Abstract
BackgroundDuring 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.ResultsLow-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’.ConclusionsThe 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.
Highlights
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
Osteopontin (OPN; known as SPP1) is a secreted extracellular matrix (ECM) protein that can bind with various integrins on the cell surface, and SPP1 has been identified as a candidate adhesion molecule for implantation in pigs and sheep [14]
The comparison of laser capture microdissection (LCM) samples and complete endometrium showed that the majority of the detectable genes (9429) could be identified in all four individual cell types as well as in the complete tissue (Upset plot, Fig. 1a)
Summary
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 surface area is increased by the presence of endometrial folds, surface epithelial folds, and microvilli between the trophoblast and dome-shaped luminal epithelium (LE) cells that are coated by a thick glycocalyx [7, 8]. Several primary molecules, such as mucins, integrins and CDs, have been shown in regulation of various cell adhesion cascades for the embryo implantation in pigs [9,10,11,12]. A related study about ITGAV in porcine trophoblast showed that ITGAV-containing integrin receptors adhere to SPP1, suggesting that mechanical forces generated by elongating conceptuses to uterine LE leads to the assembly of focal adhesions involving ITGAV and SPP1 [10]
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