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.

The process of erythropoiesis must be efficient and robust to supply the organism with red bloods cells both under condition of homeostasis and stress. The microRNA (miRNA) pathway was recently shown to regulate erythroid development. Here, we show that expression of the locus encoding miR-144 and miR-451 is strictly dependent on Argonaute 2 and is required for erythroid homeostasis. Mice deficient for the miR-144/451 cluster display a cell autonomous impairment of late erythroblast maturation, resulting in erythroid hyperplasia, splenomegaly, and a mild anemia. Analysis of gene expression profiles from wild-type and miR-144/451–deficient erythroblasts revealed that the miR-144/451 cluster acts as a “tuner” of gene expression, influencing the expression of many genes. MiR-451 imparts a greater impact on target gene expression than miR-144. Accordingly, mice deficient in miR-451 alone exhibited a phenotype indistinguishable from miR-144/451–deficient mice. Thus, the miR-144/451 cluster tunes gene expression to impart a robustness to erythropoiesis that is critical under conditions of stress.

Transposons present an acute challenge to the germline, and mechanisms that repress their activity are essential for transgenerational genomic integrity. LINE1 (L1) is the most successful retrotransposon and is epigenetically repressed by CpG DNA methylation. Here, we identify two additional important mechanisms by which L1 is repressed during spermatogenesis. We demonstrate that the Piwi protein Mili and the piRNA pathway are required to posttranscriptionally silence L1 in meiotic pachytene cells even in the presence of normal L1 DNA methylation. Strikingly, in the absence of both a functional piRNA pathway and DNA methylation, L1 elements are normally repressed in mitotic stages of spermatogenesis. Accordingly, we find that the euchromatic repressive histone H3 dimethylated lysine 9 modification cosuppresses L1 expression therein. We demonstrate the existence of multiple epigenetic mechanisms that in conjunction with the piRNA pathway sequentially enforce L1 silencing and genomic stability during mitotic and meiotic stages of adult spermatogenesis.

A fundamental principle in biology is that the program for early development is established during oogenesis in the form of the maternal transcriptome1,2. How the maternal transcriptome acquires the appropriate content and dosage of transcripts is not fully understood. Here we show that TUT4/7-mediated mRNA 3′ terminal uridylation sculpts the mouse maternal transcriptome by eliminating transcripts during oocyte growth. TUT4/7-mediated uridylation is essential for both oocyte maturation and fertility. In comparison to somatic cells, the oocyte transcriptome displays shorter poly(A) tail length and a high relative proportion of terminal oligo-uridylation. TUT4/7 deletion leads to the accumulation of a cohort of transcripts with a high frequency of very short poly(A) tails and a loss of 3′ oligo-uridylation. In contrast, TUT4/7-deficiency does not alter gene expression in a variety of somatic cells. In summary, we show essential and specific functions for poly(A) tail length and 3′ terminal uridylation in sculpting a functional maternal transcriptome.

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.