scholarly journals 236 SPATIAL GENE EXPRESSION PATTERNS OF Dnmt1, Dnmt3a, AND Hdac2 IN PREIMPLANTATION EMBRYOS

2005 ◽  
Vol 17 (2) ◽  
pp. 268
Author(s):  
C. Wrenzycki ◽  
D. Herrmann ◽  
A. Lucas-Hahn ◽  
K. Korsawe ◽  
E. Lemme ◽  
...  
Keyword(s):  
Dna Methylation ◽  
Relative Abundance ◽  
Expression Patterns ◽  
Dna Methyltransferases ◽  
Histone Deacetylation ◽  
Preimplantation Embryos ◽  
Differential Staining ◽  
Rt Pcr

In mammals, DNA methylation and the modification of histones account for the major epigenetic alterations. Usually DNA methylation is associated with transcriptional silencing, which is catalyzed by two important classes of DNA methyltransferases. DNA methyltransferase 1 (Dnmt1), a maintenance enzyme, methylates hemi-methylated DNA after DNA replication. Dnmt3a and Dnmt3b are required for de novo methylation in vivo and for establishing new DNA methylation marks during development. In addition to DNA methylation, post-translational modifications of the amino termini of core histones are thought to affect the expression or repression of transcription. Histone deacetylation catalyzsed by histone deacetylases (Hdac) often results in transcriptional repression. Perturbated epigenetic reprogramming of the genome is a likely cause of developmental abnormalities and epigenetic diseases introduced by assisted reproduction technologies. The objective of the present study was to determine the relative abundance of Dnmt1, Dnmt3a, and Hdac2 transcripts in ICM and TE cells of pre-implantation bovine embryos of different origin. Embryos were generated with standard protocols of in vitro production (IVP) and parthenogenetic activation using SOF medium supplemented with BSA (SOF d7/d8, Parth d7/d8) or TCM medium supplemented with estrus cow serum (TCM d7/d8; Wrenzycki et al. 2001 Hum. Reprod. 16, 893–901; Wrenzycki et al. 2002 Biol. Reprod. 66, 127–134). Expanded blastocysts were collected at Days 7 and Day 8 (d7/d8) of culture (Day 0 = IVF). In vivo-generated blastocysts collected from superovulated animals were included as controls (Wrenzycki et al. 1996 J. Reprod. Fert. 108, 17–24). A highly sensitive RT-PCR assay (Wrenzycki et al. 2003 Biol. Reprod. 68, 2073–2080) was used to determine the relative abundance (RA) of gene transcripts in isolated ICM and TE cells. The allocation of ICM and TE cells was analyzed using a differential staining technique (Eckert and Niemann 1998 Mol. Hum. Reprod. 4, 957–965) to calculate the RA of each transcript on a per cell basis. RT-PCR assays were repeated at least six times. Significant differences in the RA of Dnmt1 (In vivo, TCM d7, Parth d7/d8, SOF d8), Dnmt3a (In vivo, SOF d7), and Hdac2 (TCM d7) transcripts were detected between ICM and TE cells. No differences were detected for all transcripts in ICM cells of embryos collected at either Day 7 or Day 8. In TE cells, the RA of Dnmt1 transcripts was significantly reduced or increased in embryos generated in SOF or TCM medium at Day 7, respectively, and the RA of Hdac2 transcripts was significantly higher in embryos generated in TCM medium at Day 8. These results suggest that in vitro culture alters the spatial expression pattern of genes related to epigenetic modifications in the cell lineages critical for a normal embryonic and fetal development. This work was supported by the Deutsche Forschungsgemeinschaft (DFG Ni 256/18-1).

180 IDENTIFICATION AND QUANTIFICATION OF DIFFERENTIALLY REPRESENTED TRANSCRIPTS IN PREIMPLANTATION BOVINE EMBRYOS

2008 ◽  
Vol 20 (1) ◽  
pp. 169 ◽  
Author(s):  
C. E. McHughes ◽  
G. K. Springer ◽  
L. D. Spate ◽  
R. Li ◽  
R. J. Woods ◽  
...  
Keyword(s):  
Relative Abundance ◽  
Nuclear Transfer ◽  
Developmental Stages ◽  
Reproductive Technologies ◽  
Blastocyst Stage ◽  
Preimplantation Embryos ◽  
Cell Stage ◽  
Production Techniques

