scholarly journals Down-Regulation of H3K4me3 by MM-102 Facilitates Epigenetic Reprogramming of Porcine Somatic Cell Nuclear Transfer Embryos

2018 ◽  
Vol 45 (4) ◽  
pp. 1529-1540 ◽  
Author(s):  
Zhiren Zhang ◽  
Yanhui Zhai ◽  
Xiaoling Ma ◽  
Sheng Zhang ◽  
Xinglan An ◽  
...  
Keyword(s):  
Somatic Cell ◽  
Somatic Cell Nuclear Transfer ◽  
Nuclear Transfer ◽  
Epigenetic Modification ◽  
Expression Patterns ◽  
Epigenetic Reprogramming ◽  
Preimplantation Embryos ◽  
Down Regulation ◽  
Low Efficiency

Background/Aims: Aberrantly high levels of H3K4me3, caused by incomplete epigenetic reprogramming, likely cause a low efficiency of somatic cell nuclear transfer (SCNT). Smal molecule inhibitors aimed at epigenetic modification can be used to improve porcine SCNT embryo development. In this study, we examined the effects of MM-102, an H3K4 histone methyltransferase inhibitor, on porcine SCNT preimplantation embryos to investigate the mechanism by which H3K4 methylation regulated global epigenetic reprograming during SCNT. Methods: MM-102 was added to the SCNT embryos culture system and the global levels of various epigenetic modifications were measured by immunofluorescence (IF) staining and were quantified by Image J software. Relative genes expression levels were detected by quantitative real-time PCR. Results: MM-102 (75 μM) treatment reduced global H3K4, H3K9 methylation and 5mC levels especially at the zygotic gene activation (ZGA) and blastocyst stages. MM-102 treatment mainly down-regulated a series of DNA and histone methyltransferases, and up-regulated a number of hitone acetyltransferases and transcriptional activators. Furthermore, MM-102 treatment positively regulated the mRNA expression of genes related to pluripotency (OCT4, NANOG, CDX2) and apoptosis (BCL2). Conclusion: Down-regulation of H3K4me3 with MM-102 rescued aberrant gene expression patterns of a series of epigenetic chromatin modification enzymes, pluripotent and apoptotic genes at the ZGA and blastocyst stages, thereby greatly improving porcine SCNT efficiency and blastocyst quality, making them more similar to in vivo embryos (IVV).

scholarly journals Lessons Learned from Somatic Cell Nuclear Transfer

2020 ◽  
Vol 21 (7) ◽  
pp. 2314 ◽  
Author(s):  
Chantel Gouveia ◽  
Carin Huyser ◽  
Dieter Egli ◽  
Michael S. Pepper
Keyword(s):  
Somatic Cell ◽  
Somatic Cell Nuclear Transfer ◽  
Nuclear Transfer ◽  
Embryonic Stem ◽  
Donor Cell ◽  
Lessons Learned ◽  
Epigenetic Reprogramming ◽  
Area Of Interest ◽  
Legal Implications ◽  
Low Efficiency

Somatic cell nuclear transfer (SCNT) has been an area of interest in the field of stem cell research and regenerative medicine for the past 20 years. The main biological goal of SCNT is to reverse the differentiated state of a somatic cell, for the purpose of creating blastocysts from which embryonic stem cells (ESCs) can be derived for therapeutic cloning, or for the purpose of reproductive cloning. However, the consensus is that the low efficiency in creating normal viable offspring in animals by SCNT (1–5%) and the high number of abnormalities seen in these cloned animals is due to epigenetic reprogramming failure. In this review we provide an overview of the current literature on SCNT, focusing on protocol development, which includes early SCNT protocol deficiencies and optimizations along with donor cell type and cell cycle synchrony; epigenetic reprogramming in SCNT; current protocol optimizations such as nuclear reprogramming strategies that can be applied to improve epigenetic reprogramming by SCNT; applications of SCNT; the ethical and legal implications of SCNT in humans; and specific lessons learned for establishing an optimized SCNT protocol using a mouse model.

scholarly journals Combined Chaetocin/Trichostatin A Treatment Improves the Epigenetic Modification and Developmental Competence of Porcine Somatic Cell Nuclear Transfer Embryos

