Effect of delayed enucleation on the developmental potential of nuclear-transferred oocytes receiving adult and fetal fibroblast cells.

To enhance the probability of reprogramming somatic cell nuclei, fibroblast cells from an adult male rabbit and a 12-day-old fetus were fused with oocytes at the second metaphase. The chromosomes of recipient oocytes were then removed by treatment with demecolcine for 1 or 2 h after fusion. Demecolcine treatment of fused oocytes induced membrane protrusions that contained all the maternal chromosomes, thus making it possible to remove the chromosomes. The potential of nuclear-transferred oocytes to develop into blastocysts was high (48% and 59%) and the average cell number of the blastocysts was large (149 and 159) 96 h after in vitro culture. The proportions of nuclear-transferred oocytes enucleated 1 h after fusion and implanted after transfer to pseudopregnant recipients were relatively high (2.8% and 4.9%) compared with our previous reports (1.7%: Yin et al., 2000; 0.6% and 1.0%: Yin et al., 2002a) where donor cells were fused with previously enucleated oocytes. Of 34 adult somatic cell implantation sites, 6 had fetuses on day 12 or 14 of pregnancy, but none of the fetuses had a heart beat or developed to term. None of the nuclear-transferred oocytes whose chromosomes were removed 2 h after demecolcine treatment implanted after transfer to recipients. The possible reasons why the high-quality nuclear-transferred oocytes did not develop to term are discussed.