56 Spatial analysis of transcriptome changes in porcine endometrium on Day 14 of pregnancy

Abstract

During the conception cycle, the embryo undergoes a series of developmental processes including cell division, cellular reorganization, and oestrogen secretion before attaching to the uterine epithelium. The uterine endometrium is complex and consists of various layers and cell types [i.e. luminal epithelium (LE), glandular epithelium (GE), blood cells (B), and stromal areas (S)]. The objective of this study was to characterise the complex transcriptome changes in porcine endometrium during the time of conceptus attachment with respect to localization in different endometrial cell types. RNA-sequencing (RNA-Seq) was conducted for LE, GE, B, and S samples isolated from endometrial tissue collected on Day 14 of pregnancy and the oestrous cycle, respectively (each group n = 4), by laser capture microdissection (PALM LCM microscope, Zeiss, Jena, Germany). Total RNA was isolated (RNA integrity number > 6.5) and used for the preparation of 32 RNA-seq libraries (Ovation SoLo RNA-Seq System, NuGEN Technologies, San Carlos, CA, USA). Multiplexed (barcode-tagged) libraries were run on an Illumina HiSEqn 2500 (Illumina, San Diego, CA, USA). The obtained sequence data were analysed with a RNA-Seq data analysis pipeline on a local Galaxy server installation. The resulting read counts were used for statistical analysis in EdgeR to identify differentially expressed genes (DEG). Furthermore, an RNA-seq dataset for complete Day 14 endometrial tissue samples from a previous study was analysed using the same pipeline. A total of 14 297 genes were detectable in complete endometria, and 12 000, 11 903, 11 094, and 11 933 genes in LE, GE, B, and S, respectively. Differential expression analysis was performed between the pregnant and the cyclic nonpregnant group for each cell type and the complete tissue. The highest number of DEG was found for LE (1410) when compared with GE, B, and S (800, 1216, and 384, respectively). In total, 3262 DEG were obtained for the complete tissue between pregnant and nonpregnant gilts. The DEG were assigned to Gene Ontology (GO) terms to characterise overrepresented functional categories and pathways specific for the individual endometrial compartments. The GO classification revealed that most DEG in LE were involved in cell communication, such as ‘extracellular exosome’, ‘extracellular vesicle’, ‘homeostatic process’, whereas the ‘response to organic substance’ and ‘regulation of cell migration’ categories were enriched in GE. In blood vessels, categories such as ‘membrane-bounded vesicle’, ‘cell junction’, ‘cell development’, ‘cell adhesion’ and ‘blood vessel morphogenesis’ were found as overrepresented, whereas in stromal regions, most DEG were assigned to ‘cell communication’ and ‘secretion’. These results confirmed the hypothesis that conceptus signals induce specific transcriptomic regulations in the endometrial compartments/cell types related to their functions during recognition of pregnancy adding a new level of spatial gene expression regulation to endometrial transcriptome analysis.