基于液质联用技术定量分析牛体外受精胚胎中谷胱甘肽含量

doi:10.11843/j.issn.0366-6964.2018.12.011

摘要/Abstract

摘要：

旨在为探索外源谷胱甘肽（glutathione，GSH）影响胚胎发育的机制提供研究方法。将牛体外受精胚胎分别置于含GSH（3 mmol·L^-1，处理组）和不含GSH（对照组）的培养液中培养7 d，统计两组胚胎的卵裂率、桑椹胚率、囊胚率和囊胚总细胞数；并分别采用荧光染色法和液质联用技术（liquid chromatography mass spectrometry，LC/MS）对不同发育阶段的牛受精卵（受精后8、18、24、32 h）中GSH进行定量和跟踪分析，同时分析其中氧自由基（reactive oxygen species，ROS）的含量。另外，在受精卵培养液中添加GSH的同位素标记物（GSX），利用LC/MS检测受精卵及其培养液中GSX含量。结果表明：1）外源GSH可显著或极显著降低受精后18、24、32 h胚胎内ROS的含量（P<0.05，P<0.01）。2）处理组胚胎的桑椹胚率（58.67%vs. 48.46%）、囊胚率（50.83%vs. 34.88%）和囊胚总细胞数（102.26 vs. 76.27）显著高于对照组（P<0.05）。3）各发育阶段（受精后18、24、32 h）的处理组受精卵中，GSH的含量均显著高于对照组（P<0.05），且随发育进程而逐渐下降。4）外源GSX也显著提高了处理组受精卵（受精后18、24、32 h）中GSH含量（P<0.05），培养液中GSX的浓度则随培养时间的延长而持续下降。综上表明，外源GSH可清除胚胎内ROS，提高胚胎发育率和囊胚质量；LC/MS是定量分析胚胎内GSH及其同位素标记物的可靠方法，可用于外源GSH促进胚胎体外发育的机制研究。

关键词: 牛, 胚胎发育, 谷胱甘肽, 氧自由基, 液质联用

Abstract:

This study aimed to present a method to clarify the effects of exogenous glutathione (GSH) on bovine embryo development. Bovine embryos fertilized in vitro were cultured in media with 3 mmol·L^-1 GSH for 7 days in treatment group and without GSH for 7 days in control group,respectively. The rates of cleavage, morula and blastocyst formation of embryos, and the total cell number of blastocysts were calculated in both groups. The contents of reactive oxygen species (ROS) and GSH in zygotes at different stages(8,18,24,32 hours post-insemination)were detected by fluorescence staining and liquid chromatography mass spectrometry (LC/MS). Additionally, the changes of isotope labelled GSH (GSX) contents of bovine zygotes and culture media were tracked by LC/MS after GSX was added in culture medium. Results showed that:1)Exogenous GSH significantly reduced ROS contents of embryos(18,24,32 hours post-insemination)(P<0.05,P<0.01). 2)The morula rate (58.67% vs. 48.46%), the blastocyst rate (50.83% vs. 34.88%) and the total cell number of blastocysts (102.26 vs. 76.27) in treatment group were significantly higher than that in control group (P<0.05).3) GSH contents of zygotes at 3 stages in treatment group were significantly higher than that in control group (P<0.05). Meanwhile, GSH contents of zygotes in both groups decreased along with the developmental process(18,24,32 hours post-insemination). 4)After addition of GSX, the contents of GSH of zygotes in treatment group increased significantly (P<0.05). In addition, GSX concentrations in culture media decreased steadily with the extension of culture time(18,24,32 hours post-insemination). Conclusively, exogenous GSH improves the development rate of bovine embryo and blastocyst quality with scavenging of ROS. Moreover, LC/MS is a reliable method for quantitative analysis of GSH and isotope labeled GSH. Therefore LC/MS can be used to explore the mechanism of exogenous GSH improving the development of embryo cultured in vitro.

Key words: bovine, embryo development, GSH, ROS, LC/MS

中图分类号:

S823.2

李凤, 崔立欣, 郝海生, 刘岩, 赵学明, 庞云渭, 朱化彬, 杜卫华. 基于液质联用技术定量分析牛体外受精胚胎中谷胱甘肽含量[J]. 畜牧兽医学报, 2018, 49(12): 2622-2631.

LI Feng, CUI Li-xin, HAO Hai-sheng, LIU Yan, ZHAO Xue-ming, PANG Yun-wei, ZHU Hua-bin, DU Wei-hua. Quantitative Analysis of Glutathione Levels in Bovine in vitro Embryos by LC/MS[J]. ACTA VETERINARIA ET ZOOTECHNICA SINICA, 2018, 49(12): 2622-2631.

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