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Cell Death Discovery Jun 2024Despite the advances in the understanding of reproductive physiology, the mechanisms underlying ovarian aging are still not deciphered. Recent research found an...
Despite the advances in the understanding of reproductive physiology, the mechanisms underlying ovarian aging are still not deciphered. Recent research found an association between impaired ATM-mediated DNA double-strand break (DSB) repair mechanisms and oocyte aging. However, direct evidence connecting ATM-mediated pathway function decline and impaired oocyte quality is lacking. The objective of this study was to determine the role of ATM-mediated DNA DSB repair in the maintenance of oocyte quality in a mouse oocyte knockdown model. Gene interference, in vitro culture, parthenogenesis coupled with genotoxicity assay approaches, as well as molecular cytogenetic analyses based upon next-generation sequencing, were used to test the hypothesis that intact ATM function is critical in the maintenance of oocyte quality. We found that ATM knockdown impaired oocyte quality, resulting in poor embryo development. ATM knockdown significantly lowered or blocked the progression of meiosis in vitro, as well as retarding and reducing embryo cleavage after parthenogenesis. After ATM knockdown, all embryos were of poor quality, and none reached the blastocyst stage. ATM knockdown was also associated with an increased aneuploidy rate compared to controls. Finally, ATM knockdown increased the sensitivity of the oocytes to a genotoxic active metabolite of cyclophosphamide, with increased formation of DNA DSBs, reduced survival, and earlier apoptotic death compared to controls. These findings suggest a key role for ATM in maintaining oocyte quality and resistance to genotoxic stress, and that the previously observed age-induced decline in oocyte ATM function may be a prime factor contributing to oocyte aging.
PubMed: 38914566
DOI: 10.1038/s41420-024-02041-z -
PLoS Genetics Jun 2024Precise regulation of chromosome dynamics in the germline is essential for reproductive success across species. Yet, the mechanisms underlying meiotic chromosomal events...
Precise regulation of chromosome dynamics in the germline is essential for reproductive success across species. Yet, the mechanisms underlying meiotic chromosomal events such as homolog pairing and chromosome segregation are not fully understood in many species. Here, we employ Oligopaint DNA FISH to investigate mechanisms of meiotic homolog pairing and chromosome segregation in the holocentric pantry moth, Plodia interpunctella, and compare our findings to new and previous studies in the silkworm moth, Bombyx mori, which diverged from P. interpunctella over 100 million years ago. We find that pairing in both Bombyx and Plodia spermatogenesis is initiated at gene-rich chromosome ends. Additionally, both species form rod shaped cruciform-like bivalents at metaphase I. However, unlike the telomere-oriented chromosome segregation mechanism observed in Bombyx, Plodia can orient bivalents in multiple different ways at metaphase I. Surprisingly, in both species we find that kinetochores consistently assemble at non-telomeric loci toward the centers of chromosomes regardless of where chromosome centers are located in the bivalent. Additionally, sister kinetochores do not seem to be paired in these species. Instead, four distinct kinetochores are easily observed at metaphase I. Despite this, we find clear end-on microtubule attachments and not lateral microtubule attachments connecting these separated kinetochores to the meiotic spindle. These findings challenge the classical view of segregation where paired, poleward-facing kinetochores are required for accurate homolog separation in meiosis I. Our studies here highlight the importance of exploring fundamental processes in non-model systems, as employing novel organisms can lead to the discovery of novel biology.
PubMed: 38913752
DOI: 10.1371/journal.pgen.1011329 -
Reproductive Toxicology (Elmsford, N.Y.) Jun 2024Infertility affects ~12% of couples, with environmental chemical exposure as a potential contributor. Of the chemicals that are actively manufactured, very few are...
Infertility affects ~12% of couples, with environmental chemical exposure as a potential contributor. Of the chemicals that are actively manufactured, very few are assessed for reproductive health effects. Rodents are commonly used to evaluate reproductive effects, which is both costly and time consuming. Thus, there is a pressing need for rapid methods to test a broader range of chemicals. Here, we developed a strategy to evaluate large numbers of chemicals for reproductive toxicity via a yeast, S. cerevisiae high-throughput assay to assess gametogenesis as a potential new approach method (NAM). By simultaneously assessing chemicals for growth effects, we can distinguish if a chemical affects gametogenesis only, proliferative growth only or both. We identified a well-known mammalian reproductive toxicant, bisphenol A (BPA) and ranked 19 BPA analogs for reproductive harm. By testing mixtures of BPA and its analogs, we found that BPE and 17 β-estradiol each together with BPA showed synergistic effects that worsened reproductive outcome. We examined an additional 179 environmental chemicals including phthalates, pesticides, quaternary ammonium compounds and per- and polyfluoroalkyl substances and found 57 with reproductive effects. Many of the chemicals were found to be strong reproductive toxicants that have yet to be tested in mammals. Chemicals having affect before meiosis I division vs. meiosis II division were identified for 16 gametogenesis-specific chemicals. Finally, we demonstrate that in general yeast reproductive toxicity correlates well with published reproductive toxicity in mammals illustrating the promise of this NAM to quickly assess chemicals to prioritize the evaluation for human reproductive harm.
