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Journal of Animal Science and... Jun 2024Previous studies have shown that the vitrification of metaphase II (MII) oocytes significantly represses their developmental potential. Abnormally increased oxidative...
BACKGROUND
Previous studies have shown that the vitrification of metaphase II (MII) oocytes significantly represses their developmental potential. Abnormally increased oxidative stress is the probable factor; however, the underlying mechanism remains unclear. The walnut-derived peptide TW-7 was initially isolated and purified from walnut protein hydrolysate. Accumulating evidences implied that TW-7 was a powerful antioxidant, while its prospective application in oocyte cryopreservation has not been reported.
RESULT
Here, we found that parthenogenetic activation (PA) zygotes derived from vitrified MII oocytes showed elevated ROS level and delayed progression of pronucleus formation. Addition of 25 μmol/L TW-7 in warming, recovery, PA, and embryo culture medium could alleviate oxidative stress in PA zygotes from vitrified mouse MII oocytes, furtherly increase proteins related to histone lactylation such as LDHA, LDHB, and EP300 and finally improve histone lactylation in PA zygotes. The elevated histone lactylation facilitated the expression of minor zygotic genome activation (ZGA) genes and preimplantation embryo development.
CONCLUSIONS
Our findings revealed the mechanism of oxidative stress inducing repressed development of PA embryos from vitrified mouse MII oocytes and found a potent and easy-obtained short peptide that could significantly rescue the decreased developmental potential of vitrified oocytes, which would potentially contribute to reproductive medicine, animal protection, and breeding.
PubMed: 38858724
DOI: 10.1186/s40104-024-01045-0 -
The Journal of Comparative Neurology Jun 2024Mitochondria play critical roles in neural stem/progenitor cell proliferation and fate decisions. The subcellular localization of mitochondria in neural stem/progenitor...
Mitochondria play critical roles in neural stem/progenitor cell proliferation and fate decisions. The subcellular localization of mitochondria in neural stem/progenitor cells during mitosis potentially influences the distribution of mitochondria to the daughter cells and thus their fates. Therefore, understanding the spatial dynamics of mitochondria provides important knowledge about brain development. In this study, we analyzed the subcellular localization of mitochondria in the fetal human neocortex with a particular focus on the basal radial glial cells (bRGCs), a neural stem/progenitor cell subtype attributed to the evolutionary expansion of the human neocortex. During interphase, bRGCs exhibit a polarized localization of mitochondria that is localized at the base of the process or the proximal part of the process. Thereafter, mitochondria in bRGCs at metaphase show unpolarized distribution in which the mitochondria are randomly localized in the cytoplasm. During anaphase and telophase, mitochondria are still localized evenly, but mainly in the periphery of the cytoplasm. Mitochondria start to accumulate at the cleavage furrow during cytokinesis. These results suggest that the mitochondrial localization in bRGCs is tightly regulated during the cell cycle, which may ensure the proper distribution of mitochondria to the daughter cells and, thus in turn, influence their fates.
Topics: Humans; Neocortex; Mitochondria; Cell Cycle; Ependymoglial Cells; Neural Stem Cells
PubMed: 38852043
DOI: 10.1002/cne.25630 -
Fertility and Sterility Jun 2024To find a useful tool for estimating the minimum number of metaphase II (MII) oocytes needed to obtain at least one euploid blastocyst according to female age.
OBJECTIVE
To find a useful tool for estimating the minimum number of metaphase II (MII) oocytes needed to obtain at least one euploid blastocyst according to female age.
DESIGN
Retrospective analysis of in vitro fertilization (IVF) treatment cycles with preimplantational genetic testing for aneuploidies (PGT-A) performed over 5 years in IVIRMA Valencia (Spain), January 2017-March 2022. Approval from the Institutional Review Board of IVI Valencia (2204-VLC-040-CR).
SETTING
Private infertility clinic in Spain.