2021 ◽  
Vol 9 ◽  
Author(s):  
Pil-Soo Jeong ◽  
Hae-Jun Yang ◽  
Soo-Hyun Park ◽  
Min Ah Gwon ◽  
Ye Eun Joo ◽  
...  
Keyword(s):  
Somatic Cell ◽  
Somatic Cell Nuclear Transfer ◽  
Nuclear Transfer ◽  
Histone Deacetylase Inhibitors ◽  
Epigenetic Modification ◽  
Trichostatin A ◽  
Combined Treatment ◽  
Epigenetic Reprogramming ◽  
Developmental Competence ◽  
Developmental Potential

Developmental defects in somatic cell nuclear transfer (SCNT) embryos are principally attributable to incomplete epigenetic reprogramming. Small-molecule inhibitors such as histone methyltransferase inhibitors (HMTi) and histone deacetylase inhibitors (HDACi) have been used to improve reprogramming efficiency of SCNT embryos. However, their possible synergistic effect on epigenetic reprogramming has not been studied. In this study, we explored whether combined treatment with an HMTi (chaetocin) and an HDACi (trichostatin A; TSA) synergistically enhanced epigenetic reprogramming and the developmental competence of porcine SCNT embryos. Chaetocin, TSA, and the combination significantly increased the cleavage and blastocyst formation rate, hatching/hatched blastocyst rate, and cell numbers and survival rate compared to control embryos. In particular, the combined treatment improved the rate of development to blastocysts more so than chaetocin or TSA alone. TSA and combined chaetocin/TSA significantly reduced the H3K9me3 levels and increased the H3K9ac levels in SCNT embryos, although chaetocin alone significantly reduced only the H3K9me3 levels. Moreover, these inhibitors also decreased global DNA methylation in SCNT embryos. In addition, the expression of zygotic genome activation- and imprinting-related genes was increased by chaetocin or TSA, and more so by the combination, to levels similar to those of in vitro-fertilized embryos. These results suggest that combined chaetocin/TSA have synergistic effects on improving the developmental competences by regulating epigenetic reprogramming and correcting developmental potential-related gene expression in porcine SCNT embryos. Therefore, these strategies may contribute to the generation of transgenic pigs for biomedical research.

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.

51 DELAYED NUCLEOLOGENESIS IN PORCINE PREIMPLANTATION EMBRYOS DEVELOPED IN VIVO AND PRODUCED BY SOMATIC CELL NUCLEAR TRANSFER

2010 ◽  
Vol 22 (1) ◽  
pp. 183
Author(s):  
R. S. Deshmukh ◽  
O. Østrup ◽  
E. Lemme ◽  
B. Peterson ◽  
A. Lucas-Hahn ◽  
...  
Keyword(s):  
Nuclear Envelope ◽  
Somatic Cell ◽  
Somatic Cell Nuclear Transfer ◽  
Nuclear Transfer ◽  
Developmental Competence ◽  
Condensed Chromatin ◽  
Preimplantation Embryos ◽  
Metaphase Chromosomes ◽  
Cell Stage

