Determination and synchronisation of G1-phase of the cell cycle in 2-and 4-cell mouse embryos

Incorporation of [H-3]thymidine at different concentrations into mouse embryos at early developmental stages was determined by autoradiography. Methods to synchronise the G1-phase of mouse 2- and 4-cell embryos were also investigated. The results showed that the ability of embryos to incorporate [H-3]thymidine increased with development. Embryos at the 4-cell stage were not labelled when the concentration of [H-3]thymidine was lower than 5 muCi/ml, whereas the nuclei of embryos at morula and blastocyst stages began to show silver grains at a concentration of 0.1 muCi/ml of [H-3]thymidine. After 2- and 4-cell mouse embryos were synchronised at the onset of G1-phase by treatment with low temperature or nocodazole, and DNA synthesis was detected by autoradiography, the duration of G1-phase was estimated. The result showed that 43% of the 2-cell embryos had a G1-phase of S 1 h, 22% had a G1-phase of less than or equal to 2 h, 22% had a G1-phase of less than or equal to 3 h and 13% had a G1-phase of less than or equal to 4 h. The G1-phase in 85% of the 4-cell embryos was less than or equal to 3 h, that in 8% of embryos was less than or equal to 4 h and that in 7% of embryos was less than or equal to 5 h. The toxicity of nocodazole on mouse embryo development was assessed based on both blastocyst formation and the number of blastomeres, and the results indicated that the effect of nocodazole on embryo development and cell cycle block was dose-dependent. The minimum concentration of nocodazole for metaphase block of mouse late 2-cell embryos was 0.05 muM, and the appropriate concentrations which did not impair development were 0.05-0.5 muM.