INTERACTIONS OF PROTEASES AND PROTEASE INHIBITORS IN SERTOLI-GERM CELL COCULTURES PRECEDING THE FORMATION OF SPECIALIZED SERTOLI-GERM CELL-JUNCTIONS IN-VITRO

The biochemical mechanism(s) by which germ cells can form specialized junctions with Sertoli cells in the seminiferous epithelium at various stages of the spermatogenic cycle is unknown. This study sought to ex amine the biochemical changes that are involved when germ cells are co cultured with Sertoli cells in vitro preceding the establishment of sp ecialized Sertoli-germ cell junctions. While isolated germ cells were allowed to attach to Sertoli cells, media from both the apical and bas al compartments of bicameral units were collected to assess serine and cysteine protease activity, The expression of selected serine and cys teine proteases and their corresponding inhibitors in these Sertoli-ge rm cell cocultures was also examined by RT-PCR. Using an [I-125]-colla gen film assay, a transient but significant increase in serine proteas e activity was noted in both the apical and basal compartments when ge rm cells began to settle onto the Sertoli cell monolayer preceding the formation of intercellular junctions, A specific tryptase (RNK-Tryp 2 , a serine protease formerly cloned from a rat granular lymphocyte leu kemia cell line, RNK-16, cDNA expression library) was shown to be expr essed exclusively by Sertoli cells and not germ cells, Furthermore, Se rtoli cell tryptase expression as well as urokinase plasminogen activa tor (u-PA, also a serine protease) increased significantly when germ c ells were adhering to Sertoli cells. The decline in total serine prote ase activity when Sertoli-germ cell junctions were being formed was ac companied by a concomitant increase in alpha(2)-macroglobulin (alpha(2 )-MG, a nonspecific protease inhibitor) expression. No significant cha nges in cysteine protease activity in either the apical or basal compa rtment were noted. However, there was a transient but significant incr ease in cathepsin L expression when germ cells were adhering to Seriol i cells preceding cell junction formation. The subsequent reduction in cathepsin L expression after this transient increase was accompanied by a concomitant increase in cystatin C expression. These results sugg est that proteases and their corresponding inhibitors are working syne rgistically and are likely to be involved in the adherence of germ cel ls to Sertoli cells and the subsequent formation of intercellular junc tions.