LAUSR

Abstract The zona pellucida plays a crucial role in the process of fertilization to early embryonic development, including cellular arrangement and communication between blastomeres. However, little is known regarding the role of the zona pellucida in pre- and post-implantation embryonic development associated with gene expression. We investigated the effect of zona pellucida removal on pre- and post-implantation development of mouse embryos. After zona pellucida removal of two-cell stage embryos was performed by acid Tyrode's solution, which is commonly used for zona pellucida treatment, compaction occurred earlier in zona pellucida-free than zona pellucida-intact embryos. In addition, the expression of differentiation-related genes in the inner cell mass and trophectoderm was significantly altered in zona pellucida-free blastocyst compared with zona pellucida-intact embryos. After embryo transfer, the rate of implantation and live fetuses was lower in zona pellucida-free embryos than in control embryos, whereas the fetal weight at E17.5 was not different. However, placental weight significantly increased in zona pellucida-free embryos. RNA-sequencing analysis of the placenta showed that a total of 473 differentially expressed genes significantly influenced the biological process. The present study suggests that zona pellucida removal by acid Tyrode's solution at the two-cell stage not only disturbs the expression pattern of inner cell mass-/trophectoderm-related genes but affects the post-implantation development of mouse embryos. Overall, this study provides deeper insight into the role of the zona pellucida during early embryonic development and the viability of post-implantation development. Summary Sentence Zona pellucida removal by acid Tyrode's solution affects the inner cell mass/trophectoderm differentiation; a lower fetal rate, higher placental weight, and a total of 473 differentially expressed genes during post-implantation development in mice. Graphical Abstract