(78) Dynamics of the m6A Modification on the Bovine Oocytes and Early Embryo

Rajan Iyyappan¹, Yichi Niu², Chenghang Zong², Zongliang Jiang^1*

 ¹Department of Animal Sciences, Genetics Institute, University of Florida, Gainesville, FL, USA; ²Department of Molecular and Human Genetics, Baylor College of Medicine, Houston, TX, USA

Abstract Text:

Mammalian pre-implantation embryonic development is imitated by massive degradation of oocyte-stored maternal RNA/proteins and gradual activation of the embryonic genome. However, the precise mechanisms to regulate this process are not well understood. N6-methyladenosine (m6A) found in eukaryotic RNA plays key regulatory roles in gene regulation, however, mapping m6A in oocytes and early embryos using m6A antibody based methyl RNA immunoprecipitation and sequencing has been challenging due to the requirement for large amounts of materials and its inability for the genome-wide single base resolution detection.

In this study, we utilized a newly developed m6A-selective allyl chemical labeling and sequencing (m6A-SAC-seq) approach to define the landscape of m6A methylation in bovine oocytes and pre-implantation embryos at single-base resolution. Approximately 200 oocytes/embryos per replicate (n=2) was used for RNA extraction and subsequently m6A-SAC-seq library preparation. We have sequenced approximately 100 million pair-end reads per sample (60 million per SAC sample and 40 million per Input control sample) to ensure the m6A coverage. By sequencing RNA from bovine oocytes (GV and MII) and pre-implantation embryos at 2-, 4-, 8-,16-cell, morula and blastocyst stages, we identified dynamic changes in m6A abundance. Quantitative analysis of genome-wide m6A enrichment across developmental stages revealed a biphasic pattern: an initial reduction in global m6A levels from MII oocyte to the 2-cell stage, followed by an increase from the 2-cell stage to 4-cell stage then progressive reduction until morula stage. This suggests that m6A methylation may be selectively erased during maternal mRNA degradation and re-established upon EGA, potentially influencing transcript stability and translation efficiency. Integration of m6A epitranscriptomic data with previously published transcriptome and translatome datasets confirmed that m6A modification selectively influences translational regulation during oocyte maturation and early embryonic development. Furthermore, we observed significant dynamic changes of m6A site distribution in key regulatory genes in response to developmental transitions, including ribosomal protein genes. Notably, we identified a specific site in RPL12 mRNA to be highly methylated at the EGA stage, 8-cells. Using a CRISPR base editor to remove this single m6A site, we found that nearly 90% of embryos are arrested at an early stage and fail to progress to the blastocyst stage. This finding highlights the critical role of m6A modification in ensuring proper function of key regulators during early embryonic development.

In conclusion, we present the first m6A landscapes in bovine oocytes and pre-implantation embryos. Our results provide a foundation and insights for m6A mediated complex epigenetic reprogramming in early mammalian embryonic development.