60 EFFECTS OF REPETITIVE IONOMYCIN TREATMENT ON BOVINE SOMATIC CELL NUCLEAR TRANSFER (SCNT) EMBRYOS

2010 ◽  
Vol 22 (1) ◽  
pp. 188
Author(s):  
S. H. Jeong ◽  
S. Kim ◽  
M. K. Choi ◽  
J. M. Na ◽  
J. Choi ◽  
...  
Keyword(s):  
Somatic Cell ◽  
Somatic Cell Nuclear Transfer ◽  
Nuclear Transfer ◽  
Formation Rate ◽  
Essential Element ◽  
Developmental Competence ◽  
P Value ◽  
Oocyte Activation ◽  
Reconstructed Embryos ◽  
Group 3

In bovine somatic cell nuclear transfer (SCNT), oocyte activation is an essential element for the successful development of cloned embryos. Chemical treatment with ionomycin induces transient levels of Ca2+, and this has been used in the activation of reconstructed embryos. In vitro-fertilized oocytes are naturally activated by sperm following several Ca2+ transients known collectively as Ca2+ oscillations. We aimed to improve the developmental efficiency of embryos produced by SCNT by mimicking this Ca2+ oscillation artificially. SCNT was performed as follows; bovine calf skin fibroblast cells were transferred into the perivitelline space of IVM oocytes, and then these NT couplets were treated with electrical fusion (2 pulses, 1.75 kV cm-1, 15 μs). Reconstructed embryos were subsequently cultured in SOF medium (5% CO2, 5% O2, and 38°C). To mimic Ca2+ oscillation, we carried out 3 different repetitive ionomycin (10 μM) treatments at 1-h intervals. There were 3 groups: group 1 (4 min, 1 time), group 2 (30 s, 4 times), and group 3 (1 min, 4 times). The difference in embryo development amongst these experimental groups was then analyzed using the one-way ANOVA test after arcsine transformation to maintain homogeneity of variance. All analyses were performed using SAS (version 8.1, SAS Institute, Cary, NC, USA). Significant differences among the treatments were determined when the P-value was <0.05. In experiment 1, to assess developmental efficiency, the cleavage rate was investigated on Day 2 and the formation rate of blastocysts (BL) was examined on Day 7. In group 3, a significant increase in BL formation was observed [47/263 (17.8%), 50/259 (19.3%), and 67/258 (26.0%), respectively]. In experiment 2, culturing each group of embryos with different ionomycin treatments caused no significant differences among the groups in terms of the total number of BL (164.3, 158.5, and 145.1, respectively). In experiment 3, expression of apoptosis-related genes in each group was evaluated by real-time PCR and the TUNEL assay. The 3 BL within each group were analyzed for the expression of apotosis-related genes. Expression of the anti-apoptotic Bcl-2 gene was increased in group 3, whereas the expression of the pro-apoptotic Bax was decreased. A decrease in the number of apoptotic nuclei was also observed in group 3. In conclusion, the present study demonstrated that repetitive ionomycin treatment is an improved activation method that can increase the developmental competence of SCNT embryos by decreasing the incidence of apoptosis.

Parthenogenetic activation and somatic cell nuclear transfer of porcine oocytes activated by an electric pulse and AZD5438 treatment

Zygote ◽  
2017 ◽  
Vol 25 (4) ◽  
pp. 453-461 ◽  
Author(s):  
Xiao-Chen Li ◽  
Qing Guo ◽  
Hai-Ying Zhu ◽  
Long Jin ◽  
Yu-Chen Zhang ◽  
...  
Keyword(s):  
Somatic Cell ◽  
Somatic Cell Nuclear Transfer ◽  
Nuclear Transfer ◽  
Electric Pulse ◽  
Formation Rate ◽  
Developmental Competence ◽  
Oocyte Activation ◽  
Blastocyst Formation ◽  
Parthenogenetic Activation

