Fig. 1: Sod2-conditional knockout in oocytes resulted in female reproductive aging phenotype.

a Mouse breeding strategy to generate oocyte-specific Sod2-deficient mice. Sod2^L/L (L/L) male transgenic mice that expressed Cre recombinase under the control of the Gdf9 promoter were intercrossed with Sod2^L/L female mice to generate Sod2^L/L, Gdf9-Cre (L/L, Cre) female mice. Created with BioRender.com. b Western blot detection of SOD2 expression in the MII oocytes of 3-week-old L/L, Cre and L/L female mice. Each lane contains 50 MII oocytes. ACTIN was used as a loading control. The experiments were repeated 3 times, and a representative image is shown. c Representative image of HKSOX-2m staining (green) of MII oocytes from L/L and L/L, Cre mice at different ages. Scale bar: 50 µm. d Quantification of HKSOX-2m signals of MII oocytes from L/L and L/L, Cre mice at different ages. Data were mean ± SEM and were analyzed using unpaired two-tailed Student’s t-test, **p < 0.01, ***p < 0.001. e Total pup number of each female during the 6-month fertility test, wild-type (WT); L/L; +/L, Cre and L/L, Cre female mice, with each column representing all pups from one female mouse. f Number of total litters. The control group consists of mice from both the WT and L/L groups. Data were mean ± SEM and were analyzed using one-way ANOVA with Tukey’s multiple comparison test, ****p < 0.0001. g Total pup number from each group. The control group consists of mice from both the WT and L/L groups. Data were mean ± SEM and were analyzed using one-way ANOVA with Tukey’s multiple comparison test, *p <  0.05, ****p < 0.0001. h Average litter sizes from each group. The control group consists of mice from both the WT and L/L groups. Data were mean ± SEM and were analyzed using one-way ANOVA with Tukey’s multiple comparison test, *p < 0.05, **p < 0.01. i The representative morphology of two-cell stage embryos and blastocysts after in vitro fertilization. MII oocytes were obtained from 20 to 22-week-old L/L, Cre and L/L mice. After fertilization, the in vitro cultures were performed under 5% O₂ (5% O₂/5% CO₂/90% N₂). The sperm were obtained from 12 to 14-week-old wild-type C57BL/6 male mice. Scale bar: 100 μm. j The fertilization rate and blastocyst rate of L/L, Cre and L/L mice oocytes after in vitro fertilization. Data were mean ± SEM and were analyzed using unpaired two-tailed Student’s t-test, ****p < 0.0001.