24 BUFFALO (BUBALUS BUBALIS) SOMATIC CELL NUCLEAR TRANSFER EMBRYOS PRODUCED FROM FROZEN–THAWED SEMEN-DERIVED SOMATIC CELLS: EFFECT OF TRICHOSTATIN A ON THE IN VITRO AND IN VIVO DEVELOPMENTAL POTENTIAL, QUALITY, AND EPIGENETIC STATUS

2015 ◽  
Vol 27 (1) ◽  
pp. 104
Author(s):  
N. L. Selokar ◽  
M. Saini ◽  
H. Agrawal ◽  
P. Palta ◽  
M. S. Chauhan ◽  
...  
Keyword(s):  
Somatic Cell ◽  
Somatic Cell Nuclear Transfer ◽  
Nuclear Transfer ◽  
Trichostatin A ◽  
Somatic Cells ◽  
Developmental Potential ◽  
Reconstructed Embryos ◽  
Frozen Semen ◽  
Significant Difference

Cryopreservation of semen allows preservation of somatic cells, which can be used for the production of progeny through somatic cell nuclear transfer (SCNT). This approach could enable restoration of valuable high-genetic-merit progeny-tested bulls, which may be dead but the cryopreserved semen is available. We have successfully produced a live buffalo calf by SCNT using somatic cells isolated from >10 year old frozen semen (Selokar et al. 2014 PLoS One 9, e90755). However, the calf survived only for 12 h, which indicates faulty reprogramming of these cells. The present study was, therefore, carried out to study the effect of treatment with trichostatin A (TSA), an epigenetic modifier, on reprogramming of these cells. Production of cloned embryos and determination of quality and level of epigenetic markers in blastocysts were performed according to the methods described previously (Selokar et al. 2014 PLoS One 9, e90755). To examine the effects of TSA (0, 50, and 75 nM), 10 separate experiments were performed on 125, 175, and 207 reconstructed embryos, respectively. The percentage data were analysed using SYSTAT 12.0 (SPSS Inc., Chicago, IL, USA) after arcsine transformation. Differences between means were analysed by one-way ANOVA followed by Fisher's least significant difference test for significance at P < 0.05. When the reconstructed buffalo embryos produced by hand-made clones were treated with 0, 50, or 75 nM TSA post-electrofusion for 10 h, the cleavage percentage (100.0 ± 0, 94.5 ± 2.3, and 96.1 ± 1.2, respectively) and blastocyst percentage (50.6 ± 2.3, 48.4 ± 2.7, and 48.1 ± 2.6, respectively), total cell number (274.9 ± 17.4, 289.1 ± 30.1, and 317.0 ± 24.2, respectively), and apoptotic index (3.4 ± 0.9, 4.5 ± 1.4, and 5.6 ± 0.7, respectively) in Day 8 blastocysts were not significantly different among different groups. The TSA treatment increased (P < 0.05) the global level of H4K5ac but not that of H3K18a in embryos treated with 50 or 75 nM TSA compared with that in controls. In contrast, the level of H3K27me3 was significantly lower (P < 0.05) in cloned embryos treated with 75 nM TSA than in embryos treated with 50 nM TSA or controls. The ultimate test of the reprogramming potential of any donor cell type is its ability to produce live offspring. To examine the in vivo developmental potential of the 0, 50, or 75 nM TSA treated embryos, we transferred Day 8 blastocysts, 2 each to 5, 6, and 5 recipients, respectively, which resulted in 2 pregnancies from 75 nM TSA treated embryos. However, one pregnancy was aborted in the first trimester and the other in the third trimester. In conclusion, TSA treatment of reconstructed embryos produced from semen-derived somatic cells alters their epigenetic status but does not improve the live birth rate. We are currently optimizing an effective strategy to improve the cloning efficiency of semen-derived somatic cells.

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.

