Fundamentally different recombination defects trigger similar timings of spermatocyte apoptosis. Spermatocytes in Spo11−/−
, and Msh5−/−
mutants are efficiently eliminated by apoptosis during prophase of meiosis I (5
). Earlier findings indicated that cell death in all three mutants is triggered at approximately the same stages in spermatogenesis, as judged by histological criteria (described further below). To rule out subtle differences in timing, we analyzed testis sections from these mutants side by side (Fig. ).
FIG. 1. Different recombination defects trigger similar timings of spermatocyte apoptosis. (A to D) Periodic acid-Schiff-stained testis sections from wild-type and the indicated mutant mice. Examples of tubules at epithelial stage IV and the stages before (pre-IV) (more ...)
Mouse spermatogenesis is highly regular (reviewed in reference 10
). Stem cells (spermatogonia) line the periphery of the seminiferous tubule. These cells divide mitotically to replenish the stem-cell population and to give rise to differentiating cells that undergo several rounds of mitotic division before entering meiosis. Spermatocytes proceed through meiotic prophase and the two meiotic divisions to give rise to spermatids, which mature until being released into the lumen of the tubule. Within an adult testis, successive waves of spermatogenesis begin approximately once every 8 to 9 days (31
). Specific stages of spermatogenesis have stereotyped lengths, with the entire process taking approximately 34.5 days. Because the initiation of a new cycle occurs before the preceding cycles have finished, the epithelium of each tubule contains a mixture of germ cells at various steps in development, with later cells displaced toward the lumen as new waves of cells enter the pathway. A population of spermatogonia within a small region of a tubule begins a new cycle of germ cell proliferation and differentiation synchronously, but different regions of the tubules are offset from one another with respect to this cycle, so a single histological section will contain germ cells at many different steps of spermatogenesis (some examples are indicated in Fig. ).
Because both the period between cycles and the duration of individual developmental steps are so regular, each tubule section can be classified into one of 12 distinct types, referred to as stages I to XII (41
). Staging in the wild type is defined according to the constellation of germ cell types present in a tubule section, including late prophase and postmeiotic cells. In most mutants with defects in meiotic recombination, spermatocytes undergo apoptosis prior to the first division (see below) and thus lack cells undergoing postmeiotic steps in development. Nevertheless, it is possible to assign tubule sections from these mutants to stages that are equivalent to the wild-type stages by examining the division cycle of the spermatogonia and by scoring for the presence or absence of early meiotic prophase cells (11
). This staging is possible because it is based on premeiotic and early meiotic cell types whose development is not detectably affected by the mutations studied here (e.g., references 5
males, a number of tubules contained aberrant spermatocytes with condensed nuclei, shown previously by terminal deoxynucleotidyltranserase-mediated dUTP-biotin nick end labeling to be apoptotic (5
). These tubules are equivalent to those seen in stage IV in the wild type, as judged by the presence of intermediate-type spermatogonia in late G2
or mitosis and/or of early B spermatogonia (Fig. ) (10
). The same timing of apoptosis is found for Dmc1−/−
mice (Fig. ). Side-by-side comparisons demonstrate the same timing for Msh5−/−
mutant males as well (Fig. ), consistent with previous results (12
Thus, despite different molecular defects, Spo11−/−
, and Msh5−/−
spermatocytes undergo apoptosis at the same point with respect to developmental timing. This stage is inferred to be equivalent to mid-pachynema, based on the spermatocyte stage present in stage IV tubules in wild-type animals (41
). Because chromosome synapsis defects are found in all three mutants, a simple explanation is that stage IV apoptosis is a response to this common defect. Alternatively, it is possible that distinct cellular defects trigger cell death at the same developmental stages. To address these alternatives, we examined additional molecular markers for meiotic progression.
A pseudo-sex body forms in Spo11−/− mutants.
