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During meiosis, induction of DNA double strand breaks (DSB) leads to recombination between homologous chromosomes, resulting in crossovers (CO) and non-crossovers (NCO). In the mouse, only 10% of DSBs resolve as COs, mostly through a class I pathway dependent on MutSγ (MSH4/ MSH5) and MutLγ (MLH1/MLH3), the latter representing the ultimate marker of these CO events. A second Class II CO pathway accounts for only a few COs, but is not thought to involve MutSγ/ MutLγ, and is instead dependent on MUS81-EME1. For class I events, loading of MutLγ is thought to be dependent on MutSγ, however MutSγ loads very early in prophase I at a frequency that far exceeds the final number of class I COs. Moreover, loss of MutSγ in mouse results in apoptosis before CO formation, preventing the analysis of its CO function. We generated a mutation in the ATP binding domain of Msh5 (Msh5GA). While this mutation was not expected to affect MutSγ complex formation, MutSγ foci do not accumulate during prophase I. However, most spermatocytes from Msh5GA/GA mice progress to late pachynema and beyond, considerably further than meiosis in Msh5−/− animals. At pachynema, Msh5GA/GA spermatocytes show persistent DSBs, incomplete homolog pairing, and fail to accumulate MutLγ. Unexpectedly, Msh5GA/GA diakinesis-staged spermatocytes have no chiasmata at all from any CO pathway, indicating that a functional MutSγ complex is critical for all CO events regardless of their mechanism of generation.

During meiosis, induction of DNA double strand breaks (DSB) leads to recombination between homologous chromosomes, resulting in crossovers (CO) and non-crossovers (NCO). Only 10% DSBs resolve as COs, mostly through a class I pathway dependent on MutSγ (MSH4/ MSH5). Class II CO events represent a minor proportion of the total CO count and also arise from DSBs, but are not thought to involve MutSγ. However, loading of MutSγ occurs very early in prophase I at a frequency that far exceeds the final number of class I COs found in late prophase I. Moreover, loss of MutSγ in mouse results in apoptosis before CO formation, preventing analysis of its CO function. We generated a mutation in the ATP binding domain ofMsh5(Msh5GA). While this mutation was not expected to affect MutSγ complex formation, MutSγ foci do not accumulate during prophase I. Nevertheless, while some spermatocytes fromMsh5-/-animals progress into pachynema, most spermatocytes fromMsh5GA/GAmice progress to late pachynema and beyond. Some spermatocytes fromMsh5GA/GAmice complete prophase I entirely, allowing for the first time an assessment of MSH5 function in CO formation. At pachynema,Msh5GA/GAspermatocytes show persistent DSBs, incomplete homolog pairing, and fail to accumulate MutLγ (MLH1/MLH3). Unexpectedly,Msh5GA/GAdiakinesis-staged spermatocytes have no chiasmata at all from any CO pathway, indicating that a functional MutSγ complex in early prophase I is a pre-requisite for all COs.ARTICLE SUMMARYMSH4/MSH5 are critical components of the class I crossover (CO) machinery, which is responsible for >90% of the COs that arise in mammalian meiosis. We generated a point mutation in the ATP binding motif ofMsh5, and found that mutant spermatocytes lose all COs, not just those arising from the class I pathway.

CNTD1 coordinates the maturation and designation of meiotic crossover sites from an excess pool of double-strand break intermediates by regulating dynamic changes in key protein complexes associated with these sites., Meiotic crossovers (COs) are crucial for ensuring accurate homologous chromosome segregation during meiosis I. Because the double-strand breaks (DSBs) that initiate meiotic recombination greatly outnumber eventual COs, this process requires exquisite regulation to narrow down the pool of DSB intermediates that may form COs. In this paper, we identify a cyclin-related protein, CNTD1, as a critical mediator of this process. Disruption of Cntd1 results in failure to localize CO-specific factors MutLγ and HEI10 at designated CO sites and also leads to prolonged high levels of pre-CO intermediates marked by MutSγ and RNF212. These data show that maturation of COs is intimately coupled to deselection of excess pre-CO sites to yield a limited number of COs and that CNTD1 coordinates these processes by regulating the association between the RING finger proteins HEI10 and RNF212 and components of the CO machinery.

