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. 2021 Sep 28;9:733653. doi: 10.3389/fcell.2021.733653

FIGURE 7.

FIGURE 7

The soluble adenylyl cyclase (sAC) is required for [Ca2+]i increases evoked by KCl after HCO3– sensitization in epididymal mouse sperm. (A,D,F) Linear fit of the averaged normalized fluorescence in the first 20 s after KCl addition at (A) pH 7.4 and (D,F) pH 8.6. Data were obtained from epididymal (A,D) wild-type mouse sperm and from (G) sAC KO mouse sperm in control conditions (white) and after pre-incubation with HCO3– (black), HCO3– + PKA inhibitor TDI-10229 (Green), or NH4Cl (Blue). (B) Graph of slope values of the best-fit regression line for each condition. Mean values at pH 7.4 were as follows: control, 0.76 ± 0.547 amplitude units * s–1 (8 mice, 307 sperm); incubation with HCO3–, 1.35 ± 0.297 amplitude units *s–1 (15 mice, 1035 sperm); and incubation with TDI-10229, 0.75 ± 0.259 amplitude units *s–1 (6 mice, 364 sperm). (C) Graph of the percentage of responsive sperm. Mean values at pH 7.4 were as follows: control, 11.80% ± 6.9 (8 mice, 307 sperm); HCO3–, 47.6% ± 19.39 (15 mice, 1035 sperm); and HCO3– + TDI-10229,17.8% ± 16.33 (6 mice, 364 sperm). (E) Slope values per animal of the best-fit linear regression of the rise in KCl at pH 8.6 in different conditions. Mean values at pH 8.6 were as follows: control, 0.95 ± 0.237 amplitude units * s–1 (7 mice, 243 sperm); HCO3–, 1.84 ± 0.556 amplitude units *s–1 (7 mice, 281 sperm); and HCO3– + TDI-10229, 0.88 ± 0.413 amplitude units *s–1 (6 mice, 126 sperm). (G) Graph of slope values of the best-fit regression line for each condition in the sAC KO mice. Mean values at pH 8.6 were as follows: control, 0.645 ± 0.084 amplitude units * s–1 (4 mice, 123 sperm); HCO3–, 0.840 ± 0.173 amplitude units *s–1 (4 mice, 109 sperm); and NH4Cl, 1.932 ± 0.197 amplitude units *s–1 (3 mice, 74 sperm). (H) Graph of the percentage of responsive sperm. Mean values for wild-type at pH 8.6 were as follows: control, 32.62% ± 9.58 (7 mice, 243 sperm); HCO3–, 58.05% ± 21.94 (7 mice, 281 sperm); and HCO3– + TDI-10229, 40.25% ± 9.51 (6 mice, 126 sperm). Mean values for sAC knockout at pH 8.6 were as follows: control, 33.80% ± 9.44 (4 mice, 91 sperm); HCO3–, 36.36% ± 7.96 (4 mice, 83 sperm); and NH4Cl, 61.47% ± 15.20 (3 mice, 74 sperm). *P < 0.050, **P < 0.010, ***P < 0.001, and ns, non-significant by one-way ANOVA. Error bars indicate standard deviation. Iono = 5 μM Ionomycin + 2 mM Ca2+.