Effects Of Carbon Disulfide On Apoptosis And Apoptosis-related Protein Expressions In Rat Sertoli-germ Cell Co-culture

Carbon disulfide (CS2) is a kind of popular organic solvent. 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. There is little research on the mechanisms about CS2 on reproductive system. In our research, we investigate rat Sertoli-germ cell co-culture to explore the expressions of apoptosis-related protein induced by CS2 thus paveing the way for further research on mechanisms of CS2 toxicity in the structure and cells function of the testis.In our research, we established the rat Sertoli-germ cell co-culture system in vitro and identified the Sertoli cells and germ cell with Feulgen staining. Then the Sertoli-germ cells were treated with CS2 and the survive rate was determined by MTT assay. Apoptosis index of Sertoli cells and germ cell were determined by TUNEL. The expression of P53 and Bcl-2 were determined by Western blot. And the changes of Fas and FasL were examined by immunohistochemistry method.The result show that cultures consisted of a monolayer of Sertoli cells, to which clusters of germ cells were attached, and the co-cultured cells were able to survive in vitro for a long time. Survival rate decreased as the concentration of CS2 increased. The survival rate in CS2 exposure group was significantly lower than that in the control group when the concentration of CS2≥100μmol/l (P<0.05). Apoptosis index increased as the concentration of CS2 increased(P<0.05 & P<0.01)so as the expression of P53 and Bcl-2. The expression of P53 in CS2 treat groups were significantly higher than that in the control group(P<0.05). And the expression of Bcl-2 in 100μmol/L and 200μmol/L concentration group were significantly higher than the control group(P<0.05). The expressions of Fas and FasL in CS2 exposure groups were higher than that in the control group.The above results indicate that in this co-culture system in vitro, germ cell can survive and attach to Sertoli cells. The Feulgen staining used to identify the Sertoli cell and germ cell is simple but feasible. We believe that this co-culture system provide the cell model for research of the reproduction toxicity and mechanisms on Sertoli cell and germ cell induced by chemistry material; CS2 is cyotoxic to rat Sertoli-germ cell co-culture system in vitro. Cell survival rate reduced and apoptosis rate increased; the expressions of P53, Bcl-2, Fas and FasL in rat Sertoli-germ cell co-culture increased. The expressions of Fas and FasL are up-regulated, which start Fas system to makes the spermatogenic cell apoptosis, and the expression of P53 increased further promote the occurrence of apoptosis. The expression of Bcl-2 may reactive up-regulated to inhibit apoptosis. Consequently, P53, Bcl-2 and Fas system take part in the regulation of Sertoli cell and germ cell apoptosis induced by CS2. It provides scientific evidence for exploring gene regulation mechanism of apoptosis, clarifying the mechanism of male procreation injury caused by CS2 and protecting the health of the occupational people.