Meiotic crossovers are produced when programmed double-strand breaks (DSBs) are repaired by recombination from homologous chromosomes (homologues). In a wide variety of organisms, meiotic HORMA-domain proteins are required to direct DSB repair towards homologues. This inter-homologue bias is required for efficient homology search, homologue alignment, and crossover formation. HORMA-domain proteins are also implicated in other processes related to crossover formation, including DSB formation, inhibition of promiscuous formation of the synaptonemal complex (SC), and the meiotic prophase checkpoint that monitors both DSB processing and SCs. We examined the behavior of two previously uncharacterized meiosis-specific mouse HORMA-domain proteins—HORMAD1 and HORMAD2—in wild-type mice and in mutants defective in DSB processing or SC formation. HORMADs are preferentially associated with unsynapsed chromosome axes throughout meiotic prophase. We observe a strong negative correlation between SC formation and presence of HORMADs on axes, and a positive correlation between the presumptive sites of high checkpoint-kinase ATR activity and hyper-accumulation of HORMADs on axes. HORMADs are not depleted from chromosomes in mutants that lack SCs. In contrast, DSB formation and DSB repair are not absolutely required for depletion of HORMADs from synapsed axes. A simple interpretation of these findings is that SC formation directly or indirectly promotes depletion of HORMADs from chromosome axes. We also find that TRIP13 protein is required for reciprocal distribution of HORMADs and the SYCP1/SC-component along chromosome axes. Similarities in mouse and budding yeast meiosis suggest that TRIP13/Pch2 proteins have a conserved role in establishing mutually exclusive HORMAD-rich and synapsed chromatin domains in both mouse and yeast. Taken together, our observations raise the possibility that involvement of meiotic HORMA-domain proteins in the regulation of homologue interactions is conserved in mammals.

Meiotic crossover (CO) formation between homologous chromosomes (homologues) entails DNA double strand break (DSB) formation, homology search using DSB ends, and synaptonemal complex (SC) formation coupled with DSB repair. Meiotic progression must be prevented until DSB repair and homologue alignment are completed to avoid forming aneuploid gametes. Here we show that mouse HORMAD1 ensures that sufficient numbers of processed DSBs are available for successful homology search. HORMAD1 is needed for normal SC formation and for the efficient recruitment of ATR checkpoint kinase activity to unsynapsed chromatin. The latter phenomenon was proposed to be important in meiotic prophase checkpoints in both sexes.

Humans suffer from high rates of fetal aneuploidy, often arising from the absence of meiotic crossover recombination between homologous chromosomes1. Meiotic recombination is initiated by double-strand breaks (DSBs) generated by the SPO11 transesterase2. In yeast and worms, at least one buffering mechanism, crossover homeostasis, maintains crossover numbers despite variation in DSB numbers3–8. We show here that mammals display progressive homeostatic control of recombination. In wild-type mouse spermatocytes, focus numbers for early recombination proteins (RAD51, DMC1) were highly variable from cell to cell, whereas foci of the crossover marker MLH1 showed little variability. Furthermore, mice with greater or fewer copies of the Spo11 gene — with correspondingly greater or fewer numbers of early recombination foci — displayed relatively invariant crossover numbers. Homeostatic control is enforced during at least two stages, after the formation of early recombination intermediates and later while these intermediates mature toward crossovers. Thus, variability within the mammalian meiotic program is robustly managed by homeostatic mechanisms to control crossover formation, probably to suppress aneuploidy. Meiotic recombination exemplifies how order can be progressively implemented in a self-organizing system despite natural cell-to-cell disparities in the underlying biochemical processes.

Accurate chromosome segregation during meiosis requires that homologous chromosomes pair and become physically connected so that they can orient properly on the meiosis I spindle. These connections are formed by homologous recombination closely integrated with the development of meiosis-specific, higher-order chromosome structures. The yeast Pch2 protein has emerged as an important factor with roles in both recombination and chromosome structure formation, but recent analysis suggested that TRIP13, the mouse Pch2 ortholog, is not required for the same processes. Using distinct Trip13 alleles with moderate and severe impairment of TRIP13 function, we report here that TRIP13 is required for proper synaptonemal complex formation, such that autosomal bivalents in Trip13-deficient meiocytes frequently displayed pericentric synaptic forks and other defects. In males, TRIP13 is required for efficient synapsis of the sex chromosomes and for sex body formation. Furthermore, the numbers of crossovers and chiasmata are reduced in the absence of TRIP13, and their distribution along the chromosomes is altered, suggesting a role for TRIP13 in aspects of crossover formation and/or control. Recombination defects are evident very early in meiotic prophase, soon after DSB formation. These findings provide evidence for evolutionarily conserved functions for TRIP13/Pch2 in both recombination and formation of higher order chromosome structures, and they support the hypothesis that TRIP13/Pch2 participates in coordinating these key aspects of meiotic chromosome behavior.

