Serological and biochemical identification of a plasma membrane antigen specific to Leydig cells

Purified mouse Leydig cells have been prepared from interstitial cell suspensions using a Percoll gradient procedure. The isolated cells are 88-95% pure as determined by light microscopy. Staining for 3-beta-hydroxysteroid dehydrogenase indicates that over 85% of the Leydig cells recognized by differential interference microscopy are also positive for this enzyme. After separation, the Leydig cells are viable by dye exclusion assays and exhibit normal in situ morphology when examined at the ultrastructural level. Leydig cell suspensions have been used to raise a polyclonal antiserum in rabbits. This antiserum, prior to absorption, reacts in indirect immunofluorescent studies with the surfaces of isolated Leydig cells, with testicular germ cells, with spermatozoa and with somatic cells such as splenocytes. Following absorption with lymphocytes and spermatogenic cells, the antiserum binds only to Leydig cell plasma membranes. Quantitative measurements with 125I-protein A confirm the specific labeling of Leydig cells by the absorbed antiserum. Biochemical identification of the Leydig cell plasma membrane antigen has been accomplished by immunoblotting polyacrylamide gel nitrocellulose transfers. A single major band of Mr approximately 40,000 is detected on one-dimensional transfers; two weakly reactive spots of Mr 43,000 and 45,000 are detected using two-dimensional immunoblots. Blotting experiments conducted using concanavalin A have identified the major Leydig cell constituents reactive with this lectin. The Leydig cell plasma membrane antigen(s) does not bind concanavalin A.