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Premature Chromosome Condensation Is Not Essential for Nuclear Reprogramming in Bovine Somatic Cell Nuclear Transfer¹

Department of Animal Science/Center for Regenerative Biology,⁴ University of Connecticut, Storrs, Connecticut 06269 Department of Animal Science,⁵ National Pingtung University of Science and Technology, Pingtung 91201, Taiwan Evergen Biotechnologies, Inc.,⁶ Storrs, Connecticut 06269 Department of Animal Science,⁷ National Taiwan University, Taipei 10617, Taiwan Taiwan Livestock Research Institute,⁸ Hsin-hua, Tainan 71210, Taiwan Trans Ova Genetics,⁹ Sioux Center, Iowa 51250

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 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.

conceptus, embryo, gamete biology, implantation, pregnancy

³These authors contributed equally to this work.

¹Supported in part by the Research Fund from Evergen Biotechnologies, Inc., Storrs, Connecticut, awarded to F.D., a Yankee Ingenuity grant from Connecticut Innovation, Inc., Connecticut, awarded to X.Y., and a Livestock Research Institute, Council of Agriculture, Taiwan grant to S.N.L.

Correspondence: ²Fuliang Du, Evergen Biotechnologies, Inc., 1392 Storrs Rd., Unit 4213, Room 106, Storrs, CT 06269. FAX: 860 486 6628; e-mail: fuliangd{at}evergen.com

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