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 Metabolic programming of a Warburg effect-like phenotype in donor fibroblasts prior to somatic cell nuclear transfer

2017 ◽  
Author(s):  
◽  
Bethany Rae Mordhorst
Keyword(s):  
Somatic Cell ◽  
Somatic Cell Nuclear Transfer ◽  
Nuclear Transfer ◽  
Donor Cell ◽  
Nuclear Reprogramming ◽  
In Vitro Development ◽  
Fetal Fibroblasts ◽  
Pharmaceutical Agents ◽  
Vitro Development

Gene edited pigs serve as excellent models for biomedicine and agriculture. Currently, the most efficient way to make a reliably-edited transgenic animal is through somatic cell nuclear transfer (SCNT) also known as cloning. This process involves using cells from a donor (which may have been gene edited) that are typically grown in culture and using their nuclear content to reconstruct a new zygote. To do this, the cell may be placed in the perivitelline space of an enucleated oocyte and activated artificially by a calcium-containing media and electrical pulse waves. While it is remarkable that this process works, it is highly inefficient. In pigs the success of transferred embryos becoming live born piglets is only 1-3%. The creation of more cloned pigs enables further study for the benefit of both A) biomedicine in the development of prognosis and treatments and B) agriculture, whether it be for disease resistance, feed efficiency, gas emissions, etc. Two decades of research has not drastically improved the cloning efficiency of most mammals. One of the main impediments to successful cloning is thought to be due to inefficient nuclear reprogramming and remodeling of the donor cell nucleus. In the following chapters we detail our efforts to improve nuclear reprogramming of porcine fetal fibroblasts by altering the metabolism to be more blastomere-like in nature. We used two methods to alter metabolism 1) pharmaceutical agents and 2) hypoxia. After treating donor cells both methods were used in nuclear transfer. Pharmaceutical agents did not improve in vitro development of gestational survival of clones. Hypoxia did improve in vitro development and we are currently awaiting results of gestation.

48 TREATMENT OF CLONED BOVINE EMBRYOS WITH HISTONE DEACETYLASE INHIBITORS INCREASES IN VITRO DEVELOPMENT BUT NOT IN VIVO CLONING EFFICIENCY

2009 ◽  
Vol 21 (1) ◽  
pp. 124
Author(s):  
J. E. Oliver ◽  
T. Delaney ◽  
J. N. Oswald ◽  
M. C. Berg ◽  
B. Oback ◽  
...  
Keyword(s):  
Histone Deacetylase ◽  
Nuclear Transfer ◽  
Histone Deacetylase Inhibitors ◽  
Trichostatin A ◽  
Cloning Efficiency ◽  
In Vitro Development ◽  
Significant Difference ◽  
Vitro Development

Previous studies in the mouse have shown treatment of somatic cell nuclear transfer (SCNT) embryos with histone deacetylase inhibitors (HDACi) to significantly increase cloning efficiency (Kishigami S et al. 2006 BBRC 340, 183–189; van Thuan N 2007 Asian Reproductive Biology Society 4, 9 abst). Increasing histone acetylation may open donor chromatin allowing better access for oocyte cytoplasmic factors to facilitate reprogramming. Here, we determined the effect of two HDACi, Trichostatin A (TSA), and scriptaid (Sigma-Aldrich, Castle Hill, NSW, Australia), on bovine cloning efficiency. Zona-free SCNT was performed with serum starved fibroblasts fused to enucleated MII-arrested IVM oocytes. After 4 h, reconstructs were activated with 5 μm ionomycin and 2 mm 6-dimethylaminopurine (DMAP) and cultured individually in 5 μL drops of AgResearch synthetic oviduct fluid (SOF) medium. Treatment with HDACi commenced concomitant with the 4 h DMAP incubation and continued in SOF for the remainder of the treatment period; totalling either 18 or 48 h post activation (hpa). TSA concentrations examined were: 0, 5, 50, and 500 nm, with all treatments containing 0.5% DMSO (n = 1121). Following TSA treatment, increased histone (H) acetylation at lysine (K) of H4K5 was confirmed by semi-quantitative immunofluorescence at the eight-cell stage. Scriptaid concentrations examined were: 0, 5, 50, 250, and 1000 nm, with all treatments containing 0.5% DMSO during DMAP and 0.1% DMSO during IVC (n = 1059). In vitro development on Day 7 was expressed in terms of transferable quality embryos as a percentage of reconstructs cultured. Data were analyzed using a generalized linear model with binomial variation and logit link. Embryos from selected treatments were transferred singularly to recipient cows on Day 7 with pregnancy data analyzed using Fisher’s exact test. Day 7 in vitro development was significantly greater with 5 nm TSA treatment for 18 hpa compared to controls (47.1% v. 34.5%; P < 0.02). Treatment of embryos with TSA for 48 hpa had no effect at any concentration tested. In contrast, scriptaid treatment for 18 hpa had no effect in vitro, while exposure for 48 hpa at 1000 nm significantly increased the development of transferable quality embryos compared to 0 nm (44.0% v. 32.4%; P < 0.005). There was no significant difference in embryo survival rates at D150 of gestation between embryos treated with 0 or 5 nm TSA for 18 hpa (8/48 v. 10/48; 16.7% v. 20.8%). However, in vivo development at Day 150 of gestation following treatment of embryos with 1000 nm scriptaid for 48 hpa was significantly lower compared to controls (1/37 v. 6/31; 2.7% v. 19.4%; P < 0.05). Contrary to the mouse, TSA or scriptaid treatment as used in this study did not increase cloning efficiency in cattle. The use of various HDACi either alone or in combination with DNA demethylating agents may still prove beneficial for reprogramming following nuclear transfer. Supported by FRST C10X0303.

