Meiotic telomere protein Ndj1p is required for meiosis-specific telomere distribution, bouquet formation and efficient homologue pairing

We have investigated the requirements for NDJ1 in meiotic telomere redistribution and clustering in synchronized cultures of Saccharomyces cerevisiae. On induction of wild-type meiosis, telomeres disperse from premeiotic aggregates over the nuclear periphery, and then cluster near the spindle pole body (bouquet arrangement) before dispersing again. In ndj1Delta meiocytes, telomeres are scattered throughout the nucleus and fail to form perinuclear meiosis-specific distribution patterns, suggesting that Ndj1p may function to tether meiotic telomeres to the nuclear periphery. Since ndj1Delta meiocytes fail to cluster their telomeres at any prophase stage, Ndj1p is the first protein shown to be required for bouquet formation in a synaptic organism. Analysis of homologue pairing by two-color fluorescence in situ hybridization with cosmid probes to regions on III, IX, and XI revealed that disruption of bouquet formation is associated with a significant delay (>2 h) of homologue pairing. An increased and persistent fraction of ndj1Delta meiocytes with Zip1p polycomplexes suggests that chromosome polarization is important for synapsis progression. Thus, our observations support the hypothesis that meiotic telomere clustering contributes to efficient homologue alignment and synaptic pairing. Under naturally occurring conditions, bouquet formation may allow for rapid sporulation and confer a selective advantage.     

The Arabidopsis MutS homolog AtMSH5 is required for normal meiosis

MSH5, a member of the MutS homolog DNA mismatch repair protein family, has been shown to be required for proper homologous chromosome recombination in diverse organisms such as mouse, budding yeast and Caenorhabditis elegans. In this paper, we show that a mutant Arabidopsis plant carrying the putative disrupted AtMSH5 gene exhibits defects during meiotic division, producing a proportion of nonviable pollen grains and abnormal embryo sacs, and thereby leading to a decrease in fertility. AtMSH5 expression is confined to meiotic floral buds, which is consistent with a possible role during meiosis. Cytological analysis of male meiosis revealed the presence of numerous univalents from diplotene to metaphase I, which were associated with a great reduction in chiasma frequencies. The average number of residual chiasmata in the mutant is reduced to 2.54 per meiocyte, which accounts for approximately 25% of the amount in the wild type. Here, quantitative cytogenetical analysis reveals that the residual chiasmata in Atmsh5 mutants are randomly distributed among meiocytes, suggesting that AtMSH5 has an essential role during interference-sensitive chiasma formation. Taken together, the evidence indicates that AtMSH5 promotes homologous recombination through facilitating chiasma formation during prophase I in Arabidopsis.     

Identification and analysis of DYAD: a gene required for meiotic chromosome organisation and female meiotic progression in Arabidopsis

The dyad mutant of Arabidopsis was previously identified as being defective in female meiosis. We report here the analysis of the DYAD gene. In ovules and anthers DYAD RNA is detected specifically in female and male meiocytes respectively, in premeiotic interphase/meiotic prophase. Analysis of chromosome spreads in female meiocytes showed that in the mutant, chromosomes did not undergo synapsis and formed ten univalents instead of five bivalents. Unlike mutations in AtDMC1 and AtSPO11 which also affect bivalent formation as the univalent chromosomes segregate randomly, the dyad univalents formed an ordered metaphase plate and underwent an equational division. This suggests a requirement for DYAD for chromosome synapsis and centromere configuration in female meiosis. The dyad mutant showed increased and persistent expression of a meiosis-specific marker, pAtDMC1::GUS during female meiosis, indicative of defective meiotic progression. The sequence of the putative protein encoded by DYAD did not reveal strong similarity to other proteins. DYAD is therefore likely to encode a novel protein required for meiotic chromosome organisation and female meiotic progression.     

