JARB Journal of Animal Reproduction and Biotehnology

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Journal of Embryo Transfer 2014; 29(4): 345-350

Published online December 30, 2014

Copyright © The Korean Society of Animal Reproduction and Biotechnology.

Effect of L-Glutathione Treatment during Somatic Cell Nuclear Transfer Procedures on the Subsequent Embryonic Development and DNA Met Status of Cloned Bovine Embryos

Hyo-Kyung Bae, Nam-Sik Yoon, In-Sun Hwang, Choon-Keun Park, Boo-Keun Yang, Hee-Tae Cheong

College of Veterinary Medicine and Institute of Veterinary Science, Kangwon National University, College of Veterinary Medicine and Institute of Veterinary Science, Kangwon National University, College of Veterinary Medicine and Institute of Veterinary Science, Kangwon National University, College of Animal Life Science, Kangwon National University, College of Animal Life Science, Kangwon National University, College of Veterinary Medicine and Institute of Veterinary Science, Kangwon National University

Abstract

We investigate the effect of L-glutathione (GSH), an antioxidant, treatment during the somatic cell nuclear transfer (SCNT) procedures on the in vitro development and DNA methylation status of bovine SCNT embryos. Bovine in vitro matured (IVM) oocytes were enucleated and electrofused with a donor cell, then activated by a combination of Ca-ionophore and 6-dimethylaminopurine. The recipient oocytes or reconstituted oocytes were treated with 50 μM GSH during these SCNT procedures from enucleation to activation treatment. The SCNT embryos were cultured for 7 days to evaluate the in vitro development, apoptosis and DNA methylation in blastocysts. The apoptosis was measured by TUNEL assay and caspase-3 activity assay. Methylated DNA of SCNT embryos at the blastocyst stages was detected using a 5-methylcytidine (5-MeC) antibody. The developmental rate to the blastocyst stage was significantly higher (P<0.05) in GSH treatment group (32.5±1.2%, 78/235) than that of non-treated control SCNT embryos (22.3±1.8%, 50/224). TUNEL assay revealed that the numbers of apoptotic cells in GSH treatment group (2.3±0.4%) were significantly lower (P<0.05) than that of control (3.8±0.6%). Relative caspase-3 activity of GSH treated group was 0.8±0.06 fold compared to that of control. DNA methylation status of blastocysts in GSH treatment group (13.1±0.5, pixels/ embryo) was significantly lower (P<0.05) than that of control (17.4±0.9, pixels/embryo). These results suggest that antioxidant GSH treatment during SCNT procedures can improve the embryonic development and reduce the apoptosis and DNA methylation level of bovine SCNT embryos, which may enhance the nuclear reprogramming of bovine SCNT embryos.

Keywords: somatic cell nuclear transfer, GSH, in vitro development, apoptosis, DNA-methylation