蚯蚓顶体效应及其在土壤健康质量诊断中的应用研究
Earthworm Acrosome Effect and Its Application in Soil Health Quality Diagnosis
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摘要: 蚯蚓在评价土壤生态环境污染方面起着重要作用，但目前与生殖毒性相关的蚯蚓生物标志物中缺乏可以灵敏和快速对污染物生殖毒性进行诊断的生物标志物。本研究采用考马斯亮蓝法对蚯蚓精子顶体反应进行检测；采用响应面分析法优化苯甲酰精氨酸乙酯/乙醇脱氢酶（BAEE/ADH）联合测定法，以建立蚯蚓精子顶体酶活性测定法。结果表明，蚯蚓精子顶体率为（47.67±3.87）%，最优测定条件为洗涤2次，抽提时间24 h，ADH浓度1.8 mg·mL-1，氧化型辅酶Ⅰ（NAD）浓度12 mg·mL-1。为考察方法的可靠性，采用醋酸棉酚污染土壤暴露实验进行方法学考察。数据表明，醋酸棉酚污染土壤暴露3 d后，检测出顶体酶活性变化，且与精子密度具有一定的相关性（r=0.9908）。建立的蚯蚓顶体酶活性测定方法可以快速和有效的检测醋酸棉酚的生殖毒性变化，蚯蚓顶体酶活性有望作为生物标志物应用于土壤污染的生态毒理学快速诊断中。Abstract: Earthworm plays an important role in the evaluation of soil pollution, but currently there is a lack of earthworm biomarkers that could sensitively and quickly diagnose the reproductive toxicity of pollutants. In this study, the acrosomal reaction of earthworm sperm was detected by Coomassie Bright Blue Method. In order to establish a method for the determination of acrosomal activity of earthworm sperm, Response Surface Methodology was used to optimize the combined determination of benzoyl arginine ethyl ester/ethanol dehydrogenase (BAEE/ADH). The results showed that the acrosomal rate of earthworm sperm was (47.67±3.87)%. The optimal condition was washed for 2 times, extracted for 24 h, ADH concentration as 1.8 mg·mL-1, and oxidized coenzyme Ⅰ (NAD) concentration as 12 mg·mL-1. To investigate the reliability of the method, gossypol acetate soil exposure test was used to investigate the methodology. The results showed that acrosomal enzyme activity changed and correlated with sperm density (r=0.9908) in the soil exposed to gossypol acetate for 3 d. The established method for the determination of earthworm acrosomal activity could quickly and effectively detect the reproductive toxicity of gossypol acetate. The earthworm acrosomal enzyme activity is expected to be used as a biomarker for the rapid ecotoxicological diagnosis of soil pollution.
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Key words:
- gossypol acetate /
- the earthworm /
- acrosomal reaction /
- acrosomal enzyme activity /
- biomarker
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刘雅心. 土壤修复纳米材料对蚯蚓的生理生化影响及机理研究[D]. 济南:山东师范大学, 2020:14-29 张倩倩, 乔敏, 池海峰. 土壤生态毒性测试方法综述[J]. 生态毒理学报, 2017, 12(4):76-97 Zhang Q Q, Qiao M, Chi H F. Overview of soil ecotoxicity tests[J]. Asian Journal of Ecotoxicology, 2017, 12(4):76-97(in Chinese)
赵晨阳, 马俊杰, 薛璐, 等. 地质封存CO2泄漏对蚯蚓的毒性效应[J]. 生态毒理学报, 2020, 15(2):210-219 Zhao C Y, Ma J J, Xue L, et al. Toxic effects of CO2 leakage from geological storage on earthworms[J]. Asian Journal of Ecotoxicology, 2020, 15(2):210-219(in Chinese)
Capowiez Y, Rault M, Mazzia C, et al. Earthworm behaviour as a biomarker-A case study using imidacloprid[J]. Pedobiologia, 2003, 47(5-6):542-547 苏连水, 杨桂玲, 吴声敢, 等. 三唑磷和氯氟氰菊酯对蚯蚓的联合毒性效应[J]. 生态毒理学报, 2016, 11(3):294-301 Su L S, Yang G L, Wu S G, et al. The single and joint toxicity of tiazophos and cyhalothrin to earthworm[J]. Asian Journal of Ecotoxicology, 2016, 11(3):294-301(in Chinese)
