A protein designated acidic epididymal glycoprotein (AEG) was purified from rat epididymis using ion exchange chromatography on DEAE‐Sephadex, gel filtration in Sephadex G‐75 and affinity chromatography on Concanavalin‐A Sepharose. AEG is a major secretory product of the epididymis making up 2–3 per cent of total soluble protein. Antibody to AEG was raised in rabbits and purified by affinity chromatography on AEG‐Sepharose. Quantitation of AEG in cytosol using “rocket” immunoelectrophoresis showed AEG to increase in epididymal segments from caput to cauda. Ligation of the midcorpus decreased AEG in the cauda. Localization of AEG using an immunoperoxidase method revealed that it is secreted largely by the epithelium of caput and corpus beginning with the region distal to the initial segment. It appears to be secreted by a specific cell type, probably the so‐called “principal” cell. Specific staining of AEG was also noted in “clear” cells in the cauda. Spermatozoa become coated with AEG as they leave the initial segment and remain so during passage through the cauda.

A method for the isolation and culture of epididymal e ithelial cells obtained from pubertal and old adult rats is d red. This method permits the establishment of primary cultures of these cells in monolayers from aggregates isolated from whole epididymides and major epididymal anatomical segments (caput, corpus, and cauda) after trypsin and collagenase digestions. A large number of cultured epididymal cells retain a differentiated function as demonstrated by the immunocytochemical and radioimmunoassay finding of acidic epididymal glycoprotein, a spermatozoa-coating protein secreted by the principal cells of rat epididymis. The proliferative tential of cultured epididymal cells obtained from pubertal an old adult donors can be documented by [3H]thymidine labeling and mitotic indices without significant loss of gene expression for acidic epididymal glycoprotein.Results of this study demonstrate that epididymal epithelial cells, consisting of a predominant population of principal cells, can be isolated, cultured, and maintained for up to 3 months.

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