scholarly journals Epigenetic modulation of intestinal Na+/H+ exchanger-3 expression

2018 ◽  
Vol 314 (3) ◽  
pp. G309-G318 ◽  
Author(s):  
Anoop Kumar ◽  
Pooja Malhotra ◽  
Hayley Coffing ◽  
Shubha Priyamvada ◽  
Arivarasu N. Anbazhagan ◽  
...  
Keyword(s):  
Gene Expression ◽  
Dna Methylation ◽  
Dna Methyltransferase ◽  
Dna Demethylation ◽  
Epigenetic Mechanism ◽  
Mrna Levels ◽  
In Vivo Models ◽  
Epigenetic Modulation

Na+/H+ exchanger-3 (NHE3) is crucial for intestinal Na+ absorption, and its reduction has been implicated in infectious and inflammatory bowel diseases (IBD)-associated diarrhea. Epigenetic mechanisms such as DNA methylation are involved in the pathophysiology of IBD. Whether changes in DNA methylation are involved in modulating intestinal NHE3 gene expression is not known. Caco-2 and HuTu 80 cells were used as models of human intestinal epithelial cells. Normal C57/BL6, wild-type, or growth arrest and DNA damage-inducible 45b (GADD45b) knockout (KO) mice were used as in vivo models. NHE3 gene DNA methylation levels were assessed by MBDCap (MethyMiner) assays. Results demonstrated that in vitro methylation of NHE3 promoter construct (p-1509/+127) cloned into a cytosine guanine dinucleotide-free lucia vector decreased the promoter activity in Caco-2 cells. DNA methyltransferase inhibitor 5-azacytidine (10 μM, 24 h) caused a significant decrease in DNA methylation of the NHE3 gene and concomitantly increased NHE3 expression in Caco-2 cells. Similarly, 5-azacytidine treatment increased NHE3 mRNA levels in HuTu 80 cells. 5-Azacytidine treatment for 3 wk (10 mg/kg body wt ip, 3 times/wk) also resulted in an increase in NHE3 expression in the mouse ileum and colon. Small-interfering RNA knockdown of GADD45b (protein involved in DNA demethylation) in Caco-2 cells decreased NHE3 mRNA expression. Furthermore, there was a significant decrease in NHE3 mRNA and protein expression in the ileum and colon of GADD45b KO mice. Our findings demonstrate that NHE3 gene expression is regulated by changes in its DNA methylation. NEW & NOTEWORTHY Our studies for the first time demonstrate that Na+/H+ exchanger-3 gene expression is regulated by an epigenetic mechanism involving DNA methylation.

scholarly journals Differential regulation of DNA methylation versus histone acetylation in cardiomyocytes during HHcy in vitro and in vivo: an epigenetic mechanism

2014 ◽  
Vol 46 (7) ◽  
pp. 245-255 ◽  
Author(s):  
Pankaj Chaturvedi ◽  
Anuradha Kalani ◽  
Srikanth Givvimani ◽  
Pradip Kumar Kamat ◽  
Anastasia Familtseva ◽  
...  
Keyword(s):  
Dna Methylation ◽  
Histone Acetylation ◽  
Dna Methyltransferase ◽  
Epigenetic Mechanism ◽  
Dependent Manner ◽  
Histone Deacetylase 1 ◽  
H3k9 Acetylation ◽  
Dose Dependent