STAG3‐mediated stabilization of REC8 cohesin complexes promotes chromosome synapsis during meiosis

Cohesion between sister chromatids in mitotic and meiotic cells is promoted by a ring‐shaped protein structure, the cohesin complex. The cohesin core complex is composed of four subunits, including two structural maintenance of chromosome (SMC) proteins, one α‐kleisin protein, and one SA protein. Meiotic cells express both mitotic and meiosis‐specific cohesin core subunits, generating cohesin complexes with different subunit composition and possibly separate meiotic functions. Here, we have analyzed the in vivo function of STAG3, a vertebrate meiosis‐specific SA protein. Mice with a hypomorphic allele of Stag3, which display a severely reduced level of STAG3, are viable but infertile. We show that meiocytes in homozygous mutant Stag3 mice display chromosome axis compaction, aberrant synapsis, impaired recombination and developmental arrest. We find that the three different α‐kleisins present in meiotic cells show different dosage‐dependent requirements for STAG3 and that STAG3‐REC8 cohesin complexes have a critical role in supporting meiotic chromosome structure and functions.     

OsREC8 is essential for chromatid cohesion and metaphase I monopolar orientation in rice meiosis

The successful transmission of chromosomes during mitosis and meiosis relies on the establishment and subsequent release of cohesion between replicated chromatids. Cohesion is mediated by a four-subunit structural maintenance of chromosome complex, called cohesins. REC8 is a key component of this meiotic cohesion complex in most model organisms studied to date. Here, we isolated and dissected the functions of OsREC8, a rice (Oryza sativa) REC8 homolog, using two null Osrec8 mutants. We showed that OsREC8 encodes a protein that localized to meiotic chromosomes from approximately meiotic interphase to metaphase I. Homologous pairing and telomere bouquet formation were abnormal in Osrec8 meiocytes. Furthermore, fluorescent in situ hybridization experiments on Osrec8 meiocytes demonstrated that the mutation eliminated meiotic centromeric cohesion completely during prophase I and also led to the bipolar orientation of the kinetochores during the first meiotic division and accordingly resulted in premature separation of sister chromatid during meiosis I. Immunolocalization analyses revealed that the loading of PAIR2, PAIR3, OsMER3, and ZEP1 all depended on OsREC8. By contrast, the presence of the OsREC8 signal in pair2, pair3, Osmer3, and zep1 mutants indicated that the loading of OsREC8 did not rely on these four proteins. These results suggest that OsREC8 has several essential roles in the meiotic processes.     

Mouse TRIP13/PCH2 Is Required for Recombination and Normal Higher-Order Chromosome Structure during Meiosis

Accurate chromosome segregation during meiosis requires that homologous chromosomes pair and become physically connected so that they can orient properly on the meiosis I spindle. These connections are formed by homologous recombination closely integrated with the development of meiosis-specific, higher-order chromosome structures. The yeast Pch2 protein has emerged as an important factor with roles in both recombination and chromosome structure formation, but recent analysis suggested that TRIP13, the mouse Pch2 ortholog, is not required for the same processes. Using distinct Trip13 alleles with moderate and severe impairment of TRIP13 function, we report here that TRIP13 is required for proper synaptonemal complex formation, such that autosomal bivalents in Trip13-deficient meiocytes frequently displayed pericentric synaptic forks and other defects. In males, TRIP13 is required for efficient synapsis of the sex chromosomes and for sex body formation. Furthermore, the numbers of crossovers and chiasmata are reduced in the absence of TRIP13, and their distribution along the chromosomes is altered, suggesting a role for TRIP13 in aspects of crossover formation and/or control. Recombination defects are evident very early in meiotic prophase, soon after DSB formation. These findings provide evidence for evolutionarily conserved functions for TRIP13/Pch2 in both recombination and formation of higher order chromosome structures, and they support the hypothesis that TRIP13/Pch2 participates in coordinating these key aspects of meiotic chromosome behavior.     

The DUET gene is necessary for chromosome organization and progression during male meiosis in Arabidopsis and encodes a PHD finger protein

Progression through the meiotic cell cycle is an essential part of the developmental program of sporogenesis in plants. The duet mutant of Arabidopsis was identified as a male sterile mutant that lacked pollen and underwent an aberrant male meiosis. Male meiocyte division resulted in the formation of two cells instead of a normal tetrad. In wild type, male meiosis extends across two successive bud positions in an inflorescence whereas in duet, meiotic stages covered three to five bud positions indicating defective progression. Normal microspores were absent in the mutant and the products of the aberrant meiosis were uni- to tri-nucleate cells that later degenerated, resulting in anthers containing largely empty locules. Defects in male meiotic chromosome organization were observed starting from diplotene and extending to subsequent stages of meiosis. There was an accumulation of meiotic structures at metaphase 1, suggesting an arrest in cell cycle progression. Double mutant analysis revealed interaction with dyad, a mutation causing chromosome cohesion during female meiosis. Cloning and molecular analysis of DUET indicated that it potentially encodes a PHD-finger protein and shows specific expression in male meiocytes. Taken together these data suggest that DUET is required for male meiotic chromosome organization and progression.     