SFMBT1 (Scm [Sex comb on midleg] with four MBT [malignant brain tumor] domains 1) is a poorly characterized mammalian MBT domain-containing protein homologous to Drosophila SFMBT, a Polycomb group protein involved in epigenetic regulation of gene expression. Here, we show that SFMBT1 regulates transcription in somatic cells and during spermatogenesis through the formation of a stable complex with LSD1 and CoREST. When bound to its gene targets, SFMBT1 recruits its associated proteins and causes chromatin compaction and transcriptional repression. SFMBT1, LSD1, and CoREST share a large fraction of target genes, including those encoding replication-dependent histones. Simultaneous occupancy of histone genes by SFMBT1, LSD1, and CoREST is regulated during the cell cycle and correlates with the loss of RNA polymerase II at these promoters during G2, M, and G1. The interplay between the repressive SFMBT1–LSD1–CoREST complex and RNA polymerase II contributes to the timely transcriptional regulation of histone genes in human cells. SFMBT1, LSD1, and CoREST also form a stable complex in germ cells, and their chromatin binding activity is regulated during spermatogenesis.

Fancj, the gene associated with Fanconi anemia (FA) Complementation Group J, encodes a DNA helicase involved in homologous recombination repair and the cellular response to replication stress. FANCJ functions in part through its interaction with key DNA repair proteins, including MutL homolog-1 (MLH1), Breast Cancer Associated gene-1 (BRCA1), and Bloom syndrome helicase (BLM). All three of these proteins are involved in a variety of events that ensure genome stability, including the events of DNA double strand break (DSB) repair during prophase I of meiosis. Meiotic DSBs are repaired through homologous recombination resulting in non-crossovers (NCO) or crossovers (CO). The frequency and placement of COs are stringently regulated to ensure that each chromosome receives at least one CO event, and that longer chromosomes receive at least one additional CO, thus facilitating the accurate segregation of homologous chromosomes at the first meiotic division. In the present study, we investigated the role of Fancj during prophase I using a gene trap mutant allele. Fancj (GT/GT) mutants are fertile, but their testes are very much smaller than wild-type littermates, predominantly as a result of impeded spermatogonial proliferation and mildly increased apoptosis during testis development in the fetus. This defect in spermatogonial proliferation is consistent with mutations in other FA genes. During prophase I, early events of synapsis and DSB induction/repair appear mostly normal in Fancj (GT/GT) males, and the FANCJ-interacting protein BRCA1 assembles normally on meiotic chromosome cores. However, MLH1 focus frequency is increased in Fancj (GT/GT) males, indicative of increased DSB repair via CO, and is concomitant with increased chiasmata at diakinesis. This increase in COs in the absence of FANCJ is associated with increased localization of BLM helicase protein, indicating that BLM may facilitate the increased rate of crossing over in Fancj (GT/GT) males. Taken together, these results demonstrate a critical role for FANCJ in spermatogenesis at two stages: firstly in the proliferative activity that gives rise to the full complement of testicular spermatogonia and secondly in the establishment of appropriate CO numbers during prophase I.

Meiosis is a specialized form of cellular division that occurs in all sexually reproducing organisms. The goal of meiosis is to halve the genomic content of the parent cell, which contains two copies of each chromosome (i.e., diploid), so that the daughter cells, or gametes, contain only one copy of each chromosome (i.e., haploid). Two gametes can then undergo fertilization to produce an embryo that is restored to diploid chromosome status. Unlike mitosis, therefore, in which cells replicate their DNA and then divide once, during meiosis, DNA replication is followed by two consecutive divisions to result in up to four gametes. This chapter addresses the fundamental events that induce meiotic initiation, the defining events of meiosis that allow for faithful segregation of DNA content, and the molecular events that orchestrate this exquisitely choreographed and fascinating process. Also covered are the consequences for human health of inaccurate segregation of chromosomes, and how this impacts mammalian reproduction.

