Transcription profiling of pig endometrium from pregnant and non-pregnant sows
ABSTRACT: In an attempt to unveil part of the molecular processes controlling porcine placentation we have investigated the pregnancy induced gene expression in the porcine endometrium at Day 14 after insemination using the Affymetrix GeneChip® Porcine Genome Array. Experiment Overall Design: At Day 14 after insemination, samples were obtained from the endometrium of pregnant sows and sows inseminated with inactivated semen for RNA extraction and hybridization on Affymetrix microarrays. The uteri were removed and each uterine horn was flushed with PBS containing 1% fetal calf serum, and subsequently opened longitudinally at the anti-mesometrial side. The sites of embryonic attachment were macroscopically visual in the endometrium on the mesometrial side in form of hyperemic zones. Samples of the endometrium (lamina epithelialis, lamina propria and tela submucosa, but not tunica muscularis) were isolated from proximal (the end close to the ovaries), intermedial, and distal (next to the corpus uteri)) sections of each uterine horn. Samples were taken from the endometrium of the non-pregnant animals at comparable locations.
Project description:In an attempt to unveil part of the molecular processes controlling porcine placentation we have investigated the pregnancy induced gene expression in the porcine endometrium at Day 14 after insemination using the Affymetrix GeneChip® Porcine Genome Array. Overall design: At Day 14 after insemination, samples were obtained from the endometrium of pregnant sows and sows inseminated with inactivated semen for RNA extraction and hybridization on Affymetrix microarrays. The uteri were removed and each uterine horn was flushed with PBS containing 1% fetal calf serum, and subsequently opened longitudinally at the anti-mesometrial side. The sites of embryonic attachment were macroscopically visual in the endometrium on the mesometrial side in form of hyperemic zones. Samples of the endometrium (lamina epithelialis, lamina propria and tela submucosa, but not tunica muscularis) were isolated from proximal (the end close to the ovaries), intermedial, and distal (next to the corpus uteri)) sections of each uterine horn. Samples were taken from the endometrium of the non-pregnant animals at comparable locations.
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. In order to identify the uterine gene expression modified in the presence of the embryo when compared to the oocyte we used a novel model that eliminated genetic variability. Using laparoscopic insemination one oviduct was inseminated with spermatozoa and the contralateral oviduct was injected with diluent. This model allowed us to obtain samples from the oviduct and the uterine horn containing either embryos or oocytes from the same sow. Uterine horn pig samples containing embryos at blastocyst stage and and their contralateral uterine horn containing oocytes from individual sows were collected for RNA isolation and hybridization on Affymetrix microarrays. Three biological replicates were performed (n= 3 sows) and a total of 6 arrays were used for microarrays study (3 arrays for uterine horn samples containing embryos (inseminated-side) and 3 arrays for samples containing oocytes (non-inseminated side).
Project description:Maternal exposure to estrogens can induce long-term adverse effects in the offspring. This may be mediated through alterations in the endometrium affecting embryo-maternal communication as early as the preimplantational phase. Thus, we analyzed the effects of gestational estradiol-17β (E2) exposure on the endometrium. Two distinct low doses and a high dose (0.05, 10 and 1000 µg E2/kg body weight daily, respectively) were orally applied to sows from insemination until sampling at day 10 of pregnancy and compared to carrier-treated controls. RNA-sequencing revealed a dose-dependent increase of 14, 17 and 27 differentially expressed genes (DEG), respectively. Overall, the maternal E2 treatment perturbed gene expression of the endometrium, potentially altering the uterine histotroph.
Project description: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 1,933 differentially expressed genes (FDR 1%), 1,229 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 non-invasive implantation in the pig. 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: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:This study provide an opportunity to elucidate the genetic control of fetal implantation and improve our understanding of fetal implantation and gestation maintenance, thus make further improvement for litter size of pigs. Nine pregnant sows were slaughtered by electrical on day 13, day 18 and day 24 after insemination (the pregnant group, three sows every period). The non-pregnant sows were slaughtered on day 13 after inseminated (n=3).In pregnant sows, samples of the endometrium attachment sites and inter-sites were taken. Samples from the endometrium of the non-pregnant sows were taken from comparable locations.