The mechanisms of homocysteine-mediated cardiac threats are poorly understood. Homocysteine, being the precursor to S-adenosyl methionine (a methyl donor) through methionine, is indirectly involved in methylation phenomena for DNA, RNA, and protein. We reported previously that cardiac-specific deletion of N-methyl-d-aspartate receptor-1 (NMDAR1) ameliorates homocysteine-posed cardiac threats, and in this study, we aim to explore the role of NMDAR1 in epigenetic mechanisms of heart failure, using cardiomyocytes during hyperhomocysteinemia (HHcy). High homocysteine levels activate NMDAR1, which consequently leads to abnormal DNA methylation vs. histone acetylation through modulation of DNA methyltransferase 1 (DNMT1), HDAC1, miRNAs, and MMP9 in cardiomyocytes. HL-1 cardiomyocytes cultured in Claycomb media were treated with 100 μM homocysteine in a dose-dependent manner. NMDAR1 antagonist (MK801) was added in the absence and presence of homocysteine at 10 μM in a dose-dependent manner. The expression of DNMT1, histone deacetylase 1 (HDAC1), NMDAR1, microRNA (miR)-133a, and miR-499 was assessed by real-time PCR as well as Western blotting. Methylation and acetylation levels were determined by checking 5′-methylcytosine DNA methylation and chromatin immunoprecipitation. Hyperhomocysteinemic mouse models (CBS+/−) were used to confirm the results in vivo. In HHcy, the expression of NMDAR1, DNMT1, and matrix metalloproteinase 9 increased with increase in H3K9 acetylation, while HDAC1, miR-133a, and miR-499 decreased in cardiomyocytes. Similar results were obtained in heart tissue of CBS+/− mouse. High homocysteine levels instigate cardiovascular remodeling through NMDAR1, miR-133a, miR-499, and DNMT1. A decrease in HDAC1 and an increase in H3K9 acetylation and DNA methylation are suggestive of chromatin remodeling in HHcy.

scholarly journals Modulation of DNA Methylation Influences Cartilage Formation in Murine Chondrogenic Models

2021 ◽  
Author(s):  
Judit Vágó ◽  
Katalin Kiss ◽  
Edina Karanyicz ◽  
Roland Takács ◽  
Csaba Matta ◽  
...  
Keyword(s):  
Dna Methylation ◽  
In Situ Hybridization ◽  
Dna Methyltransferase ◽  
Mouse Cell ◽  
Dna Demethylation ◽  
Specific Gene ◽  
Cartilage Formation

The aim of this study was to investigate the role of DNA methylation in the regulation of in vitro and in vivo cartilage formation. Based on the data of an RNA chip-assay performed on chondrifying BMP2-overexpressing C3H10T1/2 cells, the relative expression of Tet1 (tet methylcytosine dioxygenase 1), Dnmt3a (DNA methyltransferase 3) and Ogt (O-linked N-acetylglucosamine transferase) genes was examined with RT-qPCR in mouse cell-line based and primary micromass cultures. RNA probes for in situ hybridization were used on frozen sections of 15-day-old mouse embryos. DNA methylation was inhibited with 5-azacytidine during culturing. We found very strong but gradually decreasing expression of Tet1 throughout the entire course of in vitro cartilage differentiation along with strong signals in the cartilaginous embryonic skeleton. Dnmt3a and Ogt expressions did not show significant changes with RT-qPCR and gave weak in situ hybridization signals. Inhibition of DNA methylation applied during early stages of differentiation reduced cartilage-specific gene expression and cartilage formation. In contrast, it had stimulatory effect when added to differentiated chondrocytes. Our results indicate that the DNA demethylation-inducing Tet1 is a significant epigenetic factor of chondrogenesis, and inhibition of DNA methylation exerts distinct effects in different phases of in vitro cartilage formation.

Abstract 130: Hypercholesterolemia Suppresses Carotid Artery Endothelial NOS3 Expression via Epigenetic Mechanisms

2015 ◽  
Vol 35 (suppl_1) ◽  
Author(s):  
Alex Sotolongo ◽  
Yi-Zhou Jiang ◽  
John Karanian ◽  
William Pritchard ◽  
Peter Davies
Keyword(s):  
Gene Expression ◽  
Dna Methylation ◽  
Endothelial Cells ◽  
Dna Methyltransferase ◽  
Control Group ◽  
High Cholesterol Diet ◽  
Cholesterol Diet ◽  
High Cholesterol ◽  
High Fat

