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Journal of Visualized Experiments : JoVE Jun 2023Oocytes are amongst the biggest and most long-lived cells in the female body. They are formed in the ovaries during embryonic development and remain arrested at the...
Oocytes are amongst the biggest and most long-lived cells in the female body. They are formed in the ovaries during embryonic development and remain arrested at the prophase of meiosis I. The quiescent state may last for years until the oocytes receive a stimulus to grow and obtain the competency to resume meiosis. This protracted state of arrest makes them extremely susceptible to accumulating DNA-damaging insults, which affect the genetic integrity of the female gametes and, therefore, the genetic integrity of the future embryo. Consequently, the development of an accurate method to detect DNA damage, which is the first step for the establishment of DNA damage response mechanisms, is of vital importance. This paper describes a common protocol to test the presence and progress of DNA damage in prophase-arrested oocytes during a period of 20 h. Specifically, we dissect mouse ovaries, retrieve the cumulus-oocyte complexes (COCs), remove the cumulus cells from the COCs, and culture the oocytes in Μ2 medium containing 3-isobutyl-1-methylxanthine to maintain the state of arrest. Thereafter, the oocytes are treated with the cytotoxic, antineoplasmic drug, etoposide, to engender double-strand breaks (DSBs). By using immunofluorescence and confocal microscopy, we detect and quantify the levels of the core protein γH2AX, which is the phosphorylated form of the histone H2AX. H2AX becomes phosphorylated at the sites of DSBs after DNA damage. The inability to restore DNA integrity following DNA damage in oocytes can lead to infertility, birth defects, and increased rates of spontaneous abortions. Therefore, the understanding of DNA damage response mechanisms and, at the same time, the establishment of an intact method for studying these mechanisms are essential for reproductive biology research.
Topics: Pregnancy; Female; Animals; Mice; DNA Breaks, Double-Stranded; Oocytes; Meiosis; Prophase; DNA
PubMed: 37427948
DOI: 10.3791/65494 -
Nature Structural & Molecular Biology Feb 2023In meiosis, a supramolecular protein structure, the synaptonemal complex (SC), assembles between homologous chromosomes to facilitate their recombination. Mammalian SC...
In meiosis, a supramolecular protein structure, the synaptonemal complex (SC), assembles between homologous chromosomes to facilitate their recombination. Mammalian SC formation is thought to involve hierarchical zipper-like assembly of an SYCP1 protein lattice that recruits stabilizing central element (CE) proteins as it extends. Here we combine biochemical approaches with separation-of-function mutagenesis in mice to show that, rather than stabilizing the SYCP1 lattice, the CE protein SYCE3 actively remodels this structure during synapsis. We find that SYCP1 tetramers undergo conformational change into 2:1 heterotrimers on SYCE3 binding, removing their assembly interfaces and disrupting the SYCP1 lattice. SYCE3 then establishes a new lattice by its self-assembly mimicking the role of the disrupted interface in tethering together SYCP1 dimers. SYCE3 also interacts with CE complexes SYCE1-SIX6OS1 and SYCE2-TEX12, providing a mechanism for their recruitment. Thus, SYCE3 remodels the SYCP1 lattice into a CE-binding integrated SYCP1-SYCE3 lattice to achieve long-range synapsis by a mature SC.
Topics: Animals; Mice; Chromosomal Proteins, Non-Histone; Chromosome Pairing; DNA-Binding Proteins; Mammals; Meiosis; Nuclear Proteins; Synaptonemal Complex
PubMed: 36635604
DOI: 10.1038/s41594-022-00909-1 -
Plant Science : An International... Dec 2022Meiosis plays an essential role in the production of male and female gametes. Extensive studies have elucidated that homologous chromosome association and pairing are...
