Testicular cells from chemoablated mouse after overnight culture. (A) Cells obtained after two-step enzymatic digestion of seminiferous tubules from busulphan treated mice were cultured. After overnight culture, two distinct cell types including attached polygonal Sertoli cells and few spherical stem cells (which remain unattached) were clearly visualized. (B) At higher magnification, spherical stem cells of two distinct sizes were observed. VSELs (small sized) appeared to give rise to bigger sized spermatogonial stem cells (SSCs, broken circle) and the SSCs divide rapidly and form chains (arrow). Results are in agreement with earlier proposed concept  that upon differentiation, small sized pluripotent VSELs with nuclear OCT-4 give rise to slightly bigger ‘progenitors’ with cytoplasmic OCT-4. VSELs undergo asymmetric cell division including self-renewal and giving rise to slightly bigger cells (broken circle) whereas the progenitors undergo rapid symmetrical divisions (arrow). (C-G) On day 3, the Sertoli cells appear well attached to the bottom of the culture dish as mesenchymal-like fibroblasts and apparently provide a somatic feeder support to the germ cells. Germ cells (arrow, remain unattached) were observed in close vicinity of Sertoli cells. We also observed few floating germ cell clusters which reflect clonal expansion of progenitors. Similar germ cells clusters were also reported in adult mouse ovary .