In vitro development of green fluorescent protein (GFP) transgenic bovine embryos after nuclear transfer using different cell cycles and passages of fetal fibroblasts

2000 ◽  
Vol 12 (2) ◽  
pp. 1 ◽  
Author(s):  
Sangho Roh ◽  
Hosup Shim ◽  
Woo-suk Hwang ◽  
Jong-taek Yoon
Keyword(s):  
Green Fluorescent Protein ◽  
Nuclear Transfer ◽  
Fluorescent Protein ◽  
Blastocyst Formation ◽  
In Vitro Development ◽  
Donor Cells ◽  
Fetal Fibroblasts ◽  
Green Fluorescent ◽  
Vitro Development

Nuclear transfer using transfected donor cells provides an efficient new strategy for the production of transgenic farm animals. The present study assessed in vitro development of nuclear transfer embryos using green fluorescent protein (GFP) gene-transfected bovine fetal fibroblasts. In experiment 1, bovine fetal fibroblasts (BFF) were transfected with linearized pEGFP-N1 by electroporation, and the enucleated oocytes were reconstructed by nuclear transfer of transfected cells (BFF-GFP). The rates of blastocyst formation did not differ significantly between BFF and BFF-GFP (18.2% v. 15.6%). In experiment 2, before nuclear transfer, the donor cell stage was synchronized by serum deprivation or forming a confluent monolayer. The rates of cleavage (67.1% v. 71.8%) and blastocyst formation (15.8% v. 15.5%) did not differ between confluent and serum-starved cells after nuclear transfer. In experiment 3, the effects of different passages of donor fibroblast cells on the development of nuclear transfer embryos were investigated. Donor cells from ‘early’ (at passage 8–16) showed better blastocyst development (18.9%) than those from ‘late’ (at passage 17–32; 10.5%). In conclusion, this study suggests that transgenic somatic cell nuclei from early passages can be reprogrammed more effectively than those from late passages. In addition, GFP, a non-invasive selection marker, can be used to select transgenic nuclear transfer embryos.

71 DEVELOPMENT OF CLONED DOG EMBRYOS RECONSTRUCTED WITH PRE-ACTIVATED IN VIVO OOCYTES AND DONOR FIBROBLASTS ARRESTED AT G2/M PHASE USING ROSCOVITINE

2010 ◽  
Vol 22 (1) ◽  
pp. 194
Author(s):  
H. Oh ◽  
O. J. Koo ◽  
M. J. Kim ◽  
J. Park ◽  
S. Hong ◽  
...  
Keyword(s):  
Cell Cycle ◽  
Somatic Cell ◽  
Somatic Cells ◽  
Specific Cell ◽  
In Vitro Development ◽  
Donor Cells ◽  
Fetal Fibroblasts ◽  
M Phase ◽  
Vitro Development

