27 Quisinostat, a Potent Histone Deacetylase Inhibitor, Regulates the Expression of Pluripotency- and Reprogramming-Related Genes on Somatic Cell Nuclear Transferred Porcine Embryos

2018 ◽  
Vol 30 (1) ◽  
pp. 153
Author(s):  
A. Taweechaipaisankul ◽  
J.-X. Jin ◽  
S. Lee ◽  
G. A. Kim ◽  
B. C. Lee
Keyword(s):  
Dna Methylation ◽  
Somatic Cell ◽  
Histone Acetylation ◽  
Histone Deacetylase ◽  
Histone Deacetylase Inhibitors ◽  
Formation Rate ◽  
Developmental Competence ◽  
Limited Information ◽  
Relative Expression

The low efficiency of somatic cell nuclear transfer (SCNT) has been attributed mostly to inefficient epigenetic reprogramming. Recently, various histone deacetylase inhibitors (HDACi) were used to improve developmental competence of SCNT embryos in several species. However, limited information is available on the effects of quisinostat (JNJ-26481585, JNJ), a second-generation HDACi with high cellular potency towards Class I and II histone deacetylases. Based on our previous study, among various concentrations, treatment with 100 nM JNJ could improve embryo development into blastocysts compared with the control (23.50 ± 1.30 v. 13.97 ± 1.37; P < 0.05). Thus, in the present study, treatment with 100 nM JNJ was used for further investigation into the relative expression of genes related to pluripotency and reprogramming in order to assess the quality of pre-implantation embryos cultured in media with JNJ using quantitative real-time PCR. Porcine fibroblasts isolated from kidney of adult pigs from passage 6 to 8 were used as nuclear donor cells for SCNT. After SCNT, embryos were cultured with or without 100 nM JNJ during the first 24 h of in vitro culture, and blastocysts from each experimental group were collected and kept at –80°C until analysis. Total RNAs were extracted, and transcribed into cDNA before amplification. Then, the relative expression of development-related (Oct4, Sox2, and Nanog), histone acetylation-related (HDAC1, HDAC2, and HDAC3) and DNA methylation-related (DNMT1, DNMT3a, and DNMT3b) genes between the control and 100 nM JNJ groups were compared. All experiments were repeated 4 times and results were analysed by independent t-test using SPSS 17.0K (SPSS Inc., Chicago, IL, USA). Treatment with 100 nM JNJ showed significant increases in the expression Oct4, Sox2, and Nanog compared with the control (P < 0.05). Moreover, there was significantly lower expression of HDAC1, HDAC2, HDAC3, DNMT1, DNMT3a, and DNMT3b in the 100 nM JNJ treatment than in the control (P < 0.05). These expression results moderately illustrated more active transcriptional factors, stable maintenance of embryonic pluripotency, and lesser activity of histone acetylation and DNA methylation enzymes, enhancing the blastocyst formation rate in the treatment group. In conclusion, we suggest that improvement of the in vitro developmental competence of porcine SCNT embryos might be related to positive regulations of JNJ on the expression levels of genes related to pluripotency and reprogramming. This study was supported by the NRF (#2015R1C1A2A01054373; 2016M3A9B6903410), Research Institute for Veterinary Science and the BK21 PLUS Program.

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.

17 Effect of the zona-free aggregation on the developmental competence of kodkod (Leopardus guigna) embryos generated by interspecies somatic cell nuclear transfer

2019 ◽  
Vol 31 (1) ◽  
pp. 134
Author(s):  
D. Veraguas ◽  
C. Aguilera ◽  
D. Echeverry ◽  
D. Saez-Ruiz ◽  
F. O. Castro ◽  
...  
Keyword(s):  
Gene Expression ◽  
Somatic Cell ◽  
Blastocyst Stage ◽  
Domestic Cat ◽  
Developmental Competence ◽  
Relative Expression ◽  
Standard Curve ◽  
Morula Stage ◽  
Leopardus Guigna

