RESUMEN
Lethal (3) malignant brain tumor like 2 (L3MBTL2) is a member of the MBT-domain proteins, which are involved in transcriptional repression and implicated in chromatin compaction. Our previous study has shown that L3MBTL2 is highly expressed in the testis, but its role in spermatogenesis remains unclear. In the present study, we found that L3MBTL2 was most highly expressed in pachytene spermatocytes within the testis. Germ cell-specific ablation of L3mbtl2 in the testis led to increased abnormal spermatozoa, progressive decrease of sperm counts and premature testicular failure in mice. RNA-sequencing analysis on L3mbtl2 deficient testes confirmed that L3MBTL2 was a transcriptional repressor but failed to reveal any significant changes in spermatogenesis-associated genes. Interestingly, L3mbtl2 deficiency resulted in increased γH2AX deposition in the leptotene spermatocytes, subsequent inappropriate retention of γH2AX on autosomes, and defective crossing-over and synapsis during the pachytene stage of meiosis I, and more germ cell apoptosis and degeneration in aging mice. L3MBTL2 interacted with the histone ubiquitin ligase RNF8. Inhibition of L3MBTL2 reduced nuclear RNF8 and ubH2A levels in GC2 cells. L3mbtl2 deficiency led to decreases in the levels of the RNF8 and ubH2A pathway and in histone acetylation in elongating spermatids, and in protamine 1 deposition and chromatin condensation in sperm. These results suggest that L3MBTL2 plays important roles in chromatin remodeling during meiosis and spermiogenesis.
Asunto(s)
Ensamble y Desensamble de Cromatina/genética , Cromatina/metabolismo , Proteínas Nucleares/genética , Espermatocitos/metabolismo , Espermatogénesis/genética , Factores de Transcripción/genética , Acetilación , Animales , Apoptosis/genética , Ensamble y Desensamble de Cromatina/fisiología , Histonas/metabolismo , Masculino , Profase Meiótica I/fisiología , Ratones , Ratones Noqueados , Proteínas Nucleares/metabolismo , Fase Paquiteno/fisiología , Proteínas del Grupo Polycomb/metabolismo , Recuento de Espermatozoides , Testículo/metabolismo , Factores de Transcripción/metabolismo , Ubiquitina-Proteína Ligasas/metabolismoRESUMEN
Meiotic recombination plays a critical role in sexual reproduction by creating crossovers between homologous chromosomes. These crossovers, along with sister chromatid cohesion, connect homologs to enable proper segregation at Meiosis I. Recombination is initiated by programmed double strand breaks (DSBs) at particular regions of the genome. The meiotic recombination checkpoint uses meiosis-specific modifications to the DSB-induced DNA damage response to provide time to convert these breaks into interhomolog crossovers by delaying entry into Meiosis I until the DSBs have been repaired. The meiosis-specific kinase, Mek1, is a key regulator of meiotic recombination pathway choice, as well as being required for the meiotic recombination checkpoint. The major target of this checkpoint is the meiosis-specific transcription factor, Ndt80, which is essential to express genes necessary for completion of recombination and meiotic progression. The molecular mechanism by which cells monitor meiotic DSB repair to allow entry into Meiosis I with unbroken chromosomes was unknown. Using genetic and biochemical approaches, this work demonstrates that in the presence of DSBs, activated Mek1 binds to Ndt80 and phosphorylates the transcription factor, thus inhibiting DNA binding and preventing Ndt80's function as a transcriptional activator. Repair of DSBs by recombination reduces Mek1 activity, resulting in removal of the inhibitory Mek1 phosphates. Phosphorylation of Ndt80 by the meiosis-specific kinase, Ime2, then results in fully activated Ndt80. Ndt80 upregulates transcription of its own gene, as well as target genes, resulting in prophase exit and progression through meiosis.