Meiotic recombination plays a key role in the maintenance of sequence diversity in the human genome. However, little is known about the fine–scale distribution and processes of recombination in human chromosomes, or how these impact on patterns of human diversity. We have therefore developed sperm typing systems that allow human recombination to be analysed at very high resolution. The emerging picture is that human crossovers are far from randomly distributed but instead are targeted into very narrow hot spots that can profoundly influence patterns of haplotype diversity in the human genome. These hot spots provide fundamental information on processes of human crossover and gene conversion, as well as evidence that they can violate basic rules of Mendelian inheritance.

NIMA-related kinase 1 (NEK1) is a serine/threonine and tyrosine kinase that is highly expressed in mammalian germ cells. Mutations in Nek1 induce anemia, polycystic kidney and infertility. In this study we evaluated the role of NEK1 in meiotic spindle formation in both male and female gametes. Our results show that the lack of NEK1 provokes an abnormal organization of the meiosis I spindle characterized by elongated and/or multipolar spindles, and abnormal chromosome congression. The aberrant spindle structure is concomitant with the disruption in localization and protein levels of myosin X (MYO10) and α-adducin (ADD1), both of which are implicated in the regulation of spindle formation during mitosis. Interaction of ADD1 with MYO10 is dependent on phosphorylation, whereby phosphorylation of ADD1 enables its binding to MYO10 on mitotic spindles. Reduction in ADD1 protein in NEK1 mutant mice is associated with hyperphosphorylation of ADD1, thereby preventing the interaction with MYO10 during meiotic spindle formation. Our results reveal a novel regulatory role for NEK1 in the regulation of spindle architecture and function during meiosis.

Crossover recombination facilitates the accurate segregation of homologous chromosomes during meiosis. In mammals, poorly characterized regulatory processes ensure that every pair of chromosomes obtains at least one crossover, even though most recombination sites yield non-crossovers. Designation of crossovers involves selective localization of the SUMO ligase RNF212 to a minority of recombination sites, where it stabilizes pertinent factors such as MutSγ (ref. 4). Here we show that the ubiquitin ligase HEI10 (also called CCNB1IP1) is essential for this crossover/non-crossover differentiation process. In HEI10-deficient mice, RNF212 localizes to most recombination sites, and dissociation of both RNF212 and MutSγ from chromosomes is blocked. Consequently, recombination is impeded, and crossing over fails. In wild-type mice, HEI10 accumulates at designated crossover sites, suggesting that it also has a late role in implementing crossing over. As with RNF212, dosage sensitivity for HEI10 indicates that it is a limiting factor for crossing over. We suggest that SUMO and ubiquitin have antagonistic roles during meiotic recombination that are balanced to effect differential stabilization of recombination factors at crossover and non-crossover sites.

The identification of the H3K4 trimethylase, PRDM9, as the gene responsible for recombination hotspot localization has provided considerable insight into the mechanisms by which recombination is initiated in mammals. However, uniquely amongst mammals, canids appear to lack a functional version of PRDM9 and may therefore provide a model for understanding recombination that occurs in the absence of PRDM9, and thus how PRDM9 functions to shape the recombination landscape. We have constructed a fine-scale genetic map from patterns of linkage disequilibrium assessed using high-throughput sequence data from 51 free-ranging dogs, Canis lupus familiaris. While broad-scale properties of recombination appear similar to other mammalian species, our fine-scale estimates indicate that canine highly elevated recombination rates are observed in the vicinity of CpG rich regions including gene promoter regions, but show little association with H3K4 trimethylation marks identified in spermatocytes. By comparison to genomic data from the Andean fox, Lycalopex culpaeus, we show that biased gene conversion is a plausible mechanism by which the high CpG content of the dog genome could have occurred., Updated version, with significant revisions

