【作者】 徐绘；

【导师】 宋后燕； 汤其群； 马端； 于敏；

【作者基本信息】 复旦大学 ， 生物化学与分子生物学， 2008， 博士

【摘要】 近年来,环境内分泌干扰物（Endocrine disrupting chemicals,EDCs）对人类和野生动物健康的不良影响已引起学术界和政府的广泛关注。EDCs是能干扰内分泌系统从而影响生物生殖的一大类化合物。17α-炔雌醇（17α-ethynylestradiol,EE₂）是一种常见的EDCs,它是合成避孕药的一种常用成分,通过污水处理系统进入水环境。在污水处理系统排出的水中,EE₂的浓度可达7.2—42 ng/1。约5.7%的美国河流中EE₂含量超过5 ng/l。由于EE₂的半衰期长及生物富集作用,鱼体内EE₂的含量可比自然环境中高332倍,而且EE₂的雌激素作用在生物体内为雌二醇（E₂）和雌激素酮（E₁）的10—50倍。鱼类暴露于EE₂污染的水环境中可引起卵黄生成素（Vitellogenin,Vtg）表达,性分化异常及生殖功能受损。鉴于EE₂的强效作用以及在环境中的广泛分布,EE₂可导致鱼类不育甚至影响鱼类的种群繁殖。为了解EE₂暴露对斑马鱼生长发育以及生殖系统的影响,我们将斑马鱼分成溶剂对照、0.4 ng/L、2 ng/L和10 ng/L EE₂处理组,从出生后2天（2 days post hatch,2 dph）暴露至90 dph,之后将所有斑马鱼在不含EE₂的清水中恢复至180 dph。90 dph时,2 ng/L和10 ng/L EE₂处理组斑马鱼的死亡率及心包水肿率均显著高于对照组,10 ng/L EE₂组的平均体重和体长亦显著低于对照组。这些结果显示高于10 ng/L的EE₂暴露会有较强的非特异毒性作用。我们也利用逆转录聚合酶链反应（Reverse Transcription Polymerase Chain Reaction,RT-PCR）检测了暴露于EE₂的斑马鱼出生后第2天至出生后28天Vtg基因的mRNA表达水平。结果显示10 ng/l EE₂暴露21天后（21 day post hactch,21 dph）便可诱导Vtg表达。用我们实验室构建的对雌激素敏感的Vtg-EGFP转基因斑马鱼做相同的暴露处理进一步验证了RT-PCR的结果,表明Vtg可作为检测环境雌激素（Environmentalestrogens,EEs）污染水环境的早期敏感的生物标志物。在EE₂暴露影响斑马鱼生殖功能的机理研究中,EE₂干扰斑马鱼性腺发育分化从而损害生殖功能是一个重要的原因。90 dph时的性腺组织切片显示EE₂暴露严重抑制处理组斑马鱼的正常性腺发育和分化,这些鱼的性腺处于幼稚卵巢甚至完全未分化阶段。将这批斑马鱼在清水中恢复试验3个月后,再次行性腺组织切片检查。结果提示各组斑马鱼已恢复正常性别比例,然而EE₂处理组雄性斑马鱼的精巢明显异常,表现为生精小管严重畸形和精子数量减少。雌鱼卵巢组织切片未发现明显异常。180 dph时的交配产卵实验显示EE₂处理组斑马鱼的产卵数量及12 hpf卵存活率均较对照组显著降低。为了研究EE₂对雌雄斑马鱼分别的生殖毒理作用,我们又采用了雌/雄置换交配的方法,分别研究了EE₂处理组雌雄两性的生殖功能。结果显示EE₂处理组雌雄鱼生殖功能均严重受损,表现为雌鱼产卵数量下降,卵存活率降低;进一步通过精于计数及精液分析,证实处理组卵存活率下降的原因是雄鱼的精液量下降所致。一些EDCs如硫丹（endosulfan）和壬基酚（nonylphenol,NP）可干扰斑马鱼胚胎原始生殖细胞（primordial germ cells,PGCs）的迁移和分布从而可能对斑马鱼的生殖产生不利影响。目前尚没有关于EEs对斑马鱼PGC作用的报道。我们通过用绿色荧光蛋白标记PGC以及整体原位杂交,研究了EE₂暴露对斑马鱼胚胎PGC的迁移和分布的影响。结果显示,10 ng/L和100 ng/L EE₂处理组斑马鱼PGC的迁移和分布与对照组无显著差异。分析其原因可能和早期性腺组织对EE₂不敏感有关。我们仍不能排除其他的EEs对PGC可能的不利影响。更多还原

