乙草胺对小鼠卵母细胞体外成熟的影响

Effects of Acetochlor on in vitro Maturation of Mouse Oocytes

【目的】研究乙草胺对小鼠卵母细胞体外成熟的影响,为研究体内乙草胺对卵母细胞质量的影响提供参考。【方法】将收集的卵丘-卵母细胞复合体(cumulus-oocyte complexes,COCs)置于含有不同浓度(0(对照组)、10、50、100、130、200μmol/L)乙草胺的体外成熟培养液中培养14 h,统计卵母细胞第一极体排出率,进行体外受精后,统计体外受精胚胎的囊胚率,筛选出适宜于卵母细胞体外培养的乙草胺浓度。将COCs在对照组和筛选出的适宜浓度的乙草胺组成熟培养液中培养14 h后,利用DCFH-DA检测卵母细胞活性氧(ROS)水平;利用CellTracker蓝色染料检测卵母细胞谷胱甘肽(GSH)水平;利用JC-1染色法检测卵母细胞线粒体膜电位水平(mitochondrial membrane potential,MMP);利用实时荧光定量PCR检测卵母细胞内凋亡相关基因的表达水平,利用Hoechst 33342染色检测体外受精胚胎的囊胚细胞总数,用Fluorescein-dUTP染色法检测囊胚细胞凋亡率。【结果】与对照组相比,100、130、200μmol/L乙草胺组卵母细胞的第一极体排出率显著降低(P<0.05),100和130μmol/L乙草胺组体外受精胚胎的囊胚率显著降低(P<0.05),200μmol/L乙草胺组体外受精胚胎未发育到囊胚期,因此选择100和130μmol/L乙草胺进行后续试验。与对照组相比,在100和130μmol/L乙草胺组中,卵母细胞ROS水平显著升高(P<0.05),GSH水平及MMP显著降低(P<0.05),卵母细胞中促凋亡基因Caspase-9的相对表达量显著升高(P<0.05),而抗凋亡基因Bax、Bcl-2的相对表达量显著降低(P<0.05);100和130μmol/L乙草胺组囊胚细胞总数显著降低(P<0.05),囊胚细胞凋亡率显著升高(P<0.05)。【结论】乙草胺通过提高卵母细胞内ROS及促进卵母细胞凋亡降低卵母细胞的质量,从而影响卵母细胞受精后胚胎的发育潜力。

【Objective】 The aim of study was to explore the effect of acetochlor on the maturation of mouse oocytes in vitro,in order to provide a basic reference for judging the toxicity standard of acetochlor.【Method】 The collected cumulus-oocyte complexes(COCs) were placed in the in vitro maturation solution containing different concentrations(0(control group),10,50,100,130,200 μmol/L) of acetochlor for 14 h, the first polar body excretion rate of oocytes was calculated.After in vitro fertilization(IVF),the blastocyst rate of IVF embryos was counted, suitable acetochlor concentrations for oocyte culture in vitrowereselected out.After COCs were cultured in mature medium of control group and selected appropriate concentrations acetochlor group for 14 h, the ROS levels of oocyteswere detected by DCFH-DA.The GSH levels of oocytes were detectedby CellTrackerblue dye.The mitochondrial membrane potential(MMP) of oocytes was detected by JC-1 staining method.The expression levels of apoptosis-related genes in oocytes were detected by Real-time quantitative PCR.The total number of blastocyst cells of IVF embryos was detected by Hoechst 33342 staining, and the apoptosis rate of blastocyst cells was detected by Fluorescein-dUTP.【Result】 Compared with control group, the first polar body excretion rate of oocytes in 100 and 130 μmol/L acetochlor groups was significantly decreased(P<0.05),and the blastocyst rate of VIF embryos in 100 and 130 μmol/L acetochlor groups was significantly decreased(P<0.05),and IVF embryos in 200 μmol/L acetochlor group did not develop to blastocyst stage, therefore, 100 and 130 μmol/L acetochlor were selected for follow-up tests.Compared with control group, in 100 and 130 μmol/L acetochlor groups, the level of ROS in oocytes was significantly increased(P<0.05),the level of GSH and mitochondrial membrane potential were significantly increased(P<0.05),the relative expression of the pro-apoptotic gene Caspase9 in the oocytes was significantly increased(P<0.05),while the relative expression of the anti-apoptotic genes Bax and Bcl-2 was significantly increased(P<0.05).In 100 and 130 μmol/L acetochlor groups, the total number of blastocyst cells was significantly decreased(P<0.05),the blastocyst cell apoptosis rate was significantly increased(P<0.05).【Conclusion】 Acetochlor reduced the quality of oocytes by increasing ROS and promoting apoptosis of oocytes, thus affecting the developmental potential of oocytes after fertilization.