Inter-homolog crossing-over and synapsis in Arabidopsis meiosis are dependent on the chromosome axis protein AtASY3

In this study we have analysed AtASY3, a coiled-coil domain protein that is required for normal meiosis in Arabidopsis. Analysis of an Atasy3-1 mutant reveals that loss of the protein compromises chromosome axis formation and results in reduced numbers of meiotic crossovers (COs). Although the frequency of DNA double-strand breaks (DSBs) appears moderately reduced in Atasy3-1, the main recombination defect is a reduction in the formation of COs. Immunolocalization studies in wild-type meiocytes indicate that the HORMA protein AtASY1, which is related to Hop1 in budding yeast, forms hyper-abundant domains along the chromosomes that are spatially associated with DSBs and early recombination pathway proteins. Loss of AtASY3 disrupts the axial organization of AtASY1. Furthermore we show that the AtASY3 and AtASY1 homologs BoASY3 and BoASY1, from the closely related species Brassica oleracea, are co-immunoprecipitated from meiocyte extracts and that AtASY3 interacts with AtASY1 via residues in its predicted coiled-coil domain. Together our results suggest that AtASY3 is a functional homolog of Red1. Since studies in budding yeast indicate that Red1 and Hop1 play a key role in establishing a bias to favor inter-homolog recombination (IHR), we propose that AtASY3 and AtASY1 may have a similar role in Arabidopsis. Loss of AtASY3 also disrupts synaptonemal complex (SC) formation. In Atasy3-1 the transverse filament protein AtZYP1 forms small patches rather than a continuous SC. The few AtMLH1 foci that remain in Atasy3-1 are found in association with the AtZYP1 patches. This is sufficient to prevent the ectopic recombination observed in the absence of AtZYP1, thus emphasizing that in addition to its structural role the protein is important for CO formation.     

Antibodies directed against a meiosis-specific,chromatin-associated protein identify conserved meiotic epitopes

The molecular mechanisms by which meiotic events are regulated are at present unknown. To approach this problem, we have exploited the natural synchrony of Lilium meiocytes to compare the nuclear protein profiles of a variety of stages of meiosis. This approach has facilitated the identification of a number of nuclear proteins that appear and disappear in a stagespecific fashion. Here we report the presence of an abundant nuclear protein that first appears during premeiotic interphase, a period during which the irreversible commitment to meiosis occurs. Antibodies directed against this protein demonstrate its meiosis specificity as well as conservation of the epitope(s) in both mono-and dicotyledonous plant species. Chromatin fractionation studies indicate that this protein, which we have termed meiotin-1, is associated with strings of nucleosomes. Implications for meiotic chromatin packaging and chromosome structure are discussed.by J.H. Taylor     

Differentiated function and localisation of SPO11-1 and PRD3 on the chromosome axis during meiotic DSB formation in Arabidopsis thaliana

