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Objective: This study aimed to explore the relationship between cohesin subunit REC8 reduction and meiosis chromosome segregation errors in the ovary. Methods: Rec8+/− mice were generated using CRIPSR/Cas9 gene editing. The association between age and REC8 expression levels in the ovary was determined by Western blotting. Chromosome segregation errors were investigated by immunofluorescence imaging of superovulated oocytes. Wild-type and Rec8+/− female mice at 5, 8, 20, 36, and 40 weeks were used to evaluate ovarian reserve by ovarian clearing and immunolabeling. Results: Ovary REC8 expression levels gradually decreased with age, while chromosome segregation errors increased with age. Segregation errors were more common in Rec8+/− mice, suggesting an association with REC8 expression. The ovarian reserve capacity decreased significantly with age. The ovarian reserve in Rec8+/− mice was inferior to that of age-matched wild-type mice, indicating important roles of age and REC8 levels in the ovarian reserve. Conclusions: REC8 reduction has an age-cumulative effect on meiotic chromosome segregation errors in mouse ovaries. Rec8 haploinsufficiency poses a major challenge in generating normal and reproductive oocytes in aging mice.

Homologous recombination (HR) is a major, conserved pathway of chromosome damage repair. It not only fulfills key functions in the removal of deleterious lesions such as DNA double-strand breaks (DSBs) and interstrand cross-links (ICLs), but also in replication fork repair and protection. Several familial and acquired cancer predisposition syndromes stem from defects in HR. In particular, individuals with mutations in HR genes exhibit predisposition to breast, ovarian, pancreatic, and prostate cancers, and they also show signs of accelerated aging. However, aberrant and untimely HR events can lead to the loss of heterozygosity, genomic rearrangements, and cytotoxic nucleoprotein intermediates. Thus, it is critically important that HR be tightly regulated. In addition to DNA repair, HR is also involved in meiotic chromosome segregation and telomere maintenance in cells that lack telomerase. In this review, we focus on the role of HR in DSB repair (DSBR) and summarize the current state of the field.

The synaptonemal complex (SC) is a meiosis-specific proteinaceous macromolecular structure that assembles between paired homologous chromosomes during meiosis in various eukaryotes. The SC has a highly conserved ultrastructure and plays critical roles in controlling multiple steps in meiotic recombination and crossover formation, ensuring accurate meiotic chromosome segregation. Recent studies in different organisms, facilitated by advances in super-resolution microscopy, have provided insights into the macromolecular structure of the SC, including the internal organization of the meiotic chromosome axis and SC central region, the regulatory pathways that control SC assembly and dynamics, and the biological functions exerted by the SC and its substructures. This review summarizes recent discoveries about how the SC is organized and regulated that help to explain the biological functions associated with this meiosis-specific structure.

The number and distribution of meiotic crossovers (COs) are highly regulated, reflecting the requirement for COs during the first round of meiotic chromosome segregation. CO control includes CO assurance and CO interference, which promote at least one CO per chromosome bivalent and evenly-spaced COs, respectively. Previous studies revealed a role for the DNA damage response (DDR) clamp and the clamp loader in CO formation by promoting interfering COs and interhomolog recombination, and also by suppressing ectopic recombination. In this study, we use classical tetrad analysis of Saccharomyces cerevisiae to show that a mutant defective in RAD24, which encodes the DDR clamp loader (RAD17 in other organisms), displayed reduced CO frequencies on two shorter chromosomes (III and V), but not on a long chromosome (chromosome VII). The residual COs in the rad24 mutant do not show interference. In contrast to rad24, mutants defective in the ATR kinase homolog Mec1, including a mec1 null and a mec1 kinase-dead mutant, show slight or few defects in CO frequency. On the other hand, mec1 COs show defects in interference, similar to the rad24 mutant. Our results support a model in which the DDR clamp and clamp-loader proteins promote interfering COs by recruiting pro-CO Zip, Mer, and Msh proteins to recombination sites, while the Mec1 kinase regulates CO distribution by a distinct mechanism. Moreover, CO formation and its control are implemented in a chromosome-specific manner, which may reflect a role for chromosome size in regulation.

Significance Mutations in inner kinetochore components induce centromere repositioning without alteration in the centromeric DNA sequence, revealing a feedback mechanism underlying the high epigenetic stability of the centromere. This also provides a desirable experimental system to explore the functional significance of centromere positioning in meiosis. We discovered that in a heterozygotic meiosis, a repositioned centromere generates a reproductive barrier, suggesting a functional role of evolutionary new centromeres in speciation; furthermore, in a homozygotic meiosis, chromosomes carrying repositioned centromeres frequently undergo the 2 stages of meiotic segregation in an inverted order, demonstrating high flexibility in the meiotic process., The chromosomal position of each centromere is determined epigenetically and is highly stable, whereas incremental cases have supported the occurrence of centromere repositioning on an evolutionary time scale (evolutionary new centromeres, ENCs), which is thought to be important in speciation. The mechanisms underlying the high stability of centromeres and its functional significance largely remain an enigma. Here, in the fission yeast Schizosaccharomyces pombe, we identify a feedback mechanism: The kinetochore, whose assembly is guided by the centromere, in turn, enforces centromere stability. Upon going through meiosis, specific inner kinetochore mutations induce centromere repositioning—inactivation of the original centromere and formation of a new centromere elsewhere—in 1 of the 3 chromosomes at random. Repositioned centromeres reside asymmetrically in the pericentromeric regions and cells carrying them are competent in mitosis and homozygotic meiosis. However, when cells carrying a repositioned centromere are crossed with those carrying the original centromere, the progeny suffer severe lethality due to defects in meiotic chromosome segregation. Thus, repositioned centromeres constitute a reproductive barrier that could initiate genetic divergence between 2 populations with mismatched centromeres, documenting a functional role of ENCs in speciation. Surprisingly, homozygotic repositioned centromeres tend to undergo meiosis in an inverted order—that is, sister chromatids segregate first, and homologous chromosomes separate second—whereas the original centromeres on other chromosomes in the same cell undergo meiosis in the canonical order, revealing hidden flexibility in the perceived rigid process of meiosis.

Germ cell-derived genomic structure variants not only drive the evolution of species but also induce developmental defects in offspring. The genomic structure variants have different types, but most of them are originated from DNA double-strand breaks (DSBs). It is still not well known whether DNA DSBs exist in adult mammalian oocytes and how the growing and fully grown oocytes repair their DNA DSBs induced by endogenous or exogenous factors. In this study, we detected the endogenous DNA DSBs in the growing and fully grown mouse oocytes and found that the DNA DSBs mainly localized at the centromere-adjacent regions, which are also copy number variation hotspots. When the exogenous DNA DSBs were introduced by Etoposide, we found that Rad51-mediated homologous recombination (HR) was used to repair the broken DNA. However, the HR repair caused the chromatin intertwined and impaired the homologous chromosome segregation in oocytes. Although we had not detected the indication about HR repair of endogenous centromere-adjacent DNA DSBs, we found that Rad52 and RNA:DNA hybrids colocalized with these DNA DSBs, indicating that a Rad52-dependent DNA repair might exist in oocytes. In summary, our results not only demonstrated an association between endogenous DNA DSBs with genomic structure variants but also revealed one specific DNA DSB repair manner in oocytes.

