Identification of differentially expressed genes in equine endometrium at day 16 of early pregnancy
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ABSTRACT: In order to systematically analyze the maternal, i.e. the endometrial, changes in the equine endometrium underlying the complex embryo-maternal dialogue during early pregnancy, a transcriptome study of endometrium samples from mares at day 16 of pregnancy and day 12 cyclic mares was performed. Results were compared to a previous study of samples from day 12 of pregnancy and day 12 cyclic controls.
Project description:In order to systematically analyze the maternal, i.e. the endometrial, changes in the equine endometrium underlying the complex embryo-maternal dialogue during early pregnancy, a transcriptome study of endometrium samples from mares at day 16 of pregnancy and day 12 cyclic mares was performed. Results were compared to a previous study of samples from day 12 of pregnancy and day 12 cyclic controls. Endometrial biopsies were taken from six wormblood mares at day 16 of early pregnancy after embryo recovery and day 12 cyclic controls. 8 samples were analyzed: one pregnant sample and the corresponding control sample of every mare each at a time.
Project description:In order to systematically analyze the maternal, i.e. the endometrial, changes in the equine endometrium underlying the complex embryo-maternal dialogue during early pregnancy, a transcriptome study of endometrium samples from five mares at day 8 and six mares at day 12 of early pregnancy and the corresponding non-pregnant stage was performed.
Project description:In order to systematically analyze the maternal, i.e. the endometrial, changes in the equine endometrium underlying the complex embryo-maternal dialogue during early pregnancy, a transcriptome study of endometrium samples from five mares at day 8 and six mares at day 12 of early pregnancy and the corresponding non-pregnant stage was performed. Endometrial biopsies were taken from six warmblood mares at day 12 of early pregnancy after embryo recovery and at the corresponding non-pregnant stage. 12 samples were analyzed: one pregnant sample and the corresponding control sample of every mare each at a time. Endometrial biopsies were taken from five warmblood mares at day 8 of early pregnancy after embryo recovery and at the corresponding non-pregnant stage. 10 samples were analyzed: one pregnant sample and the corresponding control sample of every mare each at a time. Two experimental groups: 8 and 12 days
Project description:While the equine oviduct clearly affects early embryo development and while the selective transport of equine embryos through the oviduct indicates a reciprocal interaction, the influence of the embryo on gene expression in the oviduct remains to be determined in the horse. The aim of this study was to examine this by means of RNA sequencing. Four days after ovulation, the oviduct epithelial cells ipsilateral and contralateral to the ovulation side from five cyclic and five pregnant mares were collected. mRNA was extracted and samples were sequenced using the Illumina Hiseq-2000 sequencer. Data analysis was performed with the CLC Genomics software and differentially expressed genes (DEGs) were determined (p-value ≤ 0.05 and absolute fold change ≥ 2). ClueGO was used for functional interpretation. A total of 26,991 genes was identified and 253 genes were found to be upregulated and 108 to be downregulated in the pregnant ipsilateral oviduct, when compared to the cyclic ipsilateral oviduct. Comparison of the ipsilateral and the contralateral oviduct indicated 164 DEGs in pregnant mares and 77 DEGs in cyclic mares. Functional enrichment analysis only detected differences in the comparison of pregnant and cyclic ipsilateral oviducts and showed that the equine embryo affects the expression of immune response related genes in the oviduct, with marked upregulation of interferon associated genes. This research represents the foundation for further assessment of the role of specific genes in the early embryo-maternal dialogue of the horse.
Project description:Extracellular vesicles (EVs) have been identified in the uterine fluid in different species and pointed as key players in the embryo-maternal dialogue, maternal recognition of pregnancy (MRP) and establishment of pregnancy. However, little is known about the uterine EVs in the mare. Therefore, the present study aimed at characterizing EVs from uterine lavage of cyclic mares by comparing five EVs isolation methods and the combination of them: (1) Ultracentrifugation (UC); (2) Concentration of lavage volume by Centricon ultrafiltration (CE); (3) the use of CE with different washing steps (PBS versus trehalose); (4) Size-exclusion chromatography with iZON-qEV columns to yield high-purity EVs and (5) a combination of the methods with best results based on EVs yield, purity, and protein cargo profiles. Transmission electron microscopy and Western blotting confirmed the isolation of EVs by all methods but with quantitative and qualitative differences. Mass spectrometry provided different protein profiles among methods with marked differences in the number of proteins and protein classes. Our results indicate that the combination of CE/trehalose/iZON/UC is an optimal method to isolate equine uterine EVs with good yield and purity that can be applied in future studies to determine the role of uterine EVs during MRP in the mare.
Project description:mRNA profiles of (1) equine endometrium collected 14, 22, and 28 days after ovulation from pregnant mares and (2) equine endometrium collected 20 days after ovulation from pregnant mares, and from non-pregnant mares which displayed and failed to display extended luteal function following the administration of oxytocin.
Project description: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.
Project description:Establishment and maintenance of pregnancy in equids is only partially understood. To provide new insights into early events of this process, we performed a systematic analysis of transcriptome changes in the endometrium at days 8 and 12 of pregnancy. Endometrial biopsy samples from pregnant and non-pregnant (Day 12) stages were taken from the same mares. Composition of the collected biopsy samples was analyzed using quantitative stereological techniques to determine proportions of surface and glandular epithelium and blood vessels. RNA-seq analysis was performed and differentially expressed genes were identified. The samples used for RNA-seq were already analyzed before by the use of Agilent microarrays (GSE21046).
Project description:The maternal tract plays a critical role in the success of early embryonic development providing an optimal environment for establishment and maintenance of pregnancy. Preparation of this environment requires an intimate dialogue between the embryo and her mother. To advance our understanding of the process by which a foreign blastocyst is accepted by the maternal endometrium and better address the clinical challenges of infertility and pregnancy failure, it is imperative to decipher this complex molecular dialogue. The objective of the present work is to define the local response(s) of the maternal tract towards the embryo during the earliest stages of pregnancy.
Project description:The protein cargos of uterine extracellular vesicles isolated from uterine lavages, collected from pregnant mares (P; day 10, 11, 12 and 13 after insemination) and cyclic control mares (C; day 10 and 13 after insemination), was analyzed.