Effect of volume of oocyte cytoplasm on embryo development after parthenogenetic activation, intracytoplasmic sperm injection, or somatic cell nuclear transfer

Zygote ◽  
2008 ◽  
Vol 16 (3) ◽  
pp. 211-222 ◽  
Author(s):  
Wakayama Sayaka ◽  
Kishigami Satoshi ◽  
Nguyen Van Thuan ◽  
Ohta Hiroshi ◽  
Hikichi Takafusa ◽  
...  
Keyword(s):  
Somatic Cell ◽  
Intracytoplasmic Sperm Injection ◽  
Somatic Cell Nuclear Transfer ◽  
Nuclear Transfer ◽  
Subsequent Development ◽  
Developmental Potential ◽  
Parthenogenetic Activation ◽  
Somatic Cell Nucleus

SummaryAnimal cloning methods are now well described and are becoming routine. Yet, the frequency at which live cloned offspring are produced remains below 5%, irrespective of the nuclear donor species or cell type. One possible explanation is that the reprogramming factor(s) of each oocyte is insufficient or not properly adapted for the receipt of a somatic cell nucleus, because it is naturally prepared only for the receipt of a gamete. Here, we have increased the oocyte volume by oocyte fusion and examined its subsequent development. We constructed oocytes with volumes two to nine times greater than the normal volume by the electrofusion or mechanical fusion of intact and enucleated oocytes. We examined their in vitro and in vivo developmental potential after parthenogenetic activation, intracytoplasmic sperm injection (ICSI) and somatic cell nuclear transfer (SCNT). When the fused oocytes were activated parthenogenetically, most developed to morulae or blastocysts, regardless of their original size. Diploid fused oocytes were fertilized by ICSI and developed normally and after embryo transfer, we obtained 12 (4–15%) healthy and fertile offspring. However, enucleated fused oocytes could not support the development of mice cloned by SCNT. These results suggest that double fused oocytes have normal potential for development after fertilization, but oocytes with extra cytoplasm do not have enhanced reprogramming potential.

19 OPTIMIZATION OF ENUCLEATION TIME AFTER IVM FOR SOMATIC CELL NUCLEAR TRANSFER IN GOAT

2009 ◽  
Vol 21 (1) ◽  
pp. 109 ◽  
Author(s):  
G. S. Ajithkumar ◽  
B. Krishnamohan ◽  
B. C. Sarkhel
Keyword(s):  
Somatic Cell ◽  
Somatic Cell Nuclear Transfer ◽  
Nuclear Transfer ◽  
Proper Time ◽  
Polar Body ◽  
Fluorescent Microscopy ◽  
Developmental Potential ◽  
Randomized Design ◽  
Significant Difference ◽  
Arcsine Transformation

For cloning by somatic cell nuclear transfer (SCNT) in goat, there are conflicting reports about the proper time of enucleation after IVM of oocytes, which varied from 20 to 27 h (Das SK et al. 2003 Small Rumin. Res. 48, 217–225; Keefer CL et al. 2002 Biol. Reprod. 66, 199–203; Daniel SM et al. 2007 Small Rumin. Res. 77, 45–50). The present investigation has been undertaken to standardize the optimum time of enucleation after IVM of oocytes. The hypothesis behind the study was that enucleation performed during early stages of maturation maintains the MII plate and polar body (PB) in a closer position and therefore makes it easy to enucleate. To test this hypothesis, caprine COCs were aspirated from slaughterhouse ovaries of goats and good quality oocytes were matured in TCM 199 containing 7.5% FBS supplemented with FSH, LH, and estradiol. Enucleation was performed in four different interval groups after IVM (20–23 h, 23–26 h, 26–29 h and 29–32 h). The enucleation of oocytes was conducted as per method described by (Du F et al. 2006 Theriogenology 65, 642–665). Briefly, IVM oocytes were enucleated by squashing and compressing out the first PB along with 10 to 15% of its surrounding cytoplasm with an enucleation needle through a slit made on the zona pellucida. Successful enucleation was confirmed by fluorescent microscopy of removed ooplasm after staining with Hoechst 33342. The enucleation percentage values after arcsine transformation was analyzed by completely randomized design ANOVA. The result of enucleation at different intervals has been summarized in Table 1. There was no significant difference (P > 0.05) in number of PB observed among the four enucleation groups, however the enucleation percentage decreased significantly (P < 0.05) with increase in enucleation time (70.29% and 70.51% in G1 and G2 v. 59.52% and 55.61% in G3 and G4 respectively). With increase in time of enucleation after maturation the size of perivitelline space increases, causing deviation of PB from spindle, thus the success rate of enucleation is reduced (Song K et al. 2007 Repro. Fertil. Dev. 19, 293–294). In G1 and G2 groups the PB and MII chromosomes are located close together with stronger spindle force that requires minimum ooplasm to be removed with higher percentage of successful enucleation. Hence, it was concluded that G1 and G2 groups may be considered as most efficient for enucleation but the developmental potential of reconstructed oocytes after nuclear transfer in each group needs to be tested (study under progress). Table 1.Enucleation results at different intervals

