Immunocytochemical localization of the SPNR protein in wild-type, azh/azh, hop/hop, tw8/tw8, and tw2./tw2 mutant testes is shown in Figure 1. Confocal micrographs of SPNR (fluorescein isothiocyanate [FITC], green) and tubulin (Texas red) immunofluorescence in wild-type and mutant testes are shown in Figures 2-4. Spermatogenesis in the mouse has been divided into 12 developmental stages based on the presence of particular ages of developing germ cells within a given seminiferous tubule. Spermiogenesis, the haploid phase of spermatogenesis, has been divided into 16 different steps, depending on histological criteria that change as the haploid spermatids develop, including the size and position of the acrosome and nuclear shape. Descriptions of SPNR immunostaining refer to spermatids at these various steps of development. Buy Asthma Inhalers Online
Males homozygous for the azh mutation produce normal numbers of sperm but are very weakly fertile. As these previous studies have described, the reduced fertility is apparently due to gross defects in nuclear head shape. Nearly all of the sperm heads are very long and cylindrical, much different from the short, compact, and hook-shaped nuclei of normal murine sperm. The manchette forms properly in step 9 spermatids, but by step 11 the microtubules in azh/azh mutants continue to lie parallel to instead of protruding from the caudal end of the nucleus at the normal 45° angle. The extending manchette continues to be conical in shape, remaining long and straight, rather than assuming the proper angular shape of the wild-type manchette. As the spermatids elongate, the mutant manchettes tend to split off from the end of the nucleus and “float” into the cytoplasm. In addition to these defects, large aggregates of microtubules are often found in the spermatid cytoplasm, with no resemblance to a manchette-type organization.
Fig. 1). SPNR could also be seen in the cytoplasm of some elongating spermatids, as well as in a Sertoli cell at the basal lamina, apparently from the phagocytosis of an elongating spermatid (arrowhead tw8 panel, Fig. 1). Confocal micrographs of SPNR and tubulin in early-stage spermatids show that the two proteins colocalized with one another (Fig 4, A-C). As the spermatids elongated, SPNR (Fig. 4D) and tubulin (Fig. 4E) staining became patchy and discontinuous (Fig. 4F, merged image).
FIG. 2. Confocal micrographs of SPNR and tubulin immunolocalization in wild-type testes. Testis sections were treated with rabbit polyclonal affinity-purified anti-SPNR and/or mouse monoclonal anti-a-tubulin primary antibodies. Sections were then treated with an anti-rabbit FITC-con-jugated and/or anti-mouse Texas red-conjugated secondary antibody. A and D) SPNR; B and E) tubulin; C and F) merged SPNR and tubulin staining.