The coordination between the cell cycle stages of nuclear donor cells and host oocytes has a critical effect on the development of embryos produced by somatic cell nuclear transfer (SCNT). Here, we investigated (1) whether roscovitine, an inhibitor of cyclin-dependent kinases (CDK) could arrest canine somatic cells at S/G2 phase of the cell cycle; (2) whether IVM metaphase II (MII) oocyte could be induced to telophase II (TII) after activation. Last, we investigated embryo development ability of nonactivated oocytes (MII) or activated oocytes (TII) fused with somatic cells at different stages of the cell cycle. Dog fetal fibroblasts were treated with roscovitine (30 or 60 μg mL-1 at 24, 48, or 72 h) and a control group of donor cells was cultured to reach confluency. The cells were then fixed and stained with 1 mg mL-1 propidium iodide for flow cytometric analysis. For SCNT, IVM dog oocytes were obtained by flushing (approximately 72 h after ovulation) from the oviducts of oocyte donor dog (Canis familiaris) and divided into 2 groups; nonactivated oocytes (MII) and activated oocytes (TII) by 10 μg mL-1 calcium ionophore for 4 min. Following preparation of each donor cell arrested in G0 and G2/M phase, cells of G0 stage were placed into enucleated MII oocytes (MII-G0) and cells of G2/M-phase were placed into enucleated TII oocytes (TII-G2/M). After fusion by electric stimulation, the MII-G0 group was chemically activated and cultured in modified SOF medium (mSOF), and the TII-G2/M group was cultured in mSOF without activation. The embryo developmental competence was estimated by assessing in vitro development under the microscope. Data were analyzed using a statistical analysis system program. Based on flow cytometry, the frequency of cells arrested at G2/M-phase in the 30 and 60 μg mL-1 roscovitine groups was significantly higher than that in control (31.95 and 25.99% v. 19.79%, respectively), but differences were not observed between the 30 and 60 μg mL-1 roscovitine groups (P > 0.05). Also, a significant increase in the proportion of cells at G2/M-phase was observed at 48 and 72 h in both roscovitine groups compared with the group not treated with roscovitine. The proportion of cells at G2/M-phase in the 60 μg mL-1 group at 48 h and the 30 μg mL-1 group at 72 h was the highest among all treatments. For the TII-G2/M group, we injected into enucleated TII oocyte and selected a large cell that arrested at G2/M-phase in cells cultured with 60 μg mL-1 roscovitine for 48 h. For the result of in vitro development of cloned embryo from MII-G0 and TII-G2/M, TII-G2/M group (39.4 and 7.8%) showed an increased cleavage rate and development to 8 cells compared with MII-G0 (23.5 and 2.9%). In the present study, we demonstrated that, in combination with nuclear donor cells at specific cell cycle stages, MII and TII dog oocytes are similarly effective in supporting the reprogramming of somatic cell nuclei. This study was supported by Korean MEST through KOSEF (grant # M10625030005-09N250300510) and BK21 program, RNL BIO, and Natural Balance Korea.

scholarly journals In Vitro Development of Reconstructed Porcine Oocytes after Somatic Cell Nuclear Transfer1

2000 ◽  
Vol 63 (4) ◽  
pp. 986-992 ◽  
Author(s):  
Deog-Bon Koo ◽  
Yong-Kook Kang ◽  
Young-Hee Choi ◽  
Jung Sun Park ◽  
Sun-Kyung Han ◽  
...  
Keyword(s):  
Somatic Cell ◽  
In Vitro Development ◽  
Vitro Development

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 28 SIMPLIFIED ACTIVATION METHOD TO IMPROVE THE IN VITRO DEVELOPMENT OF HANDMADE CLONED (HMC) PORCINE EMBRYOS

2009 ◽  
Vol 21 (1) ◽  
pp. 114
Author(s):  
Y. Du ◽  
Z. Yang ◽  
B. Lv ◽  
L. Lin ◽  
P. M. Kragh ◽  
...  
Keyword(s):  
Cell Number ◽  
Activation Method ◽  
The Other ◽  
Electrical Activation ◽  
In Vitro Development ◽  
Reconstructed Embryos ◽  
Fetal Fibroblasts ◽  
Group 2 ◽  
Vitro Development

