Han Y , Ishibashi S , Iglesias-Gonzalez J , Chen Y , Love NR , Amaya E .

Wellcome Trust , MR/L007525/1 Medical Research Council , Biotechnology and Biological Sciences Research Council

             fertilization
             mitochondrial DNA metabolic process
             cell division
             positive regulation of reactive oxygen species biosynthetic process

	Graphical Abstract:
	Figure 1. Fertilization and Injury Trigger a Substantial Increase in ROS Levels(A) HyPer ratio images (500/430 nm) showing a ROS production in transgenic embryos expressing HyPer. See also Movie S1.(B) Quantification of HyPer ratio in (A). n = 11; p = 0.001, 1 cell compared to egg, Wilcoxon matched-pairs signed-rank test.(C) Schematic diagram of oocytes experiments. Immature ovarian oocytes were injected with HyPer RNA, matured with 2 μM progesterone, and then pricked by a needle or laser wound activated.(D) HyPer images of immature oocytes expressing HyPer were captured every 20 min after pricking. There is no increase in the HyPer ratio.(E) Quantification of HyPer ratio in (D). n = 33; p = 0.2, 20 min compared to 0 min, paired t test.(F) HyPer images of mature oocytes expressing HyPer were captured every 20 min after pricking. There is an increase in the HyPer ratio.(G) Quantification of HyPer ratio in (F). n = 28; p < 0.0001, 20 min compared to 0 min, paired t test.(H) SypHer images of mature oocytes expressing Sypher were captured every 20 min after pricking.(I) Quantification of SypHer ratio in (H). n = 27; p < 0.0001, 20 min compared to 0 min, Wilcoxon matched-pairs signed-rank test.Scale bars, 200 μm (A, D, F, and H). Data are from two independent experiments. Error bars represent mean ± SEM. ∗∗∗p ≤ 0.001 and ∗∗∗∗p < 0.0001; ns, not significant. See also Figure S1 and Movie S2.
	Figure 2. ROS Production Is Generated Downstream of Ca2+ Signaling(A) HyPer images of control-treated or Ca2+ ionophore A23187 (10 μM) -treated mature oocytes injected with HyPer RNA.(B) Quantification of HyPer ratio in (A). n = 32–33; p < 0.0001, compared to control at 40 and 60 min, two-way ANOVA and Sidak post hoc tests.(C) Oocytes injected with HyPer RNA were matured and cultured in calcium-free OR2 medium with or without EGTA (100 μM), and then HyPer images were taken after prick activation.(D) Quantification of HyPer ratio in (C). n = 32–36; p < 0.0001, compared to control at 40 and 60 min, two-way ANOVA and Sidak post hoc tests.(E) Oocytes were injected with 20 ng of R-GECO as a control or inpp5a and HyPer RNAs, matured, and then imaged after prick activation.(F) Quantification of HyPer ratio in (E). n = 20–25; p < 0.0001, compared to control at 40 and 60 min, two-way ANOVA and Sidak post hoc tests.Scale bars, 200 μm (A, C, and E). Data are from three independent experiments. Error bars represent mean ± SEM. ∗∗∗∗p < 0.001. See also Movies S3, S4, S5, and S6.
	Figure 3. Mitochondrial Inhibitors Malonate, Antimycin A, and Sodium Azide Impair ROS Production after Activation(A) NOX inhibitors 10 μM DPI or 1 mM apocynin had no effect on HyPer ratio in mature oocytes from HyPer transgenic females after pricking. n = 29–42, two-way ANOVA and Tukey’s post hoc tests.(B–F) HyPer ratio on oocytes treated with 1 μM rotenone (n = 36; B), 5 mM malonate (n = 45; p < 0.0001, compared to control at each time point, two-way ANOVA and Sidak post hoc tests; C), 10 μM antimycin A (n = 46; p < 0.0001, compared to control at 40 and 60 min, two-way ANOVA and Sidak post hoc tests; D), 1 mM sodium azide (n = 25; p < 0.0001, compared to control at 40 and 60 min, two-way ANOVA and Sidak post hoc tests; E), and 6 μM oligomycin (n = 42; F).(G) Ca2+ wave was measured by fluorescent intensity of R-GECO in laser-activated mature oocytes. Ca2+ wave was not affected by any of the mitochondrial inhibitors. Each treatment, n = 6; two independent experiments; two-way ANOVA and Tukey’s post hoc tests. See also Movie S7.(H) HyPer ratio on oocytes treated with 2 μM FCCP. n = 36; p < 0.0001, compared to control at 40 and 60 min, two-way ANOVA and Sidak post hoc tests.Data for (A)–(F) and (H) are from three or four independent experiments. Error bars represent mean ± SEM; ns, not significant; ∗∗∗∗p < 0.0001.
