乙草胺对小鼠卵母细胞体外成熟的影响

doi:10.16431/j.cnki.1671-7236.2022.10.023

摘要/Abstract

摘要： 【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、200 μmol/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^TM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、200 μmol/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130 μmol/L乙草胺组体外受精胚胎的囊胚率显著降低(P＜0.05),200 μmol/L乙草胺组体外受精胚胎未发育到囊胚期,因此选择100和130 μmol/L乙草胺进行后续试验。与对照组相比,在100和130 μmol/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130 μmol/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。

关键词: 乙草胺; 小鼠; 卵母细胞; 体外成熟; 体外受精; 早期胚胎

Abstract: 【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 CellTracker^TM blue 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-2was 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.

Key words: acetochlor; mice; oocytes; in vitro maturation; in vitro fertilization; early embryos

中图分类号:

王宇, 赵予晗, 焦安惠, 王玉琪, 高青山. 乙草胺对小鼠卵母细胞体外成熟的影响[J]. 中国畜牧兽医, 2022, 49(10): 3909-3917.

WANG Yu, ZHAO Yuhan, JIAO Anhui, WANG Yuqi, GAO Qingshan. Effects of Acetochlor on in vitro Maturation of Mouse Oocytes[J]. China Animal Husbandry and Veterinary Medicine, 2022, 49(10): 3909-3917.

