The Nucleus Remolding Patern Of G1 & M Phase Somatic Cell After Nuclear Transfer

Nuclear transfer has succeeded on many species since the first somatic cell nclear transfer sheep"Dolly"was born and many gene modified animals have also been obtained by nuclear transfer from somatic cells. However the efficiency of nuclear transfer is still very low regardless of the species。There are many factors affecting efficiency of nuclear transfer, the quality of oolasm and the cell cycle of the donor cells are the most important two factors.In this experiment, the effects of brilliant cresyl blue (BCB) stainning on selection of porcine oocytes were studied. The in vitro maturation rates, parthenogenetic developmental potential and reconstructed embryos developmental potential of oocytes derived from BCB stainning group(BCB+), BCB non-stainning group(BCB-) and control group were abserved respectively. The nuclear morphological events before the first cleavage and developmental potential of embryos reconstructed with porcine fibroblasts at M or early G1 stage were compared . The nuclear morphological events of embryos reconstructed with donor cells at early G1 stage(eG1-NT embryos) at 2,4,6h post fusion (hpf) and the nuclear morphological events of embryos reconstructed with donor cells at M stage (M-NT embryos) at 2,4,6,19,24hpf were observed and the nuclear remoldling pattern of the two kinds of reconstructed embryos were analysised. The in vitro developmental potential of eG1-NT embryos and M-NT embryos to blastocysts were studied too. Results were as follows:The percentage of matured MII oocytes derived from BCB+ group (91.6%) is significantly (P<0.05) higher than that from BCB- group (64.0% ) and the control group (77.5%);The percentage of parthenogenetic embryos derived from BCB+ group (34.9%) is significantly (P<0.05) higher than that from BCB- group (9.5%) and the control group (23.1%); The percentage of nuclear transfer blastocysts derived from BCB+ group (23.0%) is significantly (P<0.05) higher than that from BCB- group (5.0%) and the control group (14.1%).About half (50.0% and 44.8%) of M-NT embryos and eG1-NT embryos'nuclei were in nuclear swelling stage at 2hpf; The proportion of M-NT embryos formed pronucleus-like nucleus (77.8%) was significantly higher (P<0.05) than that of eG1-NT embryos (55.3%) at 4hpf. Most of M-NT embryos and eG1-NT embryos (89.3% and 75.0%) formed proncleus-like nucleus at 6hpf. 77.4% of the M-NT embryos formed two nucleus at 19hpf and no polar body-like cells(PBCs) extruded. There was no difference between the in vitro developmental potential to blastocyst of M-NT embryos and eG1-NT embryos (P>0.05).The results indicate that the BCB stainning is a useful method to select high quality porcine oocytes, although pronucleus-like nuclei form earlier in M-NT embryos than eG1-NT embryos there is no difference between the in vitro developmental potential to blastocyst between them. M-NT embryos might be tetraploid due to no PBCs extruded.