The sex body is a heterochromatin domain encompassing the XY chromosomes during pachynema (reviewed in reference 20
). Sex body chromatin accumulates a number of proteins and protein modifications (see below), and its formation is accompanied by transcriptional inactivation of X- and Y-resident genes. One proposed role is to shield the unsynapsed axes of the XY pair (or persistent DSBs on these axes) from surveillance mechanisms that would otherwise sense these as aberrant. Although the sex body is most prominent during pachynema, early components can be observed at late zygonema (e.g., reference 17
A prominent marker of sex body formation is the phosphorylation of histone H2AX on Ser-139 to form γH2AX. In somatic cells, γH2AX forms rapidly in the vicinity of DSBs (39
). In spermatocytes, γH2AX is generated in response to SPO11-induced DSBs and then disappears as chromosomes synapse and a second wave of γH2AX formation occurs on chromatin surrounding unsynapsed axes (27
). In normal males, this latter signal is usually restricted to the chromatin of the sex body. γH2AX is revealed by immunofluorescence on chromosome spreads (Fig. ) and as patches of immunoreactive material at the peripheries of pachytene nuclei in testis sections (Fig. ).
FIG. 2. Spo11−/− spermatocytes form pseudo-sex bodies. (A and B) Immunofluorescence and FISH staining of spermatocyte spreads. In wild-type spermatocytes, the X and Y chromosomes colocalize with a discrete domain of γH2AX staining (the (more ...)
FIG. 3. Localized accumulation of γH2AX does not occur in recombination mutants that are defective in the repair of SPO11-generated DSBs. Testis sections were stained with anti-γH2AX (brown). Insets show higher-magnification views of individual (more ...) Spo11−/−
spermatocytes form localized accumulations of γH2AX that resemble sex bodies (27
) (Fig. and ). The formation of this γH2AX domain suggested that the X and Y chromosomes may colocalize normally even in the absence of SPO11-dependent recombination. Surprisingly, however, γH2AX accumulation in Spo11−/−
spermatocytes did not localize to the X or Y chromosomes. In wild-type pachytene cells, the γH2AX signal of the sex body colocalized completely with the XY pair, as expected (Fig. ). By contrast, the X and Y chromosomes in Spo11−/−
spermatocytes were usually separated from one another and from the γH2AX (Fig. ). Only 1 of 35 mutant spreads showed substantial overlap of γH2AX with both the X and Y chromosomes (Fig. ). This low level of overlap is most likely random. Thus, the formation of a discrete chromatin domain containing γH2AX and other sex body-associated proteins (see below) can be uncoupled from the presence of the X or the Y to form a “pseudo-sex body.” Nevertheless, the timing for pseudo-sex body formation is similar to that for the true sex body.
Absence of a sex body-like structure in mutants with impaired DSB repair.
γH2AX was observed in Dmc1−/− spermatocytes, but in contrast to what was seen in the wild type or in Spo11−/− mutants, the signal was distributed in patches across most of the chromatin and did not form an obvious sex body-like structure (compare Fig. with Fig. ). Immunostaining of testis sections confirmed this observation (Fig. ). It is straightforward to account for the Dmc1−/− spermatocyte pattern if the γH2AX signal reflects the persistence of SPO11-generated DSBs. This signal cannot be attributed to apoptosis, because Spo11−/− spermatocytes became apoptotic at the same stage but did not show this pattern.
FIG. 4. Mutants defective in the repair of SPO11-generated DSBs do not form sex body-like domains of γH2AX. Chromosome spreads were stained with anti-SCP3 (red) and anti-γH2AX (green). The γH2AX signal occurred in discrete domains in wild-type (more ...)
Since it is possible that the abundant, dispersed γH2AX signal in Dmc1−/−
spermatocytes might mask a sex body-related signal, we examined the behavior of NBS1 protein, which also localizes to the sex body (Fig. ) like its protein partners RAD50 and MRE11 (16
). Like γH2AX, NBS1 localized to one or more discrete domains in Spo11−/−
spermatocytes (Fig. ) but not in Dmc1−/−
spermatocytes (Fig. ).
