Figure 4

Immunostaining for p53, active and precursor caspase 8 and active caspase 3, and fragmented DNA. Upper panels show protein expression of p53 (A), active and precursor caspase 8 (B) and active caspase 3 (C) as detected by immunostaining (arrows) in testicular sections of mice sacrificed 2 days after the last bortezomib injection. Inserts show negative controls of corresponding sections of (A), (B) and (C). Lower panels show TUNEL staining of testicular sections in mice killed 2 days post-treatment (D) and their untreated controls (E). Germ cells were positively stained (D, arrows) suggesting DNA fragmentation while Sertoli, peritubular and Leydig cells were all TUNEL negative (open triangle, arrowheads, and solid triangle). Panel (F) shows the quantification of the numbers of TUNEL positive germ cells per seminiferous tubule in all treated and pup groups, and the respective controls. Magnification of all pictures is 60×. *P < 0.05, **P < 0.01.