A mutation in the endonuclease domain of mouse MLH3 reveals novel roles for MutLγ during crossover formation in meiotic prophase I.

During meiotic prophase I, double-strand breaks (DSBs) initiate homologous recombination leading to non-crossovers (NCOs) and crossovers (COs). In mouse, 10% of DSBs are designated to become COs, primarily through a pathway dependent on the MLH1-MLH3 heterodimer (MutLγ). Mlh3 contains an endonuclease domain that is critical for resolving COs in yeast. We generated a mouse (Mlh3DN/DN) harboring a mutation within this conserved domain that is predicted to generate a protein that is catalytically inert. Mlh3DN/DN males, like fully null Mlh3-/- males, have no spermatozoa and are infertile, yet spermatocytes have grossly normal DSBs and synapsis events in early prophase I. Unlike Mlh3-/- males, mutation of the endonuclease domain within MLH3 permits normal loading and frequency of MutLγ in pachynema. However, key DSB repair factors (RAD51) and mediators of CO pathway choice (BLM helicase) persist into pachynema in Mlh3DN/DN males, indicating a temporal delay in repair events and revealing a mechanism by which alternative DSB repair pathways may be selected. While Mlh3DN/DN spermatocytes retain only 22% of wildtype chiasmata counts, this frequency is greater than observed in Mlh3-/- males (10%), suggesting that the allele may permit partial endonuclease activity, or that other pathways can generate COs from these MutLγ-defined repair intermediates in Mlh3DN/DN males. Double mutant mice homozygous for the Mlh3DN/DN and Mus81-/- mutations show losses in chiasmata close to those observed in Mlh3-/- males, indicating that the MUS81-EME1-regulated crossover pathway can only partially account for the increased residual chiasmata in Mlh3DN/DN spermatocytes. Our data demonstrate that mouse spermatocytes bearing the MLH1-MLH3DN/DN complex display the proper loading of factors essential for CO resolution (MutSγ, CDK2, HEI10, MutLγ). Despite these functions, mice bearing the Mlh3DN/DN allele show defects in the repair of meiotic recombination intermediates and a loss of most chiasmata.

piRNA-IPdb: a PIWI-bound piRNAs database to mining NGS sncRNA data and beyond.

BACKGROUND: PIWI-interacting RNAs (piRNAs) are an abundant single-stranded type of small non-coding RNAs (sncRNAs), which initially were discovered in gonadal cells, with a role as defenders of genomic integrity in the germline, acting against the transposable elements. With a regular size range of 21-35 nt, piRNAs are associated with a PIWI-clade of Argonaute family proteins. The most widely accepted mechanisms of biogenesis for piRNAs involve the transcription of longer precursors of RNAs to be processed, by complexes of proteins, to functional size, preferentially accommodating uridine residues at the 5' end and 3' methylation to increase the stability of these molecules. piRNAs have also been detected in somatic cells, with diverse potential functions, indicating their high plasticity and pleiotropic activity. Discovered about two decades ago, piRNAs are a large and versatile type of sncRNA and that remain insufficiently identified and analyzed, through next-generation sequencing (NGS), to evaluate their processing, functions, and biogenesis in different cell types and during development. piRNAs' distinction from other sncRNAs has led to controversial results and interpretation difficulties when using existing databases because of the heterogeneity of the criteria used in making the distinction. DESCRIPTION: We present "piRNA-IPdb", a database based uniquely on datasets obtaining after the defining characteristic of piRNAs: those small RNAs bound to PIWI proteins. We selected and analyzed sequences from piRBase that exclusively cover the binding to PIWI. We pooled a total of 18,821,815 sequences from RNA-seq after immunoprecipitation that included the binding to any of the mouse PIWI proteins (MILI, MIWI, or MIWI2). CONCLUSIONS: In summary, we present the characteristics and potential use of piRNA-IPdb database for the analysis of bona fide piRNAs.

Variants in GCNA, X-linked germ-cell genome integrity gene, identified in men with primary spermatogenic failure.