Identification of transcripts that are present at key development stages of preimplantation embryos is critical for a better understanding of early embryogenesis. To that end, this project had two goals. The first was to characterize the relative abundance of multiple transcripts during several developmental stages, including metaphase II-stage oocytes (MPII), and 2-cell-stage (2-cell), precompact morula (PCM), and in vitro-produced blastocyst-stage (IVTBL) embryos. The second was to characterize differences in the relative abundance of transcripts present in in vivo- (IVVBL), in vitro-, and nuclear transfer-produced (NTBL) blastocysts. It was our hypothesis that the identification of differentially represented transcripts from these stages would reveal not only developmentally important genes, but also genes that might be aberrantly expressed due to embryo production techniques. Individual clusters from a large bovine EST project (http://genome.rnet.missouri.edu/Bovine/), which focused on female reproductive tissues and embryos, were compared using Fisher's exact test weighted by number of transcripts per tissue by gene (SAS PROC FREQ; SAS Institute, Inc., Cary, NC, USA). Of the 3144 transcripts that were present during embryogenesis, 125 were found to be differentially represented (P < 0.01) in at least one pairwise comparison (Table 1). Some transcripts found to increase in representation from the MPII to the 2-cell stage include protein kinases, PRKACA and CKS1, as well as the metabolism-related gene, PTTG1. These same transcripts were also found to decrease in representation from the 2-cell to the PCM stage. RPL15 (translation) and FTH1 (immune function) were both more highly represented in the PCM than in the 2-cell stage. From PCM to IVTBL, we saw an increase in RPS11, another translation-related transcript. When comparing blastocyst-stage embryos from different production techniques, several transcripts involved in energy production (e.g., COX7B and COX8A) were found to be more highly represented in the NTBL than in the IVTBL. COX8A was also more highly represented in the IVVBL than in the IVTBL. By investigating these differentially represented transcripts, we will be able to better understand the developmental implications of embryo manipulation. We may also be able to better develop reproductive technologies that lead to in vitro- and nuclear transfer-derived embryos which more closely follow a normal program of development. Table 1. Differentially represented transcripts between developmental stages

scholarly journals Developmental and molecular correlates of bovine preimplantation embryos

Reproduction ◽  
2006 ◽  
Vol 131 (5) ◽  
pp. 895-904 ◽  
Author(s):  
Hakan Sagirkaya ◽  
Muge Misirlioglu ◽  
Abdullah Kaya ◽  
Neal L First ◽  
John J Parrish ◽  
...  
Keyword(s):  
Gene Expression ◽  
Dna Methyltransferase ◽  
Expression Profiles ◽  
Expression Patterns ◽  
Blastocyst Stage ◽  
Culture Conditions ◽  
Preimplantation Embryos ◽  
Developmental Potential

Expression of embryonic genes is altered in different culture conditions, which influence developmental potential both during preimplantation and fetal development. The objective of this study was to define the effects of culture conditions on: bovine embryonic development to blastocyst stage, blastocyst cell number, apoptosis and expression patterns of a panel of developmentally important genes. Bovine embryos were culturedin vitroin three culture media containing amino acids, namely potassium simplex optimization medium (KSOMaa), Charles Rosenkrans 1 (CR1aa) and synthetic oviductal fluid (SOFaa). Apoptosis in blastocysts was determined by TUNEL assay and expression profiles of developmentally important genes were assayed by real-time PCR.In vivo-produced bovine blastocysts were used as controls for experiments determining gene expression patterns. While the cleavage rates did not differ, embryos cultured in SOFaa had higher rates of development to blastocyst stage (P< 0.05). Mean cell numbers and percentages of apoptotic cells per blastocyst did not differ among the groups. Expression of the heat shock protein 70 (Hsp70) gene was significantly up-regulated in both CR1aa and KSOMaa when compared with SOFaa (P< 0.001). DNA methyltransferase 3a (Dnmt3a) expression was higher in embryos cultured in CR1aa than in those cultured in SOFaa (P< 0.001). Expression of interferon tau (IF-τ) and insulin-like growth factor II receptor (Igf-2r) genes was significantly up-regulated in KSOMaa when compared with CR1aa (P< 0.001). Gene expression did not differ betweenin vivo-derived blastocysts and theirin vitro-derived counterparts. In conclusion, SOFaa supports higher development to blastocyst stage than KSOMaa and CR1aa, and the culture conditions influence gene expression.