The kodkod is considered a vulnerable species by the International Union for Conservation of Nature. Phylogenetically, the kodkod is classified in the Leopardus genus, which has only 36 chromosome pairs compared with the domestic cat, which has 38. The proposed hypothesis was that domestic cat oocytes are capable of reprogramming somatic cells from kodkod after interspecies somatic cell NT (SCNT), allowing the in vitro embryo development up to blastocyst stage. Five experimental groups were made based on the technology and culture system: (1) cat embryos generated by IVF (IVF), (2) cat embryos generated by SCNT (Ca1x), (3) aggregated cat embryos generated by SCNT (Ca2x), (4) kodkod embryos generated by interspecies SCNT (K1x), and (5) aggregated kodkod embryos generated by interspecies SCNT (K2x). Interspecies SCNT was performed using a zona-free method. Reconstructed embryos were activated by 2 electrical pulses of 140 kV cm−1 for 40 µs and then incubated for 5h in 10μg mL−1 of cycloheximide and 5μg mL−1 of cytochalasin B. Embryos were cultured in SOF media using the well of the well system in a 5% O2, 5% CO2, and 90% N2 atmosphere at 38.5°C for 8 days. The morulae and blastocysts rates were estimated, and diameter of cloned blastocysts was measured. The relative expression of OCT4, SOX2, and NANOG was evaluated in blastocysts by RT-qPCR using the standard curve method; SDHA was used for normalization. The Kruskal-Wallis test was used to evaluate the developmental parameters and gene expression. The t-test was used to evaluate blastocyst diameter. Statistical differences were considered at P&lt;0.05. The number of replicates was IVF=10, Ca1x=8, Ca2x=6, K1x=3, and K2x=8. The morulae rate was lower when clone embryos were cultured individually (IVF=97/153, 63.4%; Ca2x=28/51, 54.9%; K2x=63/110, 57.3%; Ca1x=48/126, 38.1%; K1x=22/87, 25.3%; P&lt;0.05). In the domestic cat, blastocysts rate was higher in IVF (58/153, 37.9%) and Ca2x (28/51, 29.4%) groups than in the Ca1x group (21/126, 16.7%; P&lt;0.05). No blastocysts were generated in the K1x group (0/87), whereas 5.5% of blastocysts were obtained from the K2x (6/110; 5.5%); this was not statistically different compared with the K1x group (P&gt;0.05). No differences were found in blastocyst diameter between the Ca1x (220.4µm) and Ca2x (251.2µm) groups (P&gt;0.05). However, the diameter of the blastocysts from the K2x group (172.8µm) tended to be lower than that of the blastocysts from the Ca2x group (P=0.05). Regarding gene expression, only 1 of the 6 kodkod blastocysts expressed OCT4, and none expressed SOX2 and NANOG. On the other hand, the relative expression of OCT4 tended to decrease in blastocysts from the Ca1x and Ca2x groups compared with the IVF group (P=0.09), but no differences were found in the expression of SOX2 and NANOG among groups (P&gt;0.05). In conclusion, after interspecies SCNT, domestic cat oocytes support the development of kodkod embryos until the morula stage. However, the embryo aggregation did not significantly improve the blastocyst rate and gene expression.

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.

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.

scholarly journals BIX-01294 increases pig cloning efficiency by improving epigenetic reprogramming of somatic cell nuclei

Reproduction ◽  
2016 ◽  
Vol 151 (1) ◽  
pp. 39-49 ◽  
Author(s):  
Jiaojiao Huang ◽  
Hongyong Zhang ◽  
Jing Yao ◽  
Guosong Qin ◽  
Feng Wang ◽  
...  
Keyword(s):  
Somatic Cell ◽  
Histone Acetylation ◽  
Methylation Status ◽  
Specific Inhibitor ◽  
Epigenetic Reprogramming ◽  
Developmental Competence ◽  
Abnormal Development ◽  
Aberrant Methylation ◽  
Cell Nuclei

Accumulating evidence suggests that faulty epigenetic reprogramming leads to the abnormal development of cloned embryos and results in the low success rates observed in all mammals produced through somatic cell nuclear transfer (SCNT). The aberrant methylation status of H3K9me and H3K9me2 has been reported in cloned mouse embryos. To explore the role of H3K9me2 and H3K9me in the porcine somatic cell nuclear reprogramming, BIX-01294, known as a specific inhibitor of G9A (histone-lysine methyltransferase of H3K9), was used to treat the nuclear-transferred (NT) oocytes for 14–16 h after activation. The results showed that the developmental competence of porcine SCNT embryos was significantly enhanced bothin vitro(blastocyst rate 16.4% vs 23.2%,P<0.05) andin vivo(cloning rate 1.59% vs 2.96%) after 50 nm BIX-01294 treatment. BIX-01294 treatment significantly decreased the levels of H3K9me2 and H3K9me at the 2- and 4-cell stages, which are associated with embryo genetic activation, and increased the transcriptional expression of the pluripotency genesSOX2,NANOGandOCT4in cloned blastocysts. Furthermore, the histone acetylation levels of H3K9, H4K8 and H4K12 in cloned embryos were decreased after BIX-01294 treatment. However, co-treatment of activated NT oocytes with BIX-01294 and Scriptaid rescued donor nuclear chromatin from decreased histone acetylation of H4K8 that resulted from exposure to BIX-01294 only and consequently improved the preimplantation development of SCNT embryos (blastocyst formation rates of 23.7% vs 21.5%). These results indicated that treatment with BIX-01294 enhanced the developmental competence of porcine SCNT embryos through improvements in epigenetic reprogramming and gene expression.

