摘要
三丁基锡(TBT)是一种重要的有机锡类化合物,在环境中大量使用,具有很强的毒性。近年,许多研究表明TBT对软体动物和鱼类生殖机能产生影响,但是有关对哺乳动物的生殖影响报道较少。本实验以青春期雄性小鼠为研究对象,在生态条件下和环境污染浓度范围内,采用不同浓度TBT(0.5、5、50μg/kg)对其进行灌胃染毒,每三天灌胃一次,暴露30天。通过光学显微镜观察精子质量;放射免疫法检测睾丸性激素水平;western blotting方法检测性激素受体水平以及PCNA表达水平;并利用TUNEL法检测细胞凋亡情况,最终分析TBT对雄性小鼠性腺发育的影响及其作用机理。
实验结果如下:
1.TBT暴露30天后,青春期雄性小鼠体重没有出现显著变化,性腺指数呈剂量—依赖性下降,高浓度组出现显著性差异。组织学观察发现低浓度组和中浓度组曲细精管内细胞少量脱落,而高浓度组管内细胞层数则大幅减少,精细胞脱落,分布散乱;
2.TBT暴露影响了小鼠精子发生,通过对小鼠附睾精子质量的检测,结果表明附睾中的精子数量和精子存活率与对照组相比都有显著性下降,中浓度组和高浓度组的畸形率显著上升,且都呈剂量一效应关系;
3.TBT暴露30天后,免疫组化等方法发现TBT降低PCNA在精细胞中的表达水平,表现出剂量—效应性关系,其中中浓度和高浓度与对照组相比都有显著性差异;TUNEL实验显示TBT暴露能够诱导睾丸细胞的凋亡,且凋亡主要发生在精原细胞和精母细胞中,也呈剂量—效应关系;
4.TBT暴露后小鼠睾丸中睾酮水平没有显著变化,但17β-雌二醇水平却呈剂量—依赖性下降,其中50μg/kg组的17β-雌二醇水平与对照组相比有显著性差异,同时western blotting还显示在5μg/kg和50μg/kg暴露组中,TBT显著抑制睾丸雌激素受体(ER-α和ER-β)的水平,提高小鼠睾丸中雄激素受体(AR)的表达,并都呈剂量—效应性关系。
综上所述,低剂量的TBT暴露能够抑制雄性小鼠性腺的发育,影响精子的发生。同时,TBT使睾丸中17β-雌二醇水平产生下降,表明TBT诱导的激素水平的变化是其抑制性腺发育的机制之一。另外,在本实验中,由于TBT的作用,小鼠睾丸性激素受体水平受到明显影响,精细胞凋亡增加,这也为TBT抑制性腺发育提出了新的机制。
As an important organotin compound,tributyltin(TBT) is widely used and has intense toxicity.Reproductive toxicity induced by TBT in mollusk and fish has been demonstrated extensively in recent years.However,few studies addressing the effect of TBT on reproduction of mammals have been reported.This study was conducted to investigate effects of TBT at low doses(0.5,5,and 50μg/kg,respectively) on the development of gonad in male KM mice as exposed from puberty and insighted into the mechanism.Thirty-two mice were divided into four groups randomly and orally administered by gavaged once every 3 day with TBT(lasting 30 days).The quality of sperms were observed directly by light microscope;the sex hormone levels were detected by radio-immunoassay(RIA) method;androgen receptor,endrogen receptor and PCNA expression were evaluated by western blotting method;and the cell apoptosis was detected with method of TUNEL.
The results were shown as below:
1.After exposure for 30 days,though the body weight of male mice treated with TBT was not significantly altered,the gonadosomatic indexes of the mice were decreased in a dose-dependent manner,and a significant decrease was observed in 50μg/kg TBT groups compared to the control.A small quantity of sloughed cells in seminiferous tubules was found in 0.5μg/kg and 5μg/kg TBT group,but fewer layers of seminiferous epithelium and increments of detached debris and more sloughed cells in seminiferous tubules of the testis were found in 50μg/kg TBT group by histological examination;
2.TBT treatment resulted in degeneration of spermatogenesis.According to the dectecion of sperm quality,we found TBT significantly decrease the sperm count and viability in 5μg/kg and 50μg/kg TBT groups compared to the control,while significantly elevate the sperm abnormality in 5μg/kg and 50μg/kg TBT groups both in a dose-dependent manner;
3.The positive expression of PCNA in seminiferous epithelium in the experimental male mice was caused a dose-dependant decrease after TBT treatment by immunohistochemistry analysis,and compared to the control,a significant derease was detected in 5μg/kg and 50μg/kg TBT groups.Also,an increasement of cell apoptosis a dose-dependent manner induced by TBT was observed in testes,and it showed that the TUNEL-positive cell detected were mainly in spermatogonia and spermatocyte;
4.Exposure to TBT resulted in a decrease of 17β-estradiol levels of the testes in 50μg/kg group compared to the control,but no significant alteration of testosterone level between the treated groups and the control.TBT treatment also resulted in a decrease of ER-αand ER-βexpression in a dose-dependent manner,and it has a significant decrease in 5μg/kg and 50μg/kg TBT groups compared with the control.However,AR levels were increased in a dose-dependent manner in the testes of the mice,and a significant increase was also observed in 5μg/kg and 50μg/kg TBT groups.
In conclusion,the results strongly suggest that TBT exposure at low dose suppress the development of testis,cause decrease of spermatogenesis,and cause the decrease of the 17β-estradiol levels.The changes of sex hormone induced by TBT should be one of reasons that TBT affects the gonad development.Moreover,the increase of apoptosis in testis and the disruption of sex hormone receptors expression found in this study,especially the latter may be intensively involved in the mechanism on affecting the gonad development.
引文
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