A, B Abnormal cell division of Stella-null embryos. Representative live cell imaging photographs from fertilization to 91 h (A). Developmental stages of the embryos at 91 h after in vitro fertilization were categorized into four groups based on the morphology (B). Fifteen control and 22 Stella-null zygotes were obtained from Stella^+/− and Stella^−/− female mice from a single experiment for time-lapse imaging, respectively. C–E Retarded cell division and abnormal chromosome segregation of Stella-null embryos. The numbers of nuclei visualized by exogenous H2B-mRFP in control (blue) and Stella-null (red) developing embryos were analyzed from 6–66 h after in vitro fertilization (C). Representative images of normal chromosome segregation (NCS) and abnormal chromosome segregation (ASC) in early embryos are shown (D). ACS is represented by ectopic micronuclei, indicated by yellow arrowheads. Embryos cultured until 91 h after in vitro fertilization were categorized to NCS or four ACS groups based on the timing of micronuclei formation (E). Fifteen control and 22 Stella-null zygotes were obtained from Stella^+/− and Stella^−/− female mice from a single experiment for time-lapse imaging, respectively. F, G Abnormal chromosome integrity of intracytoplasmic sperm injection (ICSI)-derived and Stella-null 2-cell embryos. Representative images of ICSI-derived and Stella-null 2-cell embryos (F). The percentages of H3K9me2-positive micronuclei are shown (G). Total of 31 ICSI embryos from a single experiment and 17 Stella-null embryos from two independent experiments were analyzed.
Data information: Scale bars: 20 μm.

A Delayed DNA replication timing in Stella-null embryos. The control and Stella-null zygotes produced by IVF were incubated in KSOM containing BrdU at the indicated intervals. BrdU signals were classified as shown in the Supplementary Materials and Methods. The experiments were repeated at least twice for each interval. Total numbers of samples in each period (4–6, 6–8, 8–10, 10–12, 12–14, 14–16, 16–18, 18–20 hpf) were 7, 13, 13, 10, 9, 4, 8, and 2 in the control and 9, 11, 10, 9, 11, 13, 5, and 2 in Stella-null embryos, respectively. B, C γH2AX accumulation in control and Stella-null embryos during zygotic development. Pronuclei were stained with an anti-Ser139-phospho-H2AX antibody and counterstained with DAPI (B). The numbers of γH2AX foci in maternal and paternal pronuclei are shown (C). The experiments were repeated twice for PN1–2 and PN5–syngamy, and three times for PN3–4. Total numbers of samples in each stage (PN1–2, PN3–4, PN5–syngamy) were 12, 32, and 9 in the control and 8, 24, and 7 in Stella-null embryos, respectively. The median was indicated with a vertical line in the interior of the box, and the maximum and minimum are at the ends of the whiskers. *P < 0.001, **P < 0.0001, n.s., not significant by t-test. D Distribution of H3K9me2 and γH2AX in 2-cell embryos. Total of 12 control and 14 Stella-null embryos from two independent experiments were analyzed. E Distribution of H3K9me2 and 5hmC in 2-cell embryos. Total of 12 control and 11 Stella-null embryos from two independent experiments were analyzed.
Data information: m, maternal pronuleus; p, paternal pronucleus; pb, polar body; Scale bars: 20 μm.

A Distribution of 5hmC, 5fC, and 5caC in early embryos. Control and Stella-null embryos at the PN3–4 stage were stained with anti-5hmC, anti-5fC, and anti-5caC antibodies. Total numbers of samples in each staining (5hmC, 5fC, and 5caC) from two independent experiments were 5, 7, and 5 in the control and 7, 7, and 7 in Stella-null embryos, respectively. B, C Distribution of 5hmC, 5fC, and 5caC in TDG-expressing zygotes. Zygotes were injected with Myc-tagged Tdg mRNA and cultured for 5 h in KSOM. Representative images of 5mC derivatives (B) and the fluorescence intensities (C) are shown. Total numbers of samples in each staining (5hmC, 5fC, and 5caC) from three independent experiments were 17, 14, and 7 in the control and 13, 15, and 8 in TDG-expressing zygotes, respectively. Bar shows the mean ± SEM. *P < 0.01, **P < 0.001, ***P < 0.0001, n.s., not significant by t-test. D, E γH2AX localization in the TDG-expressing zygotes. Representative images of γH2AX (D) and the numbers of γH2AX foci are shown (E). Total of 13 and 15 TDG-expressing zygotes from three independent experiments were analyzed. The median was indicated with a vertical line in the interior of the box, and the maximum and minimum are at the ends of the whiskers. *P < 0.005, n.s., not significant by t-test. F γH2AX localization in the Tet3 knockdown zygotes. Representative images of γH2AX and Tet3 are shown. The numbers of samples from a single experiment for each staining (γH2AX and Tet3) were 6 and 10 in the control and 11 and 4 in TDG-expressing zygotes, respectively.
Data information: m, maternal pronuleus; p, paternal pronucleus; pb, polar body; Scale bars: 20 μm.

A 5hmC induction by Tet3 in NIH-3T3 cells. Immunostaining analysis was performed at 48 h after transfection with FLAG-Tet2, FLAG-Tet3, and a catalytic domain-defective FLAG-Tet3 mutant-expressing vector. In vitro experiments were repeated at least three times, and similar results were obtained. B γH2AX induction by Tet3-induced 5hmC in NIH-3T3 cells. Immunostaining was performed at 48 h after transfection with Tet-expressing vectors. Treatment with 500 μM H₂O₂ for 3 h was used as a positive control. C, D Inhibition of DNA replication by Tet3-induced 5hmC in NIH-3T3 cells. Representative immunostaining images (C) and percentages of BrdU-positive cells (D) are shown. Two hundred and fourteen control, 29 FLAG-Tet2-, 37 FLAG-Tet3-, and 43 FLAG-Tet3 mutant-expressing cells were analyzed. *P < 0.001 by chi-square test. E Delayed cell cycle progression in the FLAG-Tet3-expressing NIH-3T3 cells. Expressions of the EGFP, FLAG-Tet3, and FLAG-Tet3 mutant were introduced by lentiviral vectors. F Growth retardation in the FLAG-Tet3-overexpressing NIH-3T3 cells. The number of cells in control (blue), FALG-Tet3-expressing (red), and FLAG-Tet3 mutant-expressing (green) were counted every 3 days in culture. Cell proliferation data were determined by the average of four independent experiments.
Data information: Scale bars: 10 μm.