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.

27 SOMATIC CELL NUCLEAR TRANSFER CLONING AND EMBRYO AGGREGATION IN PIGS

2014 ◽  
Vol 26 (1) ◽  
pp. 128
Author(s):  
C. P. Buemo ◽  
A. Gambini ◽  
I. Hiriart ◽  
D. Salamone
Keyword(s):  
In Vitro Culture ◽  
Somatic Cell ◽  
Embryo Development ◽  
Somatic Cell Nuclear Transfer ◽  
Nuclear Transfer ◽  
Electric Pulse ◽  
Blastocyst Stage ◽  
Control Group

Somatic cell nuclear transfer (SCNT) derived blastocysts have lower cell number than IVF-derived blastocysts and their in vivo counterparts. The aim of this study was to improve the blastocyst rates and quality of SCNT blastocysts by the aggregation of genetically identical free zona pellucida (ZP) porcine clones. Cumulus–oocyte complexes were recovered from slaughterhouse ovaries by follicular aspiration. Maturation was performed in TCM for 42 to 48 h at 39°C and 5% CO2. After denudation by treatment with hyaluronidase, mature oocytes were stripped of the ZP using a protease and then enucleated by micromanipulation; staining was performed with Hoechst 33342 to observe metaphase II. Ooplasms were placed in phytohemagglutinin to permit different membranes to adhere between each other; the ooplasm membrane was adhered to a porcine fetal fibroblast from an in vitro culture. Adhered membranes of the donor cell nucleus and enucleated oocyte cytoplasm were electrofused through the use of an electric pulse (80 V for 30 μs). All reconstituted embryos (RE) were electrically activated using an electroporator in activation medium (0.3 M mannitol, 1.0 mM CaCl2, 0.1 mM MgCl2, and 0.01% PVA) by a DC pulse of 1.2 kV cm–1 for 80 μs. Then, the oocytes were incubated in 2 mM 6-DMAP for 3 h. In vitro culture of free ZP embryos was achieved in a system of well of wells in 100 μL of medium, placing 3 activated oocytes per microwell (aggregation embryo), whereas the control group was cultivated with equal drops without microwells. Embryos were cultivated at 39°C in 5% O2, 5% CO2 for 7 days in SOF medium with a supplement of 10% fetal bovine serum on the fifth day. The RE were placed in microwells. Two experimental groups were used, control group (not added 1X) and 3 RE per microwell (3X). At Day 7, resulting blastocysts were classified according to their morphology and diameter to determine their quality and evaluate if the embryo aggregation improves it. Results demonstrated that aggregation improves in vitro embryo development rates until blastocyst stage and indicated that blastocysts rates calculated over total number of oocytes do not differ between groups (Table 1). Embryo aggregation improves cleavage per oocyte and cleavage per microwell rates, presenting statistical significant differences and increasing the probabilities of higher embryo development generation until the blastocyst stage with better quality and higher diameter. Table 1.Somatic cell nuclear transfer cloning and embryo aggregation

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 Influences of somatic donor cell sex on in vitro and in vivo embryo development following somatic cell nuclear transfer in pigs

2016 ◽  
Vol 30 (4) ◽  
pp. 585-592 ◽  
Author(s):  
Jae-Gyu Yoo ◽  
Byeong-Woo Kim ◽  
Mi-Rung Park ◽  
Deug-Nam Kwon ◽  
Yun-Jung Choi ◽  
...  
Keyword(s):  
Somatic Cell ◽  
Embryo Development ◽  
Somatic Cell Nuclear Transfer ◽  
Nuclear Transfer ◽  
Donor Cell

scholarly journals A modified culture method significantly improves the development of mouse somatic cell nuclear transfer embryos

Reproduction ◽  
2009 ◽  
Vol 138 (2) ◽  
pp. 301-308 ◽  
Author(s):  
Xiangpeng Dai ◽  
Jie Hao ◽  
Qi Zhou
Keyword(s):  
Somatic Cell ◽  
Embryo Development ◽  
Somatic Cell Nuclear Transfer ◽  
Nuclear Transfer ◽  
Culture Media ◽  
Culture Method ◽  
Early Embryo ◽  
Blastocyst Development

Many strategies have been established to improve the efficiency of somatic cell nuclear transfer (SCNT), but relatively few focused on improving culture conditions. The effect of different culture media on preimplantation development of mouse nuclear transfer embryos was investigated. A modified sequential media method, named D media (M16/KSOM and CZB-EG/KSOM), was successfully established that significantly improves SCNT embryo development. Our result demonstrated that while lacking any adverse effect on in vivo fertilized embryos, the D media dramatically improves the blastocyst development of SCNT embryos compared with other commonly used media, including KSOM, M16, CZB, and αMEM. Specifically, the rate of blastocyst formation was 62.3% for D1 (M16/KSOM) versus 10–30% for the other media. An analysis of media components indicated that removing EDTA and glutamine from the media can be beneficial for early SCNT embryo development. Our results suggest that in vitro culture environment plays an important role in somatic cell reprogramming, and D media represent the most efficient culture method reported to date to support mouse SCNT early embryo development in vitro.

