Extensive meiotic asynapsis in mice antagonises meiotic silencing of unsynapsed chromatin and consequently disrupts meiotic sex chromosome inactivation

Chromosome synapsis during zygotene is a prerequisite for the timely homologous recombinational repair of meiotic DNA double-strand breaks (DSBs). Unrepaired DSBs are thought to trigger apoptosis during midpachytene of male meiosis if synapsis fails. An early pachytene response to asynapsis is meiotic silencing of unsynapsed chromatin (MSUC), which, in normal males, silences the X and Y chromosomes (meiotic sex chromosome inactivation [MSCI]). In this study, we show that MSUC occurs in Spo11-null mouse spermatocytes with extensive asynapsis but lacking meiotic DSBs. In contrast, three mutants (Dnmt3l, Msh5, and Dmc1) with high levels of asynapsis and numerous persistent unrepaired DSBs have a severely impaired MSUC response. We suggest that MSUC-related proteins, including the MSUC initiator BRCA1, are sequestered at unrepaired DSBs. All four mutants fail to silence the X and Y chromosomes (MSCI failure), which is sufficient to explain the midpachytene apoptosis. Apoptosis does not occur in mice with a single additional asynapsed chromosome with unrepaired meiotic DSBs and no disturbance of MSCI.     

Meiotic silencing and fragmentation of the male germline restricted chromosome in zebra finch

During male meiotic prophase in mammals, X and Y are in a largely unsynapsed configuration, which is thought to trigger meiotic sex chromosome inactivation (MSCI). In avian species, females are ZW, and males ZZ. Although Z and W in chicken oocytes show complete, largely heterologous synapsis, they too undergo MSCI, albeit only transiently. The W chromosome is already inactive in early meiotic prophase, and inactive chromatin marks may spread on to the Z upon synapsis. Mammalian MSCI is considered as a specialised form of the general meiotic silencing mechanism, named meiotic silencing of unsynapsed chromatin (MSUC). Herein, we studied the avian form of MSUC, by analysing the behaviour of the peculiar germline restricted chromosome (GRC) that is present as a single copy in zebra finch spermatocytes. In the female germline, this chromosome is present in two copies, which normally synapse and recombine. In contrast, during male meiosis, the single GRC is always eliminated. We found that the GRC in the male germline is silenced from early leptotene onwards, similar to the W chromosome in avian oocytes. The GRC remains largely unsynapsed throughout meiotic prophase I, although patches of SYCP1 staining indicate that part of the GRC may self-synapse. In addition, the GRC is largely devoid of meiotic double strand breaks. We observed a lack of the inner centromere protein INCENP on the GRC and elimination of the GRC following metaphase I. Subsequently, the GRC forms a micronucleus in which the DNA is fragmented. We conclude that in contrast to MSUC in mammals, meiotic silencing of this single chromosome in the avian germline occurs prior to, and independent of DNA double strand breaks and chromosome pairing, hence we have named this phenomenon meiotic silencing prior to synapsis (MSPS).     

Chromosomal synapsis defects can trigger oocyte apoptosis without elevating numbers of persistent DNA breaks above wild-type levels

Generation of haploid gametes depends on a modified version of homologous recombination in meiosis. Meiotic recombination is initiated by single-stranded DNA (ssDNA) ends originating from programmed DNA double-stranded breaks (DSBs) that are generated by the topoisomerase-related SPO11 enzyme. Meiotic recombination involves chromosomal synapsis, which enhances recombination-mediated DSB repair, and thus, crucially contributes to genome maintenance in meiocytes. Synapsis defects induce oocyte apoptosis ostensibly due to unrepaired DSBs that persist in asynaptic chromosomes. In mice, SPO11-deficient oocytes feature asynapsis, apoptosis and, surprisingly, numerous foci of the ssDNA-binding recombinase RAD51, indicative of DSBs of unknown origin. Hence, asynapsis is suggested to trigger apoptosis due to inefficient DSB repair even in mutants that lack programmed DSBs. By directly detecting ssDNAs, we discovered that RAD51 is an unreliable marker for DSBs in oocytes. Further, SPO11-deficient oocytes have fewer persistent ssDNAs than wild-type oocytes. These observations suggest that oocyte quality is safeguarded in mammals by a synapsis surveillance mechanism that can operate without persistent ssDNAs.     

