Rat Testis Motor Proteins Associated with Spermatid Translocation: RESULTS(4)

RESULTS(4)

Although the IC74 antibody has been shown previously to react with a 67-kDa isoform of the intermediate chain in blots of mammalian flagella, staining in sperm tails was inconsistently observed by us and was either weak or not detectable. ventolin 100 mcg

Controls for IC74 Staining

The pattern of fluorescence described above (Fig. 8A) was not present when the primary antibody was replaced by normal mouse IgG (Fig. 8B), nor was it present when the primary antibody was replaced by buffer alone (Fig. 8C). It was also absent in the control for autofluorescence (both the primary and the secondary antibodies replaced by buffer; Fig. 8D).

Kinesin-II Was Localized in Spermatid Tails

The probe for kinesin-II was reactive only with sper-matogenic cells and was concentrated in the tails (Fig. 9). In early elongate spermatids, staining was patchy along the tails (Fig. 9, A and B), while at later stages the signal was more uniformly distributed (Fig. 9, C-H). There also occurred intense foci of fluorescence apparently associated with most proximal tail regions (small arrows in Fig. 9, C and E).
Fig8Rat Testis Motor Proteins
FIG. 8. Control series for the dynein probe. A) A fixed frozen section of rat testis incubated first with the antibody for cytoplasmic dynein and then with a secondary antibody conjugated to Texas Red. Immunoreactivity was evident in Sertoli cell regions associated with apical crypts containing spermatid heads. This pattern of staining was absent in material incubated either with normal mouse IgG (B) or with buffer alone (C) instead of the primary antibody, and in sections in which treatment with the primary and secondary antibodies was replaced by incubation with buffer alone (D).

Fig9Rat Testis Motor Proteins
FIG. 9. Stage-specific immunoreactivity with an antibody to kinesin-II. Paired fluorescence (A, C, E, and G) and phase (B, D, F, and H) micrographs of fixed frozen sections of rat seminiferous epithelium (A-F) and of a late spermatid (G and H) mechanically dissociated from a perfusion-fixed testis. Staining was first observed in the developing tails of elongate spermatids (A, B) and was present in the tails of all later-stage cells (C, E, and G) (white arrowheads). A bright signal was also emitted from a structure associated with the base of the sperm tail (small white arrows in C and E).