| Objectives:1.To explore the effect and mechanism of cyclophosphamide(CP)on testicular injury and spermatogenesis in rats.2.To explore the protective effect and mechanism of L-carnitine(LC)on CP-induced injury of testis and spermatogenesis in rats.Methods:Seventy-two 8-week-old male SD rats were randomly divided into one of three groups:1)intraperitoneal injection of normal saline(NS),2)intraperitoneal injection of CP(30 mg/kg CP),3)intraperitoneal injection of LC(100mg/kg)after injection CP one hour(CP+LC).All the injections were given for 5 consecutive days.On day 3 and week 1,2,and 4 after the last injection,the rats were sacrificed to get their serum and testis.The activity and body weight of rats before killed as well as the testicular weight after were recorded.Testicular damage was detected by haematoxylin and eosin staining and serum testosterone measured by ELISA.The expression of ?-catenin,protein phosphatase 2A(PP2A),and I2PP2A(SET)were studied through immunohistochemical staining,immunofluorescence staining,western blot,and quantitative real-time polymerase chain reaction(QPCR).The comparisons between groups were made by one-way ANOVA.The pairwise comparisons between two groups were made through Student-Newman-Keuls and Least Significant Difference test.All statistical calculations were performed using the SPSS 22.0.A P-value<0.05 was considered statistically significant.Results:1.Compared with NS group,the body weight,activity,testicular weight and the serum testosterone level were significantly reduced in CP group of rats.HE staining showed that the seminiferous tubule had a smaller diameter,the structure of spermatogenic epithelium was disordered,the spermatogenic cells were arranged at different stages in the spermatogenic epithelium,and spermatogenic cells at all levels were absent to different degrees,among which sperm cells were mainly decreased.The expressions of PP2A,SET and ?-catenin in the spermatogonial cells,primary spermatocytes,sertoli cells and Leydig cells were observed by immunohistochemistry and immunofluorescence.In CP group,the positive expression of PP2A and ?-catenin was significantly up-regulated(P<0.05),and the SET protein expression was significantly down-regulated(P<0.05).Realtime-PCR and Western Blot experiments showed that mRNA and protein expressions of PP2A and ?-catenin in CP group were significantly up-regulated(P<0.05)while mRNA and protein levels of SET were significantly down-regulated(P<0.05).2.Compared with CP group,CP+LC group of rats showed that the spermatogenic epithelial structure recovered,spermatogenic cells at different stages in the spermatogenic epithelium partially recovered,and the number of sperm cells recovered.Immunohistochemistry and immunofluorescence staining showed that the expressions of PP2A,SET and?-catenin were observed in the spermatogonial cells,primary spermatocytes,sertoli cells and Leydig cells.In the CP+LC group,the expression of PP2A and ?-catenin protein was significantly down-regulated(P<0.05),and the expression of SET protein was significantly up-regulated(P<0.05).Realtime-PCR and Western Blot experiments showed that the mRNA and protein expressions of PP2A and ?-catenin in the CP+LC group were significantly down-regulated(P<0.05),while the mRNA and protein levels of SET were significantly up-regulated(P<0.05).There was no significant difference in mRNA and protein expression of PP2A and ?-catenin between CP+LC and NS group,but the mRNA and protenin level of SET in CP+LC group was higher than NS group(P<0.05).Conclusions:1.CP caused injury to rat testis and damaged spermatogenesis through SET and ?-catenin signaling pathway.2.LC protect rat testicular injury and spermatogenesis caused by CP,and play a protective role through SET and ?-catenin signaling pathway. |
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