Nuclear-cytoplasmic interaction and development of goat embryos reconstructed by nuclear transplantation: production of goats by serially cloning embryos.

Abstract

The time of pronuclear formation of in vivo-matured oocytes was examined. Maturation-promoting factor (MPF) activity in enucleated and electrically activated oocytes was checked by assessment of nuclear envelope breakdown (NEBD) of fused blastomeres. The effect of stage of the cell cycle of donor cells and recipient oocytes on DNA synthesis and development of reconstructed embryos was studied. MPF activity declined rapidly to approximately 63.2% at 1 h, to 9.7% at 5 h, and to the level at which NEBD cannot occur at 7 h postactivation. All blastomeres that were fused at the time of recipient cytoplasm activation underwent NEBD and subsequent DNA synthesis. However, when blastomeres were fused to enucleated oocytes at 7 h postactivation, no NEBD was observed; DNA was replicated in nuclei at the G1/S border, but in G2 nuclei no DNA replication was observed. The proportion of development to blastocysts of reconstructed goat embryos increased with the decline in MPF activity in fused recipient cytoplasm when reconstruction took place at 0-6 h postactivation of enucleated oocytes. Generations 1, 2, 3, 4, 5, and 6 of cloned goat embryos were produced by a combination of nuclear transplantation and in vitro techniques. After transfer to recipients, 45 kids were obtained, including three pairs of monozygotic twins, three series of monozygotic triplets, two series of monozygotic quadruplets, three series of monozygotic quintuplets, and one series of monozygotic heptaplets. The present study indicates that normal DNA replication of goat blastomere nuclei can be accomplished in enucleated oocytes when MPF activity is low, regardless of the stage of the cell cycle of donor nuclei; induction of NEBD and prematuration chromosome condensation is not essential for further development of reconstructed goat embryos.