scholarly journals SIRT6 Promotes Osteogenic Differentiation of Adipose-Derived Mesenchymal Stem Cells Through Antagonizing DNMT1

2021 ◽  
Vol 9 ◽  
Author(s):  
Bo Jia ◽  
Jun Chen ◽  
Qin Wang ◽  
Xiang Sun ◽  
Jiusong Han ◽  
...  
Keyword(s):  
Dna Methylation ◽  
Stem Cells ◽  
Notch Signaling ◽  
Osteogenic Differentiation ◽  
Dna Methyltransferases ◽  
Luciferase Reporter ◽  
Calcium Deposition ◽  
Alizarin Red

BackgroundAdipose-derived stem cells (ADSCs) are increasingly used in regenerative medicine because of their potential to differentiate into multiple cell types, including osteogenic lineages. Sirtuin protein 6 (SIRT6) is a nicotinamide adenine dinucleotide (NAD)-dependent deacetylase that plays important roles in cell differentiation. NOTCH signaling has also been reported to involve in osteogenic differentiation. However, the function of SIRT6 in osteogenic differentiation of ADSCs and its relation to the NOTCH signaling pathways are yet to be explored.MethodsThe in vitro study with human ADSCs (hADSCs) and in vivo experiments with nude mice have been performed. Alkaline phosphatase (ALP) assays and ALP staining were used to detect osteogenic activity. Alizarin Red staining was performed to detect calcium deposition induced by osteogenic differentiation of ADSCs. Western blot, RT-qPCR, luciferase reporter assay, and co-immunoprecipitation assay were applied to explore the relationship between of SIRT6, DNA methyltransferases (DNMTs) and NOTCHs.ResultsSIRT6 promoted ALP activity, enhanced mineralization and upregulated expression of osteogenic-related genes of hADSCs in vitro and in vivo. Further mechanistic studies showed that SIRT6 deacetylated DNMT1, leading to its unstability at protein level. The decreased expression of DNMT1 prevented the abnormal DNA methylation of NOTCH1 and NOTCH2, resulting in the upregulation of their transcription. SIRT6 overexpression partially suppressed the abnormal DNA methylation of NOTCH1 and NOTCH2 by antagonizing DNMT1, leading to an increased capacity of ADSCs for their osteogenic differentiation.ConclusionThis study demonstrates that SIRT6 physical interacts with the DNMT1 protein, deacetylating and destabilizing DNMT1 protein, leading to the activation of NOTCH1 and NOTCH2, Which in turn promotes the osteogenic differentiation of ADSCs.

scholarly journals Expression of RONIN and NANOG-associated proteins in goat parthenogenetic embryos

2017 ◽  
Vol 11 (2) ◽  
pp. 145 ◽  
Author(s):  
Marcelo Tigre Moura ◽  
Pamela Ramos-Deus ◽  
José Carlos Ferreira-Silva ◽  
Priscila Germany Corrêa Silva ◽  
Ludymila Furtado Cantanhêde ◽  
...  
Keyword(s):  
Transcription Factors ◽  
Expression Patterns ◽  
Preimplantation Embryos ◽  
Rt Pcr ◽  
Cleavage Stage ◽  
Specific Expression ◽  
Early Embryos ◽  
Associated Proteins ◽  
Gene Transcripts

The expression of a subset of transcription factors is enriched in early preimplantation embryos, which contributes to their cellular plasticity. RONIN, NANOG and its associated proteins are PluripotencyAssociated Transcription Factors (PATF) that control relevant downstream pathways in pluripotent stem cells, but their activity in early embryos remained less understood. The work was aimed to determine the expression of RONIN and four NANOG-associated PATFs in goat preimplantation embryos. Goat embryos were produced in vitro by parthenogenetic activation. Gene transcripts of cleavage-stage embryos were investigated by reverse transcriptase-polymerase chain reaction (RT-PCR), while blastocysts were analyzed by both RTPCR and quantitative RT-PCR (RT-qPCR) assays. Gene transcripts of ZFP281, NAC1, and NR0B1 were detected in cleavage-stage embryos, while RONIN and OCT4 were not found expressed. Detection in blastocysts by RT-PCR confirmed the activity of NR0B1, RONIN, and OCT4. Moreover, all five PATF were detected in blastocysts by RT-qPCR (ZFP281, NAC1, RONIN, OCT4, and NR0B1). In conclusion, RONIN and NANOG-associated proteins are active during goat parthenogenetic preimplantation development and hold stage-specific expression patterns.