PATIENTS
Eligible patients were undergoing their first IVF-PGT-A treatment cycle, in which at least one MII oocyte was obtained, regardless of oocyte and semen origin. Oocyte donation cycles were included in the donor group (≤34 years old). Treatment cycles from women with their own oocytes were selected only when the oocytes were aged ≥35 years (patient group). Only trophoectoderm biopsies performed on days 5 or 6 of development and analyzed using next-generation sequencing were included. Preimplantational genetic testing for aneuploidy cycles because of a known abnormal karyotype were excluded.
INTERVENTION
Not applicable.
MAIN OUTCOME MEASURES
Number of MII oocytes needed to obtain one euploid blastocyst according to female age.
RESULTS
A total of 2,660 IVF-PGT-A treatment cycles were performed in the study period in the eligible population (patients group = 2,462; donors group =198). The mean number of MII oocytes needed to obtain one euploid blastocyst increased with age, as did the number of treatment cycles that did not get at least one euploid blastocyst. An adjusted multivariate binary regression model was designed using 80% of the patient group sample (n = 2,462; training set). A calculator for the probability of obtaining at least one euploid blastocyst was created using this model. The validation of this model in the remaining 20% of the patient group sample (n = 493; validation set) showed that it could estimate the event of having at least one euploid blastocyst with an accuracy of 72.0%.
CONCLUSIONS
Our results show a preliminary model capable of predicting the number of MII oocytes needed to obtain at least one euploid blastocyst according to female age, calculated with the largest database of IVF-PGT-A treatment cycles ever used for this purpose, including only treatment cycles using next-generation sequencing on trophoectoderm biopsies. Once this model has been properly validated, it could help with decision-making for both clinicians and patients coming to an infertility clinic.
PubMed: 38848954
DOI: 10.1016/j.fertnstert.2024.06.002 -
Scientific Reports Jun 2024The challenge of in-situ handling and high-resolution low-dose imaging of intact, sensitive and wet samples in their native state at nanometer scale, including live...
The challenge of in-situ handling and high-resolution low-dose imaging of intact, sensitive and wet samples in their native state at nanometer scale, including live samples is met by Advanced Environmental Scanning Electron Microscopy (A-ESEM). This new generation of ESEM utilises machine learning-based optimization of thermodynamic conditions with respect to sample specifics to employ a low temperature method and an ionization secondary electron detector with an electrostatic separator. A modified electron microscope was used, equipped with temperature, humidity and gas pressure sensors for in-situ and real-time monitoring of the sample. A transparent ultra-thin film of ionic liquid is used to increase thermal and electrical conductivity of the samples and to minimize sample damage by free radicals. To validate the power of the new method, we analyze condensed mitotic metaphase chromosomes to reveal new structural features of their perichromosomal layer, and the organization of chromatin fibers, not observed before by any microscopic technique. The ability to resolve nano-structural details of chromosomes using A-ESEM is validated by measuring gold nanoparticles with achievable resolution in the lower nanometre units.
Topics: Microscopy, Electron, Scanning; Humans; Gold; Metal Nanoparticles; Mitosis; Chromosomes
PubMed: 38844535
DOI: 10.1038/s41598-024-63515-9 -
Archives of Pathology & Laboratory... Jun 2024The joint College of American Pathologists/American College of Medical Genetics and Genomics Cytogenetics Committee works to ensure competency and proficiency of...
Conventional Cytogenetic Analysis of Constitutional Abnormalities: A 20-Year Review of Proficiency Test Results From the College of American Pathologists/American College of Medical Genetics and Genomics Cytogenetics Committee.
CONTEXT.—
The joint College of American Pathologists/American College of Medical Genetics and Genomics Cytogenetics Committee works to ensure competency and proficiency of clinical cytogenetics testing laboratories through proficiency testing programs for various clinical tests offered by such laboratories, including the evaluation of constitutional abnormalities.
OBJECTIVE.—
To review and analyze 20 years of constitutional chromosome analysis proficiency testing results (2003-2022), primarily utilizing G-banded karyograms.
