Regulation of autophagy and its role in late preimplantation during mouse embryo development

Autophagy is a system that contributes to cellular homeostasis by degrading intracellular proteins and organelles. Autophagy is essential for the preimplantation development of mammalian embryos, lack of which results in developmental arrest at the 4/8-cell stages. The role of autophagy beyond the compaction stage remains insufficiently explored. In this study, we investigated the role of autophagy after the 4/8-cell stages in mice using chloroquine (CQ), an autophagy inhibitor. CQ treatment from the 4/8-cell to morula stage impaired development, reducing the number of Cdx2-positive cells, an effect rescued by amino acid (AA) supplementation. CQ treatment also downregulated TFAP2C, an upstream regulator of Cdx2,which was similarly restored by AA supplementation. Consistently, autophagy at this stage showed higher activity in the outer cells and lower activity in the inner cells of the embryo. Treatment with XMU-MP-1, an MST1/2 inhibitor targeting the Hippo signaling pathway, disrupted this spatial regulation by inducing autophagy in the inner cells. Stage-specific staining revealed temporal and positional regulation of autophagy activity. These findings illustrate that autophagy during the morula stage promotes differentiation into the trophectoderm by supplying AAs, a process regulated by the Hippo signaling pathway.