赵逸昳. PFOA和Ni复合污染对蚯蚓酶活性的影响研究[D]. 泰安:山东农业大学, 2017:9-16 Zhao Y Y. Study on impacts of compound pollution of PFOA and Ni to enzyme activities in earthworms[D]. Tai'an:Shandong Agricultural University, 2017:9 -16(in Chinese)
Bundy J G, Keun H C, Sidhu J K, et al. Metabolic profile biomarkers of metal contamination in a sentinel terrestrial species are applicable across multiple sites[J]. Environmental Science & Technology, 2007, 41(12):4458-4464 Bundy J G, Lenz E M, Bailey N J, et al. Metabonomic assessment of toxicity of 4-fluoroaniline, 3,5-difluoroaniline and 2-fluoro-4-methylaniline to the earthworm Eisenia veneta (Rosa):Identification of new endogenous biomarkers[J]. Environmental Toxicology and Chemistry, 2002, 21(9):1966-1972 Spurgeon D J, Weeks J M, van Gestel C A M. A summary of eleven years progress in earthworm ecotoxicology[J]. Pedobiologia, 2003, 47(5-6):588-606 徐冬梅, 刘文丽, 刘维屏. 外源污染物对蚯蚓毒理作用研究进展[J]. 生态毒理学报, 2009, 4(1):21-27 Xu D M, Liu W L, Liu W P. Research advances in toxicological effects of external pollutants on earthworms[J]. Asian Journal of Ecotoxicology, 2009, 4(1):21-27(in Chinese)
周世萍. 两种环境激素类农药单独及混配作用的生态毒理学研究[D]. 昆明:云南大学, 2007:5-23 Zhou S P. Ecotoxicology study on the effects of two environmental hormone pesticides alone and in combination[D]. Kunming:Yunnan University, 2007:5 -23(in Chinese)
Calisi A, Zaccarelli N, Lionetto M G, et al. Integrated biomarker analysis in the earthworm Lumbricus terrestris:Application to the monitoring of soil heavy metal pollution[J]. Chemosphere, 2013, 90(11):2637-2644 宁玉翠. 镉胁迫下蚯蚓(Eisenia fetida)氧化应激反应及解毒机制研究[D]. 哈尔滨:东北农业大学, 2019:1-12 Ning Y C. Study on the detoxification mechanism and oxidative stress of Eisenia fetida under cadmium stress[D]. Harbin:Northeast Agricultural University, 2019:1 -12(in Chinese)
周世萍, 段昌群, 刘守庆, 等. 土壤中亚致死剂量毒死蜱对蚯蚓抗氧化防御系统酶活性的影响[J]. 土壤学报, 2014, 51(2):389-393 Zhou S P, Duan C Q, Liu S Q, et al. Effect of chlorpyrifos of a sublethal dose on enzyme activity of the antioxidant defense system of earthworms[J]. Acta Pedologica Sinica, 2014, 51(2):389-393(in Chinese)
李圆龙, 王树松. 精子功能检测的临床应用进展[J]. 检验医学与临床, 2019, 16(18):2738-2741 王丽, 赵杰, 刘芳, 等. 体外受精胚胎移植中异常受精的原因分析[J]. 疾病监测与控制, 2020, 14(3):237-243 Wang L, Zhao J, Liu F, et al. Analysis of the causes of abnormal human fertilization in conventional in vitro fertilization cycles[J]. Journal of Diseases Monitor & Control, 2020, 14(3):237-243(in Chinese)
张伟伟. SLO家族在中华绒螯蟹(Eriocheir sinensis)精子发生过程中的表达及功能[D]. 保定:河北大学, 2020:1-11 Zhang W W. Function and expression of SLO family during spermatogenesis in Eriocheir sinensis[D]. Baoding:Hebei University, 2020:1 -11(in Chinese)
Gray L E Jr, Ostby J, Furr J, et al. Perinatal exposure to the phthalates DEHP, BBP, and DINP, but not DEP, DMP, or DOTP, alters sexual differentiation of the male rat[J]. Toxicological Sciences, 2000, 58(2):350-365 陈其桂, 李大文. 精子顶体完整性对IVF-ET结局影响的研究进展[J]. 中国临床新医学, 2020, 13(9):945-949 Chen Q G, Li D W. Advances in research on the effect of sperm acrosome integrity on IVF-ET outcome[J]. Chinese Journal of New Clinical Medicine, 2020, 13(9):945-949(in Chinese)