258 MESSENGER RNA EXPRESSION PATTERNS OF HISTONE MODIFICATION GENES IN BOVINE EMBRYOS DERIVED FROM DIFFERENT ORIGINS

2006 ◽  
Vol 18 (2) ◽  
pp. 236 ◽  
Author(s):  
M. Nowak-Imialek ◽  
C. Wrenzycki ◽  
D. Herrmann ◽  
I. Lagutina ◽  
A. Lucas-Hahn ◽  
...  
Keyword(s):  
Histone Deacetylase ◽  
Relative Abundance ◽  
Transcriptional Activation ◽  
Messenger Rna ◽  
Expression Patterns ◽  
Rna Expression ◽  
Bovine Embryos ◽  
Different Origins

Epigenetic modifications of the genome, such as covalent modifications of histones, are crucial for transcriptional regulation during development. The N-terminal tails of the histones are subject to post-translational modifications, including acetylation, deacetylation and methylation. histone acetylation loosens chromatin packing and correlates with transcriptional activation. The enzymes Histone acetyltransferases (HATs) transfer acetyl moieties to the lysine residues of histones H2A, H2B, H3, and H4. Histone acetylation is a reversible process which is catalyzed by the histone deacetylase (HDAC) and results in transcriptional repression. Histone methyltransferase (HMT) is responsible for the methylation of arginine in histones 3 and 4, playing an important role in transcriptional activation of genes. In contrast, the H3 Lys 9 methylation is associated with a transcriptionally repressive heterochromatin. The objective of the present study was to determine the effects of different origins of embryos on the relative abundance of transcripts for the histone acetyltransferase 1 (HAT1), histone deacetylase 2 (HDAC2), histone metyltransferases (SUV39H1 and G9A), and heterochromatin protein 1 (HP1). Messenger RNA expression profiles of these genes were investigated in bovine oocytes and pre-implantation embryos up to the blastocyt stage produced either in vitro by two different culture systems, i.e. SOF+BSA or TCM+SERUM, by somatic cloning using adult male and female fibroblasts, parthenogenetic activation, and androgenetic construction, or in vivo, employing semiquantitative reverse transcription-polymerase chain reaction (RT-PCR). Significant differences are described below. HAT1, SUV39H1, G9A, and HP1 mRNA transcripts decreased in enucleated oocytes, compared with immature oocytes. The relative abundance of HAT1 and SUV39H1 transcripts was significantly increased in NT-derived zygotes produced from adult female fibroblasts, compared to their in vitro fertilized and parthenogenetic counterparts. No differences were found in the relative abundances of gene transcripts at the 8-16-cell stage, except for parthenogenetic embryos in which SUV39H1 transcripts were significantly higher than in all other 8-16 cell groups. The relative abundance of SUV39H1, G9A, and HP1 transcripts were significantly higher in NT-derived blastocysts derived from adult male fibroblasts than in their in vivo-generated counterparts. HP1 and G9A transcript levels were significantly increased in NT-derived blastocysts derived from male fibroblasts compared to NT-derived embryos produced from female fibroblasts. The results show that the in vitro environment and the nuclear transfer protocol affect mRNA expression patterns of histone modification genes in pre-implantation bovine embryos.

247 MESSENGER RNA EXPRESSION PATTERNS OF DNA AND HISTONE METHYLTRANSFERASES IN PREIMPLANTATION DEVELOPMENT OF IN VIVO- AND IN VITRO-PRODUCED BOVINE EMBRYOS

2006 ◽  
Vol 18 (2) ◽  
pp. 231 ◽  
Author(s):  
K. Höffmann ◽  
H. Niemann ◽  
K.-G. Hadeler ◽  
D. Herrmann ◽  
C. Wrenzycki
Keyword(s):  
Developmental Stage ◽  
Mrna Expression ◽  
Relative Abundance ◽  
Expression Patterns ◽  
Bovine Embryos ◽  
Histone Methyltransferases ◽  
Embryonic Genome ◽  
Uterine Flushing

