A monoclonal antibody recognizes a form of intermediate filament protein in rat Sertoli cells that is not present in seminiferous peritubular cells.
We extracted intermediate filament proteins (IFP) that were insoluble in in detergent from cultured rat Sertoli and peritubular cells after labeling with [35S] methionine and resolved them by two-dimensional polyacrylamide gel electrophoresis, autoradiography and Western blotting. We found that vimentin was the predominant IFP in both Sertoli and peritubular cells. However, while two isoelectric variants of vimentin were observed in Sertoli cells, only one was detected in peritubular cells. In addition, vimentin in Sertoli cells generated a large number of breakdown products, many differing in molecular weight and isoelectric point when compared to peritubular cells. These findings suggested that a Ca2+-activated protease, usually found in cells containing vimentin, was capable of generating proteolytic fragment patterns that were specific to cell type. Monoclonal antibodies were generated against IFP extracted from cultured Sertoli cells and their reactivity monitored by indirect immunofluorescence using Sertoli cells, peritubular cells and intact testis. One monoclonal antibody, designated IFP-SC, was an immunoglobulin (IgM) that produced a vimentin-like immunofluorescent pattern in cultured Sertoli cells. This pattern consisted of an extensive filamentous network that extended throughout the cell and surrounded the nucleus. In cultured peritubular cells, IFP-SC generated a diffuse, nonfilamentous immunoreactivity. IFP-SC reacted with components of seminiferous tubules and displayed immunoreactivity associated with Sertoli cells but not with elements of the seminiferous peritubular cell wall. Because immunofluorescent patterns were distinctly specific to cell type in cultured cells and in the intact tissue, monoclonal antibody IFP-SC is useful for the quantitative evaluation of cell cross-contamination of Sertoli and peritubular cells and, therefore, allows a precise characterization of functional events specific to cell type and monitoring of the differentiation state of cells with time in culture.
D000818:Animals; D000911:Antibodies, Monoclonal; D002478:Cells, Cultured; D004591:Electrophoresis, Polyacrylamide Gel; D007381:Intermediate Filament Proteins / Q000032:analysis* / Q000096:biosynthesis; D008297:Male; D008715:Methionine / Q000378:metabolism; D008970:Molecular Weight; D051381:Rats; D012671:Seminiferous Tubules / Q000032:analysis* / Q000166:cytology; D012708:Sertoli Cells / Q000032:analysis* / Q000166:cytology; D013462:Sulfur Radioisotopes; D013737:Testis / Q000032:analysis*
D000911:Antibodies, Monoclonal; D007381:Intermediate Filament Proteins; D013462:Sulfur Radioisotopes; D008715:Methionine