Expression And Localization Of Growth Differentiation Factor9and Its Signaling System Proteins (ALK5,Smad2and Smad4) In Adult Japanese Quail And Chicken Testes

ObjectiveGrowth differentiation factor9, a member of superfamily proteins, was the first identified growth factor secreted by oocytes. GDF9stimulates proliferation of granulosa cells, suppress differentiation of granulosa cells, regulates the expression of hormone and cytokines, and promotes the growth of preovulatory follicles, so it got a lot of attention. Recently, a series studies confirmed that GDF9was expressed in the testis of mouse, rat, human and bovine and alpaca; GDF9regulates the function of Sertoli cells in vitro. To explore the universality of GDF9expression in male animals and the concordance of the manner of GDF9action in mammal and domestic fowl testes, the present study investigated the expression and localization of growth differentiation9and its signaling system proteins (ALK5, Smad2and Smad4) in adult Japanese quail and chicken testes by Western blotting assay and immunohistochemistryMethods①The expression of GDF9and its signaling system proteins (ALK5, Smad2and Smad4) in adult Japanese quail and chicken testes was examined by Western blotting assay.②The temporal and spatial localization of GDF9and its signaling system proteins (ALK5, Smad2and Smad4) in adult Japanese quail and chicken testes was examined by immunohistochemistry.Results③Western blotting results showed that there were two protein bands about60kDa and21kDa binding with the polyclonal rabbit anti-GDF9antibody in the whole protein of adult Japanese quail and chicken testes; there was a protein band about52kDa binding with the polyclonal rabbit anti-ALK5antibody; there were two protein bands about55kDa and60kDa which respectively bound with polyclonal rabbit anti-Smad2and anti-Smad4antibody. ②The cellular localization of GDF9and its signaling system proteins (ALK5, Smad2and Smad4) in adult Japanese quail testis showed that GDF9was stage-specifically located in the cytoplasm of spermatogonia, spermatocytes and round spermatids of the adult Japanese quail seminiferous epithelium and the GDF9expression of spermatogonia and spermatocytes was higher than round spermatids; ALK5, the type I receptor of GDF9, was principally located in the cytoplasm of spermatogonia and spermatocytes; Smad2, a signal transducer of GDF9signaling pathway, which was mainly located in spermatogonia and spermatocytes and Smad4, the common transducer of GDF9signaling pathway, highly expressed in germ cells and Sertoli cells.③The cellular localization of GDF9and its signaling system proteins (ALK5, Smad2and Smad4) in adult chicken testis showed that GDF9was stage-specifically located in the cytoplasm of spermatogonia, spermatocytes and round spermatids of the adult chichken seminiferous epithelium and the GDF9expression of spermatogonia and spermatocytes was higher than round spermatids; ALK5, the type I receptor of GDF9, was principally located in the cytoplasm of spermatogonia and spermatocytes; Smad2, a signal transducer of GDF9signaling pathway, which was mainly located in spermatogonia and spermatocytes, round spermatids and Smad4, the common transducer of GDF9signaling pathway, highly expressed in germ cells and Sertoli cells.ConclusionThe present study showed that GDF9and its signaling system proteins (ALK5, Smad2and Smad4) all expressed in Japanese quail and chicken testes. The cellular localization of GDF9, ALK5, Smad2and Smad4were similar. Our research demonstrated that GDF9secreted by spermatogonia, spermatocytes and round spermatids had an important role in the proliferation and differentiation of spermatogonia by autocrine and regulating the function of Sertoli cells by paracrine.