During meiosis, DNA double-strand breaks (DSBs) occur throughout the genome, a subset of which are repaired to form reciprocal crossovers between chromosomes. Crossovers are essential to ensure balanced chromosome segregation and to create new combinations of genetic variation. Meiotic DSBs are formed by a topoisomerase-VI-like complex, containing catalytic (e.g. SPO11) proteins and auxiliary (e.g. PRD3) proteins. Meiotic DSBs are formed in chromatin loops tethered to a linear chromosome axis, but the interrelationship between DSB-promoting factors and the axis is not fully understood. Here, we study the localisation of SPO11-1 and PRD3 during meiosis, and investigate their respective functions in relation to the chromosome axis. Using immunocytogenetics, we observed that the localisation of SPO11-1 overlaps relatively weakly with the chromosome axis and RAD51, a marker of meiotic DSBs, and that SPO11-1 recruitment to chromatin is genetically independent of the axis. In contrast, PRD3 localisation correlates more strongly with RAD51 and the chromosome axis. This indicates that PRD3 likely forms a functional link between SPO11-1 and the chromosome axis to promote meiotic DSB formation. We also uncovered a new function of SPO11-1 in the nucleation of the synaptonemal complex protein ZYP1. We demonstrate that chromosome co-alignment associated with ZYP1 deposition can occur in the absence of DSBs, and is dependent on SPO11-1, but not PRD3. Lastly, we show that the progression of meiosis is influenced by the presence of aberrant chromosomal connections, but not by the absence of DSBs or synapsis. Altogether, our study provides mechanistic insights into the control of meiotic DSB formation and reveals diverse functional interactions between SPO11-1, PRD3 and the chromosome axis.     

Functional characterization of kinetochore protein,Ctf19 in meiosis I: an implication of differential impact of Ctf19 on the assembly of mitotic and meiotic kinetochores in Saccharomyces cerevisiae

Meiosis is a specialized cell division process through which chromosome numbers are reduced by half for the generation of gametes. Kinetochore, a multiprotein complex that connects centromeres to microtubules, plays essential role in chromosome segregation. Ctf19 is the key central kinetochore protein that recruits all the other non‐essential proteins of the Ctf19 complex in budding yeast. Earlier studies have shown the role of Ctf19 complex in enrichment of cohesin around the centromeres both during mitosis and meiosis, leading to sister chromatid cohesion and meiosis II disjunction. Here we show that Ctf19 is also essential for the proper execution of the meiosis I specific unique events, such as non‐homologous centromere coupling, homologue pairing, chiasmata resolution and proper orientation of homologues and sister chromatids with respect to the spindle poles. Additionally, this investigation reveals that proper kinetochore function is required for faithful chromosome condensation in meiosis. Finally, this study suggests that absence of Ctf19 affects the integrity of meiotic kinetochore differently than that of the mitotic kinetochore. Consequently, absence of Ctf19 leads to gross chromosome missegregation during meiosis as compared with mitosis. Hence, this study reports for the first time the differential impact of a non‐essential kinetochore protein on the mitotic and meiotic kinetochore ensembles and hence chromosome segregation.     

Meiotic telomere clustering requires actin for its formation and cohesin for its resolution

In diploid organisms, meiosis reduces the chromosome number by half during the formation of haploid gametes. During meiotic prophase, telomeres transiently cluster at a limited sector of the nuclear envelope (bouquet stage) near the spindle pole body (SPB). Cohesin is a multisubunit complex that contributes to chromosome segregation in meiosis I and II divisions. In yeast meiosis, deficiency for Rec8 cohesin subunit induces telomere clustering to persist, whereas telomere cluster-SPB colocalization is defective. These defects are rescued by expressing the mitotic cohesin Scc1 in rec8delta meiosis, whereas bouquet-stage exit is independent of Cdc5 pololike kinase. An analysis of living Saccharomyces cerevisiae meiocytes revealed highly mobile telomeres from leptotene up to pachytene, with telomeres experiencing an actin- but not microtubule-dependent constraint of mobility during the bouquet stage. Our results suggest that cohesin is required for exit from actin polymerization-dependent telomere clustering and for linking the SPB to the telomere cluster in synaptic meiosis.     

A puromycin-sensitive aminopeptidase is essential for meiosis in Arabidopsis thaliana

Puromycin-sensitive aminopeptidases (PSAs) participate in a variety of proteolytic events essential for cell growth and viability, and in fertility in a broad range of organisms. We have identified and characterized an Arabidopsis thaliana mutant (mpa1) from a pool of T-DNA tagged lines that lacks PSA activity. This line exhibits reduced fertility, producing shorter siliques (fruits) bearing a lower number of seeds compared with wild-type plants. Cytogenetic characterization of meiosis in the mutant line reveals that both male and female meiosis are defective. In mpa1, early prophase I appears normal, but after pachytene most of the homologous chromosomes are desynaptic, thus, by metaphase I a high level of univalence is observed subsequently leading to abnormal chromosome segregation. Wild-type plants treated with specific inhibitors of PSA show a very similar desynaptic phenotype to that of the mutant line. A fluorescent PSA-specific bioprobe, DAMPAQ-22, reveals that the protein is maximally expressed in wild-type meiocytes during prophase I and is absent in mpa1. Immunolocalization of meiotic proteins showed that the meiotic recombination pathway is disrupted in mpa1. Chromosome pairing and early recombination appears normal, but progression to later stages of recombination and complete synapsis of homologous chromosomes are blocked.     