Errors during meiosis are the leading cause of birth defects and miscarriages in humans. Thus, the coordinated control of meiotic events is critical for the faithful inheritance of the genome with each generation..., Crossovers (COs) between homologous chromosomes are critical for meiotic chromosome segregation and form in the context of the synaptonemal complex (SC), a meiosis-specific structure that assembles between aligned homologs. During Caenorhabditis elegans meiosis, central region components of the SC (SYP proteins) are essential to repair double-strand DNA breaks (DSBs) as COs. Here, we investigate the relationships between the SYP proteins and conserved pro-CO factors by examining the immunolocalization of these proteins in meiotic mutants where SYP proteins are absent, reduced, or mislocalized. Although COs do not form in syp null mutants, pro-CO factors COSA-1, MSH-5, and ZHP-3 nevertheless colocalize at DSB-dependent sites during late prophase, reflecting an inherent affinity of these factors for DSB repair sites. In contrast, in mutants where SYP proteins are present but form aggregates or display abnormal synapsis, pro-CO factors consistently track with SYP-1 localization. Further, pro-CO factors usually localize to a single site per SYP-1 structure, even in SYP aggregates or in mutants where the SC forms between sister chromatids, suggesting that CO regulation occurs within these aberrant SC structures. Moreover, we find that the meiotic cohesin REC-8 is required to ensure that SC formation occurs between homologs and not sister chromatids. Taken together, our findings support a model in which SYP proteins promote CO formation by promoting the localization of pro-CO factors to recombination events within an SC compartment, thereby ensuring that pro-CO factors identify a recombination event within an SC structure and that CO maturation occurs only between properly aligned homologous chromosomes.

Summary Faithful chromosome segregation is required for both mitotic and meiotic cell divisions and is regulated by multiple mechanisms including the anaphase‐promoting complex/cyclosome (APC/C), which is the largest known E3 ubiquitin‐ligase complex and has been implicated in regulating chromosome segregation in both mitosis and meiosis in animals. However, the role of the APC/C during plant meiosis remains largely unknown. Here, we show that Arabidopsis APC8 is required for male meiosis.We used a combination of genetic analyses, cytology and immunolocalisation to define the function of AtAPC8 in male meiosis.Meiocytes from apc8‐1 plants exhibit several meiotic defects including improper alignment of bivalents at metaphase I, unequal chromosome segregation during anaphase II, and subsequent formation of polyads. Immunolocalisation using an antitubulin antibody showed that APC8 is required for normal spindle morphology. We also observed mitotic defects in apc8‐1, including abnormal sister chromatid segregation and microtubule morphology.Our results demonstrate that Arabidopsis APC/C is required for meiotic chromosome segregation and that APC/C‐mediated regulation of meiotic chromosome segregation is a conserved mechanism among eukaryotes.

The epitranscriptome has emerged as a new fundamental layer of control of gene expression. Nevertheless, the determination of the transcriptome-wide occupancy and function of RNA modifications remains challenging. Here we have developed Rho-seq, an integrated pipeline detecting a range of modifications through differential modification-dependent rhodamine labeling. Using Rho-seq, we confirm that the reduction of uridine to dihydrouridine (D) by the Dus reductase enzymes targets tRNAs in E. coli and fission yeast. We find that the D modification is also present on fission yeast mRNAs, particularly those encoding cytoskeleton-related proteins, which is supported by large-scale proteome analyses and ribosome profiling. We show that the α-tubulin encoding mRNA nda2 undergoes Dus3-dependent dihydrouridylation, which affects its translation. The absence of the modification on nda2 mRNA strongly impacts meiotic chromosome segregation, resulting in low gamete viability. Applying Rho-seq to human cells revealed that tubulin mRNA dihydrouridylation is evolutionarily conserved.

Structural maintenance of chromosome 5/6 (SMC5/6) complex is a crucial factor for preserving genome stability. Here, we show that mutants for several Arabidopsis (Arabidopsis thaliana) SMC5/6 complex subunits produce triploid offspring. This phenotype is caused by a meiotic defect leading to the production of unreduced male gametes. The SMC5/6 complex mutants show an absence of chromosome segregation during the first and/or the second meiotic division, as well as a partially disorganized microtubule network. Importantly, although the SMC5/6 complex is partly required for the repair of SPO11-induced DNA double-strand breaks, the nonreduction described here is SPO11-independent. The measured high rate of ovule abortion suggests that, if produced, such defects are maternally lethal. Upon fertilization with an unreduced pollen, the unbalanced maternal and paternal genome dosage in the endosperm most likely causes seed abortion observed in several SMC5/6 complex mutants. In conclusion, we describe the function of the SMC5/6 complex in the maintenance of gametophytic ploidy in Arabidopsis.

Meiotic chromosome segregation relies on synapsis and crossover recombination between homologous chromosomes. These processes require multiple steps that are coordinated by the meiotic cell cycle and monitored by surveillance mechanisms. In diverse species, failures in chromosome synapsis can trigger a cell cycle delay and/or lead to apoptosis. How this key step in “homolog engagement” is sensed and transduced by meiotic cells is unknown. Here we report that inC. elegans, recruitment of the Polo-like kinase PLK-2 to the synaptonemal complex triggers phosphorylation and inactivation of CHK-2, an early meiotic kinase required for pairing, synapsis, and double-strand break induction. Inactivation of CHK-2 ends double-strand break formation and promotes crossover designation and cell cycle progression. These findings illuminate how meiotic cells ensure crossover formation and accurate chromosome segregation.SummaryAccurate chromosome segregation during meiosis requires crossovers between each pair of homologs. Zhanget al. show that meiotic progression inC. elegansinvolves inactivation of CHK-2 by PLK-2 in response to synapsis and formation of crossover precursors on all chromosomes.

DNA topoisomerase I (Top1) maintains chromatin conformation during transcription. While Top1 is not essential in simple eukaryotic organisms such as yeast, it is required for the development of multicellular organisms. In fact, tissue and cell-type-specific functions of Top1 have been suggested in the fruit fly Drosophila. A better understanding of Top1’s function in the context of development is important as Top1 inhibitors are among the most widely used anticancer drugs. As a step toward such a better understanding, we studied its localization in live cells of Drosophila. Consistent with prior results, Top1 is highly enriched at the nucleolus in transcriptionally active polyploid cells, and this enrichment responds to perturbation of transcription. In diploid cells, we uncovered evidence for Top1 foci formation at genomic regions not limited to the active rDNA locus, suggestive of novel regulation of Top1 recruitment. In the male germline, Top1 is highly enriched at the paired rDNA loci on sex chromosomes suggesting that it might participate in regulating their segregation during meiosis. Results from RNAi-mediated Top1 knockdown lend support to this hypothesis. Our study has provided one of the most comprehensive descriptions of Top1 localization during animal development.