	Figure 4. Ca2+ Mediates ROS Production by Mitochondria via MCU(A) HyPer images of oocytes injected with 0.4 pmol RuR (0.4 μM final concentration), MCU inhibitor, and 20 ng HyPer RNA.(B) Quantification of HyPer ratio in (A). ROS production was reduced by MCU inhibition. Two independent experiments; ∗∗∗∗p < 0.0001, compared to control at each time point, Mann-Whitney test. Error bars represent mean ± SEM.(C) HyPer images of oocytes injected with 20 ng of RNA for dominant-negative MCU, mcub, and 20 ng HyPer RNA compared to 20 ng HyPer RNA-injected control.(D) Quantification of HyPer ratio in (C). Inhibition of MCU by overexpression of mcub impairs ROS production. Error bars represent mean ± SEM.Two independent experiments; ∗∗p < 0.01 and ∗∗∗p < 0.001, compared to control at t30 and t60, two-way ANOVA and Sidak post hoc tests. See also Movie S8. Scale bars, 200 μm (A and D).
	Figure 5. Inhibition of mtROS Induces Cell Division Arrest in Xenopus Early Development(A) Embryos were treated at the one-cell stage with 0.1% DMSO (n = 36), 1 μM rotenone (n = 92), 10 mM malonate (n = 40), 3 mM sodium azide (n = 58), 10 μM antimycin A (n = 83), and 6 μM oligomycin (n = 88). Embryos treated with rotenone, malonate, and sodium azide were arrested at the 2- to 8-cell stage. By the blastula stage embryos treated with antimycin A were arrested.(B) 1-cell stage embryos obtained from females expressing HyPer were treated with mitochondrial inhibitors, and HyPer ratio was measured at the 4-cell and 32-cell stages. n = 12–13; ∗∗∗∗p < 0.0001, two-way ANOVA and Tukey’s multiple comparisons tests. Error bars represent mean ± SEM.(C) Embryos were treated with 0.1% DMSO or 2 μM CCCP (n = 66).(D) Embryos were injected with water as a control and 50 pmol RuR into one cell at the 2-cell stage. Injection of RuR induced cell-cycle arrest (n = 56).Pictures in (A), (C), and (D) are representative of at least three independent experiments.
	Figure 6. Removal of Inhibitors Restores Cell Division and ROS Production(A) Embryos were treated with inhibitors, 10 mM malonate, 3 mM sodium azide, and 1 μM rotenone, for 40 min until cells stopped dividing (left column), and then they were transferred to a dish containing fresh medium without inhibitors. Embryos transferred after treatment with malonate and azide retrieved cell division and divided to 8- to 16-cell stages (middle), but not in medium with inhibitors (right). Pictures are representative of three independent experiments.(B) Fertilized transgenic embryos expressing HyPer were treated with inhibitors for 40 min and imaged shown as 0 min. Then embryos were transferred to fresh medium without inhibitors, and imaged at 40 and 70 min (n = 34–36; three independent experiments).Error bars represent mean ± SD. See also Movie S9.
