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).