Meiotic homolog synapsis is essential to ensure accurate segregation of chromosomes during meiosis. In C. elegans, synapsis and a checkpoint that monitors synapsis relies on the spindle checkpoint components, Mad1 and Mad2, and Pairing Centers (PCs), cis-acting loci that interact with the nuclear envelope to mobilize chromosomes within the nucleus. Here, we show that mutations in some spindle checkpoint mutants affect PC movement early in meiotic prophase, consistent with a link between PC mobility and the regulation of synapsis. Further, we test what specific functions of Mad1 and Mad2 are required to regulate and monitor synapsis. We find that a mutation that abrogates Mad1’s localization to the nuclear periphery abolishes the synapsis checkpoint but has no effect on Mad2’s localization to the nuclear periphery or synapsis. By contrast, a mutation that prevents Mad1’s interaction with Mad2 abolishes the synapsis checkpoint, delays synapsis and fails to localize Mad2 to the nuclear periphery. These data indicate that Mad1’s primary role in regulating synapsis is through control of Mad2 and that Mad2 can bind other factors at the nuclear periphery. We also tested whether Mad2’s ability to adopt a specific conformation associated with its activity during spindle checkpoint function is required for its role in meiosis. A mutation that prevents Mad2 from adopting its active conformer fails to localize to the nuclear periphery, abolishes the synapsis checkpoint and exhibits substantial defects in meiotic synapsis. Thus, Mad2, and its regulation by Mad1, is a major regulator of meiotic synapsis in C. elegans.AUTHOR SUMMARYSexual reproduction relies on production of gametes, such as eggs and sperm, which are produced during meiosis. During this specialized cell division, chromosomes replicate, pair with their homologs, undergo synapsis and finally undergo recombination, all of which are required for correct meiotic chromosome segregation. Chromosomes are highly mobile during these steps in meiosis but the specific role of this mobility is unclear. Here, we show that spindle assembly checkpoint proteins, Mad1 and Bub3, that regulate and monitor meiotic synapsis are implicated in chromosome movement, solidifying the functional link between chromosome mobility and synapsis. Moreover, we provide additional data that another spindle checkpoint effector, Mad2, and its regulation by Mad1, plays an important role in regulating meiotic synapsis.

International audience; Meiosis is a specialized cell division that contributes to halve the genome content and reshuffle allelic combinations between generations in sexually reproducing eukaryotes. During meiosis, a large number of programmed DNA double-strand breaks (DSBs) are formed throughout the genome. Repair of meiotic DSBs facilitates the pairing of homologs and forms crossovers which are the reciprocal exchange of genetic information between chromosomes. Meiotic recombination also influences centromere organization and is essential for proper chromosome segregation. Accordingly, meiotic recombination drives genome evolution and is a powerful tool for breeders to create new varieties important to food security. Modifying meiotic recombination has the potential to accelerate plant breeding but it can also have detrimental effects on plant performance by breaking beneficial genetic linkages. Therefore, it is essential to gain a better understanding of these processes in order to develop novel strategies to facilitate plant breeding. Recent progress in targeted recombination technologies, chromosome engineering, and an increasing knowledge in the control of meiotic chromosome segregation has significantly increased our ability to manipulate meiosis. In this review, we summarize the latest findings and technologies on meiosis in plants. We also highlight recent attempts and future directions to manipulate crossover events and control the meiotic division process in a breeding perspective.

Accurate chromosome segregation during meiosis is essential for reproductive success. Yet, many fundamental aspects of meiosis remain unclear, including the mechanisms regulating homolog pairing across species. This gap is partially due to our inability to visualize individual chromosomes during meiosis. Here, we employ Oligopaint FISH to investigate homolog pairing and compaction of meiotic chromosomes in a classical model system, the silkwormBombyx mori. Our Oligopaint design combines multiplexed barcoding with secondary oligo labeling for high flexibility and low cost. These studies illustrate that Oligopaints are highly specific in whole-mount gonads and on meiotic chromosome spreads. We show that meiotic pairing is robust in both males and female meiosis. Additionally, we show that meiotic bivalent formation inB. morimales is highly similar to bivalent formation inC. elegans, with both of these pathways ultimately resulting in the pairing of chromosome ends with non-paired ends facing the spindle pole and microtubule recruitment independent of the centromere-specifying factor CENP-A.Author’s SummaryMeiosis is the specialized cell division occurring exclusively in ovaries and testes to produce egg and sperm cells, respectively. The accurate distribution of chromosomes (the genetic material) during this process is essential to prevent infertility/sterility and developmental disorders in offspring. As researchers are specifically unable to study the mechanisms regulating meiosis in depth in humans, identifying broadly conserved aspects of meiotic chromosome segregation is essential for making accurate inferences about human biology. Here, we use a sophisticated chromosome painting approach called Oligopaints to visualize and study chromosomes during meiosis in the silkworm,Bombyx mori. We illustrate that Oligopaints are highly specific inB. moriand demonstrate how Oligopaints can be used to study the dynamics of meiotic chromosomes in diverse species.

Summary: Synaptonemal complex (SC) assembly and homologous recombination, the most critical events during prophase I, are the prerequisite for faithful meiotic chromosome segregation. However, the underlying regulatory mechanism remains largely unknown. Here, we reveal that a functional RING finger E3 ubiquitin ligase, DESYNAPSIS1 (DSNP1), plays significant roles in SC assembly and homologous recombination during rice meiosis. In the dsnp1 mutant, homologous synapsis is discontinuous and aberrant SC-like polycomplexes occur independent of coaligned homologous chromosomes. Accompanying the decreased foci of HEI10, ZIP4, and MER3 on meiotic chromosomes, the number of crossovers (COs) decreases dramatically in dsnp1 meiocytes. Furthermore, the absence of central elements largely restores the localization of non-ZEP1 ZMM proteins and the number of COs in the dsnp1 background. Collectively, DSNP1 stabilizes the canonical tripartite SC structure along paired homologous chromosomes and further promotes the formation of COs.

Eukaryotic meiosis is a specialized cell cycle involving two successive nuclear divisions that lead to the formation of haploid gametes. The phosphatase Cdc14 plays an essential role in meiosis as revealed in studies of the yeastSaccharomyces cerevisiae. Cdc14 is stored in the nucleolus, a sub-nuclear domain containing the ribosomal DNA, and its release is regulated by two distinct pathways, one acting in early anaphase I of meiosis and a second at the exit from meiosis II. The early anaphase release is thought to be important for disjunction of the ribosomal DNA, disassembly of the anaphase I spindle, spindle pole re-duplication and the counteraction of CDK, all of which are required for progression into meiosis II. The release of Cdc14 from its nucleolar binding partner Net1 is stimulated by phosphorylation of cyclin-dependent kinase sites in Net1, but the importance of that phospho-regulation in meiosis is not well understood. We inducednet1-6cdkmutant cells to enter meiosis and examined the localization of Cdc14 and various indicators of meiotic progression. Thenet1-6cdkmutations inhibit, but don’t fully prevent Cdc14 release, and they almost completely prevent disjunction of the ribosomal DNA during meiosis I. Failure to disjoin the ribosomal DNA is lethal in mitosis, and we expected the same to be true in meiosis. However, the cells were able to complete meiosis II, yielding the expected four meiotic products as viable spores. Therefore, all ribosomal DNA disjunction required for meiosis can occur in meiosis II. We discuss the implications of these findings for our understanding of meiotic chromosome segregation.

The dynactin complex is an essential co-factor of the microtubule-based motor dynein. Dynein-dynactin have well-documented roles in spindle assembly and positioning during C. elegans female meiosis and embryonic mitosis, while dynein-dynactin’s contribution to male meiosis has not been investigated. Here, we characterize the G33S mutation in DNC-1’s N-terminal microtubule binding domain, which corresponds to G59S in the human dynactin subunit p150/Glued that causes motor neuron disease. In spermatocytes, dnc-1(G33S) delays spindle assembly and penetrantly inhibits anaphase spindle elongation in meiosis I, which prevents homologous chromosome segregation and generates aneuploid sperm with an extra centrosome. Consequently, embryos produced by dnc-1(G33S) hermaphrodites exhibit a high incidence of tetrapolar mitotic spindles, yet dnc-1(G33S) embryos with bipolar spindles proceed through early mitotic divisions without errors in chromosome segregation. Deletion of the DNC-1 N-terminus shows that defective meiosis in dnc-1(G33S) spermatocytes is not due to DNC-1’s inability to interact with microtubules. Rather, our results suggest that the DNC-1(G33S) protein, which is aggregation-prone in vitro, is less stable in spermatocytes than the early embryo, resulting in different phenotypic severity in the two dividing tissues. Thus, the unusual hypomorphic nature of the dnc-1(G33S) mutant reveals that dynein-dynactin drive meiotic chromosome segregation in spermatocytes and illustrates that the extent to which protein misfolding leads to loss of function can vary significantly between cell types.

