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

Abstract

Incorporation of [3H]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 [3H]thymidine increased with development. Embryos at the 4-cell stage were not labelled when the concentration of [3H]thymidine was lower than 5 μCi/ml, whereas the nuclei of embryos at morula and blastocyst stages began to show silver grains at a concentration of 0.1 μCi/ml of [3H]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 ≤ 1 h, 22% had a G1-phase of ≤ 2 h, 22% had a G1-phase of ≤ 3 h and 13% had a G1-phase of ≤ 4 h. The G1-phase in 85% of the 4-cell embryos was ≤ 3 h, that in 8% of embryos was ≤ 4 h and that in 7% of embryos was ≤ 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 μM, and the appropriate concentrations which did not impair development were 0.05-0.5 μM.

G9a co-localized with histone H3 lysine 9 monomethylation but not dimethylation in a nuclear membrane-dependent manner during mouse preimplantation embryo development

“…One study has demonstrated that high concentration and long-term nocodazole treatment Control indicates the untreated 8-cell embryos; nocodazole indicates the 8-cell embryo treated with nocodazole (0.5 μM) for 12 h; nocodazole-2 indicates 8-cell embryo treated with nocodazole (0.5 μM) for 12 h and then cultured in nocodazole-free KSOM medium for another 30 min resulted in chromosomal abnormality or even embryo-lethal mutants [29]. A 0.05-0.5 μM nocodazole treatment blocked the cell cycle, but it did not damage embryonic development [30]. Therefore, 0.5 μM nocodazole was selected in this study to treat 8-cell mouse embryos.…”

Glycogen synthase kinase‐3 regulation of chromatin segregation and cytokinesis in mouse preimplantation embryos

“…Each cell, or blastomere, in two-cell embryos contains a complete complement of chromosomes. Disruption of microtubule function during oocyte maturation or fertilization leads to aberrations in embryo chromosomal ploidy (Generoso et al, 1989;Yu et al, 2002;Sun et al, 2005). Although oocyte-derived mRNA and protein products contribute to the first few embryonic divisions, mouse embryos undergoes major zygotic genomic activation (ZGA), during the two-cell stage, to sustain early preimplantation development (Haraguchi et al, 1998).…”