64 VALPROIC ACID ENHANCES IN VITRO DEVELOPMENT OF SOMATIC CELL NUCLEAR TRANSFER EMBRYOS IN PIGS

2010 ◽  
Vol 22 (1) ◽  
pp. 190
Author(s):  
Y. J. Kim ◽  
K. S. Ahn ◽  
M. J. Kim ◽  
H. Shim
Keyword(s):  
Stem Cells ◽  
Valproic Acid ◽  
Somatic Cell Nuclear Transfer ◽  
Nuclear Transfer ◽  
Developmental Competence ◽  
Control Group ◽  
In Vitro Development ◽  
Vitro Development ◽  
And Control

Epigenetic modification influences reprogramming and subsequent development of somatic cell nuclear transfer embryos. Such modification includes an increase of histone acetylation and a decrease of DNA methylation in transferred donor nuclei. Histone deacetylase inhibitors (HDACi) such as trichostatin A (TSA) and valproic acid (VPA) have been known to maintain high cellular levels of histone acetylation. Hence, the treatment of HDACi to NT embryos may increase efficiency of cloning. Indeed, TSA treatment has significantly enhanced the developmental competence of nuclear transfer embryos in several species including pigs (Zhang et al. 2007 Cloning Stem Cells 9, 357-363; Li et al. 2008 Theriogenology 70, 800-808). Valproic acid, another type of HDACi, has often been used to assist reprogramming of somatic cells into induced pluripotent stem cells in mice. In the present study, we tested the potency of VPA compared with TSA on the enhancement of in vitro development in porcine nuclear transfer embryos. Reconstructed embryos were produced by transferring nuclei of adult ear skin fibroblasts into enucleated oocytes. After electrical activation, these embryos were cultured in PZM-3 containing no HDACi (control), 5 mM VPA, or 50 nM TSA for 24 h, and another 5 days thereafter without HDACi. At least 3 replicates were conducted for the following experiments. The rates of cleavage were not different among the VPA, TSA, and control groups. However, the rate of blastocyst development was significantly higher (P < 0.05) in embryos treated with VPA than in those treated with TSA and without HDACi (125/306, 40.8% v. 94/313, 30.0% v. 80/329, 24.3%). Differential staining of inner cell mass (ICM) and trophectoderm (TE) also supported the beneficial effect of VPA treatment in NT embryos. Compared with the control group, the number of TE cells was significantly increased (P < 0.05) in the VPA and TSA treatment groups (79.3 ± 7.4 v. 74.6 ± 9.2 v. 40.0 ± 6.7). Moreover, VPA treatment significantly increased (P < 0.05) the number of ICM cells compared with the control (15.6 ± 1.7 v. 10.8 ± 2.6), whereas no differences were observed between the TSA treatment and control group (12.9 ± 3.0 v. 10.8 ± 2.6). The present study demonstrates that VPA enhances in vitro development of nuclear transfer embryos, in particular by an increase of blastocyst formation and the number of ICM cells, suggesting that VPA may be more potent than TSA in supporting developmental competence of cloned embryos. However, long-term effects of different HDACi in the development of nuclear transfer embryos, including any adverse outcome from destabilizing epigenetic condition, remain to be determined by further in vivo embryo transfer studies.

scholarly journals Valproic Acid Improves the In Vitro Development Competence of Bovine Somatic Cell Nuclear Transfer Embryos

2012 ◽  
Vol 14 (2) ◽  
pp. 138-145 ◽  
Author(s):  
Wenbing Xu ◽  
Yongsheng Wang ◽  
Yanyan Li ◽  
Lijun Wang ◽  
Xianrong Xiong ◽  
...  
Keyword(s):  
Valproic Acid ◽  
Somatic Cell ◽  
Somatic Cell Nuclear Transfer ◽  
Nuclear Transfer ◽  
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.

