Inhibition of Reactive Oxygen Species Generation by Antioxidant Treatments during Bovine Somatic Cell Nuclear Transfer

다국어 초록 (Multilingual Abstract)

This study was conducted to examine the optimal concentration and treatment time of antioxidants for inhibition of the ROS generation in bovine somatic cell nuclear transfer (SCNT) embryos. Bovine oocytes were activated parthenogenetically, during which oocytes were treated with various antioxidants to determine the optimal concentrations and kind of antioxidants. Determined antioxidants were applied to oocytes during in vitro maturation (IVM) and/or SCNT procedures. Finally, antioxidant-treated SCNT embryos were compared with in vitro fertilized (IVF) embryos. H2O2 levels were analyzed in embryos at 20 h of activation, fusion or insemination by staining of embryos in 10 μM 2'7'-dichlorodihydrofluorescein diacetate (H2DCFDA) dye, followed by fluorescence microscopy. H2O2 levels of parthenogenetic embryos were significantly lower in 25 μM β- mercaptoethanol (β-ME), 50 μM L-ascorbic acid (Vit. C), and 50 μM L-glutathione (GSH) treatment groups than each control group (24.0±1.5 vs 39.0±1.1, 29.7±1.0 vs 37.0±1.2, and 32.9±0.8 vs 36.3±0.8 pixels/embryo, p<0.05). There were no differences among above concentration of antioxidants in direct comparison (33.6±0.9～35.2±1.1 pixels/embryo). Thus, an antioxidant of 50 μM Vit. C was selected for SCNT. H2O2 levels of bovine SCNT embryos were significantly lower in embryos treated with Vit. C during only SCNT procedure (26.4±1.1 pixels/embryo, p<0.05) than the treatment group during IVM (29.9±1.1 pixels/embryo) and non-treated control (34.3±1.0 pixels/embryo). Moreover, H2O2 level of SCNT embryos treated with Vit. C during SCNT procedure was similar to that of IVF embryos. These results suggest that the antioxidant treatment during SCNT procedures can reduce the ROS generation level of SCNT bovine embryos.

목차 (Table of Contents)

• ABSTRACT
• INTRODUCTION
• MATERIALS AND METHODS
• In Vitro Maturation of Oocytes
• Parthenogenesis
• Preparation of Donor Cells
• Nuclear Transfer
• Electrofusion and Activation
• Antioxidant Treatment
• In Vitro Fertilization
• Measurement of Intercellular ROS Content
• Statistical Analysis
• RESULS
• The H2O2 Levels of Parthenogenetic Embryos following Treatment with Various Concentrations of Antioxidants
• The H2O2 Levels of Parthenogenetic Embryos following Treatment with Different Antioxidants
• The H2O2 Levels of SCNT Embryos with Different Treatment Time of Antioxidant
• Comparison of H2O2 Levels between the SCNT Em bryosTreated with Antioxidant and IVF Embryos
• DISCUSSION
• REFERENCES