An in vitro culture system using Sertoli cells was employed to assess the expression of component genes pertinent to occluding junctions (OJ) (such as zonula occludens-1, ZO-1), anchoring junctions (AJ) (such as N-cadherin and β€-catenin), and communicating gap junctions (GJ) (such as connexin 33, Cx33) when they are being formed in vitro. Freshly isolated Sertoli cells from 20-day-old rats with a purity of greater than 90% were cultured either at low-(2.5 Ο« 10 4 cells/cm 2 ) or high-cell density (0.6 Ο« 10 6 cells/cm 2 ) on Matrigel-coated dishes for 7 days in vitro to allow the establishment of specialized junctions. In low cell density Sertoli cell cultures, specialized OJ such as tight junctions did not form during the entire culture period when assessed by the transepithelial electrical resistance (TER). In high cell density cultures, there was an increase in ZO-1 expression in days 1 to 3 preceding the establishment of tight junctions by day 4. When Sertoli cells were cultured at both cell densities, there was a transient increase in Sertoli cell N-cadherin expression, which peaked by days 4 -5, suggesting the time course for the establishment of AJ may overlap with the OJ. A significant increase in the expression of Sertoli cell β€-catenin was also detected by days 5-7 in the high but not low cell density cultures. The expression of Cx33 was also enhanced at days 4 -5 in both high and low density cultures. These results suggest that OJ, AJ, and GJ are formed between Sertoli cells in high density cultures, whereas OJ cannot be formed in low density cultures. A full-length cDNA clone coding for rat testicular β€-catenin was also isolated. The deduced amino acid sequence of rat β€-catenin yielded a 781 amino acid polypeptide which displayed a 99.9% identity with the mouse homolog. Conditioned medium of germ cells induced a dose-dependent stimulation on Sertoli cell β€-catenin expression, suggesting germ cells may affect the N-cadherin/β€-catenin-mediated signal transduction pathway. In summary, this study illustrates several target genes can be used as molecular markers to monitor the inter-Sertoli junction formation. This system should be applicable to screen new male contraceptives in vitro targeted at the interference of junction formation by disrupting the timely expression of genes necessary for junction establishment and/or maintenance.