Male infertility impacts millions of couples yet, the etiology of primary infertility remains largely unknown. A critical element of successful spermatogenesis is maintenance of genome integrity. Here, we present a genomic study of spermatogenic failure (SPGF). Our initial analysis (n = 176) did not reveal known gene-candidates but identified a potentially significant single-nucleotide variant (SNV) in X-linked germ-cell nuclear antigen (GCNA). Together with a larger follow-up study (n = 2049), 7 likely clinically relevant GCNA variants were identified. GCNA is critical for genome integrity in male meiosis and knockout models exhibit impaired spermatogenesis and infertility. Single-cell RNA-seq and immunohistochemistry confirm human GCNA expression from spermatogonia to elongated spermatids. Five identified SNVs were located in key functional regions, including N-terminal SUMO-interacting motif and C-terminal Spartan-like protease domain. Notably, variant p.Ala115ProfsTer7 results in an early frameshift, while Spartan-like domain missense variants p.Ser659Trp and p.Arg664Cys change conserved residues, likely affecting 3D structure. For variants within GCNA's intrinsically disordered region, we performed computational modeling for consensus motifs. Two SNVs were predicted to impact the structure of these consensus motifs. All identified variants have an extremely low minor allele frequency in the general population and 6 of 7 were not detected in > 5000 biological fathers. Considering evidence from animal models, germ-cell-specific expression, 3D modeling, and computational predictions for SNVs, we propose that identified GCNA variants disrupt structure and function of the respective protein domains, ultimately arresting germ-cell division. To our knowledge, this is the first study implicating GCNA, a key genome integrity factor, in human male infertility.

MicroRNA dynamics at the onset of primordial germ and somatic cell sex differentiation during mouse embryonic gonad development.

In mammals, commitment and specification of germ cell lines involves complex programs that include sex differentiation, control of proliferation, and meiotic initiation. Regulation of these processes is genetically controlled by fine-tuned mechanisms of gene regulation in which microRNAs (miRNAs) are involved. We have characterized, by small-RNA-seq and bioinformatics analyses, the miRNA expression patterns of male and female mouse primordial germ cells (PGCs) and gonadal somatic cells at embryonic stages E11.5, E12.5, and E13.5. Differential expression analyses revealed differences in the regulation of key miRNA clusters such as miR-199-214, miR-182-183-96, and miR-34c-5p, whose targets have defined roles during gonadal sexual determination in both germ and somatic cells. Extensive analyses of miRNA sequences revealed an increase in noncanonical isoforms on PGCs at E12.5 and dramatic changes of 3' isomiR expression and 3' nontemplate nucleotide additions in female PGCs at E13.5. Additionally, RT-qPCR analyses of genes encoding proteins involved in miRNA biogenesis and 3' nucleotide addition uncovered sexually and developmentally specific expression, characterized by the decay of Drosha, Dgcr8, and Xpo5 expression along gonadal development. These results demonstrate that miRNAs, their isomiRs, and miRNA machinery are differentially regulated and participate actively in gonadal sexual differentiation in both PGCs and gonadal somatic cells.

Characterization of telomeric repeat-containing RNA (TERRA) localization and protein interactions in primordial germ cells of the mouse†.