AtSPO11-1 is necessary for efficient meiotic recombination in plants

The Saccharomyces cerevisiae Spo11 protein catalyses DNA double-strand breaks (DSBs) that initiate meiotic recombination. The model plant Arabidopsis thaliana possesses at least three SPO11 homologues. T-DNA and ethyl-methane sulfonate mutagenesis allowed us to show that meiotic progression is altered in plants in which the AtSPO11-1 gene is disrupted. Both male and female meiocytes formed very few bivalents. Furthermore, no fully synapsed chromosomes were observed during prophase I. Later, in meiosis I, we observed that chromosomes segregated randomly, leading to the production of a large proportion of non-functional gametes. These meiotic aberrations were associated with a drastic reduction in meiotic recombination. Thus, our data show that initiation of meiotic recombination by SPO11- induced DSBs is a mechanism conserved in plants. Furthermore, unlike Drosophila and Caenorhabditis elegans, but like fungi, SPO11 is necessary for normal synapsis in plants.     

Defective imprint resetting in carriers of Robertsonian translocation Rb (8.12)

Meiotic silencing of unsynapsed chromatin (MSUC) occurs in the germ cells of translocation carriers and may cause meiotic arrest and infertility. We hypothesized that if bypassing meiotic checkpoints MSUC may cause epigenetic defects in sperm. We investigated the meiotic behavior of the Robertsonian translocation Rb (8.12) in mice. The unsynapsed 8 and 12 trivalent was associated with the XY body during early and mid-pachynema in heterozygous Rb (8.12) carriers, suggesting possible silencing of pericentromeric genes, such as the Dnmt3a gene. In wild-type mice, DNMT3A protein showed a dramatic accumulation in the nucleus during the mid-pachytene stage and distinct association with the XY body. In translocation carriers, DNMT3A was less abundant in a proportion of pachytene spermatocytes that also had unsynapsed pericentromeric regions of chromosomes 8 and 12. The same mice had incomplete methylation of the imprinted H19 differentially methylated region (DMR) in sperm. We propose that impaired H19 imprint establishment results from lack of synapsis in chromosomes 8 and 12 probably through transient silencing of a chromosome 8 or 12 gene during pachynema. Furthermore, our findings support the notion that imprint establishment at the H19 locus extends into pachynema.     

DNA polymerase β is critical for mouse meiotic synapsis

We have shown earlier that DNA polymerase β (Pol β) localizes to the synaptonemal complex (SC) during Prophase I of meiosis in mice. Pol β localizes to synapsed axes during zygonema and pachynema, and it associates with the ends of bivalents during late pachynema and diplonema. To test whether these localization patterns reflect a function for Pol β in recombination and/or synapsis, we used conditional gene targeting to delete the PolB gene from germ cells. We find that Pol β-deficient spermatocytes are defective in meiotic chromosome synapsis and undergo apoptosis during Prophase I. We also find that SPO11-dependent γH2AX persists on meiotic chromatin, indicating that Pol β is critical for the repair of SPO11-induced double-strand breaks (DSBs). Pol β-deficient spermatocytes yielded reduced steady-state levels of the SPO11-oligonucleotide complexes that are formed when SPO11 is removed from the ends of DSBs, and cytological experiments revealed that chromosome-associated foci of replication protein A (RPA), RAD51 and DMC1 are less abundant in Pol β-deficient spermatocyte nuclei. Localization of Pol β to meiotic chromosomes requires the formation of SPO11-dependent DSBs. Taken together, these findings strongly indicate that Pol β is required at a very early step in the processing of meiotic DSBs, at or before the removal of SPO11 from DSB ends and the generation of the 3′ single-stranded tails necessary for subsequent strand exchange. The chromosome synapsis defects and Prophase I apoptosis of Pol β-deficient spermatocytes are likely a direct consequence of these recombination defects.     

