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Effect of Cell Cycle Stage on the Development of Embryos Produced by Cumulus Cell Nuclear Transfer in Hanwoo (Korean Cattle) |
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G. S. Im, B. S. Yang, B. C. Yang, W. K. Chang, Y. J. Yi, C. S. Park |
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| Abstract |
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This study was carried out to investigate the effect of activation timing, cell cycle and passage on the development of embryos produced by cumulus cell nuclear transfer in Hanwoo (Korean cattle). Nuclear donor cumulus cells were cultured in Dulbecco`s modified Eagle medium supplemented with 10% fetal bovine serum at 38.5째C in a humidified atmosphere of 5% CO2 in air. The 1節퓈assages of serum deprived or actively dividing cumulus cells were isolated and used as donor cells. The in vitro matured oocytes were enucleated and then the isolated donor cells were introduced. One pulse of 180 volts for 15 關s was applied to induce the fusion between karyoplast and cytoplast. The activation was done before or after the fusion. To activate, oocytes were treated with 10 關M calcium ionophore for 5 min immediately followed by 2 mM 6-dimethylaminopurine for 3 h. The nuclear transfer embryos were cultured in 500 關l of modified CRlaa supplemented with 3 mg/ml BSA in four well dish covered with mineral oil. After 3 days culture, culture medium was changed into modified CRlaa medium containing 1.5 mg/ml BSA and 5% FBS for 4 days. The incubation environment was 5% CO2, 5% O2, 90% N2 at 38.5째C. There was no blastocyst formation when the nuclear transfer embryos were activated before the fusion, whereas, 29.9% of blastocyst formation was shown when the nuclear transfer embryos were activated after the fusion. When serum deprived and actively dividing cumulus cells were used as nuclear donor cells, the developmental rates to blastocyst were 38.5% and 40.6%, respectively. There was no significant difference between serum deprived and actively dividing cells in the developmental rates. The developmental rates to blastocyst according to 1節퓈assages were 37.5節풥owever, there were no significant differences among passages. These results indicate that 1節퓈assage cumulus cell irrespective of cell cycle could support development of nuclear transfer embryos activated after the fusion. |
| Keywords:
Cell Cycle; Nuclear Transfer; Donor Cumulus Cell; Blastocyst |
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