Prior to 1980, experimental induction of polyploidy led to the release of several tetraploid Brassica rapa ssp. rapa as turnip cultivars. Most experimentally induced polyploids are meiotically unstable and show reduced fertility, but we hypothesized that these commercially propagated turnip lines should have restored fertility and stabilized meiosis. We collected all available B. rapa lines listed as tetraploid from germplasm banks, and subsequently investigated chromosome karyotypes, meiotic chromosome behaviour and fertility. Unexpectedly, all accessions showed unstable meiosis: the average tetravalent frequency per meiosis per plant ranged from 4.8 to 6.4 per line. Using chromosome-specific fluorescence in situ hybridisation probes, we found that most chromosomes showed similar frequencies of tetravalent formation except for chromosomes A3 and A6, which predominantly formed tetravalents (>90%). Of the 21 individuals sequenced (one per accession), approximately half (9/21) were aneuploid (loss or gain of a whole chromosome), and two displayed additional chromosomal rearrangements. We nevertheless observed no significant phenotypic abnormalities or reductions in fertility (although all accessions were self-incompatible). Our findings indicate that stabilizing meiosis may not always be necessary to produce relatively fertile and homogeneous polyploid populations, and point at self-incompatibility as a possible mechanism helping prevent fixation of undesirable aneuploid karyotypes.

During meiosis, the programmed formation of DNA double-strand breaks (DSBs) by Spo11, a conserved topoisomerase VI family protein, initiates homologous recombination that leads to crossovers between homologous chromosomes, essential for accurate chromosome segregation and genome evolution. The DSB number, distribution and timing of formation are regulated during meiosis to ensure crossing over on all chromosomes and prevent genome instability. In S. cerevisiae, DSB interference suppresses the coincident formation of DSBs in neighboring hotspots through a Tel1/ATM dependent mechanism that remains unexplored. Here, we demonstrate that Tel1 is recruited to meiotic DSB hotspots and chromosomal axis sites in a DSB-dependent manner. This supports the tethered loop-axis complex (TLAC) model that postulates meiotic DSBs are formed within the chromosome axis environment. Tel1 recruitment to meiotic DSBs, DSB interference and the meiotic DNA damage checkpoint are all dependent on the C-terminal moiety of Xrs2, known to mediate Tel1-Xrs2 interaction in vegetative cells. However, mutation of the Xrs2 FxF/Y motif, known to stabilize Tel1 interaction with Xrs2, does not affect DSBs interference but abolishes the Tel1-dependent DNA damage checkpoint. Altogether, this work uncovers the dynamic association of Tel1 with meiotic chromosomes and highlights the critical role of its interaction with Xrs2 in fine-tuning both the meiotic DNA damage checkpoint and DSB interference.

The meiotic chromosome axis organizes chromatin and sets the stage for homolog pairing and recombination. Meiotic HORMA domain proteins (mHORMADs) are conserved axis components that conformationally transform during target binding. In C. elegans, four functionally distinct mHORMADs directly interact, but how binding between them is restricted to axis assembly is unknown. Using a mutation in the mHORMADs that delays axis assembly, we isolated a suppressor mutation in a TRiC (Tailless complex peptide 1 Ring Complex) chaperonin subunit that restored mHORMAD localization. CCT-4 associates with meiotic chromatin and forms in vivo complexes with mHORMADs, while germline disruption of TRiC results in axis defects, indicating a nuclear function for TRiC alongside meiotic chromosomes. We propose that chromosome-associated TRiC locally folds mHORMADs into the binding-competent conformation required for axis morphogenesis. More broadly, our results support the model that spatially-restricted folding by TRiC/CCT is a mechanism of controlling the assembly of multimeric complexes that function in tightly co-ordinated events.

