Changes in the expression of junctional and nonjunctional complex component genes when inter-Sertoli tight junctions are formed in vitro

Throughout spermatogenesis, germ cells move progressively from the basal to the adluminal compartment, which is accompanied by continual disassembly a nd reassembly of intercellular junctions suggesting germ cell movement is c omposed of intermittent phases of junction disassembly and reassembly. A st udy was performed to correlate the expression of junctional-complex compone nts (such as zonula occtudens-1 [ZO-1], a tight-junction component protein) and nonjunctional complex components (such as urokinase-type plasminogen a ctivator [uPA], a serine protease; cathepsin L, a cysteine protease; alpha( 2)-macroglobulin, a nonspecific protease inhibitor; and cystatin C, a cyste ine protease inhibitor) at the time when inter-Sertoli tight junctions were established in vitro. This is an attempt to investigate whether the expres sion of nonjunctional component genes also correlates with the formation of inter-Sertoli tight junctions in vitro. This is part of an effort to under stand the physiologic elements of germ cell movement in the epithelium. Ser toli cells cultured in vitro are known to undergo programmed cell death. To ensure that the changes in target gene expression were not the result of a poptosis, Sertoli cells were cultured in vitro at densities of 0.25, 0.75, and 3 x 10(6) cells/cm(2) for up to 7 days on bicameral culture units coate d with Matrigel (Collaborative Research) and were assessed by morphologic a nalysis and agarose gel electrophoresis. It was noted that many of the Sert oli cells cultured at 3 x 10(6) cells/cm(2) underwent apoptosis by day 7, i n contrast to cultures at 0.25 and 0.75 x 10(6) cells/cm(2) illustrating th e Sertoli cell number per unit of area may be an important parameter to be considered when studying Sertoli cell function in vitro. Also, it was shown that the expression of ZO-1 increased significantly between days 2 and 3 p rior to the establishment of inter-Sertoli tight junctions assessed by tran sepithelial resistance measurement (TER), which illustrates that ZO-1 can b e used as a marker to monitor this cellular event. More interestingly, ther e was also a transient increase in the expression of uPA and cathepsin L be tween days 2 and 3 at the time preceding the formation of tight junctions. In Sertoli cells cultured at low density (2 x 10(4) cells/cm(2)), when a co nfluent monolayer of cells could not form, there were no changes in the exp ression of either ZO-1, uPA, or cathepsin L throughout the 7-day culture pe riod. These results show that the establishment of specialized junctions, s uch as tight junctions between Sertoli cells in vitro, may require the part icipation of both junctional and nonjunctional complex components.