Telomeres are dynamic nucleoprotein structures capping the physical ends of linear eukaryotic chromosomes. They consist of telomeric DNA repeats (TTAGGG), the shelterin protein complex, and telomeric repeat-containing RNA (TERRA). Proposed TERRA functions are wide ranging and include telomere maintenance, telomerase inhibition, genomic stability, and alternative lengthening of telomere. However, the presence and role of TERRA in primordial germ cells (PGCs), the embryonic precursors of germ cells, is unknown. Using RNA-fluorescence in situ hybridization, we identify TERRA transcripts in female PGCs at 11.5, 12.5, and 13.5 days postcoitum. In male PGCs, the earliest detection TERRA was at 12.5 dpc where we observed cells with either zero or one TERRA focus. Using qRT-PCR, we evaluated chromosome-specific TERRA expression. Female PGCs showed TERRA expression at 11.5 dpc from eight different chromosome subtelomeric regions (chromosomes 1, 2, 7, 9, 11, 13, 17, and 18) while in male PGCs, TERRA expression was confined to the chromosome 17. Most TERRA transcription in 13.5 dpc male PGCs arose from chromosomes 2 and 6. TERRA interacting proteins were evaluated using identification of direct RNA interacting proteins (iDRiP), which identified 48 in female and 26 in male protein interactors from PGCs at 13.5 dpc. We validated two different proteins: the splicing factor, proline- and glutamine-rich (SFPQ) in PGCs and non-POU domain-containing octamer-binding protein (NONO) in somatic cells. Taken together, our data indicate that TERRA expression and interactome during PGC development are regulated in a dynamic fashion that is dependent on gestational age and sex.

FancJ (Brip1) loss-of-function allele results in spermatogonial cell depletion during embryogenesis and altered processing of crossover sites during meiotic prophase I in mice.

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.

Diversity and functional convergence of small noncoding RNAs in male germ cell differentiation and fertilization.

The small noncoding RNAs (sncRNAs) are considered as post-transcriptional key regulators of male germ cell development. In addition to microRNAs (miRNAs) and PIWI-interacting RNAs (piRNAs), other sncRNAs generated from small nucleolar RNAs (snoRNAs), tRNAs, or rRNAs processing may also play important regulatory roles in spermatogenesis. By next-generation sequencing (NGS), we characterized the sncRNA populations detected at three milestone stages in male germ differentiation: primordial germ cells (PGCs), pubertal spermatogonia cells, and mature spermatozoa. To assess their potential transmission through the spermatozoa during fertilization, the sncRNAs of mouse oocytes and zygotes were also analyzed. Both, microRNAs and snoRNA-derived small RNAs are abundantly expressed in PGCs but transiently replaced by piRNAs in spermatozoa and endo-siRNAs in oocytes and zygotes. Exhaustive analysis of miRNA sequence variants also shows an increment of noncanonical microRNA forms along male germ cell differentiation. RNAs-derived from tRNAs and rRNAs interacting with PIWI proteins are not generated by the ping-pong pathway and could be a source of primary piRNAs. Moreover, our results strongly suggest that the small RNAs-derived from tRNAs and rRNAs are interacting with PIWI proteins, and specifically with MILI. Finally, computational analysis revealed their potential involvement in post-transcriptional regulation of mRNA transcripts suggesting functional convergence among different small RNA classes in germ cells and zygotes.

Dasatinib and quercetin increase testosterone and sperm concentration in mice.

Cellular senescence is a defense mechanism to arrest proliferation of damaged cells. The number of senescent cells increases with age in different tissues and contributes to the development of age-related diseases. Old mice treated with senolytics drugs, dasatinib and quercetin (D+Q), have reduced senescent cells burden. The aim of this study was to evaluate the effects of D+Q on testicular function and fertility of male mice. Mice (n = 9/group) received D (5 mg kg-1) and Q (50 mg kg-1) via gavage every moth for three consecutive days from 3 to 8 months of age. At 8 months mice were breed with young non-treated females and euthanized. The treatment of male mice with D+Q increased serum testosterone levels and sperm concentration and decreased abnormal sperm morphology. Sperm motility, seminiferous tubule morphometry, testicular gene expression and fertility were not affected by treatment. There was no effect of D+Q treatment in ß-galactosidase activity and in lipofuscin staining in testes. D+Q treatment also did not affect body mass gain and testes mass. In conclusion, D+Q treatment increased serum testosterone levels and sperm concentration and decreased abnormal sperm morphology, however did not affect fertility. Further studies with older mice and different senolytics are necessary to elucidate the effects in the decline of sperm output (quality and quantity) associated with aging.

The Interrelationship Between Female Reproductive Aging and Survival.

