二硫化碳暴露致男（雄）性生殖细胞和精子损伤机制的研究
发布时间：2018-09-17 09:22
【摘要】：二硫化碳(Carbon Disulfide, CS_2)是重要的化工溶剂和原料,它的使用范围非常广泛,因此人群和动物很容易处于CS2暴露之中。研究报道,CS2对哺乳动物多种器官、系统具有毒性作用。但在毒性作用机制的研究方面,关于CS2对神经系统和心血管系统毒性研究的报道较多,而关于CS2对男(雄)性生殖毒性研究的报道匮乏。本研究通过体内和体外实验探索CS2诱导雄性睾丸生殖细胞损伤的机制,通过队列研究探讨CS2的男性生殖毒性,并进一步通过分子生物学实验研究精子细胞损伤的机制。为最终阐明CS2暴露诱导男(雄)性生殖细胞损伤的机制,及明确CS2职业暴露与男工性功能和性激素水平的关系提供理论基础和科学依据。 第一部分线粒体凋亡通路在CS2诱导睾丸生殖细胞损伤中的作用 目的：探讨CS2对雄性大鼠睾丸超微结构和生殖细胞的影响,探索线粒体离子通道转换在其中的作用和机制,并研究线粒体通透性转换孔(MPTP)抑制剂CsA的干预作用。 方法：48只雄性SD大鼠采用随机数法平均分为6组,前4组根据预实验以CS2递增浓度(0、50、250、1250mg/m~3)进行静式吸入染毒,共染毒10周。后两组设置为CsA(12.5mg/kg)对照组以及CS2(1250mg/m~3)+CsA(12.5mg/kg)干预组,动物染毒后6周,在吸入CS2的同时采用灌胃摄入CsA。染毒结束后,用光镜和电镜观察大鼠睾丸组织结构和超微结构的改变；使用相关试剂盒检测细胞凋亡率、细胞内钙离子浓度(Ca2+)、活性氧自由基(ROS)浓度、线粒体跨膜电位(△ψm)、胞内ATP含量和MPTP蛋白表达水平；实时定量PCR检测相关基因mRNA表达水平：Western Blot检测蛋白的表达水平。 结果：CS2暴露可造成睾丸生殖细胞超微结构损伤,线粒体肿胀、空泡化；生殖细胞凋亡显著增加,细胞内Ca2+蓄积,ROS浓度升高,线粒体呼吸链复合物活性增加。同时,△ψm、胞内ATP含量和MPTP开放水平显著下降。实时荧光PCR和Western blot分析结果显示,Bcl-2基大mRNA和蛋白1的表达显著降低,而Bax,Cyt C基因mRNA和蛋自的表达随CS2染毒浓度的增加而增加。而且,MPTP抑制剂CsA对CS2诱导睾丸组织和细胞的损伤具有一定的拮抗作用。 结论：CS2可造成睾丸组织、细胞和线粒体超微结构的损伤,并通过线粒体凋亡通路诱导睾丸生殖细胞凋亡。MPTP在其中发挥重要作用。另外,本研究结果提示CsA对CS2诱导睾丸细胞损伤可能具有潜在的治疗作用。 第二部分内质网凋亡通路在CS2诱导睾丸支持细胞损伤中的作用 目的：建立睾丸支持细胞原代培养体系,并探索内质网凋亡通路是否在CS2诱导睾丸支持细胞凋亡中发挥作用。 方法：32只雄性SD大鼠随机分为4组,以不同浓度CS2(0,50,250,1250mg/m3)静式吸入染毒,共4周。染毒结束后,取大鼠部分睾丸组织制片观察,其余部分睾丸组织进行支持细胞原代培养。采用富尔根染色法对体外培养的支持细胞进行鉴定,并在倒置显微镜下动态观察支持细胞在体外的生长情况。对原代培养支持细胞进行Caspase3活性、胞内Ca2+浓度、内质网凋亡通路相关基因(Calpain2, Cleaved-Caspase12, GRP78和CHOP) mRNA和蛋白表达的检测。 结果：暴露4周后,光学显微镜下观察发现大鼠睾丸曲细精管结构松散,细胞排列紊乱。电子显微镜观察到睾丸支持细胞超微结构损伤,染色质变性,并发现内质网肿胀形成空泡。对支持细胞进行原代培养发现,CS2暴露组细胞凋亡率与对照组相比显著增加,且凋亡率随着CS2暴露浓度增加而增加。Caspase3活性和细胞内Ca2+的浓度均显著增加，内质网凋亡相关分子(Calpain2, Cleaved-Caspase12, GRP78和CHOP)的基因mRNA和蛋白表达显著增加。 结论：本研究发现内质网凋亡通路在CS2诱导睾丸支持细胞凋亡中发挥重要作用。 第三部分职业性CS2暴露对男性工人性功能、性激素水平和精子质量的影响 一、职业性CS2暴露对男工性功能和性激素水平的影响 目的：研究职业性CS2暴露对男工性功能和性激素水平的影响。 方法：对76名CS2暴露男工和94名非CS2暴露男工的基本情况和性功能进行问卷调查：对其血清中性激素结合球蛋白(SHBG)、卵泡刺激素(FSH)、黄体生成素(LH)和睾酮(T)等激素水平进行实验室检测。并且对男工性功能和性激素水平与影响因素进行单因素分析。 结果：男性工人性功能调查结果显示,CS2暴露组男工存在性厌恶感和性功能障碍的人数比例显著高于对照组男工,差异有统计学意义(P0.05)。男性工人性激素水平检测结果显示,与对照组相比,CS2暴露组男工FSH和LH水平显著增高,血清SHBG和T水平显著降低(P0.05)。 结论：职业性CS2暴露可导致男性工人发生性功能障碍和性激素水平异常比例增高。 二、职业性CS2暴露对男工精子质量的影响 目的：探讨职业性CS2暴露对男工精子质量的影响及导致损伤的分子机制。 方法：选择人口学特征基本相似的CS2暴露男工76名和非CS2暴露男工94名,采集精液进行精子常规分析、精液抗氧化能力、精子线粒体膜电位、MPTP蛋白表达和精子线粒体呼吸酶复合物等指标的实验室研究。并且对男工精子质量与影响因素进行单因素分析。 结果：与对照组相比,职业性CS2暴露男工精液液化时间延长,精子畸形率升高,精子活率降低,精子活动力减弱,精子细胞凋亡率和染色质断裂精子比例均升高。而且精液总抗氧化能力、精子蛋白表达和精子线粒体呼吸酶复合物Ⅱ和Ⅳ活性均降低。 结论：职业性CS2暴露可导致男工精子质量显著降低,而线粒体功能损伤是导致男性工人精子质量降低的重要因素。