FIG. 5. Localized accumulation of NBS1 does not occur in recombination mutants that are defective in the repair of SPO11-generated DSBs. (A to F) Testis sections were stained with anti-NBS1 (brown). As in the wild type (panel A), the NBS1 signal occurred in discrete (more ...)
The failure to observe a localized domain of γH2AX or NBS1 suggests that sex body formation is more profoundly defective in Dmc1−/− spermatocytes than in Spo11−/− spermatocytes. Epistasis analysis revealed that Spo11−/− Dmc1−/− spermatocytes were indistinguishable from Spo11−/− single mutants (Fig. , , and ). Thus, the failure to form even a pseudo-sex body is due to the persistence of SPO11-dependent DSBs rather than to a role for DMC1 in sex body formation per se.
Previous studies revealed γH2AX patterns in Msh5−/−
mutant spermatocytes similar to those of Dmc1−/−
spermatocytes, consistent with the persistence of unrepaired or improperly repaired DSBs (27
) (Fig. ). Moreover, analysis of Msh5−/−
females demonstrated that the absence of MSH5 triggers a DNA damage-dependent response in oocytes similar to that in Dmc1−/−
). We therefore analyzed sex body formation in Msh5−/−
spermatocytes. Similar to what is seen with Dmc1−/−
spermatocytes, there was no obvious accumulation of γH2AX or NBS1 into discrete domains in Msh5−/−
spermatocytes (Fig. , , and ). As with Dmc1−/−
spermatocytes, the inability of Msh5−/−
spermatocytes to form discrete domains of sex-body-related proteins was suppressed by Spo11
mutation (Fig. , , and ).
These studies reveal that Spo11 mutation is epistatic to Dmc1 and Msh5 mutations for this marker of meiotic progression and that distinct physiological states result from recombination failure in the presence versus the absence of DSBs.
Deposition of H1t and XMR onto spermatocyte chromatin in recombination mutants.
Meiotic progression was analyzed using additional markers. H1t, a testis-specific isoform of histone H1, replaces somatic H1 on chromatin at mid-pachynema (22
). XMR protein is at first faint and dispersed on chromatin at leptonema, becomes more abundant through early zygonema, and then disappears from bulk chromatin and accumulates in the sex body in late zygonema to early pachynema (47
) (Fig. ). The accumulation of XMR in the sex body occurs after the appearance of γH2AX (compare Fig. ). The deposition of H1t occurs after XMR accumulates in the sex bodies, and H1t persists in postmeiotic cells after the sex bodies have disappeared (Fig. ).
FIG. 6. Staining patterns of γH2AX and XMR in wild-type and Spo11−/− spermatocytes in squash preparations of testicular cells. Squash preparations of testicular cells were stained with DAPI (blue), anti-γH2AX (green), and anti-XMR (more ...)
FIG. 7. Different recombination mutants show distinct patterns of markers for meiotic progression. (A to H) Squash preparations of testicular cells from mice of the indicated genotypes were stained for histone H1t and XMR. Examples of early pachytene (ep) and (more ...)
In Spo11−/− spermatocytes, diffuse XMR was observed on the chromatin, comparable to what is seen in early prophase in the wild type. In addition, a subset of spermatocytes accumulated XMR in the pseudo-sex body (34.6% of XMR-positive cells; n = 393) (Fig. and ). Even in cells containing pseudo-sex bodies, however, XMR staining persisted on bulk chromatin (Fig. and ). In these squash preparations, 19.5% (n = 393) of XMR-positive Spo11−/− spermatocytes were positive for histone H1t, although with weaker staining than is seen in mid- to late pachynema in the wild type (Fig. ). These findings indicate that Spo11−/− spermatocytes reach a stage with properties characteristic of early to mid-pachynema in the wild type.