DESIGN.—
A retrospective review of results from 2003 through 2022 was performed, identifying challenges addressing constitutional disorders. The chromosomal abnormalities and overall performance were evaluated.
RESULTS.—
A total of 184 cases from 161 proficiency testing challenges were administered from 2003 through 2022. Challenges consisted of metaphase images and accompanying clinical history for evaluation of numerical and/or structural abnormalities. Of the 184 cases, only 2 (1%) failed to reach an 80% grading consensus for recognition of the abnormality. Both cases illustrated the limitations of correctly characterizing some chromosomal abnormalities, including recombinant chromosomal abnormalities and isochromosome identification. In addition, 2 cases failed to reach a consensus for nomenclature reporting: 1 with an isochromosome and another with a duplication.
CONCLUSIONS.—
This 20-year review illustrates the high rate of competency and proficiency of cytogenetic laboratories in the correct identification of constitutional chromosome abnormalities.
PubMed: 38838342
DOI: 10.5858/arpa.2024-0048-CP -
PLoS Genetics Jun 2024Cryptococcus neoformans is an opportunistic, human fungal pathogen which undergoes fascinating switches in cell cycle control and ploidy when it encounters stressful...
Cryptococcus neoformans is an opportunistic, human fungal pathogen which undergoes fascinating switches in cell cycle control and ploidy when it encounters stressful environments such as the human lung. Here we carry out a mechanistic analysis of the spindle checkpoint which regulates the metaphase to anaphase transition, focusing on Mps1 kinase and the downstream checkpoint components Mad1 and Mad2. We demonstrate that Cryptococcus mad1Δ or mad2Δ strains are unable to respond to microtubule perturbations, continuing to re-bud and divide, and die as a consequence. Fluorescent tagging of Chromosome 3, using a lacO array and mNeonGreen-lacI fusion protein, demonstrates that mad mutants are unable to maintain sister-chromatid cohesion in the absence of microtubule polymers. Thus, the classic checkpoint functions of the SAC are conserved in Cryptococcus. In interphase, GFP-Mad1 is enriched at the nuclear periphery, and it is recruited to unattached kinetochores in mitosis. Purification of GFP-Mad1 followed by mass spectrometric analysis of associated proteins show that it forms a complex with Mad2 and that it interacts with other checkpoint signalling components (Bub1) and effectors (Cdc20 and APC/C sub-units) in mitosis. We also demonstrate that overexpression of Mps1 kinase is sufficient to arrest Cryptococcus cells in mitosis, and show that this arrest is dependent on both Mad1 and Mad2. We find that a C-terminal fragment of Mad1 is an effective in vitro substrate for Mps1 kinase and map several Mad1 phosphorylation sites. Some sites are highly conserved within the C-terminal Mad1 structure and we demonstrate that mutation of threonine 667 (T667A) leads to loss of checkpoint signalling and abrogation of the GAL-MPS1 arrest. Thus Mps1-dependent phosphorylation of C-terminal Mad1 residues is a critical step in Cryptococcus spindle checkpoint signalling. We conclude that CnMps1 protein kinase, Mad1 and Mad2 proteins have all conserved their important, spindle checkpoint signalling roles helping ensure high fidelity chromosome segregation.
Topics: Cryptococcus neoformans; Cell Cycle Proteins; Mad2 Proteins; Spindle Apparatus; Signal Transduction; Fungal Proteins; Humans; Protein Serine-Threonine Kinases; M Phase Cell Cycle Checkpoints; Mitosis; Kinetochores; Chromosome Segregation; Microtubules; Nuclear Proteins
PubMed: 38829899
DOI: 10.1371/journal.pgen.1011302 -
Mutation Research. Genetic Toxicology... 2024Cytogenetic studies have shown that human chromosomes 1, 9, and 16, with a large heterochromatic region of highly methylated classical satellite DNA, are prone to...