Asunto(s)
Reparación del ADN , Proteínas de Unión al ADN/metabolismo , MAP Quinasa Quinasa 1/metabolismo , Meiosis/fisiología , Proteínas de Saccharomyces cerevisiae/metabolismo , Saccharomyces cerevisiae/citología , Saccharomyces cerevisiae/metabolismo , Factores de Transcripción/metabolismo , Secuencia de Aminoácidos , Puntos de Control del Ciclo Celular , Secuencia Conservada , Roturas del ADN de Doble Cadena , Proteínas de Unión al ADN/química , Proteínas de Unión al ADN/genética , Genes Fúngicos , Péptidos y Proteínas de Señalización Intracelular/genética , Péptidos y Proteínas de Señalización Intracelular/metabolismo , MAP Quinasa Quinasa 1/genética , Meiosis/genética , Modelos Biológicos , Modelos Moleculares , Mutación , Fase Paquiteno/genética , Fase Paquiteno/fisiología , Fosforilación , Dominios y Motivos de Interacción de Proteínas , Proteínas Serina-Treonina Quinasas/genética , Proteínas Serina-Treonina Quinasas/metabolismo , Proteolisis , Reparación del ADN por Recombinación , Saccharomyces cerevisiae/genética , Proteínas de Saccharomyces cerevisiae/química , Proteínas de Saccharomyces cerevisiae/genética , Homología de Secuencia de Aminoácido , Factores de Transcripción/química , Factores de Transcripción/genéticaRESUMEN
Cohesins containing a meiosis-specific α-kleisin subunit, RAD21L or REC8, play roles in diverse aspects of meiotic chromosome dynamics including formation of axial elements (AEs), assembly of the synaptonemal complex (SC), recombination of homologous chromosomes (homologs), and cohesion of sister chromatids. However, the exact functions of individual α-kleisins remain to be elucidated. Here, we examined the localization of RAD21L and REC8 within the SC by super-resolution microscopy, 3D-SIM. We found that both RAD21L and REC8 were localized at the connection sites between lateral elements (LEs) and transverse filaments (TFs) of pachynema with RAD21L locating interior to REC8 sites. RAD21L and REC8 were not symmetrical in terms of synaptic homologs, suggesting that the arrangement of different cohesins is not strictly fixed along all chromosome axes. Intriguingly, some RAD21L signals, but not REC8 signals, were observed between unsynapsed regions of AEs of zygonema as if they formed a bridge between homologs. Furthermore, the signals of recombination intermediates overlapped with those of RAD21L to a greater degree than with those of REC8. These results highlight the different properties of two meiotic α-kleisins, and strongly support the previous proposition that RAD21L is an atypical cohesin that establishes the association between homologs rather than sister chromatids.
Asunto(s)
Proteínas de Ciclo Celular/metabolismo , Proteínas Cromosómicas no Histona/metabolismo , Meiosis/fisiología , Proteínas Nucleares/metabolismo , Fosfoproteínas/metabolismo , Espermatocitos/metabolismo , Complejo Sinaptonémico/metabolismo , Animales , Masculino , Ratones , Fase Paquiteno/fisiologíaRESUMEN
The XX/XY system is the rule among mammals. However, many exceptions from this general pattern have been discovered since the last decades. One of these non-conventional sex chromosome mechanisms is the multiple sex chromosome system, which is evolutionary fixed among many bat species of the family Phyllostomidae, and has arisen by a translocation between one original gonosome (X or Y chromosome), and an autosome, giving rise to a "neo-XY body." The aim of this work is to study the synaptic behavior and the chromatin remodeling of multiple sex chromosomes in different species of phyllostomid bats using electron microscopy and molecular markers. Testicular tissues from adult males of the species Artibeus lituratus, Artibeus planirostris, Uroderma bilobatum, and Vampyrodes caraccioli from the eastern Amazonia were analyzed by optical/electron microscopy and immunofluorescence of meiotic proteins involved in synapsis (SYCP3 and SYCE3), sister-chromatid cohesion (SMC3), and chromatin silencing (BRCA1, γ-H2AX, and RNApol 2). The presence of asynaptic axes-labeled by BRCA1 and γ-H2AX-at meiotic prophase in testes that have a normal development of spermatogenesis, suggests that the basic mechanism that arrests spreading of transcriptional silencing (meiotic sex chromosome inactivation (MSCI)) to the autosomal segments may be per se the formation of a functional synaptonemal complex between homologous or non-homologous regions, and thus, this SC barrier might be probably related to the preservation of fertility in these systems.
Asunto(s)
Quirópteros/genética , Ensamble y Desensamble de Cromatina/fisiología , Cromatina/metabolismo , Procesos de Determinación del Sexo/genética , Cromosoma X/genética , Cromosoma Y/genética , Animales , Emparejamiento Cromosómico/genética , Masculino , Fase Paquiteno/fisiología , Espermatocitos/metabolismo , Espermatogénesis/fisiologíaRESUMEN
Spermatogenesis is a highly regulated process that involves both mitotic and meiotic divisions, as well as cellular differentiation to yield mature spermatozoa from undifferentiated germinal stem cells. Although Gpat2 was originally annotated as encoding a glycerol-3-phosphate acyltransferase by sequence homology to Gpat1, GPAT2 is highly expressed in testis but not in lipogenic tissues and is not up-regulated during adipocyte differentiation. New data show that GPAT2 is required for the synthesis of piRNAs (piwi-interacting RNAs), a group of small RNAs that protect the germ cell genome from retrotransposable elements. In order to understand the relationship between GPAT2 and its role in the testis, we focused on Gpat2 expression during the first wave of mouse spermatogenesis. Gpat2 expression was analysed by qPCR (quantitative real-time PCR), in situ hybridization, immunohistochemistry and Western blotting. Gpat2 mRNA content and protein expression were maximal at 15 dpp (days post-partum) and were restricted to pachytene spermatocytes. To achieve this transient expression, both epigenetic mechanisms and trans-acting factors are involved. In vitro assays showed that Gpat2 expression correlates with DNA demethylation and histone acetylation and that it is up-regulated by retinoic acid. Epigenetic regulation by DNA methylation was confirmed in vivo in germ cells by bisulfite sequencing of the Gpat2 promoter. Consistent with the initiation of meiosis at 11 dpp, methylation decreased dramatically. Thus, Gpat2 is expressed at a specific stage of spermatogenesis, consistent with piRNA synthesis and meiosis I prophase, and its on-off expression pattern responds predominantly to epigenetic modifications.