参考文献

[1] ROUBEIX V,FAUVELLE V,TISON-ROSEBERY J,et al.Assessing the impact of chloroacetanilide herbicides and their metabolites on periphyton in the Leyre River via short term growth inhibition tests on autochthonous diatoms[J].Journal of Environmental Monitoring,2012,14(6):1655-1663.
[2] SHBY J,KIER L,WILSON A,et al.Evaluation of the potential carcinogenicity and genetic toxicity to humans of the herbicide acetochlor[J].Human & Experimental Toxicology,1996,15(9):702-735.
[3] 徐雄,李春梅,孙静,等.我国重点流域地表水中29种农药污染及其生态风险评价[J].生态毒理学报,2016,11(2):347-354. XU X,LI C M,SUN J,et al.Pollution of 29 pesticides in surface waters of key river basins in my country and their ecological risk assessment[J].Acta Ecotoxicology,2016,11(2):347-354.(in Chinese)
[4] 于志勇,金芬,李红岩,等.我国重点城市水源及水厂出水中乙草胺的残留水平[J].环境科学,2014,35:1694-1697. YU Z Y,JIN F,LI H Y,et al.Residue levels of acetochlor in water sources and effluents of water plants in key cities in my country[J].Environmental Science,2014,35:1694-1697.(in Chinese)
[5] 刘阳.除草剂乙草胺对花背蟾蜍肝脏的影响及机理[D].兰州:兰州大学,2006. LIU Y.The effect and mechanism of herbicide acetochlor on the liver of Toad latifolia[D].Lanzhou:Lanzhou University,2006.(in Chinese)
[6] SONG X P,ZHANG F,CHEN D Y,et al.Study on systemic and reproductive toxicity of acetochlor in male mice[J].Toxicology Research,8(1):77-89.
[7] ZHANG Y,XUE W,LONG R,et al.Acetochlor affects zebrafish ovarian development by producing estrogen effects and inducing oxidative stress[J].Environmental Science Pollution Research,2020,27(22):27688-27696.
[8] 熊显荣,王艳,李键,等.SIRT1对牦牛卵母细胞体外成熟与老化的影响[J].畜牧兽医学报,2019,50(12):2440-2448. XIONG X R,WANG Y,LI J,et al.Effects of SIRT1 on in vitro maturation and aging of yak oocytes[J].ChineseJournal of Animal and Veterinary Sciences,2019,50(12):2440-2448.(in Chinese)
[9] WATSON A J.Oocyte cytoplasmic maturation:A key mediator of oocyte and embryo developmental competence[J]. Journalof Animal Science,2007,85(13 Suppl):E1-E3.
[10] KEEFE D,KUMAR M,KALMBACH K.Oocyte competency is the key to embryo potential[J]. Fertility and Sterility,2015,103(2):317-322.
[11] 杨晓梅,谭志军,苑怡,等.乙草胺对中华大蟾蜍(Bufobufogar garizans)早期胚胎发育的影响[J].动物学报,2001,S1:125-130. YANG X M,TAN Z J,YUAN Y,et al.Effects of acetochlor on the early embryonic development of Chinese Bufo (Bufobufogar garizans)[J].Animal Journal,2001,S1:125-130.(in Chinese)
[12] FU D J,LI P,SONG J,et al.Mechanisms of synergistic neurotoxicity induced by two high risk pesticide residues―Chlorpyrifos and carbofuran via oxidative stress[J].Toxicology in Vitro,2019,54:338-344.
[13] RAMBABU L,MEGSON I L,EDDLESTON M.Does oxidative stress contribute to toxicity in acute organophosphorus poisoning―A systematic review of the evidence[J].Clinical Toxicology,2020,58(6):437-452.
[14] TAHA M,BADAWY M,ABDEL-RAZIK R K,et al.Mitochondrial dysfunction and oxidative stress in liver of male albino rats after exposing to sub-chronic intoxication of chlorpyrifos,cypermethrin,and imidacloprid[J].Pesticide Biochemistry Physiology,2021,178:104938.
[15] LIU M,WU X,CUI Y,et al.Mitophagy and apoptosis mediated by ROS participate in AlCl3-induced MC3T3-E1 cell dysfunction[J].Food and Chemical Toxicology,2021,155:112388.
[16] TRAVERSO N,RICCIARELLI R,NITTI M,et al.Role of glutathione in cancer progression and chemoresistance[J]. Oxidative Medicineand Cellular Longevity,2013,2013:972913.
[17] 蒋青桃,宋仙平,张锋,等.乙草胺对雄性小鼠GC-1精原细胞的毒性研究[J].职业与健康,2020,36(21):2920-2926. JIANG Q T,SONG X P,ZHANG F,et al.Toxicity of acetochlor on GC-1 spermatogonia in male mice[J].Occupation and Health,2020,36(21):2920-2926.(in Chinese)
[18] 李龙雪,郑晶,徐艺华,等.乙草胺对大鼠肝脏氧化性应激状态的影响[J].中华疾病控制杂志,2016,20(1):54-57. LI L X,ZHENG J,XU Y H et al.Effect of acetochlor on oxidative stress in rat liver[J].Chinese Journal of Disease Control,2016,20(1):54-57.(in Chinese)
[19] ANNESLEY S J,FISHER P R.Mitochondria in health and disease[J]. Cells,2019,8(7):680.
[20] PALMEIRA C M,ROLO A P.Mitochondrial membrane potential (ΔΨ) fluctuations associated with the metabolic states of mitochondria[J].Methods in Molecular Biology,2012,810:89-101.
[21] SAKAMURU S,ATTENE-RAMOS M S,XIA M.Mitochondrial membrane potential assay[J].Methods in Molecular, 2016,1473:17-22.
[22] TORNER H,BRUSSOW K P,ALM H,et al.Mitochondrial aggregation patterns and activity in porcine oocytes and apoptosis in surrounding cumulus cells depends on the stage of pre-ovulatory maturation[J].Theriogenology,2004,61(9):1675-1689.
[23] PISTRITTO G,TRISCIUOGLIO D,CECI C,et al.Apoptosis as anticancer mechanism:Function and dysfunction of its modulators and targeted therapeutic strategies[J].Aging,2016,8(4):603-619.
[24] 胡伟军.除草剂乙草胺对雄性小鼠生殖毒性的研究[D].长春:吉林大学,2010. HU W J.Reproductive toxicity of herbicide acetochlor on male mice[D].Changchun:Jilin University,2010.(in Chinese)