Neonatal mouse gonocyte proliferation assayed by an in vitro clonogenic method

Survival and proliferation of mouse gonocytes was studied using a single ce ll clonogenic assay in vitro. The effect of growth factors and extracellula r matrix on clonogenic development was quantitated. Fundamental requirement s for growth of neonatal gonocytes included addition of fetal calf serum an d coating culture wells with collagen IV alone or with added fibronectin. A fter 4-5 days, colonies ranged in size from four to > 128 cells, and some c ontained very elongated cells indicating migratory behaviour. Soluble stem cell factor did not have any effect on clonogenicity, although STO (subline of SIM mouse fibroblasts) cells, which produce membrane-bound stem cell fa ctor, reduced colony formation from 79 +/- 5.9% to 20 +/- 3.3% without adde d growth factor. The majority of gonocytes and type A spermatogonia express the c-kit receptor according to in situ hybridization studies. However, th e results indicate that the receptor may not be functional in neonatal gono cytes and their immediate progeny. The current assay for gonocytes can be e xtended to test new growth factors or proliferation-inducing agents directl y, as well as to study cell-cell interactions. This assay and long-term pro pagation of neonatal germ cells will provide the much needed resources to e nable greater understanding of the early development of germ cells.