Propagation of bovine spermatogonial stem cells in vitro

The access to sufficient numbers of spermatogonial stem cells (SSC) is a prerequisite for the study of their regulation and further biomanipulation. A specialized medium and several growth factors were tested to study the in vitro behaviour of bovine type A spermatogonia, a cell population that includes the SSC and can be specifically stained for the lectin DBA (Dolichos bifflorus agglutinin). During short term culture (two weeks), colonies appeared, the morphology of which varied with the specific growth factor(s) added. Whenever stem cell medium was used, round structures reminiscent of sectioned seminiferous tubules appeared in the core of the colonies. Remarkably, these round structures always contained A spermatogonia. When LIF, EGF or FGF2 were added, specific effects on the numbers and arrangement of somatic cells were observed. However, the number of A spermatogonia was significantly higher in cultures to which GDNF was added and highest when GDNF, LIF, EGF and FGF2 were all present. The latter suggests that a proper stimulation of the somatic cells is necessary for optimal stimulation of the germ cells in culture. Somatic cells present in the colonies included Sertoli cells, peritubular myoid cells and a few Leydig cells. A transplantation experiment, using nude mice, showed the presence of SSC among the cultured cells and in addition strongly suggested a more than 10000-fold increase in the number of SSC after 30 days of culture. These results demonstrate that bovine SSC self-renew in our specialized bovine culture system and that this system can be used for the propagation of these cells.