Institute of Biotechnology, Shanghai, China) containing protease inhibitors (protease inhibitor cocktail and phosphatase inhibitor cocktail, Roche, Mannheim, Germany) and 1mM phenylmethylsulfonyl fluoride (PMSF). After ultrasonication and centrifugation, the supernatant was collected. Protein sample concentrations were determined using a bicinchoninic acid assay (BCA) kit (Beyotime Institute of Biotechnology, Shanghai, China). For different treatment groups, equal amounts of sperm protein (20μg) were denatured via incubation with protein loading buffer at 100°C for 5min and separated by 10% SDS-PAGE with a pre-stained protein marker. Proteins, transferred to PVDF membranes, were blocked with 5% skim milk (m/v). The PVDF membranes were incubated with primary antibodies at 4°C overnight and then washed three times with TBS buffer supplemented with 0.01% Tween-20 (v/v). The membrane was incubated with appropriate secondary antibodies at room temperature for 2h. After washing with TBS buffer three times, protein blots were detected by an ECL kit (Thermo Fisher Scientific) using a gel imaging system (Amersham Imager 600; General Electric Company, United States). For loading control, the membranes were stripped and probed with β-tubulin antibodies. Gray intensity was analyzed using the ImageJ software.

After fixation in 4% paraformaldehyde for 30min, the sperm were mounted on Silane-Prep slides and airdried. Sperm were permeabilized with 0.1% Triton X-100 and blocked with 10% goat serum. The sperm were then incubated with primary antibodies (mouse anti-KCNQ1, rabbit anti-KCNE1 or mouse anti-p-Tyr) or normal IgG (negative control) overnight at 4°C. After washing three times with PBS, Alexa Fluor 555-conjugated anti-mouse IgG secondary antibody and Alexa Fluor 488-conjugated anti-rabbit IgG secondary antibody were applied for 1h at 37°C. Following incubation with DAPI and washing with PBS, the sperm were examined using fluorescence microscopy (Nikon Eclipse 80i; Nikon Inc., Tokyo, Japan). For the immunofluorescence studies of KCNQ1, KCNE1 and p-Tyr, both non-capacitation and capacitation for 3h samples were used.

Because only capacitated sperm undergo exocytosis, human sperm capacitation was assessed indirectly using progesterone-induced AR. Different sperm groups were treated with different reagents for 3h during capacitation, followed by treatment with 15μM progesterone for 15min to induce the AR. According to the WHO Laboratory Manual for the Examination and Processing of Human Semen (5th ed.), the AR was evaluated by PSA-FITC staining. After fixing with 95% ethanol for 30min, sperm were mounted on Silane-Prep slides, air dried, and incubated overnight at 4°C with 25mg/L PSA-FITC in the dark. Sperm were washed with PBS and analyzed by fluorescence microscopy. At least 200 sperm were counted for each sample. To detect spontaneous AR, sperm were stained with PSA-FITC immediately after discontinuous Percoll gradient centrifugation and washing.

To evaluate sperm viability, propidium iodide (PI) was used to detect dead cells. Sperm were stained with 12μM PI for 10min at 37°C before or after capacitation for 3h. After washing with PBS three times, the sperm were mounted on Silane-Prep slides, air dried, and analyzed by fluorescence microscopy. Sperm with red fluorescence at the head was considered dead sperm. At least 200 sperm were counted for each sample. The percentage of non-viable cells (NVC%) was calculated.

Sperm motility was analyzed using a computer-assisted sperm analyzer (CASA; IVOS, Hamilton-Thorne Bio-Sciences, Beverly, MA, United States) with the following parameters: acquisition frame, 30; frame rate, 60Hz; minimum cell size, 3 pixels; minimum contrast, 80; cell intensity, 40; magnification, 1.73 ×; temperature, 37°C; illumination intensity, 2,164; path velocity, 25.0μm/s; straightness threshold, 80%; slow cell, average path velocity (VAP) and straight line velocity (VSL) of less than 5.0μm/s and 11μm/s, respectively; and chamber depth, 20μm (n>200 motile sperm per sample). Briefly, a 5μL sperm sample was loaded into a 20μm deep slide chamber warmed to 37°C. The following parameters were assessed for each sample: VSL, VAP, curvilinear velocity (VCL), straightness (STR), linearity (LIN), amplitude of lateral head displacement (ALH), beat-cross frequency (BCF), and percentage of motile, progressive, and hyperactivation. Hyperactivated sperm met the following criteria: VCL≥150μm/s, ALH≥7.0μm, and LIN≤50%.