In Dmc1−/− spermatocytes, XMR was only diffuse on chromatin and never accumulated in a discrete patch (0/476 XMR-positive squashes examined) (Fig. ). This is consistent with the γH2AX and NBS1 analyses above, which indicated a profound defect in sex body formation. No histone H1t deposition was detectable (Fig. ). These patterns imply that Dmc1−/− spermatocytes arrest with physiological properties characteristic of an earlier stage than the arrest in Spo11−/− spermatocytes. As noted before, Spo11−/− Dmc1−/− double mutants were indistinguishable from Spo11−/− single mutants (Fig. ) (n = 407). We conclude that the more severe progression defect in Dmc1−/− spermatocytes is a consequence of the persistence of unrepaired DSBs.
Msh5−/− spermatocytes displayed a distinct phenotype. Similar to Spo11−/− spermatocytes, Msh5−/− spermatocytes were positive for H1t, although the intensity of the H1t staining appeared stronger than that for Spo11−/− spermatocytes (Fig. ). However, as in Dmc1−/− spermatocytes, XMR staining in Msh5−/− spermatocytes was diffuse and did not accumulate into a discrete domain (Fig. ) (0/707 XMR-positive cells examined). Consistent with the analyses above, the Msh5−/− defect in the formation of a sex body-like structure was dependent on DSBs, because mutation of Spo11 suppressed this defect (Fig. ). Thus, Msh5−/− spermatocytes respond to the presence of unrepaired DSBs, yet not in a manner identical to that of Dmc1−/− spermatocytes.
Association of TOPBP1 with meiotic chromosomes with persistent DSBs.
The differences in meiotic progression in Spo11−/−
spermatocytes compared to that in Dmc1−/−
spermatocytes reveal the existence of a previously unknown surveillance mechanism(s) for persistent DNA damage in mammalian male meiosis. In yeast (Saccharomyces cerevisiae
), factors that respond to DNA damage in mitotically dividing cells are often required for the arrest of meiotic progression caused by the defective repair of meiotic DSBs (26
). To address whether such checkpoint factors respond to persistent DSBs in mammalian meiosis, we examined the behavior of TOPBP1, a BRCT repeat containing protein homologous to S. cerevisiae
Dpb11 and Saccharomyces pombe
). These yeast proteins have roles in DNA damage checkpoint responses in vegetative cells (1
) and in meiosis (33
). In human cells, TOPBP1 interacts with other known checkpoint factors, including ATR, and forms foci on chromatin of irradiated cells, consistent with a role in the responses to DNA damage (28
). TOPBP1 also forms foci along the axes of spermatocyte chromosomes from leptonema into zygonema (33
) (Fig. ), which colocalize with ATR and are coincident with chromatin regions containing γH2AX (33
). The foci are greatly reduced on autosomes as homologous chromosomes synapse (Fig. ), but TOPBP1 protein persists on the unsynapsed axes and chromatin of the XY pair from pachynema into diplonema (33
) (Fig. ).
FIG. 8. Association of TOPBP1 with meiotic chromosomes in Dmc1−/− and Msh5−/− spermatocytes but not in Spo11−/− spermatocytes. Spermatocyte chromosome spreads were stained with anti-SCP3 (red) and anti-TOPBP1 (green). (more ...)
TOPBP1 localization was altered in Spo11−/− spermatocytes; in 69% (11/16) of spermatocytes at a stage equivalent to zygonema (i.e., with long axes but little synapsis), TOPBP1 staining was virtually absent (Fig. ), demonstrating that TOPBP1 in the wild type responds to SPO11-generated DSBs. In remaining cells of this stage and in the majority (11/15) of later cells with significant amounts of nonhomologous synapsis, TOPBP1 colocalized with γH2AX staining within pseudo-sex bodies (Fig. and data not shown).
