Monitoring of autophagic activity in the developing mouse preimplantation embryo

Background: In most animals, maternal factors including mRNA, protein, and organelles that stored during oogenesis could be rapidly degraded and newly zygotic products are generated after fertilization. Therefore, autophagy, which is cytoplasmic degradation pathway mediated by the lysosome, could be ideal way to eliminate these maternal factors and recycle them to synthesize newly products. The aim of this study is to image the autophagic activity in developing mouse embryo.Observations: To monitor autophagic actvity, we microinjected mRNA encoding GFP-LC3, which is autophagosome-marker, into 1-cell embryos and observed its fluorescence under live cell condition. We found that the fluorescence level of GFP-LC3 was constantly high at the 2-cell stages but significantly decreased between 4- to 8- cell stages. We confirmed that GFP-LC3 degradation was completely blocked by the treatment of bafilomycin A1, a specific inhibitor of vacuolar H+ ATPase (V-ATPase), which caused the increase of lysosomal pH, indicating that GFP-LC3 degradation is dependent on both autophagic activation and lysosomal acidification. Using our monitoring system, we also investigated whether autophagic activity decreased in aged embryos and found that the level of GFP-LC3 degradation decline, probably due to decrease of lysosomal activity.Conclusions: We successfully imaged autophagic activity in developing embryos by monitoring the GFP-LC3 degradation and revealed that autophagic activity decrease during aging. Since autophagy associates with cytoplasmic quality control, our method will be applied to the assessment of embryo viability.The EMBO Meeting 201