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PloS One 2020Clinical applications of oocytes cryopreservation include preservation of future fertility of young cancer patients, substitution of embryo freezing to avoid associated...
Clinical applications of oocytes cryopreservation include preservation of future fertility of young cancer patients, substitution of embryo freezing to avoid associated legal and ethical issues, and delaying childbearing years. While the outcome of oocyte cryopreservation has recently been improved, currently used vitrification method still suffer from increased biosafety risk and handling issues while slow freezing techniques yield overall low success. Understanding better the mechanism of cryopreservation-induced injuries may lead to development of more reliable and safe methods for oocyte cryopreservation. Using the mouse model, a microarray study was conducted on oocyte cryopreservation to identify cryoinjuries to transcriptionally active genome. To this end, metaphase II (MII) oocytes were subjected to standard slow freezing, and then analyzed at the four-cell stage after embryonic genome activation. Non-frozen four-cell embryos served as controls. Differentially expressed genes were identified and validated using RT-PCR. Embryos produced from the cryopreserved oocytes displayed 200 upregulated and 105 downregulated genes, associated with the regulation of mitochondrial function, protein ubiquitination and maintenance, cellular response to stress and oxidative states, fatty acid and lipid regulation/metabolism, and cell cycle maintenance. These findings reveal previously unrecognized effects of standard slow oocyte freezing on embryonic gene expression, which can be used to guide improvement of oocyte cryopreservation methods.
Topics: Animals; Cryopreservation; Embryo, Mammalian; Embryonic Development; Female; Fertilization in Vitro; Freezing; Gene Expression Regulation, Developmental; Humans; Male; Metaphase; Mice; Mice, Inbred C57BL; Mice, Inbred DBA; Oocytes; Protein Interaction Maps; Real-Time Polymerase Chain Reaction; Transcriptome
PubMed: 32251418
DOI: 10.1371/journal.pone.0231108 -
Cellular and Molecular Life Sciences :... Mar 2021Chromosomal fragile sites are described as areas within the tightly packed mitotic chromatin that appear as breaks or gaps mostly tracing back to a loosened structure... (Review)
Review
Chromosomal fragile sites are described as areas within the tightly packed mitotic chromatin that appear as breaks or gaps mostly tracing back to a loosened structure and not a real nicked break within the DNA molecule. Most facts about fragile sites result from studies in mitotic cells, mainly during metaphase and mainly in lymphocytes. Here, we synthesize facts about the genomic regions that are prone to form gaps and breaks on metaphase chromosomes in the context of interphase. We conclude that nuclear architecture shapes the activity profile of the cell, i.e. replication timing and transcriptional activity, thereby influencing genomic integrity during interphase with the potential to cause fragility in mitosis. We further propose fragile sites as examples of regions specifically positioned in the interphase nucleus with putative anchoring points at the nuclear lamina to enable a tightly regulated replication-transcription profile and diverse signalling functions in the cell. Consequently, fragility starts before the actual display as chromosomal breakage in metaphase to balance the initial contradiction of cellular overgrowth or malfunctioning and maintaining diversity in molecular evolution.
Topics: Animals; Cell Nucleus; Chromosomal Instability; Chromosome Fragile Sites; DNA; DNA Replication; Genome, Human; Humans; Interphase; Mitosis
PubMed: 33219838
DOI: 10.1007/s00018-020-03698-2 -
PLoS Genetics Jan 2021Although kinetochores normally play a key role in sister chromatid separation and segregation, chromosome fragments lacking kinetochores (acentrics) can in some cases...
Although kinetochores normally play a key role in sister chromatid separation and segregation, chromosome fragments lacking kinetochores (acentrics) can in some cases separate and segregate successfully. In Drosophila neuroblasts, acentric chromosomes undergo delayed, but otherwise normal sister separation, revealing the existence of kinetochore- independent mechanisms driving sister chromosome separation. Bulk cohesin removal from the acentric is not delayed, suggesting factors other than cohesin are responsible for the delay in acentric sister separation. In contrast to intact kinetochore-bearing chromosomes, we discovered that acentrics align parallel as well as perpendicular to the mitotic spindle. In addition, sister acentrics undergo unconventional patterns of separation. For example, rather than the simultaneous separation of sisters, acentrics oriented parallel to the spindle often slide past one another toward opposing poles. To identify the mechanisms driving acentric separation, we screened 117 RNAi gene knockdowns for synthetic lethality with acentric chromosome fragments. In addition to well-established DNA repair and checkpoint mutants, this candidate screen identified synthetic lethality with X-chromosome-derived acentric fragments in knockdowns of Greatwall (cell cycle kinase), EB1 (microtubule plus-end tracking protein), and Map205 (microtubule-stabilizing protein). Additional image-based screening revealed that reductions in Topoisomerase II levels disrupted sister acentric separation. Intriguingly, live imaging revealed that knockdowns of EB1, Map205, and Greatwall preferentially disrupted the sliding mode of sister acentric separation. Based on our analysis of EB1 localization and knockdown phenotypes, we propose that in the absence of a kinetochore, microtubule plus-end dynamics provide the force to resolve DNA catenations required for sister separation.
