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

초록

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 invitro matured (IVM) oocytes were enucleated and electrofused with a donor cell, then activated by a combination ofCa-ionophore and 6-dimethylaminopurine. The recipient oocytes or reconstituted oocytes were treated with 50 μM GSHduring these SCNT procedures from enucleation to activation treatment. The SCNT embryos were cultured for 7 daysto evaluate the in vitro development, apoptosis and DNA methylation in blastocysts. The apoptosis was measured byTUNEL assay and caspase-3 activity assay. Methylated DNA of SCNT embryos at the blastocyst stages was detectedusing 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 significantlylower (P<0.05) than that of control (3.8±0.6%). Relative caspase-3 activity of GSH treated group was 0.8±0.06fold 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 thatantioxidant GSH treatment during SCNT procedures can improve the embryonic development and reduce the apoptosisand DNA methylation level of bovine SCNT embryos, which may enhance the nuclear reprogramming of bovineSCNT embryos

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