In Drosophila melanogaster, the dual E3 Ubiquitin/SUMO-1 ligase dTopors localizes to the spermatocyte nuclear lamina and is required for nuclear morphology and meiotic chromosome transmission. To investigate the role of dTopors-mediated ubiquitination, we use CRISPR/cas9 to mutate the ubiquitination domain. Two mutants were recovered: C105A, a substitution of a conserved cysteine, and Df(114-118), a hypomorphic mutant with an altered ubiquitination domain. Whereas each mutation similarly disrupted nuclear morphology, nondisjunction was less frequent in Df(114-118) mutants, suggesting that nondisjunction is not a direct consequence of nuclear perturbation. Anaphase I bridges were observed in both C105A and Df(114-118) mutants, but at a much lower frequency in Df(114-118) mutants, suggesting that ubiquitination of targets important for chromosome segregation still occurs in Df(114-118) males. Bridge formation was independent of the sister chromatid cohesin Sunn, indicating that bridges did not result from sister chromatid attachments. In yeast two hybrid assays, dTopors interacted with 6 of the 28 E2 ubiquitin-conjugating enzymes in Drosophila: CG10862, Ubc2, CG7220, Taf1, CG8188 and Effete. Interactions were abolished by C105A, but only diminished by Df(114-118). We suggest that interaction of Df(114-118) with one or more E2s is nearly sufficient for ubiquitination of target(s) required for chromosome segregation but not nuclear structure. Comparative proteomic analysis by mass spectroscopy identified 924 proteins differentially ubiquitinated in C105A versus wildtype testis, including histones and Lamin Dm0. The largest ontology group of proteins identified, however, was components of ubiquitin-mediated proteolysis, suggesting that dTopors may also play a role in coordinating pathways that regulate protein turnover.

Accurate chromosome segregation during oocyte meiosis is essential for ensuring proper embryonic development and preventing aneuploidy-related disorders. Live-cell imaging combined with fluorescence labelling techniques have become a powerful approach for studying meiotic chromosome dynamics with high spatiotemporal resolution. In this protocol, we summarize key methodologies for visualizing chromosome segregation in live mouse oocytes, focusing on the use of histone H2B-RFP for chromatin labelling and SiR-Tubulin for spindle tracking. We describe the procedures for sample preparation, mRNA microinjection, oocyte culture, live-cell imaging chamber setup, and confocal microscopy settings, all optimized to achieve high-resolution imaging while minimizing phototoxicity. Furthermore, we highlight critical experimental considerations such as phototoxicity, image processing, and quantitative analysis of meiotic events. By providing a comprehensive evaluation of current methodologies, this protocol serves as a practical guide for researchers seeking to investigate chromosome dynamics in live oocytes and improve the accuracy and reproducibility of meiotic studies.

Sex chromosomes often evolve unique patterns of gene expression during spermatogenesis. In many species, sex-linked genes are downregulated during meiosis in response to asynapsis of the heterogametic sex chromosome pair (meiotic sex chromosome inactivation; MSCI). This process has evolved convergently across many taxa with independently derived sex chromosomes. Our understanding how quickly MSCI can evolve and whether it is connected to the degree of sequence degeneration remains limited. Teleost fish are a noteworthy group to investigate MSCI because sex chromosomes have evolved repeatedly across species, often over short evolutionary timescales. Here, we investigate whether MSCI occurs in the threespine stickleback fish (Gasterosteus aculeatus), which have an X and Y chromosome that evolved less than 26 million years ago. Using single-cell RNA-seq, we found that the X and Y chromosomes do not have a signature of MSCI, maintaining gene expression across meiosis. Using immunofluorescence, we also show the threespine stickleback do not form a condensed sex body around the X and Y, a feature of MSCI in many species. We did not see patterns of gene content evolution documented in other species with MSCI. Y-linked ampliconic gene families were expressed across multiple stages of spermatogenesis, rather than being restricted to post-meiotic stages, like in mammals. Our work shows MSCI does not occur in the threespine stickleback fish and has not shaped the evolution of the Y chromosome. In addition, the absence of MSCI in the threespine stickleback suggests this process may not be a conserved feature of teleost fish, despite overall sequence degeneration and structural evolution of the Y chromosome, and argues for additional investigation in other species. We also observed testis-dependent differences in coding and expression evolution for X-linked genes, revealing evidence of testis specific faster-X effect and gene-by-gene dosage compensation.

