Knockout of Smc2 loosens chromatin structure in fully-grown GV oocytes
To further investigate the reason for infertility, we examined whether the SMC2 deletion inhibited nucleolus maturation in oocytes isolated from the 5-week-old Smc2flox/flox Gdf9-Crefemales. The diameter of fully-grown SMC2-null oocytes was similar as that of Smc2flox/flox oocytes (Fig. 2A, B). However, among the well grown oocytes isolated from the ovaries ofSmc2flox/flox mice, approximately three quarters of the cells displayed the SN chromatin configuration, and one quarter of them displayed the NSN configuration, as revealed by DAPI staining (Fig. 2C). In contrast, the proportion of SN vs. NSN oocytes was reversed in Smc2flox/flox Gdf9-Cre miceĀ (Fig. 2D). This observation indicated that SMC2 deletion in oocytes inhibited nucleolar maturation.
Moreover, chromosome spreading of MI oocytes (Fig. 2E) showed that the SMC2-null oocytes all presented severe chromosomal structural disorder and improperly placed BUBR1, an important component of the spindle assembly checkpoint (SAC), which may account for the abnormality of oocytes escaping the SAC checkpoint and reaching the MII stage.
As SMC2 knockout impairs chromatin configuration of GV oocytes, we hypothesized that the DNA damages were accumulated in SMC2 mutant oocytes. Single-cell gel electrophoresis (comet assay) was used to quantify the extent of DNA damage. As expected, MII oocytes ofSmc2flox /flox Gdf9Cre females showed large amounts of DNA damage, up to 4-fold in the proportion of comet tail DNA, as quantified by using CASP software (Fig. 2F).