Castillo-Martín Miriam, Bonet Sergi, Morató Roser, Yeste Marc
Biotechnology of Animal and Human Reproduction (TechnoSperm), Department of Biology, Institute of Food and Agricultural Technology, University of Girona, E-17071 Girona, Spain.
Biotechnology of Animal and Human Reproduction (TechnoSperm), Department of Biology, Institute of Food and Agricultural Technology, University of Girona, E-17071 Girona, Spain.
Cryobiology. 2014 Jun;68(3):451-8. doi: 10.1016/j.cryobiol.2014.03.001. Epub 2014 Mar 19.
The present study sought to determine the effect of adding l-ascorbic acid (AC) to (1) in vitro culture medium and (2) vitrification and warming solutions on redox status and developmental ability and quality of IVP porcine embryos. In both experiments, embryo quality was analysed in terms of total cell number (TCN), DNA fragmentation, intracellular peroxide levels and expression of three oxidative stress-related genes: glutathione peroxidase 1 (GPX1), superoxide dismutase 1 (SOD1) and 2 (SOD2). In the first experiment, fresh blastocysts were found to upregulate SOD1 expression when cultured with medium supplemented 100 μM AC. No differences were found between culture groups in the other analysed parameters. In the second experiment, blastocysts cultured with or without AC were divided into two groups: vitrified and warmed with solutions containing 0 or 100 μM AC. Addition of AC during culture and vitrification-warming upregulated the expression of GPX1 and SOD1 genes, enhanced survival rates and decreased peroxide levels at 24h post-warming. In addition, peroxide levels were negatively correlated with relative GPX1- and SOD1-transcript abundances, whereas GPX1 was positively correlated with embryo survival at 24h post-warming. No effects of AC-supplementation were seen for TCN, DNA fragmentation or relative SOD2-transcript abundance in vitrified blastocysts. In conclusion, the addition of AC to culture and vitrification-warming media increases gene expression of antioxidant enzymes SOD1 and GPX1. This appears to improve redox balance and is suggested to ultimately enhance embryo cryosurvival.
本研究旨在确定向（1）体外培养基和（2）玻璃化及复温溶液中添加L-抗坏血酸（AC）对体外生产（IVP）猪胚胎的氧化还原状态、发育能力和质量的影响。在两个实验中，均根据总细胞数（TCN）、DNA片段化、细胞内过氧化物水平以及三种氧化应激相关基因（谷胱甘肽过氧化物酶1（GPX1）、超氧化物歧化酶1（SOD1）和2（SOD2））的表达来分析胚胎质量。在第一个实验中，发现新鲜囊胚在添加100μM AC的培养基中培养时会上调SOD1的表达。在其他分析参数方面，培养组之间未发现差异。在第二个实验中，将添加或未添加AC培养的囊胚分为两组：分别用含0或100μM AC的溶液进行玻璃化和复温。在培养以及玻璃化-复温过程中添加AC会上调GPX1和SOD1基因的表达，提高存活率，并在复温后24小时降低过氧化物水平。此外，过氧化物水平与相对GPX1和SOD1转录本丰度呈负相关，而GPX1与复温后24小时的胚胎存活率呈正相关。对于玻璃化囊胚的TCN、DNA片段化或相对SOD2转录本丰度，未观察到AC添加的影响。总之，在培养和玻璃化-复温培养基中添加AC可增加抗氧化酶SOD1和GPX1的基因表达。这似乎改善了氧化还原平衡，并最终可能提高胚胎冷冻存活率。
