兰州大学机构库 >生命科学学院
2,4-二氯酚暴露对斑马鱼性比及性腺发育的影响
Alternative TitleThe impact of 2,4-dichlorophenol exposure on sex ratios and gonad development of zebrafish
葛婷婷
Thesis Advisor黄德军
2017-04-20
Degree Grantor兰州大学
Place of Conferral兰州
Degree Name硕士
Keyword2,4-二氯酚 性比 sox9a amh DNA甲基化 dnmts 初级卵泡 卵巢 精巢
Abstract

2,4-二氯酚(2,4-dichlorophenol, 2,4-DCP)被广泛用于杀虫剂、除草剂、杀螺剂、杀螨剂、杀菌剂及木材防腐剂,可通过农业或工业途径被排入水体,是水环境中普遍存在的环境污染物。由于具有类似雌激素的结构,2,4-DCP能干扰生物体的生命活动,属于内分泌干扰物。越来越多的证据表明内分泌干扰物能影响鱼类种群的性别比例,进而影响并改变其在环境中的适应度。本研究探索了2,4-DCP暴露对斑马鱼种群性比的改变情况,并从基因表达及DNA甲基化方面初步研究了其可能性机制,为污染物对鱼类性别比例的影响提供新资料。

实验结果如下:1. 在斑马鱼发育的20-50 dpf(days post fertilization,受精后的天数)用2,4-DCP暴露会造成斑马鱼种群雌性比例显著升高。进一步研究发现在发育的20-30 dpf与30-40 dpf对斑马鱼进行2,4-DCP暴露均会引起斑马鱼种群性比显著偏向雌性(P<0.01),而40-50 dpf暴露则对性别没有显著改变(P>0.05)。说明2,4-DCP影响斑马鱼种群性比改变的关键时期为受精后的20-30及30-40天。2. 发育期间2,4-DCP暴露后斑马鱼性别相关基因(cyp19a1a、foxl2、esr1、amh、sox9a、dmrt1及ar)的表达水平也发生了改变。在不同的发育时间段,2,4-DCP对性别相关基因表达的影响不尽相同。但无论暴露的时间是在20-50 dpf,还是20-30 dpf,或者是30-40 dpf,2,4-DCP都可导致雄性相关基因sox9a的表达降低(P<0.05),同时也导致了一部分雌性相关基因(cyp19a1a、foxl2)的表达下降(P<0.05)。3. 2,4-DCP暴露能改变DNA甲基转移酶相关基因(dnmts)的表达。20 μg/L的2,4-DCP暴露30天后,dnmt1的表达显著升高(P<0.01),而dnmt3a1、dnmt3b1、dnmt3b2、dnmt3b3及dnmt3b4的表达量都显著下降(P<0.01)。但随着浓度的升高,dnmts的表达通常出现先降低后升高的趋势。4. 2,4-DCP暴露后雄性相关基因sox9a的甲基化水平显著增加(P<0.01)。5. 5-氮杂胞苷(5-AZA),一种甲基转移酶的抑制剂,在20-50 dpf处理斑马鱼后,5-AZA单独处理使得斑马鱼种群的性比朝向雄性发展,而5-AZA与2,4-DCP联合处理则能减缓由2,4-DCP导致的雌性化。说明DNA甲基化在斑马鱼的性别决定过程中起着作用。且用5-AZA处理后,斑马鱼雄性相关基因sox9a与amh的表达水平上升了。说明DNA甲基化调控了sox9a与amh的表达。当用甲基特异性PCR检测5-AZA与2,4-DCP联合处理后sox9a的甲基化水平,结果显示联合处理比单独用2,4-DCP暴露sox9a的甲基化水平低,说明DNA甲基化参与了对基因sox9a的表达调控。6. 通过免疫组化检测sox9a的表达发现sox9a在斑马鱼中呈现全身性表达,在精巢中呈现高表达,且主要在支持细胞中表达。7. 通过石蜡切片检测了2,4-DCP暴露对斑马鱼性腺发育的影响,发现在所有浓度下2,4-DCP暴露后初级卵泡的直径都显著增加了(P<0.01),而精母细胞与精子细胞的数量减少。这说明2,4-DCP可能促进了性原细胞发育为卵母细胞,而抑制性原细胞发生凋亡转变为初级精母细胞,即2,4-DCP能促进斑马鱼“类卵巢”发育为卵巢,而抑制其发育为精巢。

