CS2致男(雄)性下丘脑-垂体-性腺轴功能紊乱机制初探及NO的干预作用

CS2致男(雄)性下丘脑-垂体-性腺轴功能紊乱机制初探及NO的干预作用 CS2致男(雄)性下丘脑-垂体-性腺轴功能紊乱机制初探 及NO的干预作用 华中科技大学 博士学位论文 CS2致男雄性下丘脑-垂体-性腺轴功能紊乱机制初探及NO的干 预作用 姓名:季佳佳 申请学位级别:博士 专业:劳动卫生与环境卫生学 指导教师:陈国元 2011-05-26 华 中 科 技 大 学 博 士 学 位 论 文CS 致男(雄)性下丘脑-垂体-性腺 轴功能紊乱的机制初探及 NO 2 的干预作用 博士研究生:季佳佳 导师:陈国元 教授 摘 要 二硫化碳(Carbon disulfide,CS)是一种应用广泛的有机溶剂,主要用于橡 胶的 2 硫化、谷物蒸熏、石蜡、石油的精炼以及粘胶纤维的制造等。CS 为多系统毒物,其 2 对神经系统、心血管系统、视觉系统和生殖系统的影响已有报道。CS 对男(雄)性 2 生殖系统的影响主要表现为性功能由亢进到减退,精子数减少,活力下降和畸形精子 数增多等。本实验室在前期的动物实验中发现,CS 可引起雄性大鼠血清性激素水平 2 发生改变,导致雄性大鼠下丘脑-垂体-性腺轴(Hypothalamic-pituitary-gonad axis, HPGA)功能紊乱。 性激素的分泌主要受 HPGA的调节,通过一系列正反馈及负反馈作用来调节激素 的分泌,使外周血激素的水平保持相对稳定,对维持生殖系统脏器发育、生殖功能及 第二性征具有重要作用。一氧化氮(Nitric Oxide,NO)是一种兼有第二信使、神经 递质和效应分子等多种生理功能的生物信号分子,在生物体内发挥着十分重 要的作 用。NO广泛分布于男性生殖系统的各个器官,对生殖的调节作用具有双重性。研究 表明,NO参与了整个 HPGA的平衡调节,NO合成的多寡是机体生殖内分泌功能紊 乱的重要步骤。本实验室前期研究发现 CS 可造成雄性大鼠睾丸组织的氧化损伤,导 2 致睾丸组织中 NO含量、总一氧化氮合酶(NOS)活力和诱导型 NOS(iNOS)活力 下降,因而推测,NO可能在 CS 导致的 HPGA功能紊乱的过程中发挥重要作用。 2 目前,有关 CS 对男(雄)性 HPGA影响的研究鲜有报道,其具体的分子调控机 2 制尚不清楚。本项目以 CS 为受试物,以 HPGA为研究对象,将职业人群调查、整体 2 3 华 中 科 技 大 学 博 士 学 位 论 文动物实验和体外细胞培养相结合,探讨 CS 对男(雄)性 HPGA功能的影响及其机制, 2 并探讨 NO供体硝普钠(Sodium Nitroprusside,SNP)和总 NOS抑制剂 N-甲基-L-精 G 氨酸(N - Monomethyl- L- arginine,L-NMMA)的干预作用,为进一步阐明 NO对生 殖内分泌的作用,丰富对 CS 致男(雄)性生殖损伤分子机制的认识提供科学依据。 2 第一部分 CS 职业接触人群调查研究 2 一、CS 职业暴露对男工性功能和生殖结局的影响 2 目的:探讨 CS 对接触男工性功能及生殖结局的影响。 2 :对 276名 CS 暴露男工和 126名非 CS 暴露男工的基本情况、性功能及其 2 2 配偶生殖结局进行调查研究。 结果:性功能调查结果显示,CS 暴露组男工性生活和谐比例及性生活频度降低, 2 性生活厌恶感增加(P 0.05);各组间男工妻子妊娠并发症发生率无统计学差异(P 0.05)。生殖结局调查结果表明,混合组(夫妻双方均接触 CS)的自然流产率与 2 单纯组(只有男工接触 CS 而其妻子不接触)和对照组比较均具有统计学差异(P 2 0.05);混合组与对照组比较,子代低出生体重发生比例增高(P 0.05),子代智力 发育及生长发育无统计学差异(P 0.05)。 结论:CS 职业暴露可导致男性性功能障碍和不良生殖结局,但对子代的智力和 2 生长发育影响不明显。 二、CS 职业暴露对男工血清性激素水平的影响 2 目的:探讨 CS 对职业接触男工血清性激素水平的影响。 2 方法:选择工龄、文化程度、生活条件等基本情况相似的 CS 暴露男工 63名和 2 非 CS 暴露男工 69名,对其血清 GnRH、FSH、LH和 T水平进行检测。 2 结果:工龄 2~8年的 CS 暴露组男工,血清 T和 LH水平明显高于对照组(P 2 0.05);工龄 8年以上的 CS 暴露组男工,血清 T、FSH、GnRH水平明显高于对照组, 2 具有统计学差异(P 0.05)。 结论:职业接触 CS 可导致男性生殖内分泌功能紊乱,血清 LH水平首先发生改 2 变,可作为反映 CS 生殖损伤的早期指标。 2 第二部分 CS 对大鼠下丘脑-垂体-性腺轴超微病理结构的影响以及 NO的干预作用 2 4 华 中 科 技 大 学 博 士 学 位 论 文目的:探讨 CS 对雄性大鼠下丘脑-垂体-性腺轴超微病理结构的影响及 NO的干 2 预作用。 3 方法: 36 只雄性 SD大鼠随机分为 6组,以不同浓度 CS(0、 50、 250、 1250 mg/m) 2 3 3 静式吸入染毒,共 10周,另设 CS(1250 mg/m) +SNP(5mg/kg)和 CS(1250 mg/m) 2 2 +L-NMMA(2mg/kg)干预组,SNP和 L-NMMA从动物染毒结束前 10 d开始腹腔注 射,1次/d。染毒结束后,采用透射电镜观察大鼠下丘脑、垂体和睾丸组织超微病理 结构的改变。 结果:CS 染毒可造成下丘脑神经元、垂体促性腺激素细胞、生长激素细胞和睾 2 丸支持细胞线粒体肿胀,内质网扩张,NO供体对 CS 引起的下丘脑、垂体、睾丸组 2 织的损伤具有拮抗作用,而 NOS抑制剂则进一步导致病变的发生。 结论:CS 可造成下丘脑、垂体和睾丸组织超微病理结构的改变,NO 在这过程 2 中发挥重要作用。 第三部分 大鼠下丘脑神经元和垂体前叶细胞体外培养方法的建立 一、新生大鼠下丘脑神经元原代培养和鉴定 目的:探讨大鼠下丘脑神经元体外培养技术。 方法:选用新生 SD大鼠,取下丘脑组织进行单细胞原代培养,倒置显微镜下动 态观察培养细胞在体外生长的情况,并采用尼氏染色法对体外培养的神经元进行鉴 定。 结果:培养 3 d时观察发现下丘脑神经元具有双极突起,突起较短;培养 7 d时 神经元突起长度达到最高峰;尼氏染色可见胞质内有颗粒状的尼氏体,呈深蓝色。 结论:本实验室采用的大鼠下丘脑神经元原代培养方法能够为开展神经内分泌的 分子生物学研究提供理想的体外实验模型。 二、大鼠垂体前叶细胞体外培养和鉴定 目的:探讨大鼠垂体前叶细胞体外培养技术。 方法:选用成年雄性 SD大鼠,取垂体前叶进行单细胞原代培养,倒置显微镜下 动态观察培养细胞在体外生长的情况,并采用免疫细胞化学染色技术对垂体前叶细胞 LH和 FSH分泌细胞进行鉴定。5 华 中 科 技 大 学 博 士 学 位 论 文结果:垂体前叶细胞呈圆形,折光性较强,成纤维细胞生长明显,原代培养后期 成纤维细胞已为主要细胞。