Numerical constraints and feedback control of double-strand breaks in mouse meiosis

Abstract

Different organisms display widely different numbers of the programmed double-strand breaks (DSBs) that initiate meiotic recombination (e.g., hundreds per meiocyte in mice and humans vs. dozens in nematodes), but little is known about what drives these species-specific DSB set points or the regulatory pathways that control them. Here we examine male mice with a lowered dosage of SPO11, the meiotic DSB catalyst, to gain insight into the effect of reduced DSB numbers on mammalian chromosome dynamics. An approximately twofold DSB reduction was associated with the reduced ability of homologs to synapse along their lengths, provoking prophase arrest and, ultimately, sterility. In many spermatocytes, chromosome subsets displayed a mix of synaptic failure and synapsis with both homologous and nonhomologous partners ("chromosome tangles"). The X chromosome was nearly always involved in tangles, and small autosomes were involved more often than large ones. We conclude that homolog pairing requirements dictate DSB set points during meiosis. Importantly, our results reveal that karyotype is a key factor: Smaller autosomes and heteromorphic sex chromosomes become weak links when DSBs are reduced below a critical threshold. Unexpectedly, unsynapsed chromosome segments trapped in tangles displayed an elevated density of DSB markers later in meiotic prophase. The unsynapsed portion of the X chromosome in wild-type males also showed evidence that DSB numbers increased as prophase progressed. These findings point to the existence of a feedback mechanism that links DSB number and distribution with interhomolog interactions.

Figure 1.

Reduced DSB formation in Tg(Spo11β)^+/− males. (A) Immunoprecipitation/Western blot for SPO11 (top) and labeling of SPO11–oligonucleotide complexes (bottom) from testes of juvenile and adult mice. Three independent experiments were performed; one is shown here. Filled circles mark lower-mobility bands likely originating from the Spo11 knockout allele expressed in more advanced cell types. The asterisk indicates nonspecific band. (B) Numbers of RAD51 and DMC1 foci are reduced in early prophase Tg(Spo11β)^+/− spermatocytes. (Panel i) Examples of IF against RAD51 and SYCP3 in early zygotene nuclei. Bars, 10 μm. (Panels ii,iii) Quantification of RAD51 and DMC1 foci. For each stage and all three genotypes, foci were counted in nuclei with similar SYCP3 appearance. Each dot indicates the focus count from one nucleus. Error bars indicate mean ± SD. RAD51 focus numbers in Tg(Spo11β)^+/+ spermatocytes were published previously (Kauppi et al. 2011, 2012). (*) P ≤ 0.0178, two-tailed Mann-Whitney test.

Figure 2.

Delayed SC initiation in Tg(Spo11β)^+/− males. (A) Progression of chromosome axis formation (marked by SYCP3) and the initiation of synapsis (marked by SYCE2) in normal meiosis are illustrated in the left panel of the cartoon and the IF image below. Tg(Spo11β)^+/− males show delayed or defective synapsis initiation, as cartooned on the right and demonstrated by the representative IF image below. Bars, 10 μm. The arrowhead indicates the sole SYCE2 stretch present in this nucleus. (B) Quantification of short SYCE2 stretches in nuclei judged by SYCP3 staining to correspond to late leptonema or early zygonema. Error bars indicate mean ± SD. (*) P < 0.0001, two-tailed Mann-Whitney test.

Figure 3.

Tg(Spo11β)^+/− testes contain both aberrant and normal-looking spermatocytes. (A) IF examples of cell types in Tg(Spo11β)^+/− males. Aberrant classes I and II are as defined in the text. Bar, 10 μm. (B) Quantification of prophase I cell types in adult control and Tg(Spo11β)^+/− males. Approximately 200 nuclei were scored per genotype (two mice per genotype). (C) TUNEL-stained whole-testis sections of 7-wk-old mice. (Inset) Magnification of the area inside the box. Bar, 50 μm. (D) Timing of aberrant versus normal-appearing Tg(Spo11β)^+/− spermatocytes in juvenile mice at the indicated ages. The number of nuclei scored is shown in parentheses. The asterisk indicates the absence of class II nuclei at 20 dpp. (E) Schematic summarizing the timeline of synaptic progression in normal and aberrant Tg(Spo11β)^+/– spermatocytes, as inferred from analyses of juvenile and adult mice. Some class I cells may achieve normal autosomal synapsis; i.e., progress to normal pachynema (dashed arrow).

