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First published online 5 July 2005
doi: 10.1242/jcs.02466

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SPO11 is required for sex-body formation, and Spo11 heterozygosity rescues the prophase arrest of Atm^-/- spermatocytes

Marina A. Bellani, Peter J. Romanienko^*, Damian A. Cairatti and R. Daniel Camerini-Otero

Genetics and Biochemistry Branch, NIDDK, NIH, Bethesda, MD 20892, USA

View larger version (75K): [in a new window]   Fig. 1. Spo11-/- spermatocytes do not phosphorylate H2AX in the sex chromatin and fail to form a sex body. (A) Zygotene-like Spo11-/- spermatocytes display a wide range of H2AX staining patterns. Analysis of Spo11-/- surface-spread preparations stained for SCP3, SCP1 and H2AX revealed that a proportion of zygotene-like spermatocytes is devoid of H2AX (i), whereas most nuclei contain one or more localized H2AX chromatin domains. Examples are shown of nuclei containing one (ii), two (iii) or more (iv) H2AX signals. (B) Quantitative analysis of H2AX staining patterns observed in Spo11-/- zygotene-like spermatocytes. 350 nuclei were scored and classified according to their stage (based on SCP3 staining) and H2AX staining pattern. Zygotene-like spermatocytes (n=290 nuclei, two animals) were classified as nuclei devoid of H2AX (i) or nuclei displaying one or more localized H2AX signals (ii-iv). These zygotene-like spermatocytes containing one or more localized H2AX signals (n=197 nuclei) were further classified according to the number of H2AX signals: one signal (ii), two signals (iii) and more than two signals (iv). Examples of each of these nuclei are displayed in Fig. 1Ai-iv. (C) The H2AX signals frequently observed in zygotene-like spermatocytes are not on the sex chromatin. Combined FISH (whole-chromosome paint probes against the X and Y chromosomes are shown in green) and immunostaining (antibodies against SCP3 are shown in red and those against H2AX in blue) performed on structurally preserved spermatocytes from wild-type (a-c) and Spo11-/- (d,f) mice (two sets of littermates). In wild-type spermatocytes, the X-Y chromatin contains H2AX (c). In SPO11-deficient zygotene-like spermatocytes, even in those nuclei displaying a single H2AX signal, this does not overlap with the X and Y chromatin. None of the 50 Spo11-/- nuclei analysed contained a H2AX signal overlapping with either the X or the Y chromatin. Notice that the X chromosome appears extended and does not synapse with the Y chromosome. (D) Spo11-/- spermatocytes do not form a sex body. Combined SCP3 immunostaining and FISH (using differently labeled whole-chromosome probes recognizing the X and Y chromatin) was performed on methanol-acid-fixed Spo11-/- spermatocytes. 123 zygotene-like nuclei (two animals) were scored and classified according to the relative positions of the sex chromosomes as apart (a) or close (b). Almost 80% of SPO11-deficient zygotene-like nuclei displayed the X and Y chromosomes widely separated, whereas 21% contained the sex chromosomes close to one another. Nevertheless, in those nuclei containing the sex chromosomes in proximity, the X is always extended and barely contacting the Y on one end. This disposition clearly differs from the spatial configuration usually adopted by the X-Y chromosomes in the sex body in wild-type spermatocytes, providing further cytological evidence for the lack of sex body in SPO11-deficient spermatocytes.

View larger version (81K): [in a new window]   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.

View larger version (68K): [in a new window]   Fig. 3. The rescue of the prophase arrest of Atm-/- spermatocytes by Spo11 heterozygosity reveals that ATM plays no essential role in the sex body. (A) A single copy of Spo11 rescues the prophase-arrest characteristic of the ATM KO. Whereas inactivation of both copies of Spo11 in an Atm-/- background results in a complete prophase arrest (and a meiotic phenotype indistinguishable to that of the Spo11-/- single mutant), Spo11 heterozygosity rescues the prophase arrest of an ATM KO, allowing spermatocytes to complete prophase I. (B) ATM is dispensable for phosphorylation of H2AX in the sex body. Combined FISH (whole-chromosome paint probes against the X and Y chromosomes, in green) and immunostaining (antibodies against SCP3 in red and those against H2AX in blue) performed on structurally preserved spermatocytes from Atm-/- Spo11-/- (a-c) and Atm-/- Spo11+/- (d-f) mice (two animals for each genetic background). As is the case for the Spo11-/- single mutant, none of the 50 nuclei scored for the Atm-/- Spo11-/- double-mutant strain contained a H2AX signal overlapping with either the X or the Y chromatin. By stark contrast, in the rescued Atm-/-Spo11+/- background, the X-Y chromatin consistently contains H2AX. The H2AX signal coincided with the X-Y chromatin in all of the 30 nuclei scored.

