Study On The Damage Mechanism Of Low Dose Ionizing Radiation On The Reproductive System Of Male Mice

Objective:To study the effect of low-dose X-ray on testicular structure and sperm function of male mice,and to explore the mechanism of low-dose ionizing radiation on the damage of male reproductive system.Methods:1.Eight weeks of C57BL/6 mice in small animal irradiation center of suzhou university using X rays to total body irradiation,dose were 0.2,0.5 Gy,dose rate is 50 cGy/min.After irradiation,the mice were kept in905 storage room.After being euthanized at different time points,the testis and cauda epididymis were quickly removed.The testicle was used to observe pathology and the cauda epididymis was used to detect sperm function.2.8-week-old C57BL/6 mice were subjected to whole-body irradiation byX-ray,with the dose of 0.5 and 0.5 Gy and the dose rate of 50cGy/min,respectively.Testicular spermatogenic cells were isolated on the 3rd day after irradiation?IR3d?and the 7th day?IR7d?.After purification of spermatogenic cell protein,ubiquitination protein spectrum was analyzed.3.The results of protein ubiquitination were verified by immunoprecipitation and western blot.4.After 5 times of whole body irradiation,the genes of Ddx4,the mutant genes of Ddx4-334 and Ddx4-414 carried by lentivirus were transduced into mouse testis in vitro.After feeding for 2 weeks,the testis was taken out for H&E staining.5.piRNAs-sequencing was performed on spermatoplasm of infertile patients.6.RT-qPCR was used to verify the sequencing results and analyze the difference of piRNAs in sperm of infertility patients.7.By means of mouse testicular microinjection technique,piRNAs was transferred into mouse testicle,and the effect of piRNAs on testicular structure and function was observed by H&E staining.Results:1.After exposure tolow doses?0.2,0.5Gy?X-ray,Mouse testicular convoluted fine spermatinuctal cortex all had thinning,spermatogenic cells were first reduced,and then it goes to normal.Sperm motility also decreased first and then returnd to normal.2.Compared with the single irradiation of 0.5Gy,the testicular structure of 0.5Gy*5 did not recover but was damaged more seriously in the course of 1-14 days.3.A total of 1644 ubiquitination sites were identified on 737 proteins.IR3d vs NC showed 634 up-regulated sites in 348 proteins and 146 down-regulated sites in 118 proteins.IR7d vs NC showed 334 up-regulated sites in 215 proteins and 293 down-regulated sites in 194 proteins.Three RNA-related genes,Aszl,Ddx4 and Tdrd9,were ubiquitinated at different sites.4.The ubiquitination form of Ddx4 is neither K48 nor K63.5.The piRNAs sequencing results of sperm showed that there were significant differences in piRNAs in different groups.Compared with the normal group,963 piRNAs were up-regulated and 155 piRNAs were down-regulated in the vitality and morphologyabnormal.Compared with the normal group,3936 piRNAs were up-regulated and 389 piRNAs were down-regulated in thevitalityabnormal group.Compared with the normal group,2561 piRNAs were up-regulated and 355 piRNAs were down-regulated in the morphology abnormal group.6.The increased expression of hsa_piR₀16118 was strongly correlated with sperm motility and sperm morphology.7.After overexpression of hsa_piR₀16118 in the testis of mice,a large number of dermal cells were lost in the convoluted fine spermatozoa,fibroblasts proliferated in theinterstitium of testis,and collagen secretion increased.Conclusion:1.The sensitivity of testis to ionizing radiation was higher than that of epididymis.The testis returned to normal on the seventh day?IR7d?after 0.5Gy single irradiation,and failed to recover on the fourteenth day?IR14d?after 0.5Gy*5 irradiation,resulting in more serious injury.2.Leydig cells?Sertoli cells?spermatogenic cells may play a sequential role in the damage of spermatogenesis by ionizing radiation.Ionizing radiation can change the ubiquitination of spermatogenic cells,and the ubiquitination of Ddx4 may be involved in the regulation of spermatogenesis.3.Ionizing radiation can cause the changes of piRNAs,proteome and protein ubiquitination in spermatogenic cells.4.There are significant differences in the expression of pirnas in the seminal plasma of male infertility patients,such as hsa_piR₀16118.The expression of hsa_piR₀16118 in seminal plasma of patients with asthenospermia and dysspermia increased significantly,which could cause testicular tissue damage.It can be used as an index to diagnose asthenospermia and dysspermia.