A Meiotic Chromosomal Core Consisting of Cohesin Complex Proteins Recruits DNA Recombination Proteins and Promotes Synapsis in the Absence of an Axial Element in Mammalian Meiotic Cells

The behavior of meiotic chromosomes differs in several respects from that of their mitotic counterparts, resulting in the generation of genetically distinct haploid cells. This has been attributed in part to a meiosis-specific chromatin-associated protein structure, the synaptonemal complex. This complex consist of two parallel axial elements, each one associated with a pair of sister chromatids, and a transverse filament located between the synapsed homologous chromosomes. Recently, a different protein structure, the cohesin complex, was shown to be associated with meiotic chromosomes and to be required for chromosome segregation. To explore the functions of the two different protein structures, the synaptonemal complex and the cohesin complex, in mammalian male meiotic cells, we have analyzed how absence of the axial element affects early meiotic chromosome behavior. We find that the synaptonemal complex protein 3 (SCP3) is a main determinant of axial-element assembly and is required for attachment of this structure to meiotic chromosomes, whereas SCP2 helps shape the in vivo structure of the axial element. We also show that formation of a cohesin-containing chromosomal core in meiotic nuclei does not require SCP3 or SCP2. Our results also suggest that the cohesin core recruits recombination proteins and promotes synapsis between homologous chromosomes in the absence of an axial element. A model for early meiotic chromosome pairing and synapsis is proposed.     

Genetic control of chromosome synapsis in yeast meiosis

Both meiosis-specific and general recombination functions, recruited from the mitotic cell cycle, are required for elevated levels of recombination and for chromosome synapsis (assembly of the synaptonemal complex) during yeast meiosis. The meiosis-specific SPO11 gene (previously shown to be required for meiotic recombination) has been isolated and shown to be essential for synaptonemal complex formation but not for DNA metabolism during the vegetative cell cycle. In contrast, the RAD52 gene is required for mitotic and meiotic recombination but not for synaptonemal complex assembly. These data suggest that the synaptonemal complex may be necessary but is clearly not sufficient for meiotic recombination. Cytological analysis of spread meiotic nuclei demonstrates that chromosome behavior in yeast is comparable with that observed in larger eukaryotes. These spread preparations support the immunocytological localization of specific proteins in meiotic nuclei. This combination of genetic, molecular cloning, and cytological approaches in a single experimental system provides a means of addressing the role of specific gene products and nuclear structures in meiotic chromosome behavior.     

Synaptonemal complex formation: where does it start?

The synaptonemal complex is a prominent, evolutionarily conserved feature of meiotic prophase. The assembly of this structure is closely linked to meiotic recombination. A recent study in budding yeast reveals an unexpected role in centromere pairing for a protein component of the synaptonemal complex, Zip1. These findings have implications for synaptonemal complex formation.     

Dissociation of DNA polymerase alpha-primase complex during meiosis in Coprinus cinereus.

Previously, the activity of DNA polymerase alpha was found in the meiotic prophase I including non-S phase stages, in the basidiomycetes, Coprinus cinereus. To study DNA polymerase alpha during meiosis, we cloned cDNAs for the C. cinereus DNA polymerase alpha catalytic subunit (p140) and C. cinereus primase small subunit (p48). Northern analysis indicated that both p140 and p48 are expressed not only at S phase but also during the leptotene/zygotene stages of meiotic prophase I. In situ immuno-staining of cells at meiotic prophase I revealed a sub population of p48 that does not colocalize with p140 in nuclei. We also purified the pol alpha-primase complex from meiotic cells by column chromatography and characterized its biochemical properties. We found a subpopulation of primase that was separated from the pol alpha-primase complex by phosphocellulose column chromatography. Glycerol gradient density sedimentation results indicated that the amount of intact pol alpha-primase complex in crude extract is reduced, and that a smaller complex appears upon meiotic development. These results suggest that the form of the DNA polymerase alpha-primase complex is altered during meiotic development.     

