Functional Coupling of Neonatal Rat Sertoli Cells and Gonocytes in Coculture*

Interaction between Sertoli cells and germ cells is likely to be critical for normal development of the testis. We have established and characterized cocultures of neonatal Sertoli cells and gonocytes and have begun to study the physical and functional relationship between these cells in vitro. Cells were isolated from rat pups by sequential enzymatic treatment and cultured in serum-free medium. When plated on Matrigel, Sertoli cells rapidly attach, and gonocytes adhere to the underlying Sertoli cells shortly thereafter. We observed that some of these germ cells develop cytoplasmic processes and elongate during the first day of culture, essentially mimicking their behavior in vivo. Electron microscopic examination of typical cultures revealed the presence of desmosome-like adhesion sites and apparent gap junctions between Sertoli cells and gonocytes. To determine whether Sertoli cells and gonocytes are functionally coupled in the cocultures, we used the glass bead-loading technique of McNeil and Warder to introduce Lucifer yellow (LY), a gap junction-permeant probe, and Rhodamine-dextran (RD), a larger marker excluded by gap junctions, simultaneously into cultures 24 h after plating. Immediate fixation and viewing of cultures with fluorescence microscopy indicated that all bead-loaded cells received both probes. We studied other living cultures 10 min after bead-loading and located RD-negative (i.e. nonbead-loaded) gonocytes that were in obvious contact with RD- and LY-positive bead-loaded Sertoli cells; these gonocytes were scored for the presence or absence of cytoplasmic LY. This analysis revealed that many gonocytes were able to obtain LY from adjacent Sertoli cells, presumably via gap junctions maintained with these cells. In addition, we quantified the percentage of gonocytes that elongated with increasing time in vitro and correlated the morphology of these cells with their ability to acquire LY from adjacent Sertoli cells. Our findings indicate that although the absolute numbers of gonocytes present decreases, more of those remaining elongate as time in vitro increases. We can also conclude from our data that gonocytes with and without processes are equally likely to be coupled with Sertoli cells under these conditions. These observations provide the first demonstration of functional coupling between Sertoli cells and premeiotic germ cells. Together with our morphological observations, they suggest that gap junction-mediated communication between these cells may be involved in stimulating or regulating changes in the gonocyte population during postnatal development of the testis.