Specificity of Probes
On immunoblots of seminiferous epithelium, antibodies to IC74 and kinesin-II reacted predominantly with bands of the appropriate molecular weight for each of the motor proteins (Fig. 1). When gels were overloaded with protein or when blots were overexposed, the IC74 antibody also reacted weakly with two minor bands (at a lower molecular mass than 74 kDa) that did not convincingly react with normal mouse IgG in control blots. Kinesin-II appeared on the blot as a characteristic doublet. When gels were overloaded with protein, minor bands also appeared on the blot; however, these bands were reactive with normal mouse IgG. Buy Advair Diskus Online
IC74 Immunoreactivity Was Concentrated in Sertoli Cell Regions Surrounding Apical Crypts
The probe for the intermediate chain of cytoplasmic dy-nein (IC74) reacted intensely with Sertoli cell regions surrounding apical crypts. This was particularly evident in fixed frozen sections and at stages of spermatogenesis when elongate spermatids were positioned deep within the epithelium (Fig. 2). Intense tracts of fluorescence followed the contour of adjacent spermatid heads and the related junction plaques (arrowheads in Fig. 2, A-E). Although the dynein probe was reactive with regions related to spermatid heads, the fluorescence signal was not restricted to these sites. Distinct tracts of fluorescence often extended beyond areas containing junction plaques into more apical regions of the Sertoli cell cytoplasm (arrows in Fig. 2, A-C and E), and a diffuse fluorescence often was present in the basal half of Sertoli cells.
FIG. 1. Immunoreactivity of antibodies to the intermediate chain of cytoplasmic dynein (IC74) (A and B) and kinesin-II (C) with blots of isolated rat seminiferous epithelium. The IC74 antibody reacted strongly with a single band within the range of that determined for the dynein intermediate chain (74 kDa; arrowhead, lane 2 in A; lane 1 contains molecular weight standards X 10~3). When the gel was overloaded with protein, two minor bands appeared (lane 3 in A and lane 2 in B, arrows) that did not appear in normal mouse IgG controls (lanes 3 and 4 in B; lane 4 was exposed for a longer period of time than lanes 2 and 3). The antibody to kinesin-II reacted specifically with two closely related bands (arrowheads) at the appropriate molecular size for this motor protein (lane 1 in C). Minor bands on the blot were also present on the normal mouse IgG control blots (lane 2 in C).
FIG. 2. Fixed frozen sections of rat seminiferous epithelium double-labeled for cytoplasmic dynein (green) and filamentous actin (red).The filamentous actin associated with spermatid heads was predominantly in Sertoli cell junction plaques (ectoplasmic specializations). Notice that at these stages of spermatogenesis, immunoreactivity to the probe for cytoplasmic dynein occurred in Sertoli cell regions containing junction plaques (arrowheads) (areas of overlap are indicated in yellow). Also notice that this staining was particularly intense adjacent to dorsal aspects of spermatid heads and extended into apical Sertoli cell regions (little white arrows) well beyond those areas immediately associated with spermatid heads (arrows in A, bottom).