The Smc5-Smc6 complex is required to remove chromosome junctions in meiosis

Meiosis, a specialized cell division with a single cycle of DNA replication round and two consecutive rounds of nuclear segregation, allows for the exchange of genetic material between parental chromosomes and the formation of haploid gametes. The structural maintenance of chromosome (SMC) proteins aid manipulation of chromosome structures inside cells. Eukaryotic SMC complexes include cohesin, condensin and the Smc5-Smc6 complex. Meiotic roles have been discovered for cohesin and condensin. However, although Smc5-Smc6 is known to be required for successful meiotic divisions, the meiotic functions of the complex are not well understood. Here we show that the Smc5-Smc6 complex localizes to specific chromosome regions during meiotic prophase I. We report that meiotic cells lacking Smc5-Smc6 undergo catastrophic meiotic divisions as a consequence of unresolved linkages between chromosomes. Surprisingly, meiotic segregation defects are not rescued by abrogation of Spo11-induced meiotic recombination, indicating that at least some chromosome linkages in smc5-smc6 mutants originate from other cellular processes. These results demonstrate that, as in mitosis, Smc5-Smc6 is required to ensure proper chromosome segregation during meiosis by preventing aberrant recombination intermediates between homologous chromosomes.     

The Mnd1 protein forms a complex with hop2 to promote homologous chromosome pairing and meiotic double-strand break repair

The hop2 mutant of Saccharomyces cerevisiae arrests in meiosis with extensive synaptonemal complex (SC) formation between nonhomologous chromosomes. A screen for multicopy suppressors of a hop2-ts allele identified the MND1 gene. The mnd1-null mutant arrests in meiotic prophase, with most double-strand breaks (DSBs) unrepaired. A low level of mature recombinants is produced, and the Rad51 protein accumulates at numerous foci along chromosomes. SC formation is incomplete, and homolog pairing is severely reduced. The Mnd1 protein localizes to chromatin throughout meiotic prophase, and this localization requires Hop2. Unlike recombination enzymes such as Rad51, Mnd1 localizes to chromosomes even in mutants that fail to initiate meiotic recombination. The Hop2 and Mnd1 proteins coimmunoprecipitate from meiotic cell extracts. These results suggest that Hop2 and Mnd1 work as a complex to promote meiotic chromosome pairing and DSB repair. The identification of Hop2 and Mnd1 homologs in other organisms suggests that the function of this complex is conserved among eukaryotes.     

The Mre11 complex influences DNA repair, synapsis, and crossing over in murine meiosis

The Mre11 complex (consisting of MRE11, RAD50, and NBS1/Xrs2) is required for double-strand break (DSB) formation, processing, and checkpoint signaling during meiotic cell division in S. cerevisiae. Whereas studies of Mre11 complex mutants in S. pombe and A. thaliana indicate that the complex has other essential meiotic roles , relatively little is known regarding the functions of the complex downstream of meiotic break formation and processing or its role in meiosis in higher eukaryotes. We analyzed meiotic events in mice harboring hypomorphic Mre11 and Nbs1 mutations which, unlike null mutants, support viability . Our studies revealed defects in the temporal progression of meiotic prophase, incomplete and aberrant synapsis of homologous chromosomes, persistence of strand exchange proteins, and alterations in both the frequency and placement of MLH1 foci, a marker of crossovers. A unique sex-dependent effect on MLH1 foci and chiasmata numbers was observed: males exhibited an increase and females a decrease in recombination levels. Thus, our findings implicate the Mre11 complex in meiotic DNA repair and synapsis in mammals and indicate that the complex may contribute to the establishment of normal sex-specific differences in meiosis.     

