Identifying Lineage-Specific Transcripts in Different Embryonic Layers of Porcine Conceptuses During Peri-Implantation Development

Ty Montgomery¹, Mary York², and Kiho Lee^1,3

1. Division of Animal Sciences, University of Missouri, Columbia, MO, USA

2. Bioinformatics and Analytics Core, University of Missouri, Columbia, MO, USA

3. National Swine Resource and Research Center, University of Missouri, Columbia, MO, USA

Abstract Text:

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 proper in vitro models reflecting their changes. Consequently, less is known about molecular cues governing conceptus elongation, attachment, and placentation during this stage. Previous works have highlighted the global changes in the porcine conceptus during peri-implantation stage, and each embryonic lineage has unique expression patterns. For example, the genes SOX2, BMP4, and IFNG are respectively expressed in the inner cell mass, mesoderm, and trophectoderm of porcine conceptuses. However, overall transcriptomic differences between these embryonic lineages have not been elucidated. We hypothesize that dynamic changes occur within the embryonic lineages during peri-implantation development. In this study, we performed RNA-Seq to characterize lineage-specific gene expression profiles during the period of conceptus attachment. 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). An additional dissection of TE was used to sex the conceptuses through genomic amplification of sex-linked gene, DDX3X/Y, through PCR. Samples were flash frozen in PBS. Total RNA was extracted (n=8) and Illumina Stranded mRNA Prep kit 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 36,619,724 reads passed quality filtering per sample with 95% being uniquely 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. Preliminary evaluation confirmed ICM-exclusive expression of NANOG and PRDM14 on day 14 with SOX2 and FGF4 on days 14 and 16. Over the same period, IFNG, COX1/2/3, and several PAG family genes were highly expressed in TE. Lastly, BMP4 was abundant in mesodermal-containing layers. These findings demonstrate that the embryonic layers can be separated and sequenced through physical dissection. On day 14, we found 14,913 differentially expressed genes (DEG) between the embryonic layers. At least 3,073 transcripts are the most abundant in ICM, 743 are most abundant in MESO, and 3,492 are most abundant in TE. On day 16, we found 8,709 DEG between ICM and TE. There are widespread differences in gene family expression between the embryonic lineages. We identified 4,974 DEG between conceptuses from day 14 and day 16. Genes such as COL1A1, HBE1, FETUB, etc. dramatically increased in expression as the conceptuses aged while IFNG, PLET1, ATP6, etc. decreased. There are few differences between male and female conceptuses. At day 14, there were 25 DEG with EIF1AY, USP9Y, KDM5D, and KRT32 being the only autosomal genes. At day 16, there were 5 DEG which are all located on the sex chromosomes. Gene ontology and KEGG pathway analysis are underway. This study identifies lineage-specific transcription profiles during the peri-implantation conceptus growth. This work is supported in part by USDA-NIFA 2022-67015-36299.