Influence of nuclear and cytoplasmic activity on the development in vivo of sheep embryos after nuclear transplantation

Abstract

Reconstituted sheep embryos have been produced by electrofusion-mediated nuclear transplantation in which single cells derived from embryos at the 16-cell (Day 4) and the inner cell mass (ICM) of early blastocyst stage (Day 6) were fused to unfertilized enucleated secondary oocytes. Electrofusion rates were higher with 16-cell blastomeres (82%) than with ICM cells (47%) and when an alternating current (a.c.) preceded the direct current (d.c.) fusing pulse (88% vs. 47%). The addition of cytochalasin B (7.5% micrograms/ml) to the medium for 1 h after electrofusion significantly improved the development to morula-blastocyst stage of reconstituted embryos derived from both 16-cell blastomeres (11% vs. 35%) and ICM cells (0% vs. 56%), indicating that the cytoskeletal mechanisms operating at the time of oocyte activation may affect the developmental potential of exogenous nuclei. Transplantation of 22 reconstituted morulae and blastocysts from both groups into the uteri of recipient ewes led to the development of four lambs (18%) with the phenotype of the nuclear donor breed. These findings indicate that at least some nuclei derived from transcriptionally active embryos are totipotent and able to be reprogrammed to support full-term development when fused to enucleated secondary oocytes.