Nuclear Transfer of Inner Cell Mass Cells and Fetal Germ Cells at Different Cell Cycles into Enucleated Zygotes at the M Phase in the Mouse

Abstract

An attempt was made to use the zygotes arrested at the M phase of the cell cycle as the recipient cytoplasm for nuclear transfer. i) Zygotes which were collected 24, 28 and 32 h after hCG injection were incubated in M16 + nocodazole (3 μl/ml) for 10 to 14 h. After elimination of the drug, they were cultured in vitro for 4 days. ii) Blastocysts were cultured with nocodazole for 5 to 12 h and the mitotic index was examined. iii)M phase and G₁/S phase inner cell mass (ICM) cells synchronized with nocodazole and aphidicolin treatment, and fetal germ cells isolated from fetuses on days 11.5 (in mitosis) and 15.5 (G₀ phase) of pregnancy were fused with enucleated M phase zygotes. The results were as follows. i) Zygotes obtained 24 h after hCG injection and treated with nocodazole for 10 to 14 h were at the M phase. After release from the drug, they developed to blastocysts at a rate similar to that of non-treated zygotes (83∼87% vs 100%). ii) Mitotic index of blastocysts treated with nocodazole for 11 to 12 h was 94%. iii) Nuclear transfer into M phase zygotes did not improve the developmental ability of reconstituted embryos into blastocysts. In the present study, it was clear that mouse zygotes arrested at the M phase with nocodazole treatment, and after washing they developed to the blastocysts at the similar rate to non-treated zygotes. But, the developmental ability of reconstituted zygotes at the M phase did not support in vitro development.