雌激素受体在PFOS致雄性小鼠生殖毒性中的作用及机制研究

发布时间：2018-08-02 12:14

【摘要】：全氟辛烷磺酰基化合物(perfluorooctane sulfonate, PFOS)是一种代表性的全氟化合物(perfluorinated compounds, PFCs),由于其出色的稳定性及疏水疏油等特性而长久以来被广泛地应用于各种民用及工业产品中。PFOS分布于多种环境介质中,已造成全球性污染。研究表明,PFOS具有包括生殖毒性在内的多系统毒性,如影响雄性动物体内的性激素水平、减弱其生育能力等。流行病学研究还表明男性精液质量下降可能与PFOS暴露有关。虽然国内外的此类研究已经初步对PFOS的生殖毒性做了肯定性结论,但是PFOS的具体生殖毒作用有哪些尤其是PFOS的生殖毒作用靶点及其涉及的雄性生殖损伤机制均尚未阐明。此外,近年来国外相继有文献证实雌激素受体(ERs)与精子发生的调控过程密切相关,而我们的前期预实验结果表明PFOS可以影响小鼠睾丸组织内ERs的表达,那么PFOS所引起的雄性生殖损伤是否也是ERs参与调控的结果呢?鉴于此,我们开展了本研究。本研究通过分别建立体内和体外染毒模型,从睾丸功能和结构以及精子发生等方面观察PFOS对雄性小鼠生殖系统的影响,探讨ERs在PFOS影响精子发生中的作用并初步阐明其分子机制,为深入研究PFOS的毒作用尤其生殖毒性机制提供基础,同时也为PFOS环境污染的控制和政府决策提供理论依据。第一部分PFOS对雄性小鼠生殖系统的影响目的以雄性成年小鼠为染毒模型,从整体动物水平研究PFOS暴露对小鼠的生殖毒作用。方法36只雄性C57小鼠随机分为3组,分别为对照组(0.1%DMSO油溶液灌胃)、低剂量PFOS染毒组(0.5mg/kg/d PFOS灌胃)和高剂量PFOS染毒组(10mg/kg/d PFOS灌胃),染毒35d后称重并处死小鼠。采用计算机辅助精子分析(CASA)系统对附睾尾部精子进行计数分析；放射免疫法检测血清性激素水平；HE染色法观察睾丸组织的结构形态；原位末端标记术(TUNEL)观察睾丸中细胞凋亡情况；免疫组化及蛋白质印迹法检测ERα、ERβ、细胞增殖及凋亡相关蛋白的定位和表达。结果10mg/kg/d PFOS染毒可以使小鼠体重、睾丸重量、睾丸脏器系数、血清睾酮水平及精子数均显著下降,而雌二醇水平变化不明显；形态学观察结果显示生精小管出现病理性改变,表现为生精细胞层次减少及排列紊乱、生精上皮出现空泡化改变；TUNEL结果表明PFOS可以显著诱导生精细胞凋亡；PFOS还可使Bax、cleaved caspase-3、cleaved caspase-9和ERβ表达水平上升而PCNA和ERα水平下降。在0.5mg/kg/d PFOS染毒组,除ERβ表达水平上升外,其余蛋白表达水平与对照组无明显差异。结论一定剂量的PFOS暴露可使雄性小鼠产生生殖毒性作用,小鼠精子数的下降可能与睾丸组织中生精细胞凋亡的增加以及ERα、ERβ和细胞增殖与凋亡相关蛋白表达水平的改变有关。第二部分雌激素受体在PFOS影响精子发生中的作用及机制目的以生殖细胞株GC-2 spd(小鼠精母细胞系)为染毒模型,从细胞水平探讨ERs在PFOS诱导生精细胞凋亡中的作用及机制。方法分别以0μM、50μm、100μM和PFOS染毒GC-2细胞24h或48h,采用细胞毒性试验(MTT法)和流式细胞术观察PFOS对GC-2细胞增殖、凋亡及细胞周期的影响；蛋白质印迹法检测PFOS染毒对GC-2细胞中ERα、ERβ p-ERKl/2及下游细胞增殖及凋亡相关蛋白表达水平的影响,以探讨PFOS诱导细胞凋亡的机制；通过ERa和ERβ激动剂或拮抗剂的加入,探讨并验证ERa和ERβ在PFOS诱导GC-2细胞凋亡中的作用；报告基因法观察PFOS对ERa和ERβ转录活性的影响,确认PFOS是否通过ERs基因组模式发挥毒效应；最后采用RT-PCR法检测PFOS对参与调控ERs表达的相关miRNA的影响,分析PFOS如何干扰ERs的表达。结果100μM、150μM的PFOS可以显著抑制GC-2细胞增殖、使其发生G0/G1期阻滞并诱导其凋亡的发生。与对照组相比,100μM、150μM的PFOS还可引起ERβ、Bax、cleaved caspase-3和cleaved caspase-9的蛋白表达水平上调而ERα、 pERK1/2、 Bcl-2、PCNA和cyclin D1表达下调。ERβ激动剂的加入可加剧PFOS对GC-2细胞增殖、凋亡以及pERK1/2、细胞增殖和凋亡相关蛋白表达的影响,而ERa激动剂的加入则可部分逆转PFOS所致的以上改变。报告基因实验结果显示PFOS染毒对ERa和ERβ转录活性无明显影响。此外,100μM、150μM的PFOS还可以使GC-2细胞中miR-145和miR-206表达水平上调而miR-92下调。结论PFOS可以显著抑制GC-2细胞增殖并诱导其凋亡,这些效应的产生可能是通过PFOS干扰ERs表达从而启动ERs的非基因组模式,即抑制ERK1/2的磷酸化、继而干扰其下游包括Bax、Bcl、caspase-9和caspase-3在内的线粒体凋亡通路及细胞周期相关蛋白cyclin D1的表达而引起的。此外,PFOS染毒还显著影响GC-2细胞中miR-145、miR-206和miR-92的表达水平,这可能是我们所观察到的ERs表达发生变化的重要原因。第三部分PFOS对雌激素受体β基因敲除小鼠精子发生的影响目的以雌激素受体β基因敲除(ERβKO)雄性小鼠为染毒模型,从动物水平进一步验证雌激素受体在PFOS影响小鼠精子发生中的作用。方法11只ERPKO雄性小鼠,按照随机分组的原则分为2组,分别为：对照组(5只,0.1%DMSO油溶液灌胃)和PFOS染毒组(6只,8mg/kg/d PFOS灌胃)。12只野生型(WT)雄性小鼠,按照随机分组的原则分为2组,每组6只,分别为：对照组(0.1%DMSO油溶液灌胃)和PFOS染毒组(8mg/kg/d PFOS灌胃)。染毒35d后称重并处死小鼠。通过计算机辅助精子分析(CASA)系统对附睾尾部精子进行计数及分析；放射免疫法检测血清性激素水平；HE染色法观察睾丸组织的结构形态；原位末端标记术(TUNEL)观察睾丸中细胞凋亡情况；免疫组化及蛋白质印迹法检测细胞增殖及凋亡相关蛋白的定位和表达分析。结果无PFOS处理的WT小鼠和ERβKO小鼠在精子数、睾丸组织结构、生精细胞凋亡指数、血清睾酮水平及雌二醇水平方面均无显著差异。8mg/kg/d PFOS染毒可以使这两种小鼠的精子数、血清睾酮水平、PCNA蛋白表达水平显著下降而生精细胞凋亡指数及Bax和cleaved caspase-3的表达水平上升,但对小鼠的体重、睾丸重量和血清雌二醇水平无明显影响。进一步比较PFOS处理组的WT小鼠和ERβKO小鼠,发现WT小鼠的生精细胞凋亡指数显著高于ERβKO小鼠,其余指标在PFOS处理的这两种小鼠间无明显差异。结论：ERβ表达缺失对雄性小鼠的精子数、睾丸组织结构、血清睾酮水平及雌二醇水平均无明显影响；与野生型小鼠相比,PFOS诱导的ERβKO小鼠生精细胞凋亡指数显著下降,提示ERβ表达缺失对于PFOS诱导生精细胞凋亡可以起到一定的保护作用。
