CERTIFICATION I am Professor Chong-Gang Wang, hereby certify that I have read this manuscript and recommend for acceptance by the Xiamen University a

Size: px

Start display at page:

Download "CERTIFICATION I am Professor Chong-Gang Wang, hereby certify that I have read this manuscript and recommend for acceptance by the Xiamen University a"

此镇韦
5 years ago
Views:

1 学校编码 :10384 分类号密级学号 : UDC 硕士学位论文多氯联苯 (Aroclor 1254) 对小鼠精巢内精子发生的影响研究 EFFECT of PCBs (AROCLOR 1254) on SPERMATOGENESIS in TESTIS of MOUSE João Manuel Lopes Moreno 指导教师姓名 :Chong-Gang Wang 专业名称 : 生物化学与分子生物学论文提交日期 :2009 年 12 月论文答辩时间 :2009 年 12 月学位授予日期 :2009 年 12 月答辩委员会主席 : 评阅人 : 2009 年 12 月 i

2 CERTIFICATION I am Professor Chong-Gang Wang, hereby certify that I have read this manuscript and recommend for acceptance by the Xiamen University a dissertation entitled Effect of PCBs (Aroclor 1254) on Spermatogenesis in Testis of Mouse in fulfillment of degree of Master of Science of Xiamen University. Signed Date School of Life Sciences Xiamen University Xiamen, Fujian P.R. China Supervisor

3 厦门大学学位论文原创性声明兹呈交的学位论文, 是本人在导师指导下独立完成的研究成果本人在论文写作中参考的其他个人或集体的研究成果, 均在文中以明确方式标明本人依法享有和承担由此论文而产生的权利和责任声明人 ( 签名 ): 2009 年月日

4 DECLARATION I here declare that apart from the sources cited, this dissertation is my own work under the supervision of my supervisor Professor Chong-Gang Wang. The materials of the dissertation have not been presented and it will not be presented to any other university other than this university for similar or any other degree. Signature Candidate: João Manuel Lopes Moreno

5 厦门大学学位论文著作权使用声明本人完全了解厦门大学有关保留使用学位论文的规定厦门大学有权保留并向国家主管部门或其指定机构送交论文的纸质版和电子版, 有权将学位论文用于非赢利目的的少量复制并允许论文进入学校图书馆被查阅, 有权将学位论文的内容编入有关数据库进行检索, 有权将学位论文的标题和摘要汇编出版保密的学位论文在解密后适用本规定本学位论文属于 1 保密 ( ), 在年解密后适用本授权书 2 不保密 ( ) ( 请在以上相应括号内打 ) 作者签名 : 日期 :2009 年月日导师签名 : 日期 : 年月日

6 COPYRIGHT All rights reserved. No part of this dissertation may be reproduced, stored in any retrieval system, or transmitted in any from by any means: electronic, mechanical, photographing, recording or otherwise without prior written permission of the author or Xiamen University. Signature Candidate: João Manuel Lopes Moreno

7 目录摘要...v ABSTRACT... vi 1. 前言...1 I.1. PCBs 简介... 1 I.2. PCBs 来源以及在环境和人体中的暴露情况... 2 I.3. 内分泌干扰物... 3 I.4. PCBs 的毒性效应... 6 I.4.1. PCBs 的生殖毒性 I.5. 小鼠性腺的形态学 I.6. 生精上皮周期 I.6.1. 精子发生 I.6.2. 睾丸支持细胞 I.7. 睾丸间质细胞 I.8. 睾丸激素的调节与功能研究目的...21 目的研究的重要性材料和方法...24 II.1. 试剂和仪器 II.1.1. 试剂 II.1.2. 实验仪器 II.2. 动物染毒实验 II.3. 取样 II.4. 组织学观察 II.4.1. 形态测量技术 II.5. Western blot 分析 II.5.1. 样品预处理 II.5.2. 蛋白定量 II.5.3. Western blot 步骤 II 配制 SDS- 聚丙烯酰胺胶和其他 Western blot 试剂 II 电泳转膜 i

8 抗体孵育 ECL 显影 II.6. 性腺激素水平测定 II.6.1. 样品预处理 II.7. 数据处理与结果分析结果...32 III.1. 小鼠体重, 附睾重量和精子参数 III.2. 小鼠睾丸组织病理学 III.2.1. 定量测量 : III 输精管直径 III 生精细胞精原细胞精细胞支持细胞 III 定性测量组织形态学 III.3. Western blot 结果 III.3.1. 蛋白定量 III.3.2. PCBs 暴露对小鼠睾丸内 PCNA 表达的影响 III.3.3. PCBs 暴露对小鼠睾丸内雌激素受 α 和 β 雄激素受体表达的影响 III.4. PCBs 暴露对小鼠睾丸内睾酮激素和 17β-- 雌二醇水平的影响讨论...50 IV.1. PCBs 暴露对小鼠形态学参数的影响 IV.2. PCBs 暴露对小鼠睾丸组织学的影响 IV.3. PCBs 暴露对小鼠 PCNA 激素受体 (AR, ERα, ERβ) 的影响 IV.4. PCBs 暴露对小鼠睾丸内睾酮激素和 17β-- 雌二醇水平的影响结论...59 研究展望...60 致谢...61 参考文献...62 ii

