Primary Study On The Mechanism Of CS₂-induced Disorders Of Hypothalamus-pituitary-gonad Axis In Male And The Intervention Of NO

Carbon disulfide (CS2), a volatile organic solvent, is often used in various industrial processes, such as vulcanizing rubber, fumigating grain, extracting oil, and manufacturing viscose rayon fibers. CS2 adversely affects nearly all organ systems. There are many reports concerning CS2 toxicity particularly in peripheral and central nervous systems, cardiovascular system, ophthalmological system, and even reproductive system. Many epidemiological studies showed that CS2 could affect male sexual function, such as change 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 CS2 can lead to secretion disorders of hypothalamus-pituitary-gonadal axis (HPGA) 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 molecule. NO 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 important step of the reproductive endocrine dysfunction. In our previous studies, CS2 exposure could 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 CS2 on the disorders of the HPGA in male.However, the mechanism by which CS2 exposure causes disorders of HPGA in male remains unknown. The aim of the present study was to determine the effects of CS2 on 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 CS2 toxicity in male reproductive system.Part I Research of workers exposed to carbon disulfide1,Effect of carbon disulfide on male sexual function and reproductive outcomesObjective:To investigate the sexual function and reproductive outcomes of male workers exposed to CS2.Methods:In a retrospective study, the basic information, sexual function and reproductive outcomes of 276 workers exposed to CS2 were collected through a self-administered questionnaire. Another 126 unexposed male workers set as control.Results:The sexual function investigation showed that male workers exposed to CS2 had sexual unsatisfactory, decreased sexual frequency, and increased sex aversion (P<0.05). The 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 CS2) was significantly increased than that of the single exposure (male workers exposed to CS2 but their wives did not) and control 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>0.05).Conclusion:Occupational exposure to CS2 had an effect on the attitude and frequency of sex life of male workers, and may result in bad reproductive outcomes as well. But there were no significant changes of development in offsprings.2,Levels of sex hormones in male workers exposed to carbon disulfide Objective:To examine the effects of CS2 on the levels of sex hormones in male workers.Methods:The study subjects were 63 male workers exposed to CS2 and 69 non-exposed 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 for.Results:The levels of serum LH and T in the exposed workers who exposed to CS2 for 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<0.05).Conclusion:Occupational exposure to CS2 could induce endocrine dysfunction in male workers. The level of serum LH which changes primarily can be used as the early indicators reflect injury of reproductive function induced by CS2.PartⅡUltrastructure of hypothalamus-pituitary-gonad axis in carbon disulfide-treated rats and the intervention of NOObjective:To observe the ultrastructural changes of HPGA in carbon disulfide-treated rats and the intervention of NO.Methods:36 Sprague-Dawley male rats were randomly divided into 6 groups. Four groups were treated with CS2 at doses of 0,50,250 and 1250 mg/m3 by inhalation for 10 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 CS2 at a concentration of 1250 mg/m3 for 8 weeks. Ultramicropathology technique was used to detect ultrastructure of hypothalami, pituitaries and testes in rats.Results:Ultramicropathology technique analysis showed that CS2 caused ultrastructural 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 CS2 on ultrastructural changes. The effects were adverse while injected with L-NMMA.Conclusion:CS2 could induce ultrastructural changes of hypothalamus, pituitary and testis in male rat, which is mediated by NO.PartⅢCulture of rat hypothalamus neurons and anterior pituitary cells in vitro 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 vitro.Methods: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 formed.Conclusion:The method for culture of hypothalamal nerve cells were established successfully and could