Deep Sequencing of the Porcine Endometrial Transcriptome on Day 14 of Pregnancy
TL;DR: RNA-Seq analysis revealed comprehensive transcriptome differences in porcine endometrium between Day 14 of pregnancy and corresponding cyclicendometrium and highlighted new processes and pathways probably involved in regulation of noninvasive implantation in the pig.
Abstract: In pigs, conceptus attachment to the uterine surface epithelium starts around Day 14 of pregnancy preceded by a pronounced vascularization at the implantation zones, initiating the epitheliochorial placentation. To characterize the complex transcriptome changes in the endometrium in the course of initial conceptus attachment, deep sequencing of endometrial RNA samples of pregnant animals (n = 4) and corresponding cyclic controls (n = 4) was performed using Illumina RNA-Seq. The obtained sequence reads were mapped to the porcine genome, and relative expression values were calculated for the analysis of differential gene expression. Statistical analysis revealed 1933 differentially expressed genes (false discovery rate 1%), 1229 with higher and 704 with lower mRNA concentration, in the samples from pregnant animals. Expression of selected genes was validated by the use of quantitative real-time RT-PCR. The RNA-Seq data were compared to results of a microarray study of bovine endometrium on Day 18 of pregnancy and additional related data sets. Bioinformatics analysis revealed for the genes with higher mRNA concentration in pregnant samples strong overrepresentation, particularly for immune-related functional terms but also for apoptosis and cell adhesion. Overrepresented terms for the genes with lower mRNA concentration in pregnant samples were related to extracellular region, ion transport, cell adhesion, and lipid and steroid metabolic process. In conclusion, RNA-Seq analysis revealed comprehensive transcriptome differences in porcine endometrium between Day 14 of pregnancy and corresponding cyclic endometrium and highlighted new processes and pathways probably involved in regulation of noninvasive implantation in the pig.
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TL;DR: The expression of FGF7, NMB, COL5A3, S100A8 and PPP1R3D genes were significantly up-regulated at the time of maternal recognition of pregnancy (Day 12 of pregnancy), which first identified the characterization and expression profile of lncRNAs in pig endometrium during pre-implantation phases.
Abstract: Establishment of implantation in pig is accompanied by a coordinated interaction between the maternal uterine endometrium and conceptus development. We investigated the expression profiles of endometrial tissue on Days 9, 12 and 15 of pregnancy and on Day 12 of non-pregnancy in Yorkshire, and performed a comprehensive analysis of long non-coding RNAs (lncRNAs) in endometrial tissue samples by using RNA sequencing. As a result, 2805 novel lncRNAs, 2,376 (301 lncRNA and 2075 mRNA) differentially expressed genes (DEGs) and 2149 novel transcripts were obtained by pairwise comparison. In agreement with previous reports, lncRNAs shared similar characteristics, such as shorter in length, lower in exon number, lower at expression level and less conserved than protein coding transcripts. Bioinformatics analysis showed that DEGs were involved in protein binding, cellular process, immune system process and enriched in focal adhesion, Jak-STAT, FoxO and MAPK signaling pathway. We also found that lncRNAs TCONS_01729386 and TCONS_01325501 may play a vital role in embryo pre-implantation. Furthermore, the expression of FGF7, NMB, COL5A3, S100A8 and PPP1R3D genes were significantly up-regulated at the time of maternal recognition of pregnancy (Day 12 of pregnancy). Our results first identified the characterization and expression profile of lncRNAs in pig endometrium during pre-implantation phases.
147 citations
12 Oct 2020
TL;DR: A mutual influence of maternal and embryonic tissues appears to exist and it is suggested that successive steps in embryonic germ layer differentiation may induce specific developmental events and secretory activity of the embryos.
Abstract: Comparison of the timing of pig preimplantation development, alterations in the ultrastructure of embryonic germ layers, and cytological changes of the uterine epithelial cells leads to the supposition that a close relationship exists between embryonic and uterine development during early pregnancy. The results of in-vitro studies of embryonic development and of experiments concerning asynchrony between embryos and uterine environment confirm this supposition, especially as far as the post-hatching period is concerned. It is suggested that successive steps in embryonic germ layer differentiation may induce specific developmental events and secretory activity of the embryos. A mutual influence of maternal and embryonic tissues appears to exist, but we can only speculate about the causes of many of the described phenomena.
105 citations
TL;DR: A novel, dual role for PGF2α in corpora lutea is presented that depends on the acquisition of luteolytic sensitivity, based on the knowledge that P GF2α triggers pathways involved in lutelysis during the estrous cycle or/and may have an alternative function in maintaining progesterone synthesis during pregnancy.