scholarly journals Combined Chaetocin/Trichostatin A Treatment Improves the Epigenetic Modification and Developmental Competence of Porcine Somatic Cell Nuclear Transfer Embryos

2021 ◽  
Vol 9 ◽  
Author(s):  
Pil-Soo Jeong ◽  
Hae-Jun Yang ◽  
Soo-Hyun Park ◽  
Min Ah Gwon ◽  
Ye Eun Joo ◽  
...  
Keyword(s):  
Somatic Cell ◽  
Somatic Cell Nuclear Transfer ◽  
Nuclear Transfer ◽  
Histone Deacetylase Inhibitors ◽  
Epigenetic Modification ◽  
Trichostatin A ◽  
Combined Treatment ◽  
Epigenetic Reprogramming ◽  
Developmental Competence ◽  
Developmental Potential

Developmental defects in somatic cell nuclear transfer (SCNT) embryos are principally attributable to incomplete epigenetic reprogramming. Small-molecule inhibitors such as histone methyltransferase inhibitors (HMTi) and histone deacetylase inhibitors (HDACi) have been used to improve reprogramming efficiency of SCNT embryos. However, their possible synergistic effect on epigenetic reprogramming has not been studied. In this study, we explored whether combined treatment with an HMTi (chaetocin) and an HDACi (trichostatin A; TSA) synergistically enhanced epigenetic reprogramming and the developmental competence of porcine SCNT embryos. Chaetocin, TSA, and the combination significantly increased the cleavage and blastocyst formation rate, hatching/hatched blastocyst rate, and cell numbers and survival rate compared to control embryos. In particular, the combined treatment improved the rate of development to blastocysts more so than chaetocin or TSA alone. TSA and combined chaetocin/TSA significantly reduced the H3K9me3 levels and increased the H3K9ac levels in SCNT embryos, although chaetocin alone significantly reduced only the H3K9me3 levels. Moreover, these inhibitors also decreased global DNA methylation in SCNT embryos. In addition, the expression of zygotic genome activation- and imprinting-related genes was increased by chaetocin or TSA, and more so by the combination, to levels similar to those of in vitro-fertilized embryos. These results suggest that combined chaetocin/TSA have synergistic effects on improving the developmental competences by regulating epigenetic reprogramming and correcting developmental potential-related gene expression in porcine SCNT embryos. Therefore, these strategies may contribute to the generation of transgenic pigs for biomedical research.

91 PRODUCTION OF A CLONED BOER GOAT (CAPRA HIRCUS) BY SOMATIC CELL NUCLEAR TRANSFER

2007 ◽  
Vol 19 (1) ◽  
pp. 163
Author(s):  
Y. Tao ◽  
W. Han ◽  
M. Zhang ◽  
J. Ding ◽  
X. Zhang
Keyword(s):  
Somatic Cell ◽  
Somatic Cell Nuclear Transfer ◽  
Nuclear Transfer ◽  
Capra Hircus ◽  
Blastocyst Development ◽  
Tissue Slices ◽  
Boer Goat ◽  
Reconstructed Embryos ◽  
Significant Difference

