Spatial organization of endometrial gene expression at the onset of embryo attachment in pigs

Abstract

Background: During the preimplantation phase in the pig, the conceptus trophoblast elongates into a filamentous form and secretes estrogens, interleukin 1 beta 2, interferons, and other signaling molecules before attaching to the uterine epithelium. The processes in the uterine endometrium in response to conceptus signaling are complex. Thus, the objective of this study was to characterize transcriptome changes in porcine endometrium during the time of conceptus attachment considering the specific localization in different endometrial cell types.

Results: Low-input RNA-sequencing was conducted for the main endometrial compartments, luminal epithelium (LE), glandular epithelium (GE), blood vessels (BV), and stroma. Samples were isolated from endometria collected on Day 14 of pregnancy and the estrous cycle (each group n = 4) by laser capture microdissection. The expression of 12,000, 11,903, 11,094, and 11,933 genes was detectable in LE, GE, BV, and stroma, respectively. Differential expression analysis was performed between the pregnant and cyclic group for each cell type as well as for a corresponding dataset for complete endometrium tissue samples. The highest number of differentially expressed genes (DEGs) was found for LE (1410) compared to GE, BV, and stroma (800, 1216, and 384). For the complete tissue, 3262 DEGs were obtained. The DEGs were assigned to Gene Ontology (GO) terms to find overrepresented functional categories and pathways specific for the individual endometrial compartments. GO classification revealed that DEGs in LE were involved in 'biosynthetic processes', 'related to ion transport', and 'apoptotic processes', whereas 'cell migration', 'cell growth', 'signaling', and 'metabolic/biosynthetic processes' categories were enriched for GE. For blood vessels, categories such as 'focal adhesion', 'actin cytoskeleton', 'cell junction', 'cell differentiation and development' were found as overrepresented, while for stromal samples, most DEGs were assigned to 'extracellular matrix', 'gap junction', and 'ER to Golgi vesicles'.

Conclusions: The localization of differential gene expression to different endometrial cell types provided a significantly improved view on the regulation of biological processes involved in conceptus implantation, such as the control of uterine fluid secretion, trophoblast attachment, growth regulation by Wnt signaling and other signaling pathways, as well as the modulation of the maternal immune system.

Keywords: Cell type-specific; Endometrium; LCM; LMD; Pig; Preimplantation; RNA-seq; Transcriptomics.

Fig. 1

Numbers and overlaps of detectable genes (a) and differentially expressed genes (DEGs) (b) for the 4 LCM sample types and complete tissue samples illustrated using Upset plots. On the left side, the total numbers of detectable genes and the DEGs, respectively, are shown for complete tissue samples (green), luminal epithelium (red, LE), stromal cells (yellow, S), glandular epithelium (orange, GE), and blood vessel (blue, BV). The colored dots indicate the number of genes specifically detectable (a) or specific DEGs (b) for the corresponding sample type. Numbers with black dots show the numbers of genes commonly expressed (a) or differential (b) in different sample types

Fig. 2

Unsupervised clustering of endometrial LCM samples. Multidimensional scaling plots were generated in EdgeR for the genes showing the highest leading log-fold-changes between the samples in the dataset for LCM samples. Sample groups: CL (orange): cyclic, luminal epithelium; PL (dodgerblue): pregnant, luminal epithelium; CG (red): cyclic, glandular epithelium; PG (blue): pregnant, glandular epithelium; CB (purple): cyclic, blood vessels; PB (darkblue): pregnant, blood vessels; CS (brown): cyclic, stroma; PS (cyan): pregnant, stroma. a,b all LCM samples based on the 2000 genes with highest leading log-fold-changes (a) and on all detectable genes (b). c luminal epithelium samples. d glandular epithelium samples. e blood vessel samples. f stroma samples. c-f MDS plots based on the 500 genes with highest leading log-fold-changes. Red and Blue indicate samples from pregnant and cyclic groups, respectively

Fig. 3

Comparative DAVID Gene Ontology chart analysis. Overrepresentation of the most significantly overrepresented functional categories of each LCM sample type (LE: luminal epithelium, GE: glandular epithelium, BV: blood vessel, S: stroma, All: overrepresented in all sample types) was compared. Categories were filtered manually for redundancy. The word clouds on the left side indicate the main functional categories/terms for the DEGs obtained for the respective endometrial compartments. Characteristic terms and words of the overrepresented categories were used to generate word clouds where the font size indicates the frequency of the word or term. The heatmap shows a score combining fold enrichment and false discovery rate (blue = lowest score, red = score of 7 or higher). For details of the DAVID GO chart analysis see Additional file 7: Table S4

Fig. 4

Heatmap of the top 10 upregulated and downregulated genes identified for each sample type in comparison of samples derived from pregnant and cyclic gilts. The color scale indicates log2 fold change from −7 to 7 (corresponding to a linear fold change of 128). Each column represents one LCM sample type or complete tissue (luminal epithelium (LE), glandular epithelium (GE), blood vessel (BV), stromal cells (S), and complete tissue). Detailed information can be found in Additional file 8: Table S5

Fig. 5

Summary of the main findings of the study. This schematic overview is based on the results of the present study of endometrial localization of differential gene expression. Genes highlighted in red and blue color were found as up- and downregulated, respectively when comparing pregnant to nonpregnant cyclic stage