Effect of polyethylene glycol and dimethyl sulfoxide on the fusion of bovine nuclear transfer using mammary gland epithelial cells.
The effects of polyethylene glycol and dimethyl sulfoxide (PEG/DMSO) treatment of donor cells on the fusion and subsequent development of bovine nuclear transfer embryos using mammary gland epithelial (MGE) cells before electrofusion (fresh MGE cells) was studied. The same study was conducted on those cells that were frozen and stored in liquid nitrogen, and then thawed (frozen-thawed MGE cells). Experiment 1 showed that the exposure time and pH of PEG/DMSO solution affected the fusion of nuclear transfer, and that a higher fusion rate was obtained when fresh MGE cells were exposed to PEG/DMSO solution at pH 8.0 for 5 min. In Experiment 2, the proportion of fused oocytes with fresh PEG/DMSO-treated cells (70 +/- 6%) was significantly higher than that with non-treated cells (50 +/- 13%, p < 0.05). The same tendency was observed when frozen-thawed cells as donor nuclei were used (48 +/- 6% vs. 34 +/- 12%, p < 0.05). In addition, PEG/DMSO treatment has neither harmful nor beneficial effects on the cleavage and development of the blastocyst stage of reconstructed embryos (p > 0.05). The fusion and cleavage rates of frozen-thawed cells were significantly lower than those of fresh cells (p < 0.05). After 10 blastocysts, derived from fresh PEG/DMSO-treated cells, were transferred to five recipient heifers, one live female calf was obtained. Experiment 3 showed that PEG/DMSO treatment reduced the viability of both fresh and frozen-thawed MGE cells (p < 0.05). We conclude that the PEG/DMSO treatment of fresh MGE cells, as well as the frozen-thawed cells, before electrofusion has a positive effect on the fusion of nuclear transfer without decreasing the in vitro development of reconstructed embryos.
Kishi
Masao
M
Embryo Transplantation Laboratory, Snow Brand Milk Products Co. Ltd., Tomakomai, Hokkaido, Japan. masaok@seagreen.ocn.ne.jp
Itagaki
Yasuharu
Y
Takakura
Ryo
R
Sudo
Tomoko
T
Teranishi
Masatoshi
M
D000818:Animals; D001755:Blastocyst / Q000378:metabolism; D002417:Cattle; D002459:Cell Fusion; D002467:Cell Nucleus / Q000378:metabolism*; D002470:Cell Survival; D019976:Cloning, Organism / Q000379:methods*; D004121:Dimethyl Sulfoxide / Q000494:pharmacology*; D004624:Embryo Transfer; D004847:Epithelial Cells / Q000166:cytology*; D005615:Freezing; D006863:Hydrogen-Ion Concentration; D008321:Mammary Glands, Animal / Q000166:cytology*; D009584:Nitrogen / Q000494:pharmacology; D009865:Oocytes / Q000378:metabolism; D011092:Polyethylene Glycols / Q000494:pharmacology*; D012997:Solvents / Q000494:pharmacology; D013997:Time Factors
D012997:Solvents; D011092:Polyethylene Glycols; D009584:Nitrogen; D004121:Dimethyl Sulfoxide