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Most mutations that compromise meiotic recombination or synapsis in mouse spermatocytes result in arrest and apoptosis at the pachytene stage of the first meiotic prophase. Two main mechanisms are thought to trigger arrest: one independent of the double-strand breaks (DSBs) that initiate meiotic recombination, and another activated by persistent recombination intermediates. Mechanisms underlying the recombination-dependent arrest response are not well understood, so we sought to identify factors involved by examining mutants deficient for TRIP13, a conserved AAA+ ATPase required for the completion of meiotic DSB repair. We find that spermatocytes with a hypomorphic Trip13 mutation (Trip13mod/mod) arrest with features characteristic of early pachynema in wild type, namely, fully synapsed chromosomes without incorporation of the histone variant H1t into chromatin. These cells then undergo apoptosis, possibly in response to the arrest or in response to a defect in sex body formation. However, TRIP13-deficient cells that additionally lack the DSB-responsive kinase ATM progress further, reaching an H1t-positive stage (i.e., similar to mid/late pachynema in wild type) despite the presence of unrepaired DSBs. TRIP13-deficient spermatocytes also progress to an H1t-positive stage if ATM activity is attenuated by hypomorphic mutations in Mre11 or Nbs1 or by elimination of the ATM-effector kinase CHK2. These mutant backgrounds nonetheless experience an apoptotic block to further spermatogenic progression, most likely caused by failure to form a sex body. DSB numbers are elevated in Mre11 and Nbs1 hypomorphs but not Chk2 mutants, thus delineating genetic requirements for the ATM-dependent negative feedback loop that regulates DSB numbers. The findings demonstrate for the first time that ATM-dependent signaling enforces the normal pachytene response to persistent recombination intermediates. Our work supports the conclusion that recombination defects trigger spermatocyte arrest via pathways than are genetically distinct from sex body failure-promoted apoptosis and confirm that the latter can function even when recombination-dependent arrest is inoperative. Implications of these findings for understanding the complex relationships between spermatocyte arrest and apoptosis are discussed.

During meiosis in most sexually reproducing organisms, recombination forms crossovers between homologous maternal and paternal chromosomes and thereby promotes proper chromosome segregation at the first meiotic division. The number and distribution of crossovers are tightly controlled, but the factors that contribute to this control are poorly understood in most organisms, including mammals. Here we provide evidence that the ATM kinase or protein is essential for proper crossover formation in mouse spermatocytes. ATM deficiency causes multiple phenotypes in humans and mice, including gonadal atrophy. Mouse Atm−/− spermatocytes undergo apoptosis at mid-prophase of meiosis I, but Atm−/− meiotic phenotypes are partially rescued by Spo11 heterozygosity, such that ATM-deficient spermatocytes progress to meiotic metaphase I. Strikingly, Spo11+/−Atm−/− spermatocytes are defective in forming the obligate crossover on the sex chromosomes, even though the XY pair is usually incorporated in a sex body and is transcriptionally inactivated as in normal spermatocytes. The XY crossover defect correlates with the appearance of lagging chromosomes at metaphase I, which may trigger the extensive metaphase apoptosis that is observed in these cells. In addition, control of the number and distribution of crossovers on autosomes appears to be defective in the absence of ATM because there is an increase in the total number of MLH1 foci, which mark the sites of eventual crossover formation, and because interference between MLH1 foci is perturbed. The axes of autosomes exhibit structural defects that correlate with the positions of ongoing recombination. Together, these findings indicate that ATM plays a role in both crossover control and chromosome axis integrity and further suggests that ATM is important for coordinating these features of meiotic chromosome dynamics.

The evolutionary appearance of p53 likely preceded its role in tumor suppression, suggesting that there may be unappreciated functions for this protein. Using genetic reporters as proxies to follow in vivo activation of the p53 network in Drosophila, we discovered that the process of meiotic recombination instigates programmed activation of p53 in the germline. Specifically, doublestranded breaks in DNA generated by the topoisomerase Spo11 provoked functional p53 activity, which was prolonged in cells defective for meiotic DNA repair. This intrinsic stimulus for the p53 regulatory network is highly conserved because Spo11-dependent activation of p53 also occurred in mice. Our findings establish a physiological role for p53 in meiosis and suggest that tumor suppressive functions may have been co-opted from primordial activities linked to recombination.The p53 gene family mediates adaptive responses to genotoxic stress (1-3) and is broadly conserved (4,5). It is widely accepted that the p53 regulatory network is generally compromised in human cancers but several lines of evidence indicate that during evolution, animals rarely lived long enough to experience cancer (6,7). Therefore, tumor suppressive functions associated with p53 were likely derived from primordial activities that are poorly understood. We used Drosophila to investigate physiological properties of this network because, in this organism, a single p53 member exists and, like its mammalian counterparts, it coordinates stress responses and promotes genome stability (8-12).We constructed transgenes ( fig. S1) that place green fluorescence protein (GFP) under the control of an enhancer taken from sequences upstream of the Drosophila reaper (rpr) locus, which includes a p53 consensus binding site (13). Two transgenic lines were produced and designated as "p53Rps" for p53 reporter strains. One strain produces nuclear localized GFP (p53R-GFPnls) and the other does not (p53R-GFPcyt).Ionizing radiation (IR) leads to DNA damage and activates p53 in various model systems. Therefore, we exposed p53Rps transgenic embryos to IR and followed GFP expression by time-lapse live imaging. GFP was observed as early as 70min after exposure to IR, and was prominent at 180min in virtually all embryos ( Fig. 1A and movie S1). p53Rps expression was not observed in unirradiated embryos nor was it observed in flies lacking p53 or chk2 (Fig. 1B), its upstream activating kinase (14). To confirm that damaged DNA is responsible for activation, we also observed induction of GFP in response to UV radiation and injected fig. S2). Thus, the p53Rps reporters serve as authentic proxies that enable us to monitor p53 activation in live animals in real time.We surveyed reporter activity throughout development and found little or no evidence for unstimulated expression but, surprisingly, transient p53Rps expression was observed in germline precursors of all females. Activity was localized in region 2a and 2b of the germarium and was absent beyond region 3 and in all egg chambers (Fi...