Molecular study of mouse peri-implantation development using the in vitro culture of aggregated inner cell mass.

Abstract

To study the mechanisms of mouse peri-implantation development, we explored the in vitro culture of the isolated inner cell mass (ICM) of the blastocyst. As previously reported, individually cultured ICM recapitulated several early embryological events, such as the formation of primitive endoderm, epiblast, and proamniotic cavity. However, we found that the timing and efficiency of these morphogenetic processes significantly varied among the ICM. Due to this unpredictability in developmental potential, individually cultured ICM may be unsuitable for further analysis. By contrast, we found that when five ICM were fused into a single mass, such aggregates (5x ICM) underwent efficient and synchronous morphogenesis. The synchronous nature of 5x ICM development was also demonstrated by the temporal and spatial pattern of apoptotic cell death. TUNEL assay showed that a number of the epiblast cells committed apoptosis in 48 hr of culture, which took place after primitive endoderm differentiation but prior to proamniotic cavity formation. In situ hybridization analysis showed that Oct4 was downregulated and alpha-fetoprotein was upregulated in the primitive endoderm of the cultured 5x ICM. In addition, RT-PCR analysis revealed the expression of various primitive endodermal genes, but not of extraembryonic ectodermal markers in the cultured 5x ICM. Taken together, we propose that the 5x ICM is a useful in vitro tool to study the mechanisms of peri-implantation development of the mouse embryo. Mol. Reprod. Dev. 67: 83-90, 2004.