At 18 and 26 days of postnatal age. Electron-microscopic examination of the rat testis at these ages revealed degenerating spermatogonia in the vicinity of the basal membrane and nuclei with an appearance characteristic of apoptotic death. The only apoptotic cells associated with stages VII-VIII of the cycle were midpachytene spermatocytes. In general, the apoptotic midpachytene spermatocytes were stained more densely, and morphological features of the nucleus, including condensed chromatin cords, synaptonemal complexes, and sex vesicles, were still apparent (Fig. 3). The levels of activated caspase 3 (Fig. 2B) and cleaved PARP (Fig. 2C) peaked at the age of 18 days, thereafter declining with increasing age.
Levels and Immunolocalization of Members of the Bcl-2 Family of Proteins in the Maturing Rat Testis
At 8 days of postnatal age. This immature tissue expressed high levels of the prosurvival Bcl-2 protein (Fig. 4A), with the strongest immunohistochemical staining being exhibited by the gonocytes (Fig. 5A). Immunohisto-chemical staining for the Bax protein was also localized to the gonocytes and, in addition, the spermatogonia. In this case, the staining was particularly intense at the plasma membrane of large gonocytes in the vicinity of the basement membrane of the seminiferous tubule (Fig. 5B). The proapoptotic Bad protein was also expressed at a high level in the 8-day-old testis (Fig. 4C) and was localized immu-nohistochemically to the Sertoli cells (not shown). buy claritin online
At 18 and 26 days of postnatal age. At these later stages of testicular maturation, the levels of expression of the Bcl-2 protein has decreased considerably (Fig. 4A), and no im-munohistochemically positive cells could be detected. In marked contrast, the proapoptotic Bax (Fig. 4B) and Bad (Fig. 4C) proteins demonstrated a peak of expression at these ages. Spermatogonia in contact with the basal membrane and in all stages of the spermatogenic cycle were stained with the anti-Bax antibody (Fig. 5, B-D). Furthermore, distinctly stage-dependent Bax staining of pachytene spermatocytes was observed in stages VII and VIII (Fig. 5, C and D). The most intense immunostaining of the Bad protein was associated with the Sertoli cells (not shown).
FIG. 3. An electron micrograph of cells in spermatogenic stages VII and VIII in 26-day-old rat testis showing preleptotene (Pl) and pachytene (Pa) spermatocytes. Pachytene spermatocytes are easily identified on the basis of their synaptonemal complexes (arrows) and sex vesicles (arrowhead). Three pachytene spermatocytes in different phases of apoptosis (a), but still exhibiting sex vesicles and synaptonemal complexes, are visible. Bar = 10 ^m.
FIG. 4. Levels of expression profiles of the Bcl-2 proteins (A) Bcl-2 itself, (B) Bax, and (C) Bad in the immature and mature rat testis as determined by Western blot analysis. The upper strips illustrate the autoradiograms, and the bar graphs depict densitometric quantitation of the bands. The heights of the columns depict the mean densities, and the bars indicate the SEM. *P < 0.05 compared to the corresponding value for the 60-day-old testis.
FIG. 5. Immunohistochemical analysis of the Bcl-2 and Bax proteins in the maturing rat testis. A) Immunolocalization of the Bcl-2 protein (arrowheads) to gonocytes in the 8-day-old testis. B) Immunolocalization of the Bax protein (arrow) to spermatogonia and gonocytes (arrowheads) in the 8-day-old testis. C Immunolocalization of the Bax protein to spermatogonia (arrows) and midpachytene spermatocytes (arrowheads) in the 18-day-old testis. D) Immunolocalization of the Bax protein to spermatogonia (arrows) and midpachytene spermatocytes (arrowheads) in the 26-day-old testis. Roman numerals indicate stages of the spermatogenic cycle. Bar = 50 ^m.