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.