The effects of in vitro production (IVP) and/or somatic nuclear transfer on mRNA expression patterns have mostly been determined in morulae and blastocysts, i.e. after embryonic genome activation. Comparative data regarding mRNA expression patterns throughout the oviductal phase of pre-implantation development are scarce. Here we studied mRNA expression for genes related to DNA methylation and modification of histones which account for the major epigenetic reprogramming during development. Pertubated epigenetic reprogramming of the genome is a likely cause of developmental abnormalities and epigenetic diseases associated with assisted reproduction technologies. The objective of the present study was to compare mRNA expression of DNA methyltransferases Dnmt1, -3a, and -3b and histone methyltransferases SUV39-h1 and G9a between in vivo-derived bovine embryos and their IVP counterparts using a semiquantitative RT-PCR assay (Wrenzycki et al. 2002 Biol. Reprod. 66, 127-134) employing two embryos for each assay. In vivo-derived embryos were collected from 28 superovulated heifers by endoscopic flushing of oviducts (zygotes to 8-cell stages) (Besenfelder et al. 2001 Theriogenology 55, 837-845) or by uterine flushing (16-cell stages to blastocysts). Endoscopic flushing at different time points after AI (Days 1, 1.5, 2, 3, 4, and 4.5) yielded 31 zygotes; 15 two-cell, 5 three-cell, 13 four-cell, 1 five-cell, 2 six-cell, and 11 eight-cell embryos; 4 degenerated embryos; and 18 unfertilized ova. The recovery rate (corpora lutea counted per recovered embryos) was 58% and 62% for the endoscopic and uterine flushing, respectively. Differences in the relative abundance of each gene transcript between groups were tested using ANOVA with the main effects being origin (in vivo/in vitro) and developmental stage (zygote to blastocyst) and their interactions followed by multiple pairwise comparisons using a Tukey test (P < 0.05). Origin of embryos affected the relative abundance of transcripts for Dnmt1, Dnmt3a, and SUV39-h1, and developmental stage affected the relative abundance of transcripts for Dnmt1, -3a, -3b, SUV39-h1, and G9a. No interactive effects were observed for origin and developmental stage in the relative abundance of all transcripts. The relative abundance of Dnmt1 transcripts differed significantly between in vivo- and in vitro-produced morulae and blastocysts. For Dnmt3a, mRNA differences were determined between in vivo- and in vitro-produced 10-16-cell stages and morulae. Suv39-h1 transcripts differed significantly between in vivo- and in vitro-derived zygotes, 2-cell embryos, 8-cell embryos, 10-16-cell embryos, and blastocysts. The results suggest that IVP alters mRNA expression of genes related to epigenetic modifications very early in development, even before the embryonic genome has been activated.

199 IN VITRO-DEVELOPED BOVINE BLASTOCYSTS ARE MARKED WITH ABERRANT HYPER- AND HYPO-METHYLATED GENOMIC REGIONS

2015 ◽  
Vol 27 (1) ◽  
pp. 190
Author(s):  
D. Salilew-Wondim ◽  
M. Hoelker ◽  
U. Besenfelder ◽  
V. Havlicek ◽  
F. Rings ◽  
...  
Keyword(s):  
Gene Expression ◽  
Dna Methylation ◽  
Expression Patterns ◽  
Proximal Promoter ◽  
Altered Gene Expression ◽  
Genome Wide ◽  
Altered Gene ◽  
Genomic Regions

