Improvement of porcine cloning efficiency by trichostain A through early-stage induction of embryo apoptosisby Qianqian Ji, Kongju Zhu, Zhiguo Liu, Zhenwei Song, Yuankai Huang, Haijing Zhao, Yaosheng Chen, Zuyong He, Delin Mo, Peiqing Cong

Theriogenology

About

Year
2013
DOI
10.1016/j.theriogenology.2012.12.010
Subject
Food Animals / Animal Science and Zoology / Equine / Small Animals

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fici op i So g C a r t i c l e i n f o

Article history:

Received 27 June 2012

Received in revised form 24 December 2012

Accepted 27 December 2012

Keywords:

Trichostain A improving full-term development of cloned animals.

Recent studies attributed developmental failures of cloned animals to incomplete epigenetic reprogramming of the

However, the optimal concentration of TSA for SCNT is controversial. Whereas some reported that 5 nmol/L TSA improved blastocyst production and increased blastomere numbers [8] in porcine SCNT embryos, others reported 50 nmol/L TSA improved rate of blastocyst formation but did not increase cell numbers [9]. Opposing views of the final effect of TSA have also been reported, with some studies failing to obtain beneficial results [4]. * Corresponding author. Tel.: þ86 20 3994 3536; fax: þ86 20 3933 2940.

E-mail address: congpq@mail.sysu.edu.cn (P. Cong).

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Theriogenology 79 (2013) 815–8231. Introduction

Porcine somatic cell nucleus transfer (SCNT) has great potential, including producing genetically superior pig, conserving species, and providing organs for xenotransplantation to humans [1]. However, the low efficiency of SCNT severely limits its application [2]. Efforts to improve cloning efficiency have been less successful in somatic nucleus [3]. Thus, facilitating epigenetic reprogramming in SCNT might improve development of cloned embryos and animals.

Histone modification and DNA methylation are the two main epigenetic regulatory mechanisms involved in nuclear reprogramming [4]. As an inhibitor of histone deacetylase, trichostain A (TSA) promoted somatic cell reprogramming and improved cloning efficiency [3,5–7].Nuclear transfer

Apoptosis

Embryo

Porcine0093-691X/$ – see front matter  2013 Elsevier Inc http://dx.doi.org/10.1016/j.theriogenology.2012.12.01a b s t r a c t

Trichostain A (TSA), an inhibitor of histone deacetylases, improved developmental competence of SCNT embryos in many species, apparently by improved epigenetic reprogramming. The objective of the present study was to determine the effects of TSAinduced apoptosis in cloned porcine embryos. At various developmental stages, a comet assay and terminal deoxynucleotidyl transferase-mediated deoxyuridine triphosphate nickend labeling staining were used to detect apoptosis, and real-time polymerase chain reactionwas used to assess expression of genes related to apoptosis and pluripotency. In this study, TSA significantly induced apoptosis (in a dose-dependent manner) at the one-, two-, and four-cell stages. However, in blastocyst stage embryos, TSA decreased the apoptotic index (P < 0.05). Expression levels of Caspase 3 were higher in TSA-treated versus control embryos at the two-cell stage (not statistically significant). The expression ratio of antiapoptotic Bcl-xl gene to proapoptotic Bax gene, an indicator of antiapoptotic potential, was higher in TSA-treated groups at the one-, two-, and four-cell and blastocyst stages.

Furthermore, expression levels of pluripotency-related genes, namely, Oct4 and Nanog, were elevated at the morula stage (P < 0.05) in TSA treatment groups. We concluded that inducing apoptosis might be a mechanism by which TSA promotes development of reconstructed embryos. At the initial stage of apoptosis induction, abnormal cells were removed, thereby enhancing proliferation of healthy cells and improving embryo quality.  2013 Elsevier Inc. All rights reserved.State Key Laboratory of Biocontrol, School of Life Sciences, Sun Yat-sen University, Guangzhou, P.R. ChinaImprovement of porcine cloning ef early-stage induction of embryo ap

Qianqian Ji, Kongju Zhu, Zhiguo Liu, Zhenwe

Yaosheng Chen, Zuyong He, Delin Mo, Peiqin. All rights reserved. 0ency by trichostain A through tosis ng, Yuankai Huang, Haijing Zhao, ong* ology . ther io journal .com

Q. Ji et al. / Theriogenology 79 (2013) 815–823816As a novel class of antitumor agents, histone deacetylase inhibitors induced cell apoptosis and differentiation by regulating expression of relevant genes and stabilizing their gene products. Trichostain A, a member of the histone deacetylase inhibitors, inhibited growth and induced apoptosis in some types of cancer cells [10–12]. Therefore, analysis of its apoptosis-inducing effect in SCNT embryos might provide insights regarding how it affects embryo development.

Apoptosis, which occurs spontaneously in normal preimplantation embryos, has important roles in mammalian reproduction and development. Its function includes eliminating cells that are abnormal, detrimental, or superfluous, and regulating embryo cell numbers [13]. Perhaps apoptosis has a similar role in in vitro produced embryos, which are frequently mosaic. In human embryos, apoptosis removed only genetically damaged cells and concurrently enabled normal developing cells to proliferate [14]. Perhaps

TSA can enhance cloning efficiency by inducing apoptosis of abnormal cells in SCNT embryos.

Thegeneral objective of thepresent studywas to improve the efficiency of somatic cell cloning. More specifically, porcine SCNT embryos were treated with various concentrations of TSA and subsequently assessed for apoptosis and developmental competence. Degradation of DNA was detected by a comet assay and terminal deoxynucleotidyl transferase-mediated deoxyuridine triphosphate (dUTP) nick-end labeling (TUNEL) labeling. In addition, we assessed expression of some apoptosis and pluripotency-related genes, namely, Bcl-xl, Bax, Caspase 3, Oct4, and Nanog. 2. Materials and methods 2.1. Chemicals and reagents

All chemicals for embryo culture andmanipulationwere purchased from Sigma-Aldrich Corp. (St. Louis, MO, USA) unless specified otherwise. 2.2. Preparation of somatic cells for SCNT

The nuclear donor cells in our experiment were porcine embryonic fibroblasts (PEFs), obtained from a Landrace fetus on Day 32 of pregnancy. After removal of the head, limbs, and internal organs, the remaining tissues were cut into small pieces. After 3 hours of digestion in 0.25% tripsinEDTA at 37 C, the resultant cell suspension was filtered through several layers of gauze. Isolated cells were washed several times in PBS and cultured in Dulbecco’s modified