Nucleolus is known to be a well-suited morphological marker for embryo technologies. Failure in de novo nucleolar formation during embryonic genome activation (EGA) has been observed in many species. The aim of the present study was to investigate nuclear changes and nucleolar formation during EGA in the porcine preimplantation embryos developed in vivo and produced by somatic cell nuclear transfer (SCNT). Embryos were collected at early and late 1-cell stage, 2-, 4-, and 8-cell stage, early and late blastocyst stage, fixed in 3% glutaraldehyde for 1 h, and processed for transmission electron microscopy. In vivo embryos from 1- and 2-cell stages showed electron dense, spherical nucleolar precursor bodies (NPB) in centrally located nuclei with well-developed nuclear envelope and condensed chromatin. Two 1-cell-stage embryos, however, had represented metaphase chromosomes in the periphery. At the 4-cell stage, in vivo embryos displayed fibrillo-granular nucleoli containing all 3 functional nucleolar compartments: fibrillar centers (FC), dense fibrillar component (DFC), and granular component (GC). The nuclei were centrally located, round, and had complete nuclear envelopes. The same types of nuclei and nucleoli were observed for all following stages. On the other hand, embryos produced by SCNT at early 1-cell stage showed centrally located, irregular-shaped nuclei with incomplete nuclear envelopes and condensed chromatin with large intact NPB. Exceptionally, 1 out of the 5 embryos presented a peripheral nucleus with partially condensed chromatin lacking nuclear envelope and fibrillo-granular nucleolus probably persisting from donor fibroblast. Only 2 out of 5 late-1-cell SCNT embryos showed nuclear structures. The nuclei had irregular shapes, complete nuclear membranes, and contained large NPB. At the 2- and 4-cell stages, the embryos presented central nuclei with complete nuclear envelopes. Some of the embryos showed more than one nucleus of varying shapes and sizes. The fibrillo-granular nucleoli were first observed toward the 8-cell stage. The embryos from this stage contained irregularly shaped nuclei with well-developed nuclear envelopes. The nucleoli displayed fibrillar and granular compartments in SCNT 8-cell stage embryos, but lacked the typically structured functional nucleoli observed in in vivo embryos. The absence of formation of functional nucleoli at the 4-cell stage and altered nuclear ultrastructure during the EGA in SCNT embryos, thus, may be one of the main reasons for decreased developmental competence of SCNT embryos.

84 GENE EXPRESSION DURING BLASTOCYST-TO-ELONGATED STAGE IN BOVINE EMBRYOS DERIVED FROM SOMATIC CELL NUCLEAR TRANSFER

2007 ◽  
Vol 19 (1) ◽  
pp. 159
Author(s):  
K. Sawai ◽  
S. Moriyasu ◽  
H. Hirayama ◽  
A. Minamihashi ◽  
S. Onoe
Keyword(s):  
Gene Expression ◽  
Somatic Cell ◽  
Embryo Development ◽  
Gene Transcription ◽  
Somatic Cell Nuclear Transfer ◽  
Nuclear Transfer ◽  
Epigenetic Modification ◽  
Fibroblast Growth ◽  
Transcript Levels

Epigenetic modification is an important factor in the development of embryos and the production of normal offspring derived from somatic cell nuclear transfer (NTSC). Several investigators have reported aberrant gene transcription in bovine NTSC embryos at the blastocyst (BC) stage. The objectives of this study were to evaluate the gene expression in NTSC embryos, which had developed to the elongated (EL) stage, and clarify differential levels of gene transcription in the embryo disc (ED) and trophectoderm (TE) of EL embryos. Five specific mRNAs [octamer-binding transcription factor (OCT-4), interferon-τ (IFN-τ), fibroblast growth factor receptor 2 (FGF-R2), and fibroblast growth factors 2 and 4 (FGF-2 and FGF-4)] were selected. Bovine BC embryos were obtained from NTSC using calf fibroblast cells or the uterus of donor cows after AI (Vivo). Some BC embryos were transferred to recipient cows at Day 7 (Day 0 = estrus), and then EL embryos were collected by uterine flushing at Day 16. Total RNA in single BC, ED, and TE were reverse-transcribed for PCR. Quantification of mRNA abundance was performed by real-time PCR. The expression of each mRNA was normalized to the abundance of GAPDH. A total of 15 (BC) and 7 (ED and TE) samples were used in each group to analyze the gene expression. Data on mRNA expression levels were analyzed using a Kruskal–Wallis test followed by multiple pair-wise comparisons using the Scheffe method. Most embryos (87–100%) gave positive signals of OCT-4, IFN-τ, and FGF-R2, regardless of the origin and stage of the embryos. Transcript signals of FGF-4 in BC embryos derived from Vivo (100%) and NTSC (70%) were detected with higher frequencies. At the EL stage, the FGF-4 signal was detected in only ED. The transcript of FGF-2 was detected with lower frequencies (20–27%) in BC embryos, but was consistently (71–86%) detected in ED of both groups. The relative abundance of OCT-4 expression in NTSC was higher (P < 0.05) than in Vivo embryos at the BC stage. In contrast, the transcript of FGF-4 at the BC stage was lower (P < 0.01) in NTSC than in Vivo embryos. Transcript levels of IFN-�, FGF-R2, and FGF-2 were not significantly different in both groups at the BC stage. The amount of OCT-4, FGF-4, and FGF-2 transcripts in ED were significantly (P < 0.05) higher than in TE. Transcript levels of these genes did not differ between NTSC and Vivo embryos. FGF-R2 levels were not significantly different in origins and tissue of EL embryos. In Vivo embryos, the IFN-τ level of TE was significantly (P < 0.05) higher than in ED. However, the difference in the IFN-τ transcription was not observed between ED and TE in NTSC embryos. The results of an analysis of mRNA transcripts at 2 different stages of development demonstrate that bovine NTSC embryos at the BC stage show deviations in expression patterns with respect to several genes which have important roles in cell differentiation, implantation, and embryo development, but these expressions, except for IFN-τ, were modified to a normal level according to the embryo development and differentiation.