SummaryWe examined the in vitro developmental competence of parthenogenetic activation (PA) oocytes activated by an electric pulse (EP) and treated with various concentrations of AZD5438 for 4 h. Treatment with 10 µM AZD5438 for 4 h significantly improved the blastocyst formation rate of PA oocytes in comparison with 0, 20, or 50 µM AZD5438 treatment (46.4% vs. 34.5%, 32.3%, and 24.0%, respectively; P < 0.05). The blastocyst formation rate was higher in the group treated with AZD5438 for 4 h than in the groups treated with AZD5438 for 2 or 6 h (42.8% vs. 38.6% and 37.2%, respectively; P > 0.05). Furthermore, 66.67% of blastocysts derived from these AZD5438-treated PA oocytes had a diploid karyotype. The blastocyst formation rate of PA and somatic cell nuclear transfer (SCNT) embryos was similar between oocytes activated by an EP and treated with 2 mM 6-dimethylaminopurine for 4 h and those activated by an EP and treated with 10 µM AZD5438 for 4 h (11.11% vs. 13.40%, P > 0.05). In addition, the level of maturation-promoting factor (MPF) was significantly decreased in oocytes activated by an EP and treated with 10 µM AZD5438 for 4 h. Finally, the mRNA expression levels of apoptosis-related genes (Bax and Bcl-2) and pluripotency-related genes (Oct4, Nanog, and Sox2) were checked by RT-PCR; however, there were no differences between the AZD5438-treated and non-treated control groups. Our results demonstrate that porcine oocyte activation via an EP in combination with AZD5438 treatment can lead to a high blastocyst formation rate in PA and SCNT experiments.

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.

62 REPROGRAMMING EVENTS AND DEVELOPMENTAL COMPETENCE OF RHESUS MONKEY EMBRYOS PRODUCED BY SOMATIC CELL NUCLEAR TRANSFER

2006 ◽  
Vol 18 (2) ◽  
pp. 139 ◽  
Author(s):  
S. Mitalipov ◽  
Q. Zhou ◽  
J. Byrne ◽  
W.-Z. Ji ◽  
D. Wolf
Keyword(s):  
Somatic Cell ◽  
Somatic Cell Nuclear Transfer ◽  
Nuclear Transfer ◽  
Formation Rate ◽  
Embryonic Stem ◽  
Developmental Competence ◽  
Lamin A ◽  
Cell Nuclei ◽  
Blastocyst Development ◽  
Morula Stage

Successful reprogramming of somatic cell nuclei after nuclear transfer requires active remodeling by factors present in the nonactivated cytoplast. High levels of maturation promoting factor (MPF) activity are associated with this remodeling process which includes nuclear envelope breakdown (NEBD), premature chromosome condensation (PCC), and spindle formation. In this study, we examined the extent of nuclear remodeling in monkey somatic cell nuclear transfer (SCNT) embryos by monitoring the dynamics of lamin A/C appearance, as detected immunocytochemically, following fusion of donor cells with recipient cytoplasts. In the control, intracytoplasmic sperm injection (ICSI) fertilized embryos, lamin A/C was readily detected at the pronuclear stage but disappeared in early cleaving embryos only to reappear by the morula stage in association with the activation of the embryonic genome. We initially documented lack or incomplete NEBD and PCC in SCNT embryos in the form of retention of lamin A/C signal emanating from the donor nucleus. This observation was consistent with premature cytoplast activation due to the manipulation procedures. SCNT embryos produced by this approach typically arrested at the morula stage. Significant modifications in nuclear transfer protocols were then employed. Optimization of procedures resulted in robust NEBD and PCC, as indicated by loss of lamin A/C signal from the donor cell. Also, significant improvement of SCNT embryo development in vitro was observed, with a markedly improved blastocyst formation rate (21%). Several different fetal and adult somatic cell types screened as nuclear donors supported blastocyst development. SCNT blastocysts displayed a pattern of Oct-4 expression similar to that of sperm fertilized counterparts, indicative of efficient nuclear reprogramming. However, no pregnancies were established following a preliminary trial of 8 embryo transfers with 48 cloned embryos. Nevertheless, our results represent a breakthrough in efforts to produce cloned monkeys and should provide the resources required for the derivation of embryonic stem cells from SCNT blastocysts.

