Inhibition of neddylation causes early developmental arrestment in mouse embryo because of the zygotic genome activation failure

Abstract

Background Maternal protein degradation in mammalian preimplantation embryos has yet to be fully understood. One major pathway is the ubiquitin-proteasome system. Recently, new forms of ubiquitination such as neddylation have been found to play important roles in a wide variety of biological processes, including reproduction. However, the molecular mechanism of neddylation in the early embryonic development of mammals is mostly unknown. Methods The zygotes were collected through in vitro fertilization and the expression of marker genes during embryonic development and zygotic genome activation (ZGA) was monitored after 24,48,72,96 hours of culture with MLN4924 (specific inhibition of neddylation) using real-time quantitative PCR. Single-cell RNA sequencing and quantitative PCR were applied to monitor and validate the changes in the downstream transcriptome. We utilized immunofluorescence and Western blotting to detect the expression and localization of proteins in mouse embryos. Results Blocking neddylation in mouse zygotes led to a statistically significant decrease in the cleavage rate to the 2-cell stage. Transcriptional profiling showed genes differentially expressed in pathways involving cell fate determination, cell differentiation, and cytoskeletal proteins. The expressions of zygotic ZGA markers were significantly reducejiand, indicating a significant downstream alteration in relevant pathways leading to the 2-cell stage arrest phenotype. A decrease in the level of RNA polymerase II in the nucleus was detected, showing impaired gene transcription in the embryo. We also identified a decrease in methyltransferase expression and concomitant reduction in histone H3K4 trimethylation, which may be the molecular mechanism of early embryonic developmental arrest caused by neddylation inhibition. Reduction of Yap1 was detected, suggesting aberrant downstream reactions of the Hippo signaling pathway. It also addressed the problem of the neddylation inhibition caused early embryonic arrest. Our study shed light upon new forms of ubiquitination regulating mammalian embryonic development and may contribute to further investigation of female infertility pathology. Conclusions Our data suggest that blocking neddylation leads to ZGA failure, possibly due to a decrease in H3K4me3 caused by a decrease in methyltransferase (KMT2D).