be used as an ideal experimental model on neuroendocrine molecular biology research.2,Culture and identification of rat anterior pituitary cells in vitroObjective:To find out the best techniques for culture of rat anterior pituitary cells in vitro.Methods: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 FSH.Results: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 cells. Immunochemistry staining showed that LH and FSH positive product was brown in cytoplasm.Conclusion: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 cells.Part IV Mechanism research on secretion disorders of HPGA induced by CS2 in male rat 1,Effect of CS2 on hormone and hormone receptor mRNA in rat cells in vitro and the intervention of NOObjective:To investigate effect of CS2 on GnRH, ABP, FSHR and INH mRNA in rat cells in vitro and the intervention of NO.Methods:The cultured anterior pituitary cells were divided into six groups:control group; three various concentrations of CS2 groups (2.5,5.0,10.0μmol/ml);SNP group (10.0μmol/ml CS2+20μmol/ml SNP) and L-NMMA group (10.0μmol/ml CS2+50μmol/ml L-NMMA). The six groups of Sertoli cells were control group, three various concentrations of CS2 groups (1.25,2.50,5.00μmol/ml), SNP group (5.00μmol/ml CS2+10μmol/ml SNP) and L-NMMA group (5.00μmol/ml CS2+25μmol/ml L-NMMA). Real-time PCR was used to detect GnRHR mRNA in anterior pituitary cells and ABP, FSHR, INH mRNA in Sertoli cells of rat.Results:The mRNA level of GnRHR in rat anterior pituitary cells was significantly lower in 10.0μmol/ml CS2 group than that of the control group, which could be attenuated by L-NMMA (P<0.05). In Sertoli cells of rat, no significant effects of ABP and INHαmRNA were observed in various concentrations of CS2 groups (P>0.05). In the 5.0μmol/ml CS2 group, FSHR and INHβb mRNA expression level were significantly lower and INHβa 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 5.0μmol/ml CS2 group (P<0.05), but ABP, FSHR, INHβa and INHβb mRNA 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 5.0μmol/ml CS2 group (P<0.05), but no significant effects of FSHR, INHα, INHβa and INHβb mRNA were observed between these two groups (P>0.05).Conclusion:CS2 could induce secretion disorders of HPGA in male rat by inhibit synthesis and secretion of various hormones and hormone receptors including GnRHR in pituitary and FSHR, INHβb in Sertoli cells. Inhibition of GnRHR in pituitary induced by CS2 related to activity of NOS.2,Effect of CS2 on cAMP and cGMP in rat hypothalamus neurons, anterior pituitary cells and Sertoli cells, and the intervention of NOObjective:To investigate effect of CS2 on cAMP and cGMP in rat hypothalamus neurons, anterior pituitary cells and Sertoli cells, and the intervention of NO.Methods:The cultured hypothalamus neurons and anterior pituitary cells were divided into six groups:control group; three various concentrations of CS2 groups (2.5,5.0,10.0μmol/ml); SNP group (10.0μmol/ml CS2+20μmol/ml SNP) and L-NMMA group (10.0μmol/ml CS2+50μmol/ml L-NMMA). The six groups of Sertoli cells were control group, three various concentrations of CS2 groups (1.25,2.50,5.00μmol/ml), SNP group (5.00μmol/ml CS2+10μmol/ml SNP) and L-NMMA group (5.00μmol/ml CS2+25μmol/ml L-NMMA). Enzyme-linked immuno sorbent assay (ELISA) was used to detect cAMP and cGMP in rat hypothalamus neurons, anterior pituitary cells and Sertoli cells.Results:In hypothalamus neurons of rat, no significant effect of cAMP was observed in various concentrations of CS2 groups (P>0.05). The level of cGMP in rat hypothalamus neurons was significantly lower in 10.0μmol/ml CS2 group than that of the control group, 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 CS2 groups and control group (P>0.05). In the L-NMMA group, cAMP and cGMP levels were significantly higher than that of the 10.0μmol/ml CS2 group (P<0.05). In rat Sertoli cells, significant effects of cAMP and cGMP were observed neither in various concentrations of CS2 groups nor the SNP and L-NMMA group (P>0.05).Conclusion:CS2 could significantly reduce the level of cGMP in hypothalamus, which could be attenuated by SNP, but no significant effect of cAMP and cGMP was observed in pituitary and Sertoli cells. It suggested that CS2 could induce secretion disorders of hypothalamus in male rat through NO-cGMP pathway, but the effect of CS2 on endocrine function of the pituitary and Sertoli cells were not dependent on cAMP and cGMP.