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Abstract

Changes in the expression of junctional and nonjunctional complex component genes when inter-Sertoli tight junctions are formed in vitro 
Wong,Connie C.S.; Chung,Sanny S.W.; Grima,Josephine; Zhu,Li-Ji; Mruk,Dolores D.; Lee,William M.; Cheng,Chuen-yan
Journal of Andrology 21(2): 227-237
Publication date: 2000


Throughout spermatogenesis, germ cells move progressively from the basal tothe adluminal compartment, which is accompanied by continual disassemblyand reassembly of intercellular junctions suggesting germ cell movement iscomposed of intermittent phases of junction disassembly and reassembly. Astudy was performed to correlate the expression of junctional-complexcomponents (such as zonula occludens-1 [ZO-1], a tight-junction componentprotein) and nonjunctional complex components (such as urokinase-typeplasminogen activator [uPA], a serine protease; cathepsin L, a cysteineprotease; alpha2-macroglobulin, a nonspecific protease inhibitor; andcystatin C, a cysteine protease inhibitor) at the time when inter-Sertolitight junctions were established in vitro. This is an attempt toinvestigate whether the expression of nonjunctional component genes alsocorrelates with the formation of inter-Sertoli tight junctions in vitro.This is part of an effort to understand the physiologic elements of germcell movement in the epithelium. Sertoli cells cultured in vitro are knownto undergo programmed cell death. To ensure that the changes in target geneexpression were not the result of apoptosis, Sertoli cells were cultured invitro at densities of 0.25, 0.75, and 3 x 10(6) cells/cm2 for up to 7 dayson bicameral culture units coated with Matrigel (Collaborative Research)and were assessed by morphologic analysis and agarose gel electrophoresis.It was noted that many of the Sertoli cells cultured at 3 x 10(6) cells/cm2underwent apoptosis by day 7, in contrast to cultures at 0.25 and 0.75 x10(6) cells/cm2 illustrating the Sertoli cell number per unit of area maybe an important parameter to be considered when studying Sertoli cellfunction in vitro. Also, it was shown that the expression of ZO-1 increasedsignificantly between days 2 and 3 prior to the establishment ofinter-Sertoli tight junctions assessed by transepithelial resistancemeasurement (TER), which illustrates that ZO-1 can be used as a marker tomonitor this cellular event. More interestingly, there was also a transientincrease in the expression of uPA and cathepsin L between days 2 and 3 atthe time preceding the formation of tight junctions. In Sertoli cellscultured at low density (2 x 10(4) cells/cm2), when a confluent monolayerof cells could not form, there were no changes in the expression of eitherZO-1, uPA, or cathepsin L throughout the 7-day culture period. Theseresults show that the establishment of specialized junctions, such as tightjunctions between Sertoli cells in vitro, may require the participation ofboth junctional and nonjunctional complex components.