【作者】 鄢绯寰；

【导师】 陈奕欣；

【作者基本信息】 厦门大学 ， 动物学， 2008， 博士

【摘要】 本实验以青春期昆明雄鼠为研究对象,通过较低浓度的氯化三丁基锡(Tributyltin chloride,TBTC1)(0.5,5,and 50μg/kg)灌胃实验,染毒45 d后,观察TBT对生殖系统的毒性效应,探讨其对精子发生及精子成熟损伤的机理和途径。有关精子发生方面的检测指标包括睾丸的组织形态学,睾丸成熟情况、血清促性腺激素和睾丸性激素水平、睾丸细胞凋亡和细胞增殖情况,血清甲状腺激素(T3、T4)水平和睾丸甲状腺激素受体(TRs)表达量(mRNA水平);有关精子成熟方面的检测指标包括附睾组织形态,精液质量参数、附睾功能标志物活性(中性α-葡萄糖苷酶和附睾酸性磷酸酶)、精子功能指标以及附睾内特异性表达基因(MMP7和TD02)的表达量(mRNA水平)。1 TBT对睾丸精子发生的影响。TBT处理后雄性小鼠性体比指数没有显著变化,但睾丸组织结构发生明显改变,而且损伤程度随TBT剂量增加而加重,在50μg/kg剂量组,生精细胞排列杂乱、无序,各级生精细胞间出现大量的间隙,大量生精细胞异常脱落进入生精小管管腔。和对照组比较,处理组睾丸睾酮水平呈现出TBT剂量依赖性显著升高,雌激素水平呈现剂量依赖性下降趋势,而血清黄体生成素水平以及卵泡刺激素水平较对照组有所下降。运用免疫组织化学和western-blotting的方法对睾丸内增殖细胞核抗原(PCNA)表达水平进行研究,发现随着TBT浓度的增加,睾丸内PCNA表达量有下降趋势,但与对照组相比较,没有显著性变化。运用TUNEL技术检测TBT染毒45 d对小鼠睾丸生殖细胞凋亡的影响,发现各处理组凋亡阳性细胞数与对照组相比有所升高,但差异不显著。TBT处理后小鼠血清三碘甲腺原氨酸(T3)水平以及三碘甲腺原氨酸(T3)与甲状腺素(T4)比率呈现剂量依赖性显著下降,睾丸TRαmRNA表达量下调。2 TBT对附睾功能以及精子成熟的影响。TBT处理组小鼠精子密度、存活率和畸形率呈现剂量依赖性显著下降。组织形态学观察显示附睾头部未见明显变化,而体部和尾部可见不同程度的损伤,附睾损伤程度随着处理剂量的增加而逐渐加剧,呈现剂量依赖效应:体部附睾管上皮细胞逐渐稀少,排列疏松,管间间隙变大,腔内精子减少,中浓度组和高浓度组附睾管腔内可见大量精子凝聚;附睾尾部上皮细胞排列疏松,管间间隙逐渐变大,管腔内精子减少,管间结缔组织逐渐减少。TBT处理使附睾上皮特异性分泌物中性α-葡萄糖苷酶(NAG)活性有所下降,但与对照组相比,差异不显著:附睾酸性磷酸酶(ACP)活性随处理剂量的升高呈下降趋势,但与对照组相比无统计学差异。处理组精子顶体酶活性呈现剂量依赖性显著下降;精子乳酸脱氢酶同工酶-X活性在高浓度组受到显著抑制。与对照组相比较,5和50μg/kg TBT浓度组MMP7 mRNA表达量显著下降,TD02表达量呈现剂量依赖性显著下降。综上所述,本实验结果表明TBT能干扰青春期小鼠精子发生。组织学观察以及睾丸成熟度检查结果表明TBT能破坏睾丸组织的正常结构,抑制睾丸发育。TBT处理能够显著诱导睾丸睾酮水平上升,17-β雌二醇水平下降,血清LH和FSH水平有所下降,造成生殖内分泌系统紊乱,进而干扰睾丸精子发生。TBT处理显著降低血清T3含量,T3/T4比率显著下降,TRα表达水平下调,说明TBT能干扰甲状腺正常生理机能,进而诱发睾丸生精机能障碍。另外,处理组凋亡细胞数目的增加以及PCNA蛋白表达量的降低,提示TBT可在一定程度诱导睾丸生精细胞凋亡,抑制其增殖,这可能是TBT抑制精子发生的潜在机制之一。TBT干扰精子发生应该是多条途径共同作用的结果。本实验结果还显示TBT能干扰青春期小鼠精子成熟。组织形态学观察结果表明TBT对附睾组织结构造成损伤。精液常规检测结果显示处理组附睾精子密度下降,畸形率上升,同时精子存活率下降,提示TBT可影响哺乳动物精子质量。附睾酸性磷酸酶活性下降很可能与精子畸形率上升有一定关系。精子顶体酶以及乳酸脱氢酶同工酶-X活性的显著降低,提示精子成熟受到影响,这些精子质量功能指标的变化情况与精液常规检测结果一致,进一步证实TBT具有精子毒性。MMP7和TD02 mRNA表达量的显著下降,提示TBT可影响附睾内一些基因的表达,而这些基因很可能与附睾的功能或精子成熟有关。以上结果说明TBT具有附睾毒性以及精子毒性。TBT引起的精子数量和质量下降能影响动物的生殖。更多还原

