柠檬苦素对小鼠卵母细胞体外成熟及其体外受精胚胎发育的影响

doi:10.16431/j.cnki.1671-7236.2022.10.021

摘要/Abstract

摘要： 【目的】研究柠檬苦素(limonin,Lim)对小鼠卵母细胞体外成熟(IVM)及后续体外受精(IVF)胚胎发育潜能的影响,旨在为体外成熟培养系统的优化提供参考。【方法】在小鼠体外成熟培养液中添加不同浓度的Lim(0、10、20、50 μmol/L),成熟培养12 h后统计小鼠卵母细胞第一极体(PBI)排出率,筛选体外成熟培养液中添加Lim的最适浓度;在体外成熟培养液中添加最适浓度的Lim,以0 μmol/L Lim为对照组,成熟培养12 h,通过免疫荧光染色检测活性氧(ROS)、谷胱甘肽(GSH)以及线粒体膜电位(MMP)水平;通过实时荧光定量PCR检测卵母细胞抗氧化及凋亡相关基因的mRNA表达水平。将最适Lim组及对照组卵母细胞体外成熟24 h后进行体外受精,于体外受精24 h和3.5 d分别统计胚胎卵裂率和囊胚率,并用Fluorescein-dUTP和Hoechst 33342染色分别检测囊胚总细胞数及囊胚内凋亡细胞比率。【结果】与0 μmol/L Lim组相比,20 μmol/L Lim组小鼠卵母细胞PBI排出率显著升高(P＜0.05),后续试验均用20 μmol/L Lim进行处理。与对照组组相比,20 μmol/L Lim组小鼠卵母细胞内ROS水平显著降低(P＜0.05),GSH、MMP水平均显著增加(P＜0.05),抗氧化相关基因(GPx3、CAT和Prdx3)、抗凋亡相关基因(Bcl-2、Bcl-xl)表达水平均显著上调(P＜0.05),促凋亡相关基因(Caspase-3)表达水平显著下调(P＜0.05);体外受精胚胎的卵裂率、囊胚率、囊胚总细胞数均显著增加(P＜0.05),囊胚内细胞凋亡比率显著下降(P＜0.05)。【结论】在体外成熟培养液中添加20 μmol/L Lim可以通过抑制氧化应激和细胞凋亡、增加MMP水平提高小鼠卵母细胞质量,从而提高体外受精胚胎的发育潜力。

关键词: 小鼠; 卵母细胞; 柠檬苦素; 体外成熟; 氧化应激

Abstract: 【Objective】 The aim of this study was to investigate the effects of limonin (Lim) on the development potential of in vitro maturation (IVM) mouse oocytes and subsequent in vitro fertilization (IVF) embryos,in order to provide reference for the optimization of in vitro maturation culture system.【Method】 Different concentrations of Lim (0,10,20,50 μmol/L) were added into the culture medium of mouse IVM culture medium.After 12 h of maturation culture,the excretion rate of the first polar body (PBI) of mouse oocytes was calculated,and the optimal concentration of Lim was selected.The optimal concentration of Lim was added to IVM culture medium,0 μmol/L Lim was used as control group,after mature culture for 12 h,the levels of reactive oxygen species (ROS),glutathione (GSH) and mitochondrial membrane potential (MMP) were detected by immunofluorescence staining.The mRNA expression levels of antioxidant and apoptosis-related genes in oocytes were detected by Real-time quantitative PCR.The embryo cleavage rate and blastocyst rate were calculated at 24 h and 3.5 d after in vitro fertilization.The total number of blastocyst cells and the ratio of apoptotic cells in blastocysts were detected by Fluorescein-dUTP staining and Hoechst 33342 staining.【Result】 Compared with 0 μmol/L Lim group,PBI excretion rate of oocytes in 20 μmol/L Lim group was significantly increased (P＜0.05),and 20 μmol/L Lim was used in the subsequent experiments.Compared with control group,ROS levels in oocytes of mice in 20 μmol/L Lim group were significantly decreased (P＜0.05),GSH and MMP levels were significantly increased (P＜0.05).The expression of antioxidant related genes (GPx3,CAT and Prdx3) and anti-apoptotic related genes (Bcl-2 and Bcl-xl) were significantly up-regulated (P＜0.05),while the expression of pro-apoptotic related gene (Caspase-3) was significantly down-regulated (P＜0.05).After in vitro fertilization,the cleavage rate,blastocyst rate and blastocyst total cell number of embryos were significantly increased (P＜0.05),and the apoptosis rate of cells in blastocyst was significantly decreased (P＜0.05).【Conclusion】 Adding 20 μmol/L Lim to in vitro maturation medium could improve the quality of mouse oocytes by inhibiting oxidative stress and cell apoptosis and increasing MMP level,thus improving the development potential of in vitro fertilization embryos.

Key words: mouse; oocytes; limonin; in vitro maturation; oxidative stress

中图分类号:

焦安惠, 张笑梦, 赵予晗, 王宇, 高青山. 柠檬苦素对小鼠卵母细胞体外成熟及其体外受精胚胎发育的影响[J]. 中国畜牧兽医, 2022, 49(10): 3891-3900.

JIAO Anhui, ZHANG Xiaomeng, ZHAO Yuhan, WANG Yu, GAO Qingshan. Effects of Limonin on in vitro Maturation of Mouse Oocytes and Embryo Development of in vitro Fertilization[J]. China Animal Husbandry and Veterinary Medicine, 2022, 49(10): 3891-3900.

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