[Abstract]:Carbon disulfide (CS2) is an important chemical solvent and raw material. It has a wide range of applications, so people and animals are easy to be exposed to CS2. Studies have reported that CS2 has toxic effects on many mammalian organs. However, in the study of the mechanism of toxicity, CS2 on the nervous system and cardiovascular system. There are many reports on systemic toxicity, but there are few reports on the toxicity of CS2 to male (male) reproduction. This study explored the mechanism of CS2-induced male testicular germ cell damage in vivo and in vitro, explored the male reproductive toxicity of CS2 through cohort study, and further studied sperm cell damage through molecular biological experiments. The results provide theoretical and scientific basis for clarifying the mechanism of CS2 exposure induced male (male) sexual germ cell injury and the relationship between CS2 occupational exposure and sexual function and sex hormone level of male workers.
Part one the role of mitochondrial apoptotic pathway in CS2 induced testicular germ cell injury
AIM: To investigate the effects of CS2 on the ultrastructure and germ cells of testis in male rats, explore the role and mechanism of mitochondrial ion channel transition, and study the intervention of CsA, an inhibitor of mitochondrial permeability transition pore (MPTP).
Methods: 48 male SD rats were divided into 6 groups by random number method. The first 4 groups were treated with CS2 incremental concentration (0,50,250,1250 mg/m 3) by static inhalation for 10 weeks according to the preliminary experiment. After administration of CsA, the histological and ultrastructural changes of rat testis were observed by light and electron microscopy; the apoptosis rate, intracellular calcium ion concentration (Ca2+), reactive oxygen species (ROS), mitochondrial transmembrane potential (_m), intracellular ATP content and MPTP protein expression were detected by relevant kits; and the expression level of ATP and MPTP protein was measured by real-time quantitative PCR. The expression level of mRNA was detected by Western Blot.
Results: CS2 exposure could cause ultrastructural damage of testicular germ cells, mitochondria swelling and vacuolation; germ cell apoptosis increased significantly, intracellular Ca2+ accumulation, ROS concentration increased, and mitochondrial respiratory chain complex activity increased. The results showed that the expression of Bcl-2-based large mRNA and protein-1 decreased significantly, while the expression of Bax, Cyt-C gene mRNA and egg self-expression increased with the increase of CS2 concentration.
Conclusion: CS2 can damage the ultrastructure of testicular tissue, cells and mitochondria, and induce testicular germ cell apoptosis through mitochondrial apoptosis pathway. MPTP plays an important role in CS2-induced testicular germ cell apoptosis.
The second part is the role of endoplasmic reticulum apoptosis pathway in CS2 induced Sertoli cell injury.
