Effect of sex differences in donor foetal fibroblast on the early development and DNA methylation status of buffalo (Bubalus bubalis ) nuclear transfer embryos

2018 ◽  
Vol 54 (1) ◽  
pp. 11-22 ◽  
Author(s):  
Ziyun Ruan ◽  
Xin Zhao ◽  
Zhengda Li ◽  
Xiling Qin ◽  
Qiming Shao ◽  
...  
Keyword(s):  
Dna Methylation ◽  
Sex Differences ◽  
Early Development ◽  
Nuclear Transfer ◽  
Methylation Status ◽  
Bubalus Bubalis

DNA methylation status of H19 and Xist genes in lungs of somatic cell nuclear transfer bovines

2008 ◽  
Vol 53 (13) ◽  
pp. 1996-2001 ◽  
Author(s):  
Jie Chen ◽  
DongJie Li ◽  
YanQin Liu ◽  
Cui Zhang ◽  
YunPing Dai ◽  
...  
Keyword(s):  
Dna Methylation ◽  
Somatic Cell ◽  
Somatic Cell Nuclear Transfer ◽  
Nuclear Transfer ◽  
Methylation Status

Antioxidant treatment during manipulation procedures prevents mitochondrial and DNA damage and enhances nuclear reprogramming of bovine somatic cell nuclear transfer embryos

2015 ◽  
Vol 27 (7) ◽  
pp. 1088 ◽  
Author(s):  
Hyo-Kyung Bae ◽  
In-Sun Hwang ◽  
Ji-Ye Kim ◽  
Sung-Young Lee ◽  
Choon-Keun Park ◽  
...  
Keyword(s):  
Dna Methylation ◽  
Dna Damage ◽  
Membrane Potential ◽  
Somatic Cell Nuclear Transfer ◽  
Nuclear Transfer ◽  
Methylation Status ◽  
Nuclear Reprogramming ◽  
Antioxidant Treatment ◽  
Treatment Groups ◽  
H2o2 Level

We tried to prevent the mitochondrial and DNA damage caused by mechanical stress-associated reactive oxygen species (ROS), and to improve the reprogramming of bovine somatic cell nuclear transfer (SCNT) embryos by antioxidant treatment during the manipulation procedures of SCNT. Bovine recipient oocytes and reconstituted oocytes were treated with antioxidants during manipulation procedures. The H2O2 level, mitochondrial morphology, membrane potential and apoptosis at the one-cell stage, and in vitro development and DNA methylation status of blastocysts were evaluated. Antioxidant treatment during manipulation procedures reduced the H2O2 level of SCNT embryos. Antioxidant-treated SCNT embryos normally formed mitochondrial clumps, similar to IVF embryos, and showed higher mitochondrial membrane potential versus the SCNT control (P < 0.05). Apoptosis and DNA fragmentation were reduced by antioxidant treatment. The development rate to the blastocyst stage was higher (P < 0.05) in the antioxidant treatment groups (30.5 ± 2.5 to 30.6 ± 1.6%) versus the control (23.0 ± 1.9%). The DNA methylation status of blastocysts in the antioxidant treatment groups was lower (P < 0.05) than that of the control and similar to that of IVF embryos. These results indicate that antioxidant treatment during manipulation procedures can prevent cellular damage that may be caused by mechanical stress-associated ROS, and improve nuclear reprogramming.

1 ABERRANT DNA METHYLATION IN PORCINE IN VITRO-, PARTHENOGENETIC-, AND NUCLEAR TRANSFER-PRODUCED BLASTOCYSTS

2006 ◽  
Vol 18 (2) ◽  
pp. 109 ◽  
Author(s):  
A. Bonk ◽  
M. Samuel ◽  
L. Lai ◽  
Y. Hao ◽  
R. Li ◽  
...  
Keyword(s):  
Dna Methylation ◽  
Nuclear Transfer ◽  
Methylation Status ◽  
Germinal Vesicle ◽  
Blastocyst Stage ◽  
The Other ◽  
Specific Gene ◽  
Aberrant Dna Methylation

