Immunolocalization in the Epididymis
Negative controls showed no immunoreactivity in either fetal or adult epididymides (not shown). In the fetal epididymis (Fig. 3, a-c), scinderin was located along the subsurface of the principal cells, chiefly near the cellular contacts. Labeling was particularly heavy in the body (corpus) of the epididymis (Fig. 3b). In the adult epididymis (Fig. 3, d-f), scinderin immunoreactivity was observed in the subsurface region of the principal cells (Fig. 3d) in all three regions (head [caput], body [corpus], and tail [cauda]) of the epididymis. Deposits of scinderin-positive materials were observed near the base of the principal cells (Fig. 3, e and f). The deposits were most frequent and largest in the tail of the epididymis (Fig. 3f). Spermatozoa were positively labeled in all three regions of the epididymis (Fig. 3, d-f).
Immunolocalization in Spermatozoa
Negative controls in epididymal (Fig. 4a) and ejaculated (not shown) spermatozoa showed no immunoreactivity in the head; however, part of the tail, principally the middle piece, appeared slightly stained. In epididymal spermatozoa (Fig. 4b), scinderin labeling was heavy in the anterior ac-rosome and in the equatorial segment, but it was light in the postacrosomal region. Labeling was also found in part of the connecting piece situated caudally to the postacro-somal region (Fig. 4b). Furthermore, labeling occurred in the middle piece of the tail; however, because controls also showed staining in this part of the tail, some degree of false positive labeling cannot be ruled out. In ejaculated spermatozoa (Fig. 4, c and d), the acrosome and the postacro-somal region were heavily labeled, but the equatorial segment appeared negative. The results obtained were the same whether labeling was achieved using immunofluorescence (Fig. 4c) or streptavidin-HRP conjugate (Fig. 4d). Labeling was also noted in part of the tail with both techniques.
FIG. 3. Immunolocalization of scinderin in the fetal epididymides (head [a], body [b], tail [c]) and adult (head [d], body [e], tail [f]). A lumen is clearly evident in epididymides obtained during the 8th month of gestation. During this period (a-c), scinderin was localized near the lateral cell surface of the principal cells near the base (closed arrows) and close to the apical border near the intercellular junctions (open arrows). In tangential sections of adult epididymis (d-f), scinderin immunoreactivity was detected all along the subsurface of the principal cells’ lateral plasma membranes (open arrows in d). In the body and particularly in the tail of the epididymis, deposits of scinderin-positive material were detected (closed arrows in e, f) near the basal cells. The curved solid arrows in d-f point to scinderin-positive spermatozoa. X780.
FIG. 4. Immunolocalization of scinderin in epididymal (a, b) and ejaculated (c, d) spermatozoa. Controls using preimmune serum show no reaction product in the head of epididymal spermatozoa; however, staining was detected (open arrowheads in a) in portions of the tails. Immunofluorescence localization of scinderin in epididymal spermatozoa (b) shows small spots of immunoreactivity in the postacrosomal region (arrowhead); the protein is localized principally in the anterior acrosome and in the equatorial segment (arrow). Part of the connecting piece situated caudally to the postacrosomal region is also scinderin-positive. In ejaculated spermatozoa (c), the acrosome (curved arrow) and the postacrosomal region were heavily labeled when immunofluorescence labeling (c) or streptav-idin-HRP was used (d). X780 (reproduced at 82%).