Cotreatment with RepSox and LBH589 improves the in vitro developmental competence of porcine somatic cell nuclear transfer embryos

2018 ◽  
Vol 30 (10) ◽  
pp. 1342 ◽  
Author(s):  
Zhao-Bo Luo ◽  
Long Jin ◽  
Qing Guo ◽  
Jun-Xia Wang ◽  
Xiao-Xu Xing ◽  
...  
Keyword(s):  
Somatic Cell ◽  
Somatic Cell Nuclear Transfer ◽  
Nuclear Transfer ◽  
Formation Rate ◽  
Epigenetic Reprogramming ◽  
Developmental Competence ◽  
Abnormal Development ◽  
Control Group ◽  
Blastocyst Formation

Accumulating evidence suggests that aberrant epigenetic reprogramming and low pluripotency of donor nuclei lead to abnormal development of cloned embryos and underlie the inefficiency of mammalian somatic cell nuclear transfer (SCNT). The present study demonstrates that treatment with the small molecule RepSox alone upregulates the expression of pluripotency-related genes in porcine SCNT embryos. Treatment with the histone deacetylase inhibitor LBH589 significantly increased the blastocyst formation rate, whereas treatment with RepSox did not. Cotreatment with 12.5 μM RepSox and 50 nM LBH589 (RepSox + LBH589) for 24 h significantly increased the blastocyst formation rate compared with that of untreated embryos (26.9% vs 8.5% respectively; P < 0.05). Furthermore, the expression of pluripotency-related genes octamer-binding transcription factor 4 (NANOG) and SRY (sex determining region Y)-box 2 (SOX2) were found to significantly increased in the RepSox + LBH589 compared with control group at both the 4-cell and blastocyst stages. In particular, the expression of NANOG was 135-fold higher at the blastocyst stage in the RepSox + LBH589 group. Moreover, RepSox + LBH589 improved epigenetic reprogramming. In summary, RepSox + LBH589 increases the expression of developmentally important genes, optimises epigenetic reprogramming and improves the in vitro development of porcine SCNT embryos.

scholarly journals Dynamic Methylation Changes of DNA and H3K4 by RG108 Improve Epigenetic Reprogramming of Somatic Cell Nuclear Transfer Embryos in Pigs

2018 ◽  
Vol 50 (4) ◽  
pp. 1376-1397 ◽  
Author(s):  
Yanhui Zhai ◽  
Zhiren Zhang ◽  
Hao Yu ◽  
Li Su ◽  
Gang Yao ◽  
...  
Keyword(s):  
Dna Methylation ◽  
Somatic Cell ◽  
Histone Modifications ◽  
Somatic Cell Nuclear Transfer ◽  
Nuclear Transfer ◽  
Histone H3 ◽  
Dna Demethylation ◽  
Epigenetic Reprogramming ◽  
Expression Levels ◽  
Fetal Fibroblasts