Cytogenetic studies have shown that human chromosomes 1, 9, and 16, with a large heterochromatic region of highly methylated classical satellite DNA, are prone to induction of chromatid breaks and interchanges by mitomycin C (MMC). A couple of studies have indicated that material from chromosome 9, and possibly also from chromosomes 1 and 16, are preferentially micronucleated by MMC. Here, we further examined the chromosome-specific induction of micronuclei (MN; with and without cytochalasin B) and chromosomal aberrations (CAs) by MMC. Cultures of isolated human lymphocytes from two male donors were treated (at 48 h of culture, for 24 h) with MMC (500 ng/ml), and the induced MN were examined by a pancentromeric DNA probe and paint probe for chromosome 9, and by paint probes for chromosomes 1 and 16. MMC increased the total frequency of MN by 6-8-fold but the frequency of chromosome 9 -positive (9) MN by 29-30-fold and the frequency of chromosome 1 -positive (1) MN and chromosome 16 -positive (16) MN by 12-16-fold and 10-17-fold, respectively. After treatment with MMC, 34-47 % of all MN were 9, 17-20 % 1, and 3-4 % 16. The majority (94-96 %) of the 9 MN contained no centromere and thus harboured acentric fragments. When MMC-induced CAs aberrations were characterized by using the pancentromeric DNA probe and probes for the classical satellite region and long- and short- arm telomeres of chromosome 9, a high proportion of chromosomal breaks (31 %) and interchanges (41 %) concerned chromosome 9. In 83 % of cases, the breakpoint in chromosome 9 was just below the region (9cen-q12) labelled by the classical satellite probe. Our results indicate that MMC specifically induces MN harbouring fragments of chromosome 9, 1, and 16. CAs of chromosome 9 are highly overrepresented in metaphases of MMC-treated lymphocytes. The preferential breakpoint is below the region 9q12.
Topics: Humans; Mitomycin; Male; Chromosome Aberrations; Micronuclei, Chromosome-Defective; Chromosomes, Human, Pair 9; Chromosomes, Human, Pair 1; Chromosomes, Human, Pair 16; Lymphocytes; Adult; Micronucleus Tests; Cells, Cultured; Cytochalasin B; In Situ Hybridization, Fluorescence
PubMed: 38821666
DOI: 10.1016/j.mrgentox.2024.503753 -
Cureus Apr 2024This case report demonstrates the management of primary infertility in a couple: the male was 37 years old and the female was 32 years old. The female had a submucosal...
This case report demonstrates the management of primary infertility in a couple: the male was 37 years old and the female was 32 years old. The female had a submucosal uterine fibroid. Later, the female underwent a myomectomy to remove submucosal fibroids in the uterus after two failed intrauterine insemination (IUI) cycles. After six months of her recovery period, she underwent ovum pickup for an in vitro fertilization (IVF) cycle. During the process of ovum pickup (OPU), four oocytes were retrieved: three in the metaphase one (M1) stage and one in the metaphase two (M2) stage. Subsequently, the couple underwent in vitro maturation (IVM) of oocytes, where the M1 stage oocytes were cultured for six hours. The M1 stage oocytes progressed to the M2 stage. These oocytes were then injected with sperm, which resulted in the formation of two blastocysts. These blastocysts were then cryopreserved for three months, and after three months, these frozen embryos were then transferred, leading to the successful conception. The case study evaluates a couple who suffered from infertility. This study includes a treatment of myomectomy and in vitro maturation.
PubMed: 38813276
DOI: 10.7759/cureus.59257 -
Journal of Biomedical Optics Jun 2024Preparation of a recipient cytoplast by oocyte enucleation is an essential task for animal cloning and assisted reproductive technologies in humans. The femtosecond...
SIGNIFICANCE
Preparation of a recipient cytoplast by oocyte enucleation is an essential task for animal cloning and assisted reproductive technologies in humans. The femtosecond laser is a precise and low-invasive tool for oocyte enucleation, and it should be an appropriate alternative to traditional enucleation by a microneedle aspiration. However, until recently, the laser enucleation was performed only with applying a fluorescent dye.
