Lee Hwa-Yeon, Bae Hyo-Kyung, Jung Bae-Dong, Lee Seunghyung, Park Choon-Keun, Yang Boo-Keun, Cheong Hee-Tae
College of Veterinary Medicine and Institute of Veterinary Science, Kangwon National University, Chuncheon 24341, Korea.
College of Animal Life Sciences, Kangwon National University, Chuncheon 24341, Korea.
Dev Reprod. 2018 Mar;22(1):73-83. doi: 10.12717/DR.2018.22.1.073. Epub 2018 Mar 31.
This study investigates the endoplasmic reticulum (ER) stress and subsequent apoptosis in duced during somatic cell nuclear transfer (SCNT) process of porcine SCNT embryos. Porcine SCNT and fertilization (IVF) embryos were sampled at 3 h and 20 h after SCNT or IVF and at the blastocyst stage for mRNA extraction. The x-box binding protein 1 (Xbp1) mRNA and the expressions of ER stress-associated genes were confirmed by RT-PCR or RT-qPCR. Apoptotic gene expression was analyzed by RT-PCR. Before commencing SCNT, somatic cells treated with tunicamycin (TM), an ER stress inducer, confirmed the splicing of Xbp1 mRNA and increased expressions of ER stress-associated genes. In all the embryonic stages, the SCNT embryos, when compared with the IVF embryos, showed slightly increased expression of spliced Xbp1 (Xbp1s) mRNA and significantly increased expression of ER stress-associated genes (<0.05). In all stages, apoptotic gene expression was slightly higher in the SCNT embryos, but not significantly different from that of the IVF embryos except for the Bax/Bcl2L1 ratio in the 1-cell stage (<0.05). The result of this study indicates that excessive ER stress can be induced by the SCNT process, which induce apoptosis of SCNT embryos.
本研究调查了猪体细胞核移植（SCNT）胚胎在体细胞核移植过程中诱导产生的内质网（ER）应激及随后的凋亡情况。在体细胞核移植或体外受精（IVF）后的3小时和20小时以及囊胚期采集猪体细胞核移植胚胎和体外受精胚胎用于提取mRNA。通过逆转录聚合酶链反应（RT-PCR）或实时定量逆转录聚合酶链反应（RT-qPCR）确认X盒结合蛋白1（Xbp1）mRNA及内质网应激相关基因的表达。通过RT-PCR分析凋亡基因表达。在开始体细胞核移植之前，用内质网应激诱导剂衣霉素（TM）处理体细胞，确认了Xbp1 mRNA的剪接并增加了内质网应激相关基因的表达。在所有胚胎阶段，与体外受精胚胎相比，体细胞核移植胚胎的剪接型Xbp1（Xbp1s）mRNA表达略有增加，内质网应激相关基因的表达显著增加（P<0.05）。在所有阶段，体细胞核移植胚胎的凋亡基因表达略高，但除1细胞期的Bax/Bcl2L1比值外，与体外受精胚胎无显著差异（P<0.05）。本研究结果表明，体细胞核移植过程可诱导过度的内质网应激，进而诱导体细胞核移植胚胎凋亡。
