An Epigenetic Modifier Results in Improved In Vitro Blastocyst Production after Somatic Cell Nuclear Transfer

Abstract. We examined the comprehensive epigenetic status, including histone H3 and H4 acetylation, DNA methylation and level of mRNA transcripts of bovine somatic cell nuclear transfer (SCNT) embryos treated with trichostatin A (TSA), along with their full-term developmental efficacy. Treatment with 50 nM TSA enhanced early developmental competence; increased acetylation of two histones, H3K9K14 and H4K8, at the blastocyst stage; and maintained the DNA methylation status of the satellite I sequence in bovine SCNT embryos. The difference in IGFBP-3 transcript levels between in vivo and SCNT embryos disappeared in SCNT embryos after treatment with 50 nM TSA. Pregnancy, full-term developmental competence and body weight at birth of offspring did not differ between SCNT embryos treated with 50 nM TSA and untreated embryos. These results suggest that treatment with TSA improves preimplantation development and changes the epigenetic status but does not promote the full-term development competence in bovine SCNT embryos. Key words: Bovine embryo, Gene expression, Histone modification, Nuclear transfer, TSA (J. Reprod. Dev. 58: [302][303][304][305][306][307][308][309] 2012) T he efficiency of animal production by somatic cell nuclear transfer (SCNT) is still very low. In bovine cloning, high rates of embryonic, fetal, neonatal and postnatal abnormalities have consistently been observed [1][2][3][4][5][6]. Although the cause of such abnormalities is unknown, the phenomenon may be correlated with abnormal gene expression [7], because several investigators have reported aberant transcription patterns of various genes that have an important role in pre-or postimplantation development in bovine SCNT embryos [8][9][10][11][12][13]. Moreover, an aberrant pattern of gene expression is persistently observed in bovine SCNT embryos that develop to the elongated stage and into a conceptus after implantation [11,12,14,15].The characteristics of gene transcription in SCNT embryos can be attributed to abnormalities in the control system for gene expression such as DNA methylation [16][17][18][19] and acetylation of histones [20]. Kang et al. [16,18] reported that various repeated genomic sequences, including satellite I, were barely demethylated in bovine SCNT embryos at the blastocyst stage. The methylation patterns observed in such embryos closely resembled those in donor cells but were quite different from those in normal embryos produced in vivo or in vitro. These findings suggest dysregulation of epigenetic modifications, such as DNA methylation, resulting in abnormal transcription of developmentally crucial genes at the blastocyst stage.Recently, it was clearly demonstrated in mice that histone modifications are closely related to the success rate of cloning [21,22]. It was reported that treatment with trichostatin A (TSA), which is a histone deacetylase inhibitor, improves in vitro blastocyst development, embryonic stem cell derivation and full-term development of mouse SCNT embryos. Furthermore, TSA treatment also improves...

Section: Discussionsupporting

confidence: 87%

mentioning

confidence: 95%

Epigenetic Status and Full-term Development of Bovine Cloned Embryos Treated with Trichostatin A

Sawai

Fujii

Hirayama

et al. 2012

“…TSA treatment of SCNT embryos has been reported to be effective for improving the blastocyst formation rate in mice [20][21][22]38] and pigs [23,24]. In contrast, when bovine SCNT embryos were treated with TSA at 50 nM for 13 h [26] and at 5, 50 or 500 nM for 12 h [25] after NT, no significant effects on the development to the blastocyst stage were observed.…”

Section: Discussionmentioning

confidence: 98%

“…In these reports, treatment with 5 to 100 nM TSA for 9 to 20 h led to a significant increase not only in the blastocyst formation rate, but also in the full-term development rate. In pigs, treatment of SCNT embryos with 37.5 to 50 nM TSA for 24 h significantly improved the blastocyst formation rate [23,24]. These reports suggest that inhibition of histone deacetylation in SCNT embryos during a short period of culture after NT may promote reprogramming of the donor nucleus.…”

mentioning

confidence: 80%

Treatment with a Histone Deacetylase Inhibitor after Nuclear Transfer Improves the Preimplantation Development of Cloned Bovine Embryos