Topics: Animals; Cell Cycle Proteins; Chromatids; Chromosomal Proteins, Non-Histone; Chromosome Segregation; DNA Topoisomerases, Type II; Drosophila melanogaster; Kinetochores; Larva; Metaphase; Microtubules; Mitosis; Spindle Apparatus; Cohesins
PubMed: 33513180
DOI: 10.1371/journal.pgen.1009304 -
Clinical Laboratory Apr 2022Most laboratories adopt the results of metaphase fluorescent in situ hybridization (FISH) for the diagnosis of microdeletion syndromes. To investigate the discrepancy...
BACKGROUND
Most laboratories adopt the results of metaphase fluorescent in situ hybridization (FISH) for the diagnosis of microdeletion syndromes. To investigate the discrepancy between the results of interphase and metaphase, we compared the quantitative results of FISH for 5 kinds of microdeletion syndrome and gender determination disorders (SDD).
METHODS
A total of 282 (135 for DiGeorge syndrome, 20 for Kalmann syndrome, 7 for Miller-Dieker syndrome, 38 for Prader Willi/Angelman syndrome, 62 for Williams syndrome, and 20 for SDD (SRY FISH)) were enrolled. For SRY FISH, we artificially mixed fresh blood of male and female with various ratios and then compared the results of metaphase and interphase SRY FISH. Using a bio-cell chip, we performed interphase FISH in 168 patients with microdeletion syndromes and compared the results with manual interphase.
RESULTS
The concordance rate between the results of metaphase and interphase was 100% in microdeletion syndrome. In the disorders of gender development, SRY FISH showed 100% concordance between interphase and metaphase when we counted 50 metaphase cells and 100 interphase cells. Comparison with mixtures of male and female blood at various ratios also showed 100% concordance. The results of bio-cell chip showed 100% concordance between previous interphase FISH results.
CONCLUSIONS
Considering the complete concordance between interphase and metaphase in microdeletion syndrome, the application of interphase FISH without performing metaphase FISH can be a screening test for microdeletion syndrome. Confirmation by metaphase FISH can be performed only in cases with abnormal results by interphase FISH.
Topics: DiGeorge Syndrome; Female; Humans; In Situ Hybridization, Fluorescence; Interphase; Male; Prader-Willi Syndrome; Williams Syndrome
PubMed: 35443603
DOI: 10.7754/Clin.Lab.2021.210747 -
Current Protocols Mar 2023The fluorescent dyes Hoechst (HO) and Chromomycin A3 (CA3) are commonly used for bivariate flow karyotyping to distinguish individual chromosomes from one another based...
The fluorescent dyes Hoechst (HO) and Chromomycin A3 (CA3) are commonly used for bivariate flow karyotyping to distinguish individual chromosomes from one another based on differences in base composition and DNA content. However, analysis of chromosomes using this fluorescent dye combination requires a flow cytometer equipped with lasers of specific wavelengths and higher power than is typical of conventional flow cytometers. This unit presents a chromosome staining technique with a dye combination of DAPI and propidium iodide (PI). Chromosomes stained using this dye combination can be analyzed on conventional flow cytometers equipped with a typical configuration of lasers and optics. © 2023 The Authors. Current Protocols published by Wiley Periodicals LLC. Basic Protocol 1: Cell culture and metaphase harvest of suspension cell line Alternate Protocol 1: Cell culture and metaphase harvest of adherent cell line Basic Protocol 2: Preparation of chromosome suspension using polyamine isolation buffer Basic Protocol 3: Staining chromosomes with DAPI and propidium iodide Alternate Protocol 2: Staining chromosomes with Hoechst and Chromomycin A3 Basic Protocol 4: Bivariate flow karyotyping on a cell analyzer Basic Protocol 5: Bivariate flow karyotyping on a cell sorter Basic Protocol 6: Purification of flow-sorted chromosomes.
