Isolation, Culture And Identification Of Porcine Spermatogonial Stem Cells

Spermatogenesis is maintained by the self-renew and differentiation of spermatogonial stem cells.At present, long-term culture systems of germline stem cells (GSCs) have been developed for mouse,rats and human. However, we still have little knowledge about the growth characteristics and expressionpatterns of porcine GSCs because of lacking steady culture system in vitro.In the current study, we isolated the germ cells in vitro by two-step enzymatic digestion, comparedthe enrichment efficiency of gelatin and laminin according to difference adherence velocity. The resultsshowed that the laminin was superior to gelatin in germ cell enrichment, culture and proliferation. GSCsclones could be cultured for more than1month, subcultured for more than three passages, andmaintained their specific morphology and proliferation characteristics well. These cells resembled GSCsof mouse and other species in morphology, proliferation and expression patterns. We identified culturedGSCs for alkaline phosphatase activity, as well as protein and gene expression. The results showed thatGSCs remained pluripotent, and expressed alkaline phosphatase, stem cell marker proteins OCT-4andSOX2and pluripotency genes Nanog, Klf4and c-Myc. This indicated that we successfully established aculture system for GSCs derived from the gonocytes of neonatal porcine testes in vitro.In addition, we found that porcine GSCs showed some unique proliferation and expressioncharacteristics. Two kinds of clones derived from gonocytes with different proliferation patterns andexpression characteristics were observed in our culture system. The clones consisted of cells at differentstages, in which compact clone consisted of SSCs and chain-like clones consisted of Apr and Aalspermatogonia. The result explained the types of spermatogonia that gonocytes could give rise to whilecultured in vitro. The alkaline phosphatase showed different activities in various species. In porcine, APwas expressed in SSCs, but Apr and Aal spermatogonia. However, Apr and Aal spermatogonia of mousewere strongly stained by AP staining under the same condition, which was similar to that of human andgoat. In chickens, AP expression was not found in GSCs or embryonic germ cells (EGCs). Proteinexpression of OCT-4and SOX2was found in Apr and Aal spermatogonia. However, the proteinexpression in spermatogonia is different from that in embryonic stem cells (ESCs). The expression ofOCT-4and SOX2in Apr and Aal spermatogonia was restricted to the cytoplasm.Development of this culture system help us to gain further knowledge of GSCs and the stem cellniche, and provides research material and technical support for gene function research, animalconservation, and reproduction. Combining the culture system with cryopreservation technology andtransplantation technology, could help a lot in transgenic research. The phenotypic characterization ofGSCs may assist us to further our knowledge of regulation mechanism of porcine GSCs and bettercharacterize porcine spermatogonia.