BackgroundThe genome of rye, Secale cereale, is distinguished by large repetitive regions including subtelomeric heterochromatin and retrotransposon-dominant centromeres, which contrast with the satellite-repeat-based centromeres in most characterized plant genome assemblies. This study aims to decode the architecture and evolution of these elusive regions through high-resolution genome assembly, with a focus on centromere dynamics and chromatin regulation.ResultsUsing PacBio HiFi and Nanopore sequencing, we generate a chromosome-scale assembly encompassing three complete centromeres and resolving subtelomeric heterochromatin. We identify terminal tandem repeat arrays as key determinants in establishing specialized chromatin environments linked to retrotransposon deposition. Notably, rye centromeres exhibit an unconventional epigenetic signature depleted of conventional activation and repression marks but displaying unique DNA hypomethylation patterns. This retrotransposon-enriched landscape promotes both the integration of young LTR retrotransposons and the recruitment of CENH3. Cross-species CENH3 ChIP-seq analyses reveal that Cereba retrotransposons are associated with enhanced CENH3 loading in cultivated and wild rye lineages, particularly through their conserved protease and integrase domains, suggesting a potential positive feedback loop for centromere evolution.ConclusionsOur findings establish retrotransposons as autonomous organizers of centromere chromatin and identity in rye, challenging the paradigm of satellite-dependent centromere specification. The dual role of retrotransposons in maintaining CENH3 recruitment while facilitating genomic innovation provides a mechanistic basis for centromere plasticity. This work advances functional genomics of Triticeae crops and opens new avenues for centromere engineering to manipulate meiotic stability and chromosome transmission in crop breeding.Supplementary InformationThe online version contains supplementary material available at 10.1186/s13059-025-03792-3.

Multiple DNA repair pathways have evolved to safeguard genome integrity and ensure organismal viability in the face of DNA damage. Errors in DNA repair processes in meiosis can lead to aneuploidy and developmental defects, but the processes that protect the germline from DNA damage remain poorly understood. Here we report a DNA damage-induced phosphorylation of the BRC-1/BRD-1 heterodimer that is essential for germline integrity in Caenorhabditis elegans. Failure to phosphorylate BRC-1/BRD-1 in response to DNA damage results in meiotic double-strand breaks (DSBs) accumulation, chromosome breakage, catastrophic diakinesis, and loss of fecundity. We further show that these defects are driven by the activity of C. elegans Bloom and Mus81, which catalyze Holliday junction dissolution and resolution, respectively. Hence, we propose that phosphorylation of BRC-1/BRD-1 in response to ionizing radiation-induced DSBs constitutes a key regulatory step that ensures the proper resolution of recombination intermediates required to preserve germline integrity.

Holliday junctions (HJs) are branched four-way DNA structures that link recombining chromosomes during double-strand break repair1. Despite posing a risk to chromosome segregation, HJs accumulate during meiotic prophase I as intermediates in the process of crossing-over2,3. Whether HJs have additional regulatory functions remains unclear. Here we establish an experimental system in budding yeast that enables conditional nucleolytic resolution of HJs after the establishment of meiotic chromosome synapsis. We find that HJ resolution triggers complete disassembly of the synaptonemal complex without disrupting the axis-loop organization of chromosomes. Mechanistically, HJs mediate the continued association of ZMM proteins with recombination nodules that form at the axes interface of homologous chromosome pairs. ZMM proteins, in turn, promote polymerization of the synaptonemal complex while simultaneously protecting HJs from processing by non-crossover pathways. Thus, reciprocal feedback between ZMMs, which stabilize HJs, and HJs, which retain ZMM proteins at future crossover sites, maintains chromosome synapsis until HJ-resolving enzymes are activated during exit from prophase I. Notably, by polymerizing and maintaining the synaptonemal complex structure, the HJ-ZMM interplay suppresses de novo double-strandbreak formation and recombination reinitiation. In doing so, this interplay suppresses the DNAdamage response, enabling meiotic progression without unrepaired breaks and supporting crossover assurance.