[K⁺]_i in sperm was measured using PBFI-AM. Sperm were loaded with 10μM PBFI-AM in a 5% CO₂ incubator in the dark at 37°C for 30min. Excess dye in the medium was removed by washing five times with HTF. PBFI-AM-loaded sperm were resuspended in HTF and incubated at 37°C for further 20min. Sperm aliquots (10⁶ cells/mL) were exposed to vehicle control (DMSO) and chromanol 293B (20, 100, or 200μM). The K⁺ fluorescence signal was then recorded using a Synergy 2 Multi-Function Microplate Reader (Bio-Tek Instruments, Winooski, United States) with excitation at 340/380nm and emission wavelengths of 500nm. The data were acquired at 3min intervals for 30min during capacitating incubation because the effect of chromanol 293B on ion homeostasis may be compensated for by other potassium channels over time. After capacitation for 3h, a fluorescence signal was acquired. The ratio (340:380) of the two signals is directly proportional to [K⁺]_i. First recorded raw intensity values were used to normalize the other raw intensity values.

The [Ca²⁺]_i levels in human sperm were measured using Fluo3-AM according to a previously described method (Li et al., 2014). Briefly, the prepared sperm were loaded with 10μM Fluo3-AM in a 5% CO₂ incubator at 37°C for 30min in the dark and then washed five times with HTF to remove free Fluo3-AM. Fluo3-AM-loaded sperm were resuspended in HTF and incubated at 37°C for another 20min. Sperm aliquots (10⁶ cells/mL) were then exposed to vehicle control (DMSO) and chromanol 293B (20, 100, or 200μM). The Ca²⁺ fluorescence signal was then recorded using a Synergy 2 Multi-Function Microplate Reader, at 485nm excitation and 528nm emission wavelengths. The data were acquired at 3min intervals for 30min during the capacitating incubation. After capacitation for 3h, a fluorescence signal was acquired. Fluorescence intensity is directly proportional to [Ca²⁺]_i. First recorded raw intensity values were used to normalize the other raw intensity values.

The [Cl⁻]_i levels in sperm were measured using a Cl⁻- specific fluorescence probe (MQAE). Prepared sperm were loaded with 5μM MQAE in a 5% CO₂ incubator in the dark at 37°C for 30min and were then washed five times with HTF to remove free MQAE. MQAE-loaded sperm were resuspended in HTF and incubated at 37°C for another 20min. MQAE-loaded sperm aliquots (10⁶ cells/mL) were exposed to vehicle control (DMSO) and chromanol 293B (20, 100, or 200μM). The Cl⁻ fluorescence signal was recorded using a Synergy 2 Multi-Function Microplate Reader at 355nm excitation and 460nm emission wavelengths. The data were acquired at 3min intervals for 30min during the capacitating incubation. After capacitation for 3h, a fluorescence signal was acquired. Fluorescence intensity was inversely proportional to [Cl⁻]_i. First recorded raw intensity values were used to normalize the other raw intensity values.

Sperm sample pH was evaluated using BCECF-AM. The prepared sperm were loaded with 10μM BCECF-AM in a 5% CO₂ incubator in the dark at 37°C for 30min and were then washed five times with HTF to remove free BCECF-AM. BCECF-AM-loaded sperm were resuspended in HTF and incubated at 37°C for another 20min. BCECF-AM-loaded sperm aliquots (10⁶ cells/mL) were exposed to vehicle control (DMSO) and chromanol 293B (20, 100, or 200μM). To determine pH_i, fluorescence signals were recorded using a Synergy 2 Multi-Function Microplate Reader at 490/440nm excitation and 535nm emission wavelengths. The data were acquired at 3min intervals for 30min during the capacitating incubation. After capacitation for 3h, a fluorescence signal was acquired. The ratio (490:440) of the two signals was directly proportional to the pH_i. The first recorded raw intensity values were used to normalize the other raw intensity values.