16 HISTONE DEACETYLASE INHIBITORS PCI-24781 AND QUISINOSTAT IMPROVE THE IN VITRO DEVELOPMENTAL COMPETENCE OF PIG SOMATIC CELL NUCLEAR TRANSFER EMBRYOS

2016 ◽  
Vol 28 (2) ◽  
pp. 138
Author(s):  
L. Jin ◽  
H.-Y. Zhu ◽  
Q. Guo ◽  
Y.-C. Zhang ◽  
X.-C. Li ◽  
...  
Keyword(s):  
Somatic Cell ◽  
Histone Deacetylase ◽  
Histone Deacetylase Inhibitors ◽  
Control Group ◽  
Blastocyst Formation ◽  
In Vitro Development ◽  
First Polar Body ◽  
Fetal Fibroblasts ◽  
Vitro Development

The aim of this study was to examine the effects of PCI-24781 and quisinostat, two novel histone deacetylase inhibitors, on the in vitro development of pig somatic cell NT (SCNT) embryos. Pig fetal fibroblasts were used as donor cells for SCNT embryos. In vitro-matured eggs with first polar body were enucleated by aspiration using a 15-μm inner diameter glass pipette. A single donor cell was inserted into the perivitelline space of each egg and electrically fused using 2 direct pulses of 150 V mm–1 for 50 μs in 0.28 M mannitol. After 1 h, embryos were activated by 2 direct pulses of 100 V mm–1 for 20 μs and incubated with 2 mM 6-DMAP for 4 h. Subsequently, the cloned embryos were cultured in medium for 7 days. In Experiment 1, after activation and treatment with 6-DMAP for 4 h, pig SCNT embryos were treated with various concentrations of PCI-24781 or quisinostat for 24 h. In Experiment 2, NT embryos were treated with 0.5 nM PCI-24781 or 10 nM quisinostat for different durations. The rate of blastocyst formation was significantly higher in the 0.5 nM PCI-24781 group than in the control (25.3 v. 10.5%; P < 0.05; Table 1). Moreover, treatment with 10 nM quisinostat dramatically increased the proportion of embryos that reached the blastocyst stage, in comparison with the control group (18.4 v. 10.7%; P < 0.05). Table 1 shows that SCNT embryos treated with 0.5 nM PCI-24781 for 6 h had higher rates of blastocyst formation than control group (25.2 v. 10.2%; P < 0.05). However, the rate of blastocyst formation was significantly higher in the 10 nM quisinostat for 24 h group than in the control (19.8 v. 10.1%; P < 0.05). We determined the treatment conditions of PCI-24781 (0.5 nM for 6 h) and quisinostat (10 nM for 24 h) that significantly improve the in vitro development of pig SCNT embryos. Table 1.Concentration-dependent and time-dependent effects of treatment with histone deacetylase inhibitors (HDACi) PCI-24781 or quisinostat on the in vitro development of pig SCNT embryos

scholarly journals The effects of DNA methylation and histone deacetylase inhibitors upon the human papillomavirus early genes expression in cervical cancer. An in vitro and clinical study

BMC Cancer ◽  
2007 ◽  
Vol 7 (Suppl 1) ◽  
pp. A25 ◽  
Author(s):  
Erik de la Cruz-Hernandez ◽  
Adriana Contreras-Paredes ◽  
Alejandro Mohar ◽  
David Cantú ◽  
Marcela Lizano ◽  
...  
Keyword(s):  
Cervical Cancer ◽  
Dna Methylation ◽  
Human Papillomavirus ◽  
Clinical Study ◽  
Histone Deacetylase ◽  
Histone Deacetylase Inhibitors ◽  
Early Genes ◽  
Genes Expression
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