Fig. 2. (A) Mitotic index of primary spermatogonia (I SPG) in the frog R. esculenta testis at various times, 22°C and 12h:12h photoperiod. The I SPG was expressed as the number of metaphases per total primary spermatogonia counted x100 in three randomly chosen sections/animal. Values are means ± S.D. Differences were considered significant at P<0.01. (B) Western blot detection of PCNA protein in the cytosolic testicular extracts of R. esculenta at various times, 22°C and 12h:12h photoperiod. Proteins (40 µg/lane) were resolved by SDS-PAGE, transferred to nitrocellulose membranes and then incubated with antibody raised against PCNA protein; a specific band was observed of 36 kDa (determined by comparison with comigrating size markers). (C) The amount of PCNA was quantitated using ImageQuant 5.2 Program. The values shown are representative of three separate experiments.