Dissociation of the Nuf2-Ndc80 complex releases centromeres from the spindle-pole body during meiotic prophase in fission yeast

In the fission yeast Schizosaccharomyces pombe, centromeres remain clustered at the spindle-pole body (SPB) during mitotic interphase. In contrast, during meiotic prophase centromeres dissociate from the SPB. Here we examined the behavior of centromere proteins in living meiotic cells of S. pombe. We show that the Nuf2-Ndc80 complex proteins (Nuf2, Ndc80, Spc24, and Spc25) disappear from the centromere in meiotic prophase when the centromeres are separated from the SPB. The centromere protein Mis12 also dissociates during meiotic prophase; however, Mis6 remains throughout meiosis. When cells are induced to meiosis by inactivation of Pat1 kinase (a key negative regulator of meiosis), centromeres remain associated with the SPB during meiotic prophase. However, inactivation of Nuf2 by a mutation causes the release of centromeres from the SPB in pat1 mutant cells, suggesting that the Nuf2-Ndc80 complex connects centromeres to the SPB. We further found that removal of the Nuf2-Ndc80 complex from the centromere and centromere-SPB dissociation are caused by mating pheromone signaling. Because pat1 mutant cells also show aberrant chromosome segregation in the first meiotic division and this aberration is compensated by mating pheromone signaling, dissociation of the Nuf2-Ndc80 complex may be associated with remodeling of the kinetochore for meiotic chromosome segregation.     

Evolutionary conservation of recombination proteins and variability of meiosis-specific proteins of chromosomes

A comparison of amino acid sequences is performed for orthologs to the meiosis-specific proteins in humans and seven other species, including animals, fungi, and plants that serve as models for the study of molecular mechanisms of meiosis. It is demonstrated that the RAD51 recombination mediator protein is the most conserved of the studied proteins. Its meiotic homolog DMC1 is less conserved, like the MHL1 mismatch-repair protein. The meiosis-specific SPO11 endonuclease is the least conserved among the studied meiotic enzymes. Structural proteins of meiotic chromosomes are poorly conserved. REC8 meiotic cohesin has 6 times lower similarity in the organisms from different kingdoms than its somatic homolog RAD21. The intermediate conservation level is characteristic of the synaptonemal complex proteins containing HORMA domain. Two functional domains of SPO11 endonuclease and MutL Trans_MLH1 domain of MLH1 enzyme are equally or even less conserved than the whole proteins. HORMA functional domain of a number of synaptonemal complex proteins is only 2–3 times more conserved than the whole molecule. Thus, among the key meiotic proteins, the most conserved are proteins responsible for the accuracy of meiotic recombination. Cohesins, synaptonemal complex proteins, and meiosis-specific SPO11 endonuclease are less conserved even within their functional domains. Obviously, the meiosis-specific proteins have undergone independent evolution in different phylogenetic lineages of eukaryotes.     

The genetic analysis of meiosis in female Drosophila melanogaster.

The three major features of meiosis are first synapsis, then exchange, and finally, disjunction of homologous chromosomes; these phenomena occur before pachytene, during pachytene, and after pachytene respectively. The effects of meiotic mutants, or other perturbations, either endogenous or exogenous, on the meiotic process may be assigned tentatively to one of these intervals, based on the earliest discernible abnormality. Thus mutants exhibiting abnormal disjunction and normal exchange affect post-pachytene functions; mutants exhibiting abnormal disjunction and exchange but with ultrastructurally normal appearing synaptonemal complex affect pachytene functions; and mutants with abnormal disjunction, exchange, and synaptonemal complex affect prepachytene functions. This rationale is applied to the temporal seriation of effects of meiotic mutants and chromosomal abnormalities on the meiotic programme.     

In silico identification and characterization of meiotic DNA: AluJb possibly participates in the attachment of chromatin loops to synaptonemal complex

Earlier, using bioinformatic methods, we reported the identification of repeated DNA sequences (RS), presumably responsible for the attachment of chromatin loops to the lateral elements of synaptonemal complex in meiotic chromosomes. In the present study, consensus sequences for this class of RS were identified. It was demonstrated that at least part of these sequences belonged to the AluJb subfamily of Alu sequences. The Alu copies distribution along the major human histocompatibility complex (MHC) and their spatial separation from the sites of meiotic recombination was examined. It was demonstrated that simple sequences, like (GC/CA)n, were flanking meiotic recombination sites. A model of the RS organization in meiotic chromosome, most efficiently linking experimental data on the meiotic recombination in MHC and the in silico data on the RS localization (the coefficient of multiple correlation, r = 0.92) is suggested.     