Figure 4.

Chromosome configurations in class II nuclei. (A) Quantification of the number of normally synapsed autosomes in class II nuclei. Error bars indicate mean (12.4) ± SD. (B,C) Examples of immuno-FISH on class II spermatocytes to reveal the identity of chromosomes involved in nonhomologous synapsis. (B, left) IF against SYCP3, with arrows indicating chromosome tangles. The middle and right images show FISH using bacterial artificial chromosome (BAC)-based probes against centromere-distal regions of chromosomes 18 and 19, respectively. In this example, the two chromosomes 18 are in separate tangles, synapsed with nonhomologous partners, while chromosome 19 appears correctly synapsed. (C) Immuno-FISH with whole-chromosome probes against chromosome 1 and the X chromosome. The dashed lines highlight FISH signals. In this example, chromosome 1 is correctly synapsed, while the X chromosome is in the tangle (arrow). (D) Quantification of the involvement of large (chromosomes 1 and 2) and small (chromosomes 18 and 19) autosomes and sex chromosomes in nonhomologous synaptic configurations. Error bars show upper and lower 95% confidence intervals. Only class II nuclei with at least five properly synapsed autosomes were considered. (*) P = 0.037, Fisher's exact test (two-tailed). (E) Example of a class II spermatocyte nucleus with daisy chain tangles. Cartoon shows inferred configuration of homologously and nonhomologously synapsed regions, with different homologs shown in different colors.

Figure 5.

Synapsis and RAD51 focus density. (A) IF examples of nuclei with modest (panel i) or extensive (panel ii) synapsis, presumably earlier and later in prophase, respectively. Dashed lines indicate boundaries with other spermatocyte nuclei nearby. Bars, 10 μm. (B) RAD51 density on unsynapsed axes increases in late nuclei of Tg(Spo11β)^+/− mice. The genotype for controls was Spo11^+/− Tg(Spo11β)^+/−. Each dot shows the RAD51 focus density from one nucleus, ordered according to the extent of synapsis nucleus-wide. The points for the nuclei shown in panels i and ii in A are indicated. With the exception of nuclei showing 0% synapsis, all nuclei analyzed for Tg(Spo11β)^+/− were aberrant cell types. (Lines) Linear regression. The two red lines are for nuclei with <70% synapsis and >70% synapsis, to highlight the apparently biphasic RAD51 focus density in Tg(Spo11β)^+/−. (C) Examples of RAD51 foci on the X chromosome in wild-type spermatocytes. Images show overlays of IF for RAD51 and SYCP3 and FISH with an X chromosome BAC probe hybridizing to the PAR boundary. Bar, 10 μm. The insets show magnifications of the X chromosome axis, with arrows pointing to examples of RAD51 foci. (D) RAD51 foci on the 3.5 μm of X chromosome axis closest to the PAR (see example from C in the inset; counted foci are indicated by white arrows). Each dot shows the RAD51 focus count from a nucleus of the indicated stage. Error bars indicate mean ± SD. (*) P < 0.0001, two-tailed Mann-Whitney test. (E) Density of RAD51 foci along the entire measurable length of the PAR-proximal X chromosome axis (see example from C in the inset; counted foci are indicated by white arrows). Each dot shows the RAD51 focus density from a nucleus of the indicated stage. Error bars indicate mean ± SD. (*) P = 0.0008, two-tailed Mann-Whitney test.

Figure 6.

Model summarizing the chromosome-specific effects of DSB reduction and continued DSB formation on unsynapsed axes. SPO11-dependent DSBs are indicated as yellow circles. Pairing interactions and subsequent synapsis are shown as thick black lines. When DSB levels are reduced, “first phase” autosomal synapsis is partially impaired. During the later, “second phase” of synapsis, chromosome axes that remain unsynapsed can undergo nonhomologous synapsis. This process nearly always involves a synaptic interaction of the X chromosome with an autosome (frequently a small one) that, because of an insufficient number of DSBs, failed to synapse with its homolog during the “first phase.” The remaining orphan homolog, now left without a pairing partner, may be more likely to engage in additional nonhomologous synapsis. We propose that chromosome synapsis shuts down de novo DSB formation, whereas chromosome axes that remain unsynapsed continue to undergo DSB formation.