Objective: To establish a primary culture system of testicular Sertoli cells and explore whether the endoplasmic reticulum apoptosis pathway plays a role in CS2-induced apoptosis of testicular Sertoli cells.
Methods: Thirty-two male SD rats were randomly divided into four groups and exposed to CS2 (0,50,250,1250 mg/m3) for 4 weeks. After exposure, some testicular tissues were taken for observation and the rest of testicular tissues were cultured for primary culture. Caspase 3 activity, intracellular Ca2+ concentration, mRNA and protein expression of endoplasmic reticulum apoptosis pathway related genes (Calpain 2, Cleaved-Caspase 12, GRP78 and CHOP) were detected in primary cultured Sertoli cells.
Results: After 4 weeks of exposure, the structure of testicular seminiferous tubules was loosened and the cells were disordered. The ultrastructural damage of Sertoli cells, chromatin degeneration and endoplasmic reticulum swelling were observed under electron microscope. The apoptosis rate of Sertoli cells in CS2 exposed group was compared with that in control group. Caspase 3 activity and intracellular Ca 2+ concentration increased significantly, and the mRNA and protein expression of endoplasmic reticulum apoptosis-related molecules (Calpain 2, Cleaved-Caspase 12, GRP78 and CHOP) increased significantly.
CONCLUSION: The endoplasmic reticulum apoptosis pathway plays an important role in CS2-induced apoptosis of Sertoli cells.
The third part is the effect of occupational CS2 exposure on sexual function, sex hormone levels and sperm quality in male workers.
Effect of occupational exposure to CS2 on sexual function and sex hormone levels in male workers
Objective: To study the effects of occupational CS2 exposure on sexual function and sex hormone levels in male workers.
Methods: Seventy-six CS2 exposed male workers and 94 non-CS2 exposed male workers were investigated by questionnaire. The levels of sex hormone binding globulin (SHBG), follicle stimulating hormone (FSH), luteinizing hormone (LH) and testosterone (T) in their serum were measured in laboratory, and the sex function and sex hormone levels in male workers were determined. Univariate analysis was performed.
Results: The sexual function survey showed that the proportion of male workers with sexual aversion and sexual dysfunction in CS2 exposed group was significantly higher than that in control group (P And T level decreased significantly (P0.05).
CONCLUSION: Occupational CS2 exposure can lead to sexual dysfunction and abnormal sex hormone levels in male workers.
Two, the effect of occupational CS2 exposure on sperm quality of male workers.
Objective: To explore the effect of occupational CS2 exposure on sperm quality and molecular mechanism of male workers.
Methods: Seventy-six CS2 exposed male workers and 94 non-CS2 exposed male workers with similar demographic characteristics were selected to collect semen for routine sperm analysis, antioxidant capacity, mitochondrial membrane potential, MPTP protein expression and mitochondrial respiratory enzyme complex. Univariate analysis was performed.
Results: Compared with the control group, CS2 exposed male workers had longer liquefaction time, higher sperm deformity rate, lower sperm motility, lower sperm apoptosis rate and higher percentage of chromatin breakage sperm. Sex decreased.
CONCLUSION: Occupational exposure to CS2 can result in significant reduction of sperm quality in male workers, and mitochondrial dysfunction is an important factor leading to the decrease of sperm quality in male workers.
【学位授予单位】：华中科技大学
【学位级别】：博士
【学位授予年份】：2015
【分类号】：R114
本文编号：2245423
[Abstract]:Carbon disulfide (CS2) is an important chemical solvent and raw material. It has a wide range of applications, so people and animals are easy to be exposed to CS2. Studies have reported that CS2 has toxic effects on many mammalian organs. However, in the study of the mechanism of toxicity, CS2 on the nervous system and cardiovascular system. There are many reports on systemic toxicity, but there are few reports on the toxicity of CS2 to male (male) reproduction. This study explored the mechanism of CS2-induced male testicular germ cell damage in vivo and in vitro, explored the male reproductive toxicity of CS2 through cohort study, and further studied sperm cell damage through molecular biological experiments. The results provide theoretical and scientific basis for clarifying the mechanism of CS2 exposure induced male (male) sexual germ cell injury and the relationship between CS2 occupational exposure and sexual function and sex hormone level of male workers.
Part one the role of mitochondrial apoptotic pathway in CS2 induced testicular germ cell injury
AIM: To investigate the effects of CS2 on the ultrastructure and germ cells of testis in male rats, explore the role and mechanism of mitochondrial ion channel transition, and study the intervention of CsA, an inhibitor of mitochondrial permeability transition pore (MPTP).