Aberrant DNA methylation of in vitro-, parthenogenetic-, and nuclear transfer-derived embryos has been implicated in the low developmental competence of early embryos. Demethylation of the genome occurs immediately after fertilization and continues through the blastocyst stage. Remethylation or reprogramming of the genome occurs around the time of implantation and is maintained in somatic tissues. The aim of this study was to analyze DNA methylation in porcine gametes and blastocysts. Differential DNA methylation hybridization was conducted to analyze the methylation status of the Bstu I site (CGCG) in the gamete and blastocyst epigenomes. Germinal vesicle oocytes were aspirated from ovaries collected at an abattoir, sperm was isolated from a fresh ejaculate, and blastocysts were derived and collected from in vivo, in vitro, nuclear transfer, and parthenogenetic sources. Genomic clones were selected from a porcine CpG Island library based on the presence of a Bstu I site. The inserts from these clones were PCR amplified and spotted on glass slides. DNA was digested with Mse I, ligated to linkers, and digested with Bstu I. Fragments with methylated Bstu I sites remained intact whereas fragments with unmethylated Bstu I sites were cut. Intact fragments were amplified by PCR and labeled with amino allyl-dUTP. Liver DNA served as the reference and was labeled with Cy5; the other samples were labeled with Cy3. An Axon Genepix 4000B scanner (Axon Instruments, Inc., Union City, CA, USA) was used to scan the slides. Initial analysis of the microarray image was performed with GenePix Pro 4.0 software. Additional analysis, performed by using Genespring 7.0 ANOVA (P < 0.05), identified 221 clones as being significantly different in at least one of the biological conditions of the gametes or the blastocysts. Forty-six clones were sequenced and BLAST analysis identified 18 clones that were unique, 16 clones that had no similarity, and 12 clones that had similarity to multiple genes. Ribosomal (RPS20, RPL18) and protoporphyrinogen oxidase (PPOX) genes were identified in several clones. Components of the immune system (CCRs, TLRs), a transcription factor (ATF2), and an embryo-specific gene (WNT8B) were also identified. A condition tree was created according to the standard correlation similarity measure for the spots identified as significantly different. The condition tree shows that the methylation profiles are most similar in the germinal vesicle oocyte, parthenogenetic blastocyst, nuclear transfer blastocyst, in vitro-produced blastocyst, and sperm. In vivo-produced blastocysts grouped separately from the other samples. These results are consistent with previous studies that have shown that gametes undergo demethylation after fertilization on through the blastocyst stage when the genome is remethylated. Additionally, these results suggest that the reprogramming events that occur during the development of the in vivo-produced blastocysts are less likely to occur in in vitro-, nuclear transfer-, and parthenogenetic-produced blastocysts. This work was funded by a grant from the NIH (RR13438) and Food for the 21st Century.

42 DEVELOPMENTAL POTENTIAL AND REPROGRAMMING EFFICIENCY OF BOVINE EMBRYOS CLONED WITH ADULT FIBROBLASTS TREATED BY A DEMETHYLATING AGENT, S-ADENOSYL-HOMOCYSTEINE

2006 ◽  
Vol 18 (2) ◽  
pp. 130 ◽  
Author(s):  
B.-G. Jeon ◽  
S. D. Perrault ◽  
G.-J. Rho ◽  
D. H. Betts ◽  
W. A. King
Keyword(s):  
Dna Methylation ◽  
Nuclear Transfer ◽  
Methylation Status ◽  
Somatic Cells ◽  
Telomerase Activity ◽  
Dna Demethylation ◽  
Blastocyst Development ◽  
Developmental Potential ◽  
Reprogramming Efficiency ◽  
Low Efficiency

