Rho/ROCK Signaling Pathway Regulates The Reconstruction Of Seminiferous Tubule-like Structure

Objective:We evaluate the effect and mechanism of the Rho/ROCK signaling pathway in reconstruction seminiferous tubule-like structure in this study,which will provide theoretical basis for clinical diagnosis the pathogenesis and the treatment of male infertility.Methods:1.Establishment of the model of seminiferous tubules:The 10-dpp mouse(C57BL/6)testis cells were digested to obtain re-aggregates of dissociated cells,and we cultured the re-aggregates cells in 3-D collagen matrix;2.The exact role of Rho/ROCK signaling pathway in reconstruction seminiferous tubule-like structure:The experiments were randomly assigned to the following groups:negative control group(cont),Positive control group(KSR),KSR+Y27632group and Bradykinin(BK)group respectively.The re-construction of seminiferous tubule-like structure and the location of Sertoli cells(SCs),Peritubular Myoid cells(PTMs),Leydig cells(LCs),germ cells(GCs)were analyzed by immunofluorescence;3.The proliferation of Sertoli cells and germ cells were observed by immunofluorescence;4.Immunofluorescence staining was also used for determination of ZO-1 and Claudin11 expression;5.The levels of Rho GTPase and ROCK were explored by RT-q PCR;6.Western blot experiments were performed to quantify MLC,p-MLC levels in testicular cell mass;7.The 10-dpp mouse testis were digested to obtain primary Sertoli cells,and the Sertoli cells were identified by immunocytochemistry with GATA4 staining;8.The primary sertoli cells were divided into control group(cont),KSR group,KSR+Y27632 group and BK group respectively.The tubule-like structures were observed by inverted phase-contrast microscope;9.RNA sequencing was performed to identify differentially expressed genes in BK compared to cont.The effect of Rho/ROCK signaling pathway on the formation of seminiferous tubule-like structure was further investigated by Cxcl5 receptor inhibitor SB225002.Results:1.The results of immunofluorescence showed that compared with KSR,the Sertoli cells lose polarity,thin and elongated peritubular myoid cells turned to rounded.And Leydig cells,germ cells,spermatogonia,spermatocytes random distributed,the expression of Laminin decreased by pretreatment of Y27632.After administrating BK,peritubular myoid cells attached the Sertoli cells to form cord-like structures.Leydig cells were sorted out of the seminiferous tubule-like structure.Spermatogonia and spermatocytes were found in basal or lumen of the tubule-like structure.The laminin expression was increased;2.Y27632 inhibited the proliferation of Sertoli cells,and germ cells,p<0.05.On the contrary,proliferation of Sertoli cells and germ cells were observed after BK treatment,p<0.05;3.Compared with cont,BK promotes the expression of ZO-1 and Claudin11after culture for 2 wks;4.The results of RT-qPCR showed that Rho GTPase and ROCK was significantly upregulated,p<0.05;5.Western blot showed that the protein expression levels of p-MLC was significantly downregulated in the presence of Y27632,p<0.05.With BK,the protein expression levels of p-MLC was significantly upregulated,p<0.05;6.GATA4~+DAPI~+cells were accounted for more than 90%by Image J software;7.After treatment with Y27632,the tubule-like structure was gradually destroyed with the increase of Y27632 concentration,while BK tends to promote the formation of tubule-like structure by inverted phase-contrast microscope;8.After verification by qPCR,eight differentially expressed genes(Cxcl5,Myl1,CKM,Hnrnpa3,Tnnt3,Tnni2,Mfsd4a and Myot)were up-regulated,and which involved in cytoskeletal remodeling and cell migration(Cxcl5 was significantly upregulated).Eight down-regulated expressed genes(Rdh7,Cyp24a,Cyp3a11,Cyp2a5,Mup3,Mup22,Serpinald and Akr1c6)were associated with gender development.Moreover,we also found that the formation of seminiferous tubule-like structures were suppressed with the SB225002,which a CXCR2 specific inhibitor.Conclusion:1.Rho/ROCK signaling pathway is involved in the remodeling of seminiferous tubule like structure;2.Rho/ROCK singning pathway promoted the formation of Sertoli cells tubular structures;3.Cxcl5/CXCR2 may interacts with Rho/ROCK signaling pathway induceds seminiferous tubule like structure formation.