免疫细胞化学染色结果显示,LH和 FSH免疫阳性细胞胞 浆呈棕黄色。 结论:体外培养的垂体细胞中有多种细胞的生长,反复差速贴壁法能较好的去除 成纤维细胞,提高垂体前叶细胞的纯度,但 LH和 FSH免疫反应阳性细胞比例较低。 第四部分 CS 致雄性大鼠下丘脑-垂体-性腺轴功能紊乱机制的研究 2 一、CS 对大鼠下丘脑-垂体-性腺轴激素和激素受体 mRNA表达水平的影响以及 NO 2 的干预作用 目的:探讨 CS 对大鼠下丘脑-垂体-性腺轴激素和激素受体 mRNA表达水平的影 2 响以及 NO的干预作用 方法:对体外培养的大鼠垂体前叶细胞进行染毒,共设 6个染毒组,分别为对照 组、 3个不同浓度 CS 染毒组(2.5、 5.0、 10.0 mmol/ml CS )、 SNP干 预组(10.0 mmol/ml 2 2 CS +20 mmol/ml SNP)和 L-NMMA干预组(10.0 mmol/ml CS +50 mmol/ml L-NMMA), 2 2 支持细胞的 6个染毒组分别为对照组、3个不同浓度 CS 染毒组(1.25、2.50、5.00 2 mmol/ml CS)、SNP干预组(5.00 mmol/ml CS +10 mmol/ml SNP)和 L-NMMA干预 2 2 组(5.00 mmol/ml CS +25 mmol/ml L-NMMA),采用 Real-time PCR对垂体前叶细胞 2 中 GnRHR mRNA和睾丸支持细胞中 FSHR、ABP、INH mRNA表达水平进行检测。 结果:垂体前叶细胞中 GnRHR mRNA表达水平在 10.0 mmol/ml CS 染毒时显著 2 降低(P 0.05),NOS抑制剂 L-NMMA可拮抗 CS 的作用,使 GnRHR mRNA表达 2 水平显著增高(P 0.05);在睾丸支持细胞中,不同浓度 CS 染毒组与对照组比较, 2 ABP和 INHa mRNA表达水平没有明显变化(P 0.05), 5.0 mmol/ml CS 染毒组 FSHR 2 和 INH? b mRNA表达水平显著降低,INH? a mRNA表达水平显著增高(P 0.05); NO供体 SNP干预组与 5.0 mmol/ml CS 染毒组比较,INHa mRNA表达水平显著增高 2 (P 0.05), ABP、 FSHR、 INH? a和 INH? b mRNA表达水平改变均不明显(P 0.05); NOS抑制剂 L-NMMA干预组与 5.0 mmol/ml CS 染毒组比较,ABP mRNA表达水平 2 显著增高(P 0.05),FSHR、INHa、INH? a和 INH? b mRNA表达水平改变均不明 显(P 0.05)。6 华 中 科 技 大 学 博 士 学 位 论 文结论:CS 可抑制垂体前叶细胞中 GnRHR、支持细胞中 FSHR和 INH? b的表达, 2 通过抑制 HPGA中多种激素和激素受体的合成,导致 HPGA功能紊乱,CS 对垂体 2 GnRHR的抑制作用与 NOS活性有关。 二、CS对大鼠下丘脑神经元、垂体前叶细胞和睾丸支持细胞内 cAMP/cGMP的影响 2 以及 NO的干预作用 目的:探讨 CS 对大鼠下丘脑神经元、垂体前叶细胞和睾丸支持细胞内 2 cAMP/cGMP的影响以及 NO的干预作用。 方法:对体外培养的大鼠下丘脑神经元和垂体前叶细胞进行染毒,共设 6个染毒 组,分别为对照组、3个不同浓度 CS 染毒组(2.5、5.0、10.0 mmol/ml CS)、SNP 2 2 干预组(10.0 mmol/ml CS +20 mmol/ml SNP)和 L-NMMA干预组(10.0 mmol/ml CS +50 2 2 mmol/ml L-NMMA),支持细胞的 6个染毒组分别为对照组、3个不同浓度 CS 染毒 2 组(1.25、2.50、5.00 mmol/ml CS)、SNP干预组(5.00 mmol/ml CS +10 mmol/ml SNP) 2 2 和 L-NMMA干预组(5.00 mmol/ml CS +25 mmol/ml L-NMMA),采用酶联免疫吸附 2 试验对下丘脑神经元、垂体前叶细胞和睾丸支持细胞中 cAMP和 cGMP水平进行检测。 结果:下丘脑神经元中,各染毒组 cAMP水平变化不明显(P 0.05),cGMP水 平在 10.0 mmol/ml CS 染毒组显著降低,SNP可拮抗 CS 的作用,使 cGMP水平显著 2 2 增高(P 0.05);垂体前叶细胞中 cAMP和 cGMP水平不随 CS 染毒浓度的增加而发 2 生改变(P 0.05),但 L-NMMA干预组与 10.0 mmol/ml CS 染毒组比较,细胞中 cAMP 2 和 cGMP水平均显著增高(P 0.05);睾丸支持细胞中各染毒组 cAMP和 cGMP水 平均无明显改变(P 0.05)。 结论:CS 可明显降低大鼠下丘脑神经元中 cGMP水平,并能被 SNP拮抗,但对 2 垂体和睾丸 cAMP和 cGMP没有影响,提示 CS 可能通过 NO-cGMP途径影响 下丘脑 2 分泌功能,而 CS 对垂体和支持细胞分泌功能的影响则不依赖于 cAMP和 cGMP。 2关键词:二硫化碳;下丘脑-垂体-性腺轴;细胞培养技术;一氧化氮;超微结 构; 激素7 华 中 科 技 大 学 博 士 学 位 论 文Primary study on the mechanism of CS -induced disorders of 2 hypothalamus-pituitary-gonad axis in male and the intervention of NO Candidate: : Jiajia Ji Supervisor: : Prof. Guoyuan Chen Abstract Carbon disulfide CS , a volatile organic solvent, is often used in various industrial 2 processes, such as vulcanizing rubber, fumigating grain, extracting oil, and manufacturing viscose rayon fibers. CS adversely affects nearly all organ systems. There are many reports 2 concerning CS toxicity particularly in peripheral and central nervous systems, 2 cardiovascular system, ophthalmological system, and even reproductive system. Many epidemiological studies showed that CS could affect male sexual function, such as change 2 of sexual