Abstract: Porcine conceptuses secrete pregnancy-recognition signals (estrogens, including estradiol-17β) that inhibit luteolysis, thereby prolonging progesterone production by corpora lutea. The supportive mechanism by which the conceptus also inhibits luteolysis is by shifting endometrial prostaglandin (PG) synthesis to luteoprotective PGE2. Progesterone stimulates endometrial production of factors that are essential for conceptus development. Priming the uterus by progesterone and loss of progesterone receptors from the uterine epithelium by D1ay 10-12 after estrus are key for achieving endometrial receptivity for implantation. Conceptus implantation involves a series of events, many resembling the inflammatory reaction, that are greatly influenced by cytokines, growth factors, and prostaglandins. We herein present a novel, dual role for PGF2α in corpora lutea that depends on the acquisition of luteolytic sensitivity, based on the knowledge that PGF2α triggers pathways involved in luteolysis during the estrous cycle or/and may have an alternative function in maintaining progesterone synthesis during pregnancy. We also point out a new role for PGF2α that, together with PGE2, can act as embryonic signal mediators. PGF2α, which until recently was considered undesirable for promoting pregnancy, is now known to stimulate conceptus-maternal interactions and angiogenesis in the endometrium. This function is in line with other important prostaglandin functions, such as stimulating adhesion of trophoblasts (PGE2, PGI2) as well as endometrial vascular functions and trophoblast cell proliferation (PGI2). Finally, microRNAs have emerged as important post-transcriptional regulators of gene function, adding a new area of investigation that may enhance understanding of conceptus-endometrial interactions.
92 citations
Cites background from "Deep Sequencing of the Porcine Endo..."
...Analysis of changes in global endometrial gene expression suggested that different activities occur in the endometrium during the period of maternal recognition of pregnancy versus implantation (Samborski et al., 2013b)....
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...Other highly expressed genes in the endometrium on Day 14 of gestation were related to either pro- or anti-apoptotic functions (Samborski et al., 2013a)....
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...The DEGs on Day 12 of pregnancy were involved in mitosis, increased cell proliferation in the uterine glandular epithelium, immune cell infiltration, epithelial cell differentiation, tissue remodeling, prostaglandin signaling, and metabolism (Samborski et al., 2013b)....
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...Cell adhesion was also associated with DEGs in the pregnant endometrium, particularly integrins and genes belonging to the mucin family (Samborski et al., 2013a)....
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...…cell cycle, cell proliferation, cellular movement and adhesion, extracellular matrix remodeling, proteolysis, immune-related processes, and hormone activity (Kiewisz et al., 2014; Østrup et al., 2010; Ross et al., 2009; Samborski, Graf, Krebs, Kessler, & Bauersachs, 2013a; Samborski et al., 2013b)....
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TL;DR: Comprehensive transcriptome changes in porcine endometrium associated with establishment of pregnancy is identified and could be a resource for targeted studies of genes and pathways potentially involved in regulation of this process.
Abstract: 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, deep sequencing of endometrial RNA samples of Day 12 pregnant animals (n = 4) and corresponding nonpregnant controls (n = 4) was performed using RNA sequencing (RNA-Seq). Between 30 000 000 and 35 000 000 sequence reads per sample were produced and mapped to the porcine genome (Sscrofa10.2). Analysis of read counts revealed 2593 differentially expressed genes (DEGs). 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, the level of the DEG, and 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. This study identified comprehensive transcriptome changes in porcine endometrium associated with establishment of pregnancy and could be a resource for targeted studies of genes and pathways potentially involved in regulation of this process.
80 citations
Cites background or methods or result from "Deep Sequencing of the Porcine Endo..."
...[15], the IDs of the samples are, from left to right, D12_C004, D12_C006, D12_C007, D12_C011, D12_P003, D12_P008, D12_P010, D12_P012, D14_C902, D14_C912, D14_C916, D14_C917, D14_P903, D14_P909, D14_P915, and D14_P918....
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...Systematic studies of transcriptome changes in porcine endometrium during the time of implantation (Day 14) have been performed with DNA microarrays [14], and next-generation sequencing (NGS) [15]....
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...The obtained sequence reads (Fastq files) were analyzed with a locally installed version of Galaxy [16] as described recently [15]....
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...Particularly in the NGS study, a huge number of differentially expressed genes (DEGs) have been identified in comparison to pregnancy and the corresponding stage of the estrous cycle....
