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. 2021 Aug 25;118(35):e2023909118. doi: 10.1073/pnas.2023909118

Fig. 5.

Fig. 5.

SLC-30A9–mediated Zn2+ mobilization is required for sperm activation. (A) Representative confocal images of isolated spermatids costained with Zinpyr-1 green and MitoTracker red in him-5(e1490) and slc-30a9(wy50591); him-5(e1490) mutant males treated with SM (control medium), SM + TEA, or SM + CCCP + TEA. The arrowheads indicate pseudopods. (Scale bar, 5 µm.) (B) Quantification of sperm with Zinpyr-1 depleted from mitochondria in him-5(e1490) and slc-30a9(wy50591); him-5(e1490) mutants supplemented with SM, SM + TEA, or SM + CCCP + TEA. Data are shown as mean ± SEM. One-way ANOVA with Tukey correction. ***P < 0.001. Thirty-four worms or more were scored for each genotype. (C) DIC micrographs showing spermatids of him-5(e1490) and slc-30a9(wy50591); him-5(e1490) in SM, SM + TPEN, and SM + EGTA stimulated by sperm activators TEA, NH4Cl, or zinc in separate experiments. The arrowheads indicate pseudopods. (Scale bar, 5 μm.) (D) Quantification of sperm activation measured by pseudopods in C. Data are shown as mean ± SEM. One-way ANOVA with Tukey correction. ***P < 0.001. Forty or more worms were scored for each genotype. (E) Representative confocal images of spermatids stained with FM 1-43 in him-5(e1490) and slc-30a9(wy50591); him-5(e1490) mutant males treated with SM or SM + TEA. The arrows indicate the putative MO fusion site at the rear edge of the spermatozoa. (Scale bar, 5 µm.) (F) Quantification of sperm with punctate FM 1-43 staining near the plasma membrane. Data are shown as mean ± SEM. One-way ANOVA with Tukey correction. **P < 0.01. Forty or more worms were scored for each genotype. (G) Quantification of total brood size of N2 hermaphrodites (n = 18), slc-30a9(wy50591) hermaphrodites (n = 22), cross-progenies between him-5(e1490) males and slc-30a9(wy50591) hermaphrodites (n = 14), cross-progenies between him-5(e1490) males and fog-2(q71) females (n = 37), and cross-progenies between slc-30a9(wy50591); him-5(e1490) males and fog-2(q71) females (n = 40). Data are shown as mean ± SEM. One-way ANOVA with Tukey correction. ***P < 0.001.