[Abstract]:The perfluorooctane sulfonyl compound (perfluorooctane sulfonate, PFOS) is a representative perfluorocompound (perfluorinated compounds, PFCs). Due to its excellent stability and hydrophobic oil and oil properties, the perfluoroalkyl sulfonyl compound has long been widely used in various civil and industrial products with.PFOS in a variety of environmental mediums. Spherical pollution. Studies have shown that PFOS has multiple systemic toxicity including reproductive toxicity, such as the effects of sex hormone levels in the male animals and their fertility. The epidemiological study also suggests that the quality of male semen may be associated with PFOS exposure. Although this study at home and abroad has preliminarily done the reproductive toxicity of PFOS Affirmative conclusions, but the specific reproductive toxicity of PFOS, especially the target of the reproductive toxicity of PFOS and the mechanism of male reproductive injury, has not been clarified. In addition, in recent years, there have been some literatures that have confirmed that the estrogen receptor (ERs) is closely related to the regulation of the regulation of spermatogenesis, and our pretest results show that PFO S can affect the expression of ERs in the testicular tissue of mice, then whether the male reproductive damage caused by PFOS is also the result of the regulation of ERs participation? In view of this, we carried out this study. This study was conducted by establishing both in vivo and in vitro poisoned models to observe the reproduction of PFOS on male mice from the aspects of testis function, structure and spermatogenesis. The effect of ERs on the effect of PFOS on spermatogenesis was discussed and its molecular mechanism was preliminarily clarified, which provided a basis for the in-depth study of the toxic effect of PFOS, especially on the mechanism of reproductive toxicity, and also provided a theoretical basis for the control of environmental pollution in PFOS and government decision-making. The first part of the effect of PFOS to the reproductive system of male mice was aimed at males. 36 male C57 mice were randomly divided into 3 groups: the control group (0.1%DMSO oil solution gavage), the low dose PFOS group (0.5mg/kg/d PFOS gavage) and the high dose PFOS venom group (10mg/kg/d PFOS gavage), and the weight of the toxic 35d after 35d, and the weight of the mice after 35d. A computer assisted sperm analysis (CASA) system was used to count the spermatozoa in the epididymis; the level of serum sex hormone was detected by radioimmunoassay; the structure of the testicular tissue was observed by HE staining; in situ end labeling (TUNEL) was used to observe the cell apoptosis in the testis, and the immunohistochemical and Western blot methods were used to detect ER alpha, ER Results 10mg/kg/d PFOS can reduce the weight of mice, the weight of the testis, the coefficient of testicular organ, the level of serum testosterone and the number of sperm, but the level of estradiol is not