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论文题名(中文):

 氟致小鼠精子损伤的分子机理研究    

作者:

 孙子龙    

学号:

 20071023    

保密级别:

 保密    

论文语种:

 chi    

学科代码:

 090603    

学科名称:

 临床兽医学    

学生类型:

 硕士    

学位:

 农学硕士    

学校:

 山西农业大学    

院系:

 动物科技学院    

专业:

 临床兽医学    

研究方向:

 环境兽医学    

第一导师姓名:

 王俊东    

第一导师学校:

 山西农业大学    

论文完成日期:

 2010-04-20    

论文答辩日期:

 2010-06-24    

论文题名(外文):

 Study on the Molecular Mechanism of Sperm Damage in Mice Exposed to Fluoride    

关键词(中文):

  小鼠 精子 cDNA微阵列芯片 氧化应激 精子凋亡 Ca2+ CALM/CaMK2信号通路 超激活运动 DNA完整性 染色质结构损伤    

关键词(外文):

 Fluoride Mouse Sperm cDNA microarray chip Oxidative stress Sperm apoptosis Ca2+ CALM/CaMK2 Signaling pathway hyperactiviation DNA integrity Chromatin structure lesion    

论文文摘(中文):

[目的] 氟中毒是全世界广泛存在的一种人畜共患性地方病,近年来大量研究表明,氟对人类及畜禽等动物生殖系统和功能产生较强的毒副作用,本论文结合基因组学高通量和高效率的特点,从精子基因组学的变化研究氟对精子的毒性作用机理,进而探讨氟对精子超激活运动、精子凋亡及染色质结构的影响及其分子机理的研究,从而为进一步阐明氟的生殖毒性奠定理论基础,为实施科学诊断和靶标药物设计提供依据。

[方法] 360只昆明雄性小鼠随机分成4组:对照组(饮用去离子水);低氟组(饮用30mgNaF/L的蒸馏水);中氟组(饮用70mgNaF/L的蒸馏水);高氟组(饮用150mgNaF/L的蒸馏水)。49天后剖杀小鼠,取血清、股骨、门齿、睾丸、附睾、精囊腺和精子进行相关试验。采用cDNA微阵列芯片技术、生物信息学、电镜技术、Diff-Quik方法、苯胺蓝染色、染色质结构分析技术(SCSA)、荧光分光光度计、流式细胞技术、视频分析技术、QRT-PCRWestern blotting以及免疫荧光细胞技术等手段,对氟处理小鼠的生育力、精液品质、精子超微结构、精子全基因表达、ROSTAC含量、DNA完整性、组蛋白分析、总巯基含量、精子凋亡、超激活运动能力、Ca2+浓度、细胞色素ccaspase-3以及CALMCAMK2定位定量研究、CatSper1CatSper2mRNA以及Prm1Prm2 mRNA的表达等指标进行测定,从而探讨氟致小鼠精子损伤的分子机理

[结果]1)低、中、高氟组雄性小鼠日均摄氟量分别为2.84±0.296.28±0.6114.18±1.00 mg/kg/day。各试验组小鼠体重与对照组相比无显著性差异,而高氟组体增重显著低于对照组。各试验组小鼠牙齿均出现白垩样不透明性改变,从而使牙齿失去正常光泽,表面粗糙,但X光片未显示明显的牙齿密度的差异;同时,与对照组相比,不同氟使骨密度增高,骨纹模糊、紊乱,间有粗大骨斑。各试验组股骨氟含量均显著高于对照组,且呈剂量依赖性。睾丸和附睾中氟含量随摄氟剂量的增加呈剂量依赖性,且在高氟组中,氟含量显著升高。血清与精囊腺中氟含量与对照组相比未出现显著性差异。