Animal cloning by somatic cell nuclear transfer (SCNT) has been successfully applied to several species although with low efficiency and often associated with severe abnormalities. These poor outcomes are thought to be a consequence of aberrant DNA methylation patterns that result from incomplete epigenetic reprogramming of the transplanted nucleus into recipient oocytes. Telomerase, an enzyme not expressed in most somatic cells, should be expressed in cloned embryos. Therefore its activity has been used as an index of reprogramming in SCNT embryos. The objective of this study was to investigate the DNA methylation status of donor fibroblasts treated with a non-cytotoxic transmethylation inhibitor, S-adenosyl homocysteine (SAH), and to assess the relative telomerase activity (RTA) and developmental potential of SCNT embryos derived from such cells. Adult ear skin fibroblasts were cultured in DMEM supplemented with 0, 0.5, 1.0, or 2.0 mM SAH for 144 h by daily media change prior to nuclear transfer. The SAH-treated fibroblasts were immunostained with a fluorescein isothiocyanate (FITC) conjugated 5-methylcytosine antibody and the relative fluorescence intensity (RFI) was analyzed using a fluorescence microscope equipped with an Openlab" program (Improvision, Coventry, UK). RTA was measured in Day 8 SCNT blastocysts using the real-time quantitative telomeric repeat amplification protocol (RQ-TRAP). Fibroblasts treated with 0.5, 1.0, and 2.0 mM SAH showed lower levels of DNA methylation compared to nontreated controls, and the values did not differ among the treatment groups. Cleavage rates did not differ between the SCNT embryos derived from 0.5 mM SAH-treated cells and nontreated control cells (92.3% vs. 91.3%, respectively). However, the rates of blastocyst development and hatching were significantly (P < 0.05) higher in SCNT embryos derived from 0.5 mM SAH treated donor cells compared to controls (60.0 and 40.0% vs. 34.3 and 26.4%, respectively). Moreover, RTA of the 0.5 mM SAH SCNT embryos was significantly (P < 0.05) increased (1.5-fold) in relation to controls. S-adenosyl homocysteine treatment induces global DNA demethylation in donor fibroblasts and enhances the blastocyst frequencies for bovine SCNT embryos that also exhibit greater telomerase activity levels. These results suggest that use of hypomethylated donor somatic cells increases the developmental potential for SCNT embryos by enhancing the nuclear reprogramming efficiency. This work was funded by NSERC, OMAF, OCAG, and CRC.

Altered gene expression in cloned piglets

2009 ◽  
Vol 21 (1) ◽  
pp. 60 ◽  
Author(s):  
X. Cindy Tian ◽  
Joonghoon Park ◽  
Richard Bruno ◽  
Richard French ◽  
Le Jiang ◽  
...  
Keyword(s):  
Dna Methylation ◽  
Somatic Cell Nuclear Transfer ◽  
Nuclear Transfer ◽  
Expression Profiles ◽  
Methylation Status ◽  
Microarray Technology ◽  
Conventional Breeding ◽  
Altered Gene Expression ◽  
Altered Gene ◽  
Cloned Pig

Studies on cloned pigs are scant compared with those in mice and cattle. Expression profiles of cloned pig embryos on full-term cloned pigs are even more limited owing to the limited availability of DNA microarray technology in the pig. We have conducted expression profile comparisons between pigs from somatic cell nuclear transfer and pigs from conventional breeding at birth and 1 month of age. Differentially expressed genes that are subjected to DNA methylation were also examined for their DNA methylation status. These data will be presented in the 2009 Annual Meeting of the International Embryo Transfer Society in San Diego. In the present review, we focus on summarising existing findings on epigenetic and other changes in cloned embryo, cloned pigs and their offspring by conventional breeding.

scholarly journals Nuclear Transfer Alters the DNA Methylation Status of Specific Genes in Fertilized and Parthenogenetically Activated Mouse Embryonic Stem Cells

Stem Cells ◽  
2008 ◽  
Vol 26 (3) ◽  
pp. 783-788 ◽  
Author(s):  
Takafusa Hikichi ◽  
Takashi Kohda ◽  
Sayaka Wakayama ◽  
Fumitoshi Ishino ◽  
Teruhiko Wakayama
Keyword(s):  
Dna Methylation ◽  
Stem Cells ◽  
Embryonic Stem Cells ◽  
Nuclear Transfer ◽  
Methylation Status ◽  
Embryonic Stem ◽  
Mouse Embryonic Stem Cells

Changes in the DNA Methylation Status of Bovine Embryos from the Blastocyst to Elongated Stage Derived from Somatic Cell Nuclear Transfer

2010 ◽  
Vol 12 (1) ◽  
pp. 15-22 ◽  
Author(s):  
Ken Sawai ◽  
Masashi Takahashi ◽  
Satoru Moriyasu ◽  
Hiroki Hirayama ◽  
Akira Minamihashi ◽  
...  
Keyword(s):  
Dna Methylation ◽  
Somatic Cell ◽  
Somatic Cell Nuclear Transfer ◽  
Nuclear Transfer ◽  
Methylation Status ◽  
Bovine Embryos
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