PubMed: 38906490
DOI: 10.1016/j.reprotox.2024.108630 -
Developmental Cell Jun 2024Sexually reproducing eukaryotes employ a developmentally regulated cell division program-meiosis-to generate haploid gametes from diploid germ cells. To understand how...
Sexually reproducing eukaryotes employ a developmentally regulated cell division program-meiosis-to generate haploid gametes from diploid germ cells. To understand how gametes arise, we generated a proteomic census encompassing the entire meiotic program of budding yeast. We found that concerted waves of protein expression and phosphorylation modify nearly all cellular pathways to support meiotic entry, meiotic progression, and gamete morphogenesis. Leveraging this comprehensive resource, we pinpointed dynamic changes in mitochondrial components and showed that phosphorylation of the FF-ATP synthase complex is required for efficient gametogenesis. Furthermore, using cryoET as an orthogonal approach to visualize mitochondria, we uncovered highly ordered filament arrays of Ald4, a conserved aldehyde dehydrogenase that is highly expressed and phosphorylated during meiosis. Notably, phosphorylation-resistant mutants failed to accumulate filaments, suggesting that phosphorylation regulates context-specific Ald4 polymerization. Overall, this proteomic census constitutes a broad resource to guide the exploration of the unique sequence of events underpinning gametogenesis.
PubMed: 38906138
DOI: 10.1016/j.devcel.2024.05.025 -
Cell Jun 2024Gamete formation and subsequent offspring development often involve extended phases of suspended cellular development or even dormancy. How cells adapt to recover and...
Gamete formation and subsequent offspring development often involve extended phases of suspended cellular development or even dormancy. How cells adapt to recover and resume growth remains poorly understood. Here, we visualized budding yeast cells undergoing meiosis by cryo-electron tomography (cryoET) and discovered elaborate filamentous assemblies decorating the nucleus, cytoplasm, and mitochondria. To determine filament composition, we developed a "filament identification" (FilamentID) workflow that combines multiscale cryoET/cryo-electron microscopy (cryoEM) analyses of partially lysed cells or organelles. FilamentID identified the mitochondrial filaments as being composed of the conserved aldehyde dehydrogenase Ald4 and the nucleoplasmic/cytoplasmic filaments as consisting of acetyl-coenzyme A (CoA) synthetase Acs1. Structural characterization further revealed the mechanism underlying polymerization and enabled us to genetically perturb filament formation. Acs1 polymerization facilitates the recovery of chronologically aged spores and, more generally, the cell cycle re-entry of starved cells. FilamentID is broadly applicable to characterize filaments of unknown identity in diverse cellular contexts.
Topics: Saccharomyces cerevisiae; Saccharomyces cerevisiae Proteins; Cryoelectron Microscopy; Mitochondria; Gametogenesis; Meiosis; Aldehyde Dehydrogenase; Electron Microscope Tomography; Coenzyme A Ligases; Spores, Fungal; Cytoplasm; Cell Nucleus
PubMed: 38906101
DOI: 10.1016/j.cell.2024.04.026 -
BioRxiv : the Preprint Server For... Feb 2024During meiosis, homologous chromosomes segregate so that alleles are transmitted equally to haploid gametes, following Mendel's Law of Segregation. However, some selfish...
