The Role Of WDFY1 In Mouse Spermatogenesis And Testicular Inflammation Induced By PolyI:C

[Purpose]Investigate the effects of Poly I:C on the inflammatory response of wild-type mouse testes,mouse primary Sertoli cells and mouse primary Leydig cells.Explore the effects of Poly I:C on testicular inflammation and reproductive capacity of wild-type mice,and analyze the differences in the expression of inflammation-related genes in testicular tissues,mouse primary Sertoli cells and mouse primary Leydig cells after Poly I:C intervention.[Methods](1)Construction of testicular tissue infection models in mice: 6-8week C57BL/6J male mice were randomly divided into two groups: control group(Mock group)and experimental group(Poly I:C group).Poly I:C or the equal volume of PBS was injected into the testis of mice to generate Mock group and Poly I:C group,and set different time points(0h,3h,6h,9h,12 h,24h).H&E staining,IHC staining and RT-q PCR were used to detect the difference of histological morphology and the secretion of inflammatory cytokines in infected mouse testicular tissues,and the effect of Poly I:C on mouse testicular tissues was studied.(2)Construction of primary Sertoli cells and mouse primary Leydig cells infection models in mice: 6-8week C57BL/6J male mice were selected to isolate primary Sertoli cells and primary Leydig cells,which were cultured in a medium F12/DMEM supplemented with 10%FBS for 2-3 days.Then cells were subjected to serum starvation for 2h prior to transfection with 2μg/ml Poly I:C or a volume of PBS using 1μl lipofectamine 8000 according to the manufacturer’s instruction to generate Mock group and Poly I:C Group.After 6h treatment,we evaluate and compare the relative m RNA level of Tnfα,Il-1β,Ifnα,Ifnβ,Isg15 and Isg56 in Mock group and Poly I:C group.(3)We detect the differentially expressed genes between the Mock group and Poly I:C group by RT-q PCR.In particular,TLR3 signaling pathway-related genes,such as Wdfy1,Trif,Rig-1,Myd88,Mda5,Traf6,et al.[Results](1)Histological morphology: compared with the Mock group,the interstitial cells proliferated and the spaces between the seminiferous tubules became larger by 6 hours after infection.As the infection progresses,a large number of inflammatory cell infiltration appeared in the interstitium by 24 hours,and structure of some seminiferous tubule were disrupted,and some of the spermatogenic cells were lost.(2)In terms of inflammatory cytokine secretion: compared with the Mock group,the relative m RNA expression levels of Tnfα,Il-1β,Ifnα,Ifnβ,Isg15 and Isg56 in testis,LC and SC of mice increased at 3h after Poly I:C treatment,peaked at 6h,and then gradually decreased to the normal level at 24 h.(3)Signal pathway: compared with the Mock group,the m RNA expression levels of Tlr3 in testis,LC and SC of infected mice were increased,and among the differentially expressed genes between two groups,Wdfy1 was the most significantly up-regulated.[Conclusions](1)PolyI:C successfully activated the TLR3 signaling pathway in mouse testis,SCs and LCs,and induced testicular inflammation in mice.(2)Among the differentially expressed genes of Poly I:C-induced testicular inflammation in mice,Wdfy1 was the most up-regulated.[Purpose]The results of Part1 showed that Poly I:C could induce an inflammatory response in wild-type mouse testis,and Poly I:C treatment could cause changes in some gene expression,among which WDFY1 up-regulated most.Therefore,in order to further explore the role of WDFY1 in Poly I:C-induced testicular inflammation in mice.In this part,we first studied the conservation of WDFY1 protein among different species,and the expression and localization of WDFY1 in different tissues of mice,especially testis.[Methods]Bioinformatic analysis was used to study the conservation of WDFY1 among different species.The expression of WDFY1 in different organs and germ and immune cells of mice was studied by RT-q PCR and WDFY1-tag mice construction.[Results]WDFY1 was highly conserved among different species,and human WDFY1 was about 97% identical with its mouse direct homologues.In addition,WDFY1 was the highly expressed in mouse testis.WDFY1 was expressed in germ cells,Leydig cells,Sertoli cells and macrophages,and among these,the expression level of round spermatids and elongated spermatids is higher.[Conclusions]WDFY1 is highly conserved proteins in the testes of mammals.Besides,WDFY1 is expressed in mice germ cells(round spermatids and elongated spermatids),LCs and SCs.[Purpose]The results of Part2 show that WDFY1 is a conserved protein in mammalian testes,and expressed in mouse testis,LCs,and SCs.Since WDFY1 is an enhancer of the TLR3/TRIF signaling pathway,we want to explore the effect of WDFY1 on Poly I:C induced mouse testicular inflammation.[Methods]The Talen system was used to generate the Wdfy1 global knockout mouse model(KO).Firstly,we analyzed sperm fuction,fertilization process and male fertility by testicular morphological analyses,sperm transport experiments and fertility tests,respectively.Secondly,we generated the WT Poly I:C group and KO Poly I:C group by injecting equal volume of Poly I:C into testes.Then H&E staining,IHC staining and RT-q PCR were used to detect the difference of histological morphology and the secretion of inflammatory cytokines in infected mice testes.Thirdly,we isolated mouse primary Sertoli cells and primary Leydig cells,and transfection cells with equal Poly I:C supplemented lipofectamine 8000 according to Part 1 method.After 6h treatment,we evaluated and compared the relative m RNA level of Tnfα,Il-1β,Ifnα,Ifnβ,Isg15 and Isg56 in Mock group and Poly I:C group.[Results](1)Global Wdfy1 knockout mice were successfully generated.And ablation of Wdfy1 in mice does not affect normal spermatogenic process,fertilization and male fertility.(2)Histological morphology: In WT PolyI:C group,we observed a few inflammatory cells infiltrated in the interstitium of seminiferous tubules,and structure of some seminiferous tubules were disrupted,and some of the spermatogenic cells were lost,while knockout mice did not see significant changes.(3)In terms of inflammatory cytokine secretion: compared with the WT Mock group,the relative m RNA expression levels of Tnfα,Il-1β,Ifnα,Ifnβ,Isg15 and Isg56 in testis LC and SC of KO Mock group were decreased after Poly I:C treatment,and relative Tlr3 expression level was also decreased.[Conclusions]Wdfy1 is dispensable for spermatogenesis,fertilization and male fertility.And Wdfy1 knockout could protect mosue testicular cells from Poly I:C-induced inflammation and impair testicular innate immune response guided by TLR3 signaling pathway.