Immunodetection of cell adhesion molecules in rat Sertoli cell cultures.

PROBLEM: The presence of cell adhesion molecules (CAMs) in Sertoli cells has not been explored extensively. The expression of CAMs involved in cell-matrix and cell-to-cell interactions in Sertoli cell cultures was examined. METHOD OF STUDY: Immunohistochemical and Western blot techniques were applied to rat Sertoli cell cultures using specific antibodies to alpha 3, alpha 5, and alpha 6 integrin subunits; NCAM; and cadherins. RESULTS: Expression of alpha 3 and alpha 6 integrin subunits (mainly laminin receptors) and lack of expression of alpha 5 integrin subunit (fibronectin receptor) was observed in Sertoli cells by immunohistochemistry. These cells also expressed neural CAM (NCAM) and N-cadherin. By Western blot analysis, Sertoli cell extracts reacted with antibodies to alpha 3 integrin subunit revealed a band approximately 130 kDa, whereas no expression of alpha 5 integrin subunit was detected. Cell extracts incubated with antibodies to pan cadherin exhibited a band approximately 120 kDa, whereas bands of 180, 140, and 120 kDa were observed with antibodies to NCAM. CONCLUSION: New data about the expression of receptors for extracellular matrix proteins (alpha 3 and alpha 6 integrin subunits) as well as cell-to-cell adhesion molecules (NCAM and cadherins) are reported in rat Sertoli cell cultures.

Immunodetection of cell adhesion molecules and extracellular matrix proteins in rat Leydig cell cultures.

Cell adhesion molecules (CAMs) as well as extracellular matrix (ECM) proteins were identified in Leydig cell cultures using immunohistochemical and Western blot analysis. Leydig cells were isolated from 60-day-old rats and cultured for 4 days. For immunofluorescence and immunoperoxidase techniques, Leydig cells were incubated with antisera to ECM proteins (antibodies to laminin, type IV collagen and fibronectin); antisera to integrins (antibodies to beta 1, alpha 3, alpha 5 and alpha 6 integrin subunits) and antisera to cell-to-cell adhesion molecules (antibodies to N-CAM and N-cadherin). Results of the two immunohistochemical techniques were similar. Laminin and type IV collagen were detected in the perinuclear area of Leydig cell cytoplasm and cell processes as bright granular immunofluorescence or as a brown reaction product using the immunoperoxidase technique. Leydig cells expressed alpha 3 and alpha 6 integrin subunits (mainly laminin receptors), while no reaction was detected with antibodies to the alpha 5 integrin subunit (fibronectin receptor). Leydig cells also expressed cell-to-cell adhesion molecules such as N-CAM and N-cadherin. Using Western blot analysis, Leydig cell extracts incubated with antibodies to laminin revealed two bands of around 200 kDa, which is characteristic of laminin 1 light chains. A band with electrophoretic mobility similar to that of the alpha 2 (IV) collagen chain from EHS sarcoma and a band of around 230 kDa similar to fibronectin were also detected in Leydig cell extracts using specific antisera. Leydig cells incubated with antibodies to the alpha 3 integrin subunit revealed two bands below 120 kDa. Finally, Western blot results showed that Leydig cells expressed N-CAM as two 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 synthesize ECM proteins and express ECM receptors (integrins), as well as cell-to-cell adhesion molecules such as N-CAM and N-cadherin.