(A–E) Histological analysis of testis sections from 8-week-old Ing2+/+ (A, B) and Ing2−/− (C–E) mice by hematoxylin and eosin (H&E) staining. Black arrows in (B) and (D) indicate spermatocytes with large, condenced nuclei. The white arrow in (B) indicates spermatids, which are missing in (D). In (E), Leydig cell hyperplasia (white arrows), apoptotic cell (black arrow) and multinucleated giant cell (white arrowhead) are indicated. Scale bars are 200 µm in (A) and (C), 100 µm in (B), (D) and (E, left), and 50 µm in (E, right). (F) Histological analysis of epididymis from 8-week-old Ing2+/+ and Ing2−/− mice by H&E staining. Scale bars, 100 µm. Black arrows indicate degenerated round cells in Ing2−/− epididymis. (G) Flow cytometric analysis of testis cells isolated from 2- and 6-month-old Ing2+/+ and Ing2−/− mice. The flow cytograms (left) demonstrate three peaks; 1N peak representing round and elongated spermatids, 2N peak representing somatic cells, spermatogonia and secondary spermatocytes, and 4N peak representing primary spermatocytes, including leptotene, zygotene and pachytene stages. The data are shown as percentage of 1N, 2N and 4N cell fractions (right). (H–K) Phosphorylated histone H2AX (γ-H2AX) staining in testes from 8-week-old Ing2+/+ (H, I) and Ing2−/− (J, K) mice. Normal leptotene and zygotene spermatocytes with positive γ-H2AX staining [black arrows in (I) and (K)] develop into pachytene spermatocytes in Ing2+/+ testes [white arrows in (I)], which have a γ-H2AX focus corresponding to the sex body but are otherwise negative for γ-H2AX staining [21]. In Ing2−/− testes, γ-H2AX-positive, abnormal spermatocytes accumulate [white arrowheads in (K)] without development into pachytene spermatocytes. Scale bars are 100 µm in (H) and (J), and 50 µm in (I) and (K).