Role of tissue inhibitor of metalloproteases-1 in junction dynamics in the testis
Mruk,Dolores D.; Siu,Michelle K.Y.; Conway,Anne M.; Lee,Nikki P.Y.; Lau,Ann S.N.; Cheng,Chuen-yan
Journal of Andrology 24(4): 510-523
Publication date: 2003
Using multiple high-performance liquid chromatography steps,we have identified and purified a polypeptide to apparent homogeneityfrom primary Sertoli cell conditioned culture medium that consistedof 2 molecular variants of 31 and 29 kDa when electrophoresedon a sodium dodecyl sulfate-polyacrylamide gel run underreducing conditions. Partial N-terminal amino acid sequenceanalysis of these 2 proteins revealed a sequence of NH-IKMAKMLKGFDAVGNATG,which is homologous to tissue inhibitor of metalloproteases-1(TIMP-1). Studies by semiquantitative reverse transcription-polymerasechain reaction using a primer pair specific to rat TIMP-1 demonstratedthat both Sertoli and germ cells express TIMP-1. During maturation,the steady-state TIMP-1 mRNA level in the testis increased significantlyfrom 40 to 60 days of age, which suggests its role in the restructuringof the epithelium during spermiation. This increase in testicularTIMP-1 expression was apparently not due to the increase ingerm cell number, because TIMP-1 expression decreased approximatelyfivefold in germ cells isolated from testes of aging rats. UsingSertoli cells cultured at low (0.05 x 10 cells/cm) and high(0.5 x 10 cells/cm) densities, it was found that TIMP-1 expressionincreased transiently but significantly during junction assembly.A similar induction of TIMP-1 mRNA was also detected in Sertoli-germcell cocultures during germ cell adhesion onto Sertoli cells.More important, the inclusion of either -macroglobulin (a proteaseinhibitor produced by Sertoli cells) or aprotinin (a serineprotease inhibitor) into an in vitro germ cell adhesion assayfacilitated the attachment of fluorescently labeled germ cellsonto the Sertoli cell epithelium when compared to control, whichsuggests that the assembly of adherens junctions may involveprotease inhibitors.