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BioRxiv : the Preprint Server For... May 2024Advanced maternal age is associated with a decline in oocyte quality, which often leads to reproductive failure in humans. However, the mechanisms behind this...
Advanced maternal age is associated with a decline in oocyte quality, which often leads to reproductive failure in humans. However, the mechanisms behind this age-related decline remain unclear. To gain insights into this phenomenon, we applied plexDIA, a multiplexed, single-cell mass spectrometry method, to analyze the proteome of oocytes from both young women and women of advanced maternal age. Our findings primarily revealed distinct proteomic profiles between immature fully grown germinal vesicle and mature metaphase II oocytes. Importantly, we further show that a woman's age is associated with changes in her oocyte proteome. Specifically, when compared to oocytes obtained from young women, advanced maternal age oocytes exhibited lower levels of the proteasome and TRiC complex, as well as other key regulators of proteostasis and meiosis. This suggests that aging adversely affects the proteostasis and meiosis networks in human oocytes. The proteins identified in this study hold potential as targets for improving oocyte quality and may guide future studies into the molecular processes underlying oocyte aging.
PubMed: 38903107
DOI: 10.1101/2024.05.23.595547 -
Communications Biology Jun 2024AMPK is a well-known energy sensor regulating cellular metabolism. Metabolic disorders such as obesity and diabetes are considered detrimental factors that reduce...
AMPK is a well-known energy sensor regulating cellular metabolism. Metabolic disorders such as obesity and diabetes are considered detrimental factors that reduce fecundity. Here, we show that pharmacologically induced in vitro activation (by metformin) or inhibition (by dorsomorphin) of the AMPK pathway inhibits or promotes activation of ovarian primordial follicles in cultured murine ovaries and human ovarian cortical chips. In mice, activation of primordial follicles in dorsomorphin in vitro-treated ovaries reduces AMPK activation and upregulates Wnt and FOXO genes, which, interestingly, is associated with decreased phosphorylation of β-catenin. The dorsomorphin-treated ovaries remain of high quality, with no detectable difference in reactive oxygen species production, apoptosis or mitochondrial cytochrome c oxidase activity, suggesting safe activation. Subsequent maturation of in vitro-treated follicles, using a 3D alginate cell culture system, results in mature metaphase eggs with protruding polar bodies. These findings demonstrate that the AMPK pathway can safely regulate primordial follicles by modulating Wnt and FOXO genes, and reduce β-catenin phosphorylation.
Topics: Animals; Female; Mice; Ovarian Follicle; AMP-Activated Protein Kinases; Pyrimidines; Pyrazoles; Humans; Up-Regulation; Forkhead Transcription Factors; Wnt Proteins; beta Catenin; Phosphorylation; Mice, Inbred C57BL; Metformin; Wnt Signaling Pathway
PubMed: 38902324
DOI: 10.1038/s42003-024-06418-9 -
Frontiers in Endocrinology 2024Anti-Müllerian hormone (AMH) is a key paracrine/autocrine factor regulating folliculogenesis in the postnatal ovary. As antral follicles mature to the preovulatory...
Anti-Müllerian hormone (AMH) is a key paracrine/autocrine factor regulating folliculogenesis in the postnatal ovary. As antral follicles mature to the preovulatory stage, AMH production tends to be limited to cumulus cells. Therefore, the present study investigated the role of cumulus cell-derived AMH in supporting maturation and competence of the enclosed oocyte. Cumulus-oocyte complexes (COCs) were isolated from antral follicles of rhesus macaque ovaries for maturation with or without AMH depletion. Oocyte meiotic status and embryo cleavage after fertilization were assessed. maturation with AMH depletion was also performed using COCs from antral follicles of human ovarian tissue. Oocyte maturation and morphology were evaluated. The direct AMH action on mural granulosa cells of the preovulatory follicle was further assessed using human granulosa cells cultured with or without AMH supplementation. More macaque COCs produced metaphase II oocytes with AMH depletion than those of the control culture. However, preimplantation embryonic development after fertilization was comparable between oocytes derived from COCs cultured with AMH depletion and controls. Oocytes resumed meiosis in human COCs cultured with AMH depletion and exhibited a typical spindle structure. The confluency and cell number decreased in granulosa cells cultured with AMH supplementation relative to the control culture. AMH treatment did not induce cell death in cultured human granulosa cells. Data suggest that reduced AMH action in COCs could be beneficial for oocyte maturation. Cumulus cell-derived AMH is not essential for supporting oocyte competence or mural granulosa cell viability.