scholarly journals Simultaneous gene quantitation of multiple genes in individual bovine nuclear transfer blastocysts

Reproduction ◽  
2007 ◽  
Vol 133 (1) ◽  
pp. 231-242 ◽  
Author(s):  
Craig Smith ◽  
Debbie Berg ◽  
Sue Beaumont ◽  
Neil T Standley ◽  
David N Wells ◽  
...  
Keyword(s):  
Gene Expression ◽  
Nuclear Transfer ◽  
Inner Cell Mass ◽  
Cell Mass ◽  
Ectopic Expression ◽  
Cell Number ◽  
Transcript Levels ◽  
Multiple Genes

During somatic cell nuclear transfer (NT), the transcriptional status of the donor cell has to be reprogrammed to reflect that of an embryo. We analysed the accuracy of this process by comparing transcript levels of four developmentally important genes (Oct4,Otx2,Ifitm3,GATA6), a gene involved in epigenetic regulation (Dnmt3a) and three housekeeping genes (β-actin, β-tubulinandGAPDH) in 21 NT blastocysts with that in genetically half-identicalin vitroproduced (IVP,n=19) andin vivo(n=15) bovine embryos. We have optimised an RNA-isolation and SYBR-green-based real-time RT-PCR procedure allowing the reproducible absolute quantification of multiple genes from a single blastocyst. Our data indicated that transcript levels did not differ significantly between stage and grade-matched zona-free NT and IVP embryos except for Ifitm3/Fragilis, which was expressed at twofold higher levels in NT blastocysts.Ifitm3expression is confined to the inner cell mass at day 7 blastocysts and to the epiblast in day 14 embryos. No ectopic expression in the trophectoderm was seen in NT embryos. Gene expression in NTand IVP embryos increased between two- and threefold for all eight genes from early to late blastocyst stages. This increase exceeded the increase in cell number over this time period indicating an increase in transcript number per cell. Embryo quality (morphological grading) was correlated to cell number for NT and IVP embryos with grade 3 blastocysts containing 30% fewer cells. However, only NT embryos displayed a significant reduction in gene expression (50%) with loss of quality. Variability in gene expression levels was not significantly different in NT, IVP orin vivoembryos but differed among genes, suggesting that the stringency of regulation is intrinsic to a gene and not affected by culture or nuclear transfer.Oct4levels exhibited the lowest variability. Analysing the total variability of all eight genes for individual embryos revealed thatin vivoembryos resembled each other much more than did NT and IVP blastocysts. Furthermore,in vivoembryos, consisting of 1.5-fold more cells, generally contained two- to fourfold more transcripts for the eight genes than did their cultured counterparts. Thus, culture conditions (in vivoversusin vitro) have greater effects on gene expression than does nuclear transfer when minimising genetic heterogeneity.

scholarly journals Differential In Vivo Binding Dynamics of Somatic and Oocyte-specific Linker Histones in Oocytes and During ES Cell Nuclear Transfer

2005 ◽  
Vol 16 (8) ◽  
pp. 3887-3895 ◽  
Author(s):  
Matthias Becker ◽  
Antje Becker ◽  
Faiçal Miyara ◽  
Zhiming Han ◽  
Maki Kihara ◽  
...  
Keyword(s):  
Somatic Cell ◽  
Somatic Cell Nuclear Transfer ◽  
Nuclear Transfer ◽  
Somatic Cells ◽  
Linker Histone ◽  
Binding Properties ◽  
Paternal Genome ◽  
Linker Histones ◽  
In Vivo Binding

The embryonic genome is formed by fusion of a maternal and a paternal genome. To accommodate the resulting diploid genome in the fertilized oocyte dramatic global genome reorganizations must occur. The higher order structure of chromatin in vivo is critically dependent on architectural chromatin proteins, with the family of linker histone proteins among the most critical structural determinants. Although somatic cells contain numerous linker histone variants, only one, H1FOO, is present in mouse oocytes. Upon fertilization H1FOO rapidly populates the introduced paternal genome and replaces sperm-specific histone-like proteins. The same dynamic replacement occurs upon introduction of a nucleus during somatic cell nuclear transfer. To understand the molecular basis of this dynamic histone replacement process, we compared the localization and binding dynamics of somatic H1 and oocyte-specific H1FOO and identified the molecular determinants of binding to either oocyte or somatic chromatin in living cells. We find that although both histones associate readily with chromatin in nuclei of somatic cells, only H1FOO is capable of correct chromatin association in the germinal vesicle stage oocyte nuclei. This specificity is generated by the N-terminal and globular domains of H1FOO. Measurement of in vivo binding properties of the H1 variants suggest that H1FOO binds chromatin more tightly than somatic linker histones. We provide evidence that both the binding properties of linker histones as well as additional, active processes contribute to the replacement of somatic histones with H1FOO during nuclear transfer. These results provide the first mechanistic insights into the crucial step of linker histone replacement as it occurs during fertilization and somatic cell nuclear transfer.

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