Kim Eunhye, Hwang Seon-Ung, Yoon Junchul David, Jeung Eui-Bae, Lee Eunsong, Kim Dae Young, Hyun Sang-Hwan
Laboratory of Veterinary Embryology and Biotechnology, Veterinary Medical Center and College of Veterinary Medicine, Chungbuk National University, Cheongju 28644, Republic of Korea.
Institute of Stem Cell & Regenerative Medicine (ISCRM), Chungbuk National University, Cheongju 28644, Republic of Korea.
J Reprod Dev. 2017 Dec 15;63(6):581-590. doi: 10.1262/jrd.2017-020. Epub 2017 Oct 6.
Compared with the in vivo environment, porcine in vitro embryo-culture systems are suboptimal, as they induce oxidative stress via the accumulation of reactive oxygen species (ROS). High ROS levels during early embryonic development cause negative effects, such as apoptosis. In this study, we examined the effects of the antioxidant carboxyethylgermanium sesquioxide (Ge-132) during in vitro culture (IVC) on embryonic development in porcine in vitro fertilization (IVF) embryos. Zygotes were treated with different concentrations of Ge-132 (0, 100, 200 and 400 μg/ml). All of the Ge-132 treatment groups displayed greater total cell numbers after IVC (98.1, 98.5 and 103.4, respectively) compared with the control group (73.9). The 200 μg/ml Ge-132 treatment group exhibited significantly increased intracellular GSH levels compared with the control group, whereas the ROS generation levels decreased in Ge-132 dose-dependent manner (P < 0.05). The mRNA expression levels of the KEAP1 gene and proapoptotic genes BAX and CASPASE3 were lower in the Ge-132 treated blastocysts compared with the control group (P < 0.05). The percentages of apoptotic and necrotic cells in the Ge-132 treated embryos on day 2 (48 h) were significantly lower than the untreated embryos (9.1 vs. 17.1% and 0 vs. 2.7%, respectively). In the day 7 blastocysts, the percentages of apoptotic cells in 200 µg/ml Ge-132 treated group were lower compared to controls (1.6 vs. 2.5%). More KEAP1 protein was found to be localized in cytoplasm of the 200 μg/ml Ge-132 treated blastocysts, whereas KEAP1 protein was predominantly nuclei in the control blastocysts. These results indicate that the developmental competence of embryos cultured under Ge-132 treatment may be associated with KEAP1 signaling cascades involved in oxidative stress and apoptosis during porcine preimplantation embryo development.
与体内环境相比，猪的体外胚胎培养系统并不理想，因为它们会通过活性氧（ROS）的积累诱导氧化应激。早期胚胎发育过程中高水平的ROS会产生负面影响，如细胞凋亡。在本研究中，我们检测了抗氧化剂倍半氧化羧乙基锗（Ge-132）在体外培养（IVC）过程中对猪体外受精（IVF）胚胎发育的影响。将受精卵用不同浓度的Ge-132（0、100、200和400μg/ml）处理。与对照组（73.9）相比，所有Ge-132处理组在体外培养后均显示出更多的总细胞数（分别为98.1、98.5和103.4）。与对照组相比，200μg/ml Ge-132处理组的细胞内谷胱甘肽（GSH）水平显著升高，而ROS生成水平则以Ge-132剂量依赖性方式降低（P<0.05）。与对照组相比，Ge-132处理的囊胚中KEAP1基因以及促凋亡基因BAX和CASPASE3的mRNA表达水平较低（P<0.05）。在第2天（48小时），Ge-13处理的胚胎中凋亡和坏死细胞的百分比显著低于未处理的胚胎（分别为9.1%对17.1%和0对2.7%）。在第7天的囊胚中，200μg/ml Ge-132处理组的凋亡细胞百分比低于对照组（1.6%对2.5%）。发现更多的KEAP1蛋白定位于200μg/ml Ge-132处理的囊胚的细胞质中，而KEAP1蛋白在对照囊胚中主要位于细胞核中。这些结果表明，在Ge-13处理下培养的胚胎的发育能力可能与猪植入前胚胎发育过程中参与氧化应激和细胞凋亡的KEAP1信号级联有关。
