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The localization of sulfated glycoprotein-2 (clusterin; SGP-2) was investigated in the rete testis, efferent ducts, and epididymis of the rat using light (LM) and electron (EM) microscope immunocytochemistry. At the LM level, the epithelial cells of the rete testis and efferent ducts demonstrated an intense immunoperoxidase reaction over their apical and supranuclear regions, and sperm in the lumen of the efferent ducts were unreactive. In the EM, gold particles were found exclusively over the endocytic apparatus of these cells. In the proximal area of the epididymal initial segment, an insignificant immunostaining of epithelial cells and sperm was observed. However, the distal area of the initial segment showed a moderate staining over the epithelial principal cells and sperm, while in the intermediate zone of the epididymis a stronger reaction was observed over these cells. The strongest immunoperoxidase reaction was noted in the caput epididymidis, where it formed a distinct mottled pattern. Thus, while some principal cells were intensely stained, others were moderately or weakly stained; a few were completely unreactive. In the corpus and cauda epididymidis, the staining pattern was similar but not as intense. In the EM, only the secretory apparatus of these cells was found to be immunolabeled with gold particles. Sperm in the lumen of these different regions were also labeled. The epithelial clear cells were unreactive throughout the epididymis. Northern blot analysis substantiated these results and showed the presence of highest levels of SGP-2 mRNA in the caput epididymidis, especially in its proximal area, whereas increasingly lower levels were found in the corpus and cauda epididymidis. In summary, these results suggest that testicular SGP-2 dissociates from the sperm during passage through the rete testis and efferent ducts, where it is endocytosed by the epithelial cells lining these regions. In the epididymis, it is replaced by an epididymal SGP-2 that is secreted by the epithelial principal cells of the epididymis. Furthermore, in the epididymis, the principal cells appear to be in different functional states with respect to the secretion of epididymal SGP-2 within a given region of the duct as well as along the epididymal duct.

Rabbit polyclonal antibodies, directed specifically against clusterin purified from ram rete testis fluid, were employed in an investigation of the biosynthesis of clusterin by cultures of rete testis epithelial cells and by Sertoli cells prepared from testes of adult rams. Cells in serum-free medium were incubated in the presence of either [35S]methionine, [3H]leucine, or [3H]glucosamine, and radiolabeled proteins secreted were immunoprecipitated. The pellet was subjected to polyacrylamide gel electrophoresis under reducing and non-reducing conditions, and the gels were then fluorographed. In other experiments, protein bands were transferred to nitrocellulose and visualized immunochemically. Under non-reducing conditions, a single band was detected, having a molecular weight of 80,000. Under reducing conditions, doublet bands were detected, having approximate molecular weights of 40,000 (major band) and 37,000 (minor band). These properties were indistinguishable from those obtained with authentic samples of pure clusterin subjected to gel electrophoresis and Western immunoblot procedures. Amounts of clusterin synthesized by rete testis cells in culture, quantitatively determined with a sandwich enzyme-linked immunosorbent assay procedure, were approximately 4 micrograms/micrograms cell DNA/48 h. Immunocytochemical localization investigations, using monoclonal antibodies against clusterin, revealed the presence of clusterin in the perinuclear of juxtanuclear regions, in both rete testis epithelial cells and Sertoli cells in culture. The possible functions of clusterin produced by rete testis epithelial cells and by Sertoli cells are discussed.