Objective: One of the first clinically detectable changes in the vasculature during atherogenesis is the accumulation of cholesterol within the vessel wall. Hypercholesterolemia is characterized by dysfunctional endothelial-dependent vessel relaxation and impaired NOS3 function. Since DNA methylation at gene promoter regions strongly suppresses gene expression, we postulated that high-fat/high-cholesterol diet suppresses endothelial NOS3 through promoter DNA methylation. Methods: Domestic male pigs were fed control diet (CD) or isocaloric high fat and high cholesterol diet (HC; 12% fat and 1.5% cholesterol) for 2, 4, 8 or 12 weeks prior to tissue collection. Furthermore, to determine the effects of risk factor withdrawal, an additional group of swine received HC for 12 weeks and then CD for 8 weeks; a control group received HC continuously for 20 weeks. Endothelial cells were harvested from common carotid aorta. In parallel in vitro studies, cultured human aortic endothelial cells (HAEC) were treated with human LDL, GW3956 (LXR agonist) and RG108 (DNA methyltransferase [DNMT] inhibitor). In cells from both sources, DNA methylation at the NOS3 promoter was measured using methylation specific pyro sequencing, and endothelial gene expression was measured using RT PCR. Results: HC diet increased plasma cholesterol level from 75 mg/dl on CD to a plateau of about 540 mg/dl within 2 weeks. Endothelial NOS3 expression was significantly reduced (71±9 % of CD) after 4 weeks of HC, a level sustained at subsequent time points. Withdrawal of HC for 8 weeks did not recover NOS3 expression. After 12-week HC, the NOS3 promoter was hypermethylated. Withdrawal of HC did not reverse NOS3 promoter methylation. In vitro treatment of HAEC with human LDL (200 mg/dl total cholesterol) or GW3956 (5μM) suppressed NOS3 mRNA to 50% and 30% respectively, suggesting that LXR/RXR is involved in suppression of NOS3. Nitric oxide production was consistently suppressed by GW3959. Both could be reversed through inhibition of DNMTs by RG108. Conclusions: DNA methylation and LXR/RXR pathway can mediate the HC-suppression of endothelial NOS3. The study identifies novel pharmaceutical targets in treating endothelial dysfunction. Crosstalk between these pathways is under investigation.

scholarly journals Unraveling the functional role of DNA methylation using targeted DNA demethylation by steric blockage of DNA methyltransferase with CRISPR/dCas9

2020 ◽  
Author(s):  
Daniel M. Sapozhnikov ◽  
Moshe Szyf
Keyword(s):  
Gene Expression ◽  
Dna Methylation ◽  
Promoter Region ◽  
Dna Methyltransferase ◽  
Dna Demethylation ◽  
New Method ◽  
Inducible Promoters ◽  
Specific Targeting ◽  
Main Effect

AbstractAlthough associations between DNA methylation and gene expression were established four decades ago, the causal role of DNA methylation in gene expression remains unresolved. Different strategies to address this question were developed; however, all are confounded and fail to disentangle cause and effect. We developed here a highly effective new method using only deltaCas9(dCas9):gRNA site-specific targeting to physically block DNA methylation at specific targets in the absence of a confounding flexibly-tethered enzymatic activity, enabling examination of the role of DNA methylation per se in living cells. We show that the extensive induction of gene expression achieved by TET/dCas9-based targeting vectors is confounded by DNA methylation-independent activities, inflating the role of DNA methylation in the promoter region. Using our new method, we show that in several inducible promoters, the main effect of DNA methylation is silencing basal promoter activity. Thus, the effect of demethylation of the promoter region in these genes is small, while induction of gene expression by different inducers is large and DNA methylation independent. In contrast, targeting demethylation to the pathologically silenced FMR1 gene targets robust induction of gene expression. We also found that standard CRISPR/Cas9 knockout generates a broad unmethylated region around the deletion, which might confound interpretation of CRISPR/Cas9 gene depletion studies. In summary, this new method could be used to reveal the true extent, nature, and diverse contribution to gene regulation of DNA methylation at different regions.

scholarly journals Effect of the proinflammatory cytokine TNF-α on intestinal riboflavin uptake: inhibition mediated via transcriptional mechanism(s)

2018 ◽  
Vol 315 (5) ◽  
pp. C653-C663 ◽  
Author(s):  
Kasin Yadunandam Anandam ◽  
Omar A. Alwan ◽  
Veedamali S. Subramanian ◽  
Padmanabhan Srinivasan ◽  
Rubina Kapadia ◽  
...  
Keyword(s):  
Ex Vivo ◽  
Significant Inhibition ◽  
Mrna Levels ◽  
In Vivo Models ◽  
Uptake Inhibition ◽  
Tnf Α ◽  
Vitro Model ◽  
Factor Α