Asunto(s)
Metilación de ADN/fisiología , Epigénesis Genética/fisiología , Glicerol-3-Fosfato O-Aciltransferasa/metabolismo , Profase Meiótica I/fisiología , Fase Paquiteno/fisiología , Regiones Promotoras Genéticas/fisiología , Espermatocitos/metabolismo , Espermatogénesis/fisiología , Animales , Glicerol-3-Fosfato O-Aciltransferasa/genética , Masculino , Ratones , Ratones Endogámicos BALB C , ARN Mensajero/genética , ARN Mensajero/metabolismo , ARN Interferente Pequeño/genética , ARN Interferente Pequeño/metabolismo , Espermatocitos/citologíaRESUMEN
Vasa is a universal marker of the germ line in animals, yet mutations disrupting vasa cause sexually dimorphic infertility, with impaired development of the ovary in some animals and the testis in others. The basis for this sexually dimorphic requirement for Vasa is not clear; in most animals examined, both the male and female gonad express vasa throughout the life of the germ line. Here we characterized a loss-of-function mutation disrupting zebrafish vasa. We show that maternally provided Vasa is stable through the first ten days of development in zebrafish, and thus likely fulfills any early roles for Vasa during germ-line specification, migration, survival, and maintenance. Although zygotic Vasa is not essential for the development of juvenile gonads, vasa mutants develop exclusively as sterile males. Furthermore, phenotypes of vasa;p53 compound mutants are indistinguishable from those of vasa mutants, therefore the failure of vasa mutants to differentiate as females and to support germ-cell development in the testis is not due to p53-mediated apoptosis. Instead, we found that failure to progress beyond the pachytene stage of meiosis causes the loss of germ-line stem cells, leaving empty somatic tubules. Our studies provide insight into the function of zebrafish vasa during female meiosis, differentiation, and maintenance of germ-line stem cells.
Asunto(s)
Diferenciación Celular/fisiología , ARN Helicasas DEAD-box/metabolismo , Células Germinativas/metabolismo , Fase Paquiteno/fisiología , Células Madre/metabolismo , Proteínas de Pez Cebra/metabolismo , Pez Cebra/metabolismo , Animales , Apoptosis/genética , ARN Helicasas DEAD-box/genética , Femenino , Células Germinativas/citología , Infertilidad Masculina/genética , Infertilidad Masculina/metabolismo , Masculino , Mutación , Células Madre/citología , Proteína p53 Supresora de Tumor/genética , Proteína p53 Supresora de Tumor/metabolismo , Pez Cebra/genética , Proteínas de Pez Cebra/genéticaRESUMEN
In barley (Hordeum vulgare L.), chiasmata (the physical sites of genetic crossovers) are skewed towards the distal ends of chromosomes, effectively consigning a large proportion of genes to recombination coldspots. This has the effect of limiting potential genetic variability, and of reducing the efficiency of map-based cloning and breeding approaches for this crop. Shifting the sites of recombination to more proximal chromosome regions by forward and reverse genetic means may be profitable in terms of realizing the genetic potential of the species, but is predicated upon a better understanding of the mechanisms governing the sites of these events, and upon the ability to recognize real changes in recombination patterns. The barley MutL Homologue (HvMLH3), a marker for class I interfering crossovers, has been isolated and a specific antibody has been raised. Immunolocalization of HvMLH3 along with the synaptonemal complex transverse filament protein ZYP1, used in conjunction with fluorescence in situ hybridization (FISH) tagging of specific barley chromosomes, has enabled access to the physical recombination landscape of the barley cultivars Morex and Bowman. Consistent distal localization of HvMLH3 foci throughout the genome, and similar patterns of HvMLH3 foci within bivalents 2H and 3H have been observed. A difference in total numbers of HvMLH3 foci between these two cultivars has been quantified, which is interpreted as representing genotypic variation in class I crossover frequency. Discrepancies between the frequencies of HvMLH3 foci and crossover frequencies derived from linkage analysis point to the existence of at least two crossover pathways in barley. It is also shown that interference of HvMLH3 foci is relatively weak compared with other plant species.
