NCAM MEDIATES ADHESION BETWEEN GONOCYTES AND SERTOLI CELLS IN COCULTURES FROM TESTES OF NEONATAL RATS

During neonatal development of the rat testis, gonocytes resume mitosi s and display renewed motility to migrate toward the basal lamina, two events that occur in vitro when these cells are cocultured with Serto li cells. However, although substantial evidence suggests that develop ment of gonocytes depends on Sertoli cells, little is known of how the se cell types interact beyond our previous observations that they comm unicate via gap junctions and adhere avidly to each other. In the pres ent study, we utilized several approaches to examine the mechanism by which gonocytes adhere to Sertoli cells in vitro. First, we characteri zed this attachment in general by (1) determining its susceptibility t o brief trypsinization in decreasing concentrations of Ca2+, (2) asses sing the ability of gonocytes to adhere to Sertoli cells at reduced te mperature, and (3) examining the effect of phospholipase C treatment o n the number of gonocytes attached to a Sertoli cell monolayer. Becaus e the findings suggested that a non-cadherin mechanism is involved, we used immunofluorescence to identify the presence of neural cell adhes ion molecule (NCAM) at virtually all gonocyte-Sertoli cell (and Sertol i cell-Sertoli cell) boundaries and found that incubation of coculture s in the continuous presence of NCAM antibodies caused release of esse ntially all gonocytes (but not Sertoli cells) from the monolayer. We a lso found, in (3) above, that gonocyte-Sertoli cell adhesion was very susceptible to phospholipase C in cocultures isolated from newborns an d maintained in vitro for 2 hours or 1 day but not in cultures maintai ned for 3 days. Moreover, cells isolated from pups 5 days old were as resistant to enzyme treatment at 2 hours postplating as were cultures from newborns after 3 days in vitro. Thus, the way in which gonocytes adhere to Sertoli cells appears to change during the immediate postnat al period, as reflected by the observed change in phospholipase sensit ivity, perhaps indicating production of a phospholipase C-resistant NC AM isoform by several days after birth. These data constitute new info rmation on the way in which postnatal gonocytes adhere to Sertoli cell s and provide a basis for future work in our ongoing exploration of ge rm cell development in the neonatal rat testis.