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Biology of Reproduction Jun 2015Vertebrate eggs are arrested at metaphase of meiosis II, a state classically known as cytostatic factor arrest. Maintenance of this arrest until the time of...
Vertebrate eggs are arrested at metaphase of meiosis II, a state classically known as cytostatic factor arrest. Maintenance of this arrest until the time of fertilization and then fertilization-induced exit from metaphase II are crucial for reproductive success. Another key aspect of this meiotic arrest and exit is regulation of the metaphase II spindle, which must be appropriately localized adjacent to the egg cortex during metaphase II and then progress into successful asymmetric cytokinesis to produce the second polar body. This study examined the mitogen-activated protein kinases MAPK3 and MAPK1 (also known as ERK1/2) as regulators of these two related aspects of mammalian egg biology, specifically testing whether this MAPK pathway affected myosin-II function and whether myosin-II perturbation would produce some of the same effects as MAPK pathway perturbation. Inhibition of the MEK1/2-MAPK pathway with U0126 leads to reduced levels of phosphorylated myosin-regulatory light chain (pMRLC) and causes a reduction in cortical tension, effects that are mimicked by treatment with the myosin light chain kinase (MLCK) inhibitor ML-7. These data indicate that one mechanism by which the MAPK pathway acts in eggs is by affecting myosin-II function. We further show that MAPK or MLCK inhibition induces loss of normal cortical spindle localization or parthenogenetic egg activation. This parthenogenesis is dependent on cytosolic and extracellular calcium and can be rescued by hyperloading eggs with zinc, suggesting that these effects of inhibition of MLCK or the MAPK pathway are linked with dysregulation of ion homeostasis.
Topics: Animals; Azepines; Butadienes; Calcium; Chelating Agents; Egtazic Acid; Enzyme Inhibitors; Female; Metaphase; Mice; Mitogen-Activated Protein Kinase 1; Mitogen-Activated Protein Kinase 3; Myosin Type II; Myosin-Light-Chain Kinase; Naphthalenes; Nitriles; Ovum; Zinc
PubMed: 25904014
DOI: 10.1095/biolreprod.114.127027 -
Mutation Research. Genetic Toxicology... Jul 2021Chromosomal aberrations (CAs) in peripheral blood lymphocytes can be used as biomarkers of cancer risk. Cytogenetic tests were conducted on 2396 healthy Hungarian...
Chromosomal aberrations (CAs) in peripheral blood lymphocytes can be used as biomarkers of cancer risk. Cytogenetic tests were conducted on 2396 healthy Hungarian individuals and cancer incidence was followed up from 1989 to 2018. Venous blood samples were obtained from the subjects and metaphases from lymphocyte cultures were prepared. We compared the CA frequencies of the various smoking (1-5; 6-10; 11-19; or 20-40 cigarettes/day) and exposure (irradiation; chemical industry; chemical research laboratory) groups. Chromatid break (p = 0.0002), total aberration (p = 0.002), and aberrant cell (p = 0.001) frequencies were higher in smokers than in non-smokers. For very heavy smokers, total CAs were significantly higher than for non-smokers (<0.001) or less intensive smokers (p = 0.003-0.0006). Intensity of smoking was a predictor of chromosomal aberrations, while duration was not. During follow-up, 177 (7.3 %) cancer cases were found. A Cox-regression model showed that subjects with cell values ≥2 CAs developed cancer more frequently (hazard ratio = 1.39; 95 % CI, 1.02-1.90). The relative risks of cancer were 1.06 (95 % CI 0.53-2.06) for light smokers and 1.74 (95 % CI 1.08-2.77) for very heavy smokers. The distributions of cancer sites showed differences between smoker and non-smoker groups: in male smokers, lung cancer, in non-smokers, prostate, and in females (both groups) breast cancer were most common. Cancer incidence correlated with chromosome aberrations; smoking was not a confounder in this relationship.