Differentiated function and localisation of SPO11-1 and PRD3 on the chromosome axis during meiotic DSB formation in Arabidopsis thaliana

During meiosis, DNA double-strand breaks (DSBs) occur throughout the genome, a subset of which are repaired to form reciprocal crossovers between chromosomes. Crossovers are essential to ensure balanced chromosome segregation and to create new combinations of genetic variation. Meiotic DSBs are formed by a topoisomerase-VI-like complex, containing catalytic (e.g. SPO11) proteins and auxiliary (e.g. PRD3) proteins. Meiotic DSBs are formed in chromatin loops tethered to a linear chromosome axis, but the interrelationship between DSB-promoting factors and the axis is not fully understood. Here, we study the localisation of SPO11-1 and PRD3 during meiosis, and investigate their respective functions in relation to the chromosome axis. Using immunocytogenetics, we observed that the localisation of SPO11-1 overlaps relatively weakly with the chromosome axis and RAD51, a marker of meiotic DSBs, and that SPO11-1 recruitment to chromatin is genetically independent of the axis. In contrast, PRD3 localisation correlates more strongly with RAD51 and the chromosome axis. This indicates that PRD3 likely forms a functional link between SPO11-1 and the chromosome axis to promote meiotic DSB formation. We also uncovered a new function of SPO11-1 in the nucleation of the synaptonemal complex protein ZYP1. We demonstrate that chromosome co-alignment associated with ZYP1 deposition can occur in the absence of DSBs, and is dependent on SPO11-1, but not PRD3. Lastly, we show that the progression of meiosis is influenced by the presence of aberrant chromosomal connections, but not by the absence of DSBs or synapsis. Altogether, our study provides mechanistic insights into the control of meiotic DSB formation and reveals diverse functional interactions between SPO11-1, PRD3 and the chromosome axis.     

The mouse Spo11 gene is required for meiotic chromosome synapsis

The Spo11 protein initiates meiotic recombination by generating DNA double-strand breaks (DSBs) and is required for meiotic synapsis in S. cerevisiae. Surprisingly, Spo11 homologs are dispensable for synapsis in C. elegans and Drosophila yet required for meiotic recombination. Disruption of mouse Spo11 results in infertility. Spermatocytes arrest prior to pachytene with little or no synapsis and undergo apoptosis. We did not detect Rad51/Dmc1 foci in meiotic chromosome spreads, indicating DSBs are not formed. Cisplatin-induced DSBs restored Rad51/Dmc1 foci and promoted synapsis. Spo11 localizes to discrete foci during leptotene and to homologously synapsed chromosomes. Other mouse mutants that arrest during meiotic prophase (Atm -/-, Dmc1 -/-, mei1, and Morc(-/-)) showed altered Spo11 protein localization and expression. We speculate that there is an additional role for Spo11, after it generates DSBs, in synapsis.     

Meiotic sex chromosome inactivation

X chromosome inactivation is most commonly studied in the context of female mammalian development, where it performs an essential role in dosage compensation. However, another form of X-inactivation takes place in the male, during spermatogenesis, as germ cells enter meiosis. This second form of X-inactivation, called meiotic sex chromosome inactivation (MSCI) has emerged as a novel paradigm for studying the epigenetic regulation of gene expression. New studies have revealed that MSCI is a special example of a more general mechanism called meiotic silencing of unsynapsed chromatin (MSUC), which silences chromosomes that fail to pair with their homologous partners and, in doing so, may protect against aneuploidy in subsequent generations. Furthermore, failure in MSCI is emerging as an important etiological factor in meiotic sterility.     

Functional interactions between SPO11 and REC102 during initiation of meiotic recombination in Saccharomyces cerevisiae.

