In mouse testes, spermatogonial stem cells (SSCs), a subpopulation of GFR alpha 1 (GDNF family receptor-alpha 1)-positive spermatogonia, are widely distributed along the convoluted seminiferous tubules. The proliferation and differentiation of the SSCs are regulated in part by local expression of GDNF (glial cell-derived neurotorphic factor), one of major niche factors for SSCs. However, the in vivo dynamics of the GDNF-stimulated GFR alpha 1-positive spermatogonia remains unclear. Here, we developed a simple method for transplanting Dil-labeled and GDNF-soaked beads into the mouse testicular interstitium. By using this method, we examined the dynamics of GFRO-positive spermatogonia in the tubular walls close to the transplanted GDNF-soaked beads. The bead-derived GDNF signals were able to induce the stratified aggregate formation of GFR alpha 1-positive undifferentiated spermatogonia by day 3 post-transplantation. Each aggregate consisted of tightly compacted Asingle and marginal A(paired)-A(aligned) GFR alpha 1-positive spermatogonia and was surrounded by A(aligned) GFR alpha 1-negative spermatogonia at more advanced stages. These data not only provide in vivo evidence for the inductive roles of GDNF in forming a rapid aggregation of GFR alpha 1-positive spermatogonia but also indicate the usefulness of this in vivo assay system of various growth factors for the stem/progenitor spermatogonia in mammalian spermatogenesis. (C) 2016 Elsevier Inc. All rights reserved.