Riboflavin (RF), is essential for normal cellular metabolism/function. Intestinal RF absorption occurs via a specific carrier-mediated process that involves the apical transporter RFVT-3 ( SLC52A3) and the basolateral RFVT-1 (SLC52A1). Previously, we characterized different cellular/molecular aspects of the intestinal RF uptake process, but nothing is known about the effect of proinflammatory cytokines on the uptake event. We addressed this issue using in vitro, ex vivo, and in vivo models. First, we determined the level of mRNA expression of the human (h)RFVT-3 and hRFVT-1 in intestinal tissue of patients with inflammatory bowel disease (IBD) and observed a markedly lower level compared with controls. In the in vitro model, exposing Caco-2 cells to tumor necrosis factor-α (TNF-α) led to a significant inhibition in RF uptake, an effect that was abrogated upon knocking down TNF receptor 1 (TNFR1). The inhibition in RF uptake was associated with a significant reduction in the expression of hRFVT-3 and -1 protein and mRNA levels, as well as in the activity of the SLC52A3 and SLC52A1 promoters. The latter effects appear to involve Sp1 and NF-κB sites in these promoters. Similarly, exposure of mouse small intestinal enteroids and wild-type mice to TNF-α led to a significant inhibition in physiological and molecular parameters of intestinal RF uptake. Collectively, these findings demonstrate that exposure of intestinal epithelial cells to TNF-α leads to inhibition in RF uptake and that this effect is mediated, at least in part, via transcriptional mechanism(s). These findings may explain the significantly low RF levels observed in patients with IBD.

scholarly journals Differential Regulation of β-Defensin Gene Expression during Cryptosporidium parvum Infection

2004 ◽  
Vol 72 (5) ◽  
pp. 2772-2779 ◽  
Author(s):  
Tarek K. Zaalouk ◽  
Mona Bajaj-Elliott ◽  
John T. George ◽  
Vincent McDonald
Keyword(s):  
Gene Expression ◽  
Cryptosporidium Parvum ◽  
Protozoan Parasite ◽  
Immune Defense ◽  
Microbial Pathogens ◽  
In Vivo Models ◽  
Defensin Gene ◽  
Intestinal Epithelia

ABSTRACT Invasion of enterocytes by pathogenic microbes evokes both innate and adaptive immune responses, and microbial pathogens have developed strategies to overcome the initial host immune defense. β-Defensins are potentially important endogenous antibiotic-like effectors of innate immunity expressed by intestinal epithelia. In this study, the interplay between the enteric protozoan parasite Cryptosporidium parvum and host epithelial β-defensin expression was investigated. Using human and murine models of infection, we demonstrated that C. parvum infection differentially regulates β-defensin gene expression. Downregulation of murine β-defensin-1 mRNA and protein was observed in both in vitro and in vivo models of infection. Infection of the human colonic HT29 cell line with the parasite resulted in differential effects on various members of the defensin gene family. Partial reduction in human β-defensin-1 (hBD-1), induction of hBD-2, and no effect on hBD-3 gene expression was observed. Recombinant hBD-1 and hBD-2 peptides exhibited significant antimicrobial activity against C. parvum sporozoites in vitro. These findings demonstrate that C. parvum infection of enterocytes may affect the expression of various defensins in different ways and suggest that the overall outcome of the effect of antimicrobial peptides on early survival of the parasite may be complex.

