The Effect Of P, P'-DDE-induced Testicular Apoptosis In Rats On Fas/FasL And Endoplasmic Reticulum-mediated Pathways

2,2-bis (4-Chlorophenyl) -1,1,1-trichloroethane (DDT), the first widely used synthetic organochlorine pesticide, is a kind of widespread environmental endocrine disrupting chemical and had been given credit for having helped one billion people live free from malaria. However, its bioaccumulation, long-range transport, persistence in the environment and antiandrogenic properties raise the concern about its possible long-term adverse effects. Though having being banned or restricted for three decades, DDT is still being used for the control of vectors in public health in some developing countries.1,1-dichloro-2,2 bis (p-chlorophenyl) ethylene (p,p'-DDE), DDT's major metabolite with the highest persistence, is the form usually found in human tissues in the highest concentration. It is a widespread environmental endocrine disrupting chemical. It has been reported that some abnormalities in sexual development in rats and wildlife might be associated with exposure to p,p'-DDE. p,p'-DDE is antiandrogenic and can inhibit androgen binding to the androgen receptor.Cell death by apoptosis is a part of normal development and maintenance of homeostasis, but is also involved in pathological situation associated with sterility. In the testis, apoptosis is such a common programmed event that 75 % of germ cells are reduced by spontaneous apoptosis. However, excessive or inadequate apoptosis of testicular cells result in abnormal spermatogenesis or testicular tumors. In rodents heat and irradiation as well as xenohormones and testis intoxicants are known inducers of germ cell apoptosis.Although there have been some reports concerning p,p'-DDE-induced apoptosis in some cells, few studies investigated male reproductive toxicity and testicular apoptosis. The aim of the present study was to determine the effects of different concentrations of p,p'-DDE on apoptosis of Sertoli cells and testicular apoptosis in prepubertal and pubertal rats, and to investigate Fas/FasL, and endoplasmic reticulum-mediated apoptotic pathways.PartⅠStudy of the mechanism involved in p,p'-DDE-induced apoptosis of Sertoli cells1,Effect of p,p'-DDE on the apoptosis of Sertoli cellsObjective: To investigate the effect of p,p'-DDE on the apoptosis of Sertoli cells. Methods: Sertoli cells were incubated in various concentrations of p,p'-DDE (10, 30, 50μM) for 24 h. In other experiment, cells were pre-incubated with 300μM NAC for 1 h followed by incubation with 50μM p,p'-DDE for 24 h. Then apoptosis was examined by flow cytometric analysis.Results: Flow cytometric analysis showed that p,p'-DDE caused apoptotic cell death in a dose-dependent manner. After treatment of 50μM p,p'-DDE, apoptosis reached a peak value of 30 %, which could be attenuated by NAC preincubation.Conclusion: p,p'-DDE could induce apoptosis of Sertoli cells in a dose-dependent manner. Antioxidant NAC could attenuate p,p'-DDE-induced apoptosis of Sertoli cells.2,Effect of p,p'-DDE on FasL,caspase-3 and -8 mRNA and protein in rat Sertoli cellsObjective: To investigate effect of p,p'-DDE on FasL,caspase-3 and -8 mRNA and protein in rat Sertoli cells.Methods: RT-PCR and Western blotting were used to detect FasL,caspase-3 and -8 mRNA and protein in rat Sertoli cells, respectively.Results: FasL mRNA expression level in the 50μM dose group was significantly higher than that of the control group (P<0.05). The caspase-3 mRNA expression levels of Sertoli cells in the 30, 50μM p,p'-DDE groups were markedly higher than that of the control group (P<0.05). Moreover, compared with the control group, the caspase-8 mRNA expression levels in different doses of p,p'-DDE were increased, and the differences were statistically significant (P<0.05).p,p'-DDE treatment induced an increase of FasL in the 50μM group. Preincubation with NAC could attenuate this effect successfully (P < 0.05). A significant reduction was observed in procaspase-3 over 30μM p,p'-DDE treatment and procaspase-8 in different doses, suggesting the caspase activation, respectively.Conclusion: p,p'-DDE could induce apoptosis of Sertoli cells through a FasL-dependent pathway including increase of the FasL expression and activation of the caspase-8 and -3.3,Effect of p,p'-DDE on calpain-1 and caspase-12 mRNA in rat Sertoli cells Objective: To investigate effect of p,p'-DDE on calpain-1 and caspase-12 