Akagi

Matsukawa

Mizutani

et al. 2011

Abstract. We examined the effects of treatment with histone deacetylase inhibitors (HDACi), trichostatin A (TSA) and scriptaid (SCR), on the blastocyst formation rate in bovine somatic cell nuclear transferred (SCNT) embryos derived from fibroblast cells. Three fibroblast cell lines (L1, L2 and L3) were used as somatic cell donors to produce SCNT embryos (L1, L2 and L3 embryos, respectively). In Experiment 1, we compared the in vitro developmental competence of L1 embryos treated with various concentrations of TSA for different time periods following chemical activation. Embryos treated with 5 nM TSA for 20 h showed a significantly increased blastocyst formation rate compared with untreated controls. In Experiment 2, we examined the effect of TSA (5 nM) treatment of L1, L2 and L3 embryos as well as the effect of treatment of L1, L2 and L3 embryos with various concentrations of SCR on in vitro developmental competence. It was found that 5 nM TSA treatment significantly increased the blastocyst formation rate in L1 and L3 embryos but did not have an influence on the development of L2 embryos. On the other hand, 5 nM SCR treatment significantly increased the blastocyst formation rates of L1 and L2 embryos compared with controls. However, there was no significant increase in the blastocyst formation rate of L3 embryos when they were treated with SCR. In Experiment 3, acetylation of H4K12 was examined in donor cells and pronuclear-stage L1, L2 and L3 embryos treated with 5 nM TSA or 5 nM SCR by immunostaining. The level of H4K12 acetylation was different among donor cells. The staining intensities in the TSA-treated L1 and L3 embryos and SCR-treated L2 embryos were significantly higher than those of untreated embryos. These results suggest that HDACi treatment of bovine SCNT embryos improves the blastocyst formation rate; however, the optimal treatment conditions may differ among donor cell lines. Key words: Bovine, Nuclear transfer, Histone deacetylase inhibitor (J. Reprod. Dev. 57: [120][121][122][123][124][125][126] 2011) ive cloned offspring have been produced in many species by somatic cell nuclear transfer (SCNT), although the cloning efficiency is still very low. It has been reported that, in SCNT, embryo losses occur at various stages, and the anomalies in fetal and perinatal stages have been observed [1][2][3][4][5][6]. Because such abnormalities of clones are not transmitted to their progenies [7][8][9][10][11][12], most of the developmental problems of clones are believed to be the results of epigenetic defects [13]. In fact, several studies have revealed abnormal epigenetic modifications such as DNA methylation and histone modifications in SCNT embryos [14][15][16][17].A wide variety of histone deacetylase inhibitors (HDACi) of both natural and synthetic origin has been reported [18]. Trichostatin A (TSA), which is a natural product isolated from Streptomyces hygroscopicus [19], is one of the most frequently used drugs for various studies as a selective HDACi. Recently, it has been reported from two la...

“…In serial cloning, accumulation of epigenetic modifications was initially thought to make it impossible to clone past several repetitions [110,111]. However, with recent advances in SCNT, it may be possible to repair epigenetic modifications by chemical treatment [112][113][114][115][116][117][118]. Thus, somatic cell cloning may be repeated beyond previously expected limits (Wakayama, personal communication).…”

Section: Development Of Genetically Modified Pigs For Xenotransplantamentioning

confidence: 99%

Application of Genetically Modified and Cloned Pigs in Translational Research

Matsunari

Nagashima

2009

Abstract. Pigs are increasingly being recognized as good large-animal models for translational research, linking basic science to clinical applications in order to establish novel therapeutics. This article reviews the current status and future prospects of genetically modified and cloned pigs in translational studies. It also highlights pigs specially designed as disease models, for xenotransplantation or to carry cell marker genes. Finally, use of porcine somatic stem and progenitor cells in preclinical studies of cell transplantation therapy is also discussed. Key words: Cloned pig, Gene knockout, Transgenic pig, Translational research (J. Reprod. Dev. 55: [225][226][227][228][229][230] 2009) t present, pigs are not only considered important livestock but are also essential large-animal models in various types of biomedical research [1][2][3][4][5][6]. Furthermore, due to recent technological advances in genetic modification and somatic cell cloning, the range of applications for porcine models has increased markedly [7][8][9][10][11][12][13][14]. The present review focuses on development of genetically modified and cloned pigs and their use in translational studies.Translational research plays an important role as a bridge between basic science outcomes and the clinical realm, leading to development of novel therapeutics. However, disparate findings for rodent models and humans have hindered the translation of rodent data into actionable technologies for patients [15]. In contrast, pigs have many anatomical and physiological similarities to humans, including their cardiovascular systems, omnivorous gastrointestinal tracts and central nervous systems. Their physical sizes are also more comparable to that of humans, rendering pigs more appropriate for development of new surgical or endoscopic techniques.In the future, more advanced translational research may be conducted by improving the clinical relevance of pig models through genetic engineering. This article reviews the current status and future prospects of genetically modified pigs in preclinical studies of stem and progenitor cell therapy, the development of disease models and xenotransplantation.