The link between survival and reproductive function is demonstrated across many species and is under both long-term evolutionary pressures and short-term environmental pressures. Loss of reproductive function is common in mammals and is strongly correlated with increased rates of disease in both males and females. However, the reproduction-associated change in disease rates is more abrupt and more severe in women, who benefit from a significant health advantage over men until the age of menopause. Young women with early ovarian failure also suffer from increased disease risks, further supporting the role of ovarian function in female health. Contemporary experiments where the influence of young ovarian tissue has been restored in postreproductive-aged females with surgical manipulation were found to increase survival significantly. In these experiments, young, intact ovaries were used to replace the aged ovaries of females that had already reached reproductive cessation. As has been seen previously in primitive species, when the young mammalian ovaries were depleted of germ cells prior to transplantation to the postreproductive female, survival was increased even further than with germ cell-containing young ovaries. Thus, extending reproductive potential significantly increases survival and appears to be germ cell and ovarian hormone-independent. The current review will discuss historical and contemporary observations and theories that support the link between reproduction and survival and provide hope for future clinical applications to decrease menopause-associated increases in disease risks.

Senolytic treatment reverses obesity-mediated senescent cell accumulation in the ovary.

Senescent cells are in a cell cycle arrest state and accumulate with aging and obesity, contributing to a chronic inflammatory state. Treatment with senolytic drugs dasatinib and quercetin (D + Q) can reduce senescent cell burden in several tissues, increasing lifespan. Despite this, there are few reports about senescent cells accumulating in female reproductive tissues. Therefore, the aim of the study was to characterize the ovarian reserve and its relationship with cellular senescence in genetically obese mice (ob/ob). In experiment 1, ob/ob (n = 5) and wild-type (WT) mice (n = 5) at 12 months of age were evaluated. In experiment 2, 2-month-old female ob/ob mice were treated with senolytics (D + Q, n = 6) or placebo (n = 6) during the 4 months. Obese mice had more senescent cells in ovaries, indicated by increased p21 and p16 and lipofuscin staining and macrophage infiltration. Treatment with D + Q significantly reduced senescent cell burden in ovaries of obese mice. Neither obesity nor treatment with D + Q affected the number of ovarian follicles. In conclusion, our data indicate that obesity due to leptin deficiency increases the load of senescent cells in the ovary, which is reduced by treatment by senolytics. However, neither obesity nor D + Q treatment affected the ovarian reserve.

Multiple 9-1-1 complexes promote homolog synapsis, DSB repair, and ATR signaling during mammalian meiosis.

DNA damage response mechanisms have meiotic roles that ensure successful gamete formation. While completion of meiotic double-strand break (DSB) repair requires the canonical RAD9A-RAD1-HUS1 (9A-1-1) complex, mammalian meiocytes also express RAD9A and HUS1 paralogs, RAD9B and HUS1B, predicted to form alternative 9-1-1 complexes. The RAD1 subunit is shared by all predicted 9-1-1 complexes and localizes to meiotic chromosomes even in the absence of HUS1 and RAD9A. Here, we report that testis-specific disruption of RAD1 in mice resulted in impaired DSB repair, germ cell depletion, and infertility. Unlike Hus1 or Rad9a disruption, Rad1 loss in meiocytes also caused severe defects in homolog synapsis, impaired phosphorylation of ATR targets such as H2AX, CHK1, and HORMAD2, and compromised meiotic sex chromosome inactivation. Together, these results establish critical roles for both canonical and alternative 9-1-1 complexes in meiotic ATR activation and successful prophase I completion.

The naked truth: a comprehensive clarification and classification of current 'myths' in naked mole-rat biology.