Summary Polyploidy is a major driver of evolutionary change. Autopolyploids, which arise by within-species whole-genome duplication, carry multiple nearly identical copies of each chromosome. This presents an existential challenge to sexual reproduction. Meiotic chromosome segregation requires formation of DNA crossovers (COs) between two homologous chromosomes. How can this outcome be achieved when more than two essentially equivalent partners are available? We addressed this question by comparing diploid, neo-autotetraploid, and established autotetraploid Arabidopsis arenosa using new approaches for analysis of meiotic CO patterns in polyploids. We discover that crossover interference, the classical process responsible for patterning of COs in diploid meiosis, is defective in the neo-autotetraploid but robust in the established autotetraploid. The presented findings suggest that, initially, diploid-like interference fails to act effectively on multivalent pairing and accompanying pre-CO recombination interactions and that stable autopolyploid meiosis can emerge by evolution of a “supercharged” interference process, which can now act effectively on such configurations. Thus, the basic interference mechanism responsible for simplifying CO patterns along chromosomes in diploid meiosis has evolved the capability to also simplify CO patterns among chromosomes in autopolyploids, thereby promoting bivalent formation. We further show that evolution of stable autotetraploidy preadapts meiosis to higher ploidy, which in turn has interesting mechanistic and evolutionary implications., Graphical abstract, Highlights • In a neo-autotetraploid, aberrant crossover interference confers aberrant meiosis • In a stable autotetraploid, regular crossover interference confers regular meiosis • Crossover and synaptic patterns point to evolution of “supercharged” interference • Accordingly, evolution of stable autotetraploidy preadapts to higher ploidies, How does an established autopolyploid segregate its (multiple) homologous chromosomes two by two during meiosis? Morgan, White et al. show that crossover interference plays a critical role. They propose that stable autopolyploidy evolves by “supercharging” of interference and show that this also preadapts autotetraploid meiosis to higher ploidies.

Methylenetetrahydrofolate reductase (MTHFR) is a key enzyme in the folate metabolic pathway, and its loss of function through polymorphisms is often associated with human conditions, including cancer, congenital heart disease, and Down syndrome. MTHFR is also required in the maintenance of heterochromatin, a crucial determinant of genomic stability and precise chromosomal segregation. Here, we characterize the function of a fission yeast gene met11+, which encodes a protein that is highly homologous to the mammalian MTHFR. We show that, although met11+ is not essential for viability, its disruption increases chromosome missegregation and destabilizes constitutive heterochromatic regions at pericentromeric, sub-telomeric and ribosomal DNA (rDNA) loci. Transcriptional silencing at these sites were disrupted, which is accompanied by the reduction in enrichment of histone H3 lysine 9 dimethylation (H3K9me2) and binding of the heterochromatin protein 1 (HP1)-like Swi6. The met11 null mutant also dominantly disrupts meiotic fidelity, as displayed by reduced sporulation efficiency and defects in proper partitioning of the genetic material during meiosis. Interestingly, the faithful execution of these meiotic processes is synergistically ensured by cooperation among Met11, Rec8, a meiosis-specific cohesin protein, and the shugoshin protein Sgo1, which protects Rec8 from untimely cleavage. Overall, our results suggest a key role for Met11 in maintaining pericentromeric heterochromatin for precise genetic inheritance during mitosis and meiosis.

The role of the kinetochore during meiotic chromosome segregation in C. elegans oocytes has been a matter of controversy. Danlasky et al. (2020. J. Cell. Biol.https://doi.org/10.1083/jcb.202005179) show that kinetochore proteins KNL-1 and KNL-3 are required for early stages of anaphase during female meiosis, suggesting a new kinetochore-based model of chromosome segregation.

Maternal uniparental disomy of chromosome 7 is present in 5–10% of patients with Silver-Russell syndrome (SRS), and duplication of 7p including GRB10 (Growth Factor Receptor-Bound Protein 10), an imprinted gene that affects pre-and postnatal growth retardation, has been associated with the SRS phenotype. Here, we report on a 17 year old girl referred to array-CGH analysis for short stature, psychomotor delay, and relative macrocephaly. Array-CGH analysis showed two copy number variants (CNVs): a ~12.7 Mb gain in 7p13-p11.2, involving GRB10 and an ~9 Mb loss in 7q11.21-q11.23. FISH experiments performed on the proband’s mother showed a chromosome 7 pericentric inversion that might have mediated the complex rearrangement harbored by the daughter. Indeed, we found that segmental duplications, of which chromosome 7 is highly enriched, mapped at the breakpoints of both the mother’s inversion and the daughter’s CNVs. We postulate that pairing of highly homologous sequences might have perturbed the correct meiotic chromosome segregation, leading to unbalanced outcomes and acting as the putative meiotic mechanism that was causative of the proband’s rearrangement. Comparison of the girl’s phenotype to those of patients with similar CNVs supports the presence of 7p in a locus associated with features of SRS syndrome.

During meiosis, diploid organisms reduce their chromosome number by half to generate haploid gametes. This process depends on the repair of double strand DNA breaks as crossover recombination events between homologous chromosomes, which hold homologs together to ensure their proper segregation to opposite spindle poles during the first meiotic division. Although most organisms are limited in the number of crossovers between homologs by a phenomenon called crossover interference, the consequences of excess interfering crossovers on meiotic chromosome segregation are not well known. Here we show that extra interfering crossovers lead to a range of meiotic defects and we uncover mechanisms that counteract these errors. Using chromosomes that exhibit a high frequency of supernumerary crossovers in Caenorhabditis elegans, we find that essential chromosomal structures are mispatterned in the presence of multiple crossovers, subjecting chromosomes to improper spindle forces and leading to defects in metaphase alignment. Additionally, the chromosomes with extra interfering crossovers often exhibited segregation defects in anaphase I, with a high incidence of chromatin bridges that sometimes created a tether between the chromosome and the first polar body. However, these anaphase I bridges were often able to resolve in a LEM-3 nuclease dependent manner, and chromosome tethers that persisted were frequently resolved during Meiosis II by a second mechanism that preferentially segregates the tethered sister chromatid into the polar body. Altogether these findings demonstrate that excess interfering crossovers can severely impact chromosome patterning and segregation, highlighting the importance of limiting the number of recombination events between homologous chromosomes for the proper execution of meiosis., Author summary Meiosis is a process that ensures developing eggs and sperm contain the correct number of chromosomes. Failure to accurately segregate chromosomes during meiosis is one of the leading causes of birth defects and miscarriages. During meiosis, crossover events must form between the homologous chromosomes to ensure proper chromosome segregation. Although crossover events are required for proper chromosome segregation in most organisms, crossover numbers are limited even when the meiotic cell is overloaded with DNA breaks, the initiating events for crossovers. This stringent limitation of crossovers in multiple organisms suggests that there are negative consequences to having too many crossovers, but this has not been formally tested. In this study, we find that increasing crossover number negatively impacts chromosome segregation during meiosis by altering chromosome-associated structures, which leads to misalignment of the chromosomes on the meiotic spindle. Moreover, chromosomes with excess crossovers often have large chromatin bridges during the chromosome segregation process, but we find that these bridges can be corrected by at least two mechanisms. Our results thus highlight the importance of limiting crossover numbers to enable faithful chromosome segregation during sperm and egg development.