By contrast, in Dmc1−/− spermatocytes, TOPBP1 formed abundant, bright foci on chromosome axes in all SCP3-positive spreads examined, with significantly more signal than in the wild type (Fig. ). Consistent with other markers, no sex body-like accumulation of TOPBP1 was observed (n = 50). In Msh5−/− spermatocytes as well, TOPBP1 accumulated to higher levels than in the wild type and persisted on chromosome axes and surrounding chromatin without forming obvious pseudo-sex bodies (Fig. ). Unlike what was seen for Dmc1−/− spermatocytes, however, rare Msh5−/− spermatocyte spreads showed only faint (5 out of 54 zygotene-like cells examined) or no (1 out of 54) TOPBP1 foci (data not shown). In both Dmc1−/− and Msh5−/− spermatocytes, TOPBP1 foci were absent from many of the axes that had undergone at least limited synaptonemal complex formation (Fig. ). Spo11−/− Dmc1−/− double mutants were again indistinguishable from Spo11−/− single mutants (Fig. ). Thus, TOPBP1 binding to chromosomes in early prophase depends on SPO11-induced DSBs, and TOPBP1 persists on chromosomes when DSBs cannot be repaired.
Atm−/− spermatocytes show hallmarks of the presence of poorly repaired DSBs.
encodes a Ser/Thr kinase that activates cell cycle checkpoints in somatic cells in response to DSBs (43
). ATM is also required for normal meiosis, such that Atm−/−
spermatocytes have chromosome synapsis defects and undergo apoptosis at stage IV of the seminiferous epithelial cycle (4
). In the female germ line, Atm−/−
oocytes are eliminated at or prior to follicle formation similar to what is seen in Dmc1−/−
mutants, indicating that ATM is required for the proper repair of SPO11-induced DSBs in female meiosis as opposed to simply monitoring defects in DSB repair (13
To determine whether ATM provides a similar function in the male germ line, we analyzed sex body-associated proteins and histone H1t in Atm−/−
and Spo11−/− Atm−/−
males. Leptotene chromosomes from Atm−/−
males had little or no γH2AX signal, comparable to what was seen in Spo11−/−
males (Fig. ). Zygotene cells showed a γH2AX signal reduced compared to that of the wild type but slightly above that seen in Spo11−/−
cells (Fig. ). This reduction and/or delay of γH2AX formation indicates that ATM is the principal kinase responsible for H2AX phosphorylation in response to SPO11-induced DSBs, in agreement with other recent observations (18
). In addition, a subset (34/75) of Atm−/−
spermatocytes was observed with an abnormally high γH2AX signal dispersed across the chromatin (Fig. ). These nuclei usually exhibited short, fragmented synaptonemal complexes as well (26/34).
FIG. 9. Atm−/− spermatocytes show hallmarks of the presence of poorly repaired DSBs. (A and B) Collages of chromosome spreads from Atm−/− and Spo11−/− Atm−/− testes, stained with anti-SCP3 (red) (more ...)
Spreads with accumulations of γH2AX similar to sex bodies were rarely observed (1/40 SCP3-positive spreads). This defect was verified in spermatocyte squashes stained for XMR (Fig. ) and in testis sections stained for γH2AX or NBS1 (Fig. , respectively). Histone H1t deposition was nearly as defective as that in Dmc1−/− mutants (Fig. ). The Atm−/− mutant block to the deposition of histone H1t and the localization of sex body-associated proteins is dependent on the presence of SPO11-induced DSBs, because both processes in Spo11−/− Atm−/− double mutants were indistinguishable from those in Spo11−/− single mutants (Fig. and ). Thus, Atm−/− spermatocytes show characteristics similar to those of Dmc1−/− mutants, indicating that ATM is required for the proper repair of SPO11-induced DSBs in spermatocytes, as in oocytes. Importantly, ATM activity is not required for spermatocyte apoptosis in the absence of SPO11, because the Atm−/− mutation did not ameliorate cell death in Spo11−/− cells (Fig. ).