Effects of cell-cycle-arrest agents on cleavage and development of mouse embryos

Abstract

In mammals, there are no reliable methods for synchronizing cell division of early embryos without reducing their ability to develop into blastocysts and fetuses. The present study was undertaken to examine the in vitro inhibition of cell division of four‐cell mouse embryos by cell cycle arrest agents. The reversibility of the agents was also tested by examining the developmental ability of treated embryos. Four‐cell mouse embryos obtained at 54 hr post‐human chorionic gonadotrophin (post‐hCG) were cultured for 4, 8, 12, or 16 hr in media supplemented with either nocodazole, an inhibitor of tubulin polymerization, 6‐dimethylaminopurine (6‐DMAP), an inhibitor of maturation promoting factor (MPF) activation, or aphidicolin, a specific inhibitor of DNA polymerase alpha. Reversibility and toxicity of these agents were both dose and time dependent. For all three agents, prolonging cleavage arrest for 8 or 16 hr (at the effective concentrations) caused embryo lethality. Although nocodazole treatment was least cytotoxic, 6‐DMAP and aphidicolin concentrations which induce cleavage arrest were detrimental to development beyond the blastocyst stage. The results of this study show that the development of embryos treated with these three cell‐cycle‐arrest agents is dose and incubation time dependent. Toxic effects beyond the blastocyst stage could only be minimized for nocodazole by reducing the exposure time of treatment and concentration of the mitotic inhibitor. However, these results render doubt on the usefulness of 6‐DMAP and aphidicolin for synchronization studies leading to embryo transfer procedures. © 1996 Wiley‐Liss, Inc.

Characterisation of the dynamic behaviour of lipid droplets in the early mouse embryo using adaptive harmonic generation microscopy

“…As expected, both nocodazole and cytochalasin D treated embryos failed to form blastocysts and the latter underwent decompaction [28-30]. However there was no difference in the distribution of LD sizes in either nocodazole or cytochalasin D treated embryos when compared to control (DMSO) treated embryos (Figure.…”

“…5B-D). We verified the activity of nocodazole by visualising chromosomes, that arrest at metaphase as expected [28] (Figure. 5E,E' and additional file 10: M8-noc-control.mov).…”

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

“…Nocodazole has been proven to have the least side effects on mouse embryo development compared with other commonly used cell-cycle arrest agents, such as 6dimethylaminopurine (6-DMAP) and aphidicolin [20]. The treatment concentration and time of nocodazole varies significantly between the embryo and the somatic cells [21].…”

Mouse <i>in vivo</i>-derived late 2-cell embryos have higher developmental competence after high osmolality vitrification and −80°C preservation than IVF or ICSI embryos

“…Early 2-cell embryos or late 2-cell embryos collected from oviducts were cultured in CZB medium with 3 mg/ml aphidicolin (FUJIFILM Wako Pure Chemical Corporation, Osaka, Japan), a specific inhibitor of replicative DNA polymerases [ 19 ]. The next morning (1000 h), the developmental stage of the treated embryos was evaluated.…”

“…We hypothesized that the cell cycle of embryos may affect the tolerance to vitrification, particularly in the S-phase. Therefore, we examined the stage of the cell cycle in early and late 2-cell embryos using aphidicolin, in which cell division was prevented during the S-phase by inhibiting DNA polymerization [19]. As shown in Table 6, of the early 2cell embryos treated with aphidicolin, 16 of 48 (33%) stopped at the 2-cell stage.…”