Constitutive heterochromatin at the pericentric regions of chromosomes undergoes dynamic changes in its epigenetic and spatial organization during spermatogenesis. Accurate control of pericentric heterochromatin is required for meiotic cell divisions and production of fertile and epigenetically intact spermatozoa. In this study, we demonstrate that pericentric heterochromatin is expressed during mouse spermatogenesis to produce major satellite repeat (MSR) transcripts. We show that the endonuclease DICER localizes to the pericentric heterochromatin in the testis. Furthermore, DICER forms complexes with MSR transcripts, and their processing into small RNAs is compromised in Dicer1 knockout mice leading to an elevated level of MSR transcripts in meiotic cells. We also show that defective MSR forward transcript processing in Dicer1 cKO germ cells is accompanied with reduced recruitment of SUV39H2 and H3K9me3 to the pericentric heterochromatin and meiotic chromosome missegregation. Altogether, our results indicate that the physiological role of DICER in maintenance of male fertility extends to the regulation of pericentric heterochromatin through direct targeting of MSR transcripts.

In meiosis, DNA break formation and repair are essential for the formation of crossovers between homologous chromosomes. Without crossover formation, faithful meiotic chromosome segregation and sexual reproduction cannot occur. Crossover formation is initiated by the programmed, meiosis‐specific introduction of numerous DNA double‐strand breaks, after which specific repair pathways promote recombination between homologous chromosomes. Despite its crucial nature, meiotic recombination is fraud with danger: When positioned or repaired inappropriately, DNA breaks can have catastrophic consequences on genome stability of the resulting gametes. As such, DNA break formation and repair needs to be carefully controlled. Within centromeres and surrounding regions (i.e., pericentromeres), meiotic crossover recombination is repressed in organisms ranging from yeast to humans, and a failure to do so is implicated in chromosome missegregation and developmental aneuploidy. (Peri)centromere sequence identity and organization diverge considerably across eukaryotes, yet suppression of meiotic DNA break formation and repair appear universal. Here, we discuss emerging work that has used budding and fission yeast systems to study the mechanisms underlying pericentromeric suppression of DNA break formation and repair. We particularly highlight a role for the kinetochore, a universally conserved, centromere‐associated structure essential for chromosome segregation, in suppressing (peri)centromeric DNA break formation and repair. We discuss the current understanding of kinetochore‐associated and chromosomal factors involved in this regulation and suggest future avenues of research.

Infertility is one of the most important and burning issues in present times, as a marked increase in the frequency of infertile cases has been observed all over the world. Chromosomal aneuploidy is among the known factors associated with infertility, and among sex chromosome aneuploidies, 47,XXY and 47,XYY constitute the most common class of chromosome abnormality in human live births. Considerable attention has been given to the somatic abnormalities associated with these conditions, but less is known about their meiotic progression; that is, how sex chromosome imbalance influences the meiotic process. It has been documented that men with the same underlying genetic cause of infertility do not present with uniform pathology, so it is informative to find out how meiotic progression differs in patients with similar chromosomal aneuploidy having different phenotypes. The importance of studying meiotic progression in patients with sex chromosome abnormalities has increased many fold with the introduction of assisted reproductive technologies that have made it possible for infertile men to become biological parents. Hence, exploring the possible consequences of sex chromosome aneuploidy for meiotic chromosome segregation is worthwhile. The objective of this review, in the context of current knowledge, is to discuss problems associated with fertility and progression of meiosis in two relatively common sex chromosome aneuploidies, 47,XXY and 47,XYY, reported in humans.

Zhang et al. identify new synaptonemal complex (SC) central region proteins SYP-5 and SYP-6 in Caenorhabditis elegans and show that SC central region proteins form assembly units through stable interactions and weak interactions between the units that drive SC formation., The synaptonemal complex (SC) is an ordered but highly dynamic structure assembled between homologous chromosomes to control interhomologous crossover formation, ensuring accurate meiotic chromosome segregation. However, the mechanisms regulating SC assembly and dynamics remain unclear. Here, we identified two new SC components, SYP-5 and SYP-6, in Caenorhabditis elegans that have distinct expression patterns and form distinct SC assembly units with other SYPs through stable interactions. SYP-5 and SYP-6 exhibit diverse in vivo SC regulatory functions and distinct phase separation properties in cells. Charge-interacting elements (CIEs) are enriched in SC intrinsically disordered regions (IDRs), and IDR deletion or CIE removal confirmed a requirement for these elements in SC regulation. Our data support the theory that multivalent weak interactions between the SC units drive SC formation and that CIEs confer multivalency to the assembly units.

The proteinaceous zipper-like structure known as the synaptonemal complex (SC), which forms between pairs of homologous chromosomes during meiosis from yeast to humans, plays important roles in promoting interhomolog crossover formation, regulating cessation of DNA double-strand break (DSB) formation following crossover designation, and ensuring accurate meiotic chromosome segregation. Recent studies are starting to reveal critical roles for different protein modifications in regulating SC dynamics. Protein SUMOylation, N-terminal acetylation, and phosphorylation have been shown to be essential for the regulated assembly and disassembly of the SC. Moreover, phosphorylation of specific SC components has been found to link changes in SC dynamics with meiotic recombination. This review highlights the latest findings on how protein modifications regulate SC dynamics and functions.

Chromosomes that have undergone crossing over in meiotic prophase must maintain sister chromatid cohesion somewhere along their length between the first and second meiotic divisions. Although many eukaryotes use the centromere as a site to maintain cohesion, the holocentric organism Caenorhabditis elegans instead creates two chromosome domains of unequal length termed the short arm and long arm, which become the first and second site of cohesion loss at meiosis I and II. The mechanisms that confer distinct functions to the short and long arm domains remain poorly understood. Here, we show that phosphorylation of the synaptonemal complex protein SYP-1 is required to create these domains. Once crossover sites are designated, phosphorylated SYP-1 and PLK-2 become cooperatively confined to short arms and guide phosphorylated histone H3 and the chromosomal passenger complex to the site of meiosis I cohesion loss. Our results show that PLK-2 and phosphorylated SYP-1 ensure creation of the short arm subdomain, promoting disjunction of chromosomes in meiosis I., 正常な卵子を生み出す細胞分裂に必須の分子メカニズムを解明. 京都大学プレスリリース. 2017-12-28.

