Disruption of Temporal Progression and Synapsis in Meiocytes from Mre11 Complex Mutants
Although histological examination of testis morphology fromMre11 complexhyomorphs indicated that meiogenesis was not grossly disturbed (see Figure S1
in the Supplemental Data available online), subfertility in the mutants [12
] was consistent with the hypothesis that Mre11 complex hypomorphism causes perturbations in meiosis. To assess meiotic progression, we determined the distribution of meiotic prophase substages in mutants relative to controls. Oocytes enter meiosis and progress through prophase in a semisynchronous wave during fetal development. Examination of oocytes from Mre11ATLD1/ATLD1
females at 17.5– 18.5 days of gestation revealed a significant difference in prophase distribution by comparison with control littermates (Figures S2A and S2B; Mre11ATLD1/ATLD1
= 122.7, p < 0.0001; Nbs1δB/δB
= 49.4, p < 0.0001). For both mutants, more than 50% of oocytes were at zygotene, whereas fewer than 15% of oocytes from controls remained at zygotene, with the vast majority progressing to pachytene or beyond (for a description of meiotic prophase stages, see Supplemental Experimental Procedures
). The paucity of later stages in mutants suggests meiotic delay or arrest at zygotene, the stage in which DSBs are processed and strand exchange intermediates are formed.
Temporal disturbances in meiotic progression were also observed in adult male Mre11 complex hypomorphs. Mre11ATLD1/ATLD1 males exhibited an increase in the proportion of pachytene cells, from40%in controls to 68% in mutants (Figure S2C; χ2 = 109.5, p < 0.0001). Nbs1δB/δB males exhibited a slight but statistically significant increase in the proportion of zygotene cells, from 13% in controls to 20% in mutants (Figure S2D).
Examination of zygotene and pachytene cells from Mre11ATLD1/ATLD1 females (Nbs1δB/δB females were not further examined) and Mre11ATLD1/ATLD1 and Nbs1δB/δB males revealed defects in homologous chromosome synapsis. In Mre11ATLD1/ATLD1 females and males, synaptic defects were evident in 67% (85/126) and 38% (51/134) of pachytene cells, respectively, whereas only 16% (20/123) of control oocytes and 3% (2/68) of control spermatocytes exhibited defects (females: χ2 = 50.9, p < 0.0001; males: χ2 = 28.7, p < 0.0001). The most common aberration in mutant females was partial synapsis of 1 to 3 bivalents () in cells classified as pachytene; in addition to incomplete synapsis, males commonly exhibited fragmented () or gapped SCs.
SC Assembly Defects in Prophase Meiocytes from Mre11 Complex Hypomorphic Mice
A significant subset of pachytene cells in Mre11ATLD1/ATLD1 mice of both sexes exhibited end-to-end associations between the SCs of nonhomologous chromosomes. In females, associations often involved three or more SCs in a ‘‘pinwheel’’ configuration (19/119 cells versus 3/101 cells in controls, χ2 = 10.2, p < 0.01; ) and occurred exclusively at the centromere-proximal ends of SCs (identifiable by intense centromeric heterochromatin staining; ). Because no associations involved distal telomeres, the data do not support the interpretation that these associations result from telomere dysfunction. In males, associations between the X chomosome and autosomal SCs were occasionally observed () and, like females, involved the centromeric ends of chromosomes.
Nbs1δB/δB males exhibited less severe synaptic aberrations. Nevertheless, 45% (20/44) of cells at the zygotene-pachytene boundary contained an asynaptic bivalent, while all other bivalents were completely synapsed (). In contrast, only 12% (5/39) of similarly staged nuclei from control mice contained a single asynaptic bivalent (χ2 = 10.5, p < 0.01).