function from sthenic to decrease, decrease of sperm count and sperm motility and increase of the ratio of sperm deformity. Preliminary study in our laboratory showed that CS can lead to secretion disorders of hypothalamus - pituitary - gonadal axis HPGA 2 of male rat Sex hormones secretion is mainly affected by the regulation of HPGA which makes peripheral hormone levels remain relatively stable through positive feedback and negative feedback effect of HPGA to regulate endocrine hormone. HPGA plays an important role in reproductive system viscera development, reproductive function and secondary sex characteristics. Nitric oxide NO is a kind of signal molecule which may have muhiplicate physiological functionsuch as secondary messenger, neurotransmitter and effect moleculeNO may distribute in almost all kind of organs of male reproductive system, which may have the function of bifunctional regulation for reproduction. There are many reports shows that, NO participated in the whole HPGA adjustment, the amount of NO is an important8 华 中 科 技 大 学 博 士 学 位 论 文step of the reproductive endocrine dysfunction. In our previous studies, CS exposure could 2 lead to decrease of the NO concentration in testicular tissue, total nitric oxide synthase NOS activity and inducible NOS iNOS activity. Therefore, we speculated that NO plays an important roal in the effects of CS on the disorders of the HPGA in male2 However, the mechanism by which CS exposure causes disorders of HPGA in male 2 remains unknown. The aim of the present study was to determine the effects of CS on 2 HPGA in male, and the intervention of NO donor sodium nitroprusside, SNP and NOS inhibitor N-methyl - L-arginine, L -NMMA, in order to further clarify the role of NO in reproductive endocrine system and pave the way for further research on mechanisms of CS 2 toxicity in male reproductive systemPart? Research of workers exposed to carbon disulfide 1、Effect of carbon disulfide on male sexual function and reproductive outcomes Objective: To investigate the sexual function and reproductive outcomes of male workers exposed to CS 2 Methods: In a retrospective study, the basic information, sexual function and reproductive outcomes of 276 workers exposed to CS were collected through a 2 self-administered questionnaire. Another 126 unexposed male workers set as controlResults: The sexual function investigation showed that male workers exposed to CS had 2 sexual unsatisfactory, decreased sexual frequency, and increased sex aversion P 0.05The rates of pregnancy complications of the wives in all groups had no statistical significance P 0.05. The rate of spontaneous abortion of the mixed exposure group both of male workers and their wives exposed to CS was significantly increased than that 2 of the single exposure male workers exposed to CS but their wives did not and control 2 group P 0.05. The rate of low birth weight infants of the mixed exposure group had significant increased than that of