Topics: Adolescent; Adult; Aged; Aged, 80 and over; Biomarkers; Cells, Cultured; Chromosome Aberrations; Female; Healthy Volunteers; Humans; Incidence; Lymphocytes; Male; Metaphase; Middle Aged; Neoplasms; Sister Chromatid Exchange; Smoking; Young Adult
PubMed: 34266629
DOI: 10.1016/j.mrgentox.2021.503373 -
Chromosoma Sep 2019Cyclins, as regulatory partners of cyclin-dependent kinases (CDKs), control the switch-like cell cycle transitions that orchestrate orderly duplication and segregation...
Cyclins, as regulatory partners of cyclin-dependent kinases (CDKs), control the switch-like cell cycle transitions that orchestrate orderly duplication and segregation of genomes. Compared to mitosis, relatively little is known about how cyclin-CDK complexes control meiosis, the specialized cell division that generates gametes for sexual production. Mouse cyclin B3 was previously shown to have expression restricted to the beginning of meiosis, making it a candidate to regulate meiotic events. Indeed, female mice lacking cyclin B3 are sterile because oocytes arrest at the metaphase-to-anaphase transition of meiosis I. However, whether cyclin B3 functions during spermatogenesis was untested. Here, we found that males lacking cyclin B3 are fertile and show no detectable defects in spermatogenesis based on histological analysis of seminiferous tubules. Cytological analysis further showed no detectable defects in homologous chromosome synapsis or meiotic progression, and suggested that recombination is initiated and completed efficiently. Moreover, absence of cyclin B3 did not exacerbate previously described meiotic defects in mutants deficient for cyclin E2, suggesting a lack of redundancy between these cyclins. Thus, unlike in females, cyclin B3 is not essential for meiosis in males despite its prominent meiosis-specific expression.
Topics: Alleles; Amino Acid Sequence; Animals; Cyclin B; Gene Editing; Gene Expression; Immunohistochemistry; Male; Meiosis; Metaphase; Mice; Prophase; Protein Domains; Recombination, Genetic; Spermatogenesis
PubMed: 31446450
DOI: 10.1007/s00412-019-00725-5 -
Science Advances Mar 2020The meiotic prophase I to metaphase I (PI/MI) transition requires chromosome desynapsis and metaphase competence acquisition. However, control of these major meiotic...
The meiotic prophase I to metaphase I (PI/MI) transition requires chromosome desynapsis and metaphase competence acquisition. However, control of these major meiotic events is poorly understood. Here, we identify an essential role for SKP1, a core subunit of the SKP1-Cullin-F-box (SCF) ubiquitin E3 ligase, in the PI/MI transition. SKP1 localizes to synapsed chromosome axes and evicts HORMAD proteins from these regions in meiotic spermatocytes. SKP1-deficient spermatocytes display premature desynapsis, precocious pachytene exit, loss of PLK1 and BUB1 at centromeres, but persistence of HORMAD, γH2AX, RPA2, and MLH1 in diplonema. Strikingly, SKP1-deficient spermatocytes show sharply reduced MPF activity and fail to enter MI despite treatment with okadaic acid. SKP1-deficient oocytes exhibit desynapsis, chromosome misalignment, and progressive postnatal loss. Therefore, SKP1 maintains synapsis in meiosis of both sexes. Furthermore, our results support a model where SKP1 functions as the long-sought intrinsic metaphase competence factor to orchestrate MI entry during male meiosis.
Topics: Alleles; Animals; Gene Expression Regulation; Male; Meiosis; Meiotic Prophase I; Mesothelin; Metaphase; Mice; Mice, Transgenic; Oocytes; S-Phase Kinase-Associated Proteins; Sex Factors
PubMed: 32232159
DOI: 10.1126/sciadv.aaz2129 -
Taiwanese Journal of Obstetrics &... Jul 2022To investigate whether the rate of euploidy and pregnancy outcomes are affected by smooth endoplasmic reticulum clusters (SERc) and other metaphase II human oocyte...
OBJECTIVE
To investigate whether the rate of euploidy and pregnancy outcomes are affected by smooth endoplasmic reticulum clusters (SERc) and other metaphase II human oocyte dysmorphisms.
