Meiosis requires that each chromosome finds its homologous partner and undergoes at least one crossover. X-Y chromosome segregation hinges on efficient crossing-over in a very small region of homology, the pseudoautosomal region (PAR). We find that mouse PAR DNA occupies unusually long chromosome axes, potentially as shorter chromatin loops, predicted to promote double-strand break (DSB) formation. Most PARs show delayed appearance of RAD51/DMC1 foci, which mark DSB ends, and all PARs undergo delayed DSB-mediated homologous pairing. Analysis of Spo11β isoform-specific transgenic mice revealed that late RAD51/DMC1 foci on the PAR are genetically distinct from early PAR foci and global foci, and that late PAR foci promote efficient X-Y pairing, recombination and male fertility. Our findings uncover specific mechanisms that surmount the unique challenges of X-Y recombination.

Fundamentally different recombination defects cause apoptosis of mouse spermatocytes at the same stage in development, stage IV of the seminiferous epithelium cycle, equivalent to mid-pachynema in normal males. To understand the cellular response(s) that triggers apoptosis, we examined markers of spermatocyte development in mice with different recombination defects. In Spo11 ؊/؊ mutants, which lack the double-strand breaks (DSBs) that initiate recombination, spermatocytes express markers of early to mid-pachynema, forming chromatin domains that contain sex body-associated proteins but that rarely encompass the sex chromosomes. Dmc1؊/؊ spermatocytes, impaired in DSB repair, appear to arrest at or about late zygonema. Epistasis analysis reveals that this earlier arrest is a response to unrepaired DSBs, and cytological analysis implicates the BRCT-containing checkpoint protein TOPBP1. Atm ؊/؊ spermatocytes show similarities to Dmc1 ؊/؊ spermatocytes, suggesting that ATM promotes meiotic DSB repair. Msh5 ؊/؊ mutants display a set of characteristics distinct from these other mutants. Thus, despite equivalent stages of spermatocyte elimination, different recombination-defective mutants manifest distinct responses, providing insight into surveillance mechanisms in male meiosis.Recombination promotes accurate segregation of homologous chromosomes during the first meiotic division and is initiated by the formation of DNA double-strand breaks (DSBs) by the evolutionarily conserved SPO11 protein. Many factors, including the DNA strand exchange protein DMC1 and the meiosis-specific MutS homolog MSH5, act on these DSBs to catalyze recombination with a homologous, nonsister chromatid (see references 23 and 24 for reviews). Many mutations that affect the formation or repair of DSBs result in arrest and/or programmed cell death during prophase of meiosis I in mice, as in many other organisms (11,21,38). This response reveals that cellular systems monitor the recombination process, presumably to prevent the formation of gametes with damaged or aneuploid genomes. However, it is not always clear what molecular defect is responsible for triggering cell death. By examining oocyte development in several mouse mutants, we recently demonstrated that distinct DNA damage-dependent and independent responses drive the loss of recombinationdefective meiocytes in the female germ line (13). Specifically, the absence of DSB formation (in a Spo11 Ϫ/Ϫ mutant) caused only a partial defect in follicle formation at the stage of dictyate arrest, whereas defects in DSB repair (in Dmc1 Ϫ/Ϫ and Msh5 Ϫ/Ϫ mutants) caused more-severe oocyte loss earlier in meiotic prophase, which could be suppressed by eliminating DSB formation.Recombination defects cause meiocyte loss in the male germ line as well, but the situation is different than that for females. Several mutations that cause fundamentally different defects in meiotic recombination result in spermatocyte apoptosis at what appears to be roughly the same stages in meiosis, described variously as late z...

SCF ubiquitin ligases, composed of three major subunits, Skp1, Cul1, and one of many F box proteins (Fbps), control the proteolysis of important cellular regulators. We have inactivated the gene encoding the Fbp beta-Trcp1 in mice. beta-Trcp1(-/-) males show reduced fertility correlating with an accumulation of methaphase I spermatocytes. beta-Trcp1(-/-) MEFs display a lengthened mitosis, centrosome overduplication, multipolar metaphase spindles, and misaligned chromosomes. Furthermore, cyclin A, cyclin B, and Emi1, an inhibitor of the anaphase promoting complex, are stabilized in mitotic beta-Trcp1(-/-) MEFs. Indeed, we demonstrate that Emi1 is a bona fide substrate of beta-Trcp1. In contrast, stabilization of beta-catenin and IkappaBalpha, two previously reported beta-Trcp1 substrates, does not occur in the absence of beta-Trcp1 and instead requires the additional silencing of beta-Trcp2 by siRNA. Thus, beta-Trcp1 regulates the timely order of meiotic and mitotic events.

Defects in meiotic recombination in many organisms result in arrest because of activation of a meiotic checkpoint(s). The proximal defect that triggers this checkpoint in mammalian germ cells is not understood, but it has been suggested to involve either the presence of DNA damage in the form of unrepaired recombination intermediates or defects in homologous chromosome pairing and synapsis independent of DNA damage per se. To distinguish between these possibilities in the female germ line, we compared mouse oocyte development in a mutant that fails to form the double-strand breaks (DSBs) that initiate meiotic recombination (Spo11 ؊/؊ ) to mutants with defects in processing DSBs when they are formed (Dmc1 ؊/؊ and Msh5 ؊/؊ ), and we examined the epistasis relationships between these mutations. Absence of DSB formation caused a partial defect in follicle formation, whereas defects in DSB repair caused earlier and more severe meiotic arrest, which could be suppressed by eliminating DSB formation. Therefore, our analysis reveals that there are both DNA-damage-dependent and -independent responses to recombination errors in mammalian oocytes. By using these findings as a paradigm, we also examined oocyte loss in mutants lacking the DNA-damage checkpoint kinase ATM. The absence of ATM caused defects in folliculogenesis that were similar to those in Dmc1 mutants and that could be suppressed by Spo11 mutation, implying that oocyte death in Atmdeficient animals is a response to defective DSB repair.