Sperm membrane potential changes were evaluated using the potential-sensitive fluorescence probe DiSC3(5), as previously described (Xu et al., 2007). Before measurement, the prepared sperm were loaded with 1μM DisC3(5) in a 5% CO₂ incubator in the dark at 37°C for 5min. CCCP was added to a final concentration of 1μM. Sperm were incubated for 2min. Sperm aliquots (10⁶ cells/mL) were exposed to vehicle control (DMSO) and chromanol 293B (20, 100, or 200μM). The membrane potential fluorescence signal was then recorded using a Synergy 2 Multi-Function Microplate Reader, with 620nm excitation and 670nm emission wavelengths. The data were acquired at 3min intervals for 30min during the capacitating incubation. After capacitation for 3h, a fluorescence signal was acquired. The fluorescence intensity was directly proportional to V_m. The first recorded raw intensity values were used to normalize the other raw intensity values.

The Statistical Package for the Social Sciences software (SPSS, version 23; IBM Corporation, Armonk, NY, United States) was used for statistical analyses. Results are expressed as the means ± standard error of the mean (SEM). One-way analysis of variance was used to determine differences between the groups. When tests for the homogeneity of variance were not significant, the least significant difference test was used; otherwise, the data were analyzed using Dunnett’s T3 test; and p<0.05 was considered statistically significant (two-sided).

We studied the expression of KCNQ1 and KCNE1 in human sperm using Western blotting. The results showed the presence of a KCNQ1-specific band at approximately 70kDa and nonspecific band at approximately 48kDa, in addition to a KCNE1-specific band at 15kDa (Figure 1A). We examined the immunofluorescence of KCNQ1 and KCNE1 in human sperm before and after capacitation (Figure 1B). The results showed that KCNQ1 was localized mainly in the head and tail regions of human sperm, while KCNE1 was localized mainly in the neck and tail regions, which is in accordance with previous studies (Yeung and Cooper, 2008). The merging of both proteins showed that they were partially co-localized. The localization of KCNQ1 and KCNE1 in human sperm before and after capacitation was not significantly different. These results show that both KCNQ1 and KCNE1 are expressed in human sperm.

The effect of chromanol 293B on the human sperm AR was detected using a PSA-FITC staining assay. This method can easily distinguish sperm with acrosome integrity (AI) from those with AR (Figure 2A). For the AI pattern, bright and uniform fluorescence was observed in most regions of sperm heads, whereas for the AR pattern, no fluorescent staining was observed in the acrosomal zone, or only fluorescence bands in the equatorial zone were observed. The spontaneous AR (control 0h) ratio was approximately 12.1±1.4%, which was determined before capacitation incubation. After capacitation for 3h, the AR ratio of vehicle control (DMSO) was approximately 35.1±1.7%. When human sperm were treated with 100μM chromanol 293B during capacitation, the ratio of AR was significantly lower than that of the vehicle control (29.7±1.4% vs. 35.1±1.7%, p=0.017; Figure 2B). However, chromanol 293B did not affect the sperm viability. As shown in Figure 2B, before capacitation, the number of NVC% was approximately 13.0±1.1%. After capacitation for 3h, there were no significant differences in NVC% between the vehicle control group and the 100μM chromanol 293B-treated group (12.6±1.3% vs. 14.7±1.3%). These results suggest that the KCNQ1 channel is involved in human sperm capacitation.

CASA was used to analyze the effects of chromanol 293B on human sperm motility. As shown in Table 1, no significant differences were observed in human sperm hyperactivation between the chromanol 293B-treated groups and vehicle control (DMSO) for 3h capacitation. However, most sperm motility parameters in the 200μM chromanol 293B group were significantly lower than those in the vehicle control group, including sperm motility, progressive sperm, VAP, VSL, VCL, BCF, STR, and LIN (p<0.05). When sperm were treated with 100μM chromanol 293B, only sperm motility and progressive sperm showed significant differences compared to the vehicle control. These results suggest that KCNQ1 plays a role in human sperm motility during capacitation.

To further verify that the KCNQ1 potassium channel plays a role in human sperm during capacitation, we examined the effect of chromanol 293B on protein tyrosine phosphorylation levels, because capacitated sperm show high levels of protein tyrosine phosphorylation (Visconti et al., 2011). The effect of chromanol 293B on protein tyrosine phosphorylation levels was assessed by Western blotting. As shown in Figure 3A, protein tyrosine phosphorylation levels increased during human sperm capacitation. After 3h of sperm capacitation, the 100μM chromanol 293B group showed a significant decrease in protein tyrosine phosphorylation. We analyzed the ratio of protein tyrosine phosphorylation levels to that of the loading control, β-tubulin (Figure 3B). Protein tyrosine phosphorylation was enhanced over time during capacitation. The effect of 30min treatment of chromanol 293B on capacitation was negligible. However, after capacitation for 3h, chromanol 293B reduced protein tyrosine phosphorylation levels in a dose-dependent manner. The addition of 100μM chromanol 293B significantly decreased tyrosine phosphorylation, with bands at 120, 90, and 70kDa compared to the DMSO control (p=0.005, 0.012, and 0.032, respectively). We also examined the effect of chromanol 293B on phosphorylated tyrosine localization using indirect immunofluorescence. Before capacitation, phosphorylated tyrosine was localized in the head and tail regions of the sperm, and the fluorescence intensity was weak. After capacitation for 3h, the phosphorylated tyrosine was mainly localized in the equatorial and tail regions of the sperm, and the fluorescence intensity became intense. However, when the sperm were treated with 100μM chromanol 293B, phosphorylated tyrosine only localized in the tail region (Figure 3C).

