Male germline stem cells (mGSCs) are unique adult germ cells with self-renewal potential and spermatogenesis function in the testis.  However, further studies are needed to establish a long-term cultural system of mGSCs in vitro, especially for large animals such as bovine mGSCs.  In this study, we first established a stable immortalized bovine male germline stem cell line by transducing Simian virus 40 (SV40) large T antigen.  The proliferation of these cells was improved significantly.  These cells could express spermatogonial stem cell (SSC)-specific markers, such as PLZF, PGP9.5, VASA, LIN28A, and CD49F, both in the mRNA and protein levels.  Additionally, these cells could be differentiated into three germ layer cells to enter meiosis, form colonies, and proliferate in the seminiferous tubules of busulfan-induced infertile mice.  The immortalized bovine mGSCs maintain the criteria of mGSCs.  

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.

Mesenchymal stem cells (MSCs) derived from bone marrow are a well-characterized population of adult stem cells that can be maintained and propagated in culture for a long time with the capacity to form a variety of cell types. This study investigated the characteristics of dairy goat bone marrow MSCs (gMSCs) and their differentiation potential toward germ cells in vitro, and to test their potential in vivo, these cells were transplanted into seminiferous tubes of endogenous germ cells-depleted mouse models. The results showed that characteristic gMSC lines were established and a small population of gMSCs transdifferentiated into male germ cell-like cells which expressed Stra8 after induction with retinoic acid (RA), as analysed by reverse transcription-polymerase chain reaction (RT-PCR) and immunofluorescence. Further, we transplanted the gMSCs into endogenous germ cells-depleted mouse models. A variety of analysis demonstrated that gMSCs might differentiate into male germ cells and helped spermatogenesis in endogenous germ cells depleted mouse models at 30 d after transplantation. The gMSCs could be used as a potential source of cells for reproductive studies and a neoadjuvant therapy for the spermatogenesis anomaly. Moreover, these cells may offer a new strategy for male infertility and an alternative approach for production of transgenic animals.

A83-01 is a selective inhibitor of the TGF-β type I receptor ALK, which inhibits the TGF-β-induced epithelial-to-mesenchymal transition (EMT) via the inhibition of Smad2 phosphorylation. Previous studies have showed that A83-01 promoted somatic cellular reprogramming significantly. Male germline stem cells (mGSCs), as an alternative resource of pluripotent stem cells derived adult testis, have promising valuable in clinic medicine and regeneration, however, the derivation of mGSCs was complex and difficult. What the role A83-01 plays in promoting the proliferation of mGSCs is still unknown. In this study, combined with A83-01 and knockout serum replacement (KSR) medium, we obtained a relatively feeder- and serum-free system for mGSCs culturing in vitro and the optimal concentration of A83-01 was 0.25 μmol L-1. After continuous culturing, the proliferation efficiency of undifferentiated mGSCs and differentiation capacity of mGSC were examined as well. Results showed that, A83-01 dramatically increased the number of mGSCs and AP positive colonies, and the mitosis index according to the BrdU assay. A83-01 could also increase the expression of pluripotent markers including Oct4, Klf4, Nanog and c-Myc, analyzed by real-time quantative PCR. mGSCs cultured in the optimal feeder-and serum-free system combined with A83-01 could form embryoid bodies (EBs), which consisted of three embryonic layers detected by immunofluorescence and RT-PCR. Remarkably, the results demonstrated 0.25 μmol L-1 A83-01 could promote the proliferation of mouse mGSC colonies and maintain their undifferentiated status under feeder- and serum-free systems.