Altered Repair of DSBs in Mre11 Complex Mutants
Temporal changes in progression and synaptic aberrations in Mre11 complex hypomorphs suggest significant DSB repair defects. Therefore, we assessed localization patterns of RAD51, an evolutionarily conserved RecA protein required for strand exchange during meiotic DSB repair (see [17
]). In normal mice, the number of RAD51 foci peaks in leptotene and early zygotene, declining to only a few foci as cells progress to late pachytene and DSBs are resolved [18
We found no difference between Mre11ATLD1/ATLD1
female mice and controls in the number of RAD51 foci at early prophase stages (data not shown), suggesting that the frequency of DSB formation is not significantly altered. However, the mean number of RAD51 foci in pachytene oocytes was significantly increased in mutants (Table S1; U = 1381.0; p < 0.0001). A change in localization pattern was also observed: typically a few RAD51 foci were scattered across all bivalents in controls, whereas mutants exhibited a large number of foci restricted to several SCs (), giving these bivalents a ‘‘hot’’ appearance. Hot SCs were not necessarily those with synaptic aberrations, as would be expected if these bivalents were temporally out of synchrony with the rest of the cell. Additionally, phosphorylated histone γH2AX (a marker of DSBs; [25
]) colocalized to lingering RAD51 foci in mutant oocytes (), suggesting that these are sites of unrepaired breaks. Specifically, 92 of 93 SCs with 5 or more RAD51 foci (‘‘hot SCs’’) also recruited γH2AX. We observed no evidence of nonhomologous synapsis or exchange, so we do not favor the interpretation that hot SCs represent nonhomologous synapsis.
Persistent RAD51 Localization in Mre11ATLD1/ATLD1 Mice
Persistent RAD51 foci were also evident in spermatocytes from Mre11ATLD1/ATLD1 males at late pachytene, resulting in an approximate 2.5-fold increase in the mean number of foci (Table S1; U = 315.5; p < 0.05). Additionally, these foci were detected on autosomal SCs, rather than restricted to the XY bivalent as in controls (). Further, occasional RAD51 foci persisted in some cells at the diplotene stage (data not shown).
Nbs1δB/δB males did not exhibit gross changes in RAD51 foci localization during early prophase. There was, however, a slight, albeit nonsignificant, increase in the proportion of pachytene cells with 1 or 2 remaining RAD51 foci (data not shown), suggesting a similar, though less severe, defect in repair kinetics in this mutant.
Altered Crossover Formation in Mre11 Complex Hypomorphs
The temporal disruption in RAD51 localization raised the possibility of downstream consequences in the recombination pathway, including abnormalities in crossover formation. MLH1 is a MutL family protein required for crossover formation and is commonly used as a marker of exchanges [26
]. In Mre11ATLD1/ATLD1
females, counts of MLH1 foci in pachytene cells exhibiting complete or nearly complete synapsis revealed a significant decrease in mean foci number (; t = 5.6, p < 0.0001). In contrast, the average number of MLH1 foci in Mre11ATLD1/ATLD1
males was significantly increased (; t = 4.0, p < 0.0001 and t = 4.3; p < 0.0001, respectively).
Average Number of MLH1 Foci in Pachytene-Stage Meiocytes from Mre11ATLD1/ATLD1 and Nbs1δB/δB Mutant and Control Mice
To further characterize MLH1 patterns in males, the location of foci on single-exchange bivalents was examined. A significant increase in centromere-proximal foci was observed in Mre11
mutants, with a concomitant decrease in the frequency of foci in the most distal segment of the chromosome (; χ2
= 20.3, p < 0.005), the preferred region of exchange in wild-type males [29
A Change in the Position of MLH1 Foci Occurs in Mre11ATLD1/ATLD1 Spermatocytes
To verify the MLH1 data, we analyzed chiasmata in diakinesis meiocytes from Mre11ATLD1/ATLD1 mice. In both sexes, the results paralleled the findings of the MLH1 studies. Mre11ATLD1/ATLD1 females showed a significant decrease in chiasmata (25.1 ± 2.7 versus 27.9 ± 3.2 in controls, t = 3.9, p < 0.01), and males showed a slight, although nonsignificant, increase in chiasmata (23.3 ± 2.2 versus 22.9 ± 1.6 in controls). Failure to achieve statistical significance likely stems from the small number of cells analyzed (technical difficulty prevented obtaining good-quality diakinesis preparations from spermatocytes, also prohibiting analysis of chiasmata location). Additionally, we observed an increase in the frequency of univalents, presumably reflecting defects in synapsis or repair.