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First published online 24 April 2007
doi: 10.1242/jcs.004945

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Characterization of Spo11-dependent and independent phospho-H2AX foci during meiotic prophase I in the male mouse

¹ Laboratory of Differentiation and Radiobiology of the Gonads, Unit of Gametogenesis and Genotoxicity, Unité Mixte de Recherche-S 566, Commissariat à l'Energie Atomique DSV/IRCM/SEGG/LDRG, F-92265 Fontenay aux Roses, France
² Université Denis Diderot Paris 7, Unité 566, F-92265 Fontenay aux Roses, France
³ Institut National de la Santé et de la Recherche Médicale, Unité 566, F-92265 Fontenay aux Roses, France
⁴ Human Genetic Institut, CNRS UPR 1142, 141 rue de la Cardonille 34396 Montpellier Cedex 5, France
⁵ National Museum of Natural History, CNRS UMR 5202, 16 rue Buffon, 75005 Paris, France

View larger version (52K): [in this window] [in a new window]   Fig. 1. (A) Coexistence of two types of -H2AX foci on surface-spread preparations of rodent spermatocytes. -H2AX foci were revealed by fluorescein-conjugated secondary antibodies (green). Axial elements and SCs were labeled by anti-SCP3 antibody using a Cy3-conjugated secondary antibody (red). In mice, numerous S-foci (arrowhead) and L-foci (arrow) coexist at early zygotene (a). At early pachytene (b), numerous S-foci persist and only few L-foci are detected. At late pachytene (e), the number of S-foci strongly decreased whereas the number of L-foci remained steady. Finally, at diplotene (f) no more S-foci were found. Numerous S-foci were also detected on early pachytene of guinea pig (c) and chinchilla (d) (see Table 1 for quantification of S-foci number). From pachytene to diplotene stages the XY body was intensely stained (*). Bars, 10 µm. (B) Distinct -H2AX L-foci distributions at early and late pachytene, diplotene and diakinesis. Pachytene cells at early (black bars) and late (dark gray bars) stages, diplotene stage (light gray bars) and diakinesis (white bars) were distributed according to their number of L-foci per cell.

View larger version (79K): [in this window] [in a new window]   Fig. 2. (A) -H2AX-containing S-foci partially colocalize with RAD51 foci on SCs. (a) Partial mid-pachytene chromosome spread stained with anti--H2AX (green), anti-RAD51 (red) and anti-SCP3 (blue) antibodies. Examples of non-colocalized -H2AX and RAD51 foci are indicated by white arrowheads. Mixed -H2AX/RAD51 foci are marked by red arrows. (b-c) Higher magnification of one synapsed chromosome core (b) triple-labeled with anti--H2AX (green), anti-RAD51 (red) and anti-SCP3 (blue) antibodies, (c) double-labeled with anti--H2AX, and anti-RAD51 are shown. -H2AX and RAD51 foci are slightly offset to show their colocalization more clearly (d). Red and white arrows indicate sites of colocalized and juxtaposed foci, respectively. (B) -H2AX S-foci partially colocalize with MLH1 foci on SCs. (a) Partial pachytene chromosome spread stained with anti--H2AX (green), anti-MLH1 (red) and anti-SCP3 (blue) antibodies. Examples of non-colocalized -H2AX and MLH1 foci are indicated by white arrowheads. The only mixed focus on this image is indicated by a red arrow. (b-d) Enlarged images of one SC triple-labeled with (b) anti--H2AX (green), anti-MLH1 (red) and anti-SCP3 (blue) antibodies, and (c) double-labeled with anti--H2AX and anti-MLH1 antibodies. (d) -H2AX and MLH1 foci are slightly offset. White arrows indicate juxtaposed foci; red arrows indicate colocalized foci. Bars, 10 µm.

View larger version (40K): [in this window] [in a new window]   Fig. 3. (A) Spo11–/– zygotene cells are devoid of S-foci. In contrast to wild-type (a) and Spo11+/– (b) zygotene cells, surface-spread preparations of Spo11–/– zygotene cells stained for SCP3 (red) revealed the presence of L-foci and a lack of S-foci on the axial elements (c,d). Spo11–/– zygotene cells exhibited -H2AX signals that looked like L-foci (c,d). We defined two categories of zygotene Spo11–/– cells: (c) without or (d) with a pseudo sex body (*). In c' and d', zygotene stages of Spo11–/– nuclei were defined by co-labeling of SCP1 (red) and -H2AX (green): SCP1-positive nuclei with a pseudo sex body corresponded to the most advanced stage (d'). Arrows indicate L-foci. Bars, 10 µm. (B) Change in the distribution of -H2AX L-foci during zygotene stage in Spo11–/– mice. Zygotene nuclei from early (light gray bars) and late (dark gray bars) stages were classified according to their number of L-foci. A quantitative analysis of L-foci in Spo11–/– zytotenes revealed that the most advanced cells (with a pseudo sex body) contained less L-foci.

View larger version (27K): [in this window] [in a new window]   Fig. 4. Spo11+/– early pachytene cells exhibit the same number of S-foci but contain more L-foci than Spo11+/+ early pachytene cells. (A,B) Quantification of -H2AX containing S-foci (A) and L-foci (B) in early and late Spo11+/+ and Spo11+/– pachytene cells (black and white bars, respectively). Bars represent mean numbers ±s.e.m. for each experimental group (n=3). Statistical comparisons between Spo11+/+ and Spo11+/– mice at early and late pachytene were performed using the Mann-Whitney U test. Significance is ***P<0.001. (C) Distinct repartition of L-foci in Spo11+/– and Spo11+/+ pachytene spermatocytes. Pachytene nuclei of Spo11+/+ mice (black and dark gray bars) and Spo11+/– mice (light gray and white bars) were classified according to their number of L-foci.

View larger version (21K): [in this window] [in a new window]   Fig. 5. Induction of both -H2AX-containing S-foci and L-foci on late pachytene chromosome spreads 8 hours after irradiation at 2 Gy. (A) Late pachytene cell stained for -H2AX (green) and SCP3 (red) reveals additional signals that look like S-foci and L-foci in irradiated cells compared with untreated cells (see Fig. 1Ae). *, XY body. Bar, 10 µm. (B) Effect of increasing doses of -rays on -H2AX foci number in pachytene cell 8 hours after irradiation. (a,a') Dose-dependent increase was found in L-foci number (a) but not in S-foci number (a'). Each column represents the mean ±s.e.m. at early (black bars) and late (gray bars) pachytene of at least three independent experiments. Lower case letters correspond to multiple comparisons of mean number of S-foci and L-foci according to the -ray doses given at a certain pachytene stage. Lower case letters are different at significantly different P-values (P<0.05). (b,b') Time-course of disappearance of -H2AX S-foci and L-foci after 1 Gy exposure to -irradiation; L-foci (b) and S-foci (b') mean number ±s.e.m. of at least three independent experiments at early (black bars) and late (gray bars) pachytene. Counting of S-foci could not be done at 15 minutes. Statistical comparisons to the control were performed at a given pachytene stage by using the Student's t-test; **P<0.01, ***P<0.001. (c,c') Concomitant induction of -H2AX S-foci and L-foci after treatment with neocarzinostatin in vitro equivalent to a dose of 0.5 Gy; L-foci (c) and S-foci (c') mean number ±s.e.m. for each experimental group (n=2). Statistical comparisons to the control at a given pachytene stage were performed by the Student's t-test. *P<0.05. (d) MMS cell treatment does not induce -H2AX L-foci. Bars represent mean number ± s.e.m. for each experimental group (n=5) at early and late pachytene.