Asunto(s)
Cromosomas de las Plantas/genética , Hordeum/genética , Fase Paquiteno/genética , Arabidopsis/genética , Secuencia de Bases , Mapeo Cromosómico , Cromosomas de las Plantas/fisiología , Intercambio Genético/genética , Intercambio Genético/fisiología , Ligamiento Genético/genética , Ligamiento Genético/fisiología , Sitios Genéticos/genética , Sitios Genéticos/fisiología , Genoma de Planta/genética , Genoma de Planta/fisiología , Hordeum/fisiología , Hibridación Fluorescente in Situ , Datos de Secuencia Molecular , Fase Paquiteno/fisiología , Filogenia , Alineación de Secuencia , Complejo Sinaptonémico/genética , Complejo Sinaptonémico/fisiologíaRESUMEN
Reactive oxygen species (ROS) and reactive nitrogen species (RNS) like superoxide and nitric oxide are produced by testis and spermatogenic cells in response to heat stress. However, the magnitude and mechanisms of this production in spermatogenic cells have not been described. In this work, we evaluated ROS/RNS production, its pharmacology, mitochondrial oxidative metabolism, membrane potential and antioxidant capacity at different temperatures in isolated rat pachytene spermatocytes and round spermatids. Our results showed an increment in ROS/RNS production by pachytene spermatocytes when increasing the temperature to 40â°C. Instead, ROS/RNS production by round spermatids did not change at temperatures higher than 33â°C. ROS/RNS production was sensitive to NADPH oxidase inhibitor diphenylene iodonium or the mitochondrial complex I inhibitor rotenone. No additive effects were observed for these two compounds. Our results suggest an important mitochondrial ROS/RNS production in spermatogenic cells. Oligomycin-insensitive oxygen consumption (uncoupled oxygen consumption) increased with temperature and was significantly larger in round spermatids than pachytene spermatocytes, indicating a likely round spermatid mitochondrial uncoupling at high temperatures. A similar conclusion can be reached by measuring the mitochondrial membrane potential using rhodamine 123 fluorescence in permeabilized cells or JC-1 fluorescence in intact cells. The antioxidant capacity was higher in round spermatids than pachytene spermatocytes at 40â°C. Our results strongly suggest that at high temperatures (40â°C) pachytene spermatocytes are more susceptible to oxidative stress, but round spermatids are more protected because of a temperature-induced mitochondrial uncoupling together with a larger antioxidant capacity.
Asunto(s)
Frío , Calor , Fase Paquiteno/fisiología , Especies de Nitrógeno Reactivo/metabolismo , Especies Reactivas de Oxígeno/metabolismo , Espermátides/metabolismo , Espermatocitos/metabolismo , Animales , Antioxidantes/metabolismo , Temperatura Corporal/fisiología , Células Cultivadas , Respuesta al Choque Térmico/fisiología , Masculino , Ratas , Ratas Sprague-Dawley , Espermátides/fisiología , Espermatocitos/fisiología , Espermatogénesis/fisiologíaRESUMEN
During the first meiotic prophase in male mammals, sex chromosomes undergo a program of transcriptional silencing called meiotic sex chromosome inactivation (MSCI). MSCI is triggered by accumulation of proteins like BRCA1, ATR, and γH2AX on unsynapsed chromosomes, followed by local changes on the sex chromatin, including histone modifications, incorporation of specific histone variants, non-histone proteins, and RNAs. It is generally thought that MSCI represents the transition of unsynapsed chromatin from a transcriptionally active state to a repressed state. However, transcription is generally low in the whole nucleus during the early stages of the first meiotic prophase, when markers of MSCI first appear, and is then reactivated globally during pachytene. Thus, an alternative possibility is that MSCI represents the targeted maintenance and/or reinforcement of a prior repressed state, i.e., a failure to reactivate. Here, we present an analysis of the temporal and spatial appearance of transcriptional and MSCI markers, as well as chromatin modifications related to transcriptional regulation. We show that levels of RNA pol II and histone H3 acetylated at lysine 9 (H3K9ac) are low during leptotene, zygotene, and early pachytene, but increase strongly in mid-pachytene, indicating that reactivation occurs with some delay after synapsis. However, while transcription markers appear abundantly on the autosomes at mid-pachytene, they are not directed to the sex chromosomes. Interestingly, we found that chromatin modifications related to transcriptional silencing and/or MSCI, namely, histone H3 trimethylated at lysine 9 (H3K9me3), histone H3 monomethylated at lysine 4 (H3K4me1), γH2AX, SUMO1, and XMR, appear on the sex chromosomes before autosomes become reactivated. These results suggest that the onset of MSCI during late zygotene and early pachytene may prevent sex chromosome reactivation during mid-pachytene instead of promoting inactivation de novo. Additionally, we found temporal differences between the X and Y chromosomes in the recruitment of DNA repair and MSCI markers, indicating a differential regulation of these processes. We propose that many of the meiotic defects attributed to failure to silence sex chromosomes could be interpreted as a more general process of transcriptional misregulation that occurs under certain pathological circumstances in zygotene and early pachytene.