Sex-linked meiotic drivers limit the inheritance of the alternate sex chromosome in the heterogametic sex, subsequently skewing the offspring sex ratio. They consequently have large impacts on genome evolution, adaptation, and the emergence and maintenance of sexually selected traits. Despite this, our understanding of their molecular basis and consequences for gametogenesis and sex chromosome regulation more broadly has focused on a handful of model organisms, primarily Drosophila and mouse, which are not representative of the broad diversity of reproductive modes and drive systems in nature. Here, we employ single-cell RNA sequencing (scRNA-seq) to investigate a sex-linked meiotic driver in the Malaysian stalk-eyed fly, Teleopsis dalmanni. First, we produce a comprehensive single-cell atlas of the male T. dalmanni gonad and identify major testis cell types. We then provide a comprehensive profile of the cellular and transcriptional landscape of the testis, providing evidence for a lack of complete meiotic sex chromosome inactivation and complex trajectory of dosage compensation. Second, by contrasting single-cell expression data between drive and standard testes, we provide insight into the consequences of a meiotic driver for the transcriptomic landscape of the testis and sex chromosome regulation. Importantly, we show that the presence of a meiotic driver does not perturb fundamental patterns of X-linked regulation. Our results provide insight into how the meiotic driver might bias its transmission to the next generation and highlight genes with perturbed expression as a potential consequence of the disruption of spermatogenesis.

SummaryCrossover recombination supports meiotic chromosome inheritance and fertility by establishing chiasmata between homologous chromosomes prior to the first meiotic division. In addition to the physical exchange of DNA mediated by meiotic recombination, chiasma formation also involves restructuring of the underlying chromosome axis, possibly to help with chiasma maturation or to resolve chromosomal interlocks. Here, we identify condensin as an important regulator of axis remodeling inS. cerevisiae. Condensin is recruited near sites of meiotic crossover designation by pro-crossover factors but is largely dispensable for DNA exchange. Instead, condensin helps to create discontinuities in the meiotic chromosome axis by promoting removal of cohesin. In addition, chromosomes of condensin mutants exhibit unusually common parallel chromatin clouds and experience a chromosomal buildup of the conserved axis remodeler Pch2. Consistent with an important role of axis restructuring at crossover sites, the canonical anaphase-bridge phenotype of condensin mutants is partly rescued by redirecting meiotic DNA repair to sister chromatids instead of homologous chromosomes, suggesting that crossover-associated axis reorganization is important for faithful meiotic chromosome segregation.

The inherent differences between sex chromosomes in males and females create conflicts in gene expression, driving the evolution of regulatory mechanisms such as Meiotic Sex Chromosome Inactivation (MSCI), a process that transcriptionally silences the sex chromosomes during male meiosis. In this study, we explore the evolutionary dynamics of MSCI within the Drosophila genus by analyzing transcriptomes across different stages of spermatogenesis in D. melanogaster and its progressively more distant relatives, D. simulans, D. willistoni, and D. mojavensis. Stage-enriched bulk RNA sequencing, showing a strong correlation in spermatogenic gene expression patterns among these species, revealed that MSCI dates back to the early evolution of the Drosophila genus, impacting the regulation of both coding and long non-coding RNAs. Notably, for newly evolved genes, X-linked genes show higher expression levels than autosomal genes during mitosis and meiosis, indicating that MSCI predominantly regulates older genes. In contrast, newly evolved autosomal genes exhibit a gradual increase in expression throughout spermatogenesis, reaching their peak in the post-meiotic phase. During this phase, the expression of X-linked new genes decreases, eventually aligning with that of autosomal genes. This expression pattern suggests that haploid selection plays a crucial role in the regulation of new genes, with monoallelic expression of the X chromosome providing an advantage across all stages of germline development, while autosomal gene expression gains a selective edge primarily in the post-meiotic phase. Together, these findings provide new insights into the evolution of sex chromosomes and highlight the critical role of MSCI in shaping gene expression profiles in Drosophila.