mRNA in rat Sertoli cells.Methods: Real-time PCR was used to detect calpain-1 and caspase-12 mRNA in rat Sertoli cells.Results: No significant effects of calpain-1 and caspase-12 mRNA were observed in various concentrations of p,p'-DDE groups(P>0.05).Conclusion: The effect of p,p'-DDE on endoplasmic reticulum-mediated pathway needs further research.PartⅡStudy of the mechanism involved in p,p'-DDE-induced testicular apoptosis in prepubertal rats1,Effect of p,p'-DDE on organ coefficient and ultrastructure of testis in rats Objective: To investigate the effect of p,p'-DDE on organ coefficient and ultrastructure of testis in rat.Methods: 22-day-old prepubertal male rats were given different doses of p,p'-DDE (0, 20, 60, 100) mg/kg b.wt respectively in corn oil every other day by intraperitoneal injection for 10 days. Ultramicropathology technique was used to detect ultrastructure of testis in rat. Results: There were no significant differences in the organ coefficient of testis between different doses of p,p'-DDE groups and control group (P>0.05).Ultramicropathology technique analysis showed that p,p'-DDE caused many cavitates in the endochylema of Sertoli cells, Leydig cells and spermatogenic cells. Mitochondria became sweller, and nucleus was condensed.Conclusion: p,p'-DDE could induce ultrastructure changes of testis in rat.2,Effect of p,p'-DDE on testicular apoptosis and oxidative stress in rats Objective: To investigate the effect of p,p'-DDE on testicular apoptosis and oxidative stress in prepubertal rats.Methods: The method of TUNEL was used to investigate the effect of p,p'-DDE on testicular apoptosis. GSH-PX activity, SOD activity and MDA level were detected following with the protocol of assay kit.Results: Treatment with 20, 60, 100 mg/kg b.wt p,p'-DDE resulted in selective degeneration of germ cells at the seminiferous tubules. Most germ cells undergoing apoptosis were located along the periphery of the seminiferous tubules. Compared with the control group, the apoptotic index (AI) increased significantly in different p,p'-DDE-treated groups.It could be observed that 20, 60, 100 mg/kg b.wt p,p'-DDE induced a significant decreasing activities of SOD production (P < 0.05). The GSH-Px activity in 100 mg/kg b.wt p,p'-DDE group was significantly lower than that in the control group (P < 0.05), and the MDA level in 60, 100 mg/kg b.wt p,p'-DDE groups was remarkably higher than that in control group (P < 0.05).Conclusion: p,p'-DDE could induce testicular apoptosis and oxidative stress in prepubertal rats.3,Effect of p,p'-DDE on Fas,FasL,caspase-3,-8 and NF-κB of testis in rats Objective: To investigate effect of p,p'-DDE on Fas,FasL,caspase-3,-8 and NF-κB of testis in rats.Methods: Real-time PCR were used to detect Fas,FasL,caspase-3,and -8 mRNA. Western blotting was used to evaluate Fas, FasL, caspase-8 protein and NF-κB p65 nuclear protein. Caspase-3,and -8 activities were detected following with the protocol of assay kit.Results: In 100 mg/kg b.wt p,p'-DDE group, the mRNA levels of Fas, FasL, caspase-3 and -8 were significantly higher than that of the control group, and the differences were statistically significant (P<0.05).p,p'-DDE treatment was shown to result in an increase of FasL protein in 100 mg/kg b.wt p,p'-DDE group, but Fas protein in all p,p'-DDE groups was no significantly higher than that in the control group (P > 0.05) . A significant reduction was observed in procaspase-8 in 100 mg/kg b.wt p,p'-DDE group, suggesting the caspase activation. p,p'-DDE treatment induced an increase of NF-κB p65 in the nuclear extracts in 60, 100 mg/kg b.wt p,p'-DDE groups (P<0.05).Compared with the control group, the activities of caspase-3, -8 significantly increased in100 mg/kg b.wt p,p'-DDE group (P<0.05).Conclusion: p,p'-DDE could induce testicular apoptosis in prepubertal rats through the Fas/FasL dependent pathway including increase of the Fas-FasL expression, nuclear translocation of NF-κB and activation of the caspase-8 and -3.4,Effect of p,p'-DDE on calpain-1 and caspase-12 mRNA and protein of testis in ratsObjective: To investigate effect of p,p'-DDE on calpain-1 and caspase-12 mRNA and protein of testis in rats.Methods: Quantative real-time PCR and Western blotting were used to detect calpain-1 and caspase-12 mRNA and protein, respectively.Results: In 100 mg/kg b.wt p,p'-DDE group, the mRNA levels of calpain-1 and caspase-12 were significantly higher than that of the control