Study On The Mechanism Of CS₂-induced Male Germ Cells And Sperms Injury

Carbon disulfide (CS2) is an important industrial solvent and material. Animals and humans exposed to CS2easily because of its extensive usage. It has been demonstrated that CS2induces serious impairments to many systems. A number of scholars reported CS2toxicity on nervous system and cardiovascular system. But few researches were carried out on the topic of CS2induces male reproductive toxicity. And the mechanism of CS2induces male reproductive toxicity is currently unknown. In present study, animal experiments were taken to explore the molecular mechanisms by which CS2induces testes impairments and germ cells injury in male rats. Moreover, epidemiologic investigation and laboratory researches were carried out to clarify the male reproductive toxicology. Our ultimate purpose is to provide a scientific basis on the molecular mechanisms on CS2induces sperm injury, and clarify the relationship between occupational CS2exposure and male sexual behavior, sexual hormones, semens quality of male workers. Parti Ⅰ The role of Mitochondrial apoptotic pathway in CS2-induced testicular cell injury Objective:To investigate the effects of CS2on testicular ultrastructure, germ cells injury and the intervention effect of MPTP inhibitor (Cyclosporin A, CsA). Methods:Forty-eight SD male rats were randomly divided into6groups. The front four groups statically inhaled to four doses of CS2(0,50,250,1250mg/m3) for10w. The fifth group was treated with CsA. The sixth group was co-administrated with CS2and CsA. During the last6w of exposure period, the latter two groups were gavaged CsA. Light microscopy, electron microscopy, TUNEL assay, intracellular Ca2+detection kit, ROS detection kit, mitochondrial transmembrane potential (A Ψm) detection kit, ATP detection kit, MPTP proteins detection kit, RT-PCR and Western Blot were used to explore the effects of CS2on testes of rats.Results:After CS2exposure, the ultrastructure of germ cells were impaired, mitochondria were vacuolated, apoptotic germ cells were increased, intracellular Ca2+were accumulated, ROS levels were elevated and activities of complexes of respiratory chain were improved. Meanwhile, ΔΨm, levels of ATP and MPTP proteins were significantly decreased. The results of real-time PCR and western blot analyses showed that the expression of Bcl-2was remarkably decreased, while the expressions of Bax, Cyt C, were simultaneously increased. All these changes of expressions took place in a dose-dependent manner. Moreover, our results revealed that CsA reversed or alleviated the adverse effects of CS2on testicular germ cells. Conclusion:Our findings suggest that CS2induces testicular injury via mitochondrial apoptotic pathway, and MPTP plays a crucial role in this process. In addition, CsA has potential therapeutic effect on the injury of male reproductive system caused by CS2.Part ⅡThe role of Endoplasmic reticulum (ER) apoptotic pathway in CS2induced Sertoli cell injuryObjective:To establish Sertoli cell primary culture method, and investigate whether Endoplasmic reticulum apoptotic pathway takes a part in CS2induced Sertoli cell injury.Methods:Thirty-two mature male SD rats were divided into4groups, treated with CS2(0,50,250,1250mg/m3) for4w. After exposure, the testes were removed and treated into primary cells. The Sertoli cells were identified by Feulgen staining. Then, the activity of Caspase3, the concentration of intracellular Ca2+, mRNA and protein expressions of the endoplasmic reticulum apoptosis pathway-related genes (Calpain2, Cleaved-Caspase12, GRP78and CHOP) were measured.Results:After exposure for4w, loose structures of seminiferous tubules and disordered cell arrangements were observed by light microscopy. Ultrastructural lesions, deformed chromatins and vacuoles formed from swollen ER were observed by electron microscopy. After primary culture of Sertoli cells, a dose-dependent increased apoptosis were found. The increased activity of Caspase3, accumulation of intracellular Ca2+, up-regulation of mRNA and protein expressions of ER apoptotic relative molecules (Calpain2, Cleaved-Caspase12, GRP78and CHOP) were also found in this study.Conclusion:Our findings indicated that ER apoptotic pathway played an important role in CS2-induced Sertoli cell impairment. Moreover, we also clarified parts of the molecular mechanisms.Part ⅢThe effects of CS2exposure on male sexual function, sexual hormones and sperm quality of relative workers1. Effects of CS2on sexual function and sexual hormones of male workersObjective:To investigate the relationship between CS2exposure and the changes of sexual function and sexual hormones.Methods:A total of76male workers exposed to CS2were recruited for this study, while94workers who were not exposed to any toxic agent in the working environment served as controls. The effects of CS2on male sexual behavior were explored by questionnaire. While, the sexual hormones (SHBG, FSH, LH, T) of these workers were detected by lab experiments. The relationship between sexual function, sexual hormones and relative factors were analyzed with univariate methods.Results:In the present study, after CS2exposure, the rates of sexual aversion and sexual dysfunction of male workers was significantly increased. The results of sexual hormones showed that the exposed workers had significantly higher levels of FSH and LH, and lower levels of SHBG and T in serum. Conclusion:Male sexual behavior and sexual hormones of workers were disturbed by CS2exposure.2. Effects of CS2on sperm quality of male workersObjective:To investigate the effects and the relative mechanism of CS2exposure on male sperm quality.Methods:A total of76male workers exposed to CS2were recruited for this study, while94workers who were not exposed to any toxic agent in the working environment served as controls. The semens were collected for sperm routine analysis, the total antioxidant capacity, ΔΨm, expressions of MPTP and the complexes of respiratory chain. The relationship between sperm quality and relative factors were analyzed with univariate methods.Results:Compared with the control, the liquefaction time of semens and abnormality morphological sperms were statistically increased in the CS2-exposed group. While, sperm viability and sperm motility were all significantly decreased. The proportion of sperm chromatin fracture and the rate of sperm apoptosis were all increased. In addition, the total antioxidant capacity, ΔΨm, expressions of MPTP and the complexes of respiratory chain Ⅱ and Ⅳ were statistically decreased in comparison with the control.Conclusion:Occupational CS2exposure lead to significant decrease of sperm quality, and the dysfunction of mitochondria played a crucial role in this process.