the control group P 0.05. Offsprings’ intelligence and physical development had no significant difference in all groups P 0.05Conclusion: Occupational exposure to CS had an effect on the attitude and frequency of 2 sex life of male workers, and may result in bad reproductive outcomes as well. But there were no significant changes of development in offsprings2、Levels of sex hormones in male workers exposed to carbon disulfide9 华 中 科 技 大 学 博 士 学 位 论 文Objective: To examine the effects of CS on the levels of sex hormones in male workers2 Methods: The study subjects were 63 male workers exposed to CS and 69 non-exposed 2 workers. The levels of gonadotropin-releasing hormone GnRH, follicle stimulating hormone FSH, luteinizing hormone LH and testosterone T in serum were determined to assess endocrine function. Potential confounding factors were adjusted forResults: The levels of serum LH and T in the exposed workers who exposed to CS for 2 2~8 years were significantly higher than that of the control group P 0.05. The levels of serum GnRH, FSH and T in the exposed workers who exposed more than 8 years were significantly higher than that of the control group P 0.05Conclusion: Occupational exposure to CS could induce endocrine dysfunction in male 2 workers. The level of serum LH which changes primarily can be used as the early indicators reflect injury of reproductive function induced by CS 2 Part ? Ultrastructure of hypothalamus - pituitary - gonad axis in carbon disulfide - treated rats and the intervention of NO Objective: To observe the ultrastructural changes of HPGA in carbon disulfide - treated rats and the intervention of NOMethods: 36 Sprague-Dawley male rats were randomly divided into 6 groups. Four 3 groups were treated with CS at doses of 0, 50, 250 and 1250 mg/m by inhalation for 10 2 weeks. The rest two groups were intraperitoneal injection with SNP 5 mg/kg and L-NMMA 2 mg/kg once a day for 10 days after exposed to CS at a concentration of 1250 2 3 mg/m for 8 weeks. Ultramicropathology technique was used to detect ultrastructure of hypothalami, pituitaries and testes in ratsResults: Ultramicropathology technique analysis showed that CS caused ultrastructural 2 changes of hypothalamic neurons, gonadotropin cells, growth hormone cells and Sertoli cells in male rats. Mitochondria became sweller and endoplasmic reticulum widened. SNP could decrease the effects of CS on ultrastructural changes. The effects were adverse while 2 injected with L-NMMAConclusion: CS could induce ultrastructural changes of hypothalamus, pituitary and 2 testis in male rat, which is mediated by NOPart ? Culture of rat hypothalamus neurons and anterior pituitary cells in vitro10 华 中 科 技 大 学 博 士 学 位 论 文1、Culture and identification of newborn rat hypothalamus neurons in vitroObjective: To find out the best techniques for culture of nerve cells from hypothalamus in vitroMethods: We conducted primary culture of Hypothalamus neurons of newborn SD rat and identified