Immunoblot analysis done on bovine chromaffin cells using anti-scinderin showed one band at 80 kDa (Fig. 1). Immunoblots of bovine seminiferous tubules, interstitial tissue, spermatozoa, and blood vessels also showed a unique band bearing the same molecular mass as chromaffin cell scinderin (Fig. 1). The intensity of the band for immuno-reactive scinderin was lower in adult than in fetal seminiferous tubules (Fig. 1, lanes aT, fT), but it was similar in fetal interstitial cells and in adult testis (Fig. 1, lanes ait, fit). Scinderin levels were higher in epididymal (Fig. 1, lane pSpz) than in ejaculated spermatozoa (Fig. 1, lane jSpz). Scinderin expression was higher in the vena cava (line aV) than in the aorta (line aA).
Immunolocalization in the Testis
Positive controls using adrenal glands from adult bulls revealed the presence of scinderin-positive cells in the medulla (not shown). Negative controls using either primary or secondary antibody alone or preimmune serum (Fig. 2, a and b) in fetal (Fig. 2a) and adult (Fig. 2b) testes or preadsorbed scinderin antiserum #6 (not shown) showed no immunoreactivity.
FIG. 1. Immunoblot analysis of several bovine tissues with scinderin antiserum. Twenty-five micrograms of proteins of chromaffin cells (CC), seminiferous tubules isolated from adult testes (aT), interstitial tissue cells isolated from adult testes (ait), epididymal spermatozoa (pSpz), ejaculated spermatozoa (jSpz), seminiferous tubules isolated from fetal testes (fT), interstitial tissue cells isolated from fetal testes (fit), aorta from adult bovine (aA), and vena cava from adult cattle (aV) were loaded in a 10% polyacrylamide minigel. Proteins were subjected to electrophoresis and electrotransferred onto nitrocellulose membranes. Membranes were incubated with scinderin antiserum #6 and then with alkaline phosphatase-conjugated secondary antibody. Reactive bands were revealed by treatment with p-nitroblue tetrazolium chloride and 5-bromo-4-chloro-3-in-dolyl phosphate toluidine salt. The left lane shows the position of molecular weight standards (kaleidoscope; Bio-Rad). The presence of scinderin (SC) is indicated by an arrowhead. The figure is a representative immunoblot of three different experiments using samples from different bulls.
FIG. 2. Immunolocalization of scinderin in the testis. Controls using preimmune serum showed no reaction product in a) fetal and b) adult testes. In fetal testes, scinderin was localized (arrows) in minute dots aligned along the Sertoli cells’ plasma membrane regardless of whether the membrane was facing a gonocyte or a neighboring Sertoli cell (c). d-h) Material obtained from adult testes; the roman numerals identify the stage of the cycle of the seminiferous epithelium. In germ cells, scinderin (closed arrows) was associated with the acrosome of the round developing (d-g) as well as of the elongated mature spermatids (arrowheads) (d). d) Scinderin immunoreactivity in a region corresponding with the subacrosomal space in the developing round spermatids. In addition, during all the stages of the seminiferous cycle, scinderin was localized in the Sertoli cells next to the cell surface of the trunk of the cells and of their cytoplasmic processes which surrounds the germ cells (open arrows d, g, h). Within the cytoplasm of Sertoli cells, deposits of scinderin-positive material accumulated near the base of the cell in a stage-specific manner (open arrows near the limiting membrane of the tubule g,h). X780.