Transfer of nuclei from 8-cell stage mouse embryos following use of nocodazole to control the cell cycle

Mouse 2-, 4-, 8-, and 16-cell embryos were exposed to nocodazole in M16 culture medium. The effect of different concentrations and exposure times on the efficiency of cell cycle synchronization and the development of the treated embyros after release from the drug was determined. The minimum effective concentration (>95% of arrested nuclei) for 4-, 8-, and 16-cell embryos was 5 mu M nocodazole. The effect upon subsequent development of mouse embryos depended upon both the stage of development of the embryo at treatment (P <0.001) and the length of exposure to nocodazole (P <0.001). Exposure to any concentration of nocodazole within the range 2.5-10 mu M for 12 hr caused a reduction in the proportion of embryos that formed blastocysts. As the period of exposure to 5 mu M nocodazole increased from 12 to 24 hr, the proportion of embryos developing to the blastocyst stage decreased. The lower proportion of embyros developing to the blastocyst stage and to term (P <0.01) suggests that the more advanced stages were more susceptible to damage as a result of exposure to nocodazole. The rate of development of 4-cell embryos to blastocysts was not affected when an exposure time of 9 hr was used. Together these results show that it is possible to use nocodazole to arrest mouse embryonic cells in mitosis but that it is not appropriate to culture the embryos in the presence of this drug for prolonged periods. Individual blastomeres completed mitosis at 60-90 min and started DNA synthesis at 120-150 min after release from nocodazole. Nuclei from blastomeres thus synchronized were used to conduct studies on the effect of the cell cycle on nuclear transfer. A signficant effect was found. When nuclei from 8-cell embryos in G1 or S-phase were used as nuclei donors, development to blastocyst was respectively 27% and none. (C) 1994 Wiley-Liss, Inc.