2)各试验组中雌性小鼠受孕率均低于对照组,且高氟组差异显著。高氟组与对照组相比,虽然没有显著影响生殖力中的胚胎植入数,但显著降低了可用胚胎数,并显著增加了胚胎吸收数/胚胎植入数的比例。与对照组相比,各试验组睾丸、附睾、精囊腺及其脏器系数未发生显著性变化。高氟组睾丸生精上皮变薄,细胞排列不规则,精子细胞少见,提示生精功能低下。与对照组相比,70mg/L150mg/LNaF均显著降低了小鼠精子的活力。精子密度和活率在低氟组和中氟组中有降低趋势,但差异不显著,而在高氟组中均显著降低。

3)对照组与高氟组不同荧光标记的精子DNA混合后与36K小鼠全基因组芯片杂交,结果分析有86个已知基因和11个未知基因发生上调或下调,其中上调基因34个,下调基因63个,这些差异基因经过Gene Ontology分析后显示与信号转导、氨基酸磷酸化、氧化应激、细胞周期、细胞凋亡、电子传递、糖酵解、趋化性、精子发生、精子获能等生物学途径相关,信号通路分析后发现这些差异基因涉及到信号转导、核糖体蛋白、G蛋白信号通路、ATP酶活性和催化活性等信号通路。9个差异表达的基因(Atp1b3CcniDffaOlfr1031Pde4dPpp2r2bTnk2Prm2Stat2), CcniDffaPde4d的表达与对照组相比显著升高,Atp1b3Olfr1031Ppp2r2bTnk2Prm2Stat2等基因的表达显著低于对照组。Real-time荧光定量PCR验证各基因的结果与芯片结果相一致。

4)透射电镜观察高氟组中小鼠精子超微结构出现异常,包括头部有核内空泡,质膜间隙增宽,质膜不完整,线粒体排列不规则,大小不一,甚至缺失等病理变化;扫描电镜发现高氟组精子头部断裂,顶体缺乏,颈部少许颗粒粘附,尾部中段线粒体鞘脱落,以及尾部卷曲等变化。与对照组相比,ROS、精子晚期凋亡率及总凋亡率显著升高,而TAC含量显著降低。高氟组精子细胞色素Ccaspase-3蛋白表达量显著高于对照组。

5中氟组和高氟组中Ca2+浓度和精子超激活运动百分率显著低于对照组,而低氟组无显著变化。与对照组相比,中、高氟组中CaMK2蛋白表达量显著低于对照组。高氟组精子中CALMCaMK2基因的表达量显著低于对照组;70mg/LNaF显著降低了CALM基因的表达水平,但对CaMK2无显著影响;而在低氟组中两种基因表达量均未发生显著变化。与对照组相比,70mg/L150mg/LNaF显著降低了精子中CatSper1基因的表达量;而CatSper2基因表达水平在各试验组中与对照组相比均无显著性差异。

6)与对照组相比,150mg/LNaF可导致精子头部、颈部、尾部畸形率的显著升高,最终导致总畸形率增加。中氟组精子尾部及总畸形率显著高于对照组。而在低氟组中,精子各部位畸形率也出现增加趋势,但差异不显著。各试验组中染色质结构异常的精子百分率显著高于对照组。中、高氟组苯胺蓝染色精子的百分率显著高于对照组,总巯基含量显著低于对照组。与对照组相比,70mg/L150mg/LNaF显著降低了精子中Prm1Prm2基因的表达量;而在低氟组中,两种基因表达量均未发生显著变化;关于Prm1/ Prm2比值,仅高氟组显著低于对照组。

[结论] 通过微阵列芯片技术和生物信息学的方法分析了高氟对小鼠精子中转录本的影响,它们涉及了与精子形态与功能相关的信号转导、氨基酸磷酸化、氧化应激、细胞周期、细胞凋亡、电子传递、糖酵解、趋化性、精子发生、精子获能等生物学途径。进一步的研究结果揭示:①过量氟可能通过升高精子中ROS含量,降低TAC,从而引起氧化损伤,导致精子膜的完整性遭到破坏,线粒体结构受到损伤,触发细胞色素C/Caspase-3线粒体凋亡通路来诱导精子凋亡;②过量氟影响CatSper-1基因水平、Ca2+浓度及CALM/CaMK2信号通路从而导致小鼠精子超激活运动能力显著下降;③过量氟可能通过精子中Prm1Prm2mRNA含量及总巯基的降低、DNA完整性的破坏,从而导致精子核的成熟受阻,引起精子头部畸形。这些结果表明氟对生殖的影响是一个复杂的、值得重视的、需要深入研究的课题
文摘(外文):