Effect of cytochalasins B and D on the developmental competence of somatic cell nuclear transfer embryos in miniature pigs

Zygote ◽  
2008 ◽  
Vol 16 (2) ◽  
pp. 153-159 ◽  
Author(s):  
Satoshi Sugimura ◽  
Manabu Kawahara ◽  
Takuya Wakai ◽  
Ken-ichi Yamanaka ◽  
Hiroshi Sasada ◽  
...  
Keyword(s):  
Somatic Cell ◽  
Uniform Distribution ◽  
Somatic Cell Nuclear Transfer ◽  
Nuclear Transfer ◽  
Polar Body ◽  
Formation Rate ◽  
Developmental Competence ◽  
Miniature Pigs ◽  
Artificial Activation

SummaryIn many animals, cytochalasins have generally been used as cytoskeletal inhibitors for the diploid complement retention of somatic cell nuclear transfer (SCNT) embryos. However, limited information is available on the effects of cytochalasins on the in vitro development of SCNT embryos. Hence, we compared the effects of cytochalasin B (CB) and cytochalasin D (CD) on pseudo-polar body (pPB) extrusion, cortical actin filament (F-actin) distribution in porcine parthenogenetic oocytes and in vitro development of SCNT embryos that were reconstructed using foetal fibroblasts in the G0/G1 phase derived from miniature pigs. CB (7.5 μg/ml) and CD (2.5 μg/ml) treatments effectively inhibited pPB extrusion in SCNT embryos. CB (2.5 μg/ml) treatment could not inhibit pPB extrusion and insufficiently destabilized F-actin immediately following artificial activation. In parthenogenetic oocytes treated with 2.5 μg/ml CD, normal reorganization and uniform distribution of cortical F-actin at the cytoplasmic membrane were observed at 8 h after artificial activation; this finding was similar to that of control oocytes. In contrast, parthenogenetic oocytes treated with 7.5 μg/ml CB showed non-uniform distribution of F-actin at 8 h after artificial activation. On day 5 after in vitro cultivation, the blastocyst formation rate of SCNT embryos treated with 2.5 μg/ml CD was significantly higher than that of SCNT embryos treated with 2.5 and 7.5 μg/ml CB (p < 0.05). Hence, the present findings suggest that CD is more effective than CB as the cytoskeletal inhibitor for the production of SCNT embryos in miniature pigs.

Melatonin significantly improves the developmental competence of bovine somatic cell nuclear transfer embryos

2015 ◽  
Vol 59 (4) ◽  
pp. 455-468 ◽  
Author(s):  
Jianmin Su ◽  
Yongsheng Wang ◽  
Xupeng Xing ◽  
Lei Zhang ◽  
Hongzheng Sun ◽  
...  
Keyword(s):  
Somatic Cell ◽  
Somatic Cell Nuclear Transfer ◽  
Nuclear Transfer ◽  
Developmental Competence

scholarly journals Transcriptome Analyses Reveal Differential Transcriptional Profiles in Early- and Late-Dividing Porcine Somatic Cell Nuclear Transfer Embryos

Genes ◽  
2020 ◽  
Vol 11 (12) ◽  
pp. 1499
Author(s):  
Zhiguo Liu ◽  
Guangming Xiang ◽  
Kui Xu ◽  
Jingjing Che ◽  
Changjiang Xu ◽  
...  
Keyword(s):  
Somatic Cell ◽  
Rna Processing ◽  
Somatic Cell Nuclear Transfer ◽  
Nuclear Transfer ◽  
Molecular Mechanisms ◽  
Differentially Expressed Gene ◽  
Blastocyst Stage ◽  
Developmental Competence ◽  
Rna Seq ◽  
Transcriptional Profiles