174 THE EFFECT OF DIFFERENT OVARY TRANSPORT TEMPERATURES ON IN VITRO DEVELOPMENT AND POST-THAW SURVIVAL OF BOVINE EMBRYOS

2007 ◽  
Vol 19 (1) ◽  
pp. 203
Author(s):  
S. R. Cho ◽  
S. H. Choi ◽  
H. J. Kim ◽  
C. Y. Choe ◽  
H. J. Jin ◽  
...  
Keyword(s):  
Room Temperature ◽  
Development Rate ◽  
Control Group ◽  
Bovine Embryos ◽  
In Vitro Development ◽  
Embryo Freezing ◽  
Nuclear Maturation ◽  
Vitro Development ◽  
And Control

The present study was carried out to investigate the effect of different ovary transport temperatures on in vitro development and post-thaw survivability of bovine embryos. Bovine ovaries were collected at a local slaughterhouse and transported at 4 different temperature categories to the laboratory: 7–10�C (T1), 11–17�C (T2), 18–25�C (T3), and above 26�C (control group). The cumulus–oocyte complexes (COCs) were aspirated from 2–8 mm antral follicles using a syringe with an 18 gauge needle. Selected COCs were washed in HEPES-buffered tissue culture medium (TCM-199) supplemented with 5% FBS. Sets of 50 COCs were matured for 22 h in 4-well dishes of TCM-199 supplemented with 5% FBS, 10 �g mL-1 LH, and 10 �g mL-1 FSH, that had been previously covered with mineral oil and equilibrated in an atmosphere of 5% CO2 in air at 39�C. Mature COCs were fertilized with frozen–thawed semen treated with BO medium. To evaluate nuclear maturation to the metaphase II stage, the matured COCs were fixed in 1 : 3 acetic acid–ethanol for 30 s and stained with 3% basic Fuchsin. For embryo freezing, Day 7 and 8 blastocysts were equilibrated for 15 min in 1.8 M ethylene glycol as a cryoprotectant. Embryos were loaded into 0.25-mL straws at room temperature, plunged directly into a cooling chamber, kept at -7�C for 10 min, including time for seeding, and further cooled to -35�C at -0.3�C min-1; after 2 min at this temperature, they were plunged into liquid nitrogen. Thawing was performed by keeping straws at room temperature for 10 s, followed by immersion in a water bath at 37�C. The appearance of the embryos was evaluated immediately after warming and again at 24-h intervals for at least 3 days. The development rate was assessed by the re-expansion of the blastocoel and the hatching of blastocysts. Results were compared by ANOVA. The rates of maturation (to metaphase II), cleavage, and development to blastocysts were compared among treatment groups. Furthermore, frozen–thawed blastocysts were in vitro cultured to compare the survivability among groups. The maturation rates in the T1, T2, and T3 groups (24/40, 60.0%; 25/41, 61.0%; and 30/44, 68.2%, respectively) were significantly lower than that in the control group (36/44, 81.8%; P &lt; 0.05). The cleavage rates in the T1 and T2 groups (61/116, 52.6% and 66/121, 54.5%) were significantly lower than that in the control group (112/134, 83.6%; P &lt; 0.05). However, there was no difference in the development rate to blastocysts among all groups (27.9–33.0%; P &gt; 0.05). The survivability of frozen–thawed embryos was significantly lower in the T1 group (6/13, 46.2%) than in the T2 (11/16, 68.8), T3 (13/18, 72.2%), and control groups (19/26, 73.1%; P &lt; 0.05). In conclusion, the results suggest that ovary transport at 26�C may be optimal for better in vitro development and survival of frozen–thawed embryos produced in vitro. Furthermore, exposure of ovaries to temperatures below 10�C during transport may significantly decrease both in vitro development and survivability of frozen-thawed blastocysts.

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.