Most often, in vitro produced embryos display poor quality and altered gene expression patterns compared to their in vivo counterparts. Aberrant DNA methylation occurring during in vitro embryo development is believed to be one of the multifaceted factors which may cause altered gene expression and poor embryo quality. Here, we investigated the genome-wide DNA methylation patterns of in vitro derived embryos using the recently developed Bovine EmbryoGENE Methylation Platform (BEGMP) array (Shojaei Saadi et al. BMC Genomics 2014 15, 451. doi: 10.1186/1471-2164-15-451) to unravel the aberrantly methylated genomic region in in vitro developed embryos. For this, in vitro and in vivo produced blastocysts were produced and used for genome-wide DNA methylation analysis. In vitro blastocysts were produced from oocytes retrieved from ovaries collected from the local abattoir and matured, fertilized, and cultured in vitro using SOF media. The in vivo blastocysts were produced by superovulation and AI of Simmental heifers followed by uterine flushing. Genomic DNA (gDNA) was then isolated from four replicates (each 10 blastocysts) of in vivo and in vitro derived blastocysts using Allprep DNA/RNA micro kit (Qiagen, Valencia, CA, USA) and the gDNA was then fragmented using the MseI enzyme. Following this, MseLig21 and MseLig were ligated to the MseI-digested genomic fragments in the presence of Ligase enzyme. Methyl-sensitive enzymes, HpaII, AciI, and Hinp1I, were used to cleave unmethlayted genomic regions within the MseI-MseI region of the fragmented DNA. The gDNA was subjected to two rounds of ligation-mediated polymerase chain reaction (LM-PCR) amplification. After removal of the adapters, the amplified gDNA samples from in vivo or in vitro groups were labelled either Cy-3 or Cy-5 dyes in dye-swap design using ULS Fluorescent gDNA labelling kit (Kreatech Biotechnology BV, Amsterdam, The Netherlands). Hybridization was performed for 40 h at 65°C. Slides were scanned using Agilent's High-Resolution C Scanner (Agilent Technologies Inc., Santa Clara, CA, USA) and features were extracted with Agilent's Feature Extraction software (Agilent Technologies Inc.). The results have shown that from a total of 414 566 probes harboured by the BEGMP array, 248 453 and 253 147 probes were detected in in vitro and in vivo derived blastocysts, respectively. Data analysis using the linear modelling for microarray (LIMMA) package and R software (The R Project for Statistical Computing, Vienna, Austria) revealed a total of 3434 differentially methylated regions (DMRs; Fold change ≥1.5, P-value <0.05), of which 42 and 58% were hyper- and hypo-methylated, respectively, in in vitro derived blastocysts compared to their in vivo counterparts. The DMRs were found to be localised in the intronic, exonic, promoter, proximal promoter, and distal promoter, and some of the probes did not have nearby genes. In addition, 10.8% of the DMRs were found to be stretched in short, long, or intermediate CpG islands. Thus, this study demonstrated genome-wide dysregulation in the epigenome landscape of in vitro-derived embryos by the time they reach to the blastocysts stage.

38 DNA METHYLATION IN PORCINE PREIMPLANTATION EMBRYOS DEVELOPED IN VIVO AND PRODUCED BY IN VITRO FERTILIZATION, PARTHENOGENETIC ACTIVATION, AND SOMATIC CELL NUCLEAR TRANSFER

2011 ◽  
Vol 23 (1) ◽  
pp. 125
Author(s):  
R. S. Deshmukh ◽  
O. Oestrup ◽  
E. Oestrup ◽  
M. Vejlsted ◽  
H. Niemann ◽  
...  
Keyword(s):  
Dna Methylation ◽  
Somatic Cell ◽  
Somatic Cell Nuclear Transfer ◽  
Nuclear Transfer ◽  
Cell Mass ◽  
Epifluorescence Microscopy ◽  
Preimplantation Embryos ◽  
Parthenogenetic Activation

DNA de- and re-methylation are crucial for reprogramming of the differentiated parental/somatic genome in the ooplasm. The presented research was aimed at analysis of the DNA methylation dynamics in porcine preimplantation embryos developed in vivo (IV) and produced in vitro by IVF, somatic cell nuclear transfer (SCNT), and parthenogenetic activation (PA). Embryos of early and late 1-cell, 2-, 4-, and 8-cell, and early and late blastocysts stages obtained by the mentioned methods were fixed in 4% paraformaldehyde and subjected to immunocytochemistry using anti-5MetC (Mouse monoclonal, Abcam, Cambridge, MA, USA) antibody. DNA was labelled using Hoechst 33258 (Sigma, Copenhagen, Denmark). Epifluorescence microscopy (Leica Microsystems, Wetzlar, Germany) images were subjected to NIH imageJ software to measure the DNA methylation/DNA content signal by manually outlining the nuclei (n = 2003) of the embryos. The data were analysed using PROC-GLM statistical procedure in SAS 9.1 (SAS Institute Inc., Cary, NC, USA), least square means were compared and P-values were used to decide the significant differences within and between different groups of embryos. The 1-cell stages lacked active demethylation of paternal genome in IV and IVF embryos. Embryos produced under in vitro conditions presented higher levels of DNA methylation than IV. A lineage specific DNA methylation (hypermethylation of inner cell mass and hypomethylation of trophectoderm) observed in porcine IV late blastocysts was absent in PA and SCNT blastocysts despite the occurrence of de novo methylation in early blastocysts. SCNT early (50%) and late (14%) blastocysts presented DNA methylation pattern similar to IV early and late blastocysts, respectively. Concluding, DNA methylation patterns are strongly impaired under in vitro conditions in porcine preimplantation embryos.

