Immunocytochemical Localization of 2D6-Reactive Antigens in Epididymal Tissue
The epithelium lining the efferent ductules, initial segment, and proximal caput epididymidis did not stain with 2D6 McAb/FITC-RAM (Fig. 1A). However, a strong signal was detected in the microvilli and apical cytoplasm of principal cells in the distal caput epididymidis and thereafter in the epithelium throughout the corpus and cauda epididymidis (Fig. 1, B and C). Only background fluorescence was present in basal cells and peritubular connective tissue. A noteworthy feature was the abrupt transition between the negatively staining epithelium in the proximal caput region and the positively staining epithelium in the adjoining distal caput. Despite the strong reaction in the epithelium, only a weak reaction was observed in the duct lumen in the distal caput (Fig. 1B).
The strength of the fluorescent signal associated with spermatozoa increased rapidly in a distal direction so that a very intense reaction was present in the lumen of the distal corpus and cauda epididymidis (Fig. 1C).
At the ultrastructural level, gold-conjugated RAM IgG-2D6 McAb complexes were associated with stacked arrays of membranes in the Golgi region of principal cells throughout the distal caput epididymidis (Fig. 1D). Gold particles were also associated with small membrane-bound vesicles in the apical cytoplasm that were either empty or weakly electron dense (Fig. 1, D and E).
FIG. 1. Immunofluorescence (A-C) and immunogold (D-H) staining of the epidid-ymal epithelium with 2D6 McAb. A) Frozen section of the proximal caput epidi-dymidis; only background fluorescence is present in the epithelium and peritubular connective tissue. B) Distal caput region showing positive fluorescence in apical cytoplasm of principal cells. C) Section of cauda region. A strong reaction is present in the lumen (L) and epithelium. D and E) Gold particles are present in Golgi cister-nae (G) and surrounding small vesicles in the apical cytoplasm of a principal cell in the distal caput epididymidis. Large empty vesicles are unlabeled. M = mitochondrion; L = lumen. F) The surface of microvilli on principal cells in the distal caput is encrusted with gold particles, which are also present in the lumen (L). G) Control section showing the apical region of a principal cell in the distal caput stained with second-layer antibodies only. Only a few gold particles are present. H) Cross section of a sperm tail in the lumen of the cauda epididymidis. There is heavy labeling of the sperm tail with numerous small clusters of gold particles free in the lumen. Bars: 50 ^m (A-C); 1 ^m (D-H).