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Cell type-specific endometrial transcriptome changes in comparison of equine endometrium samples collected on Day 12 of pregnancy and Day 12 of the estrous cycle

ABSTRACT: The results of endometrial gene expression studies during the time of conceptus migration (Days 8 to 16) did not provide final conclusions on possible mechanisms of maternal recognition of pregnancy (MRP) in the mare. This finding called for the analysis of cell type-specific gene expression in the endometrium in response to the equine embryo, which could be the key for improved understanding of gene expression regulation in context of MRP. In a pilot study, we used laser capture microdissection (LCM) to collect samples of the luminal epithelium (LE), glandular epithelium (GE), and stroma from endometrial biopsies taken from five mares on Day 12 of pregnancy and Day 12 of the estrous cycle, respectively. RNA was isolated from the LCM samples in order to perform RNA sequencing. Data analysis showed that gene expression differences were greater between cell types than between pregnant and cyclic state. Differential gene expression between pregnant and cyclic state was mainly found in the LE but also in GE and stroma. The comparison with a previous RNA-Seq data set for whole biopsy samples derived from Day 12 of pregnancy revealed the specific origin of the observed gene expression differences. Furthermore, we found that a number of genes are specifically differentially expressed (DE) in only one cell type. Some genes not DE when looking at the whole biopsy were DE in one cell type and showed similar levels in the other cell types in comparison of pregnancy and cyclic stage. Overall, this study revealed new and important spatial information about endometrial gene expression during the phase of initial recognition of pregnancy in the mare. The results indicated induction of angiogenesis and blood vessel development by the presence of the conceptus, a specific spatial regulation of the immune system, spatial regulation of growth factors controlling cell differentiation and endometrial remodeling, and regulation of genes involved in production of prostaglandin (PG) precursors, PG transport, and PG receptors, specifically PTGFR in context of prevention of luteolysis. Furthermore, these results support our hypothesis of the existence of a specific response in different endometrial cell types in the mare and call for further studies.

ORGANISM(S): Equus caballus

PROVIDER: GSE112236 | GEO | 2018/09/30

REPOSITORIES: GEO

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Project description:The porcine conceptus undergoes rapid differentiation and expansion of its trophoblastic membranes between Days 11 and 12 of gestation. Concomitant with trophoblast elongation, production of conceptus estrogen, the porcine embryonic pregnancy recognition signal increases. Conceptus attachment to the uterine surface epithelium starts after Day 13 initiating epitheliochorial placentation. To analyze the transcriptome changes in the endometrium in the course of maternal recognition of pregnancy (MRP), deep sequencing of endometrial RNA samples of Day 12 pregnant animals (n=4) and corresponding non-pregnant controls (n=4) was performed using Illumina RNA-Seq. Between 30 and 35 million sequence reads per sample were produced and mapped to the porcine genome (Sscrofa10.2). Analysis of read counts revealed 2,593 differentially expressed genes (DEG). Expression of selected genes was validated by the use of quantitative real-time RT-PCR. Bioinformatics analysis identified several functional terms specifically overrepresented for up-regulated or down-regulated genes. Comparison of the RNA-Seq data from Days 12 and 14 of pregnancy was performed at the level of all expressed genes, of the DEG, and at the level of functional categories. This revealed specific gene expression patterns, reflecting the different functions of the endometrium during these stages, i.e. recognition of pregnancy and preparation for conceptus attachment. Genes related to mitosis, immune response, epithelial cell differentiation and development, proteolysis, and prostaglandin signaling and metabolism are discussed in detail. In conclusion, this study identified comprehensive transcriptome changes in porcine endometrium associated with MRP and initiation of implantation and a number of genes and pathways potentially involved in regulation of these processes. In total, 8 samples were analyzed, 4 biological replicates for pregnant animals (samples from 4 different animals) and 4 biological replicates for cyclic controls (samples from 4 different animals)

Project description:Interferon tau (IFNT), a Type I IFN similar to alpha IFNs (IFNA), is the pregnancy recognition signal, produced by the ruminant conceptus. To elucidate specific effects of bovine IFNT and of other conceptus-derived factors, endometrial gene expression changes during early pregnancy were compared to gene expression changes after intrauterine application of human IFNA2. In study one, endometrial tissue samples were obtained on days (D) 12, 15, and 18 post-mating from nonpregnant or pregnant heifers. In study two, heifers were treated from D14 to D16 of the estrous cycle with an intrauterine device releasing IFNA2 or placebo lipid extrudates or PBS only as controls. Endometrial biopsies were collected after flushing the uterus. All samples from both experiments were analyzed with an Affymetrix Bovine Genome Array. Study one revealed differential gene expression between pregnant and nonpregnant endometria on D15 and D18. In study two, IFNA2 treatment resulted in differential gene expression in the bovine endometrium. Comparison of the datasets from both studies identified genes that were differentially expressed in response to IFNA2 but not in response to pregnancy on D15 or D18. Vice versa, genes were found as differentially expressed during pregnancy but not after IFNA2 treatment. In study three, spatiotemporal alterations in expression of selected genes were determined in uteri from nonpregnant and early pregnant heifers using in situ hybridization. The findings of this study suggest differential effects of bovine IFNT compared to human IFNA2 and that some pregnancy-specific changes in the endometrium are elicited by conceptus-derived factors other than IFNT. Study I: Early pregnancy; day 12 of pregnancy (n=5 heifers), day 15 of pregnancy (n=3), day 18 of pregnancy (n=4), day 12 cyclic controls (n=5), day 15 cyclic controls (n=3), day 18 cyclic controls (n=4). Study II: Treatment with human interferon alpha (IFNA); IFNA treatment group (IFNA, n=3 heifers), placebo group (PLAC, n=3 heifers), control group (CONT, n=3 heifers).

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Cell type-specific endometrial transcriptome changes in comparison of equine endometrium samples collected on Day 12 of pregnancy and Day 12 of the estrous cycle

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