Scriptaid affects histone acetylation and the expression of development-related genes at different stages of porcine somatic cell nuclear transfer embryo during early development

ZHOU Yan 1 HUANG YongYe 1 XIE WanHua 1 SONG Qi 1 JI Yuan 1 ZHANG YanPing 0 OUYANG HongSheng 1 LAI LiangXue 1 PANG DaXin 1 TANG XiaoChun 1 0 College of Life Science and Technology, Dalian University , Dalian 116622, China 1 Jilin Provincial Key Laboratory of Animal Embryo Engineering, College of Animal Science and Veterinary Medicine, Jilin University , Changchun 130062, China Although the somatic cell nuclear transfer (SCNT) technique has been used extensively for cloning and generating transgenic pigs, the cloning efficiency is still very low. It has been proposed that the low efficiency of this technique is the result of incomplete epigenetic reprogramming and abnormal gene expression during early embryonic development. In this study, we investigate the effect of Scriptaid, a low-toxicity histone deacetylase inhibitor, on the developmental competence of porcine SCNT embryos. We found that treating SCNT embryos with 500 nmol/L Scriptaid for 15 h after activation significantly enhanced the blastocyst formation rate (27.7%) compared with the untreated group (control) (12.2%, P<0.05). Using an immunofluorescence technique to measure the average fluorescence intensity, we also found that treating SCNT embryos with Scriptaid increased the level of histone acetylation on histone H3 at lysine 14 (acH3K14). Furthermore, treating embryos with Scriptaid increased the expression level of three genes that play important roles during embryonic development (Oct4, Klf4 at the blastocyst stage and Nanog at the 4-cell stage). Moreover, the expression level of the apoptosis-related gene Caspase-3 was significantly lower in the Scriptaid-treated SCNT embryos compared with the control SCNT embryos at the 4-cell and blastocyst stages. In conclusion, these results indicate that Scriptaid treatment improves the development and nuclear reprogramming of porcine SCNT embryos. - In 2000, the birth of the worlds first cloned pig using somatic cell nuclear transfer (SCNT) created a new range of applications for somatic cell-cloned pigs in biomedical research [1,2]. Because of its similarity to humans in terms of breeding characteristics and physiology, the pig is considered an excellent donor for xenotransplantation and has been extensively used in human disease models [3]. A low cloning efficiency (<1%), however, has restricted the extensive use of porcine SCNT in biomedical research, and it has been proposed that this low efficiency is primarily caused These authors contributed equally to this work. *Corresponding author (email: ) by inadequate epigenetic reprogramming [4]. Previous studies have shown that the incomplete reprogramming of SCNT embryos results in abnormal gene expression patterns [5]. As a consequence of these abnormal expression patterns, the cloned embryos may stop developing and be aborted [6]. Epigenetic modifications made to the genome include genomic methylation and histone modifications [7,8]. Histone acetylation can alter chromosome conformation, which can increase gene transcription and translation [9]. Histone deacetylase inhibitor (HDACi) can increase the acetylation level of core histones in cloned bovine embryos [10]. This finding demonstrates that HDACi induces hyperacetylation and the expression of key genes that are important for The Author(s) 2013. This article is published with open access at Springerlink.com SCNT embryonic development. Currently, several types of HDACi have been used successfully to enhance the nuclear reprogramming of SCNT embryos. Valproic acid, for example, can significantly improve the epigenetic reprogramming of SCNT embryos in vitro [11,12]. After Trichostatin A (TSA) treatment, the developmental ability of pre-implantation SCNT embryos is enhanced in several species, such as mice [13], cattle [14] and pigs [15]. These findings demonstrate that TSA can improve cloning efficiency, but TSA treatments at higher concentrations or over longer durations can have harmful effects on SCNT embryonic development in vitro and may cause developmental defects [16,17]. Scriptaid is a synthetic HDACi that induces histone acetylation and chromosome remodeling and enhances gene transcription [18]. A previous study has shown that the development of cloned inbred mouse embryos improved significantly in vitro and in vivo after Scriptaid treatment and that nascent mRNA expression levels were higher in the treated group compared with the control groups [19]. Furthermore, treating porcine SCNT embryos with Scriptaid also improved their in vitro developmental capability and their nuclear reprogramming [16,20]. Similar results were also obtained in bovine embryos [21]. Previous studies have shown that Scriptaid has a positive effect on the in vitro and in vivo development of porcine embryos, but its underlying mechanism remains to be elucidated. For instance, the effect of Scriptaid treatment on gene expression remains unknown. In the present study, we aimed to determine the optimal concentration of Scriptaid for treatment. We also investigated the relationship among histone acetylation (acH3K14), pluripotent gene expression (Oct4, Sox2, Klf4 and Nanog), apoptosis-related gene expression (Bcl-xl, Caspase-3 and Bak) and Scriptaid treatment at different early developmental stages in porcine SCNT embryos. We investigate whether treating embryos with Scriptaid can change the expression levels of these genes and make them similar to the expression levels observed in the conventional in vitro fertilized (IVF) embryos. Materials and methods All of the chemicals used in this study were purchased from the Sigma Chemical Company (St Louis, MO, USA) unless otherwise noted. All of the solutions and media mentioned were filtered through a 0.22-m filter. All of the animal experiments were approved by the Animal Care and Use Committee of Jilin University and performed in accordance with the Animal Welfare and Ethics Guidelines. Establishment of donor cell All of the pigs used in this study were from the Jilin University swine herd. Landrace fetal fibroblast cells (FFCs) were used for SCNT and were prepared as previously described [22]. First, fetuses were collected from pregnant sows at approximately day 35. The head, viscera and limbs were removed, and the remaining fetal tissues were cut into 3 very fine pieces (1 mm ) with ophthalmic scissors. The tissue pieces were digested with Collagenase IV (200 U/mL) and DNase I (25 U/mL) in DMEM containing 20% fetal bovine serum for 46 h at 39C with 5% CO2 in the atmosphere. Following the digestion, the cell pellet was pooled in a 10-cm culture dish and allowed to propagate until confluent approximately 12 h. The cells were cultured for several passages. The cells were frozen and used between passages 2 and 6 for SCNT. The cultured cells were separated into single cells using a trypsin digestion and used as donor cells 0.5 h before performing SCNT. Preparation of SCNT embryos For the preparation of SCNT embryos, a slightly mo (...truncated)