Significance Meiosis is the specialized cell division that generates haploid sperm and eggs. Proper segregation of chromosomes in meiosis is required to prevent pregnancy loss and birth defects. This requires that the replicated copies of each chromosome remain attached as the homologous copies of each chromosome segregate in the first meiotic division. The replicated copies of each chromosome then segregate in the second meiotic division. The Shugoshin proteins protect attachments between the replicated chromosome copies in meiosis I. This paper shows that, in Drosophila meiosis, a phosphatase and the Shugoshin MEI-S332 reciprocally regulate each other’s localization to centromeres and together they thus function to ensure accurate segregation., Proper segregation of chromosomes in meiosis is essential to prevent miscarriages and birth defects. This requires that sister chromatids maintain cohesion at the centromere as cohesion is released on the chromatid arms when the homologs segregate at anaphase I. The Shugoshin proteins preserve centromere cohesion by protecting the cohesin complex from cleavage, and this has been shown in yeasts to be mediated by recruitment of the protein phosphatase 2A B′ (PP2A B′). In metazoans, delineation of the role of PP2A B′ in meiosis has been hindered by its myriad of other essential roles. The Drosophila Shugoshin MEI-S332 can bind directly to both of the B′ regulatory subunits of PP2A, Wdb and Wrd, in yeast two-hybrid experiments. Exploiting experimental advantages of Drosophila spermatogenesis, we found that the Wdb subunit localizes first along chromosomes in meiosis I, becoming restricted to the centromere region as MEI-S332 binds. Wdb and MEI-S332 show colocalization at the centromere region until release of sister-chromatid cohesion at the metaphase II/anaphase II transition. MEI-S332 is necessary for Wdb localization, but, additionally, both Wdb and Wrd are required for MEI-S332 localization. Thus, rather than MEI-S332 being hierarchical to PP2A B′, these proteins reciprocally ensure centromere localization of the complex. We analyzed functional relationships between MEI-S332 and the two forms of PP2A by quantifying meiotic chromosome segregation defects in double or triple mutants. These studies revealed that both Wdb and Wrd contribute to MEI-S332’s ability to ensure accurate segregation of sister chromatids, but, as in centromere localization, they do not act solely downstream of MEI-S332.

Loss of the FPC protein Swi1 or Swi3 results in reduced spore viability, delayed replication, changes in recombination, reduced centromeric cohesion stability, and chromosome missegregation in meiosis I and II. These phenotypes reveal a crucial link between meiotic replication fork stability and chromosome segregation., The replication fork protection complex (FPC) coordinates multiple processes that are crucial for unimpeded passage of the replisome through various barriers and difficult to replicate areas of the genome. We examine the function of Swi1 and Swi3, fission yeast’s primary FPC components, to elucidate how replication fork stability contributes to DNA integrity in meiosis. We report that destabilization of the FPC results in reduced spore viability, delayed replication, changes in recombination, and chromosome missegregation in meiosis I and meiosis II. These phenotypes are linked to accumulation and persistence of DNA damage markers in meiosis and to problems with cohesion stability at the centromere. These findings reveal an important connection between meiotic replication fork stability and chromosome segregation, two processes with major implications to human reproductive health.

Summary Reproductive aging is characterized by a marked decline in oocyte quality that contributes to infertility, miscarriages, and birth defects. This decline is multifactorial, and the underlying mechanisms are under active investigation. Here, we performed RNA‐Seq on individual growing follicles from reproductively young and old mice to identify age‐dependent functions in oocytes. This unbiased approach revealed genes involved in cellular processes known to change with age, including mitochondrial function and meiotic chromosome segregation, but also uncovered previously unappreciated categories of genes related to proteostasis and organelles required for protein metabolism. We further validated our RNA‐Seq data by comparing nucleolar structure and function in oocytes from reproductively young and old mice, as this organelle is central for protein production. We examined key nucleolar markers, including upstream binding transcription factor (UBTF), an RNA polymerase I cofactor, and fibrillarin, an rRNA methyltransferase. In oocytes from mice of advanced reproductive age, UBTF was primarily expressed in giant fibrillar centers (GFCs), structures associated with high levels of rDNA transcription, and fibrillarin expression was increased ~2‐fold. At the ultrastructural level, oocyte nucleoli from reproductively old mice had correspondingly more prominent fibrillar centers and dense fibrillar centers relative to young controls and more ribosomes were found in the cytoplasm. Taken together, our findings are significant because the growing oocyte is one of the most translationally active cells in the body and must accumulate high‐quality maternally derived proteins to support subsequent embryo development. Thus, perturbations in protein metabolism are likely to have a profound impact on gamete health.

Cairo et al. show that in S. cerevisiae meiosis, spindle checkpoint proteins Bub1 and Bub3 have a critical role in preventing chromosome missegregation and setting the normal duration of anaphase I and II onset by regulating the kinetochore-localization of Ipl1 and PP1., Accurate chromosome segregation depends on the proper attachment of kinetochores to spindle microtubules before anaphase onset. The Ipl1/Aurora B kinase corrects improper attachments by phosphorylating kinetochore components and so releasing aberrant kinetochore–microtubule interactions. The localization of Ipl1 to kinetochores in budding yeast depends upon multiple pathways, including the Bub1–Bub3 pathway. We show here that in meiosis, Bub3 is crucial for correction of attachment errors. Depletion of Bub3 results in reduced levels of kinetochore-localized Ipl1 and concomitant massive chromosome missegregation caused by incorrect chromosome–spindle attachments. Depletion of Bub3 also results in shorter metaphase I and metaphase II due to premature localization of protein phosphatase 1 (PP1) to kinetochores, which antagonizes Ipl1-mediated phosphorylation. We propose a new role for the Bub1–Bub3 pathway in maintaining the balance between kinetochore localization of Ipl1 and PP1, a balance that is essential for accurate meiotic chromosome segregation and timely anaphase onset.

Translesion synthesis polymerases (TLSPs) are non-essential error-prone enzymes that ensure cell survival by facilitating DNA replication in the presence of DNA damage. In addition to their role in bypassing lesions, TLSPs have been implicated in meiotic double strand break repair in several systems. Here we examine the joint contribution of four TLS polymerases to meiotic progression in the fission yeast S. pombe. We observed the dramatic loss of spore viability in fission yeast lacking all four TLSPs which is accompanied by disruptions in chromosome segregation during meiosis I and II. Rec8 cohesin dynamics are altered in the absence of the TLSPs. These data suggest that the TLSPs contribute to multiple aspects of meiotic chromosome dynamics.

Epitranscriptomic has emerged as a fundamental control of gene expression. Nevertheless, the determination of the transcriptome-wide occupancy of RNA modifications remains challenging. We have developed Rho-seq, an integrated pipeline detecting a range of modifications through differential modification-dependent Rhodamine labeling. Using Rho-seq, we confirm that the reduction of uridine to dihydrouridine targets tRNAs in E. coli. Unexpectedly, we find that the D modification expands to mRNAs in fission yeast.The modified mRNAs are enriched for cytoskeleton-related encoding proteins. We show that the α-tubulin encoding mRNA nda2 undergoes dihydrouridylation, which affects its protein expression level. The absence of the modification onto the nda2 mRNA impacts meiosis by inducing a metaphase delay or by completely preventing the formation of spindles during meiosis I and meiosis II, resulting in low gamete viability. Collectively these data show that the codon-specific reduction of uridine within specific mRNA is required for proper meiotic chromosome segregation and gamete viability.

In the nematode Caenorhabditis elegans, the mammalian tumor suppressor p53 ortholog CEP-1 (C. elegans p53-like protein) is associated not only with the stress response, germline apoptosis, and meiotic chromosome segregation but also with longevity through the modification of energy metabolism during aging. The mitochondrial respiration-related gene sco-1 in C. elegans is orthologous to the human SCO1 gene and a target of p53/CEP-1. Using quantitative real-time polymerase chain reaction (PCR) analysis, we recently found that the expression levels of sco-1 gene were increased in wild-type C. elegans in an aging-related manner and decreased in long-lived cep-1 mutants. Here, we describe the relative quantitative strategy using a commercial real-time PCR system to detect more accurately differences in the levels of expressed genes between long-lived and wild-type C. elegans strains. To estimate the expression levels of target genes compared with wild-type using relative quantification, we used the expression levels of an endogenous control gene, such as a housekeeping gene. In addition, it is critical to normalize differences in the expression levels of the common housekeeping gene among the strains analyzed for an accurate comparison of the quantitative expression levels of target genes.