The RAD9-RAD1-HUS1 (9-1-1) complex is a heterotrimeric PCNA-like clamp that responds to DNA damage in somatic cells by promoting DNA repair as well as ATR-dependent DNA damage checkpoint signaling. In yeast, worms, and flies, the 9-1-1 complex is also required for meiotic checkpoint function and efficient completion of meiotic recombination; however, since Rad9, Rad1, and Hus1 are essential genes in mammals, little is known about their functions in mammalian germ cells. In this study, we assessed the meiotic functions of 9-1-1 by analyzing mice with germ cell-specific deletion of Hus1 as well as by examining the localization of RAD9 and RAD1 on meiotic chromosomes during prophase I. Hus1 loss in testicular germ cells resulted in meiotic defects, germ cell depletion, and severely compromised fertility. Hus1-deficient primary spermatocytes exhibited persistent autosomal γH2AX and RAD51 staining indicative of unrepaired meiotic DSBs, synapsis defects, an extended XY body domain often encompassing partial or whole autosomes, and an increase in structural chromosome abnormalities such as end-to-end X chromosome-autosome fusions and ruptures in the synaptonemal complex. Most of these aberrations persisted in diplotene-stage spermatocytes. Consistent with a role for the 9-1-1 complex in meiotic DSB repair, RAD9 localized to punctate, RAD51-containing foci on meiotic chromosomes in a Hus1-dependent manner. Interestingly, RAD1 had a broader distribution that only partially overlapped with RAD9, and localization of both RAD1 and the ATR activator TOPBP1 to the XY body and to unsynapsed autosomes was intact in Hus1 conditional knockouts. We conclude that mammalian HUS1 acts as a component of the canonical 9-1-1 complex during meiotic prophase I to promote DSB repair and further propose that RAD1 and TOPBP1 respond to unsynapsed chromatin through an alternative mechanism that does not require RAD9 or HUS1., Author Summary Meiosis is a specialized cell division process in which germ cells undergo two cell divisions to produce haploid progeny. Two processes, genetic recombination and chromosome pairing/synapsis, are critical for successful meiosis and the production of gametes with high chromosomal integrity. The RAD9-RAD1-HUS1 (9-1-1) complex has been proposed to play critical roles in recombination as well as in the checkpoint-dependent monitoring of chromosomal synapsis by facilitating activation of the ATR checkpoint kinase. Our data indicate that HUS1 is required for normal germ cell development and fertility, for efficient completion of a subset of meiotic DNA recombination events, and for proper exclusion of the non-sex chromosomes from a specialized, repressive chromatin domain containing the X and Y chromosomes. However, HUS1 is not required for the meiotic functions of ATR in responding to chromosome synapsis defects. Furthermore, RAD1 localizes to sites along asynapsed chromosomes that lack detectable RAD9, and does so in the absence of Hus1, implicating RAD1 in a novel response to unsynapsed chromatin that is independent of the canonical 9-1-1 complex. Since mice lacking Hus1 in germ cells exhibit chromosomal abnormalities and severely reduced fertility, this work has broad implications for the maintenance of genome stability in the germline and for human reproductive health.

Crossing-over ensures accurate chromosome segregation during meiosis, and every pair of chromosomes obtains at least one crossover, even though the majority of recombination sites yield non-crossovers. A putative regulator of crossing-over is RNF212, which is associated with variation in crossover rates in humans. We show that mouse RNF212 is essential for crossing-over, functioning to couple chromosome synapsis to the formation of crossover-specific recombination complexes. Selective localization of RNF212 to a subset of recombination sites is shown to be a key early step in the crossover designation process. RNF212 acts at these sites to stabilize meiosis-specific recombination factors, including the MutSγ complex (MSH4-MSH5). We infer that selective stabilization of key recombination proteins is a fundamental feature of meiotic crossover control. Haploinsufficiency indicates that RNF212 is a limiting factor for crossover control and raises the possibility that human alleles may alter the amount or stability of RNF212 and be risk factors for aneuploid conditions.