79 DEVELOPMENTAL COMPETENCE OF OVINE OOCYTES VITRIFIED AT GERMINAL VESICLE STAGE: IN VITRO FERTILIZATION, PARTHENOGENETIC ACTIVATION, AND SOMATIC CELL NUCLEAR TRANSFER

2011 ◽  
Vol 23 (1) ◽  
pp. 145
Author(s):  
A. R. Moawad ◽  
I. Choi ◽  
J. Zhu ◽  
K. H. S. Campbell
Keyword(s):  
Somatic Cell ◽  
Somatic Cell Nuclear Transfer ◽  
Nuclear Transfer ◽  
Germinal Vesicle ◽  
Developmental Competence ◽  
Control Groups ◽  
High Frequencies ◽  
Parthenogenetic Activation ◽  
And Control

Oocyte cryopreservation represents an important development in the field of assisted reproductive technologies. This study investigated the effects of vitrification on spindle morphology following subsequent in vitro maturation (IVM), cleavage, and development following IVF and parthenogenetic activation. The developmental competence of ovine oocytes vitrified at the germinal vesicle (GV) stage, matured, and used as cytoplast recipients for somatic cell nuclear transfer (SCNT) was also determined. Cumulus–oocyte complexes obtained at slaughter were divided into 3 groups: 1) untreated (control), 2) toxicity (exposed to vitrification solutions without freezing), and 3) vitrified (2008 Reprod. Fertil. Dev. 20, 122). At 24 hpm (hours post onset of maturation), oocytes were subjected to 1) immunostaining, 2) IVF, or 3) activation by 2 different protocols [calcium ionophore, cycloheximide, and cytochalasin B (CA+CHX/CB), or strontium and CB (Sr/CB)]. The SCNT was performed as previously described (2010 Reprod. Fertil. Dev. 22, 1000–1014). Presumptive zygotes were cultured in vitro for 7 days. No significant differences (P > 0.05; chi-square) were observed in the frequencies of oocytes with normal spindle configuration between vitrified, toxicity, and control groups (50.0, 54.9, and 70.4%, respectively). Cleavage 24, 48 hpi, and morula development (5 days pi) were significantly decreased (P < 0.01) in the vitrified group (17.3, 42.9, and 36.4%) compared with toxicity (47.0, 85.3, and 60.7%) and control (68.9, 89.7, and 62.6%) groups. Blastocyst development significantly decreased (P < 0.01) in the vitrified group (12.3%) compared with toxicity (42.7%) and control (40.4%) groups. Based on cleaved embryos, no significant difference was observed between vitrified and control groups (29.4 v. 45.1%). Post-activation, cleavage 24 hpa (hours post-activation, 6.2 v. 3.8%) and 48 hpa (28.4 v. 27.5%) was significantly lower (P < 0.05) in vitrified oocytes activated by (CA+CHX/CB and Sr/CB) than other groups. No blastocyst developed from vitrified oocytes activated by CA+CHX/CB; however, 3.8% developed from Sr/CB oocytes. This was significantly (P < 0.05) lower than toxicity and control (20.0 and 27.3%) groups. Following SCNT, high frequencies of enucleation (99%) and fusion (98%) were achieved in vitrified and control groups. Cleavage 24 and 48 hpa significantly decreased (P < 0.05) in the vitrified group (31.0 and 48.0%) compared with the control (55.1 and 85.0%). No significant differences were observed in morula (38.0 v. 46.7%) and blastocyst (13.0 v. 23.4%) development. The proportion of cleaved embryos that developed to blastocyst stages was similar in both groups (27.0%). No significant differences (t-test) were observed in total cell numbers, apoptotic nuclei, and proportion of diploid embryos. In conclusion, ovine oocytes vitrified at GV stage can be matured, fertilized, and develop in vitro with high developmental potential. Strontium can be used effectively for activation of vitrified/thawed ovine oocytes. Vitrified/thawed ovine oocytes were used successfully for the first time as recipient cytoplasts for SCNT and produced high frequencies of good-quality blastocyst stage embryos.

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