Gametogenesis Research Center, Kashan University of Medical Sciences, Kashan, Iran.
Anatomical Sciences Research Center, Kashan University of Medical Sciences, Kashan, Iran.
Theriogenology. 2020 Mar 15;145:59-66. doi: 10.1016/j.theriogenology.2020.01.008. Epub 2020 Jan 10.
In vitro developed embryos are inevitably exposed to various reactive oxygen species (ROS) which may decrease the embryo's competence in assisted reproductive technology (ART) procedures. Optimization of embryo culture media using antioxidant agents could help to improve embryo quality and could overcome failures in current ART. The aim of this study was to evaluate the effects of l-carnitine (LC), an enhancer of mitochondrial activity and free radical scavenger, in culture media on early embryo competence and expression of ErbB1 and ErbB4 implantation related genes. Two-cell mouse embryos were cultured in the following four conditions: 1. LC group in media containing LC; 2.H O group exposed to HO for 30 min and then transferred into a simple media; 3.HO+LC group exposed to HO for 30 min and then transferred into a simple media containing LC; 4.the control group kept throughout in simple media. All groups were allowed to develop until the blastocyst stage. ErbB1 and ErbB4 expression were evaluated by Real-time PCR and immunocytochemistry. The expression of Sirt3 gene was also evaluated. Intracellular ROS levels were examined by DCFH-DA fluorescence intensity. In order to assess the morphological quality of the embryos, ICM and OCM number blastocyst cells were evaluated by using Hoechst and propidium iodide (PI) staining. ErbB1, ErbB4, ROS levels and cell number were compared across all in vitro groups. Our data reveal that LC significantly increases ErbB1 and ErbB4 gene and protein expression with intracellular ROS levels and Sirt3 gene expression significantly decreased after LC treatment. It is worth noting that an elevated cell number was observed in the LC-treated group compared with the other groups suggesting increased viability and/or proliferation. Our findings suggest that the use of LC could be helpful to improve preimplantation embryo culture media through its effects in decreasing ROS levels and the increase of implantation-related genes.
体外培养的胚胎不可避免地会接触到各种活性氧（ROS），这可能会降低胚胎在辅助生殖技术（ART）程序中的能力。使用抗氧化剂优化胚胎培养基可以帮助提高胚胎质量，并克服当前 ART 中的失败。本研究旨在评估左旋肉碱（LC），一种增强线粒体活性和自由基清除剂的物质，在培养基中的应用对早期胚胎能力以及 ErbB1 和 ErbB4 植入相关基因表达的影响。将两细胞期小鼠胚胎在以下四种条件下进行培养：1. LC 组，培养基中含有 LC；2. H2O2 组，暴露于 H2O2 30 分钟，然后转移到简单培养基中；3. H2O2+LC 组，暴露于 H2O2 30 分钟，然后转移到含有 LC 的简单培养基中；4. 对照组，整个过程都保持在简单培养基中。所有组均允许发育至囊胚阶段。通过实时 PCR 和免疫细胞化学评估 ErbB1 和 ErbB4 的表达。还评估了 Sirt3 基因的表达。通过 DCFH-DA 荧光强度检测细胞内 ROS 水平。为了评估胚胎的形态质量，使用 Hoechst 和碘化丙啶（PI）染色评估囊胚细胞的 ICM 和 OCM 数。通过比较所有体外组来比较 ErbB1、ErbB4、ROS 水平和细胞数。我们的数据表明，LC 处理后显著增加了 ErbB1 和 ErbB4 基因和蛋白的表达，同时细胞内 ROS 水平和 Sirt3 基因表达显著降低。值得注意的是，与其他组相比，LC 处理组观察到细胞数量增加，表明活力和/或增殖增加。我们的研究结果表明，LC 的使用可能有助于通过降低 ROS 水平和增加植入相关基因来改善植入前胚胎培养基。