9 TABLE OF CONTENTS ABSTRACT(IN CHINESE)...v ABSTRACT... vi CHAPTER I. INTRODUCTION...1 I.1. PCB... 1 I.2. SOURCE, ENVIRONMENTAL & HUMAN EXPOSURE OF PCBS... 2 I.3. ENDOCRINE DISRUPTORS... 3 I.4. TOXICITY OF PCBs... 6 I.4.1. PCBs and reproductive toxicity I.5. PHYSIOLOGY OF TESTIS I.6. CYCLE OF SEMINIFEROUS EPITHELIUM I.6.1. Spermatogenesis I.6.2. Sertoli cell I.7. LEYDIG CELL I.8. HORMONAL CONTROL and FUNCTION of TESTIS AIMS OF STUDY...21 Objectives SIGNIFICANCE OF STUDY CHAPTER II. MATERIAL AND METHOD...24 II.1. CHEMICALS and EQUIPMENT II.1.1. Chemical II.1.2. Experimental equipment II.2. ANIMALS and TREATMENT II.3. COLLECTION OF TISSUE SAMPLE II.4. HISTOLOGICAL EXAMINATION II.4.1. Morphometric Techniques II.5. WESTERN BLOT ANALYSIS II.5.1. Sample pre-treatment II.5.2. Protein quantification...27 II.5.3. Western blot procedure II Poly Acrylamide Gel Electrophoresis and western blot reagent formulations II Performance of Gel Electrophoresis and western blot Electrophorectic transfer of proteins on to PVDF membrane Immunodetection Enhanced chemiluminescente detection II.6. HORMONES LEVELS MEASUREMENT iii

10 II.6.1. Sample pre-treatment II.7. STATISTICAL ANALYSIS CHAPTER III. RESULTS...32 III.1. BODY WEIGHT, EPIDIDYMIS WEIGHT AND SPERM PARAMETERS III.2. HISTOPATHOLOGY III.2.1. Quantitative Measurement: III Seminiferous tubule diameter III Spermatogenic cell Spermatogonia Spermatides Sertoli cell III Qualitative measurement Histomorphology III.3. WESTERN BLOT III.3.1. Determination of protein quantification III.3.2. Effect of PCBs on the PCNA expression in testes III.3.3. Effect of PCBs on the expression of ER-α, ER- β, AR in testicle III.4. EFFECT OF AROCLOR 1254 ON THE LEVEL OF TESTOSTERONE AND 17Β- ESTRADIOL OF TESTIS IN MALE MICE CHAPTER IV- DISCUSSION...50 IV.1. EFFECT OF AROCLOR 1254 ON MORPHOLOGICAL PARAMETERS OF MALE MOUSE IV.2. EFFECT of AROCLOR 1254 on HISTOPATHOLOGICAL PARAMETERS of TESTIS IV.3. EFFECT OF AROCLOR 1254 ON PCNA AND STEROID HORMONES (AR, ER ALPHA/BETA) IN TESTIS IV.4. EFFECT OF AROCLOR 1254 ON TESTOSTERONE AND 17Β-ESTRADIOL IN TESTIS OF MALE MICE CONCLUSION...59 FURTHER STUDY...60 ACKNOWLEDGEMENT...61 REFERENCE...62 iv