Effects of ten–eleven translocation 1 (Tet1) on DNA methylation and gene expression in chicken primordial germ cells

2019 ◽  
Vol 31 (3) ◽  
pp. 509 ◽  
Author(s):  
Minli Yu ◽  
Dongfeng Li ◽  
Wanyan Cao ◽  
Xiaolu Chen ◽  
Wenxing Du
Keyword(s):  
Gene Expression ◽  
Dna Methylation ◽  
Germ Cells ◽  
Dna Methyltransferase ◽  
Primordial Germ Cells ◽  
Dna Demethylation ◽  
Caveolin 1 ◽  
Expression Of Genes ◽  
Deleted In Azoospermia

Ten–eleven translocation 1 (Tet1) is involved in DNA demethylation in primordial germ cells (PGCs); however, the precise regulatory mechanism remains unclear. In the present study the dynamics of 5-methylcytosine (5mC) and 5-hydroxymethylcytosine (5hmC) in developing PGCs and the role of Tet1 in PGC demethylation were analysed. Results show that 5mC levels dropped significantly after embryonic Day 4 (E4) and 5hmC levels increased reaching a peak at E5–E5.5. Interestingly, TET1 protein was highly expressed during E5 to E5.5, which showed a consistent trend with 5hmC. The expression of pluripotency-associated genes (Nanog, PouV and SRY-box 2 (Sox2)) and germ cell-specific genes (caveolin 1 (Cav1), piwi-like RNA-mediated gene silencing 1 (Piwi1) and deleted in azoospermia-like (Dazl)) was upregulated after E5, whereas the expression of genes from the DNA methyltransferase family was decreased. Moreover, the Dazl gene was highly methylated in early PGCs and then gradually hypomethylated. Knockdown of Tet1 showed impaired survival and proliferation of PGCs, as well as increased 5mC levels and reduced 5hmC levels. Further analysis showed that knockdown of Tet1 led to elevated DNA methylation levels of Dazl and downregulated gene expression including Dazl. Thus, this study reveals the dynamic epigenetic reprogramming of chicken PGCs invivo and the molecular mechanism of Tet1 in regulating genomic DNA demethylation and hypomethylation of Dazl during PGC development.

3 IN VITRO MATURATION ALTERS GENE EXPRESSION IN MOUSE OOCYTES

2008 ◽  
Vol 20 (1) ◽  
pp. 82
Author(s):  
M. Paczkowski ◽  
C. Bidwell ◽  
D. Spurlock ◽  
J. Waddell ◽  
R. L. Krisher
Keyword(s):  
Gene Expression ◽  
Cell Death ◽  
Dna Methyltransferase ◽  
In Vitro Maturation ◽  
Fold Increase ◽  
Developmental Competence ◽  
Differentially Expressed ◽  
Dna Methyltransferase 1

The in vitro culture environment significantly impacts nuclear maturation, fertilization, embryonic development, and epigenetic competence; however, our knowledge of the effects of in vitro maturation on oocyte developmental competence, and specifically cytoplasmic maturation, is limited. The objective of this experiment was to identify alterations in the transcriptome of oocytes matured in vitro compared to those matured in vivo that correlate to developmental competence. Immature oocytes were collected from Day 26 and 7-8-week-old B6D2F1 mice 48 h post-pregnant mare serum gonadotropin (PMSG) administration and matured for 16 h in Gmat supplemented with 0.5 mm citric acid, 0.5 mm cysteamine, 100 ng mL–1 epidermal growth factor (EGF), 0.05% insulin-transferrin-selenium (ITS; v/v), 0.01% recombumin (v/v) and 2 mg mL–1 fetuin. In vivo-matured oocytes from females of the same ages were collected from the oviducts 62 h post-PMSG and 14 h post-hCG and mating to vasectomized males. In vivo- and in vitro-matured oocytes were identified visually by the presence of the first polar body. Mature oocytes were pooled into three groups of 150 oocytes per treatment and lysed; poly A+ RNA was extracted. Samples were processed through two cycles of linear amplification and hybridized to the GeneChip� Mouse Genome 430 2.0 Array (Affymetrix, Inc., Santa Clara, CA, USA), with three arrays per treatment. Microarray data were sorted and filtered to include genes that were classified as having two present calls per treatment. The data were then normalized to the chip median and analyzed using a one-way analysis of variance; the level of significance was calculated at P < 0.01. In total, 2.17% (482/22170) and 1.61% (358/22170) of genes were differentially expressed between in vitro- and in vivo-matured oocytes in Day 26 and 7–8-week-old mice, respectively. However, 72.82% (351/482) and 67.87% (243/358) of differentially expressed genes had increased abundance in the in vitro- and in vivo-matured oocytes, respectively. Transcripts involved in gene expression, cellular growth and proliferation, and cellular development were increased in in vivo-matured oocytes from both age groups compared to those matured in vitro. Cell death was one of the higher ranking functional groups increased in the 7–8-week-old in vitro-matured oocytes compared to the 7–8-week-old in vivo-matured oocytes. Specific genes altered by in vitro maturation conditions in Day 26 oocytes were DNA methyltransferase 1 (>7-fold increase in vivo), caspase 8 (>4-fold increase in vivo), and eukaryotic translation initiation factor 1B (>4-fold increase in vivo). DNA methyltransferase 1 and ubiquitin-conjugating enzyme E2T were significantly increased in in vivo-matured 7–8-week-old oocytes (>3-fold and >5-fold, respectively). These results indicate that gene expression is altered in oocytes matured in vitro compared to those matured in vivo. Based on the functional annotations of genes differentially expressed, dysregulation of gene expression in the oocyte resulting in altered DNA methylation and an up-regulation in cell death pathways are potential developmental mechanisms influenced by in vitro culture conditions that correlate to reduced embryonic developmental potential.