We reported the birth of a goat clone produced by somatic cell nuclear transfer. The fusion and activation protocols of reconstructed oocytes and embryo transfer procedure were optimized. The donors of somatic cells were fibroblasts derived from ear skin of a Boer goat while the recipient ooplasm was in vitro-matured oocytes of Huanghuai white goat, an Anhui native goat species. The reconstructed embryos were activated by ionomycin, 6-dimethylaminopurine (6-DMAP), and cytochalasin B (CB) singly or simultaneously (termed as Ionomycin, Ionomycin+6-DMAP, and Ionomycin+6-DMAP+CB). The result showed that the cleavage rate in single ionomycin was significantly lower than that in Ionomycin+6-DMAP and 6-DMAP+CB (34.38% vs. 69.85% and 72.02%; P &lt; 0.05). However, the cleavage rates and blastocyst rates had no significant difference after in vitro culture (P &gt; 0.05). When the cloned embryos were co-cultured with fetal mouse fibroblast monolayer, the blastocyst development rate increased. The reconstructed embryos were equilibrated 1–3 h, 3–6 h, and 6–9 h after fusion, and then activation was undertaken by ionomycin+6-DMAP. We found that the cleavage rates had no significant difference during 1–3 h and 3–6 h (72.58% vs. 72.97%; P &gt; 0.05), but both were significantly higher than during 6–9 h (64.40%) (P &lt; 0.05). A total of 491 reconstructed embryos were surgically transferred into 37 recipient surrogates, Huanghuai white goats with natural estrus. One of those who were treated with hCG after transfer was pregnant and gave birth to a live kid on 153 days. The lamb died accidentally 8 h after birth. The cloned offspring was then dissected and proved well in all organs. Staining of paraffin tissue slices of the viscera suggested that the organs developed well. Microsatellite analysis indicated that the lamb was derived from the somatic cell donor doe genetically.

Effect of melatonin treatment on developmental potential of somatic cell nuclear-transferred mouse oocytes in vitro

Zygote ◽  
2013 ◽  
Vol 22 (2) ◽  
pp. 213-217 ◽  
Author(s):  
Mohammad Salehi ◽  
Yoko Kato ◽  
Yukio Tsunoda
Keyword(s):  
Somatic Cell ◽  
Somatic Cell Nuclear Transfer ◽  
Nuclear Transfer ◽  
Culture Medium ◽  
Blastocyst Stage ◽  
Control Group ◽  
Developmental Potential ◽  
Significant Difference ◽  
Mouse Somatic Cell

SummaryThe beneficial effect of supplementing culture medium with melatonin has been reported during in vitro embryo development of species such as mouse, bovine and porcine. However, the effect of melatonin on mouse somatic cell nuclear transfer remains unknown. In this study, we assessed the effects of various concentrations of melatonin (10−6 to 10−12 M) on the in vitro development of mouse somatic cell nuclear transfer embryos for 96 h. Embryos cultured without melatonin were used as control. There was no significant difference in cleavage rates between the groups supplemented with melatonin, dimethyl sulphoxide (DMSO) and the control. The rate of development to blastocyst stage was significantly higher in the group supplemented with 10−12 M melatonin compared with the control group (P < 0.05). Thus, our data demonstrated that adding melatonin to pre-implantation mouse nuclear-transferred embryos can accelerate blastocyst formation.

The developmental potential of mouse somatic cell nuclear-transferred oocytes treated with trichostatin A and 5-aza-2′-deoxycytidine

Zygote ◽  
2009 ◽  
Vol 17 (2) ◽  
pp. 109-115 ◽  
Author(s):  
Yuta Tsuji ◽  
Yoko Kato ◽  
Yukio Tsunoda
Keyword(s):  
Somatic Cell ◽  
Treatment Duration ◽  
Dna Methyltransferase ◽  
Trichostatin A ◽  
Somatic Cells ◽  
Cell Stage ◽  
Developmental Potential ◽  
Morula Stage