The mammalian ortholog of yeast Slx4, BTBD12, is an ATM substrate that functions as a scaffold for various DNA repair activities. Mutations of human BTBD12 have been reported in a new sub-type of Fanconi anemia patients. Recent studies have implicated the fly and worm orthologs, MUS312 and HIM-18, in the regulation of meiotic crossovers arising from double-strand break (DSB) initiating events and also in genome stability prior to meiosis. Using a Btbd12 mutant mouse, we analyzed the role of BTBD12 in mammalian gametogenesis. BTBD12 localizes to pre-meiotic spermatogonia and to meiotic spermatocytes in wildtype males. Btbd12 mutant mice have less than 15% normal spermatozoa and are subfertile. Loss of BTBD12 during embryogenesis results in impaired primordial germ cell proliferation and increased apoptosis, which reduces the spermatogonial pool in the early postnatal testis. During prophase I, DSBs initiate normally in Btbd12 mutant animals. However, DSB repair is delayed or impeded, resulting in persistent γH2AX and RAD51, and the choice of repair pathway may be altered, resulting in elevated MLH1/MLH3 focus numbers at pachynema. The result is an increase in apoptosis through prophase I and beyond. Unlike yeast Slx4, therefore, BTBD12 appears to function in meiotic prophase I, possibly during the recombination events that lead to the production of crossovers. In line with its expected regulation by ATM kinase, BTBD12 protein is reduced in the testis of Atm−/− males, and Btbd12 mutant mice exhibit increased genomic instability in the form of elevated blood cell micronucleus formation similar to that seen in Atm−/− males. Taken together, these data indicate that BTBD12 functions throughout gametogenesis to maintain genome stability, possibly by co-ordinating repair processes and/or by linking DNA repair events to the cell cycle via ATM., Author Summary Mutations in genes essential for genome maintenance during meiosis can result in severe disruptions to spermatogenesis and subsequent low fertility and/or birth defects in mammals. The mammalian homolog of yeast Slx4, BTBD12, plays a critical role in somatic cell repair in mice. Here, we show that this critical function extends to mammalian germ cells, by examining the effects of a Btbd12 gene disruption in mice. Btbd12 mutant mice show severely reduced fertility, as a result of both pre-meiotic spermatogonial proliferation defects and impairment of proper meiotic progression. BTBD12 appears to be required for normal progression of double-strand break repair events that result in the formation of crossovers between maternal and paternal homologous chromosomes, with Btbd12 mutants displaying an increase in unrepaired breaks, impaired homologous chromosome interactions, and a slight increase in the number of crossover intermediates. BTBD12 protein is also down-regulated in the testes of Atm null mice, supporting previous studies showing that BTBD12 is a target of ATM kinase. These data provide new evidence about the role of BTBD12 in mammalian gametogenesis and are critical to furthering the understanding of the molecular processes involved in meiotic DNA repair.

Meiosis is a highly conserved process, which is stringently regulated in all organisms, from fungi through to humans. Two major events define meiosis in eukaryotes. The first is the pairing, or synapsis, of homologous chromosomes and the second is the exchange of genetic information in a process called meiotic recombination. Synapsis is mediated by the meiosis-specific synaptonemal complex structure in combination with the cohesins that tether sister chromatids together along chromosome arms through prophase I. Previously, we identified FKBP6 as a novel component of the mammalian synaptonemal complex. Further studies demonstrated an interaction between FKBP6 and the NIMA-related kinase-1, NEK1. To further investigate the role of NEK1 in mammalian meiosis, we have examined gametogenesis in the spontaneous mutant, Nek1kat2J. Homozygous mutant animals show decreased testis size, defects in testis morphology, and in cohesin removal at late prophase I of meiosis, causing complete male infertility. Cohesin protein SMC3 remains localized to the meiotic chromosome cores at diplonema in the Nek1 mutant, and also in the related Fkbp6 mutant, while in wild type cells SMC3 is removed from the cores at the end of prophase I and becomes more diffuse throughout the DAPI stained region of the nucleus. These data implicate NEK1 as a possible kinase involved in cohesin redistribution in murine spermatocytes.

