Our long-term goal is to study the biology of spermatogonial stem cells, the A/single spermatogonia. There is great interest in the A/single spermatogonia because of their importance for understanding basic mechanisms of cell self-renewal versus differentiation, for the treatment of infertility, for the development of contraceptives and for the understanding of the etiology of testicular cancer, in particular seminoma. However, studies on the biology of these cells have been severely hampered because their number is low, no unique marker exists and it has been difficult to physically separate them from the other more mature spermatogonia. We recently developed a method that allows us to isolate pure populations of A/single spermatogonia, and we have started to study their behavior in vitro. Using microarrays, we also have analyzed the transcriptional program of the A/single spermatogonia in the presence of glial cell line-derived neurotrophic factor (GDNF), a growth factor produced by Sertoli cells. In the first aim, we will isolate Asingle spermatogonia and evaluate their clonogenic potential by germ cell transplantations. In the second aim, we will determine which signaling pathways are induced by GDNF, leading to Asingle spermatogonia self-renewal or differentiation. In the third aim, we will study how GDNF interacts with other growth factors/receptors to promote self-renewal or differentiation. In summary, this line of work will expand our knowledge of the functional characteristics of the A/single spermatogonia and give us better insight into the molecular mechanisms that drive the first steps of spermatogenesis. In particular, the role of the growth factor GDNF in regulating self-renewal and/or differentiation will be elucidated.