RESUMEN
The effects of two antioxidants, superoxide dismutase (SOD) and the flavonoid 3,4-dihydroxyflavone (DHF), on bovine embryo development in vitro were examined. Blastocyst development, total cell and inner cell mass (ICM) numbers, intracellular levels of reactive oxygen species (ROS), apoptotic indices and gene expression levels were examined before and after treatment of day 2 bovine embryos (≥2-4 cells) with various concentrations of 3,4-DHF or SOD for 6 days. Statistical analysis was performed using analysis of variance, with significance defined at the P<0.05 level. SOD had no significant effect on bovine embryo development at any tested concentration (control, 32.8%; 300 U/ml, 33.9%; 600 U/ml, 24.2%). In contrast, 10 µM 3,4-DHF promoted higher blastocyst development (39.3%) than any other concentration (control, 26.7%; 1 µM, 30.3%; 50 µM, 29.5%; 100 µM, 20.5%). Compared with 300 U/ml SOD, 10 µM 3,4-DHF resulted in significantly higher blastocyst development (44.2%) (control, 31.5%; SOD 300 U/ml, 33.6%). Treatment with 3,4-DHF increased the ICM cell number and reduced intracellular ROS production and apoptotic cell numbers. When O(2) tension was decreased from 20% (high tension) to 5% (low tension), embryo development rates were doubled regardless of 3,4-DHF treatment. Under high O(2) tension, 10 µM 3,4-DHF treatment may render bovine embryo development similar to a low O(2) tension environment. The best blastocyst development was obtained under low O(2) tension plus 10 µM 3,4-DHF treatment. The relative expression levels of antioxidant (MnSOD), antiapoptotic (Survivin, Bax inhibitor) and growth-related genes (IFN-τ, Glut-5) were significantly increased after 3,4-DHF treatment, while the expression levels of oxidant (Sox) and apoptotic genes (Caspase-3 and Bax) were reduced. These results suggest that 3,4-DHF may promote the in vitro development of bovine embryos through its antioxidant and antiapoptotic effects.
Asunto(s)
Antioxidantes/farmacología , Apoptosis/efectos de los fármacos , Blastocisto/efectos de los fármacos , Blastocisto/fisiología , Ectogénesis/efectos de los fármacos , Flavonas/farmacología , Animales , Proteínas Reguladoras de la Apoptosis/genética , Proteínas Reguladoras de la Apoptosis/metabolismo , Blastocisto/citología , Masa Celular Interna del Blastocisto/citología , Masa Celular Interna del Blastocisto/efectos de los fármacos , Masa Celular Interna del Blastocisto/metabolismo , Bovinos , Recuento de Células , Técnicas de Cultivo de Embriones , Regulación de la Expresión Génica/efectos de los fármacos , Transportador de Glucosa de Tipo 5/genética , Transportador de Glucosa de Tipo 5/metabolismo , Interferón Tipo I/genética , Interferón Tipo I/metabolismo , Cinética , Oxígeno/efectos adversos , Proteínas Gestacionales/genética , Proteínas Gestacionales/metabolismo , ARN Mensajero/metabolismo , Especies Reactivas de Oxígeno/metabolismo , Factores de Transcripción SOX/genética , Factores de Transcripción SOX/metabolismo , Superóxido Dismutasa/genética , Superóxido Dismutasa/metabolismoRESUMEN
Like embryonic stem cells, induced pluripotent stem cells (iPSCs) can differentiate into all three germ layers in an in vitro system. Here, we developed a new technology for obtaining neural stem cells (NSCs) from iPSCs through chimera formation, in an in vivo environment. iPSCs contributed to the neural lineage in the chimera, which could be efficiently purified and directly cultured as NSCs in vitro. The iPSC-derived, in vivo-differentiated NSCs expressed NSC markers, and their gene-expression pattern more closely resembled that of fetal brain-derived NSCs than in vitro-differentiated NSCs. This system could be applied for differentiating pluripotent stem cells into specialized cell types whose differentiation protocols are not well established.