During meiosis, homologous chromosomes segregate so that alleles are transmitted equally to haploid gametes, following Mendel's Law of Segregation. However, some selfish genetic elements drive in meiosis to distort the transmission ratio and increase their representation in gametes. The established paradigms for drive are fundamentally different for female vs male meiosis. In male meiosis, selfish elements typically kill gametes that do not contain them. In female meiosis, killing is predetermined, and selfish elements bias their segregation to the single surviving gamete (i.e., the egg in animal meiosis). Here we show that a selfish element on mouse chromosome 2, , drives using a hybrid mechanism in female meiosis, incorporating elements of both male and female drivers. If is destined for the polar body, it manipulates segregation to sabotage the egg by causing aneuploidy that is subsequently lethal in the embryo, so that surviving progeny preferentially contain . In heterozygous females, orients randomly on the metaphase spindle but lags during anaphase and preferentially remains in the egg, regardless of its initial orientation. Thus, the egg genotype is either euploid with or aneuploid with both homologs of chromosome 2, with only the former generating viable embryos. Consistent with this model, heterozygous females produce eggs with increased aneuploidy for chromosome 2, increased embryonic lethality, and increased transmission of . In contrast to a male meiotic driver, which kills its sister gametes produced as daughter cells in the same meiosis, eliminates "cousins" produced from meioses in which it should have been excluded from the egg.
PubMed: 38903120
DOI: 10.1101/2024.02.22.581453 -
BioRxiv : the Preprint Server For... Mar 2024When germ cells transition from the mitotic cycle into meiotic prophase I (MPI), chromosomes condense into an array of chromatin loops that are required to promote...
When germ cells transition from the mitotic cycle into meiotic prophase I (MPI), chromosomes condense into an array of chromatin loops that are required to promote homolog pairing and genetic recombination. To identify the changes in chromosomal conformation, we isolated nuclei on a trajectory from spermatogonia to the end of MPI. At each stage along this trajectory, we built genomic interaction maps with the highest temporal and spatial resolution to date. The changes in chromatin folding coincided with a concurrent decline in mitotic cohesion and a rise in meiotic cohesin complexes. We found that the stereotypical large-scale A and B compartmentalization was lost during meiotic prophase I alongside the loss of topological associating domains (TADs). Still, local subcompartments were detected and maintained throughout meiosis. The enhanced Micro-C resolution revealed that, despite the loss of TADs, higher frequency contact sites between two loci were detectable during meiotic prophase I coinciding with CTCF bound sites. The pattern of interactions around these CTCF sites with their neighboring loci showed that CTCF sites were often anchoring the meiotic loops. Additionally, the localization of CTCF to the meiotic axes indicated that these anchors were at the base of loops. Strikingly, even in the face of the dramatic reconfiguration of interphase chromatin into a condensed loop-array, the interactions between regulatory elements remained well preserved. This establishes a potential mechanism for how the meiotic chromatin maintains active transcription within a highly structured genome. In summary, the high temporal and spatial resolution of these data revealed previously unappreciated aspects of mammalian meiotic chromatin organization.
PubMed: 38903112
DOI: 10.1101/2024.03.25.586627 -
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 -
BioRxiv : the Preprint Server For... Jun 2024The fission yeast is a single-celled eukaryote that can be cultured as a haploid or as a diploid. Scientists employ mating, meiosis, and the plating of ascospores and...
The fission yeast is a single-celled eukaryote that can be cultured as a haploid or as a diploid. Scientists employ mating, meiosis, and the plating of ascospores and cells to generate strains with novel genotypes and to discover biological processes. Our two laboratories encountered independently sudden-onset, major impediments to such research. Spore suspensions and vegetative cells no longer plated effectively on minimal media. By systematically analyzing multiple different media components from multiple different suppliers, we identified the source of the problem. Specific lots of agar, from different suppliers, were toxic. Interestingly, the inhibitory effect was attenuated on rich media. Consequently, quality control checks that use only rich media can provide false assurances on the quality of the agar. Lastly, we describe likely sources of the toxicity and we provide specific guidance for quality control measures that should be applied by all vendors as preconditions for their sale of agar.
PubMed: 38895319
DOI: 10.1101/2024.06.06.597796 -
MicroPublication Biology 2024Several strains of with mutations in or are readily available to aid in elucidating the functions of these two genes in DNA damage repair, meiosis, and gene...
Several strains of with mutations in or are readily available to aid in elucidating the functions of these two genes in DNA damage repair, meiosis, and gene repression. DW102 is the only strain to our knowledge with mutations in both and . However, several groups have reported the DW102 strain is indistinguishable from wild-type when observing levels of embryonic lethality, sensitivity to radiation, and rates of male progeny, while strains with mutations in either or display increased occurrence of these phenotypes. Here, RT-qPCR analysis of the gene family, reveals distinctive and aberrant expression patterns in DW102 compared to other or mutant strains underscoring the need for caution in choosing this strain to draw conclusions about and functions.
PubMed: 38894809
DOI: 10.17912/micropub.biology.001152