Meiosis plays an essential role in the production of male and female gametes. Extensive studies have elucidated that homologous chromosome association and pairing are essential for crossing-over and recombination of chromosomal segments. However, the molecular mechanism of chromosome recognition and pairing remains elusive. Here, we identified a rice male-female sterility mutant plant. Cytological observations showed that the development of both pollen and embryo sacs of the mutant were abnormal due to defects in homologous chromosome recognition and pairing during prophase I. Map-based cloning revealed that Os06g0473000 encoding a poor homologous synapsis 1 (PHS1) protein is the candidate target gene, which was confirmed by knockout using CRISPR/Cas9 technology. Sequence analysis revealed a single base mutation (G > A) involving the junction of the fourth exon and intron of OsPHS1, which is predicted to alter splicing, resulting in an Osphs1 mutant. Expression pattern analysis indicated that OsPHS1 expression levels were mainly expressed in panicles at the beginning of meiosis. Subcellular localization analysis demonstrated that the OsPHS1 protein is situated in the nucleus and cytoplasm. Taken together, our results suggest an important role for OsPHS1 in homologous chromosome pairing in both male and female gametogenesis in rice.
Topics: Oryza; Plant Proteins; Chromosome Pairing; Meiosis; Germ Cells
PubMed: 36183810
DOI: 10.1016/j.plantsci.2022.111480 -
Current Genetics Aug 2016The pairing and recombination of homologous chromosomes during the meiotic prophase is necessary for the accurate segregation of chromosomes in meiosis. However, the... (Review)
Review
The pairing and recombination of homologous chromosomes during the meiotic prophase is necessary for the accurate segregation of chromosomes in meiosis. However, the mechanism by which homologous chromosomes achieve this pairing has remained an open question. Meiotic cohesins have been shown to affect chromatin compaction; however, the impact of meiotic cohesins on homologous pairing and the fine structures of cohesion-based chromatin remain to be determined. A recent report using live-cell imaging and super-resolution microscopy demonstrated that the lack of meiotic cohesins alters the chromosome axis structures and impairs the pairing of homologous chromosomes. These results suggest that meiotic cohesin-based chromosome axis structures are crucial for the pairing of homologous chromosomes.
Topics: Cell Cycle Proteins; Chromosomal Proteins, Non-Histone; Chromosome Pairing; Chromosomes, Fungal; Meiosis; Schizosaccharomyces; Cohesins
PubMed: 26856595
DOI: 10.1007/s00294-016-0570-x -
Molecular Cell Sep 2020A long-standing conundrum is how mitotic chromosomes can compact, as required for clean separation to daughter cells, while maintaining close parallel alignment of...
A long-standing conundrum is how mitotic chromosomes can compact, as required for clean separation to daughter cells, while maintaining close parallel alignment of sister chromatids. Pursuit of this question, by high resolution 3D fluorescence imaging of living and fixed mammalian cells, has led to three discoveries. First, we show that the structural axes of separated sister chromatids are linked by evenly spaced "mini-axis" bridges. Second, when chromosomes first emerge as discrete units, at prophase, they are organized as co-oriented sister linear loop arrays emanating from a conjoined axis. We show that this same basic organization persists throughout mitosis, without helical coiling. Third, from prophase onward, chromosomes are deformed into sequential arrays of half-helical segments of alternating handedness (perversions), accompanied by correlated kinks. These arrays fluctuate dynamically over <15 s timescales. Together these discoveries redefine the foundation for thinking about the evolution of mitotic chromosomes as they prepare for anaphase segregation.
Topics: Adenosine Triphosphatases; Anaphase; Animals; Cell Cycle Proteins; Chromatids; Chromosomal Proteins, Non-Histone; Chromosomes; DNA Topoisomerases, Type II; DNA-Binding Proteins; Imaging, Three-Dimensional; Mammals; Metaphase; Mitosis; Prophase
PubMed: 32768407
DOI: 10.1016/j.molcel.2020.07.002 -
Proceedings of the National Academy of... Nov 2022In the early stages of meiosis, maternal and paternal chromosomes pair with their homologous partner and recombine to ensure exchange of genetic information and proper...