obvious. Morphological observation shows the pathological changes in the seminiferous tubules, which are spermatogenic cells. TUNEL results showed that PFOS could induce apoptosis of spermatogenic cells, and PFOS also increased the level of Bax, cleaved Caspase-3, cleaved caspase-9 and ER beta, while PCNA and ER alpha levels decreased. The expression level was not significantly different from that of the control group. Conclusion a certain dose of PFOS exposure could produce reproductive toxicity in male mice. The decrease of sperm count in mice may be related to the increase of spermatogenic cell apoptosis in the testis tissue and the changes of ER alpha, ER beta and cell proliferation and apoptosis related protein expression. The second part of estrogen receptor is in P The effect and mechanism of FOS on spermatogenesis and the mechanism aim to use the germ cell line GC-2 SPD (mouse spermatocyte line) as a poisoned model to explore the role and mechanism of ERs in the apoptosis of spermatogenic cells induced by PFOS from the cell level. The methods were 0 u M, 50 mu m, 100 mu M and PFOS GC-2 cells 24h or 48h, and the cytotoxicity test and flow cytometry were used. The effects of PFOS on the proliferation, apoptosis and cell cycle of GC-2 cells were observed, and the effect of PFOS on the expression of ER alpha, ER beta p-ERKl/2 and downstream cell proliferation and apoptosis related protein in GC-2 cells was detected by Western blot, in order to explore the mechanism of PFOS induced apoptosis, and the addition of ERa and ER beta agonists or antagonists was discussed. The effect of ERa and ER beta on the apoptosis of GC-2 cells induced by PFOS was verified. The effect of PFOS on the transcriptional activity of ERa and ER beta was observed and the toxic effect of PFOS was confirmed through the ERs genome pattern. Finally, RT-PCR method was used to detect the effect of PFOS on the related regulation of ERs expression. PFOS can inhibit the proliferation of GC-2 cells and induce G0/G1 phase block and induce apoptosis of GC-2 cells. Compared with the control group, 100 u M, 150 mu PFOS can also cause ER beta, Bax, cleaved caspase-3 and protein expression up regulation. The addition of the agent could aggravate the effect of PFOS on the proliferation, apoptosis and the expression of pERK1/2, cell proliferation and apoptosis related proteins, while the addition of ERa agonists could partly reverse the above changes caused by PFOS. The results of the reported gene experiment showed that PFOS had no obvious effect on the transcriptional activity of ERa and ER beta. In addition, 100 u M, the PFOS of 150 mu M was also possible. The expression level of miR-145 and miR-206 in GC-2 cells is up regulated and miR-92 is down regulated. Conclusion PFOS can significantly inhibit the proliferation and induce apoptosis of GC-2 cells. These effects may be induced by PFOS interfering ERs expression to initiate the non genomic pattern of ERs, that is, to inhibit the phosphorylation of ERK1/2, and then interfere with