69 BLASTOCYSTS DEVELOPED FROM EMBRYOS THAT SPENT UP TO 2-CELL STAGE IN VIVO EXHIBITED MASSIVE DNA METHYLATION DYSREGULATION INCLUDING IMPRINTED GENES AND DNA METHYLTRANSFERASES

2017 ◽  
Vol 29 (1) ◽  
pp. 142 ◽  
Author(s):  
D. Salilew-Wondim ◽  
M. Hoelker ◽  
U. Besenfelder ◽  
V. Havlicek ◽  
E. Held ◽  
...  
Keyword(s):  
Dna Methylation ◽  
Culture Condition ◽  
Dna Methyltransferases ◽  
Cell Stage ◽  
Embryonic Genome ◽  
Genomic Regions ◽  
Methylation Patterns ◽  
Genome Activation

Suboptimal culture condition before minor or major genome activation is believed to affect the quality and the transcriptome landscape of the resulting blastocysts. Thus, we hypothesised that exposure of bovine embryos to suboptimal culture condition before minor embryonic genome activation could affect the genome methylation patterns of the resulting blastocysts. Therefore, here we aimed to investigate the genome wide DNA methylation patterns of blastocysts derived from embryos developed up to 2-cell stages in vivo followed by in vitro culture. For this, Simmental heifers were superovulated and artificially inseminated. The 2-cell stage embryos were then flushed using a state-of-the-art nonsurgical endoscopic early-stage embryo flushing technique and in vitro cultured until the blastocyst stage. The DNA methylation patterns of these blastocysts were then determined with reference to blastocysts derived from embryos developed completely under in vivo condition. For this, the genomic DNA isolated from each blastocyst group were fragmented, and unmethylated genomic regions were cleaved using methylation sensitive restriction enzymes. The samples were then amplified using ligation mediated PCR and labelled either with Cy-3 or Cy-5 dyes in a dye-swap design using the ULS Fluorescent genomic DNA labelling kit (Kreatech Biotechnology) and hybridized on an EmbryoGENE DNA Methylation Array as described previously (Saadi 2014 BMC Genomics 15, 451; Salilew-Wondim 2015 PLoS ONE 10, e0140467). Array hybridization was performed for 40 h at 65°C, and 4 hybridizations were preformed to represent 4 biological replicates. The slides were scanned using Agilent’s High-Resolution C Scanner (Agilent Technologies, Santa Clara, CA, USA), and Agilent’s Feature Extraction software (Agilent Technologies) was used to extract data features. Differentially methylated regions with fold change ≥1.5 and P-value < 0.05 were identified using linear modelling for microarray and R software. The results have shown that including imprinted genes (PEG3, IGF1, RASGRF1, IGF2R, GRB10, SNRPN, and PLAGL1) and DNA methyltransferases (DNMT1, DNMT3A, and DNMT3B), a total of 10,388 genomic regions were differentially methylated, of which 6393 genomic regions were hypermethylated in blastocysts derived from 2-cell flush compared with the complete vivo group. In addition, comparative analysis of the current DNA methylation data with our previous transcriptome profile data have shown that including DNMT3A, CTSZ, ElF3E, and PPP2R2B, the expression patterns of 603 genes was inversely correlated with the methylation patterns. Moreover, canonical pathways including gap junction, adherens junction, axon guidance, focal adhesion, and calcium signalling were affected by differentially methylated regions. Therefore, this study indicated that exposure of embryos to suboptimal culture condition before embryonic genome activation would lead to a massive dysregulation of methylation pattern of genes involved in developmentally relevant pathways in the resulting blastocysts.

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