Piwi proteins and Piwi-interacting RNAs (piRNAs) have conserved functions in transposon silencing. The murine Piwi proteins Mili and Miwi2 (also called Piwil2 and Piwil4, respectively) direct epigenetic LINE1 and intracisternal A particle transposon silencing during genome reprogramming in the embryonic male germ line. Piwi proteins are proposed to be piRNA-guided endonucleases that initiate secondary piRNA biogenesis; however, the actual contribution of their endonuclease activities to piRNA biogenesis and transposon silencing remain unknown. To investigate the role of Piwi-catalysed endonucleolytic activity, we engineered point mutations in mice that substitute the second aspartic acid to an alanine in the DDH catalytic triad of Mili and Miwi2, generating the Mili(DAH) and Miwi2(DAH) alleles, respectively. Analysis of Mili-bound piRNAs from homozygous Mili(DAH) fetal gonadocytes revealed a failure of transposon piRNA amplification, resulting in the marked reduction of piRNA bound within Miwi2 ribonuclear particles. We find that Mili-mediated piRNA amplification is selectively required for LINE1, but not intracisternal A particle, silencing. The defective piRNA pathway in Mili(DAH) mice results in spermatogenic failure and sterility. Surprisingly, homozygous Miwi2(DAH) mice are fertile, transposon silencing is established normally and no defects in secondary piRNA biogenesis are observed. In addition, the hallmarks of piRNA amplification are observed in Miwi2-deficient gonadocytes. We conclude that cycles of intra-Mili secondary piRNA biogenesis fuel piRNA amplification that is absolutely required for LINE1 silencing.

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...

SummaryYTHDF2 binds and destabilizes N6-methyladenosine (m6A)-modified mRNA. The extent to which this branch of m6A RNA-regulatory pathway functions in vivo and contributes to mammalian development remains unknown. Here we find that YTHDF2 deficiency is partially permissive in mice and results in female-specific infertility. Using conditional mutagenesis, we demonstrate that YTHDF2 is autonomously required within the germline to produce MII oocytes that are competent to sustain early zygotic development. Oocyte maturation is associated with a wave of maternal RNA degradation, and the resulting relative changes to the MII transcriptome are integral to oocyte quality. The loss of YTHDF2 results in the failure to regulate transcript dosage of a cohort of genes during oocyte maturation, with enrichment observed for the YTHDF2-binding consensus and evidence of m6A in these upregulated genes. In summary, the m6A-reader YTHDF2 is an intrinsic determinant of mammalian oocyte competence and early zygotic development.

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.