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...Day 14 of pregnancy in porcine endometrium [15] and to...
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TL;DR: The global analysis of the fluid proteomes revealed a general pattern of endocrine regulation of the tract, with the cervix and the oviduct showing an increased differential proteins abundance mainly at oestrus while the uterus showed an increased abundance mainly during the luteal phase.
76 citations
References
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TL;DR: The 2-Delta Delta C(T) method as mentioned in this paper was proposed to analyze the relative changes in gene expression from real-time quantitative PCR experiments, and it has been shown to be useful in the analysis of realtime, quantitative PCR data.
139,407 citations
TL;DR: The Gene Set Enrichment Analysis (GSEA) method as discussed by the authors focuses on gene sets, that is, groups of genes that share common biological function, chromosomal location, or regulation.
Abstract: Although genomewide RNA expression analysis has become a routine tool in biomedical research, extracting biological insight from such information remains a major challenge. Here, we describe a powerful analytical method called Gene Set Enrichment Analysis (GSEA) for interpreting gene expression data. The method derives its power by focusing on gene sets, that is, groups of genes that share common biological function, chromosomal location, or regulation. We demonstrate how GSEA yields insights into several cancer-related data sets, including leukemia and lung cancer. Notably, where single-gene analysis finds little similarity between two independent studies of patient survival in lung cancer, GSEA reveals many biological pathways in common. The GSEA method is embodied in a freely available software package, together with an initial database of 1,325 biologically defined gene sets.
34,830 citations
TL;DR: A method based on the negative binomial distribution, with variance and mean linked by local regression, is proposed and an implementation, DESeq, as an R/Bioconductor package is presented.
Abstract: High-throughput sequencing assays such as RNA-Seq, ChIP-Seq or barcode counting provide quantitative readouts in the form of count data. To infer differential signal in such data correctly and with good statistical power, estimation of data variability throughout the dynamic range and a suitable error model are required. We propose a method based on the negative binomial distribution, with variance and mean linked by local regression and present an implementation, DESeq, as an R/Bioconductor package.
13,356 citations
TL;DR: DAMID is a web-accessible program that integrates functional genomic annotations with intuitive graphical summaries that assists in the interpretation of genome-scale datasets by facilitating the transition from data collection to biological meaning.
Abstract: The distributed nature of biological knowledge poses a major challenge to the interpretation of genome-scale datasets, including those derived from microarray and proteomic studies. This report describes DAVID, a web-accessible program that integrates functional genomic annotations with intuitive graphical summaries. Lists of gene or protein identifiers are rapidly annotated and summarized according to shared categorical data for Gene Ontology, protein domain, and biochemical pathway membership. DAVID assists in the interpretation of genome-scale datasets by facilitating the transition from data collection to biological meaning.
8,849 citations
"Deep Sequencing of the Porcine Endo..." refers methods in this paper
...A functional classification of the obtained DEG was performed with the DAVID ‘‘functional annotation clustering’’ and ‘‘functional annotation chart’’ tools (Supplemental Table S4)....
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...A comparison of the DAVID result for typical overrepresented functional terms found for genes with higher expression and for genes with lower expression in Day 14 pregnant endometrium is shown in Table 4....
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...Comparison of the results from DAVID functional annotation chart analysis of genes with lower and genes with higher mRNA concentrations in Day 14 pregnant porcine endometrium for selected functional terms....
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...Integrated analysis of different functional databases was done using the ‘‘functional annotation clustering’’ tool of the Database for Annotation, Visualization, and Integrated Discovery (DAVID) [24]....
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...DAVID Functional Annotation Clustering and ClueGO analysis confirmed the results obtained from GSEA for the genes with lower mRNA concentrations in pregnant endometrium on Day 14 of pregnancy (see discussion above)....
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TL;DR: ClueGO is an easy to use Cytoscape plug-in that strongly improves biological interpretation of large lists of genes and creates a functionally organized GO/pathway term network.
Abstract: We have developed ClueGO, an easy to use Cytoscape plug-in that strongly improves biological interpretation of large lists of genes. ClueGO integrates Gene Ontology (GO) terms as well as KEGG/BioCarta pathways and creates a functionally organized GO/pathway term network. It can analyze one or compare two lists of genes and comprehensively visualizes functionally grouped terms. A one-click update option allows ClueGO to automatically download the most recent GO/KEGG release at any time. ClueGO provides an intuitive representation of the analysis results and can be optionally used in conjunction with the GOlorize plug-in.
4,768 citations