Dhh Signaling Pathway Regulates The Reconstruction Of Seminiferous Tubule-like Structure

Objective:Utilizing the inhibitor Cyclopamine and agonist SAG to investigate the effect of Dhh signaling pathway on the reconstruction of seminiferous tubule-like structure and the mechanism.Methods:The dissociated cells of 10-dpp testes were cultured in a 3D system and treated with inhibitor Cyclopamine or agonist SAG respectively.Immunofluorescence was used to observe the reconstruction of seminiferous tubule-like structure through detect the behavior of Sertoli cells,peritubular myoid cells,Leydig cells,germ cells and the expression of laminin.While,live cell imaging system was used to record the process of the formation of tubule-like structure.Meanwhile,the proliferation of Sertoli cells,peritubular myoid cells,Leydig cells and germ cells could be detected by using immunofluorescence.Finally,Western blot and Real-time PCR were used to detect the expression of the components of Dhh singling pathway.Results:After treating with Cyclopamine,the Sertoli cells didn't show polarity,rounded peritubular myoid cells and Leydig cells scattered.Live image showed that the cells moved slowly or even didn't move,and there was no aggregate formed.And the secretion of laminin was inhibited by Cyclopamine.While in the present of SAG,we could see that polarized Sertoli cells gathered and trended to form monolayer;these elongated peritubular myoid cells surrounded Sertoli cells.While at the outer part of peritubular myoid cells,a lot of Leydig cells gathered.In addition,SAG promoted the secretion of laminin to assist in the formation of basal membrane.The data of live image showed that some thin and elongated PTM cells moved fast to surround re-aggregates cells.In addition,Cyclopamine inhibited the proliferation of Leydig cells(P<0.05),peritubular myoid cells(P<0.05),germ cells(P<0.05)and Sertoli cells(P<0.05).On the contrary,SAG promoted the proliferation of Leydig cells(P<0.05),peritubular myoid cells(P<0.05)and germ cells(P<0.05).The results of RT-PCR showed Ptch1 and Smo had no obverse difference,Gli1 was significantly downregulated(P<0.05)after treating with Cyclopamine.While in the presence of SAG,Ptch1 and Smo also showed no obverse difference,Gli1 was significantly upregulated(P<0.05).Western blot showed the expression of protein Gli1 was significantly downregulated in the presence of Cyclopamine(P<0.05).With SAG,the expression of protein Gli1 was significantly upregulated(P<0.05).Conclusion: 1.Dhh signaling pathway play roles in the proliferation of peritubular myoid cells,Leydig cells and germ cells.2.Dhh signaling pathway regulates the reconstruction of tubule-like structure through regulating the expression of Gli1.