Depletion of maternal SMC2 impaired pronuclear activity
Since the pronuclear morphology was altered, we hypothesized that the pronuclear function in SMC2 depleted zygotes might be affected. We performed BrdU staining to show DNA replication. SMC2-/+ zygotes underwent the DNA replication process, however, the activity decreased by more than half compared with the control (Fig. 4D and E).
To assess whether the transcription activity was affected by the lack of maternal SMC2, we selected several embryo development related genes that should be transcription-activated in zygotes. After fertilization, new transcripts need to be generated along with protein synthesis corresponding to minor ZGA (Yu et al., 2016). This progress appears in the late zygote and two-cell stage. Therefore, we chose fertilized PN5 zygotes to detect the transcription levels.
First, we investigated the expression of transcription-related genes (pre-rRNA, FBL, and B23 ) (Liu et al., 2019). We found that all of the selected gene expressions decreased at the late zygote stage in SMC2 depleted embryos compared to the control group (Fig. 4F).
Then, we selected several translation-related genes (Rpl26, Rps13, and Rps20 ), and the results showed that these gene expressions were also significantly lower in the SMC2−/ + group, while they were activated normally in the control along with embryonic development (Fig. 4G).
The major aim for transcription in zygotes is the preparation for successful ZGA (Li et al., 2013). Further, we detected several representative genes that were highly transcribed during minor ZGA in late zygotes (MuERV-L , Dppa4, Sertad1 ) (Kigami et al., 2003). Notably, expressions of all these genes was down-regulated in SMC2 −/ + embryos (Fig. 4H). The disorganized zygotic genome activation may lead to the zygote arrest in SMC2-deficient embryos.