The synaptonemal complex (SC) is a meiosis-specific structure that aligns homologous chromosomes and promotes the repair of meiotic DNA double-strand breaks (DSBs). To investigate how defects in SC formation affect gametogenesis in zebrafish, we analyzed mutations in two genes encoding core SC components: syce2 and sycp1. In syce2 mutants, chromosomes exhibit partial synapsis, primarily at sub-telomeric regions, whereas sycp1 mutant chromosomes display early prophase co-alignment but fail to synapse. Both mutants exhibit reduced efficiency in repairing meiotic DSBs compared to wild type. Despite these defects, syce2 and sycp1 mutant females are fertile. However, sycp1 mutant females produce a higher proportion of malformed progeny, correlating with increased univalent formation. While syce2 mutant males are fertile and produce normal offspring, sycp1 mutant males are sterile, with spermatocytes that transit prophase I but arrest at metaphase I or II. Additionally, sycp1 mutants display a male-biased sex ratio that can be suppressed by extending the developmental window for sex determination, suggesting that the absence of synapsis delays-but does not completely block-meiotic progression. Notably, embryos from syce2 and sycp1 mutant females exhibit widespread somatic mosaic aneuploidy, indicating that impaired meiotic chromosome dynamics can compromise genome stability during early development. In contrast to mouse SC mutants, the zebrafish syce2 and sycp1 mutants examined in this study progress through meiotic prophase I with minimal disruption, suggesting a less stringent surveillance mechanism for synapsis errors in zebrafish.

Crossover designation recruits condensin to reorganize the meiotic chromosome axis.

The role of the mitochondrial dynamic distribution in oocyte development arrest induced by F-53B.

In mouse early pachytene spermatocytes, the X and Y chromosomes undergo rapid non-homologous (NH) synapsis and desynapsis, but the functional significance remains unknown. Here, we report that pachynema-specific knockout of Speedy A (SpdyA) from telomeres caused persistent Y-X NH synapsis, with the entire Y axis synapsed onto the X axis. This persistent Y-X NH synapsis did not interrupt meiotic sex chromosome inactivation, recombination, or sex body formation, but it disrupted X-Y loop-axis organization and homologous X-Y desynapsis, leading to spermatocyte death. Similarly, persistent Y-X NH synapsis was also observed in pachytene spermatocytes lacking TRF1, where SpdyA was frequently lost from the X-Y non-pseudoautosomal region (non-PAR) telomeres. Mechanistic studies revealed that Serine 48 of SUN1 is a key SpdyA/CDK2 phosphorylation site required for Y-X NH desynapsis. We propose that SpdyA governs Y-X NH desynapsis by stabilizing the linkage between the X-Y non-PAR telomeres and their LINC complexes, and that this process is regulated independently from other aspects of pachynema progression. Our findings suggest a key role for Y-X NH desynapsis in establishing proper X-Y loop-axis organization.

INSIGHT INTO MEIOTIC DNA END RESECTION: MECHANISMS AND REGULATION

IntroductionMre11 is a multisubunit nuclease involved in DNA repair, and its dysfunction often causes DNA damage sensitivity, genomic instability, telomere shortening, and aberrant meiosis. However, the specific roles of Mre11 in porcine oocyte meiosis remain unclear.MethodsIn this study, porcine oocytes were treated with the Mre11-specific inhibitor mirin to investigate the function of Mre11 during meiotic maturation. Meiotic progression, spindle and chromosome structure, spindle migration, cytoplasmic actin polymerization, and DNA damage levels were assessed using immunofluorescence and relevant molecular markers including BubR1 and γH2A.X.ResultsInhibition of Mre11 activity led to failure of first polar body extrusion, with sustained BubR1 presence at kinetochores, indicating activation of the spindle assembly checkpoint (SAC). Mre11-inhibited oocytes showed disrupted spindle and chromosome organization due to decreased microtubule stability. Additionally, spindle migration to the oocyte cortex was impaired, correlating with reduced cytoplasmic actin polymerization. Elevated DNA damage levels were observed in treated oocytes as evidenced by increased γH2A.X staining.DiscussionThese findings demonstrate that Mre11 is essential for porcine oocyte meiotic progression by maintaining normal spindle assembly, actin cytoskeleton dynamics, and SAC activity. DNA damage accumulation following Mre11 inhibition likely contributes to meiotic failure, highlighting its critical role in ensuring oocyte quality.

Meiotic chromosome segregation in oocytes often relies on meiosis-specific modifications of mitotic molecular mechanisms to respond to the unique challenges of this asymmetric division. In this issue, Narula and Wignall (https://doi.org/10.1083/jcb.202503080) demonstrate how the conserved polo-like kinase in Caenorhabditiselegans, PLK-1, has been repurposed in unexpected ways to ensure accurate meiotic chromosome segregation during oogenesis.

Non-homologous sequence interactions during meiosis: meiotic challenges and evolutionary opportunities.