AIM
This work is aimed to (1) achieve femtosecond laser oocyte enucleation without applying a fluorescent dye and (2) to study the effect of laser destruction of chromosomes on the structure and dynamics of the spindle.
APPROACH
We applied polarized light microscopy for spindle visualization and performed stain-free mouse and human oocyte enucleation with a 1033 nm femtosecond laser. Also, we studied transformation of a spindle after metaphase plate elimination by a confocal microscopy.
RESULTS
We demonstrated a fundamental possibility of inactivating the metaphase plate in mouse and human oocytes by 1033 nm femtosecond laser radiation without applying a fluorescent dye. Irradiation of the spindle area, visualized by polarized light microscopy, resulted in partly or complete metaphase plate destruction but avoided the microtubules impairment. After the metaphase plate elimination, the spindle reorganized, however, it was not a complete depolymerization.
CONCLUSIONS
This method of recipient cytoplast preparation is expected to be useful for animal cloning and assisted reproductive technologies.
Topics: Animals; Mice; Oocytes; Humans; Female; Lasers; Spindle Apparatus; Microscopy, Confocal; Metaphase; Microscopy, Polarization
PubMed: 38812963
DOI: 10.1117/1.JBO.29.6.065002 -
Cell Biochemistry and Function Jun 2024Cancer is the second leading cause of mortality worldwide. The development of anticancer therapy plays a crucial role in mitigating tumour progression and metastasis....
Cancer is the second leading cause of mortality worldwide. The development of anticancer therapy plays a crucial role in mitigating tumour progression and metastasis. Epithelioid hemangioendothelioma is a very rare cancer, however, with a high systemic involvement. Kynurenine metabolites which include l-kynurenine, 3-hydroxykynurenine, 3-hydroxyanthranilic acid and quinolinic acid have been shown to inhibit T-cell proliferation resulting in a decrease in cell growth of natural killer cells and T cells. Furthermore, metabolites such as l-kynurenine have been shown to inhibit proliferation of melanoma cells in vitro. Considering these metabolite properties, the present study aimed to explore the in vitro effects of l-kynurenine, quinolinic acid and kynurenic acid on endothelioma sEnd-2 cells and on endothelial (EA. hy926 cells) (control cell line). The in vitro effect at 24, 48, and 72 h exposure to a range of 1-4 mM of the respective kynurenine metabolites on the two cell lines in terms of cell morphology, cell cycle progression and induction of apoptosis was assessed. The half inhibitory concentration (IC), as determined using nonlinear regression, for l-kynurenine, quinolinic acid and kynurenic acid was 9.17, 15.56, and 535.40 mM, respectively. Optical transmitted light differential interference contrast and hematoxylin and eosin staining revealed cells blocked in metaphase, formation of apoptotic bodies and compromised cell density in l-kynurenine-treated cells. A statistically significant increase in the number of cells present in the sub-G phase was observed in l-kynurenine-treated sample. To our knowledge, this was the first in vitro study conducted to investigate the mechanism of action of kynurenine metabolites on endothelioma sEnd-2 cells. It can be concluded that l-kynurenine exerts an antiproliferative effect on the endothelioma sEnd-2 cell line by decreasing cell growth and proliferation as well as a metaphase block. These hallmarks suggest cell death via apoptosis. Further research will be conducted on l-kynurenine to assess the effect on cell adhesion in vitro and in vivo as cell-cell adhesion has been shown to increase metastasis to distant organs therefore, the inhibition of adhesion may lead to a decrease in metastasis.
Topics: Kynurenine; Humans; Apoptosis; Cell Proliferation; Quinolinic Acid; Kynurenic Acid; Cell Cycle; Cell Line, Tumor; Antineoplastic Agents; Endothelial Cells; Dose-Response Relationship, Drug
PubMed: 38807444
DOI: 10.1002/cbf.4065