In the early stages of meiosis, maternal and paternal chromosomes pair with their homologous partner and recombine to ensure exchange of genetic information and proper segregation. These events can vary drastically between species and between males and females of the same species. In in contrast to females, males do not form synaptonemal complexes (SCs), do not recombine, and have no crossing over; yet, males are able to segregate their chromosomes properly. Here, we investigated the early steps of homolog pairing in males. We found that homolog centromeres are not paired in germline stem cells (GSCs) and become paired in the mitotic region before meiotic entry, similarly to females. Surprisingly, male germline cells express SC proteins, which localize to centromeres and promote pairing. We further found that the SUN/KASH (LINC) complex and microtubules are required for homolog pairing as in females. Chromosome movements in males, however, are much slower than in females and we demonstrate that this slow dynamic is compensated in males by having longer cell cycles. In agreement, slowing down cell cycles was sufficient to rescue pairing-defective mutants in female meiosis. Our results demonstrate that although meiosis differs significantly between males and females, sex-specific cell cycle kinetics integrate similar molecular mechanisms to achieve proper centromere pairing.
Topics: Animals; Male; Female; Chromosome Pairing; Drosophila; Synaptonemal Complex; Centromere; Meiosis; Chromosomes; Chromosome Segregation
PubMed: 36375065
DOI: 10.1073/pnas.2207660119 -
Journal of Molecular Cell Biology Dec 2021Meiosis produces the haploid gametes required by all sexually reproducing organisms, occurring in specific temperature ranges in different organisms. However, how...
Meiosis produces the haploid gametes required by all sexually reproducing organisms, occurring in specific temperature ranges in different organisms. However, how meiotic thermotolerance is regulated remains largely unknown. Using the model organism Caenorhabditis elegans, here, we identified the synaptonemal complex (SC) protein SYP-5 as a critical regulator of meiotic thermotolerance. syp-5-null mutants maintained a high percentage of viable progeny at 20°C but produced significantly fewer viable progeny at 25°C, a permissive temperature in wild-type worms. Cytological analysis of meiotic events in the mutants revealed that while SC assembly and disassembly, as well as DNA double-strand break repair kinetics, were not affected by the elevated temperature, crossover designation, and bivalent formation were significantly affected. More severe homolog segregation errors were also observed at elevated temperature. A temperature switching assay revealed that late meiotic prophase events were not temperature-sensitive and that meiotic defects during pachytene stage were responsible for the reduced viability of syp-5 mutants at the elevated temperature. Moreover, SC polycomplex formation and hexanediol sensitivity analysis suggested that SYP-5 was required for the normal properties of the SC, and charge-interacting elements in SC components were involved in regulating meiotic thermotolerance. Together, these findings provide a novel molecular mechanism for meiotic thermotolerance regulation.
Topics: Animals; Animals, Genetically Modified; Caenorhabditis elegans; Caenorhabditis elegans Proteins; Computational Biology; Meiosis; Synaptonemal Complex; Thermotolerance
PubMed: 34081106
DOI: 10.1093/jmcb/mjab035 -
Nucleic Acids Research Sep 2023RAD54 family DNA translocases partner with RAD51 recombinases to ensure stable genome inheritance, exhibiting biochemical activities both in promoting recombinase...