the neurons by Nissl staining. We made dynamic observation on cultured cells by inverted microscope examination Results: The processus of nerve cells isolated from the rat hypothalamus were found on the third day of culture, and then reached their highest levels on the 7th day. Nissl staining showed Nissl's body dyeing was thick blue and granule formedConclusion: The method for culture of hypothalamal nerve cells were established successfully and could be used as an ideal experimental model on neuroendocrine molecular biology research2、Culture and identification of rat anterior pituitary cells in vitro Objective: To find out the best techniques for culture of rat anterior pituitary cells in vitroMethods: Anterior pituitary of adult SD male rats were isolated for single cell primary culture. We made dynamic observation on cultured cells by inverted microscope examination. Immunocytochemistry method was used to identify expression of LH and FSHResults: Anterior pituitary cells were round and had strong refraction. Fibroblasts grew obviously and became the cell in majority in the late stage of the primary culture cellsImmunochemistry staining showed that LH and FSH positive product was brown in cytoplasmConclusion: There were variety hormone cells in cultured pituitary cells. The repeated differential adhesion method could remove fibroblasts and sulted in better purification of anterior pituitary cells. LH and FSH positive cells accounted low proportion in cultured pituitary cellsPart ? Mechanism research on secretion disorders of HPGA induced by CS in male 2 rat 11 华 中 科 技 大 学 博 士 学 位 论 文1、Effect of CS on hormone and hormone receptor mRNA in rat cells in vitro and the 2 intervention of NO Objective: To investigate effect of CS on GnRH, ABP, FSHR and INH mRNA in rat 2 cells in vitro and the intervention of NOMethods: The cultured anterior pituitary cells were divided into six groups: control group; three various concentrations of CS groups 2.5, 5.0, 10.0 mmol/ml; SNP group 10.0 2 mmol/ml CS + 20 mmol/ml SNP and L-NMMA group 10.0 mmol/ml CS + 50 mmol/ml 2 2 L-NMMA. The six groups of Sertoli cells were control group, three various concentrations of CS groups 1.25, 2.50, 5.00 mmol/ml, SNP group 5.00 mmol/ml CS + 10 mmol/ml 2 2 SNP and L-NMMA group 5.00 mmol/ml CS + 25 mmol/ml L-NMMA. Real-time PCR 2 was used to detect GnRHR mRNA in anterior pituitary cells and ABP, FSHR, INH mRNA in Sertoli cells of ratResults: The mRNA level of GnRHR in rat anterior pituitary cells was significantly lower in 10.0 mmol/ml CS group than that of the control group, which could be attenuated 2 by L-NMMA P 0.05. In Sertoli cells of rat, no significant effects of ABP and INHa mRNA were observed in various concentrations of CS groups P 0.05. In the 5.0 2 mmol/ml CS group, FSHR and INH?b mRNA expression level w ere significantly lower 2 and INH?a mRNA expression level was significantly higher than that of the control group P 0.05. In the SNP group, INHa