Fig 2: Oocyte maturation is impaired in oocytes from Xrcc5-cKO mice following etoposide-induced DNA damage.A Fully grown GV oocytes were exposed to 0, 50, or 100 µg/ml etoposide for 3 h, then released from meiotic arrest and in vitro maturation (IVM) monitored. B Percentage of oocytes that underwent GVBD transition after etoposide treatment over a 6-h time period. (i) Data were generated from three independent experiments; (ii)–(iv) are drawn from (i); number of oocytes analyzed is shown in parentheses. Student’s t-test was used for statistical analyses. Error bars are mean ± SEM, *p < 0.05, **p < 0.01. C Representative immunofluorescence images showing the chromosome alignment of WT and Xrcc5-cKO oocytes 16 h after treatment with 50 µg/ml etoposide.

Fig 3: Loss of Ku80 does not increase DNA damage in oocytes, or affect oocyte development and maturation, in mice at 6 months of age.A Xrcc5-cKO mice were constructed by deleting exon 4 of xrcc5 gene. To confirm conditional deletion of Xrcc5 in oocytes, RT-PCR was performed on GV-stage oocytes from wildtype (Xrcc5fl/fl; Gdf9+/+) and Xrcc5-cKO (Xrcc5fl/fl; Gdf9cre/+) mice (n = 3 mice/genotype). ß-actin was used as a loading control. Xrcc5 gene expression was detected using both pairs of primers in WT oocytes; whereas it was not detected using P1 and P2 in Xrcc5-cKO oocytes. B Ku80 protein (red) localization in isolated GV oocytes from WT and Xrcc5-cKO mice. A white dotted outline indicates the oocyte and nucleus. C Ku80 protein (red) localization in ovarian tissue sections from WT (arrows indicate staining in oocyte nuclei) and Xrcc5-cKO (arrowheads indicate the absence of staining in oocyte nuclei) mice at 6 months of age. Representative images of primordial, primary, secondary, and antral follicles from top to bottom. Scale bars: 10 µm. D Representative images of GV-stage oocytes from 6-month-old WT and Xrcc5-cKO mice stained with Hoechst (DNA; blue) and ?H2AX (yellow) (n = 3 mice/genotype). ?H2AX foci are indicated by arrows. E Number of ?H2AX foci in GV stage oocytes collected from WT and Xrcc5-cKO mice at 6-month-old (n = 10 oocytes/genotype). F Representative images DNA (blue), f-actin (red), aß-tubulin (green), and ?H2AX (yellow) in MII-stage oocytes from WT and Xrcc5-cKO mice at 6 months of age (n = 10 oocytes/genotype). G Number of ovulated MII-stage oocytes collected from WT and XRCC5 cKO mice at 6 months of age (n = 6 mice/genotype). Student’s t-test was used for statistical analyses (B and D). Error bars are mean ± SEM, ns no significant difference, p > 0.05.

Fig 4: Ku80 is required for the repair of etoposide-induced DNA damage in fully grown GV oocytes.Fully grown GV oocytes were exposed to 0, 5, 10, 50, or 100 µg/ml etoposide for 3 h, then DNA damage and repair were monitored by ?H2AX staining. T = 0, T = 3, and T = 6 represent 0, 3, and 6 h post-etoposide treatment, respectively. A Representative image of ?H2AX staining (yellow). B and C Quantification of ?H2AX pixel intensity. Data were generated from three independent experiments; number of oocytes analyzed is shown in parentheses. Student’s t-test was used for statistical analyses (B and C). Error bars are mean ± SEM, *p < 0.05, **p < 0.01, ***p < 0.001, ****p < 0.0001.