Background/Aims: DNA methylation and histone modifications are essential epigenetic marks that can significantly affect the mammalian somatic cell nuclear transfer (SCNT) embryo development. However, the mechanisms by which the DNA methylation affects the epigenetic reprogramming have not been fully elucidated. Methods: In our study, we used quantitative polymerase chain reaction (qPCR), Western blotting, immunofluorescence staining (IF) and sodium bisulfite genomic sequencing to examine the effects of RG108, a DNA methyltransferase inhibitor (DNMTi), on the dynamic pattern of DNA methylation and histone modifications in porcine SCNT embryos and investigate the mechanism by which the epigenome status of donor cells’ affects SCNT embryos development and the crosstalk between epigenetic signals. Results: Our results showed that active DNA demethylation was enhanced by the significantly improving expression levels of TET1, TET2, TET3 and 5hmC, and passive DNA demethylation was promoted by the remarkably inhibitory expression levels of DNMT1, DNMT3A and 5mC in embryos constructed from the fetal fibroblasts (FFs) treated with RG108 (RG-SCNT embryos) compared to the levels in embryos from control FFs (FF-SCNT embryos). The signal intensity of histone H3 lysine 4 trimethylation (H3K4me3) and histone H3 lysine 9 acetylation (H3K9Ac) was significantly increased and the expression levels of H3K4 methyltransferases were more than 2-fold higher expression in RG-SCNT embryos. RG-SCNT embryos had significantly higher cleavage and blastocyst rates (69.3±1.4%, and 24.72±2.3%, respectively) than FF-SCNT embryos (60.1±2.4% and 18.38±1.9%, respectively). Conclusion: Dynamic changes in DNA methylation caused by RG108 result in dynamic alterations in the patterns of H3K4me3, H3K9Ac and histone H3 lysine 9 trimethylation (H3K9me3), which leads to the activation of embryonic genome and epigenetic modification enzymes associated with H3K4 methylation, and contributes to reconstructing normal epigenetic modifications and improving the developmental efficiency of porcine SCNT embryos.

Aberrant Epigenetic Reprogramming in the First Cell Cycle of Bovine Somatic Cell Nuclear Transfer Embryos

2021 ◽  
Vol 23 (2) ◽  
pp. 99-107
Author(s):  
LiJun Wang ◽  
LiXiu Liu ◽  
YongSheng Wang ◽  
Nan Li ◽  
HongLi Zhu ◽  
...  
Keyword(s):  
Cell Cycle ◽  
Somatic Cell ◽  
Somatic Cell Nuclear Transfer ◽  
Nuclear Transfer ◽  
Epigenetic Reprogramming

Mitochondria and the success of somatic cell nuclear transfer cloning: from nuclear - mitochondrial interactions to mitochondrial complementation and mitochondrial DNA recombination

2005 ◽  
Vol 17 (2) ◽  
pp. 69 ◽  
Author(s):  
Stefan Hiendleder ◽  
Valeri Zakhartchenko ◽  
Eckhard Wolf
Keyword(s):  
Mitochondrial Dna ◽  
Somatic Cell ◽  
Somatic Cell Nuclear Transfer ◽  
Nuclear Transfer ◽  
Dna Recombination ◽  
Epigenetic Reprogramming ◽  
Research Activities ◽  
Mitochondrial Functions ◽  
New Findings ◽  
Mitochondrial Dna Recombination

The overall success of somatic cell nuclear transfer (SCNT) cloning is rather unsatisfactory, both in terms of efficacy and from an animal health and welfare point of view. Most research activities have concentrated on epigenetic reprogramming problems as one major cause of SCNT failure. The present review addresses the limited success of mammalian SCNT from yet another viewpoint, the mitochondrial perspective. Mitochondria have a broad range of critical functions in cellular energy supply, cell signalling and programmed cell death and, thus, affect embryonic and fetal development, suggesting that inadequate or perturbed mitochondrial functions may adversely affect SCNT success. A survey of perinatal clinical data from human subjects with deficient mitochondrial respiratory chain activity has revealed a plethora of phenotypes that have striking similarities with abnormalities commonly encountered in SCNT fetuses and offspring. We discuss the limited experimental data on nuclear–mitochondrial interaction effects in SCNT and explore the potential effects in the context of new findings about the biology of mitochondria. These include mitochondrial fusion/fission, mitochondrial complementation and mitochondrial DNA recombination, processes that are likely to be affected by and impact on SCNT cloning. Furthermore, we indicate pathways that could link epigenetic reprogramming and mitochondria effects in SCNT and address questions and perspectives for future research.

Sign in / Sign up

Export Citation Format

Share Document