Asunto(s)
Silenciador del Gen , Profase Meiótica I/genética , Cromosoma X/metabolismo , Cromosoma Y/metabolismo , Animales , Proteínas Portadoras , Proteínas de Ciclo Celular , Cromatina/metabolismo , Emparejamiento Cromosómico/fisiología , Roturas del ADN de Doble Cadena , Reparación del ADN , Proteínas de Unión al ADN , Histonas/metabolismo , Masculino , Ratones , Proteínas Nucleares/metabolismo , Fase Paquiteno/fisiología , ARN Polimerasa II/metabolismo , Proteínas de Unión al ARN , Proteína SUMO-1/metabolismo , Transcripción GenéticaRESUMEN
We review the critical events in early meiotic prophase in Drosophila melanogaster oocytes. We focus on four aspects of this process: the formation of the synaptonemal complex (SC) and its role in maintaining homologous chromosome pairings, the critical roles of the meiosis-specific process of centromere clustering in the formation of a full-length SC, the mechanisms by which preprogrammed double-strand breaks initiate meiotic recombination, and the checkpoints that govern the progression and coordination of these processes. Central to this discussion are the roles that somatic pairing events play in establishing the necessary conditions for proper SC formation, the roles of centromere pairing in synapsis initiation, and the mechanisms by which oocytes detect failures in SC formation and/or recombination. Finally, we correlate what is known in Drosophila oocytes with our understanding of these processes in other systems.
Asunto(s)
Cromosomas/fisiología , Drosophila/fisiología , Meiosis/fisiología , Profase Meiótica I/fisiología , Oocitos/fisiología , Animales , Núcleo Celular/fisiología , Centrómero/fisiología , Emparejamiento Cromosómico/fisiología , Roturas del ADN de Doble Cadena , Daño del ADN , Femenino , Humanos , Oocitos/crecimiento & desarrollo , Fase Paquiteno/fisiología , Complejo Sinaptonémico/fisiología , Telómero/fisiologíaRESUMEN
Gonadotropin-regulated testicular RNA helicase (GRTH/DDX25), a testis-specific member of the DEAD-box family, is an essential post-transcriptional regulator of spermatogenesis. Failure of expression of Transition protein 2 (TP2) and Protamine 2 (Prm2) proteins (chromatin remodelers, essential for spermatid elongation and completion of spermatogenesis) with preservation of their mRNA expression was observed in GRTH-null mice (azoospermic due to failure of spermatids to elongate). These were identified as target genes for the testis-specific miR-469, which is increased in the GRTH-null mice. Further analysis demonstrated that miR-469 repressed TP2 and Prm2 protein expression at the translation level with minor effect on mRNA degradation, through binding to the coding regions of TP2 and Prm2 mRNAs. The corresponding primary-microRNAs and the expression levels of Drosha and DGCR8 (both mRNA and protein) were increased significantly in the GRTH-null mice. miR-469 silencing of TP2 and Prm2 mRNA in pachytene spermatocytes and round spermatids is essential for their timely translation at later times of spermiogenesis, which is critical to attain mature sperm. Collectively, these studies indicate that GRTH, a multifunctional RNA helicase, acts as a negative regulator of miRNA-469 biogenesis and consequently their function during spermatogenesis.
Asunto(s)
ARN Helicasas DEAD-box/metabolismo , Silenciador del Gen/fisiología , MicroARNs/biosíntesis , Proteínas Nucleares/biosíntesis , Sistemas de Lectura Abierta/fisiología , Protaminas/metabolismo , ARN Mensajero/biosíntesis , Espermátides/metabolismo , Espermatocitos/metabolismo , Espermatogénesis/fisiología , Animales , ARN Helicasas DEAD-box/genética , Proteínas de Unión al ADN , Masculino , Ratones , Ratones Noqueados , MicroARNs/genética , Proteínas Nucleares/genética , Especificidad de Órganos/fisiología , Fase Paquiteno/fisiología , Protaminas/genética , Proteínas/genética , Proteínas/metabolismo , Estabilidad del ARN/fisiología , ARN Mensajero/genética , Proteínas de Unión al ARN , Ribonucleasa III/genética , Ribonucleasa III/metabolismo , Espermátides/citología , Espermatocitos/citología , Testículo/citología , Testículo/metabolismo , Regulación hacia Arriba/fisiologíaRESUMEN
The transcriptional regulation of mammalian meiosis is poorly characterized, owing to few genetic and ex vivo models. From a genetic screen, we identify the transcription factor MYBL1 as a male-specific master regulator of several crucial meiotic processes. Spermatocytes bearing a novel separation-of-function allele (Mybl1(repro9)) had subtle defects in autosome synapsis in pachynema, a high incidence of unsynapsed sex chromosomes, incomplete double-strand break repair on synapsed pachytene chromosomes and a lack of crossing over. MYBL1 protein appears in pachynema, and its mutation caused specific alterations in expression of diverse genes, including some translated postmeiotically. These data, coupled with chromatin immunoprecipitation (ChIP-chip) experiments and bioinformatic analysis of promoters, identified direct targets of MYBL1 regulation. The results reveal that MYBL1 is a master regulator of meiotic genes that are involved in multiple processes in spermatocytes, particularly those required for cell cycle progression through pachynema.
