Protective Effect Of Inhibiting MTORC1 Signaling Pathway On Testicular Injury Induced By X-Ray Irradiation In Mice

Objective:To study the changes of morphology and function of testis-and the status of mTORC1 signaling pathway after X-ray irradiation,and to investigate the protective effect of inhibition of and mTORC1 signaling pathway rapamycin(Rap)treatment on in mice.Methods:Seventy-two 8-week C57B1/6 male mice were randomly divided into four groups:control group,irradiation group,rapamycin group and rapamycin treatment group(n=18).Whole body irradiation with 8Gy X-ray was performed,and Rap was injected subcutaneously at 6 hours,2d,4d,and 6d after irradiation.The observation time of each group was 1d,3d,and 7d after irradiation.The testicular volume,testicular index and sperm function in different groups were examined.The pathological damage of testis was observed by HE staining.The positive cells of pS6,PLZF,BrdU and PCNA in testicular tissues of each group were detected by immunohistochemistry.TUNEL assay was used to detect testicular apoptotic cells and analyzed by image analyzer.The mRNA of P27,P57,CyclinD1,CyclinD2,Puma,Bcl2,Bcl-xl,Bax and Bak in 7d testis of each group was measured by qRT-PCR.Results:1.Compared with the control group,the testicular volume decreased and the testicular index decreased in the irradiation group,and there was a significant difference at 7d(P<0.01).Compared with the irradiation group,the testicular volume and testicular index in the rapamycin treatment group-increased with a statistical difference at 7d(P<0.05 or P<0.01).2.Compared with the control group,the sperm density,decreased,the survival rate and motility rate decreased at day 7 after irradiation,and the sperm deformity rate increased,(P<0.01).Compared with the irradiation group,the sperm survival rate,sperm motility rate and sperm abnormality rate decreased in the rapamycin group,and there was a significant difference at 7d(P<0.05).3.Compared with the normal group,the injury at day 7 after irradiation is more serious.For example,the seminiferous tubules are loosely arranged,the basement membrane is inferior in integrity,the lumen is enlarged,the mature spermatozoa in the lumen structure is less,and the spermatogenic cells are arranged in disorder.The number of layers was reduced.Compared with the irradiation group,the basement membrane integrity of the testicular seminiferous tubules was improved and the spermatogenic cells increased at 7 days from the rapamycin treatment group.4.Compared with the control group,the expression of pS6 positive protein increased at each time point in the irradiation group(P<0.01 or P<0.05).Compared with the irradiation group,the expression of pS6 positive protein was significantly reduced at each time point in the rapamycin treatment group(P<0.01).5.Compared with the control group,numbers of PLZF~+spermatogonial stem cells at day 1 post irradiation were significantly decreased(P<0.05),and further decreased at 3d and 7d(P<0.01).6.Compared with the control group,numbers of BrdU and PCNA positive cells were significantly decreased at day 7 post irradiation(P<0.01).Compared with the irradiation group,numbers of BrdU~+and PCNA~+cells in the rapamycin treatment group were significantly increased(P<0.01).7.Compared with the control group,the number of apoptotic cells in the irradiation group was significantly increased(P<0.01).Compared with the irradiation group,the number of apoptotic cells was significantly decreased in the rapamycin treatment group(P<0.05)8.Compared with the control group,the expression of P57 mRNA in day 7 post irradiation was significantly increased(P<0.05)while,the level of CyclinD1 mRNA was decreased(P<0.05),and the level of Puma mRNA was significantly increased(P<0.01).Compared with the irradiated group,the expression of P57 mRNA in the rapamycin treatment group was significantly decreased(P<0.05)and the puma mRNA expression level was significantly decreased(P<0.01).Conclusion:1.X-ray whole body irradiation can activate mTORC1 signaling pathway in the spermatogonial stem cell,leading to testicular structure and functional damage;2.Rapamycin can inhibit the over-activation of mTORC1 signaling pathway and protect the testicular injury induced by X-ray whole body irradiation.Inhibition of mTOR signaling reduces the apoptosis of spermatogonial stem cells and promote their proliferation.