mRNA expression level was significantly higher than that of the 5.0 mmol/ml CS group P 0.05, but ABP, FSHR, INH? a and INH?b mRNA 2 were no significant differences between these two groups P 0.05. In the L-NMMA group, ABP mRNA expression level was significantly higher than that of the 5.0 mmol/ml CS group P 0.05, but no significant effects of FSHR, INHa, INH?a and INH? b mRNA 2 were observed between these two groups P 0.05 Conclusion: CS could induce secretion disorders of HPGA in male rat by inhibit 2 synthesis and secretion of various hormones and hormone receptors including GnRHR in pituitary and FSHR, INH?b in Sertoli cells. Inhibition of GnRHR in pituitary induced by CS related to activity of NOS2 2、Effect of CS on cAMP and cGMP in rat hypothalamus neurons, anterior pituitary 2 12 华 中 科 技 大 学 博 士 学 位 论 文cells and Sertoli cells, and the intervention of NO Objective: To investigate effect of CS on cAMP and cGMP in rat hypothalamus neurons, 2 anterior pituitary cells and Sertoli cells, and the intervention of NOMethods: The cultured hypothalamus neurons and anterior pituitary cells were divided into six groups: control group; three various concentrations of CS groups 2.5, 5.0, 10.0 2 mmol/ml; SNP group 10.0 mmol/ml CS + 20 mmol/ml SNP and L-NMMA group 10.0 2 mmol/ml CS + 50 mmol/ml L-NMMA. The six groups of Sertoli cells were control group, 2 three various concentrations of CS groups 1.25, 2.50, 5.00 mmol/ml, SNP group 5.00 2 mmol/ml CS + 10 mmol/ml SNP and L-NMMA group 5.00 mmol/ml CS + 25 mmol/ml 2 2 L-NMMA. Enzyme-linked immuno sorbent assay ELISA was used to detect cAMP and cGMP in rat hypothalamus neurons, anterior pituitary cells and Sertoli cellsResults: In hypothalamus neurons of rat, no significant effect of cAMP was observed in various concentrations of CS groups P 0.05. The level of cGMP in rat hypothalamus 2 neurons was significantly lower in 10.0 mmol/ml CS group than that of the control group, 2 which could be attenuated by SNP P 0.05. There were no significant differences in the levels of cAMP and cGMP in rat anterior pituitary cells between different doses of CS 2 groups and control group P 0.05. In the L-NMMA group, cAMP and cGMP levels were significantly higher than that of the 10.0 mmol/ml CS group P 0.05. In rat Sertoli cells, 2 significant effects of cAMP and cGMP were observed neither in various concentrations of CS groups nor the SNP and L-NMMA group P 0.052 Conclusion: CS could significantly reduce the level of cGMP in hypothalamus, which 2 could be attenuated by SNP, but no significant effect of cAMP and cGMP was observed in pituitary and Sertoli cells. It suggested that CS could induce secretion disorders of 2 hypothalamus in male rat through NO-cGMP pathway, but the effect of CS on endocrine 2 function of the pituitary and Sertoli cells were not dependent on cAMP and cGMP Key words: carbon disulfide; hypothalamus-pituitary-gonadal axis; cell culture techniques; Nitric oxide; ultrastructure; hormones13