Asunto(s)
Regulación del Desarrollo de la Expresión Génica , Meiosis/fisiología , Proteínas Proto-Oncogénicas c-myb/metabolismo , Espermatocitos/fisiología , Transactivadores/metabolismo , Factores de Transcripción/metabolismo , Secuencia de Aminoácidos , Animales , Roturas del ADN de Doble Cadena , Femenino , Perfilación de la Expresión Génica , Humanos , Infertilidad Masculina/genética , Masculino , Ratones , Ratones Endogámicos C3H , Ratones Endogámicos C57BL , Análisis por Micromatrices , Datos de Secuencia Molecular , Mutación , Fase Paquiteno/fisiología , Proteínas Proto-Oncogénicas c-myb/genética , Alineación de Secuencia , Espermatocitos/citología , Espermatogénesis/fisiología , Transactivadores/genética , Factores de Transcripción/genética , Transcripción GenéticaRESUMEN
During prophase of meiosis I, genetic recombination is initiated with a Spo11-dependent DNA double-strand break (DSB). Repair of these DSBs can generate crossovers, which become chiasmata and are important for the process of chromosome segregation. To ensure at least one chiasma per homologous pair of chromosomes, the number and distribution of crossovers is regulated. One system contributing to the distribution of crossovers is the pachytene checkpoint, which requires the conserved gene pch2 that encodes an AAA+ATPase family member. Pch2-dependent pachytene checkpoint function causes delays in pachytene progression when there are defects in processes required for crossover formation, such as mutations in DSB-repair genes and when there are defects in the structure of the meiotic chromosome axis. Thus, the pachytene checkpoint appears to monitor events leading up to the generation of crossovers. Interestingly, heterozygous chromosome rearrangements cause Pch2-dependent pachytene delays and as little as two breaks in the continuity of the paired chromosome axes are sufficient to evoke checkpoint activity. These chromosome rearrangements also cause an interchromosomal effect on recombination whereby crossing over is suppressed between the affected chromosomes but is increased between the normal chromosome pairs. We have shown that this phenomenon is also due to pachytene checkpoint activity.
Asunto(s)
Cromosomas de Insectos , Intercambio Genético , Drosophila/genética , Meiosis/fisiología , Animales , Roturas del ADN de Doble Cadena , Reparación del ADN , Proteínas de Drosophila/genética , Proteínas de Drosophila/metabolismo , Proteínas de Drosophila/fisiología , Femenino , Fase Paquiteno/genética , Fase Paquiteno/fisiologíaRESUMEN
Fluorochrome-labeled oligonucleotides (n = 44) corresponding to mouse genome repetitive sequences were hybridized in situ with pachytene nuclei of mouse spermatocytes. Signals of the repetitive sequences MaLR, MER, and (GT)22 were found to be dispersed through chromatin, and signals of BI 1 repeats and minisatellites were mostly attached to synaptonemal complexes immunostained with anti-SYCP3 antibodies. These results suggest that B 1 repeats and minisatellites are candidates for sequences anchoring chromatin to synaptonemal complexes.
Asunto(s)
Cromatina/metabolismo , Cromosomas de los Mamíferos/metabolismo , Fase Paquiteno/fisiología , Espermatocitos/metabolismo , Complejo Sinaptonémico/metabolismo , Animales , Cromatina/genética , Hibridación Fluorescente in Situ , Masculino , Ratones , Repeticiones de Minisatélite/fisiologíaRESUMEN
HORMA domain-containing proteins regulate interactions between homologous chromosomes (homologs) during meiosis in a wide range of eukaryotes. We have identified a mouse HORMA domain-containing protein, HORMAD1, and biochemically and cytologically shown it to be associated with the meiotic chromosome axis. HORMAD1 first accumulates on the chromosomes during the leptotene to zygotene stages of meiotic prophase I. As germ cells progress into the pachytene stage, HORMAD1 disappears from the synapsed chromosomal regions. However, once the chromosomes desynapse during the diplotene stage, HORMAD1 again accumulates on the chromosome axis of the desynapsed homologs. HORMAD1 thus preferentially localizes to unsynapsed or desynapsed chromosomal regions during the prophase I stage of meiosis. Analysis of mutant strains lacking different components of the synaptonemal complex (SC) revealed that establishment of the SC is required for the displacement of HORMAD1 from the chromosome axis. Our results therefore strongly suggest that also mammalian cells use a HORMA domain-containing protein as part of a surveillance system that monitors synapsis or other interactions between homologs.
