【作者】 房燕；

【导师】 汝少国；

【作者基本信息】 中国海洋大学 ， 海洋生物学， 2007， 硕士

【摘要】 久效磷能够诱导雄性金鱼卵黄原蛋白的产生,具有环境雌激素活性,但关于久效磷生殖毒性的研究报道还很少。本文以金鱼精巢超显微结构、精巢特征性酶活性、生殖腺指数（GSI）、精子存活率、运动率、运动时间、精子彗星尾部DNA含量、尾长、尾矩为指标,研究了久效磷对雄性金鱼生殖系统、精子运动力及精子DNA的损伤情况,探讨了久效磷对雄性金鱼的生殖毒性效应及其致毒机理。以期将该研究结果应用到人类,为环境激素对人类生殖健康的研究提供参考资料。0.01、0.10、1.00 mg·L^-1的久效磷暴露金鱼21d,随暴露浓度的升高GSI逐渐降低,1.00 mg·L^-1的久效磷可以造成GSI的显著下降,表明久效磷对金鱼精巢发育有一定程度延迟作用。久效磷的暴露导致精小叶基膜断裂、Leydig氏细胞水肿、精原细胞膨胀、间质扩大,这可能会引起精子质量下降。久效磷暴露21 d,支持细胞细胞膜和核膜溶解,脂滴和髓磷脂象数量增加,吞噬的精子数量增多;金鱼精子头部变形、线粒体和质膜溶解以及鞭毛断裂。且随着暴露浓度的升高,损伤越严重。对照组、0.01、0.10、1.00 mg·L^-1暴露组精巢乳酸脱氢酶（LDH）的活性分别为: 1879.58±141.69、1614.06±233.97、1409.20±86.93、1111.81±199.13 U·mg^-1pro。γ-谷氨酰转移酶（γ-GTP）活性分别为:6.03±1.09、4.33±0.87、3.47±0.44和3.08±0.56U·mg-1pro。可见久效磷对LDH、γ-GTP均有抑制作用。不同浓度久效磷暴露条件下,金鱼精巢山梨醇脱氢酶（SDH）、酸性磷酸酶（ACP）和碱性磷酸酶（ALP）活性下降,氧化物歧化酶（SOD）活性升高,结果表明久效磷可能通过损伤精巢膜系统结构,干扰精巢能量供应和代谢等生理过程,影响生精细胞的成熟,造成生殖毒性。雄性生殖系统最主要的功能是产生精子,精子质量是世代健康繁衍的重要保障。0.01 mg·L^-1,0.10 mg·L^-1和1.00 mg·L^-1的久效磷体外暴露金鱼精子3h,对精子存活率没有影响。0.10和1.00mg·L^-1久效磷暴露金鱼精子1h即可引起精子运动时间和运动率的下降。对照组精子运动时间为92±3s,运动率为92.73±2.48%;0.10 mg·L^-1暴露组,精子运动时间为70±3s,运动率为37.74±4.04%;1.00mg·L^-1暴露组,精子运动时间为63±5s,运动率为29.17±2.06%。久效磷体外暴露金鱼精子3h,对照组、0.01、0.10和1.00 mg·L^-1暴露组彗星尾部DNA含量分别为:1.17±0.81%、4.55±1.24%、14.60±2.17%、17.50±1.98%;彗星尾长分别为:3.94±1.29μm 6.80±2.78μm、9.21±1.51μm和12.89±3.09μm;彗星尾矩分别为:0.39±0.27μm、0.70±0.38、1.90±0.45、4.47±0.33μm。久效磷的暴露导致精子彗星尾长、尾部DNA含量和尾矩的增加,表明久效磷可以损伤金鱼精子的遗传物质。本文在组织水平、生化水平和DNA水平上,通过对精巢超显微结构的观察、特征性酶活性的检测和精子活力、DNA损伤的分析,首次提出了久效磷对雄性金鱼的生殖毒性是通过损伤精巢支持细胞、Leydig氏细胞及精子线粒体结构,影响了多种特征性酶活性,最终导致生精障碍的;久效磷还能降低精子运动力,损伤精子DNA,可能造成对受精过程、胚胎发育和子代健康的危害。更多还原

