PSXI-14 Characterization of lineage-specific small non-coding RNA expression between embryonic layers of porcine conceptuses during peri-implantation development.

Abstract The in vitro culture system is a powerful tool to study pre-implantation development of embryos. In contrast, studying the peri-implantation period is limited because of the lack of suitable in vitro models reflecting embryonic changes in vivo. Consequently, less is known about molecular cues governing conceptus elongation, attachment, and placentation during this stage, especially in large animal models such as pigs. Previous works have highlighted the role of small non-coding RNA (sncRNA) during peri-implantation. They primarily function to induce post-transcriptional gene silencing but appear to be critical for embryo-maternal communication. Global changes in embryonic sncRNA expression occur throughout this period and are implicated to regulate a variety of cellular functions. However, differences in sncRNA between embryonic lineages have not been investigated. In this study, we performed sncRNA sequencing to characterize lineage-specific sncRNA expression profiles during the period of conceptus attachment in pigs. Multiparous sows were artificially inseminated with semen from a boar constitutively expressing GFP. Conceptuses were collected 14 and 16 days after artificial insemination. Transverse dissections were made to separate the embryonic layers: inner cell mass (ICM), extraembryonic mesoderm (MESO), and trophectoderm (TE). A portion of TE was used to sex the conceptuses through genomic PCR. Total RNA was extracted (n=8) and NEBNext Small RNA Library Prep Set was used for cDNA library preparation. Sequencing was performed following standard Illumina procedures. Raw data was demultiplexed, cleaned, and trimmed then mapped to Sscrofa11.1 genome assembly using STAR. An average 7,859,877 reads passed quality filtering per sample with 62% being uniquely mapped and 30% being multi-mapped to reference genome. Raw count matrix was generated followed by differential expression analysis with DESeq2. Differences in gene expression were assessed using likelihood ratio test or Wald test for pairwise comparisons with significance determined by genes with Padj< 0.05 or |log2fc| >1, respectively. On day 14, we found 970 differentially expressed sncRNA between the embryonic layers. At least 44 sncRNAs were most abundant in ICM, 3 are most abundant in MESO, and 134 are abundant in TE. On day 16, we found 869 differentially expressed sncRNA between ICM and TE. Several sncRNAs were exclusive to ICM during these days, such as ssc-mir-105-1, MIR135B, and MIR219A2, etc. Others were exclusive to TE but only ssc-mir-129b has been annotated. We identified 4,221 differentially expressed sncRNA between conceptuses from day 14 and day 16. As the conceptus aged, sncRNAs such as ssc-mir-217-1, ssc-mir-216-1, ssc-mir-181a-1, etc. dramatically increased in expression while ssc-mir-1224, ssc-mir-582, etc. decreased. This study identifies lineage-specific sncRNA profiles during the peri-implantation period, and the sncRNA data sets are being analyzed to identify genes that are regulated by the sncRNAs. This work is supported in part by USDA-NIFA 2022-67015-36299.