MATERIALS AND METHODS
Retrospective analysis of the morphologies of metaphase II (MII) human oocytes, which had developed into 590 biopsied blastocysts derived from 109 patients that received preimplantation genetic testing for aneuploidies (PGT-A) cycles between March 2013 and December 2017. The euploid rate of blastocysts that originated from morphologically abnormal or normal oocytes were analyzed. The chromosome status of the blastocysts was determined and analyzed by array comparative genomic hybridization (aCGH) or next generation sequencing (NGS) following trophectoderm biopsy.
RESULTS
According to the odds ratios obtained for each oocyte morphotype, no statistically significant relationship was found between oocyte dysmorphisms and euploid rate. Specifically, although SERc-positive oocytes had a higher rate of arrest at two pronuclei, or 2 PN (26.7% vs. 19.4%, p > 0.05), the blastocyst formation rate was not affected as compared with SERc-negative oocytes (40.0% vs. 38.6%, p > 0.05). Among nine euploid embryos derived from oocytes with SERc, three single euploid embryo transfers were performed, of which one resulted in blighted ovum, and two resulted in the births of two healthy, singleton term babies.
CONCLUSION
The results presented here suggest that oocyte dysmorphisms do not affect the euploidy rate of the blastocyst. The occurrence of SERc in the oocyte does not seem to impair the developing blastocyst nor does it interfere with good embryo formation rate and euploid rate. Thus, the embryos derived from SERc-positive oocytes could still be considered for embryo transfer if there are no other embryos available.
Topics: Betahistine; Blastocyst; Comparative Genomic Hybridization; Endoplasmic Reticulum, Smooth; Female; Humans; Metaphase; Oocytes; Pregnancy; Retrospective Studies
PubMed: 35779904
DOI: 10.1016/j.tjog.2021.03.044 -
Genes Nov 2019R.H. Chang & C.S. Ding is a good horticultural tree because of its beautiful yellow flowers and evergreen leaves. In this study, fluorescence in situ hybridization...
R.H. Chang & C.S. Ding is a good horticultural tree because of its beautiful yellow flowers and evergreen leaves. In this study, fluorescence in situ hybridization (FISH) was used to analyse mitotic metaphase chromosomes of with 5S rDNA and (AGT) oligonucleotides. Twenty-two small chromosomes were observed. Weak 5S rDNA signals were observed only in proximal regions of two chromosomes, which were adjacent to the (AGT) proximal signals. Weak (AGT) signals were observed on both chromosome ends, which enabled accurate chromosome counts. A pair of satellite bodies was observed. (AGT) signals displayed quite high diversity, changing in intensity from weak to very strong as follows: far away from the chromosome ends (satellites), ends, subtelomeric regions, and proximal regions. Ten high-quality spreads revealed metaphase dynamics from the beginning to the end and the transition to anaphase. Chromosomes gradually grew larger and thicker into linked chromatids, which grew more significantly in width than in length. Based on the combination of 5S rDNA and (AGT) signal patterns, ten chromosomes were exclusively distinguished, and the remaining twelve chromosomes were divided into two distinct groups. Our physical map, which can reproduce dynamic metaphase progression and distinguish chromosomes, will powerfully guide cytogenetic research on and other trees.
Topics: Chromosomes, Plant; In Situ Hybridization, Fluorescence; Laurales; Metaphase; Physical Chromosome Mapping; RNA, Ribosomal, 5S
PubMed: 31703401
DOI: 10.3390/genes10110904 -
Journal of Assisted Reproduction and... Oct 2012
Topics: Female; Humans; Membrane Potential, Mitochondrial; Metaphase; Oocytes; Vitrification
PubMed: 23080275
DOI: 10.1007/s10815-012-9879-7 -
Scientific Data Feb 2023Chromosomes are a principal target of clinical cytogenetic studies. While chromosomal analysis is an integral part of prenatal care, the conventional manual...
