The present study was carried out to examine the effects of valproic acid (VPA), a histone deacetylase inhibitor, on in vitro development of miniature pig somatic cell nuclear transfer (SCNT) embryos and on expression of a mouse Oct-3/4 promoter-driven enhanced green fluorescent protein (EGFP) gene (EGFP expression only detected in Oct-3/4-expressing cells) introduced into donor cells for SCNT during their development. The addition of 4 mM VPA to embryo culture medium for 48 h after activation significantly (p < 0.01) increased the blastocyst formation rate of SCNT embryos compared with the control, whereas VPA did not affect their cleavage rate. The rate of SCNT embryos expressing EGFP at 5 days of culture was not affected by the presence or absence of VPA treatment. At 7 days of culture, however, the addition of 4 mM VPA to embryo culture medium for 48 h after activation significantly (p < 0.05) increased the rate of SCNT embryos expressing EGFP compared with the control. The results indicate that VPA enhances the ability of miniature pig SCNT embryos to develop into blastocysts and maintains the ability of them to express Oct-3/4 gene.
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"For example, treatment of donor cells with TSA, markedly improved in vitro development of SCNT embryos in rabbits  and cattle . In addition, the use of NaBu in cattle improved SCNT  whereas the use of VPA enhanced gene reactivation and reprogramming efficiency in pig SCNT  and mouse induced pluripotent stem cells (iPSCs) experiments . VPA, a short-chain fatty acid, is widely used in humans as an anticonvulsant and mood stabilizer . "
[Show abstract][Hide abstract] ABSTRACT: Cloning of mammals by somatic cell nuclear transfer (SCNT) is still plagued by low efficiency. The epigenetic modifications established during cellular differentiation are a major factor determining this low efficiency as they act as epigenetic barriers restricting reprogramming of somatic nuclei. In this regard, most factors that promote chromatin decondensation, including histone deacetylase inhibitors (HDACis), have been found to increase nuclear reprogramming efficiency, making their use common to improve SCNT rates. Herein we used valproic acid (VPA) in SCNT to test whether the treatment of nuclear donor cells with this HDACi improves pre- and post-implantation development of cloned cattle. We found that the treatment of fibroblasts with VPA increased histone acetylation without affecting DNA methylation. Moreover, the treatment with VPA resulted in increased expression of IGF2R and PPARGC1A, but not of POU5F1. However, when treated cells were used as nuclear donors no difference of histone acetylation was found after oocyte reconstruction compared to the use of untreated cells. Moreover, shortly after artificial activation the histone acetylation levels were decreased in the embryos produced with VPA-treated cells. With respect to developmental rates, the use of treated cells as donors resulted in no difference during pre- and post-implantation development. In total, five clones developed to term; three produced with untreated cells and two with VPA-treated cells. Among the calves from treated group, one stillborn calf was delivered at day 270 of gestation whereas the other one was delivered at term but died shortly after birth. Among the calves from the control group, one died seven days after birth whereas the other two are still alive and healthy. Altogether, these results show that in spite of the alterations in fibroblasts resulting from the treatment with VPA, their use as donor cells in SCNT did not improve pre- and post-implantation development of cloned cattle.
