Fig. 2. ATM is strictly required for chromatin-wide phosphorylation of H2AX during leptotene. (A) Leptotene Atm^-/- spermatocytes are devoid of H2AX. Quantitative analysis of Atm^-/- surface-spread preparations shows that 100% of the Atm^-/- spermatocytes undergoing leptonema are devoid of H2AX (n=64 nuclei, two animals). (B) Leptotene ATM-deficient spermatocytes can not phosphorylate H2AX in response to radiation-induced DSBs: Spo11^-/- (a,b) and Atm^-/- Spo11^-/- (c,d) double-mutant mice were irradiated (3 Gy) or mock treated. 1 hour after irradiation, mice were sacrificed and their testes used to prepare structurally preserved nuclei, which were subsequently immunolabeled with antibodies against SCP3 (in red) and H2AX (in green). 50 nuclei were scored for each condition. Irradiation-induced DSBs can restore H2AX phosphorylation in Spo11^-/- spermatocytes undergoing leptotene (b), whereas, in the absence of functional ATM, no alternative PIKK can phosphorylate H2AX in leptotene spermatocytes in response to radiation-induced DSBs (d). The bright red spots represent nucleolar SCP3 aggregates.