[Objective] Fluorosis is a kind of endemic disease widely spread all over the world. Recently, a large number of studies have shown that fluoride plays relatively strong toxic effects on reproductive system in both human and animals. To investigate the underling mechanism of fluoride toxicity in sperm, effects on sperm genomics, sperm hyperactiviation, sperm apoptosis and chromatin organization were evaluated in mice treated with different doses of fluoride, for further consummating rationale of fluoride toxicity and providing scientific basis of diagnosis and target drug designation.

[Method] 360 health Kunming male mice were randomly divided into 4 groups: control group (distilled water); low dose of fluoride group (30mgNaF/L); middle dose of fluoride group (70mgNaF/L); high dose of fluoride group (150mgNaF/L). After 49days exposure, all mice were sacrificed by cervical dislocation, and serum, femur, incisor tooth, testis, epididymis, seminal vesicle and sperm were collected for further analysis. With the application of diverse methods as cDNA microarray chip, bioinformatics, electron microscopy, Diff-Quik test, Aniline blue staining, sperm chromatin structure assay (SCSA), fluorospectrophotometer, flow cytometry, video analysis, QRT-PCR, Western blotting and fluoroimmunocyte technique, male fertility, sperm quality, sperm ultrastructure, sperm genomics, ROS and TAC content, DNA integrity, histone analysis, total sulfhydryl level, sperm apoptosis, hyperactiveation, Ca2+ concentration, protein expression of Cytochrome C, caspase-3, CALM and CAMK2, and mRNA expression of CatSper1, CatSper2, Prm1 and Prm2 were determined to elucidate the molecular mechanism by which fluoride induces mice sperm lesion.

[Result] (1) Fluoride ingestion of mice in low, middle, and high fluoride groups were 2.84±0.29, 6.28±0.61, and 14.18±1.00 mg/kg/day, respectively. Compared with the control, body weight gain of mice in high fluoride group was significantly decreased, whereas there is no statistical difference in body weight between control and experimental groups. Teeth of mice exposed to fluoride presented chalky and opaque surface, leading to crude appearance without the gloss, however, no significant difference in dental density was observed from X ray films. As for femur, fluoride with various doses enhanced the bone density, causing disarranged bone grains with the macula between them. Fluoride concentrations in all experimental groups were significantly higher than that in control group with a dose-dependent relationship. In testis and epididymis, fluoride levels were increased with the increased in fluoride exposure, and in high fluoride group it was significantly higher than control group. However, in serum and seminal vesicle fluoride concentration showed no obvious difference with the comparison to control group.

(2) Conception rate in female mice exposed to high fluoride was lower than controls, with a significant difference in high fluoride group. Compared to the control group, fluoride obviously decreased the number of viable fetuses, increased the ratio of resorption/implantations, however, no influence was observed in the number of implantations. The ratios of organic weights to body in testis, epididymis, and seminal vesicle presented no difference from those in control. With the comparison to controls, seminiferous epithelium was thinner, and cells in testis were irregularly arranged with less spermatid, indicating lowered function in spermatogenesis. 70 and 150mg/LNaF were significantly reduced the abilities of mice sperm compared with the controls. Sperm density and sperm survival ratio showed a decreased trend in low and middle doses of fluoride groups with no statistical changes, and significant change was observed in high fluoride group.

