Ultrastructural and biochemical evidence for gap junction and connexin 43 expression in a clonal Sertoli cell line: a potential model in the study of junctional complex formation

To clarify the exact role of Sertoli cells in testicular intercellular communications, a murine Sertoli cell line (42GPA9) has recently been established. Electronmicroscopy studies indicate that the morphology of these immortalized cells strongly resembles that of mouse Sertoli cells in vivo with an indentend nucleus, elongated mitochondria and numerous lysosome-like structures. Ultrastructure analysis has also revealed that 42GPA9 cells form gap junctions as demonstrated by the presence of small electron-dense bridges that connect the plasma membranes of adjacent cells. The gap junction protein connexin 43 (Cx43) has been identified in cultured 42GPA9 cells by immunofluorescence and Western blot analysis. No immunostaining is detected in the absence of apparent intercellular contact. The anti-Cx43 antibody labels the contacts between 42GPA9 cells at confluency. This specific staining appears as small dots forming isolated rows of dots or surrounding the entire cell, suggesting that Cx43 is assembled into membrane plaques. The gap junctional communication capacity of the 42GPA9 cell line has been demonstrated by the dye-transfer technique. Exposure of 42GPA9 cells for 24 h to cAMP and 12-O-tetradecanoylphorbol-13-acetate greatly reduces the Cx43 staining at cell-cell contacts and concomitantly increases the cytoplasmic staining, suggesting that these agents alter the trafficking of Cx43 to the plasma membrane. Thus, the 42GPA9 line may provide a useful in vitro model for studying gap junction communication between Sertoli cells.