【Abstract】 In recent years, the effects of environmental endocrine disrupting chemicals （EDCs） on the health of humans and wildlife have been a growing concern among researchers and policy makers. EDCs have the capacity to modulate the endocrine system and could possibly interfere with the reproductive system of wild life and even humans. The synthetic estrogen EE₂ is a common component of oral contraceptives. It enters the aquatic environment through domestic STWs. In two independent studies, concentrations of EE₂ in municipal STWs were reported to range from non-detectable to 7 ng/Lnd non-detectable to 42 ng/L A recent study conducted by the U.S. Geological Survey reported that 5.7% of rivers in the United States had EE₂ concentrations > 5 ng/L. Due to the long half-life of EE₂ and its bioconcentration in biota, the concentration of EE₂ in a fish body can be 332-fold higher than that in the environment and the potency of EE₂ can be 10- to 50-fold higher than that of E₂ and E₁ in vivo. Numerous studies have shown that fish exposed to EE₂ experienced vitellogenin （Vtg） induction, disrupted sex differentiation, and reproductive failure. Thus, EE₂ could potentially contribute to reproductive dysfunction in wild fish populations, given its high potency and wide distribution.To investigate the impact of EE₂ exposure on development and reproduction, zebrafish were exposed to 0.4, 2,10 ng/L EE₂ or solvent control from 2 dph to 90 dph. Fish were then transferred to clean water to recovery for 3 months. The mortality and pericardial edema rate in 2 ng/L and 10 ng/L EE₂-treated groups were significantly higher than that of the solvent control at 90 dph. The mean body weight and length of fish exposed to 10 ng/L EE₂ were also obviously lower than the control group. These results indicate there is a general toxic effect in exposure to concentration of EE₂ higher than 10 ng/L. We also investigated whether the Vtg mRNA expression could be induced in the first four weeks after hatch. RT-PCR indicates the Vtg mRNA could be induced earlier than 21 dph at 10 ng/l EE₂. The vtg-egfp transgenic zebrafish also shows a similar green fluorescent expression pattern at the same exposure period which is consistent with the RT-PCR results. Our findings suggest Vtg could be possibly used as a sensitive biomarker for estrogen even at early developmental stages.In the mechanism that EE₂ interferes with sex differentiation and development, the disrupted gonad development may be an important part. In this study, zebrafish were exposed to 0.4, 2 and 10 ng/L EE₂ or solvent control （ethanol） from 2 to 90 dph. Histological sections at 90 dph indicates a severe disruption of sex differentiation and development at 2 and 10 ng/L EE₂ in which the gonad of fish contained only immature ovary type tissue or even undifferentiated gonads. After a 3-month recovery period, growth and sex ratio were partially recovered. However, malformation of the sperm duct and reduced number of spermatozoa were still found in fish exposed to 2 ng/L and 10 ng/L EE₂. No obvious differences in gonad histological sections between EE₂ treated female fish and solvent control was found.Breeding studies at 180 dph revealed significant reproductive dysfunctions in all EE₂-treated groups. Female zebrafish produced fewer eggs and the egg viability at 12 hpf were lower than that of the control. To further investigate the respective effects of EE₂ on both sexes, we used a male/female replacement experiment. In the male replacement trial, female fecundity of EE₂-treated fish did not improve, but their egg viability had recovered. In the female replacement experiment, egg viability in EE₂-treated groups did not improve. However, female fecundity had partially recovered. Interestingly, a dose dependent decrease of fecundity was still found in these fish. Considering the results of sperm examination, these findings clearly indicate that EE₂ exposure impairs the reproductive functions of both male and female zebrafish.Some previous studies reported zebrafish exposed to endosulfan and nonylphenol （NP） resulted in disrupted primordial germ cells （PGCs） migration and distribution during embryo stages. However, no study has been carried out regarding the effects of EEs exposure on PGCs migration and distribution of zebrafish. In this study, we examined the effects of 10 ng/L and 100 ng/L EE₂ exposure on zebrafish PGC migration and distribution by labeling PGCs with GFP and in situ hybridization. The results were negative. We suspect the reason should be gonads at early stages are not sensitive to environmental estrogens. However, we can not exclude the possibility of adverse effects of other EEs on PGC.更多还原