【Abstract】 Monocrotophos is one of the environment hormones, because it can induce the production of vitellogenin in male goldfish. However, very few reports about the reproduction toxicity of monocrotophos exist in the literature. So the gonadosomatic index（GSI）of male goldfish （Carassius auratus） under monocrotophos treatment was tested in this paper. The activities of several characteristic enzymes in testis were then measured; the micro- and ultrastructure of testis were observed as well. The toxicity effect of monocrotophos to DNA and motility of sperm was evaluated via the investigation of the viability, the percentage and duration of motility of sperms by microscope, and the percentage of DNA in comet tail（Tail DNA%）, the tail length（TL） and tail moment（TM） by comet assay of sperm comets. All the works aim at exploring the effect and mechanism of reproduction toxicity of monocrotophos on male goldfish. The data might give useful information to toxicological research of environment hormones on reproduction system of human.Exposure to monocrotophos led to the change of GSI and microstructure of male goldfish. After exposed to monocrotophos of various concentration(0.01、0.10、1.00 mg·L^-1) for 21 days, the GSI of adult male goldfish decreased gradually with the increase of concentration of monocrotophos. The GSI of male goldfish in exposure concentration of 1.00 mg·L^-1 decreased significantly compared to the control, indicating that the monocrotophos delayed the development of testis. As a result of monocrotophos exposure, dissolution of ground membrane, swelling of Leydig’s and spermatogenous cell and enlargement of interstitial tissue were clearly observed. The changes in microstructure of testis may lead to the decline of sperm quantity.The alteration of ultrastructure and activities of several characteristic enzymes in testis of male goldfish treated by monocrotophos were further studied. In Sertoli’s cell, monocrotophos induced the dissolution of nuclear membrane, accumulation of lipid droplet, increase of myelin-like figure and necrosis of sperm. To the sperms, the deformation of head, the dissolution of mitochondrial and plasma membrane and the fragment of tail were observed. The damage became severer with the increase of exposure concentration. In the control and treated goldfish which were exposed to 0.01, 0.10, 1.00 mg·L^-1 monocrotophos, the lactate dehydrogenase （LDH） activities were1879.58±141.69, 1614.06±233.97, 1409.20±86.93, 1111.81±199.13U·mg-1pro separately, and the activities ofγ-glutamyl transpeptidase （γ-GTP） were 6.03±1.09, 4.33±0.87, 3.47±0.44, 3.08±0.56 U·mg-1pro respectively. So the activities of LDH andγ-GTP were inhibited by monocrotophos. The activities of acid phosphatase, alkaline phosphatase, sorbitol dehydrogenase declined, and the activity of superoxide dismutase increased as a result of monocrotophos exposure. It is supposed that the changes of ultrastructure and enzyme activities in testis of goldfish treated by monoccrotophos will interrupt the physiological courses such as energy metabolism, and further disturb the seminiferous process.The main function of male reproduction system is to produce sperms whose quantity is essential to healthy reproduction for generations. The exposure of sperms to monocrotophos(0.01 mg·L^-1, 0.10 mg·L^-1, 1.00 mg·L^-1) in vitro for 3h didn’t have any effect on viability of sperms. After exposed to monocrotophos at concentration of 0.10 and 1.00 mg·L^-1 for 1h, the percentage of motility of sperms was 37.74±4.04% and 29.17±2.06%; the duration of motility was 70±3s and 63±5s, while that of control was 92.73±2.48% and 92±3s, respectively. The result showed that the monocrotophos reduced the motility of sperms. In the sperms of control and treated goldfish which were exposed to 0.01, 0.10, 1.00 mg·L^-1 monocrotophos for 3h, the Tail DNA% was 1.17±0.81%、4.55±1.24%、14.60±2.17%、17.50±1.98% respectively; the TL of each group was 0.39±0.27, 0.70±0.38, 1.90±0.45, 4.47±0.33μm, and the TM was 0.39±0.27, 0.70±0.38、1.90±0.45、4.47±0.33μm respectively. The increase of DNA% in comet tail, tail length and TM implied that the DNA of sperm was damaged by monocrotophos.The micro- and ultrastructure of testis were observed at the histological level, the activities of characteristic enzymes in testis were measured at the biochemical level and the damage of DNA of sperms was tested at the DNA level. Finally, it is concluded that monocrotophos inhibits the seminiferous process via the damage to Leydig’s cell, Sertoli’s cell and mitochondrial of sperm and the changes in activities of characteristic enzymes. The monocrotophos can damage the motility and DNA of sperm as well, resulting in the harm to fertilization, development of embryo and the health of offspring.更多还原