View larger version (45K): [in a new window]   Fig. 4. Localization and transcription kinetics of ATR are consistent with a role for ATR in the phosphorylation of H2AX in the sex body. (A) ATR colocalizes with H2AX in all of the mutants tested. Triple labeling of structurally preserved nuclei from wild-type (a-c), Atm-/- Spo11+/- (d-f), Spo11-/- (g-i) and Atm-/- Spo11-/- (j-l) mice (two sets of littermates were analysed), with antibodies against SCP3 (red), ATR (green) and H2AX (blue). Images show the focal plane in which the H2AX-chromatin domain can be visualized. ATR consistently localizes to H2AX-positive chromatin domains, whether these correspond to the X-Y chromatin (as in wild-type and Atm-/- Spo11+/- spermatocytes) or to non-sex-chromatin regions (as in Spo11-/- and Atm-/- Spo11-/- mutants). (B) ATR transcription peaks at the stage in prophase when the sex body can first be visualized. The curves represent absolute levels of the mRNAs encoding the three PIKKs (ATM, ATR and DNA-PKcs) and SCP1 (Sycp1, as a marker of meiotic progression) during the first wave of spermatogenesis. Data were extracted from previously published microarray data (Schultz et al., 2003). Triplicates from each developmental time point were averaged and normalized to 1 dpp. The table shows prophase-stage distribution at different times during the first wave of spermatogenesis determined on meiotic spreads [data from Goetz et al. (Goetz et al., 1984)].

View larger version (101K): [in a new window]   Fig. 5. Atm-/- Spo11+/- testis sections show severe defects in spermatogenesis and substantial levels of apoptosis. (A) Atm-/- Spo11+/- testes show severe defects in spermatogenesis in spite of the prophase-I rescue. Paraffin-embedded testis sections from wild-type (a,b) and Atm-/-Spo11+/- (c-i) mice stained with hematoxylin and eosin. In wild-type sections, many round or elongated spermatids are found in the most lumenal part of the tubules. By contrast, in Atm-/- Spo11+/- testes, very few tubules contain elongated spermatids (d,e, arrows) and, even in positive tubules, only groups of two to 15 spermatids can be found. Examples of aberrant metaphases are frequently observed (f-i). (B) Metaphases frequently stain positive for TUNEL in Atm-/- Spo11+/- testes sections. Apoptotic cells were detected by TUNEL on paraffin-embedded testes sections. The DNA was stained with DAPI. (C) Aberrant metaphases of the first meiotic division can be observed in metaphase spreads from the Atm-/- Spo11+/- mutant. Metaphase spreads show metaphases of the first meiotic division containing autosomal univalents (marked with arrows), chromosomal fragments [asterisk (*)], achiasmatic sex chromosomes and broken bivalents [hash (#)].

View larger version (86K): [in a new window]   Fig. 6. In Atm-/- Spo11+/- spermatocyte spreads, pachytene and diplotene nuclei frequently display RAD51/DMC1 foci on the cores in chromosome regions containing H2AX on the chromatin loops. Spermatocyte spreads from Atm-/- Spo11+/- mice and wild-type littermates were stained for SCP3, RAD51 and H2AX. (c,e) Triple staining of two pachytene (c) and two diplotene (e) Atm-/- Spo11+/- nuclei. (d,f) Rad51 and H2AX staining of the same nuclei shown in c,e. In wild-type spreads, RAD51/DMC1 foci can be detected in leptotene and zygotene spermatocytes; by pachynema, hardly any foci remain on the synaptonemal complex of autosomal bivalents, but some foci can be frequently observed on the asynapsed X axial element (Moens et al., 1997; Tarsounas et al., 1999) (a,b). In the Atm-/- Spo11+/- mutant, we frequently observe RAD51/DMC1 foci on the chromosomal cores of pachytene (c) and diplotene (e) nuclei, suggesting a delay in either the introduction of DSBs or in the ensuing strand invasion and exchange process catalysed by these recombinases. Rad51 foci are located on the cores of chromosomal regions containing H2AX (also see d,f).

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