group, and the differences were statistically significant (P<0.05).p,p'-DDE treatment was shown to result in an increase of caspase-12 protein in 100 mg/kg b.wt p,p'-DDE group, but calpain-1 protein in all p,p'-DDE groups was no significantly higher than that in the control group (P > 0.05).Conclusion: p,p'-DDE could induce testicular apoptosis in prepubertal rats through endoplasmic reticulum-mediated pathway including activation of the caspase-12. PartⅢStudy of the mechanism involved in p,p'-DDE-induced testicular apoptosis in pubertal rats1,Effect of p,p'-DDE on organ coefficient of testis in ratsObjective: To investigate the effect of p,p'-DDE on organ coefficient of testis in rat.Methods: 50-day-old pubertal male rats were given different doses of p,p'-DDE (0, 20, 60, 100) mg/kg b.wt respectively in corn oil every other day by intraperitoneal injection for 10 days. After 10-day treatment, the rats were sacrificed, the testis were removed immediately and weighed.Results: There were no significant differences in the organ coefficient of testis between different doses of p,p'-DDE groups and control group (P>0.05). Conclusion: p,p'-DDE could not induce organ coefficient changes of testis in rat.2,Effect of p,p'-DDE on testicular apoptosis and serum oxidative stress in ratsObjective: To investigate the effect of p,p'-DDE on testicular apoptosis and serum oxidative stress in pubertal rats.Methods: The method of TUNEL was used to investigate the effect of p,p'-DDE on testicular apoptosis. GSH-PX activity, SOD activity and MDA level were detected following with the protocol of assay kit.Results: Treatment with 20, 60, 100 mg/kg b.wt p,p'-DDE resulted in selective degeneration of germ cells at the seminiferous tubules. Most germ cells undergoing apoptosis were located along the periphery of the seminiferous tubules. Compared with the control group, the apoptotic index increased significantly in different p,p'-DDE-treated groups.It could be observed that 60 mg/kg b.wt p,p'-DDE induced a significant decreasing activities of SOD production (P < 0.05). The GSH-Px activity in 60 mg/kg b.wt p,p'-DDE group was significantly lower than that in the control group (P < 0.05), but the MDA level in different doses of p,p'-DDE groups was not remarkably higher than that in control group (P > 0.05).Conclusion: p,p'-DDE could induce testicular apoptosis and serum oxidative stress in pubertal rats. 3,Effect of p,p'-DDE on Fas,FasL,caspase-3 and -8 mRNA of testis in ratsObjective: To investigate effect of p,p'-DDE on Fas,FasL,caspase-3 and -8 mRNA of testis in rats.Methods: Real-time PCR were used to detect Fas,FasL,caspase-3,and -8 mRNA. Results: The mRNA levels of Fas and FasL in 60 mg/kg b.wt p,p'-DDE group and Fas in 100 mg/kg b.wt p,p'-DDE group were significantly higher than that of the control group, and the differences were statistically significant (P<0.05). Compared with the control group, the mRNA levels of caspase-3 significantly increased in 60 mg/kg b.wt p,p'-DDE group. The mRNA levels of caspase-8 in 20, 60, 100 mg/kg b.wt p,p'-DDE group were significantly higher than that of the control group (P<0.05) .Conclusion: p,p'-DDE might induce testicular apoptosis in pubertal rats through the Fas/FasL dependent pathway including increase of the Fas-FasL expression and activation of the caspase-8 and -3.4,Effect of p,p'-DDE on calpain-1 and caspase-12 mRNA of testis in rats Objective: To investigate effect of p,p'-DDE on calpain-1 and caspase-12 mRNA of testis in rats.Methods: Real-time PCR were used to detect calpain-1 and caspase-12 mRNA .Results: The mRNA levels of calpain-1 and caspase-12 in 60 mg/kg b.wt p,p'-DDE group and caspase-12 in 100 mg/kg b.wt p,p'-DDE group were significantly higher than that of the control group, and the differences were statistically significant (P<0.05).Conclusion: p,p'-DDE might induce testicular apoptosis in pubertal rats through endoplasmic reticulum-mediated pathway.5,Effect of p,p'-DDE on rats sperm motilityObjective: To investigate effect of p,p'-DDE on rats sperm motility.Methods: CASA were used to detect rats sperm motility.Results: In 60 mg/kg b.wt p,p'-DDE group, significant decreases in sperm motility parameters of motile sperm, VCL, ALH and BCF were seen in comparison with the control group (P<0.05). Motile sperm was decreased significantly in 20 mg/kg b.wt p,p'-DDE group.Conclusion: p,p'-DDE could induce disruption of sperm motility in pubertal rats.