Project description:Co-ordinated regulation of endometrial gene expression is essential for successful pregnancy establishment. A non-receptive uterine environment may be a key contributor to pregnancy loss, as the majority of pregnancy losses occur prior to embryo implantation. DNA methylation has been highlighted as a potential contributor in regulating early pregnancy events in the uterus. It was hypothesized that DNA methylation regulates expression of key genes in the uterus during pregnancy. To gain support for this hypothesis the correlation between DNA methylation and gene expression was tested. Endometrial samples from fertile and sub-fertile dairy cow strains were obtained at day 17 of pregnancy or the reproductive cycle. Microarrays were used to characterize genome-wide DNA methylation profiles and data compared with transcription profiles which have been previously reported. 39% of DNA methylation probes assayed mapped to RefSeq genes with transcription measurements. The 1,000 most significant correlations were used for subsequent analysis. Of these, 52% percent were negatively correlated with gene expression. When this gene list was compared with previously reported gene expression studies on the same tissues, 42% were differentially expressed when comparing pregnant and cycling animals and 11% were differentially expressed comparing pregnant fertile and sub-fertile animals. DNA methylation status was correlated with gene expression in several pathways implicated in early pregnancy events. Although these data do not provide direct evidence of a causative association between DNA methylation and gene expression, this study provides critical support for an effect of DNA methylation in early pregnancy events and highlights candidate genes for future studies. The estrous cycles of 24 lactating dairy cows were synchronized (at 58.8 (SEM 3.77) and 60.2 (SEM 1.51) days post calving in dairy cows of sub-fertile and fertile strains, respectively) and 14 received a single embryo transferred on day 7 of the estrous cycle. Animals were slaughtered at day 17 of the reproductive cycle and endometrial tissues (both caruncular and intercaruncular) were sampled. Selection criteria for the study included strain and calving date, and health postcalving was an exclusion criterion (cows with severe uterine infections or mastitis were excluded before being enrolled in the embryo transfer round). Cows in each strain were matched for calving number and age. A total of 10 cycling and 12 pregnant animals enrolled in the study were utilized, due to the associated costs of slaughtering the cows. These animals represented fertile (six pregnant and five cycling Holstein-Friesian cows with New Zealand ancestry/M-bM-^IM-$30% North American genetics, n=11, NZ) and sub-fertile (six pregnant and five cycling Holstein-Friesian cows with >87% North American ancestry, n=11, NA) phenotypes of Holstein-Friesian dairy cows

Project description:Peripheral blood mononuclear cells (PBMC) from cows with experimentally induced hypocalcaemia or spontaneous milk fever were subject of an oligo-microarray analysis following quantitative real-time reverse transcription PCR (q-PCR). In experimental hypocalcaemia induced by 10 % Na2EDTA infusion (n = 4), the microarray revealed that 32 genes were significantly up- or down-regulated compared to control treatment by 11 % CaEDTA infusion (n = 4). In milk fever cases (n = 8), the microarray demonstrated that 98 genes were expressed differentially higher or lower compared to healthy parturient cows (n = 5). From both data, five genes were selected to be strongly related to both experimental hypocalcaemia and milk fever. Additionally, another gene was judged to be specific for milk fever independent of hypocalcaemia. The mRNA expressions of these six genes in milk fever cases were verified by q-PCR, which were significantly higher or lower than those in healthy parturient cows. In the experimental hypocalcemia study, four healthy nonpregnant, non-lactating ovariectomised Holstein cows were used. The experiment was performed according to a 2 M-CM-^W 2 crossover design with 2 weeks intervals. Cows were infused with either Na2EDTA (hypocalcaemic treatment) or CaEDTA (control treatment) solution during a period of 4 h. The animals were monitored for clinical signs of hypocalcaemia (dry nose, staggering, astasia, reduced ruminal activity, increased heart rate, increased respiratory frequency, sweating, and ear cold). Infusion of EDTA solution was temporarily suspended when hypocalcaemia induced astasia was represent. If affected cow stood again, EDTA infusion was resumed. PBMC samples were collected before the start of infusion (0 h; pre-treatment) and at 4 and 24 h after the start of infusion 24h and utilized for gene expression analysis of PBMC. In the spontaneous milk fever study, a total of 13 Holstein dairy cows, which were 8 parturient cows diagnosed with milk fever within 2 days postpartum and 5 clinically healthy parturient cows within 2 days after parturition (Group H), were used. All spontaneous milk fever cases showed hypocalcaemia (< 1.8 mmol/l) in the blood samples obtained before the first treatment, and were treated by an intravenous infusion of a 20 % Ca borogluconate solution (500 ml). The milk fever cases were divided into two categories according to the reponse of the cows to the first Ca treatment: an immediate response in which the cow could stand within 1 day after a single Ca treatment (Group A, n = 4) and a requirement further treatment over 1 day after a single Ca treatment (Group B, n = 4). PBMC samples were collected vein just before the first Ca treatment in groups A and B and within 2 days postpartum in group H and utilized for gene expression analysis of PBMC.

Project description:Background: Physiological inflammation of the uterus postpartum is essential for the reparative processes of involution after calving. In the majority of cows, this inflammation is resolved and homeostasis is restored. However, in a significant subset, inflammation persists and contributes to tissue damage, pathology and subfertility. Transcriptomic differences of immune genes between cattle that resolve inflammation and those that develop uterine disease have been detected as early as 7 days postpartum (DPP) suggesting that the host immune response plays an important role in disease outcome. Results: Here, we extensively characterise the immune response at the transcriptomic level in endometrial epithelial cells from post-partum dairy cows phenotyped for both clinical and sub-clinical forms of uterine disease. We address the hypothesis that excessive expression of endometrial inflammatory molecules contributes to development of endometritis. Classification of cattle (n=112) as healthy or with uterine disease (purulent vaginal discharge; PVD and cytological endometritis; CYTO) was based on vaginal mucus score and >18% polymorphonuclear cell infiltrate into the endometrium at 21 DPP. RNA-seq analysis of endometrial epithelial cells collected using cytobrushes identified differential expression of 294 genes (FDR <0.05) between cows that subsequently resolved inflammation (n=10) and those that developed disease (n=20). Pathway over-representation analysis of differentially expressed genes (DEG) identified significant changes in immune-related pathways, including the NOD-like receptor signalling pathway, cytokine-cytokine receptor interaction pathway and the Toll-like receptor signalling pathway which were up-regulated in cattle that subsequently developed disease. The majority of the DEG were upregulated in cows that developed PVD, and included all genes upregulated in CYTO cows, suggesting a core inflammatory gene signature early post-partum contributes to the onset of uterine disease. This inflammatory signature was validated by qPCR in an independent group of cows (n=56) and included upregulation of pro-inflammatory genes (including TLR2, TLR4, NLRP3, IL1A, IL1B, IL8, and S100A8) at day 7 postpartum in cows that failed to resolve inflammation. Conclusions: Despite a large amount of inter-animal heterogeneity, these results suggest that excessive activation and inappropriate regulation of the inflammatory response early postpartum is a key feature of the subsequent development of uterine disease. Keywords: Endometritis, Inflammation, Transcriptome, Next generation sequencing, Dairy cattle, Uterine involution, Immune response

			Action	DRS
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	SCFP01.dat.gz	Other
	SCFP01.fasta.gz	Fasta.gz
	SCFP01.master.dat	Other
	SCFQ01.dat.gz	Other

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Helcococcus

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