Delayed activation is commonly used in pig somatic cell nuclear transfer (SCNT) where electrical activation is followed by chemical activation. However, chemical incubation of several hours (up to 4 or 6) is logistically not very convenient even though handmade cloning (HMC) could improve the overall efficiency of pig cloning (Du et al. 2007 Theriogenology 68, 1104–1110). It was reported that a brief exposure of cycloheximide (CX) before electrical activation could significantly increase developmental rate and total blastocyst cell number when simultaneous activation was performed in micromanipulator-based pig cloning (Naruse et al. 2007 Theriogenology 68, 709–716). The purpose of our present work is to investigate whether such activation method is also applicable for pig HMC. Data were analyzed by t-test using SPSS (11.0, SPSS Inc., Chicago, IL, USA). After 42 h in vitro maturation, cumulus cells were removed. In vitro-cultured porcine fetal fibroblasts were used as donor cells. Cytoplast-fibroblast pairing, electrical fusion and activation of fused cytoplast-fibroblast pairs were performed as described previously (Kragh et al. 2005 Theriogenology 64, 1536–1545; Du et al. 2005 Cloning Stem Cells 7, 199–205). Three groups were compared due to different activation protocol. In Group 1 (control), reconstructed embryos were cultured in porcine zygote medium 3 (PZM3) supplemented with 4 mg mL–1 BSA, 5 μg mL–1 cytochalasin B (CB), and 10 μg mL–1 CX for 4 h. In Group 2 (CX priming), fused pairs and the other halves of cytoplasts were incubated in HEPES-buffered TCM-199 medium supplemented with 10% calf serum, 10 μg mL–1 CX for 10 min just before the second fusion or electrical activation. In Group 3 (CB + CX priming), treatment similar to Group 2 was performed except that additional 5 μg mL–1 CB was added for the 10-min incubation. Reconstructed embryos were in vitro cultured in the well of the well (WOW) system for 6 days. Blastocyst rates and total cell numbers of Day 6 blastocysts were evaluated. As illustrated in Table 1, embryos pretreated with both CB and CX gave the best results, with better blastocyst formation (53.8 ± 4.8%; mean ± SEM) and higher cell number (77.2 ± 5.4) compared to the other 2 groups. Our data suggested that CX and CB priming could be used as a solution to the long chemical incubation in porcine SCNT by HMC, making the embryos more receptive to electrical activation. Table 1.In vitro development of HMC reconstructed embryos with different activation protocols

scholarly journals Effect of Melatonin on the In Vitro Maturation of Porcine Oocytes, Development of Parthenogenetically Activated Embryos, and Expression of Genes Related to the Oocyte Developmental Capability

Animals ◽  
2020 ◽  
Vol 10 (2) ◽  
pp. 209 ◽  
Author(s):  
Ling Yang ◽  
Qingkai Wang ◽  
Maosheng Cui ◽  
Qianjun Li ◽  
Shuqin Mu ◽  
...  
Keyword(s):  
In Vitro Maturation ◽  
Polar Body ◽  
Melatonin Receptors ◽  
Melatonin Treatment ◽  
In Vitro Development ◽  
First Polar Body ◽  
Mitochondrial Distribution ◽  
Polar Body Extrusion ◽  
Vitro Development

Melatonin treatment can improve quality and in vitro development of porcine oocytes, but the mechanism of improving quality and developmental competence is not fully understood. In this study, porcine cumulus–oocyte complexes were cultured in TCM199 medium with non-treated (control), 10−5 M luzindole (melatonin receptor antagonist), 10−5 M melatonin, and melatonin + luzindole during in vitro maturation, and parthenogenetically activated (PA) embryos were treated with nothing (control), or 10−5 M melatonin. Cumulus oophorus expansion, oocyte survival rate, first polar body extrusion rate, mitochondrial distribution, and intracellular levels of reactive oxygen species (ROS) and glutathione of oocytes, and cleavage rate and blastocyst rate of the PA embryos were assessed. In addition, expression of growth differentiation factor 9 (GDF9), tumor protein p53 (P53), BCL2 associated X protein (BAX), catalase (CAT), and bone morphogenetic protein 15 (BMP15) were analyzed by real-time quantitative PCR. The results revealed that melatonin treatment not only improved the first polar body extrusion rate and cumulus expansion of oocytes via melatonin receptors, but also enhanced the rates of cleavage and blastocyst formation of PA embryos. Additionally, melatonin treatment significantly increased intraooplasmic level of glutathione independently of melatonin receptors. Furthermore, melatonin supplementation not only significantly enhanced mitochondrial distribution and relative abundances of BMP15 and CAT mRNA, but also decreased intracellular level of ROS and relative abundances of P53 and BAX mRNA of the oocytes. In conclusion, melatonin enhanced the quality and in vitro development of porcine oocytes, which may be related to antioxidant and anti-apoptotic mechanisms.

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