Fig 2: Aurkb cKO oocytes from older females have reduced expression of MAD2, ZW10 and Securin. (a) Representative Western blot images of prophase I-arrested oocytes from older wild-type (WT) or conditional Aurkb knockout (B cKO) females detecting MAD2, ZW10 and Securin. (b) Quantification of MAD2 (**p < 0.0030), (c) Quantification of ZW10 (** p < 0.0016). (d) Quantification of Securin (*p < 0.0468). (e, g) Representative Western blots of prophase I-arrested oocytes from young females detecting MAD2, Securin (d) and ZW10 (g). (f) Quantification of MAD2 (p = 0.9774). Graph shows individual values plus the mean ±SEM from 3 independent experiments. (h) Quantification of ZW10 (p = 0.1166). (i) Quantification of Securin (*p < 0.7505), MAD2, ZW10 and Securin signals were normalized to a-Tubulin. Each lane contained 100 prophase I-arrested oocytes. n.s.: not significant. Graph shows mean ±SEM from 2–3 independent experiments. (j) Representative confocal images of pro-metaphase I oocytes expressing Gfp or MAD2-gfp (green) DNA (blue). Scale bar: 10 µm. (k) Representative images of timing of PBE of Aurkb cKO oocytes from older females, matured in nocodazole expressing either Gfp or MAD2-gfp. Red star indicates a PB. Scale bar: 20 µm. (l) Quantification of % of oocytes from (k) that undergo PBE in nocodazole. (*p = 0.0.0160; number of oocytes examined Gfp: 73, MAD2-gfp: 59; 6 mice). Graph shows the mean ±SEM from 3 experiments. (m) Representative images of timing of PBE of Aurkb cKO oocytes from older females, matured in nocodazole expressing Securin-gfp (green). Numbers of oocytes that expelled a PB relative to total number of oocytes analyzed is indicated to the right of the images. Unpaired Students t-Test, two-tailed was used

Fig 3: Mad2-mediated SAC inactivation in Spc24-depleted oocytesControl and Spc24-depleted oocytes were fixed at 5 h following release from IBMX. (A) Oocytes were immunostained with anti-Mad2 antibody (green) and Hoechst 33342 (red). Scale bars: 20 µm. (B) Oocytes were immunostained with anti-Bub3 antibody (green) and Hoechst 33342 (red). Scale bars: 20 µm. Quantification of fluorescent intensity of Mad2 or Bub3 is shown in the right panel of images, respectively. Data are expressed as mean ± SEM of at least 3 independent experiments. *Significantly different (P < 0.05). The total numbers of analyzed oocytes are indicated (n).

Fig 4: Spindle assembly checkpoint activity is not abrogated in tetraploid embryos. a and b Quantification of mitosis duration in 8-cell control (without AZ 3146 n = 47 divisions from seven embryos; with AZ 3146 n = 63 divisions from eight embryos), 4-cell binucleated embryos (without AZ 3146 n = 30 divisions from eight embryos; with AZ 3146 n = 54 divisions from 15 embryos) ***P = 0.0009; ****P < 0.0001 (unpaired Kruskal–Wallis test with Dunn’s test for multiple comparisons) a; 16-cell control (without AZ 3146 n = 61 divisions from seven embryos; with AZ 3146 n = 59 divisions from seven embryos) and 8-cell tetraploid embryos (without AZ 3146 n = 50 divisions from nine embryos; with AZ 3146 n = 63 divisions from 12 embryos) ***P = 0.0005; ****P < 0.0001 (unpaired Kruskal–Wallis test with Dunn’s test for multiple comparisons) b. c Representative z projections of MAD2-positive and MAD2-negative kinetochores demonstrating co-localisation of MAD2 (magenta) and CREST (cyan) in positively stained kinetochores and no co-localisation in negatively stained kinetochores. d and e Proportion of MAD2-positive kinetochores in 8-cell control, 4-cell binucleated d, 16-cell control and 8-cell tetraploid embryos e at 10 mins (8-cell control n = 6 blastomeres; 4-cell binucleated n = 6 blastomeres; 16-cell control n = 5 blastomeres; 8-cell tetraploid n = 5 blastomeres), 20 mins (8-cell control n = 5 blastomeres; 4-cell binucleated n = 6 blastomeres; 16-cell control n = 5 blastomeres; 8-cell tetraploid n = 6 blastomeres) and 30 mins (8-cell control n = 4 blastomeres; 4-cell binucleated n = 6 blastomeres; 16-cell control n = 5 blastomeres; 8-cell tetraploid n = 5 blastomeres) after nuclear envelope breakdown (NEBD). Error bars represent SEM. f and g Representative time-lapse images of live 4-cell binucleated f and 8-cell tetraploid embryos g co-expressing H2B:RFP and MAD1:2EGFP. Note that MAD1:2EGFP signal is clearly observed at the kinetochores shortly after nuclear envelope breakdown (NEBD) and is gradually lost following chromosome alignment at the metaphase plate. Scale bars = 10 µm except for figure c, where scale bars = 1 µm. Where box plots are shown, the centre line represents the median, the bounds of box represent the upper and lower quartiles and the whiskers represent minimum and maximum values

Fig 5: ROS levels are increased in Aurkb cKO oocytes from older females. (a) Representative images of CM-H2DCFDA fluorescence in prophase I-arrested oocytes from wild-type (WT), conditional Aurkb knockouts (B cKO), or Aurkc knockout (C KO) females at different ages. (b) Quantification of CM-H2DCFDA intensity showed in (a) ****p < 0.0001; number of oocytes examined: positive control: 29; young WT: 58, B cKO: 77, C KO: 92; older: WT: 60, B cKO: 36, C KO: 70; 3 mice/genotype). (c) Representative images of CM-H2DCFDA fluorescence in prophase I-arrested oocytes from wild-type (WT), conditional Aurkb knockouts (B cKO), older females treated with and without NAC. Positive control: WT oocytes incubated with 200 µM H2O2. Scale bars: 20 µm. (d) Quantification of CM-H2DCFDA intensity showed in (c) (**p = 0.0035; * p < 0.05; number of oocytes examined: WT: 20, B cKO: 25, B cKO +NAC:29; 3 mice/genotype). (e) Representative confocal images of WT and Aurkb cKO Met I oocytes from older females treated with or without NAC, immunostained with antibodies against ACA (red), MAD2 (gray) and DAPI (blue). (f) Quantification of MAD2 intensity at kinetochores in (e) (*p = 0.0464; **** p < 0.0001; n.s. not significant; number of oocytes examined: WT: 35, B cKO: 32, B cKO +NAC:28; 3 mice/genotype). Graph shows individual oocyte values plus the mean ±SEM from 3 experiments. One-way ANOVA used