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| 2. |
- Guo, Yongzhi, et al.
(author)
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Differential gene expression in bovine endometrial epithelial cells after challenge with LPS; specific implications for genes involved in embryo maternal interactions
- 2019
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In: PLoS ONE. - : Public Library of Science (PLoS). - 1932-6203. ; 14
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Journal article (peer-reviewed)abstract
- Lipopolysaccharide (LPS) expressed on the surface of Gram-negative bacteria activates pro-inflammatory pathways, dys-regulates the function of endometrial cells and is a key player in the mechanisms involved in endometritis. This study aimed to investigate the effects of LPS on bovine endometrial epithelial cells (bEEC) from whole transcriptome with a special focus on genes involved in embryo-maternal interactions. Following in vitro culture, bEEC from three cows were exposed to 0, 2, and 8 mu g/mL LPS for 24h. RNA samples extracted at 0 and 24 hours were analyzed by RNA sequencing (RNA-seq). At 24h, 2035 differentially expressed genes (DEGs) were identified between controls and samples treated with 2 mu g/mL LPS. Gene ontology analysis showed that over-expressed DEGs were associated to immune response, response to stress and external stimuli, catalytic activity, and cell cycle. Genes associated with cell membrane and cell adhesion pathways were under-expressed. LPS induced changes in expression of specific genes related to embryo-maternal interactions including under-expression of eight members of the cadherin superfamily, over-expression of six members of the mucin family, and differential expression of a large set of genes binding the above molecules and of more than 20 transcripts coding for cyto-kines and their receptors. Type I interferon-tau dependent genes were also over-expressed. From a sub-set of 19 genes, (biological replicates of bEEC from cows taken at time 6 (n = 3), 24 (n = 6) and 48 hours (n = 3), and 2 technical replicates per sample) differential gene expression was confirmed by RT2-qPCR (r(2) between fold changes at 24 hours by RT2 qPCR and RNA-seq = 0.97). These results indicate that LPS affects the function of bEEC in many ways by differential transcription, glycolytic metabolism and oxidative stress. Many transcriptomic signatures related to implantation and embryo maternal interactions were strongly affected by LPS. These results pave the way for further studies to investigate the duration of these changes and their possible impact on endometrial function and fertility.
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- Jhamat, Naveed
(author)
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Comparative bioinformatics analyses of transcriptome and epigenome data using bovine model systems
- 2019
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Doctoral thesis (other academic/artistic)abstract
- Bioinformatics methodology is used to understand complex biological processes in molecular biology, genetics and epigenetics. This thesis performed analysis of the complex biological processes controlling reproduction in mammals including dairy cattle. Successful fertility in dairy cattle is required for farm sustainability as it supports milk productivity and longevity. Disorder in metabolism due to higher levels of insulin during pregnancy and disturbance of immune response due to bacterial infections after calving are detrimental in reproduction. Determinants controlling these molecular mechanisms are poorly understood. To investigate these mechanisms, we performed bioinformatics analyses on data obtained from two experimental studies: Insulin is a regulator of metabolism and conditions such as hyperinsulinemia is known to impair fertility especially during embryonic development. This study investigates the potential effect of insulin treatment on gene expression and DNA methylation patterns of bovine embryos during in vitro oocyte maturation by using the EmbryoGENE DNA Methylation Array. The results of Paper I revealed that the identified differentially methylated regions (DMRs) were correlated with differentially expressed genes involved in metabolic regulation. Uterine diseases impair fertility in dairy cattle and Lipopolysaccharide (LPS) from gram-negative Escherichia coli, is a major source of uterine diseases by activating proinflammatory pathways. This study investigates in vitro the effects of infection by LPS on bovine endometrial epithelial cells (bEEC) mimicking in vivo processes. In Paper II, RNA Sequencing analysis revealed that LPS has significantly affected the transcriptome of bEEC, identifying more than 2000 differentially expressed genes, involved in immune response, proliferation, cell adhesion, and implantation. In Paper III, Bisulfite sequencing revealed that LPS has profoundly affected the DNA methylation pattern of bEEC. 1291 DMRs were found and their associated genes were involved in molecular processes related to proliferation, apoptosis and embryo development. In Paper IV, the enrichment of motifs within these DMRs revealed transcription factor binding sites for immunologically important transcription factors. Thus, the transcriptomics, epigenomics and bioinformatics results obtained from these analyses revealed the complexity of the regulatory transcriptional network activated during inflammation.
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- Jhamat, Naveed, et al.
