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. 2011 Apr-Jun;1(2):123–136. doi: 10.4161/spmg.1.2.16393

Figure 1.

Figure 1

Drebrin E is expressed stage-specifically in the seminiferous epithelium of adult rat testes. (A) Using immunoblot analysis with ∼50 µg total protein per sample, only the drebrin E isoform was found to be expressed in the testis while both drebrin A and E isoforms were detected in brain lysate. Drebrin E was predominantly expressed by Sertoli cells (SC) in the seminiferous tubule and virtually no drebrin E was found in total germ cells (GC) isolated from adult rat testes. Actin served as the protein loading control. (B) The specificity of the antidrebrin E antibody was illustrated by immunoblotting using lysates of Sertoli cells (∼50 µg protein) since only a prominent immunoreactive band corresponding to the apparent Mr of drebrin E at 110 kDa was detected. These results also support the staining shown in (C and D) was specific for drebrin E. A study using immunohistochemistry (C) and immunofluorescence microscopy (D; drebrin E, red fluorescence; cell nuclei were visualized by DAPI in blue, 4′,6-diamidino-2-phenylindole, staining) showed that drebrin E localized near the basement membrane in the basal compartment, consistent with its localization at the bloodtestis barrier (BTB), and drebrin E also localized at the apical ectoplasmic specialization (apical ES). The expression of drebrin E at the BTB is most prominent at stage V of the seminiferous cycle using both staining techniques (C and D), which diminished gradually thereafter and was almost non-visible at the BTB by stages VIII–XIV. In (C), the boxed areas enclosed by a “solid-line” rectangle notes an area of the epithelium that was magnified; this is shown in the same micrograph but enclosed within a “broken-line” rectangle. Ctrl illustrates sections that were stained with normal rabbit IgG which was substituted in place of the anti-drebrin E antibody, confirming the reddish-brown immunoreactive drebrin E shown in (C) was specific for drebrin E. Drebrin E was localized at the apical ES, surrounding the entire head of the elongating spermatid at stages IV and V (C). However, the localization of drebrin E shifted and localized predominantly to the concave side of the head of elongating/elongated spermatids at stage VII (see also the magnified images in C), but its level was drastically reduced at stage VIII and these observations were consistent using either immunohistochemistry (C) or immunofluorescence microscopy (D). Roman numerals denote stages of the seminiferous epithelial cycle. Bar = 50 µm in the first micrograph in (C and D), which also applies to the other micrographs; bar = 25 µm in the inset in the first micrograph in (C), which also applies to the other insets in (C). These micrographs are representative results from a single set of experiments, which were repeated at least 3 times using different sets of sections from different rats and similar results were obtained.