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Effects of cell-cycle-arrest agents on cleavage and development of mouse embryos
1Centre de Recherche en Reproduction Animale, Université de Montréal, Québec, Canada.
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.
Insights
Researchers investigated cell cycle arrest agents for synchronizing early mammalian embryo division. While nocodazole showed minimal toxicity, 6-DMAP and aphidicolin proved detrimental to embryo development, limiting their use in synchronization studies.
Area of Science:
- Reproductive Biology
- Developmental Biology
- Cell Biology
Background:
- Synchronizing early mammalian embryo cell division is crucial for developmental studies and assisted reproduction.
- Current methods often compromise embryo viability and developmental potential.
- Reliable synchronization techniques are needed to improve outcomes in embryo transfer procedures.
Purpose of the Study:
- To evaluate the efficacy and reversibility of cell cycle arrest agents in synchronizing four-cell mouse embryo division in vitro.
- To assess the impact of these agents on subsequent embryo development to the blastocyst stage and beyond.
- To determine the dose- and time-dependency of toxicity for selected cell cycle arrest agents.
Main Methods:
- Four-cell mouse embryos were cultured in media containing nocodazole, 6-dimethylaminopurine (6-DMAP), or aphidicolin for varying durations (4, 8, 12, 16 hours).
- Embryos were assessed for cleavage arrest, reversibility of arrest, and developmental progression to the blastocyst stage.
- Toxicity and developmental capacity following treatment were evaluated based on dose and incubation time.
Main Results:
- All three agents (nocodazole, 6-DMAP, aphidicolin) induced cell cycle arrest in a dose- and time-dependent manner.
- Prolonged exposure (8-16 hours) to effective concentrations of any agent resulted in embryo lethality.
- Nocodazole exhibited the least cytotoxicity, but high concentrations or prolonged exposure remained detrimental.
- 6-DMAP and aphidicolin at concentrations causing arrest were toxic, significantly impairing development beyond the blastocyst stage.
- Minimizing toxic effects of nocodazole required reduced exposure time and concentration.
Conclusions:
- The use of 6-DMAP and aphidicolin for embryo synchronization in procedures like embryo transfer is questionable due to their significant toxicity.
- Nocodazole offers potential for embryo synchronization, but careful optimization of concentration and exposure time is necessary to mitigate adverse effects.
- Developing safe and effective methods for early embryo cell cycle synchronization remains a significant challenge in mammalian reproductive biology.