Study on the formation of specialized inter-Sertoli cell junctions in vitro

An in vitro culture system using Sertoli cells was employed to assess the e xpression of component genes pertinent to occluding junctions (OJ) (such as zonula occludens-l, ZO-1), anchoring junctions (AJ) (such as N-cadherin an d beta-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 x 10(4) cells/cm(2)) or high-cell density (0.6 x 10(6) cells/ cm(2)) on Matrigel-coated dishes for 7 days in vitro to allow the establish ment of specialized junctions. in low cell density Sertoli cell cultures, s pecialized OJ such as tight junctions did not form during the entire cultur e 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 S ertoli cells were cultured at both cell densities, there was a transient in crease in Sertoli cell N-cadherin expression, which peaked by days 4-5, sug gesting the time course for the establishment of Al may overlap with the OJ . A significant increase in the expression of Sertoli cell beta-catenin was also detected by days 5-7 in the high but not low cell density cultures. T he expression of Cx33 was also enhanced at days 4-5 in both high and low de nsity cultures. These results suggest that OJ, Al, and GJ are formed betwee n Sertoli cells in high density cultures, whereas OJ cannot be formed in lo w density cultures. A full-length cDNA clone coding for rat testicular beta -catenin was also isolated. The deduced amino acid sequence of rat beta-cat enin yielded a 781 amino acid polypeptide which displayed a 99.9% identity with the mouse homolog. Conditioned medium of germ cells induced a dose-dep endent stimulation on Sertoli cell beta-catenin expression, suggesting germ cells may affect the N-cadherin/beta-catenin-mediated signal transduction pathway. In summary, this study illustrates several target genes can be use d as molecular markers to monitor the inter-Sertoli junction formation. Thi s system should be applicable to screen new male contraceptives in vitro ta rgeted at the interference of junction formation by disrupting the timely e xpression of genes necessary for junction establishment and/or maintenance. (C) 1999 Wiley-Liss, Inc.