The naked mole-rat (Heterocephalus glaber) has fascinated zoologists for at least half a century. It has also generated considerable biomedical interest not only because of its extraordinary longevity, but also because of unusual protective features (e.g. its tolerance of variable oxygen availability), which may be pertinent to several human disease states, including ischemia/reperfusion injury and neurodegeneration. A recent article entitled 'Surprisingly long survival of premature conclusions about naked mole-rat biology' described 28 'myths' which, those authors claimed, are a 'perpetuation of beautiful, but falsified, hypotheses' and impede our understanding of this enigmatic mammal. Here, we re-examine each of these 'myths' based on evidence published in the scientific literature. Following Braude et al., we argue that these 'myths' fall into four main categories: (i) 'myths' that would be better described as oversimplifications, some of which persist solely in the popular press; (ii) 'myths' that are based on incomplete understanding, where more evidence is clearly needed; (iii) 'myths' where the accumulation of evidence over the years has led to a revision in interpretation, but where there is no significant disagreement among scientists currently working in the field; (iv) 'myths' where there is a genuine difference in opinion among active researchers, based on alternative interpretations of the available evidence. The term 'myth' is particularly inappropriate when applied to competing, evidence-based hypotheses, which form part of the normal evolution of scientific knowledge. Here, we provide a comprehensive critical review of naked mole-rat biology and attempt to clarify some of these misconceptions.

ATR signaling in mammalian meiosis: From upstream scaffolds to downstream signaling.

In germ cells undergoing meiosis, the induction of double strand breaks (DSBs) is required for the generation of haploid gametes. Defects in the formation, detection, or recombinational repair of DSBs often result in defective chromosome segregation and aneuploidies. Central to the ability of meiotic cells to properly respond to DSBs are DNA damage response (DDR) pathways mediated by DNA damage sensor kinases. DDR signaling coordinates an extensive network of DDR effectors to induce cell cycle arrest and DNA repair, or trigger apoptosis if the damage is extensive. Despite their importance, the functions of DDR kinases and effector proteins during meiosis remain poorly understood and can often be distinct from their known mitotic roles. A key DDR kinase during meiosis is ataxia telangiectasia and Rad3-related (ATR). ATR mediates key signaling events that control DSB repair, cell cycle progression, and meiotic silencing. These meiotic functions of ATR depend on upstream scaffolds and regulators, including the 9-1-1 complex and TOPBP1, and converge on many downstream effectors such as the checkpoint kinase CHK1. Here, we review the meiotic functions of the 9-1-1/TOPBP1/ATR/CHK1 signaling pathway during mammalian meiosis.

The antiandrogenic vinclozolin induces differentiation delay of germ cells and changes in energy metabolism in 3D cultures of fetal ovaries.

Vinclozolin is a pesticide with antiandrogenic activity as an endocrine disruptor compound. Its effects upon the progression of primordial follicles were assessed in cultures of mouse fetal ovaries from the onset of meiotic differentiation of germ cells (13.5 days post coitum) and from both in vivo exposed mice and in vitro exposed ovaries. Exposure of ovaries to vinclozolin-at in vitro dosages ranging from 10 to 200 µM and in 3D ex vivo culture following in vivo exposure to 50 mg/kg bw/day-showed delays in meiocyte differentiation and in follicle growth, even at the lowest in vitro dose exposure. Immunofluorescent analysis showed the presence of the proteins MSY2 and NOBOX in the primary follicles but no difference in the level of protein signals or in the number of follicles in relation to treatment. However, assessing the cytological differentiation of germ cells by detecting the synaptonemal complex protein SYCP3, the exposure to vinclozolin delayed meiotic differentiation from both in vitro- and in vivo-exposed ovaries. These effects were concomitant with changes in the energy metabolism, detected as a relative increase of glycolytic metabolism in live-cell metabolic assays in exposed ovaries.

SIRT1 accelerates the progression of activity-based anorexia.

Food consumption is fundamental for life, and eating disorders often result in devastating or life-threatening conditions. Anorexia nervosa (AN) is characterized by a persistent restriction of energy intake, leading to lowered body weight, constant fear of gaining weight, and psychological disturbances of body perception. Herein, we demonstrate that SIRT1 inhibition, both genetically and pharmacologically, delays the onset and progression of AN behaviors in activity-based anorexia (ABA) models, while SIRT1 activation accelerates ABA phenotypes. Mechanistically, we suggest that SIRT1 promotes progression of ABA, in part through its interaction with NRF1, leading to suppression of a NMDA receptor subunit Grin2A. Our results suggest that AN may arise from pathological positive feedback loops: voluntary food restriction activates SIRT1, promoting anxiety, hyperactivity, and addiction to starvation, exacerbating the dieting and exercising, thus further activating SIRT1. We propose SIRT1 inhibition can break this cycle and provide a potential therapy for individuals suffering from AN.