220 THE ESTROGEN-LIKE EFFECT OF 2-METHOXYESTRADIOL (2-ME) IN IN VITRO AND IN VIVO MODELS

2015 ◽  
Vol 27 (1) ◽  
pp. 200
Author(s):  
J.-S. Lee ◽  
E.-B. Jeung
Keyword(s):  
Mrna Expression ◽  
Therapeutic Effects ◽  
Mrna Levels ◽  
Oestrogen Receptors ◽  
In Vivo Models ◽  
Ru 486 ◽  
Final Injection ◽  
M 10

2-Methoxyestradiol (2-ME), an endogenous metabolite of 17β-oestradiol, interacts with oestrogen receptors and microtubules and has a low affinity for oestrogen receptors (ER). It has attracted considerable interest due to its potential anti-cancer therapeutic effects. 2-ME is also recognised for its unique and profound actions on various tumour cell lines and cancer independent of the hormone receptor status. Regardless of differences in function, 2-ME has an affinity for ER, however, the exact mechanisms of 2-ME action via the ER are not fully understood. In the current study, we examined the estrogenic effect of 2-ME on mRNA levels of CaBP-9k, ER, and progesterone receptor (PR) in the absence or presence of the 17β-oestradiol (E2) and progesterone (P4) in both in vivo and in vitro models by real-time RT–PCR. In vitro, cells (n = 3 per group) were exposed to a single dose of E2 (10–9 M), P4 (10–6 M), 2-ME (10–8 M, 10–7 M, 10–6 M). The mechanism of CaBP-9k induction by these chemicals pre-treated with 10–7 M ICI 182, 780 and 10–6 M RU 486 for 30 min before exposure to E2 and 2-ME were analysed. In vivo, 35 female ICR mice (PND 14 days) were divided into 7 groups (n = 5 per group), and each group was administered subcutaneously with 24% DMSO, 38% ethanol, and 38% sterile saline as a vehicle, E2 [40 μg kg–1 of body weight (BW)] a physiological dose level), 2-ME (4, 40, and 80 mg kg–1 of BW) for 3 days. The mice were killed 24 h after the final injection. To investigate the effect of antagonism, 10 mice were injected SC with ICI 182 780 (10 mg kg–1 of BW) and RU 486 (10 mg kg–1 of BW) at 30 min before injection with 2-ME (40 mg kg–1 of BW) for 3 days and killed 24 h after the final injection. Results are presented as mean ± s.e.m.; P-values were calculated using one-way ANOVA. In GH3 cells, the mRNA level of CaBP-9k was induced in the E2 (10–9 M) treatment group, and expression of CaBP-9k was also up-regulated in the 2-ME (10–7 M)-treated group. Uterine lactoferrin (Ltf) mRNA expression was also increased in the 2-ME (40 mg kg–1 of BW) group, similar to the response with E2 (40 μg kg–1 of BW) in mice. As a blocker for ER and PR activity, ICI 182 780 and RU 486 reversed the E2 or 2-ME mediated increase of CaBP-9k and Ltf mRNA expression. We found that 2-ME significantly increased the levels of ERa and PR transcripts. In parallel with in vitro results, the mRNA levels of ERa and PR were induced by treatment with E2 and 2-ME. Taken together, our findings demonstrated that expression of estrogenic markers, CaBP-9k and Ltf, was regulated by 2-ME in both in vitro and in vivo, which may increase their estrogenic activities in female during the cycle through ER and/or PR-mediated pathway.

