Lin Tao, Lee Jae Eun, Oqani Reza K, Kim So Yeon, Cho Eun Seok, Jeong Yong Dae, Baek Jun Jong, Jin Dong Il
Department of Animal Science & Biotechnology, Research Center for Transgenic Cloned Pigs, Chungnam National University, Daejeon 34134, Republic of Korea.
Department of Animal Resource Development, National Institute of Animal Science, Cheonan, 31001, Republic of Korea.
Reprod Biol. 2016 Dec;16(4):269-278. doi: 10.1016/j.repbio.2016.10.003. Epub 2016 Oct 17.
The aim of this study is to investigate whether endoplasmic reticulum (ER) stress attenuation could improve porcine somatic cell nuclear transfer (SCNT) embryo developmental competence. We treated porcine SCNT embryos with TUDCA (tauroursodeoxycholic acid, an inhibitor of ER stress) and/or TM (tunicamycin, an ER stress inducer), and examined embryonic developmental potential, embryo quality, the levels of ER stress markers (XBP1 protein and mRNA) and apoptosis-related-genes (BAX and BCL2 mRNA). Immunostaining detected X-box-binding protein (XBP1), a key gene regulator during ER stress, at all stages of SCNT embryo development. Embryo development analysis revealed that TUDCA treatment markedly increased (p<0.05) blastocyst formation rate, total cell number and inner cell mass (ICM) cell number compared to untreated control group. The TUDCA and TM groups showed significant alterations in XBP1 protein and XBP1-s mRNA levels compared to controls (lower and higher, respectively; p<0.05). Also, TUDCA treatment reduced oxidative stress by up-regulation of the antioxidant, GSH. TUNEL assay showed that TUDCA treatment significantly reduced apoptosis in porcine SCNT blastocysts confirmed by decreased pro-apoptotic BAX and increased anti-apoptotic BCL2 mRNA levels. Collectively, our results indicated that TUDCA can enhance the developmental potential of porcine SCNT embryos by attenuating ER-stress and reducing apoptosis.
本研究的目的是调查内质网（ER）应激减弱是否能提高猪体细胞核移植（SCNT）胚胎的发育能力。我们用牛磺熊去氧胆酸（TUDCA，一种内质网应激抑制剂）和/或衣霉素（TM，一种内质网应激诱导剂）处理猪SCNT胚胎，并检测胚胎发育潜力、胚胎质量、内质网应激标记物（XBP1蛋白和mRNA）以及凋亡相关基因（BAX和BCL2 mRNA）的水平。免疫染色在SCNT胚胎发育的各个阶段检测到内质网应激期间的关键基因调节因子X盒结合蛋白（XBP1）。胚胎发育分析显示，与未处理的对照组相比，TUDCA处理显著提高了（p<0.05）囊胚形成率、总细胞数和内细胞团（ICM）细胞数。与对照组相比，TUDCA组和TM组的XBP1蛋白和XBP1-s mRNA水平有显著变化（分别降低和升高；p<0.05）。此外，TUDCA处理通过上调抗氧化剂谷胱甘肽（GSH）来降低氧化应激。TUNEL检测表明，TUDCA处理显著降低了猪SCNT囊胚中的细胞凋亡，这通过促凋亡BAX的减少和抗凋亡BCL2 mRNA水平的增加得到证实。总的来说，我们的结果表明，TUDCA可以通过减弱内质网应激和减少细胞凋亡来提高猪SCNT胚胎的发育潜力。