(3) The results of 36K mouse whole genomic gene chip hybridized with the fluorescence labeled DNA mixture from control and experimental samples showed that expression of 97 genes with 11 unknown genes were changed, among which 34 genes were up-regulated and 63 genes were down-regulated. After Gene Ontology analysis, the affected genes are identified to relate with divers biological processes including signal transduction, amino acid phosphorylation, oxidative stress, cell cycle, cell apoptosis, electron transfer, glycolysis, chemiotaxis, spermatogenesis, and sperm capacitation. Pathway analysis revealed that the differently expressed genes involved in signal transduction, ribosomal protein, G protein signaling pathway, and the enzymic and catalytic activity of ATP. 9 genes (Atp1b3,Ccni, Dffa, Olfr1031, Pde4d, Ppp2r2b, Tnk2, Prm2, Stat2) in these 97 differently expressed genes were validated by QRT-PCR. It were found that gene expressions of Ccni, Dffa, and Pde4d were increased, and expressions of Atp1b3, Olfr1031, Ppp2r2b, Tnk2, Prm2, and Stat2 were decreased significantly compared with the control groups. The results of real time PCR was coincidence with that of gene chips.

(4) Alterations of sperm ultrastructure were observed by transmission electron microscope, which included vacuoles in head, widened plasma membrane interspaces, impaired plasma membrane, and irregularly arranged mitochondrion. Scanning electron microscope observation found the head abruption, acrosome absence, grains attached neck, shedded mitochondrial sheath, and curved tail in sperm in high fluoride group. With the comparison to control group, ROS level, late apoptotic sperm, and total sperm apoptosis were significantly increased, while TAC content was reduced with statistical significance. In addition, protein expression of Cytochrome C and caspase-3 were significantly higher than controls.

(5) In middle and high fluoride groups, Ca2+ concentration and sperm hyperactiviation proportion were notably lower than that in control, while no significance was observed in low fluoride group. Compared with the control group, CaMK2 protein levels in middle and high fluoride groups was obviously lower. In high fluoride group, gene expressions of CALM and CALM decreased significantly. Meanwhile, 70mg/LNaF markedly reduced CALM gene expression, with no effect on CALM expression, whereas low fluoride showed no influence with the above two. Additionally, compared with the controls, 70mg/LNaF and 150mg/LNaF significantly decreased the CatSper1 expression, while fluoride at different levels had no effects on CatSper2 expression.

(6) With the comparison to control group, 150mg/LNaF obviously induced the abnormality increases in sperm head, neck, tail, leading to the total sperm abnormality enhancement. In middle fluoride group, the total and tail abnormalities were higher than those in control, while in low fluoride group sperm abnormalities presented increasing trend with no statistical difference. In all experimental groups, the percent of sperm with abnormal chromatin structure was significantly higher than control group. Compared with the controls, the percentage of sperm with aniline blue staining markedly increased, and the total sulfhydryl level significantly decreased in the middle and high doses of fluoride groups. Besides the above, 70mg/L and150mg/LNaF also significantly decreased the expression levels of Prm1and Prm2, and the ratio of Prm1to Prm2 was reduced in high fluoride group with statistical difference.

[Conclusion] Effects of high fluoride on mice sperm transcript were evaluated through microarray chip technique and bioinformatics analysis, and the differently expressed genes related with signal transduction, amino acid phosphorylation, oxidative stress, cell cycle, cell apoptosis, electron transfer, glycolysis, chemiotaxis, spermatogenesis, and sperm capacitation were identified. The further investigations revealed that (1) excessive fluoride enhanced the sperm ROS content, reduced the TAC level, leading to the oxidative stress, plasma membrane impairment, mitochondrion structure lesion, and sperm apoptosis induced by triggering Cytochrome C/caspase-3 mitochondrion apoptosis pathway; (2) high fluoride adversely affected CatSper1 expression, Ca2+ concentration, and CALM/CaMK2 signaling pathway, resulting in the decrease in sperm hyperacitivarion; (3) excessive fluoride may reduce the levels of Prm1and Prm2mRNA and total sulfhydryl, and impair DNA integrity, finally cause sperm nucleus maturation obstruction and sperm head abnormality. These findings indicated that there remains a complex underlying mechanism of fluoride reproductive toxicity which requires further investigation.
论文目录:

 

中图分类号:

 X503.22    

馆藏号:

 Y3851    

开放日期:

 2015-04-20    

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