Topics: Chromomycin A3; DNA; Propidium; Chromosomes; Karyotyping; Fluorescent Dyes
PubMed: 36920094
DOI: 10.1002/cpz1.718 -
EMBO Reports Apr 2024Stabilization of microtubule plus end-directed kinesin CENP-E at the metaphase kinetochores is important for chromosome alignment, but its mechanism remains unclear....
Stabilization of microtubule plus end-directed kinesin CENP-E at the metaphase kinetochores is important for chromosome alignment, but its mechanism remains unclear. Here, we show that CKAP5, a conserved microtubule plus tip protein, regulates CENP-E at kinetochores in human cells. Depletion of CKAP5 impairs CENP-E localization at kinetochores at the metaphase plate and results in increased kinetochore-microtubule stability and attachment errors. Erroneous attachments are also supported by computational modeling. Analysis of CKAP5 knockout cancer cells of multiple tissue origins shows that CKAP5 is preferentially essential in aneuploid, chromosomally unstable cells, and the sensitivity to CKAP5 depletion is correlated to that of CENP-E depletion. CKAP5 depletion leads to reduction in CENP-E-BubR1 interaction and the interaction is rescued by TOG4-TOG5 domain of CKAP5. The same domain can rescue CKAP5 depletion-induced CENP-E removal from the kinetochores. Interestingly, CKAP5 depletion facilitates recruitment of PP1 to the kinetochores and furthermore, a PP1 target site-specific CENP-E phospho-mimicking mutant gets stabilized at kinetochores in the CKAP5-depleted cells. Together, the results support a model in which CKAP5 controls mitotic chromosome attachment errors by stabilizing CENP-E at kinetochores and by regulating stability of the kinetochore-attached microtubules.
Topics: Humans; Kinetochores; Chromosomal Proteins, Non-Histone; Microtubules; Metaphase; Kinesins; HeLa Cells; Mitosis; Chromosome Segregation; Microtubule-Associated Proteins
PubMed: 38424231
DOI: 10.1038/s44319-024-00106-9 -
The Journal of Cell Biology Jun 2022Errors in mitosis that cause chromosome missegregation lead to aneuploidy and micronucleus formation, which are associated with cancer. Accurate segregation requires the...
Errors in mitosis that cause chromosome missegregation lead to aneuploidy and micronucleus formation, which are associated with cancer. Accurate segregation requires the alignment of all chromosomes by the mitotic spindle at the metaphase plate, and any misalignment must be corrected before anaphase is triggered. The spindle is situated in a membrane-free "exclusion zone"; beyond this zone, endomembranes (mainly endoplasmic reticulum) are densely packed. We investigated what happens to misaligned chromosomes localized beyond the exclusion zone. Here we show that such chromosomes become ensheathed in multiple layers of endomembranes. Chromosome ensheathing delays mitosis and increases the frequency of chromosome missegregation and micronucleus formation. We use an induced organelle relocalization strategy in live cells to show that clearance of endomembranes allows for the rescue of chromosomes that were destined for missegregation. Our findings indicate that endomembranes promote the missegregation of misaligned chromosomes that are outside the exclusion zone and therefore constitute a risk factor for aneuploidy.
Topics: Anaphase; Aneuploidy; Cell Membrane; Chromosome Segregation; Chromosomes; Endoplasmic Reticulum; Humans; Metaphase; Mitosis; Spindle Apparatus
PubMed: 35486148
DOI: 10.1083/jcb.202203021 -
Molecular Cell Nov 2021Structural heterogeneity of nucleosomes in functional chromosomes is unknown. Here, we devise the template-, reference- and selection-free (TRSF) cryo-EM pipeline to...
