In the present study, we investigated the effect of TSA and Scriptaid on the in vitro and in vivo development potential of SCNT embryos using various landrace FFCs and EFCs. We found that treatment with a novel HDACi, Scriptaid, enhanced the developmental potential of reconstructed embryos in vitro and in vivo. This may be a result of improved reprogramming from increased histone acetylation in the somatic nuclei.
In Experiment 1, the development of SCNT embryos to the blastocyst stage after treatment with TSA after activation was twice than that of untreated groups. There was no difference of embryo quality as judged by total cell number at the blastocyst stage. From the conditions used in this study, we found treatment of 50
nM for 10
h achieved the best results in the SCNT embryos. Embryo transfer results showed that TSA treatment can produce viable cloned piglets and, although not significant, numerically increased the cloning efficiency from 0.4 to 0.8%.
In addition to TSA, Scriptaid is a novel HDAC inhibitor that belongs to an existing class of hydroxamic acid-containing HDAC inhibitors (Su et al., 2000
). When 500
nM Scriptaid was used on SCNT embryos using same donor cells as Experiment 1, the blastocyst rate was more than twice as much as those of untreated groups (25 vs. 11%). Embryo transfer results showed that the cloning efficiency was 1.6 or 3.7% in the Scriptaid treatment group when FFCs or EFCs used as donor cells, respectively (). However, the cloning efficiency in the untreated group when FFCs or EFCs were used as donor cells is only 0.4 or 0%, respectively (). In total, 23 piglets were obtained from seven litters of the Scriptaid treatment group. Thus, both Scriptaid and TSA teatment improve the developmental potential of SCNT embryos.
We observed some abnormalities during the cloning treated with HDACi in the current study, 3 of 4 cloned piglets died of heart failure in the TSA treatment group, and 3 of 23 from the Scriptaid treatment were abnormal. Despite some abnormal piglets from the TSA and Scriptaid treatment, it is difficult to make any conclusions about abnormalities caused by HDACi because a significant percentage of cloned offspring are normal and healthy and a piglet from the control TSA group was also abnormal. However, it still needs to note that there was a higher percentage (75%, three of four pigs) of abnormalities in the TSA treatment group.
Some cloned animals with abnormal phenotypes that reach sexual maturity can be naturally bred, and all of their offspring show normal phenotypes, suggesting that the abnormalities of the clones are due to epigenetic aberrancies rather than genetic mutations (Prather, 2006
; Tamashiro et al., 2002
). Besides DNA methylation, histone modification (acetylation, methylation, phosphorylation, and ubiquitination) is another important epigenetic modification to the chromatin structure. Acetylation, the introduction of an acetyl group, usually occurs on the lysine residues of core histones (Kouzarides, 2007
; Surani et al., 2007
; Wang et al., 2007
). Changes in DNA methylation, histone methylation, and histone acetylation are tightly linked to the transcriptional state of genes in those modified regions (Armstrong et al., 2006
; Li, 2002
). Histone acetylation–deacetylation is a dynamic process during embryogenesis and differentiation, and is related to transcriptional regulation by the status of the chromosome structure. Histone acetylation emerges as a central switch that allows interconversion between permissive and repressive chromatin structures and domains. Increased histone acetylation levels on most amino acid residues leads to relaxed binding of the nucleosome to DNA and/or linker histones, relaxation of the chromatin structure, and formation of a transcriptionally permissive state (Hebbes et al., 1988
; Hong et al., 1993
; Lee et al., 1993
; Zlatanova et al., 2000
). Histone deacetylation, frequently followed by histone methylation, establishes a base for highly repressive chromatin structures, such as heterochromatin (Eberharter and Becker, 2002
). These principles are not only the heart of transcriptional regulation but are also likely to govern other processes involving chromatin substrates, including replication, site-specific recombination, and DNA repair (Roth et al., 2001
; Wolffe and Hayes, 1999
). Thus, transcriptional activation within a permissive domain frequently correlates with additional targeted acetylation of histone at promoter nucleosomes (Brown et al., 2000
; Forsberg and Bresnick, 2001
Trichostatin A can induce the hyperacetylation of the core histones, resulting in structural changes in the chromatin that permit transcription. These changes include decreasing DNA methylation and thus activation of genes, which are key for development (Cervoni and Szyf, 2001
), and we believe the Scriptaid may have a similar function. Scriptaid treatment could increase the AcH4k8 intensity in both the “pro”-nuclear area of SCNT embryos and also somatic donor cells ( and ). However, the intensity AcH4K8 in the untreated SCNT embryos was lower when compared to IVF cohort embryos. After treatment with Scriptaid for 14–16
h, the histone acetylation level is increased in the SCNT embryos where it more closely resembles the IVF embryos. Similar results also have been observed in several studies that treatment of SCNT embryos with HDACi altered the histone acetylation in a manner similar to that in normal embryos (Iager et al., 2008
; Shi et al., 2008a
; Wang et al., 2007
). TSA treatment produced eight-cell stage bovine embryos with levels of acetylation on histone H4 at lysine 5 (AcH4K5) similar to fertilized counterparts and significantly greater than in control SCNT embryos (p
0.05) (Iager et al., 2008
). Similar results after TSA treatment in histone acetylation of SCNT embryos were also observed in the mouse (Wang et al., 2007
) and rabbit (Shi et al., 2008a
). Thus, the histone acetylation of a somatic genome in the cloned embryos may not be sufficient, and HDACi treatment increased the histone acetylation level, which induces a more open chromatin configuration, allowing access to transcription factors (Li, 2002
) and enhancing the DNA demethylation of the somatic nuclei after SCNT, which is a necessary step of nuclear reprogramming (Armstrong et al., 2006
; Simonsson and Gurdon, 2004
). One study showed that treatment of cloned embryos with TSA causes them to transcribe mRNA similar to in vivo
derived embryos (Li et al., 2008b
). Additionally, valproic acid, another HDAC inhibitor, improves reprogramming efficiency of mouse embryonic fibroblasts (MEFs) into induced pluripotent stem (iPS) cells by more than 100-fold over the untreated group (Huangfu et al., 2008a
In this study, we focused on the application and optimization of a novel HDACi, Scriptaid, on improving pig cloning efficiency and partially explained the mechanism. However, further studies are still needed to elucidate which cluster of genes are affected by HDACi treatment, thus improving the cloning efficiency. The effect of Scriptaid on the cloning efficiency in other species should also be investigated.
In conclusion, Scriptaid improves the in vitro and in vivo development of pig SCNT embryos, and could produce healthy cloned offspring. We also suggest that epigenetic reprogramming of the somatic nuclei after SCNT is deficient and hypoacetylation of the core histone might be a limiting factor for successful reprogramming.