Comprehensive Solution of Ultrasensitive Proteome Technology (CS-UPT) combined with low-input multi-omics analysis provides new insights and clues for mouse maternal-to-zygotic transition

Single-cell and low-input multi-omics techniques have revolutionized the study of preimplantation embryo development. However, single-cell and low-input proteomics researches are relatively underdeveloped. To get enough depth proteome, hundreds of mouse embryos often need to be collected for each sample.

Maternal-to-zygotic transition (MZT) is a fundamental and conserved process, during which the maternal environment of the oocyte transitions to the zygotic genome driven expression program. N⁶-methyladenosine (m⁶A) is the most prevalent internal modification present in the mRNA across higher eukaryotes. However, the potential regulation role of m⁶A modification on RNA metabolism during MZT remains largely unknown.

Therefore, we developed a Comprehensive Solution of Ultrasensitive Proteome Technology (CS-UPT) for single-cell and low-input mouse oocytes and preimplantation embryos. Both deep coverage and high-throughput routes significantly reduced the starting material and provided a full set of options for investigators" demands.

Using the deep coverage route, we provided a large-scale snapshot of the very early stage of mouse MZT, including almost 5,500 protein groups from 20 mouse oocytes or zygotes for each sample. Moreover, significant protein regulatory networks centered on transcription factors and kinases between the MII oocyte and 1-cell embryo provided rich insights into minor zygotic genome activation.

We also investigated m⁶A dynamics during mouse MZT through low-input multi-omics analysis. Our results showed that m⁶A could be maternally inherited or de novo gained after fertilization. By integrating SLIM-seq, LiRibo-seq, low-input proteomics and RIP-qRT-PCR data, we demonstrated that a group of maternal mRNAs with m⁶A mark were maintained throughout the MZT, and most of these genes were actively translated, indicating a role of m⁶A in safeguarding RNA stability for early embryo development.

All these findings provided new insights and clues for the study of critical biological events during mouse preimplantation development.