Couples,pairs, and clusters: mechanisms and implications of centromere associations in meiosis

Observations of a wide range of organisms show that the centromeres form associations of pairs or small groups at different stages of meiotic prophase. Little is known about the functions or mechanisms of these associations, but in many cases, synaptonemal complex elements seem to play a fundamental role. Two main associations are observed: homology-independent associations very early in the meiotic program—sometimes referred to as centromere coupling—and a later association of homologous centromeres, referred to as centromere pairing or tethering. The later centromere pairing initiates during synaptonemal complex assembly, then persists after the dissolution of the synaptonemal complex. While the function of the homology-independent centromere coupling remains a mystery, centromere pairing appears to have a direct impact on the chromosome segregation fidelity of achiasmatic chromosomes. Recent work in yeast, Drosophila, and mice suggest that centromere pairing is a previously unappreciated, general meiotic feature that may promote meiotic segregation fidelity of the exchange and non-exchange chromosomes.     

Asymmetric division: a kinesin for spindle positioning

The meiotic spindles of animal eggs move to extremely asymmetric positions, close to the cell cortex. A recent paper has identified a motor complex that may move the meiotic spindle toward the cortex in Caenorhabditis elegans eggs.     

Arp2/3 complex regulates asymmetric division and cytokinesis in mouse oocytes

Mammalian oocyte meiotic maturation involves oocyte polarization and a unique asymmetric division, but until now, the underlying mechanisms have been poorly understood. Arp2/3 complex has been shown to regulate actin nucleation and is widely involved in a diverse range of processes such as cell locomotion, phagocytosis and the establishment of cell polarity. Whether Arp2/3 complex participates in oocyte polarization and asymmetric division is unknown. The present study investigated the expression and functions of Arp2/3 complex during mouse oocyte meiotic maturation. Immunofluorescent staining showed that the Arp2/3 complex was restricted to the cortex, with a thickened cap above the meiotic apparatus, and that this localization pattern was depended on actin. Disruption of Arp2/3 complex by a newly-found specific inhibitor CK666, as well as by Arpc2 and Arpc3 RNAi, resulted in a range of effects. These included the failure of asymmetric division, spindle migration, and the formation and completion of oocyte cytokinesis. The formation of the actin cap and cortical granule-free domain (CGFD) was also disrupted, which further confirmed the disruption of spindle migration. Our data suggest that the Arp2/3 complex probably regulates oocyte polarization through its effect on spindle migration, asymmetric division and cytokinesis during mouse oocyte meiotic maturation.     

Chromatin organization at meiosis

From 1956, when the complex ultrastructure of meiotic chromosomes was discovered, ¹ until 1985, when the isolation of meiotic chromosome cores was reported, knowledge of the molecular structure of the meiotic chromosome was at best a dream. The dissection of meiotic chromosome structures has become a realistic challenge through the arrival of isolated symptonemal complexes (SCs), monoclonal and polyclonal antibodies against SCs, the possibility for screening expression libraries for genes that encode SC proteins, the isolation of SC-associated DNA, and the development of techniques for the in situ recognition of DNA sequences in the context of the meiotic chromosome structure.     

精子发生过程中组蛋白甲基化和乙酰化

精子发生(Spermatogenesis)这一高度复杂的独特分化过程包括精原细胞发育为精母细胞、单倍体精细胞的形成和精子成熟,并以阶段特异性和睾丸特异性基因的表达、有丝分裂和减数分裂以及组蛋白向鱼精蛋白的转变为特征。表观遗传修饰在减数分裂重组、联会复合物的形成、姊妹染色体的结合、减数分裂后精子的变态、基因表达阻遏和异染色质形成过程中发挥着重要作用。其中具有一定组成形式、起抑制作用和/或激活作用的组蛋白甲基化和乙酰化标记,不仅保证了正确的染色体配对和二价染色体的成功分离,并且精确调节减数分裂特异性基因的适时表达。精子发生过程中组蛋白甲基化和/或乙酰化错误会直接影响表观遗传修饰的建立和维持,导致生精细胞异常甚至引发不育。文章旨在对精子发生过程中组蛋白甲基化和乙酰化表观遗传修饰的动态变化及其相关酶的调节机制进行综述,为进一步研究精子发生的表观遗传调控,预防男性不育疾病的发生提供基础资料。     