11 ABSTRACT(IN CHINESE) 摘要 : 多氯联苯 (PCBs) 是一类由人工合成的氯代芳烃类化合物, 被称作潜在的内分泌干扰物由于他们具有类雌激素和抗雄激素的性质, 对雄性生殖系统造成极大危害, 导致不育和一些性激素依赖的生殖功能丧失他们能够引起内源性系统改变, 类似天然性激素, 并抑制性激素实现其功能本文目的是研究多氯联苯对雄性小鼠生殖系统的睾丸生长与发育的影响雄性小鼠随机分成五组, 每组 21 只采用不同浓度 Aroclor 1254(0, 0.5, 5, 50, 500 µg/kg) 对小鼠进行灌胃染毒, 每星期三次暴露 50 天后处死小鼠, 称其体重, 睾丸重量和附睾重量, 并进行精子计数和精子形态畸形实验结果表示, 处理组与对照组相比的小鼠体重与性腺指数并没有出现显著性差异精子死亡率在 500 μg/kg 处理组表现出显著的上升精子数量在 0.5 μg/kg, 5 μg/kg, 50 μg/kg 和 500 μg/kg 处理组都呈现出显著的下降精子畸形率在 50 μg/kg 和 500 μg/kg 处理组表现出显著的上升用放射免疫 (RIA) 方法检测睾丸内性激素水平变化结果表明, 与对照组相比, PCBs 处理组的睾酮和 17β 雌二醇均没有出现显著性变化对睾丸进行组织学观察发现, 输精管的直径和支持细胞的数量与对照组相比均呈现显著性上升 (p< 0.05), 精细胞的数量呈现显著性下降, 并呈剂量 - 效应关系, 而精原细胞没有出现显著性变化用 Western blot 方法检测处理组与对照组之间的细胞增殖核抗原 (PCNA) 雌激素受体 (ER α/β) 和雄激素受体 (AR) 蛋白水平变化统计分析发现, 处理理与对照组的 PCNA,AR, ER α 和 ER β 水平均没有出现显著性变化在本实验中, 精子质量和组织学上的变化表明环境浓度的 Aroclor 1254 能对引起雄性小鼠精子和精巢发生变化, 接下来我们将对 Aroclor 1254 对精子形成的影响及其机制做进一步研究关键词 :Aroclor 1254;PCBs; 精子发生 ; 雌激素受体 ; 小鼠 v

12 ABSTRACT Polychlorinated biphenyls (PCBs) are a group of halogenated aromatic hydrocarbons, synthetic chemicals which do not occur naturally in the environment. PCBs are considered potential endocrine disruptors. They are estrogen-like and anti-androgenic chemicals in the environment contain potentially hazardous effects on male reproductive axis resulting in infertility and other hormonal dependent reproductive functions. These toxic substance cause alteration of the endocrine systems, mimic natural hormones and inhibit the action of hormones. The aim of this study is to examine the effect of Polychlorinated biphenyls (PCBs) on testicular development of male reproductive system in mice. The male mice were randomly assigned to five groups with each group comprising twenty-one members. In those mice were administered 0 µg/kg (control group) and 0.5, 5, 50, 500 µg/kg Aroclor 1254 (treated group) by gavages three time per week. Treatment was carried out for 50 days after which the mouse was sacrificed and the body weight, testicular weight; epedidymis weight, sperm mortality, sperm count and sperm abnormality were taken. However, there was no significant difference in testicular/body weight and epididymis/body weight ratio in treated group compared with the control group. According to the analysis of sperm quality, Aroclor 1254 treated group demonstrated significant increased in sperm mortality in 500 μg/kg; decreased the sperm count in 0.5 μg/kg, 5 μg/kg, 50 μg/kg and 500 μg/kg; and significantly elevate the sperm abnormality in 50 μg/kg and 500 μg/kg compared to the control in a dose-dependent manner. The sex hormone levels in the testes were detected by radio-immunoassay (RIA) method. The levels of testosterone and 17β-estradiol did not reveal significant alteration (p< 0.05) in PCBs treated groups compared to the control in a dose-dependent manner. The testis were obtained and subjected to routine histopathology following exposure to PCBs in supplement diet. The diameter of the seminiferous tubule and the number of Sertoli cells in the treated group increased significantly (p< 0.05) in comparison to the control group. For the spermatogenic cell, the number of germ cell in high concentration decreased significantly (p< 0.05). However, spermatogonia cells in PCB treated group showed non-significant difference (p< 0.05) compared to the control. vi

13 Western blot analysis was used to determine the level of protein between the control and treated group. The level of Proliferating cell nuclear antigen (PCNA) was determined and the results have shown no significant alteration between the treated groups and the control. the level of sex hormone receptor (ER α/β); Androgen receptor (AR) were identified in the testes to detect the proliferative effect induced by PCBs. Statistical analyses of AR, ER α and ER β did not reveal significant difference between the control and the treated groups. In the present study, we continue to investigate adverse effect of Aroclor 1254 and their mechanism on spermatogenesis. The result of Sperm quality and histopathology showed that Aroclor 1254 at low concentration induce inhibitory effect on testicular function of male mouse. KEYWORDS: Aroclor 1254; PCBs; Spermatogenesis; Estrogen receptor; Mice. vii