(author)
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Identification of Differentially Methylated Regions (DMR) in the genome of bovine endometrial epithelial cells (bEEC)challenged by E.Coli LPS and its effect on transcription
- 2016
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Conference paper (other academic/artistic)abstract
- Lipopolysaccharide (LPS) from Gram-negative bacteria induces the activation of pro-inflammatory pathways in the endometrium and such reactions potentially affect fertility and more specifically the implantation process. Gene expression studies based on RNA sequencing [Guo et al., Epiconcept Conference 2015, Abstract O-23, 34p] have shown that more than 2000 genes were differentially expressed (DEGs) in bEEC exposed to LPS. This study aimed to identify epigenetic correlates of these DEGs by detecting Differentially Methylated Regions (DMRs) in same cells. bEEC were exposed in vitro to 0, 2, and 8 μg/ml LPS for 24 hours. DNA libraries were prepared at The Babraham Institute, UK for reduced representation bisulfite sequencing (RRBS) using MspI digestion followed by end-repair/A-tailing and 5mC adaptor ligation and bisulfite conversion plus PCR. Sequences were analyzed using BS-Seeker2 for alignment of reads and MethylKit v0.9.5 for finding significant DMRs. AliBaba2 was used to predict potential transcription factor binding sites (TFBS) at DMRs using the TRANSFAC database. Analysis of RRBS data revealed 78 DMRs with q-value<0.05 between controls and 2 μg/ml LPS samples (34 hyper- and 44 hypo-methylated) and 109 DMRs between controls and 8 μg/ml LPS samples (47 hyper- and 62 hypo-methylated). 17 common DMRs (in 2 μg/ml and 8μg/ml LPS groups vs. controls) were identified. A reciprocal relationship was found between methylation and gene expression data for a sub-set of loci known to be key regulators of endometrial function. For example, LIF (Leukemia Inhibitory Factor) gene was found to contain a hypo-methylated DMR and its expression was up-regulated. Predicted binding sites in this DMR have 100% conservation and gene synteny in the human genome. These analyses allow the identification of regions harbouring candidates for key regulatory elements of endometrial function, thus contributing to the understanding of LPS-induced deregulation that may impact implantation.
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| 5. |
- Jhamat, Naveed, et al.
(author)
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LPS-treatment of bovine endometrial epithelial cells causes differential DNA methylation of genes associated with inflammation and endometrial function
- 2020
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In: BMC Genomics. - : Springer Science and Business Media LLC. - 1471-2164. ; 21
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Journal article (peer-reviewed)abstract
- BackgroundLipopolysaccharide (LPS) endotoxin stimulates pro-inflammatory pathways and is a key player in the pathological mechanisms involved in the development of endometritis. This study aimed to investigate LPS-induced DNA methylation changes in bovine endometrial epithelial cells (bEECs), which may affect endometrial function. Following in vitro culture, bEECs from three cows were either untreated (0) or exposed to 2 and 8 mu g/mL LPS for 24h.ResultsDNA samples extracted at 0h and 24h were sequenced using reduced representation bisulfite sequencing (RRBS). When comparing DNA methylation results at 24h to time 0h, a larger proportion of hypomethylated regions were identified in the LPS-treated groups, whereas the trend was opposite in controls. When comparing LPS groups to controls at 24h, a total of 1291 differentially methylated regions (DMRs) were identified (55% hypomethylated and 45% hypermethylated). Integration of DNA methylation data obtained here with our previously published gene expression data obtained from the same samples showed a negative correlation (r=-0.41 for gene promoter, r=-0.22 for gene body regions, p<0.05). Differential methylation analysis revealed that effects of LPS treatment were associated with methylation changes for genes involved in regulation of immune and inflammatory responses, cell adhesion, and external stimuli. Gene ontology and pathway analyses showed that most of the differentially methylated genes (DMGs) were associated with cell proliferation and apoptotic processes; and pathways such as calcium-, oxytocin- and MAPK-signaling pathways with recognized roles in innate immunity. Several DMGs were related to systemic inflammation and tissue re-modelling including HDAC4, IRAK1, AKT1, MAP3K6, Wnt7A and ADAMTS17.ConclusionsThe present results show that LPS altered the DNA methylation patterns of bovine endometrial epithelial cells. This information, combined with our previously reported changes in gene expression related to endometrial function, confirm that LPS activates pro-inflammatory mechanisms leading to perturbed immune balance and cell adhesion processes in the endometrium.
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| 6. |
- Laskowski, Denise, et al.
(author)
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DNA methylation pattern of bovine blastocysts associated with hyperinsulinemia in vitro
- 2018
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In: Molecular Reproduction and Development. - : Wiley. - 1040-452X .- 1098-2795. ; 85, s. 599-611
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Journal article (peer-reviewed)abstract
- Insulin functions as a regulator of metabolism and plays an important role in reproduction. Hyperinsulinemia is often observed in patients with obesity and diabetes type 2 and is known to impair fertility, but the underlying molecular mechanisms are only partly understood. Metabolic programming through epigenetic mechanisms such as DNA methylation during embryonic development can lead to health implications for the offspring later in life. Our aim was to study the potential effect of hyperinsulinemia on gene expression and DNA methylation of embryos by adding insulin (0.1 mu g/ml=INS0.1 or 10 mu g/ml=INS10) during in vitro oocyte maturation by using the EmbryoGENE DNA methylation array for a study of the bovine epigenome. Our results showed significant differences between blastocysts originating from insulin-treated oocytes compared with untreated control blastocysts. In total, 13,658 and 12,418 probes were differentially methylated (DM) in INS0.1 and INS10, respectively, with an overlap of 3,233 probes in the DM regions (DMR) for both insulin groups. Genes related to pathways such as lipid metabolism, growth and proliferation, mitochondrial function, and oxidative stress responses were influenced at both the epigenetic and transcriptomic levels. In addition, imprinted genes and genes with functions in the epigenetic machinery were among the DMRs. This study identified DMRs correlated to differential expression of genes involved in metabolic regulation and should help to improve our knowledge of the underlying molecular mechanisms of metabolic imbalance.
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