SummaryMore than 95% of fertilizedDrosophilaoocytes from outbred stocks develop fully regardless of maternal age, in contrast to human oocytes, which frequently generate non-viable aneuploid embryos. SinceDrosophilaoocytes are normally stored only briefly prior to ovulation, unlike their human counterparts, we investigated the effects of storage on oocyte quality. Using a novel system to acquire oocytes held for known periods, we analyzed by ribosome profiling how translation and cellular function change over time. Oocyte developmental capacity decays in a precise temperature-dependent manner over 1-4 weeks, due to a progressive inability to complete meiosis. Meiotic metaphase genes, theFmr1translational regulator, and the small heat shock protein chaperonesHsp26andHsp27are preferentially translated during storage, and oocytes lackingHsp26andHsp27age prematurely. However translation falls generally 2.3-fold with age despite constant mRNA levels, and this inability to maintain translational equilibrium correlates with oocyte functional decline. These findings show that meiotic chromosome segregation inDrosophilaoocytes is uniquely sensitive to prolonged quiescence, and suggest that the extended storage of mature human oocytes contributes to their chromosome instability. If so, then these problems may be more amenable to intervention than previously supposed.

SUMMARYCharacteristics of heteroduplex DNA illuminate how strands exchange during homologous recombination, but mismatch correction can obscure them. To investigate recombination mechanisms, meiotic crossover products were analyzed at two hotspots inMsh2–/–mice containing homologous chromosomes derived from inbred strains. Recombination frequencies were unchanged in the mutant, implying that MSH2-dependent recombination suppression does not occur at this level of diversity. However, a substantial fraction of crossover products retained heteroduplex DNA in the absence of MSH2, and some also had multiple switches between parental markers suggestive of MSH2-independent correction. Recombinants appeared to reflect a biased orientation of crossover resolution, possibly stemming from asymmetry at DNA ends established in earlier intermediates. Many crossover products showed no evidence of heteroduplex DNA, suggesting dismantling by D-loop migration. Unlike the complexity of crossovers in yeast, these two modifications of the original double-strand break repair model may be sufficient to explain most meiotic crossing over in mice.

Summary Interhomolog crossovers (COs) are a prerequisite for achieving accurate chromosome segregation during meiosis [ 1 , 2 ]. COs are not randomly positioned, occurring at distinct genomic intervals during meiosis in all species examined [ 3 , 4 , 5 , 6 , 7 , 8 , 9 , 10 ]. The role of CO position as a major determinant of accurate chromosome segregation has not been previously directly analyzed in a metazoan. Here, we use spo-11 mutants, which lack endogenous DNA double-strand breaks (DSBs), to induce a single DSB by Mos1 transposon excision at defined chromosomal locations in the C. elegans germline and show that the position of the resulting CO directly affects the formation of distinct chromosome subdomains during meiotic chromosome remodeling. CO formation in the typically CO-deprived center region of autosomes leads to premature loss of sister chromatid cohesion and chromosome missegregation, whereas COs at an off-centered position, as in wild type, can result in normal remodeling and accurate segregation. Ionizing radiation (IR)-induced DSBs lead to the same outcomes, and modeling of IR dose-response reveals that the CO-unfavorable center region encompasses up to 6% of the total chromosome length. DSBs proximal to telomeres rarely form COs, likely because of formation of unstable recombination intermediates that cannot be sustained as chiasmata until late prophase. Our work supports a model in which regulation of CO position early in meiotic prophase is required for proper designation of chromosome subdomains and normal chromosome remodeling in late meiotic prophase I, resulting in accurate chromosome segregation and providing a mechanism to prevent aneuploid gamete formation.

Accurate chromosome segregation depends on proper attachment of kinetochores to spindle microtubules prior to anaphase onset. The Ipl1/Aurora B kinase corrects improper attachments by phosphorylating kinetochore components and so releasing aberrant kinetochore-microtubule interactions. The localization of Ipl1 to kinetochores in budding yeast depends upon multiple pathways, including the Bub1/Bub3 pathway. We show here that in meiosis, Bub3 is crucial for correction of attachment errors. Depletion of Bub3 results in reduced levels of kinetochore-localized Ipl1, and concomitant massive chromosome mis-segregation caused by incorrect chromosome-spindle attachments. Depletion of Bub3 also results in shorter metaphase I and metaphase II due to premature localization of protein phosphatase 1 (PP1) to kinetochores, which antagonizes Ipl1-mediated phosphorylation. We propose a new role for the Bub1-Bub3 pathway in maintaining the balance between kinetochore-localization of Ipl1 and PP1, a balance that is essential for accurate meiotic chromosome segregation and timely anaphase onset.SummaryCairoet alshow that inS. cerevisiaemeiosis, spindle checkpoint proteins Bub1 and Bub3 have an essential role in preventing chromosome mis-segregation and setting the normal duration of anaphase I and anaphase II onset by regulating the kinetochore-localization of Ipl1 and PP1.

The linker of nucleoskeleton and cytoskeleton (LINC) complex is an essential multi-protein structure spanning the eukaryotic nuclear envelope. The LINC complex functions to maintain nuclear architecture, positioning, and mobility, along with specialized functions in meiotic prophase and chromosome segregation. Members of the LINC complex were recently identified in maize, an important scientific and agricultural grass species. Here we characterized Maize LINC KASH AtSINE-like2, MLKS2, which encodes a highly conserved SINE-group plant KASH protein with characteristic N-terminal armadillo repeats (ARM). Using a heterologous expression system, we showed that actively expressed GFP-MLKS2 is targeted to the nuclear periphery and colocalizes with F-actin and the endoplasmic reticulum, but not microtubules in the cell cortex. Expression of GFP-MLKS2, but not GFP-MLKS2ΔARM, resulted in nuclear anchoring. Genetic analysis of transposon-insertion mutations, mlks2-1 and mlks2-2, showed that the mutant phenotypes were pleiotropic, affecting root hair nuclear morphology, stomatal complex development, multiple aspects of meiosis, and pollen viability. In male meiosis, the mutants showed defects for bouquet-stage telomere clustering, nuclear repositioning, perinuclear actin accumulation, dispersal of late prophase bivalents, and meiotic chromosome segregation. These findings support a model in which the nucleus is connected to cytoskeletal F-actin through the ARM-domain, predicted alpha solenoid structure of MLKS2. Functional conservation of MLKS2 was demonstrated through genetic rescue of the misshapen nuclear phenotype of an Arabidopsis (triple-WIP) KASH mutant. This study establishes a role for the SINE-type KASH proteins in affecting the dynamic nuclear phenomena required for normal plant growth and fertility. Abbreviations: FRAP: Fluorescence recovery after photobleaching; DPI: Days post infiltration; OD: Optical density; MLKS2: Maize LINC KASH AtSINE-like2; LINC: Linker of nucleoskeleton and cytoskeleton; NE: Nuclear envelope; INM: Inner nuclear membrane; ONM: Outer nuclear membrane

Cell proliferation and sexual reproduction require the faithful segregation of chromosomes. Chromosome segregation is driven by the interaction of chromosomes with the spindle, and the attachment of chromosomes to the proper spindle poles is essential. Initial attachments are frequently erroneous due to the random nature of the attachment process; however, erroneous attachments are selectively eliminated. Proper attachment generates greater tension at the kinetochore than erroneous attachments, and it is thought that attachment selection is dependent on this tension. However, studies of meiotic chromosome segregation suggest that attachment elimination cannot be solely attributed to tension, and the precise mechanism of selective elimination of erroneous attachments remains unclear. During attachment elimination, chromosomes oscillate between the spindle poles. A recent study on meiotic chromosome segregation in fission yeast has suggested that attachment elimination is coupled to chromosome oscillation. In this review, the possible contribution of chromosome oscillation in the elimination of erroneous attachment is discussed in light of the recent finding.

