Expression of ZIP6 and ZIP10 in mouse ovary.

(A) In situ hybridization in the mouse ovary showed ZIP10 expression in oocyte and granulosa cell from primordial, primary, secondary and antral follicle. Arrow indicates primordial follicular oocyte. (B) Immunofluorescent staining for ZIP10 (green) in the mouse ovary showed ZIP10 expression in oocyte membrane. Arrow indicates primordial follicular oocyte. (C) Immunofluorescent staining for ZIP6 (green) in the mouse ovary showed ZIP6 expression in oocyte nucleus and granulosa cells. Arrow indicates primordial follicular oocyte. (D) Immunofluorescent staining showed ZP2 (green; zona pellucida) and FOXL2 (red; granulosa cells) in the mouse ovary. ZP2 was not stain in primordial follicle, but primary, secondary and antral follicles stained. FOXL2 observed at granulosa cells of all stage follicles. The scale bar represents 20 µm of primordial-secondary follicle and 150 µm of antral follicle (A-D).

Number of collected oocytes and dynamics of labile zinc ion in Zip10d/d mice.

(A) The results of average number of oocytes in each group. Data represents the average ± SEM. These experiments were repeated at least thrice. Statistical differences were calculated according to student’s t-test (p > 0.05; no significant difference). (B) The percentage of extrusion of first polar body at 10, 12 and 14 h after IVM. These experiments were repeated at least thrice. Statistical differences were calculated according to student’s t-test (p > 0.05; no significant difference). (C) The morphology of spindle and chromosome organization in Zip10f/f and Zip10d/d MII oocytes at 14 h after IVM. Anti–α-tubulin antibody (green) was used to stain the spindles. Chromosomes were stained with DAPI (blue). The scale bar represents 10 μm. (D) Comparison with the fluorescence intensity of intracellular labile zinc ion in GV, MII and 2PN. The upper images showed the fluorescence of intracellular labile zinc ion of oocyte or embryo treated with 2 µM FluoZin-3AM for 1h. Representative images are shown. The white dotted circles indicate the positions of oocytes and embryos. Scale bars denote 10 μm. The lower part showed the fluorescence intensity of labile zinc ions in oocytes or embryos. Data represent the average ± SE of the experiments. For each experiment, 10–20 oocytes/embryos were stained and used for the measurement in each stage of the experiment, and these experiments were repeated three times. Statistical differences were calculated according to the welch’s t-test. Different letters represent significant differences (p < 0.05).

Measurement of calcium spike and zinc spark in Zip10d/d mice.

(A) The representative images of before or after IVF in mouse oocytes. Left side images showed before fertilization. Right side images showed after fertilization. The oocytes increased calcium ion and released zinc ion shortly after fertilization. The white dotted circles indicate the positions of oocytes. Successful fertilization was confirmed by simultaneously monitoring intracellular calcium oscillations with Calbryte 590 AM and excellular zinc ions with FluoZin-3 every 4 s. Capacitated frozen-thawed sperm was added to MII at 2 min after imaging start. Orange line showed calcium ion and dark blue line showed zinc ion. Intracellular calcium increases immediately before a zinc spark. Scale bars denote 20 μm. (B) The representative images of an MII egg activated with 5 μM ionomycin followed by monitoring of intracellular calcium oscillations with Calbryte 590 AM and extracellular zinc using 20 μM FluoZin-3. The ionomycin was added to MII at 2 min after imaging start. Orange line showed calcium ion and dark blue line showed zinc ion. Intracellular calcium increases immediately before a zinc spark. Scale bars denote 20 μm.

Presence of a mechanism to prevent multisperm fertilization in Zip10 cKO mice.

(A) The percentages of oocytes with each number of PN at 6 h after insemination. Yellow region showed other including degeneration, degression and fragmentation. Gray region showed unfertilization, namely MII oocytes. Orange showed 2PN2PB, namely embryo possessed one female and male pronucleous (2PN) and second polar body (2PB). Blue region showed multisperm fertilization (3PN2PB). (B) The percentage of fertilized oocytes and developmental embryos. Data represent the average ± SE of the experiments. The embryo development was observed at 6 (2PN) 24 (2 cell), 48 (4-8 cell), 72 (Morula), and 96 (Blastocyst) hours after IVF. The oocytes used for IVF were calculated as the parameter for the fertilization rate and the rate of embryo development. These experiments were repeated at least thrice. Statistical differences were calculated according to the chi-square test. Different letters represent significant differences (p < 0.05). (C) The cell number of blastocyst derived from IVF. Blastocysts were fixed, immunostained, and physically flattened between a slide and coverslip. Photographs represent a single plane of focus. Nuclei are indicated by DAPI staining. The count used inverted fluorescence microscope. These experiments were repeated three times, and each group counted total 46 embryos. Statistical differences were calculated according to the student’s t-test (p < 0.05; significant difference). (D) Western blot of oocytes from Zip10f/f and Zip10d/d mice at 0 or 6 h after insemination using rat anti-ZP2 antibody. Intact ZP2 and the cleaved C-terminal fragment of ZP2 measured 120-130 kD and undetected, respectively. Expression level of +-actin serves as a protein loading control. Molecular mass is indicated at the left. (E) MII oocytes and 2PN embryos from Zip10f/f and Zip10d/d mice were imaged by confocal microscopy after staining with rabbit anti-ovastacin (green). Chromosomes were stained with DAPI (blue). The scale bar represents 10 μm. (F) MII oocytes and 2PN embryos from Zip10f/f and Zip10d/d mice were imaged by BZ-X700 microscopy after staining with rat anti-mouse FR4 (JUNO; green). Chromosomes were stained with DAPI (blue). The scale bar represents 10 μm.