the downstream of Bax, Bcl, etc. The apoptosis pathway of mitochondria and the expression of cell cycle related protein cyclin D1, including the expression of spase-3 and the expression of miR-145, miR-206 and miR-92 in GC-2 cells, may be an important reason for the changes in the expression of ERs in the GC-2 cells. The third part PFOS knocks on the estrogen receptor beta gene. The effect of spermatogenesis in mice was based on the estrogen receptor beta gene knockout (ER beta KO) male mice as a poisoned model. The role of estrogen receptor in the spermatogenesis of mice was further verified from the animal level. Methods 11 male ERPKO mice were divided into 2 groups according to the principle of random grouping: the control group (5, 0.1%DMSO oil soluble). The male mice of.12 only wild type (WT) were divided into 2 groups according to the principle of random grouping, and 6 rats in each group were divided into 2 groups according to the principle of randomization. The control group (0.1%DMSO oil solution gavage) and PFOS poisoning group (8mg/kg/d PFOS gavage) were weighed and executed after 35d. The system of computer assisted sperm analysis (CASA) system was used. The sperm of epididymis tail was counted and analyzed; the level of serum sex hormone was detected by radioimmunoassay; the structure of the testicular tissue was observed by HE staining; in situ end labeling (TUNEL) was used to observe the cell apoptosis in the testis, and the localization and expression analysis of cell proliferation and apoptosis related proteins were detected by immunohistochemistry and Western blot. There were no significant differences in sperm count, testicular tissue structure, spermatogenic cell apoptosis index, serum testosterone level and estradiol level in the WT mice without PFOS treatment and ER beta KO mice. The number of sperm, the level of serum testosterone, the level of PCNA protein and the apoptotic index of spermatogenic cells were significantly decreased in the two mice. The expression level of Bax and cleaved caspase-3 increased, but had no significant influence on the weight of the mice, the weight of the testis and the level of serum estradiol. Further comparison of the WT mice and ER beta KO mice in the PFOS treatment group showed that the apoptosis index of spermatogenic cells in WT mice was significantly higher than that of the ER beta KO mice, and the other indexes were not significantly worse among the two mice treated PFOS. Conclusion: the loss of ER beta expression has no significant effect on sperm count, testicular tissue structure, serum testosterone level and estradiol level in male mice. Compared with wild type mice, the apoptosis index of spermatogenic cells induced by PFOS in ER beta KO mice decreased significantly, suggesting that the deletion of ER beta expression may play a certain role in the apoptosis of spermatogenic cells induced by PFOS. Protective effect.
【学位授予单位】：南京医科大学
【学位级别】：博士
【学位授予年份】：2014
【分类号】：R114

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