Asunto(s)
Proteínas de Ciclo Celular/metabolismo , Emparejamiento Cromosómico/fisiología , Cromosomas de los Mamíferos/metabolismo , Meiosis/fisiología , Animales , Proteína BRCA1/metabolismo , Células COS , Proteínas de Ciclo Celular/química , Núcleo Celular/metabolismo , Chlorocebus aethiops , Proteínas Cromosómicas no Histona/genética , Proteínas Cromosómicas no Histona/metabolismo , Proteínas de Unión al ADN , Embrión de Mamíferos/metabolismo , Femenino , Histonas/metabolismo , Masculino , Profase Meiótica I/fisiología , Ratones , Ratones Noqueados , Proteínas Nucleares/genética , Proteínas Nucleares/metabolismo , Oocitos/metabolismo , Fase Paquiteno/fisiología , Isoformas de Proteínas/genética , Isoformas de Proteínas/metabolismo , Espermatocitos/metabolismo , Complejo Sinaptonémico/metabolismo , Testículo/metabolismo , Transfección , CohesinasRESUMEN
Male fertility relies on the highly specialized process of spermatogenesis to continually renew the supply of spermatozoa necessary for reproduction. Central to this unique process is meiosis that is responsible for the production of haploid spermatozoa as well as for generating genetic diversity. During meiosis I, there is a dramatic increase in the number of mitochondria present within the developing spermatocytes, suggesting an increased necessity for ATP production and utilization. Essential for the utilization of ATP is the translocation of ADP and ATP across the inner mitochondrial membrane, which is mediated by the adenine nucleotide translocases (Ant). We recently identified and characterized a novel testis specific Ant, ANT4 (also known as SLC25A31 and Aac4). The generation of Ant4-deficient animals resulted in the severe disruption of the seminiferous epithelium with an apparent spermatocytic arrest of the germ cell population. In the present study utilizing a chromosomal spread technique, we determined that Ant4-deficiency results in an accumulation of leptotene spermatocytes, a decrease in pachytene spermatocytes, and an absence of diplotene spermatocytes, indicating early meiotic arrest. Furthermore, the chromosomes of Ant4-deficient pachytene spermatocyte occasionally demonstrated sustained gammaH2AX association as well as synaptonemal complex protein 1 (SYCP1)/SYCP3 dissociation beyond the sex body. Large ATP supplies from mitochondria may be critical for normal progression of spermatogenesis during early stages of meiotic prophase I, including DNA double-strand break repair and chromosomal synapsis.
Asunto(s)
Meiosis/genética , Proteínas de Transporte de Membrana/genética , Espermatozoides/fisiología , Animales , Puntos de Control del Ciclo Celular/genética , Proteínas de Ciclo Celular , Proteínas de Unión al ADN , Femenino , Células Germinativas/citología , Células Germinativas/metabolismo , Células Germinativas/fisiología , Histonas/metabolismo , Masculino , Meiosis/fisiología , Proteínas de Transporte de Membrana/deficiencia , Proteínas de Transporte de Membrana/fisiología , Ratones , Ratones Endogámicos C57BL , Ratones Noqueados , Proteínas Nucleares/metabolismo , Fase Paquiteno/genética , Fase Paquiteno/fisiología , Espermatocitos/metabolismo , Espermatocitos/fisiología , Espermatogénesis/genética , Espermatogénesis/fisiología , Espermatozoides/citología , Espermatozoides/enzimología , Espermatozoides/metabolismo , Testículo/citología , Testículo/metabolismo , Factores de TiempoRESUMEN
Instability of B-chromosomes was estimated in somatic and germline cells of samples Apodemus peninsulae from different localities of the species range. In 84 out of 188 animals (45%), in cells assessed for B-chromosome mosaicism, bone marrow cells with different B-chromosome number were observed. The numbers of B-chromosomes in spermatocytes at the pachytene stage were estimated in ten males. It was shown that the average number of B-chromosomes and the number of cell clones in germline cells was higher than the corresponding numbers in bone marrow cells. The higher number of B-chromosomes and their higher variability in germline cells than in somatic cells suggest the existence of a mechanism of premeiotic accumulation of B-chromosomes in spermatogenesis of A. peninsulae.
