ZIP9 expression in the zebrafish ovary. (A) mRNA expression of ZIP9 in different stage wildtype oocytes and follicle cells (n = 4–6). (B, C) Western blot analysis of ZIP9 protein expression on the plasma membrane of different stage wildtype oocytes/eggs (B) and the plasma membrane fraction of wildtype and zip9^-/- fish ovaries (C) (n = 3–4). All data represents means ± SEM. Significance was determined by one-way ANOVA with Bonferroni multiple comparison post-test. Different letters indicate significant differences between treatment groups in the post hoc test (P < 0.05). EMV early-mid vitellogenic oocytes, FG full grown oocytes, Ov ovulated eggs, GT (EMV) granulosa/theca cells from early-mid vitellogenic follicles, GT (FG) granulosa/theca cells from full grown follicles.

Characterization of zip9-mutant phenotype. (A) Percent of viable eggs ovulated by zip9^-/- and zip9^+/+ females 2 h post fertilization (hpf); n = 13–14 clutches. (B) Representative image of zip9^+/+ and zip9^-/- embryos at 4 hpf and 1 and 3 days post fertilization (dpf). Scale bars: 500 µm. Arrows donate the yolk sac in WT and mutants (3 dpf) and asterisks donate edema in zip9^-/- larvae (3 dpf). (C) Chorion diameter of eggs spawned by zip9^-/- and zip9^+/+ females; n = 40. (D) Yolk volume of 3 dpf zip9^-/- and zip9^+/+ larvae; n = 16–18. (E) Length of zip9^-/- and zip9^+/+ larvae without exogenous feeding between 3–10 dpf; n = 10–21. (F) Incidence of pericardial/yolk sac edema in 6 dpf zip9^-/- and zip9^+/+ larvae; n = 5 clutches. All data represents means ± SEM. Significance was determined by Welch's t-test (*, P < 0.05; **, P < 0.001; ***, P < 0.0001).

Morphology of cortical vesicles of zip9^+/+ and zip9^-/- oocytes throughout oogenesis. Representative images and average cortical vesicle diameters of (A) cortical alveoli, (B) early vitellogenic, (C) mid vitellogenic, and (D) late vitellogenic stage zip9^+/+ and zip9^-/- oocytes. Arrows indicate location of cortical vesicles. Scale bars: 12 µm. All data represents means ± SEM; n = 5 (average diameter of cortical vesicles for 5 females). Significance was determined by Welch's t-test (**, P < 0.01; ***, P < 0.001).

Meiosis II-arrested wildtype zebrafish eggs have cortically-localized, zinc-containing vesicles. (A, B) colocalization (A) of FluoZin-3-AM signal (green fluorescence) and cortically-located vesicles by brightfield and fluorescent microscopy. Images represent optical slices, with relative location to bottom (z: 0) of egg indicated.

Zinc is stored in cortically-localized vesicles that undergo exocytosis upon egg activation. (A, B)zip9^+/+(A) and zip9^-/-(B) eggs show intracellular zinc staining (arrows, 0 min) in cortically-localized vesicles that are reduced in number at 2.5–10 min post-activation. (C, D) Extracellular zinc is observed 2.5–10 min post-activation of zip9^+/+(C) and zip9^-/-(D) eggs. (E) Time trace of normalized extracellular zinc fluorescence by ROI analysis (ROI denoted by yellow circle in C, 0 min) 0–10 min post-activation of zip9^+/+ and zip9^-/- eggs. Visualization was performed on eggs from a minimum of 3 females/strain. For (E), data represents means ± SEM; n = 5–6. Significance was determined by Welch's t-test (*, P < 0.05; **, P < 0.01).

A rise in intracellular calcium proceeds zinc exocytosis in WT eggs. (A) Representative time-course images from concurrent intracellular calcium (purple) and extracellular zinc (green) monitoring. (B) Representative time trace of normalized calcium (black) and zinc (green) fluorescence signal.

Effect of sustained zinc elevation on chorion elevation. (A) Representative micrographs of the effects of pre-treatment with zinc pyrithione (ZnPy) and TPEN on meiosis II-arrested eggs 10 min post-activation. (B) Quantification of ZnPy and TPEN-treated eggs that underwent chorion elevation post-activation. All data represents means ± SEM; n = 9–12. Each treatment was repeated in triplicate with similar results obtained for each, and the experiment was repeated with 3–4 fish. Significance was determined by one-way ANOVA with Bonferroni multiple comparison post-test. Different letters indicate significant differences between treatment groups in the post hoc test (P < 0.05).