IMMUNODETECTION OF CELL-ADHESION MOLECULES AND EXTRACELLULAR-MATRIX PROTEINS IN RAT LEYDIG-CELL CULTURES

Cell adhesion molecules (CAMs) as well as extracellular matrix (ECM) p roteins were identified in Leydig cell cultures using immunohistochemi cal and Western blot analysis. Leydig cells were isolated from 60-day- old rats and cultured for 4 days. For immunofluorescence and immunoper oxidase techniques, Leydig cells were incubated with antisera to ECM p roteins (antibodies to laminin, type IV collagen and fibronectin); ant isera to integrins (antibodies to beta(1), alpha(3), alpha(5) and alph a(6) integrin subunits) and antisera to cell-to-cell adhesion molecule s (antibodies to N-CAM and N-cadherin). Results of the two immunohisto chemical techniques were similar. Laminin and type IV collagen were de tected in the perinuclear area of Leydig cell cytoplasm and cell proce sses as bright granular immunofluorescence or as a brown reaction prod uct using the immunoperoxidase technique. Leydig cells expressed alpha (3) and alpha(6) integrin subunits (mainly laminin receptors), while n o reaction was detected with antibodies to the alpha 5 integrin subuni t (fibronectin receptor). Leydig cells also expressed cell-to-cell adh esion molecules such as N-CAM and N-cadherin. Using Western blot analy sis, Leydig cell extracts incubated with antibodies to laminin reveale d two bands of around 200 kDa, which is characteristic of laminin 1 li ght chains. A band with electrophoretic mobility similar to that of th e alpha(2) (IV) collagen chain from EHS sarcoma and a band of around 2 30 kDa similar to fibronectin were also detected in Leydig cell extrac ts using specific antisera. Leydig cells incubated with antibodies to the alpha(3) integrin subunit revealed two bands below 120 kDa. Finall y, Western blot results showed that Leydig cells expressed N-CAM as tw o faint bands of around 140 kDa and N-cadherin as a 120 kDa band. The present data suggest that Leydig cells in culture are able to synthesi ze ECM proteins and express ECM receptors (integrins), as well as cell -to-cell adhesion molecules such as N-CAM and N-cadherin.