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

  • Ju, Jyh-Cherng (Department of Animal Science, National Chung Hsing University) ;
  • Yang, Jyh-Shyu (Department of Animal Science, National Chung Hsing University) ;
  • Liu, Chien-Tsung (Department of Medical Research, Mackay Memorial Hospital) ;
  • Chen, Chien-Hong (Department of Animal Science, National Chung Hsing University) ;
  • Tseng, Jung-Kai (Department of Animal Science, National Chung Hsing University) ;
  • Chou, Po-Chien (Department of Animal Science, National Chung Hsing University) ;
  • Cheng, San-Pao (Department of Animal Science, National Chung Hsing University)
  • Received : 2002.02.26
  • Accepted : 2002.06.10
  • Published : 2003.01.01


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 $38^{\circ}C$ 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 ${\mu}m$). Significant differences were observed in cleavage rates ($\geq$3 divisions) in the isolated single blastomeres (54 vs 48 for 16-cell; 28 vs 14 for 32-cell, p<0.05), but the fusion rates of oocytes with transferred nuclei were similar between small and large single blastomeres derived from either 16-cell or 32-cell stage embryos. When 16-cell stage blastomeres were used as nuclear donors, cleavage rates ($\geq$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 G1 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.


Nuclear Transfer;Cell Cycle Stage;Blastomere;Cloned Embryo;Rabbit


Supported by : National Science Council


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