【作者】 卞建春；

【导师】 康世良； 王宗元；

【作者基本信息】 东北农业大学 ， 临床兽医学， 2003， 博士

【摘要】 镉是一种公认的对动物健康有害的金属元素。研究证明，镉中毒可引起家畜及人发生呼吸机能不全、运动失调、贫血、心血管疾病、实质器官损伤、骨质疏松、免疫力低下及癌症等多种疾病甚至急性中毒死亡。工业和日常生活活动造成镉对自然界的污染有日渐严重的趋势。在我国，某些污染区产的饲料中镉浓度达1.13mg／kg～5.12mg／kg，这已严重超过了饲料中镉的允许浓度。由于镉有不可降解性和极长半衰期的特性，镉污染对动物健康已经构成了巨大的潜在性威胁。近几年来，镉污染对雄性动物包括水生动物及人类生殖功能的潜在危害作用越来越为科学家所重视。研究发现，镉中毒可以引起家畜尤其是雄性家畜繁殖功能下降，人类男性“勃起功能障碍”(ED；Erectile Dysfunction)也可能与镉暴露有关。为深入揭示镉的雄性生殖毒性作用及其机理，本课题在本研究领域首次系统地研究了镉暴露特别是慢性消化道(饮水)染镉条件下，镉对雄性动物繁殖机能的影响、镉的器官分布、镉对睾丸和副性腺的毒性、镉对生殖激素的影响、镉对睾丸间质细胞(Leydig cell)功能的影响等镉的雄性生殖毒性作用；并从生殖激素变化、生化指标改变、氧化与抗氧化系统变化、DNA单链断裂程度、诱导细胞凋亡、镉在睾丸中的存在状态(与MT的关系)等多方面探索了镉致雄性生殖机能障碍的机理。主要研究方法包括动物繁殖试验、显微和超微结构观察、放射免疫法和细胞化学法激素分析、特异或标志酶生化测定、石墨炉原子吸收分光光度法(GFAAS)元素分析、单细胞凝胶电泳法(SCGE)DNA单链断裂检验、TdT介导的原位末端标记法(TUNEL)细胞凋亡鉴定和凝胶层析法蛋白分离、氨基酸组成分析蛋白鉴定等。本实验研究表明：慢性饮水染镉对动物精子的发生具有毒性作用，可明显影响雄性动物的生育功能，且存在剂量效应和时间效应关系。表现为：随着染镉剂量的增加，大鼠附睾尾的重量、附睾尾中精子的活力、精子的密度和活精子的比例均呈下降趋势，附睾尾中精子的畸形率呈升高趋势，并且性成熟前开始染镉大鼠精子发生受到的毒性影响要大于性成熟后开始染镉大鼠；受配母鼠的受胎率、平均产活仔数随染镉剂量的增加和染镉时间的延长呈下降趋势；但雄性大鼠慢性饮水镉暴露对后代的生长发育影响不明显。雄性动物饮水慢性镉暴露对后代的性别分布没有显著性影响。认为在慢性饮水染镉条件下，镉致雄性动物精子发生障碍和精子形态改变的主要原因并非是镉对精子细胞的直接毒性作用；镉的控性(对后代性别比例的影响)并非是通过直接影响雄性动物的生殖系统功能或精子实现的，镉的控性可能发生在雌性动物体内的受精或合子发育过程而非发生在雄性动物精子发生过程中。慢性饮水镉暴露下，镉对雄性大鼠性腺和副性腺具有毒性作用，可以造成性腺和副性腺组织结构发生损伤变化，并且损伤程度随染镉剂量的加大而加重。比较而言，雄性动物生殖器官对镉毒性作用的敏感性要高于肝、肾等主要脏器，睾丸要高于附睾、精囊和前列腺，性未成熟开始染镉受到的影响要比性已成熟开始染镉大。认为慢性镉中毒的睾丸损伤机制不能完全用血管内皮细胞损伤机制来解释。镉对附睾、精囊腺和前列腺的损伤作用既有镉的直接破坏作用也有因睾酮合成减少而致的继发性改变。慢性镉中毒可以抑制大鼠睾东北农业大学农华博士学位论文丸组织中LDH、LDH一x、ALP、G石一PD和SDH等酶的活性，引起相应生理功能的变化并表现为生精功能的障碍。 饮水慢性染福情况下，辜丸的氧化损伤是福雄性生殖毒性的一个重要机制。从辜丸中GsH一Px、SOD和MDA等指标的变化情况可以看出，性未成熟开始染福受到的影响要比性成熟开始染福大，而且均存在着剂量一效应关系。 慢性饮水染锅大鼠血浆和攀丸组织中辜酮(T)的水平极显著下降，且剂量效应明显;相反，与对照组比较，染锚大鼠血浆中卵泡刺激素(FSH)和黄体生成素(LH)的水平均极显著地升高，也存在明显剂量效应。说明慢性饮水染福条件下，福对雄性动物辜丸翠酮的合成和分泌具有抑制作用。至于福暴露大鼠血液中FSH和LH的升高，可能是H川，反馈调节的结果。体外试验进一步发现:福可使原代培养辜丸Leydig细胞的活率明显降低，并对Leydig细胞的辜酮分泌功能也具有直接的抑制作用.说明福对辜丸Leydig细胞具有直接的损伤作用，锅对体内辜酮水平的影响是福直接作用于辜丸Leydig细胞的结果。 单细胞凝胶电泳(sCGE)试验证实，随着培养液中锅浓度的升高，Leydig细胞洲A的彗星率和DNA的迁移度呈明显上升趋势，说明福对Leydig细胞的DNA有较强的损伤作用。这不仅是铺损害辜丸细胞的分子毒理学证据，因为Leydig细胞DNA的完整性是其功能正常发挥的前提，Leydig细胞DNA一旦受损，其分泌翠酮的能力势必下降，最后使精子的发生受到影响，并进一步导致雄傲素依救器官的损害，而且，锅对Leydig细胞DNA的损伤也是福诱导Leydig细胞凋亡的一个重要证据。锡引起DNA损伤的机理尚待进一步研究，而锅诱导体内氧自由基和脂质过氧化反应产生的自由基的作?更多还原