SummaryTo facilitate nuclear reprogramming, somatic cells or somatic cell nuclear-transferred (SCNT) oocytes have been treated with the histone deacetylase inhibitor trichostatin A (TSA), or the DNA methyltransferase inhibitor, 5-aza-2′-deoxycytidine (5-aza-dC), to relax epigenetic marks of differentiated somatic cells. TSA-treated SCNT oocytes have increased developmental potential, but the optimal treatment period is unknown. Reduced methylation levels in somatic cells have no positive effect on SCNT oocytes, but the treatment of SCNT embryos with 5-aza-dC has not been investigated. We examined the effect of TSA treatment duration on the developmental potential of mouse SCNT oocytes and the effect of 5-aza-dC treatment on their in vitro and in vivo developmental potential. To determine the effects of TSA treatment duration, nuclear-transferred (NT) oocytes were cultured for 0 to 26 h with 100 nM TSA. SCNT oocytes treated with TSA for 8 to 12 h had the higher rate of development to blastocysts and full-term fetuses were obtained after treatment for 8 to 12 h. When oocytes were treated for 14 h and 26 h, blastocyst rates were significantly decreased and fetuses were not obtained. To examine the effect of 5-aza-dC, 2-cell stage SCNT embryos were cultured with 10 or 100 nM 5-aza-dC for 48 h to the morula stage and transferred. The potential of embryos treated with 5-aza-dC to develop into blastocysts was decreased and no fetuses were obtained after transfer. The findings demonstrated that long-term TSA treatment of SCNT mouse oocytes and treatment with 5-aza-dC inhibit the potential to develop into blastocysts and to fetuses after transfer.

45 EFFECT OF TRICHOSTATIN A TREATMENT ON THE TERM DEVELOPMENT OF SOMATIC CELL NUCLEAR TRANSFER RABBIT EMBRYOS

2008 ◽  
Vol 20 (1) ◽  
pp. 103 ◽  
Author(s):  
Q. Meng ◽  
Z. Polgar ◽  
J. Liu ◽  
A. Dinnyes
Keyword(s):  
Somatic Cell ◽  
Somatic Cell Nuclear Transfer ◽  
Nuclear Transfer ◽  
Trichostatin A ◽  
Cumulus Cell ◽  
Cell Number ◽  
Pregnant Female ◽  
Rabbit Embryos

The efficiency of somatic cell nuclear transfer (SCNT) is low in the rabbit. So far, there have been few live births reported and most clones died within the first 3 weeks after birth. It has been shown that treatment with trichostatin A (TSA), a histone deacetylase inhibitor, improved cloning efficiency in cattle (Enright et al. 2003 Biol. Reprod. 69, 896–901) and mice (Kishigami et al. 2006 Biochem. Biophys. Res. Commun. 340, 183–189; Rybouchkin et al. 2006 Biol. Reprod. 74, 1083–1089). Although a recent report indicated that TSA treatment could increase the cell number of rabbit SCNT blastocysts (Xu et al. 2007 Reprod. Fertil. Dev. 19, 165), term development of TSA-treated cloned embryos in this species has not been reported. In this study we investigated the effect of TSA treatment on the term development of somatic cell nuclear transfer (SCNT) rabbit embryos. The oocytes and cumulus cells were collected from superovulated Hycole hybrid rabbits. After staining with Hoechst 33342 and locating following 1–2 s of UV illumination, the nuclei of oocytes were removed by micromanipulation, A cumulus cell was then inserted into the perivitelline space and fused with the cytoplast with three 20 μs 3.2 kV cm–1 DC pulses. Fused embryos were activated using the same electrical parameters 1 h later, treated with 2 mm 6-dimethylaminopurine and 5 μg mL–1 cycloheximide for 1 h, and then subsequently cultured in Earles Balanced Salt Solution (EBSS) with or without 5 nm TSA for 10 h. The embryos were then cultured in EBSS either overnight (before ET) or for 4.5 days. Embryos were transferred at the 2- to 4-cell stages to the recipients 22 h after collection of the oocytes from the donors. Caesarean sections were performed on Day 30 post-ET. In vitro developmental data (Table 1) showed no differences in the cleavage, blastocyst rates, and blastocyst cell numbers between the TSA-treated or untreated cloned embryos. After ET in the TSA group, one pregnant female delivered 7 live and 3 stillborn pups, but all of the live pups died within 1 h to 19 days later. In the untreated group, one pregnant female gave birth to 2 live and 1 stillborn pup. One pup died within 1 h after birth; the other survived (2.5 months old when this abstract was submitted). In conclusion, the results indicate that TSA treatment has a limited effect on in vitro development of SCNT embryos, and both TSA-treated and untreated SCNT clones can develop to term in rabbit. The effects of TSA treatment on the health of clones need further investigation. Table 1. In vitro and in vivo development of SCNT rabbit embryos with or without TSA treatment This study was supported by Wellcome Trust (Grant No. 070246), EU FP6 (MEXT-CT-2003-509582, MRTN-CT-2006-035468), and Chinese-Hungarian Bilateral projects (TET CHN-28/04, CHN-41/05).