To determine whether the KCNQ1 channel mediates K⁺ currents during human sperm capacitation, the effect of chromanol 293B on intracellular K⁺ concentration ([K⁺]_i) was examined. [K⁺]_i was analyzed at 3min intervals over 30min and once after sperm were capacitated for 3h. The intracellular K⁺ concentration was detected using the K⁺ fluorescence probe PBFI-AM, and the fluorescence intensity was directly proportional to [K⁺]_i. As shown in Figure 4A, [K⁺]_i decreased during human sperm capacitation in the vehicle control (DMSO), suggesting that potassium channels open and result in K⁺ efflux. However, [K⁺]_i increased when sperm were treated with chromanol 293B. The results showed dose-dependent changes, suggesting that chromanol 293B inhibits the KCNQ1 potassium channel, blocks K⁺ efflux through KCNQ1, and increases [K⁺]_i.

Because KCNQ1 inhibition increases [K⁺]_i in sperm and potassium channels are always involved in the regulation of membrane potential (V_m), we further examined whether the membrane was depolarized when treated with chromanol 293B. V_m was detected using the fluorescence probe DisC3(5). The fluorescence intensity was directly proportional to V_m. As shown in Figure 4B, V_m of the chromanol 293B-treated groups increased during human sperm capacitation. The depolarized sperm V_m values for the groups treated with 100 and 200μM chromanol 293B were much higher than those for the vehicle control group (p<0.05). These results suggest that chromanol 293B increases [K⁺]_i and depolarizes V_m in human sperm.

To explore whether the KCNQ1 channel indirectly influences other intracellular ion homeostasis, the effect of chromanol 293B on intracellular Ca²⁺ concentration ([Ca²⁺]_i) was detected using the Ca²⁺ fluorescence probe Fluo3-AM, fluorescence intensity of which was directly proportional to [Ca²⁺]_i. When human sperm were treated with chromanol 293B, the [Ca²⁺]_i decreased compared to the vehicle control at the beginning of 25min (p<0.05; Figure 4C). The effect of 200μM chromanol 293B on [Ca²⁺]_i decrease was negligible compared to that of 100μM chromanol 293B. However, both 100 and 200μM chromanol 293B groups showed a significant decrease in [Ca²⁺]_i compared to the vehicle control group (p<0.05). These results indicate that KCNQ1 indirectly regulates [Ca²⁺]_i during human sperm capacitation. After capacitation for 3h, [Ca²⁺]_i increased significantly compared to the beginning of the experiment. However, there were no statistically significant differences between the groups at 3h. The explanation for this result may be that chromanol 293B initially inhibits KCNQ1, while other potassium channels compensate over time.

The intracellular Cl⁻ concentration ([Cl⁻]_i) was analyzed using the Cl⁻ fluorescence probe MQAE. The fluorescence intensity is inversely proportional to [Cl⁻]_i. As shown in Figure 4D, within the first 25min, treatment with 200μM chromanol 293B significantly decreased the fluorescence intensity of Cl⁻, which indicating that [Cl⁻]_i increased (p<0.05; Figure 4D). Although there were no significant differences between all the groups when sperm were capacitated for 3h, the groups treated with 100 and 200μM chromanol 293B groups tended to have higher [Cl⁻]_i. These results indicate that KCNQ1 indirectly regulates [Cl⁻]_i during human sperm capacitation.

The intracellular pH (pH_i) of human sperm during capacitation was measured using the pH fluorescence probe BCECF-AM. The fluorescence intensity was directly proportional to pH_i. The chromanol 293B-treated groups showed a decrease in pH_i compared to that of the vehicle control group (Figure 4E).