【Abstract】 This study was conducted to investigate the toxic effects of tributyltin chloride(TBTCl) at low doses(0.5,5,and 50μg/kg,respectively) on reproductive system in KM male mice as orally garaged from puberty for 45 days and gain insight into the mechanisms.The evaluation indexes of spermatogenetic disorder included testicular histology,testicular mature degree,the levels of serum gonadotropin-releasing hormones and testicular sex hormones,testicular cell apoptosis and proliferation, serum triiodothyronine(T3) and thyroxine(T4) levels,and thyroid hormone receptors(TRs) expression levels in the testes.The methods used for evaluating sperm maturation included histopathology of epididymis,sperm parameters,the activities of epididymal biochemical markers(neutralα-G]ucosidase and acid phosphatase),sperm functional parameters (acrosin and LDH-X activities),and the gene expression profiles of matrix metallopeptidase 7(or matrilysin,MMP7) and tryptophan 2,3- dioxygenase (TD02) in the epididymides.1 The effect of TBT on the spermatogenesis of testis.After exposure for 45 days,histological damage occurred in the testes of mice treated with TBT,although the gonadosomatic index(GSI) was not altered.The severity of histopathologic damage observed correlated positively with the increase of TBT concentration.Spermatogenic cells in 50μg/kg TBT group were arranged chaoticly.Large space between germ cells,increments of detached debris and some sloughed cells in seminiferous tubules were also observed in this group.Compared to the control group,TBT treatment resulted in a significant dose-dependent increase of testosterone level and a moderate dose-dependent decrease of 17β-estradiol level in the testicle.Exposure to TBT also resulted in a slight decrease of serum LH and FSH levels.TBT treatment reduced PCNA expression in a dose-dependent manner by Western blotting assay,but no significant differnce was observed compared to the control.The results of the Western blotting were compatible with the immunocytochemical data.There was a moderate increase in apoptotic-cells in the testes of mice after TBT exposure as assessed by TUNEL method,but no significant difference between the treated groups and the control was observed.TBT exposure significantly decreased serum T3 levels and the ratio of T3 to T4 in a dose-dependent manner,while the serum T4 levels were not significantly altered compared to the control.Testicular TRαtranscript levels in the treatment groups declined compared to the control.2 The effect and potential mechanism of TBT on the epididymal function and sperm maturation.TBT treatment resulted in a significant decrease in sperm count,viability and a significant increase in abnormality compared to the control in a dose-dependent manner.No obvious histological damage was found in caput epididymis,but in corpus and cauda epididymis,the severity of histological damage increased dose-dependently after treatment.The primary pathological changes observed in the treatment groups were listed as follows:The epithelial cells of corpus epididymal tubules were arranged loosely,and the inter-tubule space became large.Sperm amount in the lumens of epididymal duct decreased.A large amount of sperm was agglutinated in the epididymal lumen area in the middle and high dose groups.Loosely arranged epithelial cells,large inter-tubule space and decreased sperm amount were also found in cauda epididymis,furthermore,connective tissue between tubules in cauda epididymis lessened.No significant alteration was observed in neutralα- glucosidase activity in the epididymides of the treatment groups compared to the control,even though there is a slight decline after TBT exposure.There was a dose-dependent decline trend in the acid phosphatase activity,compared with the control.Acrosin activity from the cauda epididymal spermatozoa showed a dose-dependent decrease,which reach significance in 50μg/kg TBT group.Lactate dehydrogenase-x isoenzyme(LDH-X) activity was also significantly inhibited in the high dose group.The transcript expression of MMP7 in the epididymides of mice exposed to TBT was significantly decreased in 5 and 50μg/kg groups, and the expression of TD02 transcript was significantly reduced in a dose-dependent manner.In conclusion,the results in this study strongly suggest that TBT exposure perturb spermatogenesis of pubertal mice.By histological observation and mature degree assessment,we confirmed that normal tissue structure of testis was impaired by TBT,and the development of testis was suppressed.Elevated testosterone level and decreased 17β-estradiol, LH and FSH levels suggested that TBT adminstration induced reproductive endocrine dysfunction,and thereby affected the spermatogenesis in the testes.TBT exposure significantly decreased serum T3 level and T3/T4 ratio.The TRαtranscript in the testes was decreased after TBT treatment. These results indicated that TBT could affect the normal function of thyroid gland,which can further interrupt spermatogenesis.Moreover,TBT can induce cell apoptosis and inhibit cell proliferation,which would be one of the mechanisms of TBT-induced spermatogenetic disorder.The spermatogenetic abnormality present in this study should result from the interaction of many factors and pathways mentioned above.The results in the present study show that TBT has a potential role in affecting the epididymal function and sperm maturation.The normal tissue structure of epididymis was impaired by this compound.The decline of sperm count and quality caused by TBT suggests that this chemical compound could affect sperm qualtity.The decline of acid phosphatase activity might somewhat relate with the TBT- induced increase in sperm abnormality. Acrosin and lactate dehydrogenase-x isoenzyme(LDH-X) activities from the cauda epididymal spermatozoa significantly decreased after TBT treatment, which favor the conclusion drawn from the results of sperm parameters detection that TBT could cause a spermatotoxic effects.TBT also significantly inhibited the expression of some genes(MMP7 and TD02) which might be correlated with epididymal function and sperm maturation.The results mentioned above indicate that TBT could cause epididymal and sperm toxicity.The decline of sperm count and quality caused by TBT could further impair fertility in animals.更多还原