	Figure 7. Inhibition of mtROS Causes Misregulation of Cdc25C Activity Resulting in Mitotic Arrest(A) Immunoblots of Xenopus Cdc25C in embryos treated with mitochondrial inhibitors. 10 mM malonate and 3 mM sodium azide, which reduced ROS generation, caused misregulation of Cdc25C (asterisks in the blots).(B) Embryos were treated with inhibitors at the 1-cell stage (90 min after fertilization) and fixed every 10 min for immunohistochemistry. The staining for α-tubulin, Lamin B1, and DNA was used to identify the phase of cell cycle. The numbers in parentheses indicate numbers of embryos examined. A defect in mitotic entry was observed in embryos treated with malonate and sodium azide. Data are from two to three independent experiments.(C) Immunoblots of Xenopus cyclin B2 in embryos treated with 10 mM malonate or 3 mM sodium azide. Cyclin B2 degraded before cell-cycle arrest (asterisks in the blots), but its accumulation seemed to be impaired afterward.(D) Immunoblots of Xenopus Cdc25C, Cdk-Y15, pan-Cdk, cyclin B2, and α-tubulin in activated eggs treated with 0.1% DMSO, 10 mM malonate, or 3 mM sodium azide at 0, 5, 30, and 80 min after prick activation.(E) Immunoblots detecting human Cdc25C and human Cdc25C-pS216 in activated mature oocytes.Oocytes injected with human Cdc25C WT or C330S/C377S RNA were treated with DMSO or mtROS inhibitors, prick-activated, and extracted at 0, 30, 60, and 90 min.All blots are representative of at least three independent experiments.(F) Plots of measurement of HyPer ratio shown in black (left y axis) and the raw fluorescence intensities at 500 nm of HyPer and YFP embryos shown in the plot as green and yellow lines, respectively (right y axis). Embryos were imaged every 30 s from the beginning of fertilization (0 min) throughout the cleavage stage in Movie S10. Data are representative of at least two independent experiments. See Movie S11.(G) YFP (500 nm)/CFP (430 nm) ratio of embryos expressing HyPer (green) or SypHer (black and pink) obtained by nuclear transplantation of in vitro-matured oocytes. Embryos were imaged every 30 s when it started dividing (0 min) throughout the cleavage stage. Data are representative of at least two independent experiments (n = 2–4).
	Figure 1. Fertilization and Injury Trigger a Substantial Increase in ROS Levels(A) HyPer ratio images (500/430 nm) showing a ROS production in transgenic embryos expressing HyPer. See also Movie S1.(B) Quantification of HyPer ratio in (A). n = 11; p = 0.001, 1 cell compared to egg, Wilcoxon matched-pairs signed-rank test.(C) Schematic diagram of oocytes experiments. Immature ovarian oocytes were injected with HyPer RNA, matured with 2 μM progesterone, and then pricked by a needle or laser wound activated.(D) HyPer images of immature oocytes expressing HyPer were captured every 20 min after pricking. There is no increase in the HyPer ratio.(E) Quantification of HyPer ratio in (D). n = 33; p = 0.2, 20 min compared to 0 min, paired t test.(F) HyPer images of mature oocytes expressing HyPer were captured every 20 min after pricking. There is an increase in the HyPer ratio.(G) Quantification of HyPer ratio in (F). n = 28; p < 0.0001, 20 min compared to 0 min, paired t test.(H) SypHer images of mature oocytes expressing Sypher were captured every 20 min after pricking.(I) Quantification of SypHer ratio in (H). n = 27; p < 0.0001, 20 min compared to 0 min, Wilcoxon matched-pairs signed-rank test.Scale bars, 200 μm (A, D, F, and H). Data are from two independent experiments. Error bars represent mean ± SEM. ∗∗∗p ≤ 0.001 and ∗∗∗∗p < 0.0001; ns, not significant. See also Figure S1 and Movie S2.
	Figure 2. ROS Production Is Generated Downstream of Ca2+ Signaling(A) HyPer images of control-treated or Ca2+ ionophore A23187 (10 μM) -treated mature oocytes injected with HyPer RNA.(B) Quantification of HyPer ratio in (A). n = 32–33; p < 0.0001, compared to control at 40 and 60 min, two-way ANOVA and Sidak post hoc tests.(C) Oocytes injected with HyPer RNA were matured and cultured in calcium-free OR2 medium with or without EGTA (100 μM), and then HyPer images were taken after prick activation.(D) Quantification of HyPer ratio in (C). n = 32–36; p < 0.0001, compared to control at 40 and 60 min, two-way ANOVA and Sidak post hoc tests.(E) Oocytes were injected with 20 ng of R-GECO as a control or inpp5a and HyPer RNAs, matured, and then imaged after prick activation.(F) Quantification of HyPer ratio in (E). n = 20–25; p < 0.0001, compared to control at 40 and 60 min, two-way ANOVA and Sidak post hoc tests.Scale bars, 200 μm (A, C, and E). Data are from three independent experiments. Error bars represent mean ± SEM. ∗∗∗∗p < 0.001. See also Movies S3, S4, S5, and S6.
