Ablation of Sam68 increases germ cell apoptosis and impairs spermiogenesis. (A) TUNEL staining of testicular sections from wild-type or knockout 25 dpp (top) and adult (bottom) mice. Sam68^−/− testes show increased apoptosis. Nuclear brown signal indicates apoptotic cells. In adult testis, apoptotic cells mainly reside in the luminal pole of the tubule where the haploid cells are localized. Bars, 100 µm. (B) Flow cytometry analysis of TUNEL-positive germ cells. Staining with propidium iodine (x axis) allowed us to identify haploid (1C), diploid (2C), or tetraploid (4C) germ cells. TUNEL staining is represented on the y axis. (C) Bar graph of the flow cytometry analyses from three independent experiments. Data are represented as mean ± SD. *, P < 0.01. (D) Electron microscopy of testicular sections from Sam68^−/− mice. Images refer to different abnormalities that were frequently observed during spermiogenesis, such as abnormally divided spermatid nuclei (a), elongating spermatids with two flagella (b, arrows), binucleated elongated spermatids (c), and delayed spermatid differentiation, with step 9–10 spermatids (arrow) in stage XII tubules (d). Bars: (a) 0.4 µm; (b) 1 µm; (c) 0.7; (d) 2 µm. (E) Analysis of the number of spermatozoa found in the epididymis of Sam68^+/+ (n = 7), Sam68^+/− (n = 5), and Sam68^−/− (n = 7) mice. Approximately half of the 13 Sam68^−/− mice (n = 6) analyzed had empty epididymis with no detectable spermatozoa and were not included in the experiment. Only the knockout mice that had residual spermatozoa were taken into account for this analysis. Data are represented as the mean ± SD. *, P < 0.001 in a t test and an ANOVA test.