Project description:Purpose: characterize the uterine transcriptome profiles of pregnant (P) versus non-pregnant (NP) cows during early pregnancy and attempted to define a potential set of marker genes that can be valuable for predicting pregnancy outcome. Methods: beef cows were synchronized and artificially inseminated at detected estrus. Six days after AI, jugular blood samples and a biopsy from the uterine horn contralateral to the ovary containing the corpus luteum were collected. Based on pregnancy outcome on day 30, samples were retrospectively allocated to the following groups: Pregnant and Non-Pregnant. Both groups had similar plasma progesterone concentrations on D6. Uterine biopsies were submitted to RNA-Seq analysis in a Illumina HiScanSQ platform. Results: The 272,685,768 million filtered reads were mapped to the Bos taurus reference genome and 14,654 genes were analyzed for differential expression between groups. Transcriptome data showed that 216 genes are differently expressed when comparing NP versus P uterine tissue (Padj≤0.1). More specifically, 36 genes were up-regulated in P cows and 180 are up-regulated in NP cows. Conclusions: this study characterized a unique set of genes, expressed in the uterus on 6 days after insemination, that indicate a receptive state leading to pregnancy success. Furthermore, expression of such genes can be used as potential markers to efficiently predict pregnancy success. endometrial mRNA profiles of pregnant and non-pregnant cows were generated by deep sequencing using Illumina HiScanSQ platform BioProject PRJNA268916 SRA Study SRP05036
Project description:To identify the molecular markers of early pregnancy in pigs, we compared global gene expression profiles of the maternal peripheral blood in pregnant sows with the non-pregnant sows. Peripheral blood sample was collected at 14 days after insemination from the submandibular vein of pregnant and non-pregnant sows respectively, and total RNA was isolated, purified and sent for microarray analysis. This study identified 127 up-regulated and 56 down-regulated genes (FC >= 1.5 and P < 0.05) in peripheral blood from pregnant sows versus non-pregnant sows. Of the differently expressed genes, nine (including LPAR3, RXFP4, GALP, CBR1, CBR2, GPX6, USP18, LHB and NR5A1) were found to exert function related to early pregnancy processes. Seven differentially expressed genes (CHGB, USP18, VWF, LPAR3, NR5A1, PPARD, BIN1) were selected to perform qRT-PCR in the same RNA samples.The expression profiles of these genes detected by qRT-PCR were consistent with those by microarray, which confirmed the reliability of our microarray data. Overall design: To minimize the adverse effects of genetic heterogeneity in microarray experiments, three half-sib pairs and one unrelated pair were selected with four sows from the pregnancy group and the other four from the non-pregnancy group.
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).
Project description:Placentation of the conceptus to the surface epithelium is governed through a tightly regulated temporal and spatial window. Premature exogenous steriod exposure causes a shift in the maternal tissue's receptivity and prevents proper placentation. We used Affymetrix microarrays to detail the aberrant changes in uterine gene expression during this window, while removing all conceptus contribution to this dysfunction. Experiment Overall Design: Porcine endometrium was selected at chronological stages of psuedopregnancy and psuedopregnancy post endocrine disrupted early for RNA extraction and hybridization on Affymetrix microarrays. We sought to obtain homogeneous populations of normal endometrium at days 13 and 15 of the pseudo pregnant, and endometrium at days 13 and 15 of the endocrine disrupted pseudo pregnant in order to identify the temporal resolution of expression profiles. In brief, days 13 through 15 accounts for the window of attachment during pregnancy in the pig, so we created pseudo pregnant endometrium and obtained samples from days 13 (PIG13UN_TX)and 15 (PIG15UN_TX) of pseudo pregnancy. In the same, we created pseudo pregnant endometrium that was subjected to endocrine disruption of the pseudo pregnancy and collected samples on days 13 (PIG13_TX)and 15 (PIG15_TX) of pseudo pregnancy.