PLoS ONE 06/2014; 9(6):e101022. DOI:10.1371/journal.pone.0101022 · 3.23 Impact Factor
"Recently, VPA was used as a reprogramming drug for mouse embryonic fibroblasts (MEFs) and human fibroblasts (Huangfu et al., 2008a, b). VPA usage also resulted in improvement of cloning efficiency in mouse, miniature pig, and cattle (Costa-Borges et al., 2010; Miyoshi et al., 2010; Xu et al., 2012), thereby suggesting that VPA treatment can be useful for the improvement of reprogramming events in the cloned embryos. However, the mechanisms of action of VPA in modulating reprogramming events have not been investigated. "
[Show abstract][Hide abstract] ABSTRACT: Abstract In this study, we tested the effects of valproic acid (VPA), a known histone deacetylase inhibitor (HDACi), on the growth characteristics, apoptosis, and cell cycle stages distribution of donor cells, as well as cloning efficiency, embryo development, and histone methylation. Our results showed that treatment of donor cells with VPA (2.5 mM, 5.0 mM, 7.5 mM, or 10 mM) for 24 h resulted in altered cell proliferation, extent of apoptosis and necrosis, and cell cycle stage distribution, whereas no changes in cell viability and chromosomal complements were observed. Measurement of relative gene expression using real-time PCR of a few developmentally important genes in treated donor cells showed decreased expression of HDAC1 and increased expression of BAX (p<0.05). No change in relative expression of HDAC2 and Bcl2 was noticed. Treatment of donor cells with VPA for 24 h before electrofusion significantly (p<0.05) increased the blastocyst formation rate of somatic cell nuclear transfer (SCNT) embryos compared to the control embryos. Terminal deoxynucleotidyl transferase dUTP nick end labeling (TUNEL)-positive nuclei in SCNT blastocysts derived from VPA-treated donor cells were significantly decreased compared to the control blastocysts (p<0.05). Immunolocalization studies revealed that the levels of histone H3 at lysine 9 (H3K9me3) were lower in VPA-treated donor cells derived cloned blastocysts than nontreated cloned embryos, and was at the level of in vitro fertilization (IVF) counterparts, although no effects of treatments were found in donor cells. Our study demonstrates that the use of VPA in SCNT has been beneficial for efficient reprogramming of donor cells. Its effect on histone methylation in cloned embryos correlates with their developmental potential and may be a useful epigenetic marker to predict the efficiency of SCNT.
"In contrast, Costa-borges et al. 
reported that VPA treatment before (2–3 h) and during (6 h) oocyte activation in B6CBAF1 mouse
SCNT embryos improved in vitro and full-term development in comparison with
an untreated control. Interestingly, it was recently found that treatment with VPA of
miniature pig SCNT embryos for 48 h starting immediately after oocyte activation enhanced the
in vitro development and expression of Oct4 (also known as Pou5f1)  and that when fertilized mouse embryos were treated
with 1 mM VPA during progression from the 8-cell to morula stage, the expression of Oct4 was
moderately enhanced in the morula stage . Therefore, it seems likely that the effect of VPA on the in vitro development
as well as Oct4 expression of SCNT embryos varies with the timing of the treatment. "
[Show abstract][Hide abstract] ABSTRACT: We examined effects of treatment with valproic acid (0, 0.2, 1 or 2 mM, VPA), an inhibitor of class I and IIa histone deacetylases (HDACs), of mouse somatic cell nuclear transfer (SCNT) embryos for 24 h from 48 h (4-cell stage), 24 h (2-cell stage) or immediately after oocyte activation on blastocyst formation rates and qualities of the resultant blastocysts. Blastocyst formation rates (33.4-37.0%) were not improved by VPA treatments compared with the untreated control (35.1-36.4%). However, immunofluorescence staining revealed that Oct4 expression levels, evaluated from percentages of embryos expressing Oct4 strongly and having more than 10 Oct4-positive cells, in blastocysts from SCNT embryos treated with 1 mM VPA for 24 h from the 4-cell stage (VPA-4C) were highest among all the groups and that the proportion of cells with a normal nuclear distribution of histone H3 trimethylated at lysine 27 (H3K27me3), a marker of the state of X-chromosome inactivation, significantly increased in the VPA-4C group (36.6%) compared with the control group (12.4%, P<0.05). Treatments with scriptaid and sodium butyrate, inhibitors of class I and IIa/b HDACs, for 24 h from the 4-cell stage also had beneficial effects on SCNT blastocysts. These findings indicate that treatment with 1 mM VPA from the 4-cell stage improves the Oct4 expression and nuclear distribution of H3K27me3 in mouse SCNT blastocysts and suggest that the inhibition of class I and IIa HDACs from the 4-cell stage plays an important role in these effects.
Journal of Reproduction and Development 01/2013; 59(2). DOI:10.1262/jrd.2012-156 · 1.52 Impact Factor