【Abstract】 Cadmium is known as a harmful metal to animal health. Researches demonstrated that cadmium poison could lead torespiratory hypofunction, ataxia, anaemia, cardiovascular diseases, organ damage, osteoporosis, hypoimmunity, cancer and death, etc. Nowadays, the pollution of cadmium to nature is widely spread. In some polluted region in China, the cadmium content of feed was up to 1.13mg/kg-5.12mg/kg, severely more than the permitted content of feed. Additionally, due to the undegradability and long half-times, cadmium will throw great threat on animal health. Recently, scientists paid more attention to the latent effects of cadmium on male fertilities, including aquatic animal and human being. Studies indicated that cadmium poison could lead to lower fertilities of male livestocks, and it might be related to the ED(Erectile Dysfunction) of men.To deeply discover the effects and mechanism of cadmium poison on reproductive system of male animals, the effects of cadmium on male fertilities, cadmium distribution in the organs, the cadmium toxicity on testicle and accessory sex gland, the effects of cadmium on sex hormone, the effects of cadmium on Leydig cell were first systemically studied in this paper in China, under the exposure of cadmium, especially the cadmium exposure of chronic digestive tract. And more, the mechanism of cadmium on male fertility dysfunction was studied from the following aspects: the changes of sex hormone, the changes of biochemical indice, the changes of oxidative and antioxidative system, the breakdown degree of DNA single chain, the profiles of induced apoptosis, the existing status of cadmium in testicle (relationship with MT), etc. The main research methods: animal reproduction tests, the observation of microstructure and ultra-microstructure, the changes of sex hormone by radioimmunosorbent and histochemical methods, the determination of special enzymes by biochemical method, the determination of cadmium levels by atomic absorption spectrophotometry, the analysis of the breakdown degree of DNA single chain by single cell gel electrophoresis, the analysis of apoptosis by morphology and TUNEL methods, the protein isolation by gel chromatography, and the protein identification by amino acid analysis. The results showed that:chronic cadmium exposure via drinking could significantly affect fertilibility of male animals due to the toxicity to animal sperm, and the toxicity depended on the doses and time: with the increased doses of cadmium, the weight of epididymis tail, the motility of sperm, the sperm density and living sperm ratio decreased, whereas the abnormal sperm in epididymis tail of rat increased, and the effects of cadmium toxicity on the the Spermatogenesis of the rat which was exposed by cadmium beginning from sex immature were more than from sex mature . the pregnant rate and average live new-born offspring of female rat inseminated by the cadmium-treated male rats were decrease, with the increase of cadmium doses and time.however, no significant effects on the growth of offspring were observed when male rats were exposed chronically to cadmium via drinking. The research demonstrated that there were no significant effects on the offspring sex distribution when male animals were exposed chronically to cadmium via drinking. Under the condition of chronic cadmium exposure via drinking, the main reasons of the effects on the change of sperm morphology and the sperm formation dysfunction were not the direct toxic effects of sperm cells.The effects of sex control of cadmium were not achieved by directly affect the reproductive system of male animals or sperms, but at the process the fertilization in female animals.Under the chronic exposure to cadmium via drinking water, cadmium was poisonous to rat sex and accessory sex gland, and could lead to structural changes of them, and the change was dose-dependent. Comparatively, male reproductive organs were more sensitive to the cadmium than livers and kinneys; testicles were more sensitive than epididymis, glandula ves更多还原