Gene function prediction has proven valuable in formulating testable hypotheses. It is particularly useful for exploring biological processes that are experimentally intractable, such as meiotic initiation and progression in the human fetal ovary. In this study, we developed the first functional gene network for the human fetal ovary, HFOnet, by probabilistically integrating multiple genomic features using a naïve Bayesian model. We demonstrated that this network could accurately recapture known functional connections between genes, as well as predict new connections. Our findings suggest that known meiosis-specific genes (i.e., with functions only in meiotic processes in the germ cells) make either no or a few functional connections but are highly clustered with neighbor genes. In contrast, known nonspecific meiotic genes (i.e., with functions in both meiotic and nonmeiotic processes in the germ cells and somatic cells) exhibit numerous connections but low clustering coefficients, indicating their role as central modulators of diverse pathways, including those in meiosis. We also predicted novel genes that may be involved in meiotic initiation and DNA repair. This global functional network provides a much-needed framework for exploring gene functions and pathway components in early human female meiosis that are difficult to tackle by traditional in vivo mammalian genetics.

Meiotic recombination is of fundamental importance in creating haplotype diversity in the human genome and has the potential to cause genomic rearrangements by ectopic recombination between repeat sequences and through other changes triggered by recombination-initiating events. However, the relationship between allelic recombination and genome instability in the human germline remains unclear. We have therefore analysed recombination and DNA instability in the delta-, beta-globin gene region and its associated recombination hotspot. Sperm typing has for the first time accurately defined the hotspot and shown it to be the most active autosomal crossover hotspot yet described, although unusually inactive in non-exchange gene conversion. The hotspot just extends into a homology block shared by the delta- and beta-globin genes, within which ectopic exchanges can generate Hb Lepore deletions. We developed a physical selection method for recovering and validating extremely rare de novo deletions in human DNA and used it to characterize the dynamics of these Hb Lepore deletions in sperm as well as other deletions not arising from ectopic exchanges between homologous DNA sequences. Surprisingly, both classes of deletion showed breakpoints that avoided the beta-globin hotspot, establishing that it possesses remarkable fidelity and does not play a significant role in triggering these DNA rearrangements. This study also provides the first direct analysis of de novo deletion in the human germline and points to a possible deletion-controlling element in the beta-globin gene separate from the crossover hotspot.