Asunto(s)
Inestabilidad Cromosómica/fisiología , Cromosomas de los Mamíferos/genética , Murinae/genética , Fase Paquiteno/fisiología , Espermatogénesis/fisiología , Animales , Cromosomas de los Mamíferos/metabolismo , Femenino , Masculino , Murinae/metabolismo , Espermatocitos/citología , Espermatocitos/metabolismoRESUMEN
The transition from pachytene to Meiosis I is a key regulatory point in yeast meiosis. This transition requires Ndt80, a transcription factor that commits cells to complete meiosis by expression of a diverse set of genes. In this issue of Genes & Development, Sourirajan and Lichten (2627-2632) report that CDC5, an NDT80-regulated gene encoding yeast polo-like kinase, is sufficient for Holliday junction resolution and exit from pachytene. Determining the meiotic targets of Cdc5 therefore provides a new approach for identifying a eukaryotic Holliday resolvase.
Asunto(s)
Meiosis/genética , Meiosis/fisiología , Fase Paquiteno/genética , Fase Paquiteno/fisiología , Proteínas Quinasas/genética , Proteínas Quinasas/fisiología , Proteínas de Ciclo Celular/genética , Proteínas de Ciclo Celular/fisiología , Cromosomas Fúngicos/genética , Cromosomas Fúngicos/ultraestructura , Proteínas de Unión al ADN/genética , Proteínas de Unión al ADN/fisiología , Genes Fúngicos , Modelos Biológicos , Proteínas Serina-Treonina Quinasas , Saccharomyces cerevisiae/citología , Saccharomyces cerevisiae/genética , Saccharomyces cerevisiae/fisiología , Proteínas de Saccharomyces cerevisiae/genética , Proteínas de Saccharomyces cerevisiae/fisiología , Factores de Transcripción/genética , Factores de Transcripción/fisiologíaRESUMEN
In budding yeast, exit from the pachytene stage of meiosis requires the mid-meiosis transcription factor Ndt80, which promotes expression of approximately 200 genes. Ndt80 is required for meiotic function of polo-like kinase (PLK, Cdc5) and cyclin-dependent kinase (CDK), two cell cycle kinases previously implicated in pachytene exit. We show that ongoing CDK activity is dispensable for two events that accompany exit from pachytene: crossover formation and synaptonemal complex breakdown. In contrast, CDC5 expression in ndt80Delta mutants efficiently promotes both events. Thus, Cdc5 is the only member of the Ndt80 transcriptome required for this critical step in meiotic progression.
Asunto(s)
Proteínas de Ciclo Celular/genética , Proteínas de Ciclo Celular/fisiología , Meiosis/genética , Meiosis/fisiología , Fase Paquiteno/genética , Fase Paquiteno/fisiología , Proteínas Quinasas/genética , Proteínas Quinasas/fisiología , Proteínas de Saccharomyces cerevisiae/genética , Proteínas de Saccharomyces cerevisiae/fisiología , Cromosomas Fúngicos/genética , Proteínas de Unión al ADN/genética , Proteínas de Unión al ADN/fisiología , Perfilación de la Expresión Génica , Genes Fúngicos , Modelos Biológicos , Mutación , Proteínas Serina-Treonina Quinasas , Saccharomyces cerevisiae/citología , Saccharomyces cerevisiae/genética , Saccharomyces cerevisiae/fisiología , Complejo Sinaptonémico/genética , Complejo Sinaptonémico/fisiología , Factores de Transcripción/genética , Factores de Transcripción/fisiologíaRESUMEN
Programmed double-strand breaks at prophase of meiosis acquire immunologically detectable RAD51-DMC1 foci or early nodules (ENs) that are associated with developing chromosome core segments; each focus is surrounded by a gammaH2AX-modified chromosome domain. The 250-300 ENs per nucleus decline in numbers during the development of full-length cores and the remaining foci are relatively evenly distributed along the mature cores (gamma distribution of nu=2.97). The ENs become transformed nodules (TNs) by the acquisition of RPA, BLM, MSH4 and topoisomerases that function in repair and Holliday junction resolution. At the leptotene-zygotene transition, TNs orient to positions between the aligned cores where they initiate structural interhomolog contacts prior to synaptonemal complex (SC) formation, possibly future crossover sites. Subsequently, TNs are associated with SC extension at the synaptic forks. Dephosphorylation of TN-associated histone gammaH2AX chromatin suggests annealing of single strands or repair of double-strand breaks DSBs at this time. Some 200 TNs per pachytene nucleus are distributed proportional to SC length and are evenly distributed along the SCs (nu= approximately 4). At this stage, gammaH2AX-modified chromatin domains are associated with transcriptionally silenced sex chromosomes and autosomal sites. Immunogold electron microscope evidence shows that one or two TNs of the 10-15 TNs per SC acquire MLH1 protein, the hallmark of reciprocal recombination, whereas the TNs that do not acquire MLH1 protein relocate from their positions along the midline of the SCs to the periphery of the SCs. Relocation of TNs may be associated with the conversion of potential crossovers into non-crossovers.