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Abstract
Premature chromosome condensation (PCC) was believed to
promote nuclear reprogramming and to facilitate cloning by
somatic cell nuclear transfer (NT) in mammalian species.
However, it is still uncertain whether PCC is necessary
for successful reprogramming of an introduced donor
nucleus in cattle. In our study, fused NT embryos were
subjected to immediate activation (IA, simultaneous fusion
and activation), delayed activation (DA, activation
applied 4 h post fusion) and immediate activation with
aged oocytes (IAA, activation at the same oocyte age with
Group DA). The morphological changes, such as nuclear
swelling, the occurrence of PCC, and microtubule/aster
formation were analyzed in detail by laser scanning
confocal microscopy. When embryos were subjected to
immediate activation in both IA and IAA groups, the
introduced nucleus gradually became swollen, and a
pronuclear-like structure formed within the oocyte, but
PCC was not observed. In contrast, delaying embryo
activation resulted in 46.5-91.2% of NT embryos exhibiting
PCC. This PCC was observed beginning at 4 h post cell
fusion shown as one, two, or multiple chromosomal
complexes. Subsequently, a diversity of pronuclear-like
structures existed in NT embryos, characterized as single,
double and multiple nuclei. In the oocytes exhibiting PCC,
the assembled spindle structure was observed to be an
interactive mass, closely associated with condensed
chromosomes, but no aster had formed. Regardless of
whether they were subjected to IA, IAA or DA treatments,
if the oocytes contained pronuclear-like structures,
either one or two asters, were observed in proximity to
the nuclei. A significantly higher rate of development to
blastocysts was achieved in embryos that were immediately
activated (IA, 59.1%; IAA, 40.7%), compared to those in
which activation was delayed (14.2%). The development rate
was higher in Group IA to Group IAA, but it was not
significant (P=0.089). Following embryo transfer, there
was no statistically significant difference in the
pregnancy rates (d 70) between two groups (Group IA, 11.7
%, n=94 vs. Group DA, 12.3 % n=130; P>0.05), as well as
live term development (Group IA, 4.3 % vs. Group DA, 4.6
%, P>0.05). Our study has demonstrated that immediate
activation of bovine NT embryos results in embryos with
increased competence for preimplantational development.
Moreover, PCC was shown to be unnecessary for the
reprogramming of a transplanted somatic genome in a cattle
oocyte.
Key words:
Embryo •
Gamete Biology •
Pregnancy •
Conceptus •
Implantation
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