	Figure 3. Mitochondrial Inhibitors Malonate, Antimycin A, and Sodium Azide Impair ROS Production after Activation(A) NOX inhibitors 10 μM DPI or 1 mM apocynin had no effect on HyPer ratio in mature oocytes from HyPer transgenic females after pricking. n = 29–42, two-way ANOVA and Tukey’s post hoc tests.(B–F) HyPer ratio on oocytes treated with 1 μM rotenone (n = 36; B), 5 mM malonate (n = 45; p < 0.0001, compared to control at each time point, two-way ANOVA and Sidak post hoc tests; C), 10 μM antimycin A (n = 46; p < 0.0001, compared to control at 40 and 60 min, two-way ANOVA and Sidak post hoc tests; D), 1 mM sodium azide (n = 25; p < 0.0001, compared to control at 40 and 60 min, two-way ANOVA and Sidak post hoc tests; E), and 6 μM oligomycin (n = 42; F).(G) Ca2+ wave was measured by fluorescent intensity of R-GECO in laser-activated mature oocytes. Ca2+ wave was not affected by any of the mitochondrial inhibitors. Each treatment, n = 6; two independent experiments; two-way ANOVA and Tukey’s post hoc tests. See also Movie S7.(H) HyPer ratio on oocytes treated with 2 μM FCCP. n = 36; p < 0.0001, compared to control at 40 and 60 min, two-way ANOVA and Sidak post hoc tests.Data for (A)–(F) and (H) are from three or four independent experiments. Error bars represent mean ± SEM; ns, not significant; ∗∗∗∗p < 0.0001.
	Figure 4. Ca2+ Mediates ROS Production by Mitochondria via MCU(A) HyPer images of oocytes injected with 0.4 pmol RuR (0.4 μM final concentration), MCU inhibitor, and 20 ng HyPer RNA.(B) Quantification of HyPer ratio in (A). ROS production was reduced by MCU inhibition. Two independent experiments; ∗∗∗∗p < 0.0001, compared to control at each time point, Mann-Whitney test. Error bars represent mean ± SEM.(C) HyPer images of oocytes injected with 20 ng of RNA for dominant-negative MCU, mcub, and 20 ng HyPer RNA compared to 20 ng HyPer RNA-injected control.(D) Quantification of HyPer ratio in (C). Inhibition of MCU by overexpression of mcub impairs ROS production. Error bars represent mean ± SEM.Two independent experiments; ∗∗p < 0.01 and ∗∗∗p < 0.001, compared to control at t30 and t60, two-way ANOVA and Sidak post hoc tests. See also Movie S8. Scale bars, 200 μm (A and D).
	Figure 5. Inhibition of mtROS Induces Cell Division Arrest in Xenopus Early Development(A) Embryos were treated at the one-cell stage with 0.1% DMSO (n = 36), 1 μM rotenone (n = 92), 10 mM malonate (n = 40), 3 mM sodium azide (n = 58), 10 μM antimycin A (n = 83), and 6 μM oligomycin (n = 88). Embryos treated with rotenone, malonate, and sodium azide were arrested at the 2- to 8-cell stage. By the blastula stage embryos treated with antimycin A were arrested.(B) 1-cell stage embryos obtained from females expressing HyPer were treated with mitochondrial inhibitors, and HyPer ratio was measured at the 4-cell and 32-cell stages. n = 12–13; ∗∗∗∗p < 0.0001, two-way ANOVA and Tukey’s multiple comparisons tests. Error bars represent mean ± SEM.(C) Embryos were treated with 0.1% DMSO or 2 μM CCCP (n = 66).(D) Embryos were injected with water as a control and 50 pmol RuR into one cell at the 2-cell stage. Injection of RuR induced cell-cycle arrest (n = 56).Pictures in (A), (C), and (D) are representative of at least three independent experiments.
	Figure 6. Removal of Inhibitors Restores Cell Division and ROS Production(A) Embryos were treated with inhibitors, 10 mM malonate, 3 mM sodium azide, and 1 μM rotenone, for 40 min until cells stopped dividing (left column), and then they were transferred to a dish containing fresh medium without inhibitors. Embryos transferred after treatment with malonate and azide retrieved cell division and divided to 8- to 16-cell stages (middle), but not in medium with inhibitors (right). Pictures are representative of three independent experiments.(B) Fertilized transgenic embryos expressing HyPer were treated with inhibitors for 40 min and imaged shown as 0 min. Then embryos were transferred to fresh medium without inhibitors, and imaged at 40 and 70 min (n = 34–36; three independent experiments).Error bars represent mean ± SD. See also Movie S9.

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