Fig. 5

a-e Immunohistochemical localization of ZO-1, its mRNA, and protein expression in testes of control and flutamide-treated rats. a-b Immunohistochemical staining in testes of control (a, a’) and flutamide-treated rats (b, b’). Nomarski interference contrast. Scale bars represent 15 μm. Arrows point to ZO-1 signal at the base of the tubule (a, a’, b, b’). Moderate to strong ZO-1 signal at the level of the BTB is visible as continuous wavy line between adjacent Sertoli cells (a, a’). Note ZO-1 signal which lines the cytoplasmic face of Sertoli cells (a, insert in a). Following flutamide treatment weak to moderate ZO-1 signal is visible mainly in the cell cytoplasm (b, b’). Rectangles indicate the location of the higher magnification views (a, b). No ZO-1 signal was detected when anti-ZO-1 antibody was substituted by normal goat serum (a”). Representative RT-PCR (C) and quantitative real-time PCR (D) analyses of ZO-1 mRNA expression in testes of control and flutamide-treated males. As an internal control, the GAPDH mRNA level was measured in the samples (c). Lane N1 – negative control without cDNA template, lane N2 – negative control without reverse-transcribed RNA. The relative amount of ZO-1 transcript levels (d). Relative quantification (RQ) is expressed as mean ± SD. Significant difference from control values is denoted as *p < 0.05. Control (n = 6) and flutamide (n = 6) group. Representative Western blot analysis of ZO-1 protein expression levels in testes of control and flutamide-treated males (e). The relative level of ZO-1 protein normalized to β-actin which served as an internal protein loading control. Data obtained from three separate analyses are expressed as mean ± SD. Significant difference from control values is denoted as *p < 0.05. Control (n = 6) and flutamide (n = 6) group