Topics: Anti-Mullerian Hormone; Oocytes; Female; Cumulus Cells; Animals; Humans; In Vitro Oocyte Maturation Techniques; Macaca mulatta; Oogenesis; Cells, Cultured; Fertilization in Vitro; Meiosis; Granulosa Cells; Ovarian Follicle; Embryonic Development
PubMed: 38887270
DOI: 10.3389/fendo.2024.1365260 -
Reproductive Toxicology (Elmsford, N.Y.) Jun 2024Ketoconazole (KTZ) is widely used as a fungicide, but it is also known to target steroid hormone formation which may affect female reproductive health. Our study aims to...
Ketoconazole (KTZ) is widely used as a fungicide, but it is also known to target steroid hormone formation which may affect female reproductive health. Our study aims to investigate the effects of KTZ on in vitro matured bovine cumulus-oocyte complexes (COCs), as a model for female reproductive toxicity. Cumulus cells of in vitro maturing COCs produce progesterone and pregnenolone, but exposure to 10 M KTZ effectively blocked the synthesis of these hormones. Exposure to lower concentrations of KTZ (i.e. 10 M and 10 M) had no such effect on steroidogenesis compared to the 0.1 % v/v DMSO vehicle control. Classical parameters of in vitro COC maturation, such as oocyte nuclear maturation to the metaphase II stage and expansion of the cumulus investment, were not affected by any KTZ concentration tested. Apoptosis and necrosis levels were also not altered in cumulus cells or oocytes exposed to KTZ. Moreover, oocytes exposed to KTZ during maturation showed normal cleavage and early embryo development up to day 8 post fertilization; albeit a statistically significant decrease was observed in day 8 blastocysts produced from oocytes exposed to the lowest concentration of 10 M KTZ. When unexposed mature oocytes were fertilized, followed by embryo culture for 8 days under KTZ exposure, no adverse effects in embryo cleavage and blastocyst formation were observed. In conclusion, KTZ has no major impact on in vitro bovine oocyte maturation and blastocyst formation in our study, even at concentrations blocking steroidogenesis.
PubMed: 38876429
DOI: 10.1016/j.reprotox.2024.108637 -
Ecotoxicology and Environmental Safety Jul 20242-Ethylhexyl diphenyl phosphate (EHDPP) is a representative organophosphorus flame retardant (OPFR) that has garnered attention due to its widespread use and potential...
2-Ethylhexyl diphenyl phosphate (EHDPP) is a representative organophosphorus flame retardant (OPFR) that has garnered attention due to its widespread use and potential adverse effects. EHDPP exhibits cytotoxicity, genotoxicity, developmental toxicity, and endocrine disruption. However, the toxicity of EHDPP in mammalian oocytes and the underlying mechanisms remain poorly understood. Melatonin is a natural free radical scavenger that has demonstrated cytoprotective properties. In this study, we investigated the effect of EHDPP on mouse oocytes in vitro culture system and evaluated the rescue effect of melatonin on oocytes exposed to EHDPP. Our results indicated that EHDPP disrupted oocyte maturation, resulting in the majority of oocytes arrested at the metaphase I (MI) stage, accompanied by cytoskeletal damage and elevated levels of reactive oxygen species (ROS). Nevertheless, melatonin supplementation partially rescued EHDPP-induced mouse oocyte maturation impairment. Results of single-cell RNA sequencing (scRNA-seq) analysis elucidated potential mechanisms underlying these protective effects. According to the results of scRNA-seq, we conducted further tests and found that EHDPP primarily disrupts mitochondrial distribution and function, kinetochore-microtubule (K-MT) attachment, DNA damage, apoptosis, and histone modification, which were rescued upon the supplementation of melatonin. This study reveals the mechanisms of EHDPP on female reproduction and indicates the efficacy of melatonin as a therapeutic intervention for EHDPP-induced defects in mouse oocytes.
Topics: Animals; Melatonin; Mice; Oocytes; Mitochondria; Female; Flame Retardants; Reactive Oxygen Species; Organophosphates; DNA Damage; Apoptosis; Organophosphorus Compounds
PubMed: 38865937
DOI: 10.1016/j.ecoenv.2024.116559 -
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 -
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 -
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