Live-cell imaging is a powerful tool that allows investigators to directly observe the dynamics of cellular processes. Live imaging has proven particularly useful in studying mitotic and meiotic chromosome segregation, where the assembly of spindles and movement of chromosomes can be quantified in ways not possible with fixed cells. This protocol describes how to image live meiosis in the agriculturally important plant, maize. The creation of fluorescently tagged tubulin allows visualization of maize spindles, and nucleic acid dyestain chromosomes. This protocol describes all steps required for live imaging, including how to grow plants, screen for relevant genotypes, harvest meiotic cells, and collect live movies of meiosis. While this protocol was developed for imaging fluorescently tagged tubulin, it can be easily modified to observe the meiotic dynamics of any fluorescently labeled protein of interest. © 2016 by John Wiley & Sons, Inc.

Research over the last two decades has identified a group of meiosis-specific proteins, consisting of budding yeast Spo13, fission yeast Moa1, mouse MEIKIN, and Drosophila Mtrm, with essential functions in meiotic chromosome segregation. These proteins, which we call meiosis I kinase regulators (MOKIRs), mediate two major adaptations to the meiotic cell cycle to allow the generation of haploid gametes from diploid mother cells. Firstly, they promote the segregation of homologous chromosomes in meiosis I (reductional division) by ensuring that sister kinetochores face towards the same pole (mono-orientation). Secondly, they safeguard the timely separation of sister chromatids in meiosis II (equational division) by counteracting the premature removal of pericentromeric cohesin, and thus prevent the formation of aneuploid gametes. Although MOKIRs bear no obvious sequence similarity, they appear to play functionally conserved roles in regulating meiotic kinases. Here, the known functions of MOKIRs are reviewed and their possible mechanisms of action are discussed. Also see the video abstract here https://youtu.be/tLE9KL89bwk.

Crossovers (COs) between homologous chromosomes are critical for meiotic chromosome segregation and form in the context of the synaptonemal complex (SC), a meiosis-specific structure that assembles between aligned homologs. DuringCaenorhabditis elegansmeiosis, central region components of the SC (SYP proteins) are essential to repair double-strand DNA breaks (DSBs) as COs, but the roles of these SYP proteins in promoting CO formation are poorly understood. Here, we investigate the relationships between the SYP proteins and conserved CO-promoting factors by examining the immunolocalization of these factors in meiotic mutants where SYP proteins are absent, reduced, or mis-localized. Although COs do not form insypnull mutants, CO-promoting proteins COSA-1, MSH-5, and ZHP-3 nevertheless become co-localized at a variable number of DSB-dependent sites during late prophase, reflecting an inherent affinity of these factors for DSB repair sites. In contrast, in mutants where SYP proteins are present but form aggregates or display abnormal synapsis, CO-promoting proteins consistently track with SYP-1 localization. Moreover, CO-promoting proteins usually localize to a single site per SYP-1 structure, even in SYP aggregates or in mutants where SC forms between sister-chromatids, suggesting that CO regulation occurs within these structures. Further, we find that sister chromatids in the meiotic cohesin mutantrec-8require both CO-promoting proteins and the SC to remain connected. Taken together, our findings support a model in which SYP proteins promote CO formation by directing and constraining the localization of CO-promoting factors to ensure that CO maturation occurs only between properly aligned homologous chromosomes.Article SummaryErrors during meiosis are the leading cause of birth defects and miscarriages in humans. Thus, the coordinated control of meiosis events is critical for the faithful inheritance of the genome each generation. The synaptonemal complex (SC) is a meiosis-specific structure that assembles between homologs chromosomes and is critical for the establishment and regulation of crossovers, which ensure the accurate segregation of the homologous chromosomes at meiosis I. Here we show that the SC proteins function to regulate crossovers by directing and constraining the localization of proteins involved in promoting the formation of crossovers.

Although Theodor Boveri linked abnormal chromosome numbers and disease more than a century ago, an in-depth understanding of the impact of mitotic and meiotic chromosome segregation errors on cell proliferation and diseases is still lacking. This review reflects on the efforts and results of a large European research network that, from the 1980′s until 2004, focused on protection against aneuploidy-inducing factors and tackled the following problems: 1) the origin and consequences of chromosome imbalance in somatic and germ cells; 2) aneuploidy as a result of environmental factors; 3) dose-effect relationships; 4) the need for validated assays to identify aneugenic factors and classify them according to their modes of action; 5) the need for reliable, quantitative data suitable for regulating exposure and preventing aneuploidy induction; 6) the need for mechanistic insight into the consequences of aneuploidy for human health. This activity brought together a consortium of experts from basic science and applied genetic toxicology to prepare the basis for defining guidelines and to encourage regulatory activities for the prevention of induced aneuploidy. Major strengths of the EU research programmes on aneuploidy were having a valuable scientific approach based on well-selected compounds and accurate methods that allow the determination of precise dose-effect relationships, reproducibility and inter-laboratory comparisons. The work was conducted by experienced scientists stimulated by a fascination with the complex scientific issues surrounding aneuploidy; a key strength was asking the right questions at the right time. The strength of the data permitted evaluation at the regulatory level. Finally, the entire enterprise benefited from a solid partnership under the lead of an inspired and stimulating coordinator. The research programme elucidated the major modes of action of aneugens, developed scientifically sound assays to assess aneugens in different tissues, and achieved the international validation of relevant assays with the goal of protecting human populations from aneugenic chemicals. The role of aneuploidy in tumorigenesis will require additional research, and the study of effects of exposure to multiple agents should become a priority. It is hoped that these reflections will stimulate the implementation of aneuploidy testing in national and OECD guidelines.

Oocyte meiotic spindles in most species lack centrosomes and the mechanisms that underlie faithful chromosome segregation in acentrosomal meiotic spindles are not well understood. In C. elegans oocytes, spindle microtubules exert a poleward force on chromosomes that is dependent on the microtubule-stabilising protein CLS-2, the orthologue of the mammalian CLASP proteins. The checkpoint kinase BUB-1 and CLS-2 localise in the central spindle and display a dynamic localisation pattern throughout anaphase, but the signals regulating their anaphase-specific localisation remains unknown. We have shown previously that SUMO regulates BUB-1 localisation during metaphase I. Here, we found that SUMO modification of BUB-1 is regulated by the SUMO E3 ligase GEI-17 and the SUMO protease ULP-1. SUMO and GEI-17 are required for BUB-1 localisation between segregating chromosomes during early anaphase I. We also show that CLS-2 is subject to SUMO-mediated regulation; CLS-2 precociously localises in the midbivalent when either SUMO or GEI-17 are depleted. Overall, we provide evidence for a novel, SUMO-mediated control of protein dynamics during early anaphase I in oocytes.