RAD54 family DNA translocases partner with RAD51 recombinases to ensure stable genome inheritance, exhibiting biochemical activities both in promoting recombinase removal and in stabilizing recombinase association with DNA. Understanding how such disparate activities of RAD54 paralogs align with their biological roles is an ongoing challenge. Here we investigate the in vivo functions of Caenorhabditis elegans RAD54 paralogs RAD-54.L and RAD-54.B during meiotic prophase, revealing distinct contributions to the dynamics of RAD-51 association with DNA and to the progression of meiotic double-strand break repair (DSBR). While RAD-54.L is essential for RAD-51 removal from meiotic DSBR sites to enable recombination progression, RAD-54.B is largely dispensable for meiotic DSBR. However, RAD-54.B is required to prevent hyperaccumulation of RAD-51 on unbroken DNA during the meiotic sub-stage when DSBs and early recombination intermediates form. Moreover, DSB-independent hyperaccumulation of RAD-51 foci in the absence of RAD-54.B is RAD-54.L-dependent, revealing a hidden activity of RAD-54.L in promoting promiscuous RAD-51 association that is antagonized by RAD-54.B. We propose a model wherein a division of labor among RAD-54 paralogs allows germ cells to ramp up their capacity for efficient homologous recombination that is crucial to successful meiosis while counteracting potentially deleterious effects of unproductive RAD-51 association with unbroken DNA.
Topics: Animals; Caenorhabditis elegans; Caenorhabditis elegans Proteins; Carrier Proteins; DNA; DNA Repair; Germ Cells; Meiosis; Prophase; Rad51 Recombinase; DNA Helicases
PubMed: 37548405
DOI: 10.1093/nar/gkad638 -
Cytogenetic and Genome Research 2016The cytological analysis of meiotic chromosomes is an exceptional tool to approach complex processes such as synapsis and recombination during the division. Chromosome... (Review)
Review
The cytological analysis of meiotic chromosomes is an exceptional tool to approach complex processes such as synapsis and recombination during the division. Chromosome studies of meiosis have been especially valuable in birds, where naturally occurring mutants or experimental knock-out animals are not available to fully investigate the basic mechanisms of major meiotic events. This review highlights the main contributions of synaptonemal complex and lampbrush chromosome research to the current knowledge of avian meiosis, with special emphasis on the organization of chromosomes during prophase I, the impact of chromosome rearrangements during meiosis, and distinctive features of the ZW pair.
Topics: Animals; Birds; Crossing Over, Genetic; Female; Genetic Markers; Meiosis; Meiotic Prophase I; Sex Chromosomes; Synaptonemal Complex
PubMed: 28030854
DOI: 10.1159/000453541 -
PLoS Genetics Aug 2018In meiosis I, homologous chromosomes segregate away from each other-the first of two rounds of chromosome segregation that allow the formation of haploid gametes. In... (Review)
Review
In meiosis I, homologous chromosomes segregate away from each other-the first of two rounds of chromosome segregation that allow the formation of haploid gametes. In prophase I, homologous partners become joined along their length by the synaptonemal complex (SC) and crossovers form between the homologs to generate links called chiasmata. The chiasmata allow the homologs to act as a single unit, called a bivalent, as the chromosomes attach to the microtubules that will ultimately pull them away from each other at anaphase I. Recent studies, in several organisms, have shown that when the SC disassembles at the end of prophase, residual SC proteins remain at the homologous centromeres providing an additional link between the homologs. In budding yeast, this centromere pairing is correlated with improved segregation of the paired partners in anaphase. However, the causal relationship of prophase centromere pairing and subsequent disjunction in anaphase has been difficult to demonstrate as has been the relationship between SC assembly and the assembly of the centromere pairing apparatus. Here, a series of in-frame deletion mutants of the SC component Zip1 were used to address these questions. The identification of a separation-of-function allele that disrupts centromere pairing, but not SC assembly, has made it possible to demonstrate that centromere pairing and SC assembly have mechanistically distinct features and that the centromere pairing function of Zip1 drives disjunction of the paired partners in anaphase I.
Topics: Alleles; Anaphase; Centromere; Chromosome Pairing; Chromosome Segregation; Meiosis; Nuclear Proteins; Recombination, Genetic; Saccharomyces cerevisiae Proteins; Saccharomycetales; Synaptonemal Complex
PubMed: 30091974
DOI: 10.1371/journal.pgen.1007513