Somatic cell nuclear transfer (SCNT) is not only a valuable tool for understanding nuclear reprogramming, but it also facilitates the generation of genetically modified animals. However, the development of SCNT embryos has remained an uncontrollable process. It was reported that the SCNT embryos that complete the first cell division sooner are more likely to develop to the blastocyst stage, suggesting their better developmental competence. Therefore, to better understand the underlying molecular mechanisms, RNA-seq of pig SCNT embryos that were early-dividing (24 h postactivation) and late-dividing (36 h postactivation) was performed. Our analysis revealed that early- and late-dividing embryos have distinct RNA profiles, and, in all, 3077 genes were differentially expressed. Gene ontology (GO) and Kyoto Encyclopedia of Genes and Genomes (KEGG) analyses revealed that early-dividing embryos exhibited higher expression in genes that participated in the meiotic cell cycle, while enrichment of RNA processing- and translation-related genes was found in late-dividing embryos. There are also fewer somatic memory genes such as FLRT2, ADAMTS1, and FOXR1, which are abnormally activated or suppressed in early-dividing cloned embryos. These results show that early-dividing SCNT embryos have different transcriptional profiles than late-dividing embryos. Early division of SCNT embryos may be associated with their better reprogramming capacity, and somatic memory genes may act as a reprogramming barrier in pig SCNT reprogramming.

Early development of reconstructed embryos after somatic cell nuclear transfer in a non-human primate

2006 ◽  
Vol 66 (5) ◽  
pp. 1300-1306 ◽  
Author(s):  
Naiqing Chen ◽  
Swee-Lian Liow ◽  
Wan-Yue Yip ◽  
Lay-Geok Tan ◽  
Guo-Qing Tong ◽  
...  
Keyword(s):  
Somatic Cell ◽  
Early Development ◽  
Somatic Cell Nuclear Transfer ◽  
Nuclear Transfer ◽  
Reconstructed Embryos ◽  
Human Primate

Overexpression of MicroRNA-29b Decreases Expression of DNA Methyltransferases and Improves Quality of the Blastocysts Derived from Somatic Cell Nuclear Transfer in Cattle

2018 ◽  
Vol 24 (1) ◽  
pp. 29-37 ◽  
Author(s):  
Shuang Liang ◽  
Zheng-Wen Nie ◽  
Jing Guo ◽  
Ying-Jie Niu ◽  
Kyung-Tae Shin ◽  
...  
Keyword(s):  
Somatic Cell ◽  
Somatic Cell Nuclear Transfer ◽  
Nuclear Transfer ◽  
Cellular Proliferation ◽  
Dna Methyltransferases ◽  
Blastocyst Stage ◽  
Developmental Competence ◽  
Somatic Cell Reprogramming

AbstractMicroRNA (miR)-29b plays a crucial role during somatic cell reprogramming. The aim of the current study was to explore the effects of miR-29b on the developmental competence of bovine somatic cell nuclear transfer (SCNT) embryos, as well as the underlying mechanisms of action. The expression level of miR-29b was lower in bovine SCNT embryos at the pronuclear, 8-cell, and blastocyst stages compared within vitrofertilized embryos. In addition, miR-29b regulates the expression of DNA methyltransferases (Dnmt3a/3bandDnmt1) in bovine SCNT embryos. We further investigated SCNT embryo developmental competence and found that miR-29b overexpression during bovine SCNT embryonic development does not improve developmental potency and downregulation inhibits developmental potency. Nevertheless, the quality of bovine SCNT embryos at the blastocyst stage improved significantly. The expression of pluripotency factors and cellular proliferation were significantly higher in blastocysts from the miR-29b overexpression group than the control and downregulation groups. In addition, outgrowth potential in blastocysts after miR-29b overexpression was also significantly greater in the miR-29b overexpression group than in the control and downregulation groups. Taken together, these results demonstrated that miR-29b plays an important role in bovine SCNT embryo development.

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