Feasibility of producing porcine nuclear transfer embryos by using G2/M-stage fetal fibroblasts as donors

The type of donor cell most suitable for producing cloned animals is one of the topics under debate in the field of nuclear transfer. To provide usefu l information to answer this question, G2/M- and G0/G1-stage fetal fibrobla sts were used as donor cells for nuclear transfer. In vitro-matured oocytes derived from abattoir ovaries were used as recipient cytoplasts. In both g roups, nuclear envelope breakdown and premature chromosome condensation wer e completed within 1-2 h after donor cells were injected into the cytoplasm of oocytes. Microtubules were organized around condensed chromosomes and f ormed a spindle within 1-1.5 h after activation. Decondensation of chromoso mes could be seen within 2-4 h after activation. Reformation of the new nuc lear envelope occurred 4-6 h after activation and was followed by nuclear s welling and formation of a pronucleus-like structure (PN) 8-12 h after acti vation. Most (80.6%) of the reconstructed oocytes derived from G2/M cells e xtruded polar body-like structures (PB). However, a much lower frequency of PB (21.7%) was observed in the reconstructed oocytes derived from G0/G1 do nors. A variety of PN and PB combinations were observed in reconstructed oo cytes derived from G2/M-stage donors, including 1PN+0PB, 1PN+1PB, 1PN+2PB, 2PN+0PB, 2PN+1PB, 2PN+2PB, and 3PN+1PB. Chromosomes of most embryos (10/13) derived from G2/M stage were diploid. The percentage of cleavage and blast ocysts and the average nuclear number of blastocysts in the G2/M and G0/G1 groups were not different. These results demonstrate that the G2/M stage ca n be morphologically remodeled by cytoplasm of Mill oocytes in pigs. To mai ntain normal ploidy, the extra chromosomes derived from G2/M-stage cells co uld be expelled by oocytes as a second polar body. G2/M-stage fibroblast nu clei could direct reconstructed embryos to develop to the blastocyst stage.