Fig. 3. Immunolocalization of PtdIns(4,5)P₂ and PLC in rat ectoplasmic specializations. (A) Phase and fluorescence images of isolated spermatids with attached ectoplasmic specializations that have been labeled with an antibody raised against PtdIns(4,5)P₂. The spermatids have also been treated with fluorescent phallotoxin to label actin. Notice that the probe for PtdIns(4,5)P₂ stains the region surrounding the head containing an ectoplasmic specialization that labels with the probe for actin. Specific staining for PtdIns(4,5)P₂ was not observed in any of the controls (not shown). Bar, 5 µm. (B,C) Immunofluorescence localization of PLC in fixed frozen sections of rat seminiferous epithelium at stage V (B) and stage VII (C) of spermatogenesis. The locations of apical and basal ectoplasmic specializations are indicated by the `a' and `b', respectively, in the actin panels. At stage V, the probe for PLC reacts weakly at ectoplasmic specializations. The situation is much different at stage VII when apical ectoplasmic specializations are disassembling as part of the sperm release process and basal ectoplasmic specializations are turning over to allow the next generation of spermatocytes through junction complexes between Sertoli cells. At this stage, apical and basal regions containing ectoplasmic specializations, indicated by the actin staining, also react with the probe for PLC. Specific staining was not observed in any of the controls (data not shown). The intense staining of the tubule wall (asterisk) is caused by nonspecific staining by the secondary antibody. Bar, 10 µm. (D) Immunoblot of rat testis and rat seminiferous epithelium. The PLC antibody reacts specifically with a single band in each lane (148 kDa). The minor bands indicated by the asterisk are nonspecific and are present in blots treated with normal mouse IgG instead of primary antibody. The lines indicate the top and bottom of the gel. Loading densities were 80 µg of testis homogenate and 80 µg of seminiferous epithelium.

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