Fig. 6: Diminished mitochondrial function and oocyte quality in Sod2-deficient oocytes exacerbated by mtSSB heterozygous knockout.

a Generation strategy for oocyte-specific Sod2 knockout and mtSSB heterozygous mice, using Gdf9-Cre-driven recombination in Sod2^L/L males (L/L, Cre) crossed with Sod2^L/L; mtSSB^L/L females to generate Sod2^L/L, mtSSB^+/L; Gdf9-Cre (L/L, M; Cre) female mice. Created with BioRender.com. b Total pup number of each female during the 6-month fertility test, data were mean ± SEM and were analyzed using unpaired two-tailed Student’s t-test, **p < 0.01. c Number of total litters. Data were mean ± SEM and were analyzed using unpaired two-tailed Student’s t-test, ***p < 0.001. d Average litter sizes from each group. Data were mean ± SEM and were analyzed using unpaired two-tailed Student’s t-test. e Statistical analysis of superovulated oocyte number in females aged 3 weeks and 3 months. Data were mean ± SEM and were analyzed using one-way ANOVA with Tukey’s multiple comparison test, *p < 0.05, **p < 0.01, ****p < 0.0001. Sample sizes: control (n = 16, for the age of 3w and 3 m), L/L, Cre (n = 13 for 3w; n = 16 for 3 m), L/L, M; Cre (n = 11 for 3w; n = 16 for 3 m). f Representative image of the spindle morphology (red, tubulin; blue, chromosome), including oocytes with normal spindle (L/L) and oocytes with aberrant spindles (L/L, M; Cre). g Proportion of oocytes with aberrant spindle from (f), across three independent experiments, 3-4 mice per group. Data were mean ± SEM and were analyzed using unpaired two-tailed Student’s t-test, **p < 0.01. h Representative immunofluorescence images of MII oocytes from 3-month-old mice, stained for dsDNA (green) and TOMM20 (red). i mtDNA copy number semi-quantification with the ratio of yellow to red fluorescence intensity in L/L, M (n = 4), L/L, M; Cre_normal (n = 5), and L/L, M; Cre_aberrant (n = 6) oocytes from (j). Data were mean ± SEM and were analyzed using one-way ANOVA with Tukey’s multiple comparison test, *p < 0.05. j Quantitative PCR analysis of single oocyte mtDNA copy number from females of different ages. Data were mean ± SEM and were analyzed using unpaired two-tailed Student’s t-test, **p <  0.01, ****p < 0.0001. k Representative image of TMRM staining (red) of MII oocytes from control; L/L, Cre and L/L, M; Cre mice at 3-week-old and 3-month-old. l Quantification of TMRM signals in (k). Data were mean ± SEM and were analyzed using one-way ANOVA with Tukey’s multiple comparison test, **p < 0.01, ****p < 0.0001. m ATP level of single oocytes from L/L, M and L/L, M; Cre mice at 3-month-old. Data were mean ± SEM and were analyzed using unpaired two-tailed Student’s t-test, *p < 0.05. n Mutation spectrum of de novo mutations and shared variations in control (L/L; L/L, M) and L/L, M; Cre 3-week-old and 6-month-old mice, respectively.