马征兵, 薛力. 精子的顶体酶活性与受精能力的临床研究[J]. 中国性科学, 2019, 28(7):33-36 Ma Z B, Xue L. Clinical study on the relationship between sperm acrosin activity and fertilization ability[J]. Chinese Journal of Human Sexuality, 2019, 28(7):33-36(in Chinese)
Moller C C, Bleil J D, Kinloch R A, et al. Structural and functional relationships between mouse and hamster zona Pellucida glycoproteins[J]. Developmental Biology, 1990, 137(2):276-286 Aarons D, Boettger-Tong H, Holt G, et al. Acrosome reaction induced by immunoaggregation of a proteinase inhibitor bound to the murine sperm head[J]. Molecular Reproduction and Development, 1991, 30(3):258-264 Aarons D, Battle T, Boettger-Tong H, et al. Role of monoclonal antibody J-23 in inducing acrosome reactions in capacitated mouse spermatozoa[J]. Journal of Reproduction and Fertility, 1992, 96(1):49-59 江一平, 卓丹心, 陈勇. 考马斯亮蓝染色法检测人精子顶体反应的评价[J]. 解剖学报, 1998, 29(3):332 Larson J L, Miller D J. Simple histochemical stain for acrosomes on sperm from several species[J]. Molecular Reproduction and Development, 1999, 52(4):445-449 马静静, 钱新春, 张伟, 等. 土壤菲多次叠加污染对蚯蚓的毒性效应[J]. 土壤学报, 2015, 52(6):1374-1382 Ma J J, Qian X C, Zhang W, et al. Toxic effect of multiple-time overlying pollution of phe in soil on Eisenia fetida[J]. Acta Pedologica Sinica, 2015, 52(6):1374-1382(in Chinese)
余俊里, 张健, 汤智群, 等. 一个年龄序列巨桉人工林土壤对赤子爱胜蚓生长和生理指标的影响[J]. 生态学杂志, 2017, 36(1):124-131 Yu J L, Zhang J, Tang Z Q, et al. Variations of growth and physiological indicators of Eisenia foetida in soils across a chronosequence of Eucalyptus grandis plantations[J]. Chinese Journal of Ecology, 2017, 36(1):124-131(in Chinese)
Zhou S P, Duan C Q, Michelle W H, et al. Individual and combined toxic effects of cypermethrin and chlorpyrifos on earthworm[J]. Journal of Environmental Sciences, 2011, 23(4):676-680 卓丹心, 林典梁, 江一平. 哺乳动物精子顶体反应检测考马斯亮蓝染色法[J]. 福建医科大学学报, 1999, 33(3):346-348 Henkel R, Müller C, Miska W, et al. Determination of the acrosin activity of human spermatozoa by means of a gelatinolytic technique. A simple, predictive method useful for IVF[J]. Journal of Andrology, 1995(16):272-277 Zalata A A, Ahmed A H, Allamaneni S S R, et al. Relationship between acrosin activity of human spermatozoa and oxidative stress[J]. Asian Journal of Andrology, 2004, 6(4):313-318 Zhao Y K, Zhao E Y, Zhang C Y, et al. Study of the changes of acrosomal enzyme, nitric oxide synthase, and superoxide dismutase of infertile patients with positive antisperm antibody in seminal plasma[J]. Cell Biochemistry and Biophysics, 2015, 73(3):639-642 Zhang H F, Zhao E Y, Zhang C Y, et al. The change of semen superoxide dismutase and acrosin activity in the sterility of male patients with positive antisperm antibody[J]. Cell Biochemistry and Biophysics, 2015, 73(2):451-453 李钰琪, 王彩蝶, 屯妮萨·麦提赛伊迪, 等. 醋酸棉酚对绵羊瘤胃微生物数量及消化代谢的影响[J]. 动物营养学报, 2019, 31(7):3421-3430 Li Y Q, Wang C D, Tunisa·Maitisaiyidi, et al. Effects of gossypol acetate on rumen microbial population and digestion and metabolism in sheep[J]. Chinese Journal of Animal Nutrition, 2019, 31(7):3421-3430(in Chinese)
