三氯生对斑马鱼幼鱼性别分化的影响及其机制

Effects and Mechanism of Triclosan on Sexual Differentiation of Juvenile Zebrafish(Danio rerio)

【目的】探讨三氯生(Triclosan,TCS)对斑马鱼早期发育阶段的性别干扰机制。【方法】将斑马鱼胚胎暴露于不同质量浓度的TCS(0、2、10、50μg/L)中至受精后50 d,采用HE染色、实时荧光定量PCR和酶联免疫技术分析斑马鱼幼鱼的性别比、下丘脑-垂体-性腺(HPG)轴相关基因表达以及性激素和卵黄蛋白原(Vtg)含量。【结果】与对照组比较,2μg/L和10μg/L组斑马鱼幼鱼雌性比例升高,50μg/L组性比不变,但出现雌雄间体。2μg/LTCS使促性腺激素释放激素2基因(gnrh2)、雌激素受体α基因(erα)、促卵泡激素受体基因(fshr)极显著上调(P<0.01),促性腺激素释放激素3(gnrh3)、脑型芳香化酶基因(cyp19b)、促卵泡激素β基因(fshβ)显著上调(P<0.05);10μg/L TCS使雌激素受体β基因(erβ)、fshβ和黄体生成素受体基因(lhr)极显著上调(P<0.01),gnrh3、cyp19b、erα、fshr显著上调(P<0.05);50μg/LTCS使gnrh3显著上调(P<0.05),cyp19b、fshr和lhr极显著上调(P<0.01)。各TCS处理组Vtg含量显著升高(P<0.05);2μg/L组雌二醇(E2)和11-酮基睾酮(11KT)含量极显著降低(P<0.01);10μg/L TCS组睾酮(T)显著降低(P<0.05),11KT/E2极显著降低(P<0.01);50μg/L组T极显著降低(P<0.01),11KT/E2显著降低(P<0.05)。【结论】TCS影响斑马鱼早期阶段的雌性比例以及HPG轴相关基因表达,有雌激素效应,但这种性别分化干扰效应无明显的剂量依赖关系;斑马鱼雌性化与TCS的雌激素效应有关(E2/11KT和Vtg升高),而该效应的产生主要通过对HPG轴相关基因的表达调控来实现。

【Objective】To explore the sex interference mechanism of triclosan(TCS)on zebrafish(Danio rerio)in the early development stage.【Method】Zebrafish embryos were exposed to different concentrations of TCS(0,2,10 and 50μg/L)for consecutive 50 days after fertilization and the sex ratio,sex hormones and vitellogenin(Vtg)contents,and the expression of genes related to hypothalamus pituitary gonad(HPG)axis in zebrafish juveniles were analyzed using HE staining,real time fluorescent quantitative PCR,and enzyme-linked immunosorbent assay(ELISA)methods.【Result】Compared to the control group,the proportion of females of zebrafish was increased obviously in 2μg/L and 10μg/L TCS treatment groups,while the proportion was not changed and the intersexes appeared in the 50μg/L TCS treatment group.In 2μg/L TCS treatment group,the gonadotropin releasing hormone 2(gnrh2),estrogen receptorα(erα),follicle stimulating hormoneβ(fshβ)genes were highly significantly increased(P<0.01)and brain aromatase gene(cyp19b),gonadotropin releasing hormone 3(gnrh3),and follicle-stimulating hormoneβGene(fshβ)were significantly increased(P<0.05).In 10μg/L TCS treatment group,the estrogen receptorβ(erβ),luteinizing hormone receptor(lhr),and fshβgenes were highly significantly increased(P<0.01)and the erα,cyp19b,gnrh3,and fshr genes were significantly increased(P<0.05).In 50μg/L TCS treatment group,the cyp19b,fshr,and lhr genes were highly significantly increased(P<0.01)and the gnrh3 gene were significantly increased(P<0.05).The content of Vtg in TCS treatment groups was increased significantly(P<0.05).The content of estradiol(E2)and 11 ketotestosterone(11KT)in 2μg/L TCS treatment group were highly significantly decreased(P<0.01);in 10μg/L TCS treatment group,testosterone(T)was significantly decreased(P<0.05),the ratio of 11KT to E2 was extremely significantly decreased(P<0.01).While in 50μg/L TCS treatment group,testosterone(T)was highly significantly decreased(P<0.01),the ratio of 11KT to E2 was significantly decreased(P<0.05).【Conclusion】TCS affects the female proportion and the expression of the genes related to HPG axis at the early stage of zebrafish,and has estrogen effect.But this interference effect of gender differentiation has no obvious dose dependence.The feminization of zebrafish is related to the estrogen effect of TCS(the ratio of E2 to 11KT and Vtg increase),which is mainly achieved by regulating the expression of the genes related to HPG axis.