Castillo-Martín Miriam, Yeste Marc, Pericuesta Eva, Morató Roser, Gutiérrez-Adán Alfonso, Bonet Sergi
Biotechnology of Animal and Human Reproduction (TechnoSperm), Department of Biology, Institute of Food and Agricultural Technology, University of Girona, Campus Montilivi, E-17071 Girona, Spain.
Unit of Animal Reproduction, Department of Animal Medicine and Surgery, Faculty of Veterinary Medicine, Autonomous University of Barcelona, E-08193 Bellaterra, Spain.
Reprod Fertil Dev. 2015 Sep;27(7):1072-81. doi: 10.1071/RD13405.
The aims of the present study were to: (1) evaluate the effect of vitrification and warming on quality parameters and expression levels of pluripotency, apoptotic and stress genes in in vitro-produced (IVP) porcine blastocysts; and (ii) determine the correlation between these parameters. To this end, total cell number, DNA fragmentation, peroxide levels and the relative transcript abundance of BCL-2 associated X protein (BAX), BCL2-like 1 (BCL2L1), heat shock protein 70 (HSPA1A), POU class 5 homeobox 1 (POU5F1), superoxide dismutase 1 (SOD1) and superoxide dismutase 2 (SOD2) were analysed in fresh and vitrified IVP blastocysts. The results suggest that vitrification procedures have no effect on total cell number and gene expression of BAX, BCL2L1, SOD1 and SOD2 or the BAX:BCL2L1 ratio. Nevertheless, a significant increase in DNA fragmentation (2.9±0.4% vs 11.9±2.0%) and peroxide levels (80.4±2.6 vs 97.2±3.1) were seen in vitrified compared with Day 7 fresh blastocysts. In addition, after blastocyst vitrification, relative transcript abundance was downregulated for POU5F1 and upregulated for HSPA1A. Finally, there was a significant correlation of POU5F1 and HSPA1A with DNA fragmentation (POU5F1, r=-0.561; HSPA1A, r=0.604) and peroxide levels (POU5F1, r=-0.590; HSPA1A, r=0.621). In conclusion, under the conditions of the present study, vitrification and warming of IVP porcine blastocysts resulted in altered expression of POU5F1 and HSPA1A, but had no effect on BAX, BCL2L1, SOD1 and SOD2 expression.
（1）评估玻璃化和复温对体外生产（IVP）猪囊胚的质量参数以及多能性、凋亡和应激基因表达水平的影响；（2）确定这些参数之间的相关性。为此，分析了新鲜和玻璃化IVP囊胚的总细胞数、DNA片段化、过氧化物水平以及BCL-2相关X蛋白（BAX）、BCL2样1蛋白（BCL2L1）、热休克蛋白70（HSPA1A）、POU第5类同源盒1蛋白（POU5F1）、超氧化物歧化酶1（SOD1）和超氧化物歧化酶2（SOD2）的相对转录丰度。结果表明，玻璃化程序对总细胞数、BAX、BCL2L1、SOD1和SOD2的基因表达或BAX:BCL2L1比值没有影响。然而，与第7天的新鲜囊胚相比，玻璃化囊胚的DNA片段化（2.9±0.4%对11.9±2.0%）和过氧化物水平（80.4±2.6对97.2±3.1）显著增加。此外，囊胚玻璃化后，POU5F1的相对转录丰度下调，HSPA1A的相对转录丰度上调。最后，POU5F1和HSPA1A与DNA片段化（POU5F1，r=-0.561；HSPA1A，r=0.604）和过氧化物水平（POU5F1，r=-0.590；HSPA1A，r=0.621）存在显著相关性。总之，在本研究条件下，IVP猪囊胚的玻璃化和复温导致POU5F1和HSPA1A表达改变，但对BAX、BCL2L1、SOD1和SOD2表达没有影响。