In Saccharomyces cerevisiae, formation of the DNA double-strand breaks (DSBs) that initiate meiotic recombination requires the products of at least 10 genes. Spo11p is thought to be the catalytic subunit of the DNA cleaving activity, but the roles of the other proteins, and the interactions among them, are not well understood. This study demonstrates genetic and physical interactions between the products of SPO11 and another early meiotic gene required for DSB formation, REC102. We found that epitope-tagged versions of SPO11 and REC102 that by themselves were capable of supporting normal or nearly normal levels of meiotic recombination conferred a severe synthetic cold-sensitive phenotype when combined in the same cells. DSB formation, meiotic gene conversion, and spore viability were drastically reduced in the doubly tagged strain at a nonpermissive temperature. This conditional defect could be partially rescued by expression of untagged SPO11, but not by expression of untagged REC102, indicating that tagged REC102 is fully dominant for this synthetic phenotype. Both tagged and wild-type Spo11p co-immunoprecipitated with tagged Rec102p from meiotic cell extracts, indicating that these proteins are present in a common complex in vivo. Tagged Rec102p localized to the nucleus in whole cells and to chromatin on spread meiotic chromosomes. Our results are consistent with the idea that a multiprotein complex that includes Spo11p and Rec102p promotes meiotic DSB formation.     

Compartmentalization of Spo11p in vegetative cells of yeast Saccharomyces cerevisiae

Homologous recombination is initiated in meiotic eukaryotic cells at DNA double-strand breaks, which are generated by several proteins, Spo11p playing a key role. The protein products of SPO11 orthologs are highly conserved, are found in most eukaryotes from plants to human, and are structurally similar to subunit A of archaeal DNA topoisomerase VI. Saccharomyces cerevisiae SPO11 is expressed in meiotic prophase I. Spo11p acts as topoisomerase II and is presumably active as a dimer. Experimental data on Spo11p compartmentalization in vegetative yeast cells are unavailable. The SPO11 coding region and its fragments were fused in frame with the egfp reporter and expressed in vegetative yeast cells. The Spo11p-EGFP fusion was localized in the nucleus, while cytoplasmic localization was observed for Spo11Δ-EGFP devoid of the 25 N-terminal residues. N-terminal Spo11p region 7–25 fused with EGFP ensured the nuclear targeting of the reporter protein and was assumed to harbor the nuclear localization signal.     

Association of Mre11p with double-strand break sites during yeast meiosis

The repair of DNA double-strand breaks (DSBs) requires the activity of the Mre11/Rad50/Xrs2(Nbs1) complex. In Saccharomyces cerevisiae, this complex is required for both the initiation of meiotic recombination by Spo11p-catalyzed programmed DSBs and for break end resection, which is necessary for repair by homologous recombination. We report that Mre11p transiently associates with the chromatin of Spo11-dependent DSB regions throughout the genome. Mutant analyses show that Mre11p binding requires the function of all genes required for DSB formation, with the exception of RAD50. However, Mre11p binding does not require DSB formation itself, since Mre11p transiently associates with DSB regions in the catalysis-negative mutant spo11-Y135F. Mre11p release from chromatin is blocked in mutants that accumulate unresected DSBs. We propose that Mre11p is a component of a pre-DSB complex that assembles on the DSB sites, thus ensuring a tight coupling between DSB formation by Spo11p and the processing of break ends.     

The Drosophila zinc finger protein trade embargo is required for double strand break formation in meiosis

Homologous recombination in meiosis is initiated by the programmed induction of double strand breaks (DSBs). Although the Drosophila Spo11 ortholog Mei-W68 is required for the induction of DSBs during meiotic prophase, only one other protein (Mei-P22) has been shown to be required for Mei-W68 to exert this function. We show here that the chromatin-associated protein Trade Embargo (Trem), a C2H2 zinc finger protein, is required to localize Mei-P22 to discrete foci on meiotic chromosomes, and thus to promote the formation of DSBs, making Trem the earliest known function in the process of DSB formation in Drosophila oocytes. We speculate that Trem may act by either directing the binding of Mei-P22 to preferred sites of DSB formation or by altering chromatin structure in a manner that allows Mei-P22 to form foci.