【作者】 邴欣；

【导师】 汝少国； 姜明；

【作者基本信息】 中国海洋大学 ， 生态学， 2003， 硕士

【摘要】 1996年以来，环境雌激素对野生生物和人类的影响受到了各国政府和科学界的广泛重视。欧盟、美国和日本政府都已启动了环境雌激素筛选体系的国家级课题，着手建立环境雌激素的离体和活体筛选方法。但目前为止，主要集中于环境中持久性有机污染物如有机氯农药和烷基酚类化合物的筛选上，而我国是农业大国，大量地使用了有机磷农药，其残留毒性及环境雌激素效应的研究还尚未见报道。因此，本文选择国内广泛使用的有机磷农药——久效磷，以金鱼为模型生物，以雄性金鱼卵黄原蛋白产生、精巢支持细胞γ-GTP酶活性，并辅以生殖轴线器官——垂体和精巢超微结构的变化，在国内首先建立了有机磷农药环境雌激素效应的筛选体系，并首次确认久效磷具有环境雌激素活性。 本文采用腹腔注射17β—雌二醇诱导雄性金鱼卵黄原蛋白的产生，注射浓度为0.05mg g^-1BW；利用卵黄原蛋白富含磷酸、脂和糖基团的特点，进行血浆样品的常规聚丙烯酰氨凝胶电泳，通过对卵黄原蛋白特性基团磷、脂和糖蛋白的染色，确定了卵黄原蛋白在电泳图谱上的位置，开发了常规聚丙烯酰胺凝胶电泳法定性卵黄原蛋白的简便方法。电泳结果表明，经过2周的17β—雌二醇暴露，诱导雄性金鱼产生了卵黄原蛋白，并通过ELISA检测卵黄原蛋白的平均含量为690.2ng ml^-1，和对照组雄性金鱼平均含量为10.7ng ml^-1的差异极显著（p＜0.01），比雌性对照组检出量285.5ng ml^-1高1倍多，并且17β—雌二醇可以诱导雄性金鱼血浆钙离子和血浆总蛋白含量增加。 0.01，0.1和0.5mg L^-1久效磷暴露雄性金鱼21天，各暴露组均诱导了雄性金鱼卵黄原蛋白的产生；并利用酶链免疫吸附法定量了卵黄原蛋白的平均含量分别为244.0、833.1和479.5ng ml^-1，卵黄原蛋白的含量随着浓度的增加呈倒“U”型曲线。并且3个浓度的久效磷均可以诱导雄性金鱼血浆总蛋白含量的增加。 久效磷对金鱼精巢组织结构的破坏主要表现为：精小叶基膜断裂；Leydig氏细胞出现肥大现象；精原细胞膨胀。随着久效磷暴露浓度的增加对支持细胞γ—谷氨酰转移酶活性的抑制作用明显加强。精小叶组织结构的损伤和γ—谷氨酰转移酶活性的降低，表明久效磷具有一定的生殖毒性即环境雌激素效应。 久效磷对中腺垂体促性腺激素（GTH）、生长激素（STH）和促甲状腺激素（TSH）三种分泌细胞、生精细胞和精子的影响主要表现为对膜系统的损伤。三种中腺垂体分泌细胞都出现核膜水肿、内质网水肿，甚至出现局部溶解现象。久效磷对精原细胞的损伤最为严重，细胞膜和核膜水肿，高浓度暴露组的细胞膜和核膜溶解，线粒体的崎也有溶解现象;但久效磷对精母细胞和精子细胞的损伤并不明显，主要表现为膜系统的轻微水肿。久效磷可以使金鱼精子细胞质膜溶解，出现间断性断裂;有的精子头部变形，顶部出现凹陷;少量精子颈部中心粒复合体溶解;精子颈部线粒体有溶解现象，精子尾部的膜结构断裂。支持细胞核染色质凝聚程度增加并出现不规则低电子密度区;核膜溶解，细胞间连接进一步拉开。更多还原

【Abstract】 Since 1996, the effects of environmental estrogens on wild lives and human beings have attracted more and more attention of scientists in the world. Recently, several research plans of environmental estrogens were started in European Union, United States and Japan, and some in vitro and in vivo screening technology have been built up by now. But the previous research works mainly focused on the screening of environment persistent pollutants, such as organochlorine insecticides and alkylphenol, to our knowledge, no report on the residual toxicity and environmental estrogens effects has been existed in the literature yet. Since China is a big agricultural country and usually applies a large amount of organophosphorous pesticides every year, it is very necessary to do some relative research on the estrogen effects of organophosphorous. In this paper, we selected a widely used organophosphorous pesticide --monocrotophos, used goldfish as model animal, and chose the vitellogenin induction of male fish as the main indicator, -GTP of sertoli cell in testes, the changes of microstructures of testis and ultrastructures of pituitary and testis as assistant indicators, we finally built up a screening system to effectively detect the environmental estrogen effect of organophosphorous pesticides, and reported that the monocrotophos had the activity of environmental estrogen for the first time in the world.The vitellogenin in male goldfish was induced by 17/?-estradiol dissolved in ethanol (intraperitoneal injection, 0.05mg g-1BW). After two weeks induction, the blood was collected from caudal vessel, then centrifuged at 8,000 g for 5 min. After serum native-PAGE, the gel was stained with the method of CBB G250-, phospho-, lipo- and glyco-staining respectively. Then we identified the location of vitellogenin in the gel. This method provided a practical and effective way of vitellogenin identification. Theresults of native-PAGE showed that the male fish could synthesis vitellogenin after the injection of 0.05mg g"’BW of 17 -estradiol for two weeks. The average content of vitellogenin in Ej exposed group was 690.2ng ml-1 by ELISA, it was significantly different from male control group (p<0.01), which average content was 10.7 ng ml-1, and it was also much higher than the female control group, which average content was 285.5 ng ml-1. Compared with control male goldfish, both the Ca2+ content and total protein content in serum of the E2 exposed male goldfish showed significant increase.After exposing to 0.01, 0.1 and 0.5mg L-1 monocrotophos for 21 days, the inductions of vitellogenin in male goldfish were detected in all exposed group. By the method of ELISA, the vitellogenin contents were determined as 244.0, 833.1 and 479.5ng ml-1 respectively. The dose-response curve showed a reversed U-shape as long as the increasing concentration of monocrotophos, and the total protein contents in serum exhibited marked increase in all the three exposed groups.The damages of monocrotophos on tnicrostructure of testis were that it caused the deficiency of ground membrane, hypertrophied Leydig cell and spermatogenous. Besides, the Y -GTP activity was inhibited by monocrotophos. The results suggested that monocrotophos had not only environmental estrogen effects, but also reproductive toxicity.Monocrotophos could destroy the membrane systems of three kinds of pituitary secretary cell, spermatogenous cell and sperm. In three kinds of pituitary secretary cell, nuclear membrane and endoplasmic reticulum showed dilation, and endoplasmic reticulum even exhibited fusion under exposure in high concentration. In spermatogenous cell, we could find dilated cytolemma, dilated nuclear membrane and fused crista of mitochondria. Most of the sperm’s cellular membrane dissolved and broke down into pieces. The central granule and mitochondrion showed little dissolving. The chromatin of Sertoli cell condensed tightly and formed some low electron density areas, nuclear membrane dissolved, the intercellular junction dilated.更多还原