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. 2018 May 9;293(25):9924–9936. doi: 10.1074/jbc.RA118.003166

Figure 6.

Figure 6.

HCO3−-induced a rapid Em hyperpolarization in human sperm sensitive to S0859, inh-172, and H89. A, human sperm were incubated in the presence (CAP) or absence of HCO3− for 3 h. After that, cells were loaded with DISC3(5) and BCECF-AM to evaluate Em and viability, respectively. Cells that were not exposed to HCO3− displayed more depolarized Em compared with the condition incubated with HCO3−. Addition of HCO3− (25 mm) induced a rapid Em hyperpolarization in human sperm. B, human sperm were incubated in the absence of HCO3− for 3 h. After that, the cells were loaded with DISC3(5) to evaluate Em. DISC3(5) fluorescence median was recorded before (time = 0 min) and after (black arrow) addition of 25 mm HCO3−. In each experiment, the fluorescence median was normalized to fluorescence maximum reached after 4 min of addition of 1 μm valinomycin to sperm incubated in capacitating medium. Values represent the mean ± S.E. of at least 3 experiments. Two-way ANOVA with Dunnett's post test. ***, p < 0.001; **, p < 0.01 versus NC at 4 min, ns at 0 min (before HCO3− addition). C, human sperm were incubated in the presence (CAP) or absence of HCO3− for 3 h. Then, the cells were loaded with DISC3(5) and BCECF-AM to evaluate Em and viability, respectively. Cells that were not exposed to HCO3− displayed more depolarized Em than those incubated with HCO3−. Addition of cAMP analogs (1 mm Br-cAMP and 0.2 mm IBMX, dotted line) could rescue Em depolarization. D, fluorescence median of DISC3(5) shown in A and C was normalized with respect to CAP condition. Values represent the mean ± S.E. of at least 3 experiments. ***, p < 0.001 versus CAP condition. E, human sperm were incubated in the presence (CAP) or absence of HCO3− for 3 h. Then, the cells were loaded with DISC3(5) and BCECF-AM to evaluate Em and viability, respectively. Cells that were not exposed to HCO3− displayed more depolarized Em that could be rescued with increasing concentrations of amiloride (Ami). F, fluorescence median of DISC3(5) shown in E was normalized with respect to CAP condition. Values represent the mean ± S.E. *, p < 0.05 (n = 4) versus CAP condition.