Role of Tissue Inhibitor of Metalloproteases‐1 in Junction Dynamics in the Testis

Abstract

Using multiple high-performance liquid chromatography steps, we have identified and purified a polypeptide to apparent homogeneity from primary Sertoli cell conditioned culture medium that consisted of 2 molecular variants of 31 and 29 kDa when electrophoresed on a sodium dodecyl sulfate-polyacrylamide gel run under reducing conditions. Partial N-terminal amino acid sequence analysis of these 2 proteins revealed a sequence of NH(2)-IKMAKMLKGFDAVGNATG, which is homologous to tissue inhibitor of metalloproteases-1 (TIMP-1). Studies by semiquantitative reverse transcription-polymerase chain reaction using a primer pair specific to rat TIMP-1 demonstrated that both Sertoli and germ cells express TIMP-1. During maturation, the steady-state TIMP-1 mRNA level in the testis increased significantly from 40 to 60 days of age, which suggests its role in the restructuring of the epithelium during spermiation. This increase in testicular TIMP-1 expression was apparently not due to the increase in germ cell number, because TIMP-1 expression decreased approximately fivefold in germ cells isolated from testes of aging rats. Using Sertoli cells cultured at low (0.05 x 10(6) cells/cm(2)) and high (0.5 x 10(6) cells/cm(2)) densities, it was found that TIMP-1 expression increased transiently but significantly during junction assembly. A similar induction of TIMP-1 mRNA was also detected in Sertoli-germ cell cocultures during germ cell adhesion onto Sertoli cells. More important, the inclusion of either alpha(2)-macroglobulin (a protease inhibitor produced by Sertoli cells) or aprotinin (a serine protease inhibitor) into an in vitro germ cell adhesion assay facilitated the attachment of fluorescently labeled germ cells onto the Sertoli cell epithelium when compared to control, which suggests that the assembly of adherens junctions may involve protease inhibitors.

Intercellular adhesion molecule-1 is a regulator of blood–testis barrier function

“…Sertoli cells were isolated from testes of 20-day-old rats and cultured in DMEM/ F12 (Sigma-Aldrich, St Louis, MO) supplemented with growth factors and an antibiotic (Cheng et al, 1986;Mruk et al, 2003). Cells were seeded at a density of 0.5610 6 cells/cm 2 in 12-well plates for lysate preparation, RNA isolation and overexpression experiments, at a density of 1.2610 6 cells/cm 2 in Millicell inserts (Millipore Corp., Billerica, MA) for transepithelial electrical resistance (TER) measurements and overexpression experiments, or at a density of 0.05610 6 cells/ cm 2 on round glass coverslips (18 mm; Thomas Scientific, Swedesboro, NJ) for immunofluorescent staining.…”

Actin-binding protein drebrin E is involved in junction dynamics during spermatogenesis

“…Cells were cultured in serum-free DMEM/F12 containing 10 μg/ml bovine insulin, 5 μg/ml transferrin (Calbiochem/ EMD Chemicals), 2.5 ng/ml epidermal growth factor and 5 μg/ml bacitracin at 35°C in a humidified atmosphere of 5% CO 2 /95% air (v/v) as earlier described. 74 Cells were hypotonically treated with 20 mM Tris (Invitrogen) (pH 7.4 at 22°C) on day 2 to lyse residual germ cells. 75 Sertoli cell purity in these cultures was >98% with negligible contamination of germ (e.g., spermatogonia and spermatocytes), peritubular myoid and Leydig cells, as earlier characterized by RT-PCR and immunoblotting using specific primer pairs or antibodies against corresponding marker genes/proteins and electron microscopy.…”

Regulation of blood-testis barrier dynamics: an in vivo study

“…Immunofluorescence microscopy Co-localization of occludin and ZO-1 to the same site in the BTB of the rat seminiferous epithelium was performed by immunofluorescence microscopy as earlier described Mruk et al, 2003). In brief, frozen sections of testes (~8 µm) from normal rats and those treated with CdCl2 (3 mg/kg b.w., i.p.)…”

“…the influence of cytokines (Mruk et al, 1997;Mruk et al, 2003). Furthermore, we have examined whether TGF-β3 can also utilize the p38 MAP kinase pathway to regulate AJ dynamics.…”