Discussion
Chromosome condensation is a process during which the dispersed chromatin in interphase is resolved and conversed into the compacted chromosomes during mitosis. SMC2 is the core component of the condensin complex (Hirano, 2006b), and Smc2 deletion resulted in highly expanded chromosomes and disrupted structural integrity in mitosis. So far, its participation in oocyte and embryo development in vivowas unknown, owing to the early mortality of SMC2 knockout embryos (Nishide & Hirano, 2014). By generating a maternal SMC2 deletion mouse strain, we investigated the function of SMC2 in oocytes and fertilized eggs for the first time.
In the process of final oocyte growth, the chromatin undergoes condensation during the NSN-SN transition, and is further condensed during oocyte meiotic maturation. After fertilization, the zygote undergoes the reverse process; chromatin structure starts to loosen in the pronuclei, in order to render the whole genome activated. We showed that SMC2 deletion caused defects of chromatin tightening in the GV stage. Though the diameters of fully grown oocytes were normal, oocytes isolated from the ovaries of Smc2flox/floxGdf9Cre mice displayed an increasing proportion of NSN configuration. Thus, SMC2 in growing oocytes is required for the compaction of chromatin during the NSN-SN transition. And this change contributed to limited early embryo development as demonstrated by the zygote arrest after either fertilization or PA of oocytes. Previously it was reported that SN oocytes have better developmental competence than NSN oocytes after meiotic maturation and fertilization (Tan et al., 2009).
To clarify the reason underlying 1-cell embryo arrest, we showed that SMC2- depleted zygotes displayed abnormal nuclear morphology. Under normal conditions, the male pronucleus is larger than the female pronucleus in the early zygote stage due to asynchronous pronuclear formation, as the male pronucleus appears earlier than the female pronucleus (Wiker et al., 1990). But in maternal SMC2 depleted zygotes female pronuclei were larger than male pronuclei. The smaller male pronuclei may be caused by insufficient sperm chromatin loosening after fertilization. The disorganized oocyte chromatin might result in increased disarray in female pronuclear formation and enlarged nuclear area.
The abnormal nuclear morphology including chromatin bridge and micronuclei were also detected in maternal SMC2 null zygotes, which might be linked with severe genomic rearrangements and chromosome fragmentation (Zhang et al., 2015), and in turn, caused abnormal nuclear function and zygote developmental arrest. DNA replication was reduced and expressions of all the selected embryo development related genes related to DNA binding, transcription, and minor ZGA activation were decreased in maternal SMC2 depleted zygotes, compared to the control group. Thus, the abnormal chromosome structure may interrupt the process of DNA replication, DNA binding and transcription, causing failure of embryonic development beyond the zygote stage.
Moreover, malfunctions in the pronuclei render the genome more prone to DNA damage (Zhang et al., 2015). Maternal SMC2 depleted zygotes suffered more severe degrees of DNA damage than the WT zygotes, even in the PN-3 stage, and failed to be repaired at the PN-5 stage. The accumulation of DNA damage may be the other major reason for the developmental arrest of SMC2-/+ zygotes.
In conclusion, activity of SMC2 is required for chromosome condensation throughout oocyte maturation and early embryonic development. Maternal SMC2 knockout caused NSN–SN transition failure in the GV oocytes, and pronuclear reformation defects in fertilized eggs. As a result, nuclear function was impaired and DNA damage was accumulated, which prevented the development of embryos beyond the zygote stage.