Tang J, Wang Z Y, Chen L Y, et al. Gossypol acetate induced apoptosis of pituitary tumor cells by targeting the BCL-2 via the upregulated microRNA miR-15a[J]. International Journal of Clinical and Experimental Medicine, 2015, 8(6):9079-9085 Zhou S P, Duan C Q, Wang X H, et al. Assessing cypermethrin-contaminated soil with three different earthworm test methods[J]. Journal of Environmental Sciences, 2008, 20(11):1381-1385 张淑敏. 离子液体BF4、BF4和Br对蚯蚓的氧化胁迫及DNA损伤[D]. 泰安:山东农业大学, 2014:12-13 Zhang S M. Oxidative stress and DNA damage induced by ionic liquids BF4, BF4 and Br exposure in earthworm exposure in earthworm[D]. Tai'an:Shandong Agricultural University, 2014:12-13(in Chinese)
邓智标, 黎子辉, 麦贤昌. 男性不育因素的临床研究[J]. 中国医学创新, 2016, 13(18):72-75 Deng Z B, Li Z H, Mai X C. Clinical study of male infertility factors[J]. Medical Innovation of China, 2016, 13(18):72-75(in Chinese)
秦惠珍, 曹其义, 宁莞权, 等. 响应面优化华腺萼木总黄酮和总多糖的超声波辅助提取工艺[J]. 广西科学, 2020, 27(4):368-379 Qin H Z, Cao Q Y, Ning G Q, et al. Optimization of ultrasonic assisted extraction of total flavonoids and total polysaccharides from Mycetia sinensis by response surface methodology[J]. Guangxi Sciences, 2020, 27(4):368-379(in Chinese)
李莉, 张赛, 何强, 等. 响应面法在试验设计与优化中的应用[J]. 实验室研究与探索, 2015, 34(8):41-45 Li L, Zhang S, He Q, et al. Application of response surface methodology in experiment design and optimization[J]. Research and Exploration in Laboratory, 2015, 34(8):41-45(in Chinese)
郭垠利. 蛋白质含量测定方法研究[J]. 生物化工, 2018, 4(4):144-146 ,149 Guo Y L. Research progress on methods for determination of protein content[J]. Biological Chemical Engineering, 2018, 4(4):144-146,149(in Chinese)
陈军, 韩东, 曾黎, 等. 哺乳动物精子顶体反应检测方法研究进展[J]. 黑龙江动物繁殖, 2012, 20(5):1-3 ,6
张旭成. 不同处理对猪精子蛋白磷酸化的位点分布、水平及质量的影响[D]. 延吉:延边大学, 2010:7-9 Zhang X C. The effects of different treatments on the expression and distribution of protein typrosine phosphorylation quality of pig sperm[D]. Yanji:Yanbian University, 2010:7 -9(in Chinese)
张孟丹. 醋酸棉酚对鲤肝脏毒性及致毒机理的研究[D]. 新乡:河南师范大学, 2019:1-7 Zhang M D. The toxicity and mechanism of hepatotoxicity induced by gossypol acetate in Cyprinus carpio[D]. Xinxiang:Henan Normal University, 2019:1 -7(in Chinese)
袁彦波. 棉酚致小鼠睾丸生精细胞凋亡及Bax、Bcl-2表达相关性研究[D]. 哈尔滨:东北农业大学, 2015:1-9 郭春丽, 赵成键, 华坚, 等. 吲哚-2,3-二酮对醋酸棉酚致生精障碍小鼠的影响[J]. 中国新药与临床杂志, 2018, 37(8):484-487 Guo C L, Zhao C J, Hua J, et al. Effects of indole-2,3-dione on spermatogenesis impairment mice induced by gossypol acetate[J]. Chinese Journal of New Drugs and Clinical Remedies, 2018, 37(8):484-487(in Chinese)
方选, 朱爱民, 于德志, 等. 邻苯二甲酸二丁酯对大鼠精子顶体酶活性的影响[J]. 中国药事, 2015, 29(7):738-741 Fang X, Zhu A M, Yu D Z, et al. Effects of dibutyl phthalate on sperm acrosin activity in rats[J]. Chinese Pharmaceutical Affairs, 2015, 29(7):738-741(in Chinese)
Cui Y H, Zhao R L, Wang Q, et al. Determination of sperm acrosin activity for evaluation of male fertility[J]. Asian Journal of Andrology, 2000, 2(3):229-232 Howes L, Jones R. Interactions between zona pellucida glycoproteins and sperm proacrosin/acrosin during fertilization[J]. Journal of Reproductive Immunology, 2002, 53(1-2):181-192 -
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