Chromosomes are a principal target of clinical cytogenetic studies. While chromosomal analysis is an integral part of prenatal care, the conventional manual identification of chromosomes in images is time-consuming and costly. This study developed a chromosome detector that uses deep learning and that achieved an accuracy of 98.88% in chromosomal identification. Specifically, we compiled and made available a large and publicly accessible database containing chromosome images and annotations for training chromosome detectors. The database contains five thousand 24 chromosome class annotations and 2,000 single chromosome annotations. This database also contains examples of chromosome variations. Our database provides a reference for researchers in this field and may help expedite the development of clinical applications.
Topics: Female; Humans; Pregnancy; Chromosomes; Metaphase
PubMed: 36823215
DOI: 10.1038/s41597-023-02003-7 -
Molecules and Cells Dec 2000Flow cytometric analysis was systematically performed to optimize the concentration and duration of hydroxyurea (DNA synthesis inhibitor) and trifluralin (metaphase...
Flow cytometric analysis was systematically performed to optimize the concentration and duration of hydroxyurea (DNA synthesis inhibitor) and trifluralin (metaphase blocking reagent) treatments for synchronizing the cell cycle and accumulating metaphase chromosomes in barley root tips. A high metaphase index (76.5% in the root tip meristematic area) was routinely achieved. Seedlings of about 1.0-cm length were treated with 1.25 mM hydroxyurea for 14 h to synchronize the root tip meristem cells at the S/G2 phase. After rinsing with hydroxyurea, the seedlings were incubated in a hydroxyurea-free solution for 2 h and were treated with 1 microM trifluralin for 4 h to accumulate mitotic cells in the metaphase. The consistent high metaphase index depended on the uniform germination of seeds prior to treatment. High-quality and high-quantity isolated metaphase chromosomes were suitable for flow cytometric analysis and sorting. Flow karyotypes of barley chromosomes were established via univariate and bivariate analysis. A variation of flow karyotypes was detected among barley lines. Two single chromosome types were identified and sorted. Bivariate analysis showed no variation among barley individual chromosomes in AT and GC content.
Topics: Analysis of Variance; Cell Cycle; Cell Separation; Chromosomes; DNA, Plant; Flow Cytometry; Hordeum; Hydroxyurea; Karyotyping; Metaphase; Plant Roots; Trifluralin
PubMed: 11211865
DOI: 10.1007/s10059-000-0619-y -
Scientific Reports Oct 2018Ovarian follicular development and ovulation are complex and tightly regulated processes that involve regulation by microRNAs (miRNAs). We previously identified...
Ovarian follicular development and ovulation are complex and tightly regulated processes that involve regulation by microRNAs (miRNAs). We previously identified differentially expressed mRNAs between human cumulus granulosa cells (CGCs) from immature early antral follicles (germinal vesicle - GV) and mature preovulatory follicles (metaphase II - M2). In this study, we performed an integrated analysis of the transcriptome and miRNome in CGCs obtained from the GV cumulus-oocyte complex (COC) obtained from IVM and M2 COC obtained from IVF. A total of 43 differentially expressed miRNAs were identified. Using Ingenuity IPA analysis, we identified 7288 potential miRNA-regulated target genes. Two hundred thirty-four of these target genes were also found in our previously generated ovulatory gene library while exhibiting anti-correlated expression to the identified miRNAs. IPA pathway analysis suggested that miR-21 and FOXM1 cooperatively inhibit CDC25A, TOP2A and PRC1. We identified a mechanism for the temporary inhibition of VEGF during ovulation by TGFB1, miR-16-5p and miR-34a-5p. The linkage bioinformatics analysis between the libraries of the coding genes from our preliminary study with the newly generated library of regulatory miRNAs provides us a comprehensive, integrated overview of the miRNA-mRNA co-regulatory networks that may play a key role in controlling post-transcriptomic regulation of the ovulatory process.
Topics: Adult; Cumulus Cells; Female; Forkhead Box Protein M1; Genes, cdc; Humans; Metaphase; MicroRNAs; Ovarian Follicle; Ovulation; RNA, Messenger; Transcriptome
PubMed: 30353018
DOI: 10.1038/s41598-018-33807-y