Adhesions between different cells and extracellular matrix have been studied extensively in vitro, but little is known about their functions in testicular tissue counterparts. Spermatogonia and their companion somatic cells maintain a close association throughout spermatogenesis and this association is necessary for normal spermatogenesis. In order to keep the relative integrity of the testicular tissues, and to detect the development in vitro, culture testicular tissues in a threedimensional (3D) agarose matrix was examined. Testicular tissues isolated from 6.5 d postpartum (dpp) mouse were cultured on the top of the matrix for 26 d with a medium height up to 4/5 of the 3D agarose matrix. The results showed that in this 3D culture environment, each type of testicular cells kept the same structure, localization and function as in vivo and might be more biologically relevant to living organisms. After culture, germ cell marker VASA and meiosis markers DAZL and SCP3 showed typical positive analysed by immunofluorescence staining and RT-PCR. It demonstrated that this 3D culture system was able to maintain the number of germ cells and promote the meiosis initiation of male germ cells.

Studies have demonstrated that regulation of GDNF on male germline stem cells (mGSCs) mainly through Ras/Erk1/2, Src family kinase and PI3K/Akt signaling pathways, but the signaling pathways GDNF-mediated are different when the species and cell lines varied. Whether GDNF regulates self-renewal of mGSCs isolated from livestock has not been reported. Here, we purified mGSCs from dairy goat testis using mixed enzymes and fibronectin. Immunofluoresce staining revealed the cultured dairy mGSCs expressed Vasa, Nanos2, Ngn3, Tert, Dazl, Lin28, Oct4, CD49f, Stra8 and GFRa1, reflecting that these cells were mGSCs phenotype. Then we cultured these dairy goat mGSCs in different concentrations of GDNF (0, 5, 10, or 20 ng mL-1) to optimize the best concentration of GDNF to sustain the dairy goat mGSCs self-renewal, after that the inhibitor of PI3K (LY294002, 10 μmol L-1) was added to the medium which contains the optimal concentration of GDNF we obtained by experiments. The mGSCs cultured in different media were compared through the population doubling time (PDT), capacity of cell proliferation evaluated by PCNA and BrdU immunofluorescence staining, RT-PCR, QRT-PCR, Western blotting and flow cytometry. Results showed that 10 ng mL-1 was the optimal concentration of GDNF to maintain goat mGSCs self-renewal and GDNF up-regulates c-Myc transcription via the PI3K/Akt pathway to promote goat mGSCs proliferation. This study provides us an efficient model to study the mechanism in mGSCs proliferation and differentiation in goat, and has important implications in unveiling signaling pathways in livestock GSCs.