Structural heterogeneity of nucleosomes in functional chromosomes is unknown. Here, we devise the template-, reference- and selection-free (TRSF) cryo-EM pipeline to simultaneously reconstruct cryo-EM structures of protein complexes from interphase or metaphase chromosomes. The reconstructed interphase and metaphase nucleosome structures are on average indistinguishable from canonical nucleosome structures, despite DNA sequence heterogeneity, cell-cycle-specific posttranslational modifications, and interacting proteins. Nucleosome structures determined by a decoy-classifying method and structure variability analyses reveal the nucleosome structural variations in linker DNA, histone tails, and nucleosome core particle configurations, suggesting that the opening of linker DNA, which is correlated with H2A C-terminal tail positioning, is suppressed in chromosomes. High-resolution (3.4-3.5 Å) nucleosome structures indicate DNA-sequence-independent stabilization of superhelical locations ±0-1 and ±3.5-4.5. The linker histone H1.8 preferentially binds to metaphase chromatin, from which chromatosome cryo-EM structures with H1.8 at the on-dyad position are reconstituted. This study presents the structural characteristics of nucleosomes in chromosomes.
Topics: Animals; Cell Communication; Cell Cycle; Cell Division; Chromatin; Chromosomes; Computer Simulation; Cryoelectron Microscopy; DNA; Humans; Hydrophobic and Hydrophilic Interactions; Interphase; Metaphase; Nucleosomes; Protein Conformation; Protein Domains; Protein Processing, Post-Translational; Xenopus
PubMed: 34478647
DOI: 10.1016/j.molcel.2021.08.010 -
Cell Reports Jan 2021During mitotic chromosome segregation, the protease separase severs cohesin between sister chromatids. A probe for separase activity has shown that separase undergoes...
During mitotic chromosome segregation, the protease separase severs cohesin between sister chromatids. A probe for separase activity has shown that separase undergoes abrupt activation shortly before anaphase onset, after being suppressed throughout metaphase; however, the relevance of this control remains unclear. Here, we report that separase activates precociously, with respect to anaphase onset, during prolonged metaphase in multiple types of cancer cell lines. The artificial extension of metaphase in chromosomally stable diploid cells leads to precocious activation and, subsequently, to chromosomal bridges in anaphase, which seems to be attributable to incomplete cohesin removal. Conversely, shortening back of a prolonged metaphase restores the activation of separase and ameliorates anaphase bridge formation. These observations suggest that retarded metaphase progression affects the separase activation profile and its enzymatic proficiency. Our findings provide an unanticipated etiology for chromosomal instability in cancers and underscore the relevance of swift mitotic transitions for fail-safe chromosome segregation.
Topics: Animals; Chromosome Segregation; Humans; Mice; Mitosis; Rabbits; Separase
PubMed: 33472072
DOI: 10.1016/j.celrep.2020.108652 -
Journal of Ovarian Research Nov 2023The oocyte and its surrounding cumulus cells (CCs) exist as an inseparable entity. The maturation of the oocyte relies on communication between the oocyte and the... (Review)
Review
BACKGROUND
The oocyte and its surrounding cumulus cells (CCs) exist as an inseparable entity. The maturation of the oocyte relies on communication between the oocyte and the surrounding CCs. However, oocyte evaluation is primarily based on morphological parameters currently, which offer limited insight into the quality and competence of the oocyte. Here, we conducted transcriptomic profiling of oocytes and their CCs from 47 patients undergoing preimplantation genetic testing for aneuploidy (PGT-A). We aimed to investigate the molecular events occurring between oocytes and CCs at different stages of oocyte maturation (germinal vesicle [GV], metaphase I [MI], and metaphase II [MII]). Our goal is to provide new insights into in vitro oocyte maturation (IVM).
RESULTS
Our findings indicate that oocyte maturation is a complex and dynamic process and that MI oocytes can be further classified into two distinct subtypes: GV-like-MI oocytes and MII-like-MI oocytes. Human oocytes and cumulus cells at three different stages of maturation were analyzed using RNA-seq, which revealed unique transcriptional machinery, stage-specific genes and pathways, and transcription factor networks that displayed developmental stage-specific expression patterns. We have also identified that both lipid and cholesterol metabolism in cumulus cells is active during the late stage of oocyte maturation. Lipids may serve as a more efficient energy source for oocytes and even embryogenesis.
CONCLUSIONS
Overall, our study provides a relatively comprehensive overview of the transcriptional characteristics and potential interactions between human oocytes and cumulus cells at various stages of maturation before ovulation. This study may offer novel perspectives on IVM and provide a reliable reference data set for understanding the transcriptional regulation of follicular maturation.
Topics: Female; Humans; Metaphase; Transcriptome; Cumulus Cells; Oocytes; In Vitro Oocyte Maturation Techniques; Ovulation
PubMed: 37993893
DOI: 10.1186/s13048-023-01291-2