Immunomagnetic isolation and long-term culture of mouse type A spermatogonia

In the mammalian testis, type A spermatogonia proliferate and differentiate into sperm under the tight control of both endocrine and paracrine factors , in order to study the complex process of spermatogenesis at the molecular level, an in vitro system must be devised in which type A spermatogonia ca n be cultured for a prolonged period of time. Therefore, cocultures includi ng type A spermatogonia and Sertoli cells, which act as nurse cells to the developing germ cells, are desirable. We have developed a method for the sp ecific isolation of type A spermatogonia using magnetic beads and antibodie s that recognize the c-kit receptor or the hemophilic adhesion molecule, Ep -CAM. Purified spermatogonia could survive for a period of 25 days when coc ultivated on Sertoli cell monolayers. Moreover, we recently established Ser toli cell lines that produce growth factors that are essential for the main tenance of spermatogonia in a proliferative state. Some of these Sertoli ce ll lines are able to reorganize into tubular structures when cultivated on a layer of Matrigel as extracellular matrix. We show here that type A sperm atogonia associate specifically with the Sertoli cell tubules, and are able to replicate their DNA in this environment. Thus, these in vitro culture s ystems could be used for the long-term culture of primary, nonimmortalized type A spermatogonia.