LAUSR

The low efficiency of somatic cell nuclear transfer (SCNT) has been attributed mostly to inefficient epigenetic reprogramming. Recently, various histone deacetylase inhibitors (HDACi) were used to improve developmental competence of SCNT embryos in several species. However, limited information is available on the effects of quisinostat (JNJ-26481585, JNJ), a second-generation HDACi with high cellular potency towards Class I and II histone deacetylases. Based on our previous study, among various concentrations, treatment with 100 nM JNJ could improve embryo development into blastocysts compared with the control (23.50 ± 1.30 v. 13.97 ± 1.37; P < 0.05). Thus, in the present study, treatment with 100 nM JNJ was used for further investigation into the relative expression of genes related to pluripotency and reprogramming in order to assess the quality of pre-implantation embryos cultured in media with JNJ using quantitative real-time PCR. Porcine fibroblasts isolated from kidney of adult pigs from passage 6 to 8 were used as nuclear donor cells for SCNT. After SCNT, embryos were cultured with or without 100 nM JNJ during the first 24 h of in vitro culture, and blastocysts from each experimental group were collected and kept at –80°C until analysis. Total RNAs were extracted, and transcribed into cDNA before amplification. Then, the relative expression of development-related (Oct4, Sox2, and Nanog), histone acetylation-related (HDAC1, HDAC2, and HDAC3) and DNA methylation-related (DNMT1, DNMT3a, and DNMT3b) genes between the control and 100 nM JNJ groups were compared. All experiments were repeated 4 times and results were analysed by independent t-test using SPSS 17.0K (SPSS Inc., Chicago, IL, USA). Treatment with 100 nM JNJ showed significant increases in the expression Oct4, Sox2, and Nanog compared with the control (P < 0.05). Moreover, there was significantly lower expression of HDAC1, HDAC2, HDAC3, DNMT1, DNMT3a, and DNMT3b in the 100 nM JNJ treatment than in the control (P < 0.05). These expression results moderately illustrated more active transcriptional factors, stable maintenance of embryonic pluripotency, and lesser activity of histone acetylation and DNA methylation enzymes, enhancing the blastocyst formation rate in the treatment group. In conclusion, we suggest that improvement of the in vitro developmental competence of porcine SCNT embryos might be related to positive regulations of JNJ on the expression levels of genes related to pluripotency and reprogramming. This study was supported by the NRF (#2015R1C1A2A01054373; 2016M3A9B6903410), Research Institute for Veterinary Science and the BK21 PLUS Program.