The Saccharomyces cerevisiae MUM2 gene interacts with the DNA replication machinery and is required for meiotic levels of double strand breaks

The Saccharomyces cerevisiae MUM2 gene is essential for meiotic, but not mitotic, DNA replication and thus sporulation. Genetic interactions between MUM2 and a component of the origin recognition complex and polymerase alpha-primase suggest that MUM2 influences the function of the DNA replication machinery. Early meiotic gene expression is induced to a much greater extent in mum2 cells than in meiotic cells treated with the DNA synthesis inhibitor hydroxyurea. This result indicates that the mum2 meiotic arrest is downstream of the arrest induced by hydroxyurea and suggests that DNA synthesis is initiated in the mutant. Genetic analyses indicate that the recombination that occurs in mum2 mutants is dependent on the normal recombination machinery and on synaptonemal complex components and therefore is not a consequence of lesions created by incompletely replicated DNA. Both meiotic ectopic and allelic recombination are similarly reduced in the mum2 mutant, and the levels are consistent with the levels of meiosis-specific DSBs that are generated. Cytological analyses of mum2 mutants show that chromosome pairing and synapsis occur, although at reduced levels compared to wild type. Given the near-wild-type levels of meiotic gene expression, pairing, and synapsis, we suggest that the reduction in DNA replication is directly responsible for the reduced level of DSBs and meiotic recombination.     

Arp2/3 Complex Inhibition Prevents Meiotic Maturation in Porcine Oocytes

The Arp2/3 complex regulates actin nucleation, which is critical for a wide range of cellular processes, such as cell polarity, cell locomotion, and endocytosis. In the present study, we investigated the possible roles of the Arp2/3 complex in porcine oocytes during meiotic maturation. Immunofluorescent staining showed the Arp2/3 complex to localize mainly to the cortex of porcine oocytes, colocalizing with actin. Treatment with an Arp2/3 complex specific inhibitor, CK666, resulted in a decrease in Arp2/3 complex localization at the oocyte cortex. The maturation rate of porcine oocytes decreased significantly after CK666 treatment, concomitant with the failure of cumulus cell expansion and oocyte polar body extrusion. The fluorescence intensity of F-actin decreased in the cytoplasm, and CK666 also disrupted actin cap formation. In summary, our results illustrate that the Arp2/3 complex is required for the meiotic maturation of porcine oocytes and that actin nucleation is critical for meiotic maturation.     

Mammalian meiotic telomeres: protein composition and redistribution in relation to nuclear pores

Mammalian telomeres consist of TTAGGG repeats, telomeric repeat binding factor (TRF), and other proteins, resulting in a protective structure at chromosome ends. Although structure and function of the somatic telomeric complex has been elucidated in some detail, the protein composition of mammalian meiotic telomeres is undetermined. Here we show, by indirect immunofluorescence (IF), that the meiotic telomere complex is similar to its somatic counterpart and contains significant amounts of TRF1, TRF2, and hRap1, while tankyrase, a poly-(ADP-ribose)polymerase at somatic telomeres and nuclear pores, forms small signals at ends of human meiotic chromosome cores. Analysis of rodent spermatocytes reveals Trf1 at mouse, TRF2 at rat, and mammalian Rap1 at meiotic telomeres of both rodents. Moreover, we demonstrate that telomere repositioning during meiotic prophase occurs in sectors of the nuclear envelope that are distinct from nuclear pore-dense areas. The latter form during preleptotene/leptotene and are present during entire prophase I.