122 SUPPRESSION OF EPIGENETIC MODIFIERS ALTERS THE BOVINE EMBRYONIC DEVELOPMENTAL PROGRAM DURING IN VITRO CULTURE

2014 ◽  
Vol 26 (1) ◽  
pp. 175 ◽  
Author(s):  
M. D. Snyder ◽  
J. H. Pryor ◽  
M. D. Peoples ◽  
G. L. Williamson ◽  
M. C. Golding ◽  
...  
Keyword(s):  
Gene Expression ◽  
Dna Methylation ◽  
Embryonic Development ◽  
Dna Methyltransferase ◽  
Test Statistic ◽  
Gene Target ◽  
Blastocyst Rate ◽  
Green Fluorescent ◽  
Fluorescent Dextran

During early bovine embryogenesis, the regular establishment of DNA methylation and histone modification patterns is essential for proper gene expression and continuation of embryonic development. Epigenome patterns established during this period, if improperly maintained, can lead to developmental anomalies and may partially explain the lower pregnancy rates of in vitro-produced embryos. We hypothesised that the suppression of translation of the genes euchromatic histone-lysine N-methyltransferase 2 (EHMT2), DNA methyltransferase 3A (DNMT3A), absent, small, or homeotic-like (ASH2L), and SET domain, bifurcated 1 (SETDB1) would provide insightful information on the importance of these genes during early embryonic development in an in vitro setting. In order to define the roles of these genes, small interfering RNA (siRNA) targeting the gene of interest were synthesised and target verified in bovine cell culture using quantitative real-time RT-PCR (RT-qPCR). We acquired matured bovine oocytes from commercial suppliers, followed by IVF by standard laboratory procedures. Eighteen hours post IVF, cumulus cells were removed and zygotes separated into 3 different treatment groups: non-injected controls (CNTL), non-targeting siRNA injected controls (siNULL), and injection with siRNA targeting the gene of interest (si “gene target”). Each siRNA was mixed with a green fluorescent dextran at a concentration of 20 μM and ~100 pL injected cytoplasmically. The green fluorescent dextran was used to give visual confirmation that zygotes were indeed injected. Post-injection, fluorescent embryos were separated and cultured in Bovine Evolve (Zentih Biotech) medium supplemented with 4 mg mL–1 of BSA (Probumin, Millipore). Cleavage rates were monitored on Day 2, and only cleaved embryos were cultured further. On Day 8 post-IVF, embryos were morphologically examined and numbers of blastocysts recorded. Mean development rates between siNULL and targeting siRNA were compared using a t-test statistic. Over the course of these experiments the mean blastocyst rate for CNTL zygotes was 34.5% ± 2.6 s.e.m. (n = 1647). None of the zygotes injected with siEHMT2 (n = 1184) or siSETDB1 (n = 361) reached the blastocyst stage and these rates differed from the siNULL rate (21.0% ± 2.5 s.e.m., n = 1587; P < 0.05). Morphologically, embryos from both groups developed to the morula stage before they exhibited fragmentation. Injection of siDNMT3A also resulted in significant loss of viability at the 8-cell stage and few zygotes injected (n = 1057) developed to blastocyst (2.1% ± 0.5 s.e.m.; P < 0.001). Inhibiting gene expression of ASH2L showed little variation in blastocyst rate from our siNULL embryos (31.3% ± 2.0 s.e.m., n = 466 v. 34.8% ± 1.9 s.e.m., n = 418, respectively, P > 0.2). It is unknown at this time if inhibition of ASH2L translation will have effects later in development. Ongoing experiments analysing DNA methylation and histone modifications through immunocytochemistry and global gene expression via RT-qPCR will further explore the establishment and maintenance of these genes in the embryonic epigenome.

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