Other Abstract

2,4-dichlorophenol (2,4-DCP) whicih is widely used as pesticide, herbicide, molluscicide, bactericide and wood treatment agents, and is the ubiquitous contaminants in the aquatic environment mainly released from agricultural and industrial wastewater. Owning the similar molecular structure with estrogens, 2,4-DCP could distrupt the life process of the organisms and belongs to one of the endocrine disrupters. More and more studies demonstrate that endocrine disrupters could have influence on the sex ratios of fish and affect the reproductive system. This study explored the impact of 2,4-DCP exposure during sex differentiation time on the sex ratio and gonad structure of zebrafish and provides data for the influence of environmental pollutants on sex differentiation of fish.

The results of this study are listed as follow:1. 2,4-DCP exposure during sex differentiation time of 20-50 dpf (days post fertilization) could alter sex ratios of zebrafish and resulted in female biased sex ratios. Further study demonstrated that the critical time that 2,4-DCP impacts on sex ratios is 20-40 dpf, in which time 2,4-DCP exposure would resulted in female biased ratios (P<0.01). However, 40-50 dpf exposure of zebrafish to 2,4-DCP would not induced female biased ratios (P>0.05).2. 2,4-DCP treatment during zebrafish development could alter the expression of sex related genes. 2,4-DCP treatment during 20-50 dpf, 20-30 dpf and 30-40 dpf could dowmregulate the expression of sox9a, which is a male related gene, and resulted in the decrease of female related genes (cyp19a1a, foxl2).3. 2,4-DCP exposure could alter the expression of dnmts. When exposure of zebrafish to 20 μg/L 2,4-DCP during 20-50 dpf, dnmt1 expression was upregulated, while the expression of dnmt3a1, dnmt3b1, dnmt3b2, dnmt3b3,and dnmt3b4 was downregulated. With the increasing concentration of 2,4-DCP, the expression of dnmts exhibited a trend of first decrease and then increase.4. 2,4-DCP could increase the methylation level of sox9a (P<0.01).5. 5- azacytidine (5-AZA), an inhibitor of DNMTs, was co-treated with 2,4-DCP on zebrafish to explore the impact of DNA methylation on sex differentiation of zebrafish. Combined treatment of 2,4-DCP with 5-AZA could alleviate the female biased ratios resulted from 2,4-DCP exposure, which indicated that DNA methylation may play an importment role in sex differentiation of zebrafish. And after treatment with 5-AZA, the expression levels of sox9a and amh was increased, which indicates that DNA methylation was involved in the expression of male related genes. Meanwhile, after the combined treatment with 5-AZA, sox9a methylation level was decreased, which demonstrates the DNMA methylation was involved in sox9a expression.6. Using immunohistochemical staining assay, we found that sox9a is expressed broadly in zebrafish and had higher expression in testis.7. 2,4-DCP induced the increased diameter of follicles, however, the number of spermatocyte was decreased after 2,4-DCP exposure. This results indicate that 2,4-DCP could promote the formation of primary follicles and suppress the formation of spermatocyte. It maybe that 2,4-DCP could promote the development of “ juvenile ovary” of zebrafish to ovaries other than testis.

URL查看原文
Language中文
Document Type学位论文
Identifierhttps://ir.lzu.edu.cn/handle/262010/222095
Collection生命科学学院
Recommended Citation
GB/T 7714
葛婷婷. 2,4-二氯酚暴露对斑马鱼性比及性腺发育的影响[D]. 兰州. 兰州大学,2017.
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