NIMA-related kinase 1 (NEK1) regulates meiosis I spindle assembly by altering the balance between α-Adducin and Myosin X.

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.

Cohesin Removal along the Chromosome Arms during the First Meiotic Division Depends on a NEK1-PP1γ-WAPL Axis in the Mouse.

Mammalian NIMA-like kinase-1 (NEK1) is a dual-specificity kinase highly expressed in mouse germ cells during prophase I of meiosis. Loss of NEK1 induces retention of cohesin on chromosomes at meiotic prophase I. Timely deposition and removal of cohesin is essential for accurate chromosome segregation. Two processes regulate cohesin removal: a non-proteolytic mechanism involving WAPL, sororin, and PDS5B and direct cleavage by separase. Here, we demonstrate a role for NEK1 in the regulation of WAPL loading during meiotic prophase I, via an interaction between NEK1 and PDS5B. This regulation of WAPL by NEK1-PDS5B is mediated by protein phosphatase 1 gamma (PP1γ), which both interacts with and is a phosphotarget of NEK1. Taken together, our results reveal that NEK1 phosphorylates PP1γ, leading to the dephosphorylation of WAPL, which, in turn, results in its retention on chromosome cores to promote loss of cohesion at the end of prophase I in mammals.

Double trouble in human aneuploidy.

Crossing over, or reciprocal recombination, is essential for accurate segregation of homologous chromosomes at the first meiotic division, resulting in gametes containing the correct chromosome number. A new study in human oocytes analyzes the genome-wide recombination and segregation patterns in all the products of female meiosis, providing experimental support for existing theories about the origin of human aneuploidies and highlighting a novel reverse segregation mechanism of chromosome segregation during meiosis.

Exposure to endocrine disruptor induces transgenerational epigenetic deregulation of microRNAs in primordial germ cells.

In mammals, germ cell differentiation is initiated in the Primordial Germ Cells (PGCs) during fetal development. Prenatal exposure to environmental toxicants such as endocrine disruptors may alter PGC differentiation, development of the male germline and induce transgenerational epigenetic disorders. The anti-androgenic compound vinclozolin represents a paradigmatic example of molecule causing transgenerational effects on germ cells. We performed prenatal exposure to vinclozolin in mice and analyzed the phenotypic and molecular changes in three successive generations. A reduction in the number of embryonic PGCs and increased rate of apoptotic cells along with decrease of fertility rate in adult males were observed in F1 to F3 generations. Blimp1 is a crucial regulator of PGC differentiation. We show that prenatal exposure to vinclozolin deregulates specific microRNAs in PGCs, such as miR-23b and miR-21, inducing disequilibrium in the Lin28/let-7/Blimp1 pathway in three successive generations of males. As determined by global maps of cytosine methylation, we found no evidence for prominent changes in DNA methylation in PGCs or mature sperm. Our data suggest that embryonic exposure to environmental endocrine disruptors induces transgenerational epigenetic deregulation of expression of microRNAs affecting key regulatory pathways of germ cells differentiation.

MicroRNA biogenesis and variability.

MicroRNAs (miRNAs) are cell-endogenous small noncoding RNAs that, through RNA interference, are involved in the posttranscriptional regulation of mRNAs. The biogenesis and function of miRNAs entail multiple elements with different alternative pathways. These confer a high versatility of regulation and a high variability to generate different miRNAs and hence possess a broad potential to regulate gene expression. Here we review the different mechanisms, both canonical and noncanonical, that generate miRNAs in animals. The 'miRNome' panorama enhances our knowledge regarding the fine regulation of gene expression and provides new insights concerning normal, as opposed to pathological, cell differentiation and development.