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 the successful reprogramming of an introduced donor nucleus in cattle. In the present study, fused NT embryos were subjected to immediate activation (IA, simultaneous fusion and activation), delayed activation (DA, activation applied 4 h postfusion), and IA with aged oocytes (IAA, activation at the same oocyte age as group DA). The morphologic 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 IA 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 postcell fusion and was 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%) than in those for which activation was delayed (14.2%). The development rate was higher in group IA than in group IAA, but it was not significant (P = 0.089). Following embryo transfer, there was no statistically significant difference in the pregnancy rates (Day 70) between two of the groups (group IA, 11.7%, n = 94 vs. group DA, 12.3%, n = 130; P > 0.05) or live term development (group IA, 4.3% vs. group DA, 4.6%; P > 0.05). Our study has demonstrated that the IA 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.
1One of the several factors that contribute to the low efficiency of mammalian somatic 2 cloning is the poor fusion between the small somatic donor cell and the large recipient 3 oocyte. This study was designed to test phytohemagglutinin (PHA) agglutination activity 4 on fusion rate, and subsequent developmental potential of cloned bovine embryos. The 5 toxicity of PHA was established by examining its effects on the development of 6 parthenogenetic bovine oocytes treated with different dosages (Expt 1), and for different 7 durations (Expt 2). The effective dosage and duration of PHA treatment (150 µg/mL, 20 8 min incubation) was selected and used to compare membrane fusion efficiency and 9 embryo development following somatic cell nuclear transfer (Expt 3). Cloning with 10 somatic donor fibroblasts vs. cumulus cells was also compared, both with and without 11 PHA treatment (150 µg/mL, 20 min). Our results showed that the fusion rate of nuclear 12 donor fibroblasts, after phytohemagglutinin treatment, was increased from 33 to 61 % 13 (P<0.05), and from 59 to 88% (P<0.05) with cumulus cell nuclear donors. The nuclear 14 transfer (NT) efficiency per oocyte used was improved following PHA treatment, for 15 both fibroblast (13 vs. 22%), as well as cumulus cell (17 vs. 34%) (P<0.05). The cloned 16 embryos, both with and without PHA treatment, were subjected to vitrification and 17 embryo transfer testing, and resulted in similar survival (approximately 90% hatching) 18 and pregnancy rates (17 to 25%). Three calves were born following vitrification and 19 embryo transfer of these embryos; two from the PHA-treated group, and one from non-20 PHA control group. We conclude that PHA treatment can significantly improve the 21 fusion efficiency of somatic NT in cattle, and therefore, increase the development of 22 cloned blastocysts. Furthermore, within a determined range of dosage and duration, PHA 23 3 has no detrimental effect on embryo survival post vitrification, nor on pregnancy or 1 calving rates following embryo transfer. 2 3 4
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