Chromosome synapsis defects and sexually dimorphic meiotic progression in mice lacking Spo11

Spo11, a protein first identified in yeast, is thought to generate the chromosome breaks that initiate meiotic recombination. We now report that disruption of mouse Spo11 leads to severe gonadal abnormalities from defective meiosis. Spermatocytes suffer apoptotic death during early prophase; oocytes reach the diplotene/dictyate stage in nearly normal numbers, but most die soon after birth. Consistent with a conserved function in initiating meiotic recombination, Dmc1/Rad51 focus formation is abolished. Spo11(-/-) meiocytes also display homologous chromosome synapsis defects, similar to fungi but distinct from flies and nematodes. We propose that recombination initiation precedes and is required for normal synapsis in mammals. Our results also support the view that mammalian checkpoint responses to meiotic recombination and/or synapsis defects are sexually dimorphic.     

MULTIPOLAR SPINDLE 1 (MPS1), a novel coiled-coil protein of Arabidopsis thaliana, is required for meiotic spindle organization

The spindle is essential for chromosome segregation during meiosis, but the molecular mechanism of meiotic spindle organization in higher plants is still not well understood. Here, we report on the identification and characterization of a plant-specific protein, MULTIPOLAR SPINDLE 1 (MPS1), which is involved in spindle organization in meiocytes of Arabidopsis thaliana . The homozygous mps1 mutant exhibits male and female sterility. Light microscopy showed that mps1 mutants produced multiple uneven spores during anther development, most of which aborted in later stages. Cytological analysis showed that chromosome segregation was abnormal in mps1 meiocytes. Immunolocalization showed unequal bipolar or multipolar spindles in mps1 meiocytes, which indicated that aberrant spindles resulted in disordered chromosome segregation. MPS1 encodes a 377-amino-acid protein with putative coiled-coil motifs. In situ hybridization analysis showed that MPS1 is strongly expressed in meiocytes.     

Homologous chromosome interactions in meiosis: diversity amidst conservation

Proper chromosome segregation is crucial for preventing fertility problems, birth defects and cancer. During mitotic cell divisions, sister chromatids separate from each other to opposite poles, resulting in two daughter cells that each have a complete copy of the genome. Meiosis poses a special problem in which homologous chromosomes must first pair and then separate at the first meiotic division before sister chromatids separate at the second meiotic division. So, chromosome interactions between homologues are a unique feature of meiosis and are essential for proper chromosome segregation. Pairing and locking together of homologous chromosomes involves recombination interactions in some cases, but not in others. Although all organisms must match and lock homologous chromosomes to maintain genome integrity throughout meiosis, recent results indicate that the underlying mechanisms vary in different organisms.     

TopBP1 deficiency impairs the localization of proteins involved in early recombination and results in meiotic chromosome defects during spermatogenesis

Topoisomerase IIβ-binding protein 1 (TopBP1) is BRCT domain-containing protein that is required for DNA double-strand break (DSB) repair and DNA damage responses; however, its function during the early stage of spermatogenesis is still unclear. To investigate the physiological role of TopBP1, we have generated germ cell-specific TopBP1-depleted mouse model. TopBP1-deleted mice were infertile, showed a loss of germ cells and had meiotic defects. Conditional TopBP1 deletion resulted in reduced testis size, reduced number of epididymal sperm, increased apoptosis, and severely compromised fertility. TopBP1 deficiency caused defects in DMC1 and Rad51 foci formation, abnormal synaptonemal complexes and meiotic chromosome defects. Collectively, these results suggest that TopBP1 deficiency during spermatogenesis impairs the localization of proteins involved in early recombination at DSBs, results in meiotic chromosome defects and leads to infertility.