The Huntington's disease gene (HTT) CAG repeat mutation undergoes somatic expansion that correlates with pathogenesis. Modifiers of somatic expansion may therefore provide routes for therapies targeting the underlying mutation, an approach that is likely applicable to other trinucleotide repeat diseases. Huntington's disease HdhQ111 mice exhibit higher levels of somatic HTT CAG expansion on a C57BL/6 genetic background (B6.HdhQ111) than on a 129 background (129.HdhQ111). Linkage mapping in (B6x129).HdhQ111 F2 intercross animals identified a single quantitative trait locus underlying the strain-specific difference in expansion in the striatum, implicating mismatch repair (MMR) gene Mlh1 as the most likely candidate modifier. Crossing B6.HdhQ111 mice onto an Mlh1 null background demonstrated that Mlh1 is essential for somatic CAG expansions and that it is an enhancer of nuclear huntingtin accumulation in striatal neurons. HdhQ111 somatic expansion was also abolished in mice deficient in the Mlh3 gene, implicating MutLγ (MLH1–MLH3) complex as a key driver of somatic expansion. Strikingly, Mlh1 and Mlh3 genes encoding MMR effector proteins were as critical to somatic expansion as Msh2 and Msh3 genes encoding DNA mismatch recognition complex MutSβ (MSH2–MSH3). The Mlh1 locus is highly polymorphic between B6 and 129 strains. While we were unable to detect any difference in base-base mismatch or short slipped-repeat repair activity between B6 and 129 MLH1 variants, repair efficiency was MLH1 dose-dependent. MLH1 mRNA and protein levels were significantly decreased in 129 mice compared to B6 mice, consistent with a dose-sensitive MLH1-dependent DNA repair mechanism underlying the somatic expansion difference between these strains. Together, these data identify Mlh1 and Mlh3 as novel critical genetic modifiers of HTT CAG instability, point to Mlh1 genetic variation as the likely source of the instability difference in B6 and 129 strains and suggest that MLH1 protein levels play an important role in driving of the efficiency of somatic expansions., Author Summary The expansion of a CAG repeat underlies Huntington's disease (HD), with longer CAG tracts giving rise to earlier onset and more severe disease. In individuals harboring a CAG expansion the repeat undergoes further somatic expansion over time, particularly in brain cells most susceptible to disease pathogenesis. Preventing this repeat lengthening may delay disease onset and/or slow progression. We are using mouse models of HD to identify the factors that modify the somatic expansion of the HD CAG repeat, as these may provide novel targets for therapeutic intervention. To identify genetic modifiers of somatic expansion in HD mouse models we have used both an unbiased genetic mapping approach in inbred mouse strains that exhibit different levels of somatic expansion, as well as targeted gene knockout approaches. Our results demonstrate that: 1) Mlh1 and Mlh3 genes, encoding components of the DNA mismatch repair pathway, are critical for somatic CAG expansion; 2) in the absence of somatic expansion the pathogenic process in the mouse is slowed; 3) MLH1 protein levels are likely to be a driver of the efficiency of somatic expansion. Together, our data provide new insight into the factors underlying the process of somatic expansion of the HD CAG repeat.

Two eukaryotic pathways for processing double-strand breaks (DSBs) as crossovers have been described, one dependent on the MutL homologs Mlh1 and Mlh3, and the other on the structure-specific endonuclease Mus81. Mammalian MUS81 has been implicated in maintenance of genomic stability in somatic cells; however, little is known about its role during meiosis. Mus81-deficient mice were originally reported as being viable and fertile, with normal meiotic progression; however, a more detailed examination of meiotic progression in Mus81-null animals and WT controls reveals significant meiotic defects in the mutants. These include smaller testis size, a depletion of mature epididymal sperm, significantly upregulated accumulation of MLH1 on chromosomes from pachytene meiocytes in an interference-independent fashion, and a subset of meiotic DSBs that fail to be repaired. Interestingly, chiasmata numbers in spermatocytes from Mus81−/− animals are normal, suggesting additional integrated mechanisms controlling the two distinct crossover pathways. This study is the first in-depth analysis of meiotic progression in Mus81-nullizygous mice, and our results implicate the MUS81 pathway as a regulator of crossover frequency and placement in mammals., Author Summary Failure to undergo faithful meiotic chromosome segregation during mammalian meiosis can result in aneuploidy in the offspring and is a major cause of pregnancy loss and birth defects in humans. One essential component of meiotic prophase I is the exchange of genetic information between maternal and paternal chromosomes, known as recombination or crossing over, and is mediated, at least in part, by the mismatch repair proteins MSH4–MSH5 and MLH1–MLH3. A distinct subset of crossovers in lower organisms is generated by an alternate pathway involving Mus81 endonuclease. Previous studies into the impact of Mus81 mutations in mice revealed no adverse effect on the fertility of these animals. In this study, we report subtle, yet significant, defects in meiotic progression in male and female Mus81 mice, coupled with intriguing results showing that MUS81 protein is essential for crossover control in mammals. MUS81 appears to be required for correct localization of MLH1–MLH3 complexes to paired homologous chromosomes, however, not for the maintenance of physical crossovers, visualized as chiasmata. These results show a complex interplay between the MUS81 and MLH1–MLH3 pathways for generation of crossovers and, as such, are critical to the further understanding of the intricacies of crossover control with a view to reducing meiotic error rate in humans.