Abstract
Somatic cell nuclear transfer (SCNT) is a well-known laboratory technique. The principle of the SCNT involves the reprogramming a somatic nucleus by injecting a somatic cell into a recipient oocyte whose nucleus has been removed. Therefore, the nucleus donor cells are considered as a crucial factor in SCNT. Cell cycle synchronization of nucleus donor cells at G0/G1 stage can be induced by contact inhibition or serum starvation. In this study, acteoside, a phenylpropanoid glycoside compound, was investigated to determine whether it is applicable for inducing cell cycle synchronization, cytoprotection, and improving SCNT efficiency in canine fetal fibroblasts. Primary canine fetal fibroblasts were treated with acteoside (10, 30, 50 μM) for various time periods (24, 48 and 72 hours). Cell cycle synchronization at G0/G1 stage did not differ significantly with the method of induction: acteoside treatment, contact inhibition or serum starvation. However, of these three treatments, serum starvation resulted in significantly increased level of reactive oxygen species (ROS) (99.5 ± 0.3%) and apoptosis. The results also revealed that acteoside reduced ROS and apoptosis processes including necrosis in canine fetal fibroblasts, and improved the cell survival. Canine fetal fibroblasts treated with acteoside were successfully arrested at the G0/G1 stage. Moreover, the reconstructed embryos using nucleus donor cells treated with acteoside produced a healthy cloned dog, but not the embryos produced using nucleus donor cells subjected to contact inhibition. In conclusion, acteoside induced cell cycle synchronization of nucleus donor cells would be an alternative method to improve the efficiency of canine SCNT because of its cytoprotective effects.
Highlights
Somatic cell nuclear transfer (SCNT) technique has been used to produce genetically superior or manipulated animals for agricultural purposes and as biomedical resources
Canine fetal fibroblasts were treated with various concentrations of acteoside (10, 30, 50 μM) for different durations (24, 48, 72 h), and the cells were separated into G0/G1 stage, S stage and G2/M stage by fluorescence activated cell sorting (FACS)
Serum starvation resulted in the highest rate of cell cycle synchronization (88.2%) at the G0/G1 stage compared to those of the contact inhibition (84.6%) and acteoside treatment (80.8–84.5%)
Summary
SCNT technique has been used to produce genetically superior or manipulated animals for agricultural purposes and as biomedical resources. The causes of the low SCNT embryo developmental competence include many factors which are related to the quality of recipient oocytes, quality of the nucleus donor cells and the condition of the surrogate mother [7,8,9,10]. Supporting studies have reported that reducing ROS improves SCNT embryo development competence during in vitro culture [15,16,17]. Apoptosis is a physiological process that occurs spontaneously during normal preimplantation embryo development to maintain cellular homeostasis by removing DNA damaged/malfunctioning cells. Many studies reported that SCNT embryo development competence is rescued by reducing the levels of ROS and apoptosis by using antioxidants such as selenium [20], insulin-transferrin-selenium [17, 21], melatonin [22] and glutathione [23]
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