Differential Influences in Sizes and Cell Cycle Stages of Donor Blastomeres on the Development of Cloned Rabbit Embryos

Abstract

Experiments were conducted to evaluate the effect of blastomere diameters and cell cycle stages on the subsequent development of nuclear transplant rabbit embryos (NT-embryos) using nuclei derived from the 16-or 32-cell stage embryos. All blastomeres and NT-embryos were cultured individually in modified Ham's F-10 medium supplemented with 10% rabbit serum (RS) at 38°C and 5% CO 2 in air. The diameter of blastomeres from 16-cell stage embryos was found twice of those from 32-cell stage (51 vs 27 µm). Significant differences were observed in cleavage rates (≥3 divisions) in the isolated single blastomeres (54 vs 48 for 16-cell; 28 vs 14 for 32-cell, p<0.05), but the fusion rates of oocytes with transferred nuclei were similar between small and large single blastomeres derived from either 16-cell or 32-cell stage embryos. When 16-cell stage blastomeres were used as nuclear donors, cleavage rates (≥3 divisions) of the NT-embryos were greater in the small nuclear donors than in the large donors (73 vs 55%, p<0.05). On the contrary, significantly higher cleavage (43 vs 6%, p<0.05) and developmental rates (14 vs 0%, p<0.05) were observed in the large blastomere nuclear donor group of the 32-cell stage embryos. When the cell cycle stages were controlled by a microtubule polymerization inhibitor (Demicolcine, DEM) or the combined treatment of DEM and Aphidicolin (APH), a DNA polymerase inhibitor, fusion rates were 88-96% for the 16-cell donor group (without DEM treatment), which were greater than the 32-cell donor group (54-58%). Cleavage rates were also greater in the transplants derived from G 1 nuclear donor group (93-95%) than those from the DEM and APH combined treatment (73%) for the 16-cell donor group (p<0.05). No significant difference was detected in the morula/blastocyst rates in either donor cell stage (p>0.05). In conclusion, it appeared that no difference in the developmental competence between large and small isolated blastomeres was observed. When smaller 16-cell stage blastomeres were used as nuclear donor, the cleavage rate or development of NT-embryos was improved and was compromised when 32-cell stage blastomeres were used. Therefore, control nuclear stage of the donor cell at G 1 phase in preactivated nuclear recipients seemed to be beneficial for the cleavage rate of the reconstructed embryo in the 16-cell transplant, but not for subsequent morula or blastocyst development.

Cell Cycle Stage Analysis of Rabbit Foetal Fibroblasts and Cumulus Cells

“…Demicolcine and aphidicolin have been used to control cell cycle stages of embryonic blastomeres in M and G 1 /S phases, respectively (Collas et al. 1992a,b; Ju et al. 2000, 2003).…”

“…Demicolcine and aphidicolin have been used to control cell cycle stages of embryonic blastomeres in M and G 1 /S phases, respectively (Collas et al 1992a,b;Ju et al 2000Ju et al , 2003. Demicolcine, a cocemid or colchicine analog, inhibits polymerization of microtubules, which in turn arrests cell cycle in mitotic M-phase; whereas aphidicolin, a DNA synthesis inhibitor, blocks the activity of DNA polymerases, which in turn arrests the cell cycle at the G 1 /S boundary.…”

“…As described previously, demicolcine were added to the culture medium to maintain the cell cycle stage in M‐phase and aphidicolin was used for controlling the cells in G 1 /S phase. For the combined treatment, cells were pre‐treated with demicolcine for 10 h and followed by aphidicolin for 6 h to arrest the cells at the G 1 /S boundary as reported previously (Ju et al. 2003).…”

“…Chemicals used for this purpose are demicolcine (0.5 lg/ml, 10 h; Sigma D-7385, St. Louis, MO, USA) and aphidicolin (0.1 lg/ml, 6 h; Sigma A-0781). A combination of these two chemicals was also tested as described in a previous study (Ju et al 2003). Briefly, fibroblasts were first treated with demicolcine (0.5 lg/ ml) for 10 h in DMEM + 10% FBS.…”

The Influence of Microinjection of Foreign Gene into the Pronucleus of Fertilized Egg on the Preimplantation Development, Cell Number and Diameter of Rabbit Embryos

“…Mori et al (2002) have reported a high positive correlations between the cell number and the diameter in bovine embryos at each stage collected on days 7-9 after in vitro fertilization. Embryo diameter is positively dependent on size of blastomeres, which may contribute to the efficiency of embryo cloning in rabbit, using blastomeres as donors of nuclei (Ju et al, 2003). Therefore, the diameter of the embryo may be potentially used for the viability testing of the expanded blastocysts.…”