14 ABP: Androgen binding protein AMH: Antimullerian hormone AR: Androgen receptor Ah receptor: BSA: EDC: ER: FSH: GnRH: HTF: LH: NBF: OCPs: PCB: PCNA: POPs: PVDF: SER: LIST OF ABBREVIATIONS Hydrocarbon receptor Bovine Serum Albumin Endocrine disrupting chemical Estrogen receptor Follicle stimulating hormone Gonadotrophin realising hormone The human tubule fluid Luteinizing hormone Neutral buffered formalin Organochlorine pesticides Polychlorinated biphenyl Proliferating cell nuclear antigen Persistent organic pollutants Polyvinylidene difluoride Endoplasmic reticulum viii

15 Effect of PCBs (Aroclor 1254) on Spermatogenesis in Testis of Mouse CHAPTER I. INTRODUCTION I.1. PCB Polychlorinated biphenyls (PCBs) are a group of halogenated aromatic hydrocarbons, synthetic chemicals which do not occur naturally in the environment. They are a group of biphenyl ring chemicals consisting of chlorine, carbon and hydrogen (C 12 H 10-R Cl R ), composed of 209 congeners however only approximately 130 congeners have actually been used in chemical formulations [1] [2] [3] [4] (figure 1). Their production significantly increased between begun of 1929 and peaked in the 1970 because of their useful physicochemical properties and large industrial and commercial use in hydraulic fluids, heat-transfer fluids, dielectric fluids, lubricants, inks, laminating oils, paints, adhesives, dedusting agents, fire retardants, wax extenders etc. Commercial PCBs and their environmental residues contain complex mixtures of congeners and elicit a large spectrum of biological responses [2]. They are lipophilic and have very low water solubilities, and their lipophilicity increases with increasing degree of chlorination. Congeners of PCB with a lower degree of chlorination are more volatile than those with a higher degree. Pure individual PCB congeners are colorless and often crystalline. Commercial PCB mixtures are clear to light yellow oils or resins and they do not crystallize, even at low temperatures. These chemical substances are practically fire resistant because of their high flash points ( C). They form vapors which are heavier than air, but are not explosive. They have low electrical conductivity, high thermal conductivity and high resistance to thermal degradation. On the basis of these properties they have been used as dielectric isolators in electrical equipment. Similar to many organochlorine compounds, many of the congeners are highly persistent and accumulate within food chains [5]. Figure 1 Chemical structure of PCBs[6]. 1

16 Effect of PCBs (Aroclor 1254) on Spermatogenesis in Testis of Mouse I.2. SOURCE, ENVIRONMENTAL & HUMAN EXPOSURE OF PCBs Various commercial PCB mixtures are known in the United States by the trade name Aroclor that have no known smell or taste [7]. The manufacture of PCBs was stopped in the U.S. in 1970 through the US EPA Toxic Substance Control Act (US Environmental Protection Agency). However, they continue to be detected as major global pollutants in the environment and cause harmful health effects [8]. Recent evidences have shown that PCBs enter the environment from the soil, although volatilization that begins in the soil is not a major source of outdoor air PCB concentrations. Rather, outdoor air concentrations in urban areas appear to be on the basis of venting of indoor air. While the use of PCB was banned many years ago, they are still found in the environment today. This is because of their thermal stability, resistance to microbial degradation, and chemical inertness [8]. These toxic substances have a tendency to persist in the environment, with half-lives for most congeners ranging from months to years. The discharge of PCBs from the soil is slow, particularly for the more highly chlorinated congeners, and translocation to plants via soil is insignificant. Cycling of PCBs through the environment involves volatilization from land and water surfaces into the atmosphere, with subsequent removal from the atmosphere by wet or dry deposition, then revolatilization [9]. PCBs belong to a group of chemicals known as persistent organic pollutants (POPs); these can cause global environmental contamination. The contamination of PCB (POPs) is a significant health problem because POPs can be accumulated and magnified through the food web or food chain. They can then have several adverse effects on human health and wildlife survival. Many examples of the accidental contamination by POPs abound and the risk assessment of POPs in food is important and necessary for human health [10]. Occupational exposure to PCBs occurs mainly via the inhalation and dermal routes. Commercial PCB mixtures are colorless to dark brown oils, viscous liquids, or sticky resinous semisolids. Although they evaporate slowly at room temperature, the volatility of PCBs increases dramatically with even a small increase in temperature. 2

17 Degree papers are in the Xiamen University Electronic Theses and Dissertations Database. Full texts are available in the following ways: 1. If your library is a CALIS member libraries, please log on and submit requests online, or consult the interlibrary loan department in your library. 2. For users of non-calis member libraries, please mail to etd@xmu.edu.cn for delivery details.

2

10384 -------- 9601008 UDC---------------------- 1999 1999 1999 ------------------------ ------------------------ 1 2 .4...4 7 14.14..18..22..22..24 3 4.27.32.39 1 2 3 4 5 6 7 8 9 10 11 5 12 6 1 2 3 4