Effects of sucrose treatment on the development of mouse nuclear transfer embryos with morula blastomeres as donors

Zygote ◽  
2008 ◽  
Vol 16 (1) ◽  
pp. 15-19 ◽  
Author(s):  
Gang Zhang ◽  
Qing-Yuan Sun ◽  
Da-Yuan Chen
Keyword(s):  
Nuclear Transfer ◽  
Fusion Rate ◽  
Alternative Source ◽  
Kunming Mouse ◽  
Developmental Potential ◽  
Reconstructed Embryos ◽  
Significant Difference ◽  
The Mean

SummaryIn this study, nuclear transfer (NT) embryos were produced by using C57Bl/6 mouse morula blastomeres and Kunming mouse metaphase II (MII) oocytes as donors and recipients, respectively, to investigate the effects of sucrose treatment of MII oocytes with different concentrations on the manipulation time of NT, electrofusion and the in vitro and in vivo development of reconstructed embryos. The results demonstrated that: (i) when the oocytes were enucleated with 1, 2 and 3% sucrose treatment, respectively, the enucleating rates were not affected by the different sucrose concentrations, but the manipulation time had significant difference and the mean nuclear transfer manipulation times of every oocyte were 180 ± 10 s, 130 ± 10 s and 120 ± 10 s, respectively; (ii) different sucrose concentrations had no significant effects on the fusion rate and the in vitro developmental potential of the NT embryos (p > 0.05). Furthermore, 59 embryos were transplanted into the oviducts of two recipients. In the end, three dead full-term developed fetuses were obtained on 21 days post coitus (dpc). These results suggested that the mouse MII oocytes enucleated via sucrose treatment might be an alternative source for mouse cloning and could support the embryonic NT embryos developed to term in vivo.

Zebularine significantly improves the preimplantation development of ovine somatic cell nuclear transfer embryos

2019 ◽  
Vol 31 (2) ◽  
pp. 357 ◽  
Author(s):  
Hui Cao ◽  
Jun Li ◽  
Wenlong Su ◽  
Junjie Li ◽  
Zhigang Wang ◽  
...  
Keyword(s):  
Dna Methylation ◽  
Somatic Cell ◽  
Somatic Cell Nuclear Transfer ◽  
Nuclear Transfer ◽  
Cumulus Cells ◽  
Control Group ◽  
Developmental Potential ◽  
Reconstructed Embryos ◽  
Donor Cells ◽  
Pluripotency Genes

Aberrant DNA methylation reduces the developmental competence of mammalian somatic cell nuclear transfer (SCNT) embryos. Thus, hypomethylation-associated drugs are beneficial for improving reprogramming efficiency. Therefore, in the present study we investigated the effect of zebularine, a relatively novel DNA methyltransferase inhibitor, on the developmental potential of ovine SCNT embryos. First, reduced overall DNA methylation patterns and gene-specific DNA methylation levels at the promoter regions of pluripotency genes (octamer-binding transcription factor 4 (Oct4), SRY (sex determining region Y)-box 2 (Sox2) and Nanog) were found in zebularine-treated cumulus cells. In addition, the DNA methylation levels in SCNT embryos derived from zebularine-treated cumulus cells were significantly reduced at the 2-, 4-, 8-cell, and blastocyst stages compared with their corresponding controls (P&lt;0.05). The blastocyst rate was significantly improved in SCNT embryos reconstructed by the cumulus donor cells treated with 5nM zebularine for 12h compared with the control group (25.4±1.6 vs 11.8±1.7%, P&lt;0.05). Moreover, the abundance of Oct4 and Sox2 mRNA was significantly increased during the preimplantation stages after zebularine treatment (P&lt;0.05). In conclusion, the results indicate that, in an ovine model, zebularine decreases overall DNA methylation levels in donor cumulus cells and reconstructed embryos, downregulates the DNA methylation profile in the promoter region of pluripotency genes in donor cells and ultimately elevates the expression of pluripotency genes in the reconstructed embryos, which can lead to improved development of SCNT embryos.

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