Methods: 48 male SD rats were divided into 6 groups by random number method. The first 4 groups were treated with CS2 incremental concentration (0,50,250,1250 mg/m 3) by static inhalation for 10 weeks according to the preliminary experiment. After administration of CsA, the histological and ultrastructural changes of rat testis were observed by light and electron microscopy; the apoptosis rate, intracellular calcium ion concentration (Ca2+), reactive oxygen species (ROS), mitochondrial transmembrane potential (_m), intracellular ATP content and MPTP protein expression were detected by relevant kits; and the expression level of ATP and MPTP protein was measured by real-time quantitative PCR. The expression level of mRNA was detected by Western Blot.
Results: CS2 exposure could cause ultrastructural damage of testicular germ cells, mitochondria swelling and vacuolation; germ cell apoptosis increased significantly, intracellular Ca2+ accumulation, ROS concentration increased, and mitochondrial respiratory chain complex activity increased. The results showed that the expression of Bcl-2-based large mRNA and protein-1 decreased significantly, while the expression of Bax, Cyt-C gene mRNA and egg self-expression increased with the increase of CS2 concentration.
Conclusion: CS2 can damage the ultrastructure of testicular tissue, cells and mitochondria, and induce testicular germ cell apoptosis through mitochondrial apoptosis pathway. MPTP plays an important role in CS2-induced testicular germ cell apoptosis.
The second part is the role of endoplasmic reticulum apoptosis pathway in CS2 induced Sertoli cell injury.
Objective: To establish a primary culture system of testicular Sertoli cells and explore whether the endoplasmic reticulum apoptosis pathway plays a role in CS2-induced apoptosis of testicular Sertoli cells.
Methods: Thirty-two male SD rats were randomly divided into four groups and exposed to CS2 (0,50,250,1250 mg/m3) for 4 weeks. After exposure, some testicular tissues were taken for observation and the rest of testicular tissues were cultured for primary culture. Caspase 3 activity, intracellular Ca2+ concentration, mRNA and protein expression of endoplasmic reticulum apoptosis pathway related genes (Calpain 2, Cleaved-Caspase 12, GRP78 and CHOP) were detected in primary cultured Sertoli cells.
Results: After 4 weeks of exposure, the structure of testicular seminiferous tubules was loosened and the cells were disordered. The ultrastructural damage of Sertoli cells, chromatin degeneration and endoplasmic reticulum swelling were observed under electron microscope. The apoptosis rate of Sertoli cells in CS2 exposed group was compared with that in control group. Caspase 3 activity and intracellular Ca 2+ concentration increased significantly, and the mRNA and protein expression of endoplasmic reticulum apoptosis-related molecules (Calpain 2, Cleaved-Caspase 12, GRP78 and CHOP) increased significantly.
CONCLUSION: The endoplasmic reticulum apoptosis pathway plays an important role in CS2-induced apoptosis of Sertoli cells.
The third part is the effect of occupational CS2 exposure on sexual function, sex hormone levels and sperm quality in male workers.
Effect of occupational exposure to CS2 on sexual function and sex hormone levels in male workers
Objective: To study the effects of occupational CS2 exposure on sexual function and sex hormone levels in male workers.
Methods: Seventy-six CS2 exposed male workers and 94 non-CS2 exposed male workers were investigated by questionnaire. The levels of sex hormone binding globulin (SHBG), follicle stimulating hormone (FSH), luteinizing hormone (LH) and testosterone (T) in their serum were measured in laboratory, and the sex function and sex hormone levels in male workers were determined. Univariate analysis was performed.
Results: The sexual function survey showed that the proportion of male workers with sexual aversion and sexual dysfunction in CS2 exposed group was significantly higher than that in control group (P And T level decreased significantly (P0.05).
CONCLUSION: Occupational CS2 exposure can lead to sexual dysfunction and abnormal sex hormone levels in male workers.
Two, the effect of occupational CS2 exposure on sperm quality of male workers.
Objective: To explore the effect of occupational CS2 exposure on sperm quality and molecular mechanism of male workers.
Methods: Seventy-six CS2 exposed male workers and 94 non-CS2 exposed male workers with similar demographic characteristics were selected to collect semen for routine sperm analysis, antioxidant capacity, mitochondrial membrane potential, MPTP protein expression and mitochondrial respiratory enzyme complex. Univariate analysis was performed.
Results: Compared with the control group, CS2 exposed male workers had longer liquefaction time, higher sperm deformity rate, lower sperm motility, lower sperm apoptosis rate and higher percentage of chromatin breakage sperm. Sex decreased.
CONCLUSION: Occupational exposure to CS2 can result in significant reduction of sperm quality in male workers, and mitochondrial dysfunction is an important factor leading to the decrease of sperm quality in male workers.
【学位授予单位】：华中科技大学
【学位级别】：博士
【学位授予年份】：2015
【分类号】：R114
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