Effect Of X-ray Irradiation On The Expression Of RNF146 In Mouse Testis

Ionizing radiation has an important effect on the human reproductive system,and the testis is highly sensitive to ionizing radiation.In an ionizing radiation dose above,there will be different degrees of structural and functional damage,impaired spermatogenesis and decreased the count of sperm,resulting in temporary or even permanent infertility[1],which seriously affects the male reproductive health,and thus attracted wide attention,especially for patients receiving radiotherapy treatment.It is generally accepted that the damage of spermatogenic cells is an important cause of testicular spermatogenic dysfunction after irradiation [2].Therefore,it is helpful to understand the molecular mechanism of the testis damage caused by ionizing radiation.In previous studies,we found a protein RNF146 expressed in mouse testis.Ubiquit ination is a post-translational modification of proteins widely found in eukaryotes.Different E3 ubiquit in ligase can act on different protein substrates.Specific amino acid residues are labeled by ubiquitin molecules,which are then identified by proteasome and degrade specifically [3].As a DN A damage response protein,RNF146 is highly conserved.Studies indicate that RNF146 can induce the ubiquitination of AIF in cytoplasm through the specific recognition and binding of it,repairing DN A damage of drug toxicity via inhibiting cell apoptosis[5].In previous studies,we found that RNF146 was highly expressed in the testis of mice,and low expressed in the testis of newborn mice.The expression level of RNF146 increased rapidly with sexual development[4].Histopathological studies show that RNF146 is localized mainly in spermatogonia and spermatocytes in testicular tissue.What is the significance of high expression of RNF146 in testicular tissue in which DN A replication is very active? What is the effect of the down regulation of RNF146 expression in testicular tissue after high dose X-ray irradiation on testicular function? What is the molecular mechanism?The above questions will help us to understand the role and significance of RNF146 in the process of testicular irradiatio n injury.To the end,we conducted the following two parts of the research work.Part? Testicular tissue injury and the expression change of RNF146induced by X-ray irradiation in miceObjective To investigate the effect of X-ray radiation on RNF146 in normal male mice testis.Methods Adult male C57 mice were randomly divided into six groups(n = 40,each with a dose of 0,0.1,0.5,1,4 Gy irradiation).After 16h?48h and 45 d,HE staining was used to observe the morphological changes of testis in mice exposed to the X-ray;Tunel staining was used to determine the apoptosis of testicular cells in mice after 16 h and 48h;And to detect the expression of RNF146 m RN A after 16 h and 48 h using q-PC R.Male: female(1?2)mating experiment to evaluate the fertility of irradiated mice.Adult male C 57 mice were divided into five groups: Blank,2Gy 16 h,2Gy 48 h,4Gy 16 h,4Gy 48 h.All of them were removed from one side of the testis and stored at-80?,for 0,2 and 4 Gy X irradiation after one week of the operation respective ly,and the other sid e of testis was taken at the time of 16 hours and 48 hours.Western-blot was used to evaluate the changes of RNF146 protein in the testis of mice X-ray irradiation.Results HE staining showed that,after 16 h,there were no morphological changes in the testis tissue exposed to X ray irradiation;After 48 h,1Gy group showed deep staining,pyknosis and fragmentation of nuclei,2Gy and 4Gy group observed abnormal morphology and reduced number of spermatogenic cells in testicular tissue;And after 45 days,below 2Gy X ray irradiation,testicular tissue morphology changes little;2Gy group have obvious damage;And in 4Gy group,seminiferous tubule have severe empty tube,lumen shrink and becom smaller.The mating results showed that the radiation damage below 2Gy X ray irradiation did not result in a significant decrease in the fertility of mice,and the fertility was significantly decreased after 4Gy irradiation.Tunel staining showed that the apoptosis of testicular cells occurred mainly in the spermatogonia and spermatocytes outer.The results of q-PC R and Western-blot showed that,after 48 h,the expression of RNF146 decreased in mice testis exposed to 2 and 4 Gy X ray irradiation.Conclus ion The mice testicular tissue damged after X ray irradiation above 2Gy,and could not be repaired in the short term.After 4Gy irradiation,the damage was severe,the sterilization rate of male mice was as high as 96%.The apoptosis of spermatogenic cel s was induced by irradiation,and expression of RNF146 was down regulated.Part? Effect of X-ray irradiation on the expression of apoptosisinducing factor in mouse sperm cellsObjective To study the effect of X-ray irradiation on the localization of apoptosis inducing factor(AIF)in mouse sperm cel s GC1.Methods The spermatogonia l cell line GC1 cells were cultured for several days,and then inoculated into the culture flask and culture plate respectively.After GC1 cells were treated with 0,3,6 and 9 Gy X irradiation,Brd U incorporation assay were performed to detect the proliferation rate of GC1 cells.After irradiation 48 h,the condensation of cells was observed by DAPI staining;the subcellular localization of AIF was showed using an immunofluorescence staining;Analysis the expression level of AIF protein in total cell protein,cytoplasmic protein and nuclear extracts and the expression level of RNF146 in total protein by Western blot;overexpression RNF146 in GC1 cells for 9Gy ray irradiation,study the effects of RNF146 on AIF using Western blot.Results With the increase of X-ray irradiation dose,the proliferation rate of GC1 cells was significantly decreased,and the activity of cells was weakened.After 6 Gy irradiation,in nuclear extracts,but not in the whole cells extracts,the protein AIF was upregulated significantly.It meant the nuclear translocation of protein AIF occurred.The expression of RN F146 decreased with the increase of radiation dose,and the 9Gy irradiation group was the most obvious,it could inhibit the nuclear translocation of AIF after overexpression of RNF146.Conclus ion X-ray irradiation induces apoptosis of mouse sperm cells GC1,meanwhile the nuclear translocation of AIF occurs.RNF146 can inhibit the nuclear translocation of AIF.