Abnormal Chromosome Segregation Research Articles

Effects of rpl1001 Gene Deletion on Cell Division of Fission Yeast and Its Molecular Mechanism.

The rpl1001 gene encodes 60S ribosomal protein L10, which is involved in intracellular protein synthesis and cell growth. However, it is not yet known whether it is involved in the regulation of cell mitosis dynamics. This study focuses on the growth, spore production, cell morphology, the dynamics of microtubules, chromosomes, actin, myosin, and mitochondria of fission yeast (Schizosaccharomyces pombe) to investigate the impact of rpl1001 deletion on cell mitosis. RNA-Seq and bioinformatics analyses were also used to reveal key genes, such as hsp16, mfm1 and isp3, and proteasome pathways. The results showed that rpl1001 deletion resulted in slow cell growth, abnormal spore production, altered cell morphology, and abnormal microtubule number and length during interphase. The cell dynamics of the rpl1001Δ strain showed that the formation of a monopolar spindle leads to abnormal chromosome segregation with increased rate of spindle elongation in anaphase of mitosis, decreased total time of division, prolonged formation time of actin and myosin loops, and increased expression of mitochondrial proteins. Analysis of the RNA-Seq sequencing results showed that the proteasome pathway, up-regulation of isp3, and down-regulation of mfm1 and mfm2 in the rpl1001Δ strain were the main factors underpinning the increased number of spore production. Also, in the rpl1001Δ strain, down-regulation of dis1 caused the abnormal microtubule and chromosome dynamics, and down-regulation of hsp16 and pgk1 were the key genes affecting the delay of actin ring and myosin ring formation. This study reveals the effect and molecular mechanism of rpl1001 gene deletion on cell division, which provides the scientific basis for further clarifying the function of the Rpl1001 protein in cell division.

LSM14B coordinates protein component expression in the P-body and controls oocyte maturation

The generation of mature and healthy oocytes is the most critical event in the entire female reproductive process, and the mechanisms regulating this process remain to be studied. Here, we demonstrate that Smith-like (LSM) family member 14B (LSM14B) regulates oocyte maturation, and the loss of LSM14B in mouse ovaries leads to abnormal oocyte MII arrest and female infertility. Next, we find the aberrant transcriptional activation, indicated by abnormal non-surrounded nucleolus and surrounded nucleolus oocyte proportions, and abnormal chromosome assembly and segregation in Lsm14b-deficient mouse oocytes. The global transcriptome analysis suggests that many transcripts involved in cytoplasmic processing body (P-body) function are altered in Lsm14b-deficient mouse oocytes. Deletion of Lsm14b results in the expression and/or localization changes of P-body components (such as LSM14A, DCP1A, and 4E-T). Notably, DDX6, a key component of the P-body, is downregulated and accumulates in the nuclei in Lsm14b-deficient mouse oocytes. Taken together, our data suggest that LSM14B links mouse oocyte maturation to female fertility through the regulation of the P-body.

Abstract 1555: CDK2 inhibition & disruption of centrosome stoichiometry: a clinically tractable death program for aneuploid cancer cells

Abstract Genomic instability is a cancer hallmark. Aberrant centrosome numbers often occur in cancer and is associated with genomic instability. Mitosis with supernumerary centrosomes leads to abnormal chromosome segregation and aneuploidy in progeny cells. While many cells with abnormal chromosomes are eliminated through apoptosis, some gain a growth advantage and acquire resistance to antineoplastic agents. This aneuploidy is associated with aggressive tumor biology and unfavorable clinical outcomes. Cyclin-dependent kinase 2 (CDK2) plays a major role regulation of cell cycle progression, centrosome duplication and clustering. We previously reported that CDK2 inhibition prevented supernumerary centrosomes clustering, causing multipolar mitosis and anaphase catastrophe in lung and other aneuploid cancers. To elucidate engaged mechanisms focused ion beam scanning electron microscope (FIB-SEM) and immunofluorescent staining were each used to interrogate the ultrastructure of multipolar mitosis after CDK2 inhibition by CYC065-treatment (a CDK2/9 inhibitor). CYC065-treatment induced distinct ultrastructural changes in aneuploid lung cancer cells including formation of chromosome rings and multipolarity. As compared to vehicle controls, CYC065-treatment markedly extended the duration of chromosome rings in lung cancer cells and conferred aberrant mitosis in live cell imagining. Intriguingly, this treatment disrupted centrosome and centriole stoichiometry in cells with chromosome rings or multipolarity. These findings were confirmed using immunofluorescence assays of lung and other cancer cells having supernumerary centrosomes. To elucidate if CDK2 or CDK9 activity directly altered centriole stoichiometry, chromosome rings or multipolarity, immunofluorescent assays were performed in lung cancer cells that independently knocked down CDK2 or CDK9 using siRNAs relative to controls. Centriole stoichiometry abnormalities in cancer cells having chromosome rings or multipolarity were statistically significantly higher in CDK2 knock-down but not in CDK9 knock-down transfectants or controls. Engineered gain of CDK2 but not CDK9 expression rescued CYC065 mediated chromosome rings and multipolarity in aneuploid lung cancer cells. Taken together, this study uncovered that CDK2 inhibition disrupted centrosome stoichiometry, causing apoptotic death of aneuploid cancer cells. This is a clinically-tractable pharmacologic mechanism that conferring cancer cell death. Current work determines if similar mechanisms are engaged in vivo in syngeneic mouse lung cancer models undergoing CDK2 antagonism. Citation Format: Zibo Chen, Xi Liu, Masanori Kawakami, Xiuxia Liu, Allison Baker, Aayush Bhatawadekar, Liliya Tyutyunyk-Massey, Ethan Dmitrovsky, Kedar Narayan. CDK2 inhibition & disruption of centrosome stoichiometry: a clinically tractable death program for aneuploid cancer cells [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2023; Part 1 (Regular and Invited Abstracts); 2023 Apr 14-19; Orlando, FL. Philadelphia (PA): AACR; Cancer Res 2023;83(7_Suppl):Abstract nr 1555.

NuMA deficiency causes micronuclei via checkpoint-insensitive k-fiber minus-end detachment from mitotic spindle poles

Micronuclei resulting from improper chromosome segregation foster chromosome rearrangements.1,2 Toprevent micronuclei formation in mitosis, the dynamic plus ends of bundled kinetochore microtubules (k-fibers) must establish bipolar attachment with all sister kinetochores on chromosomes,3 whereas k-fiber minus ends must be clustered at the two opposing spindle poles, which are normally connected with centrosomes.4 The establishment of chromosome biorientation via k-fiber plus ends is carefully monitored by the spindle assembly checkpoint (SAC).5 However, how k-fiber minus-end clustering near centrosomes is maintained and monitored remains poorly understood. Here, we show that degradation of NuMA by auxin-inducible degron technologies results in micronuclei formation through k-fiber minus-end detachment from spindle poles during metaphase in HCT116 colon cancer cells. Importantly, k-fiber minus-end detachment from one pole creates misaligned chromosomes that maintain chromosome biorientation and satisfy the SAC, resulting in abnormal chromosome segregation. NuMA depletion also causes minus-end clustering defects in non-transformed Rpe1 cells, but it additionally induces centrosome detachment from partially focused poles, resulting in highly disorganized anaphase. Moreover, we find that NuMA depletion causes centrosome clustering defects in tetraploid-like cells, leading to an increased frequency of multipolar divisions. Together, our data indicate that NuMA is required for faithful chromosome segregation in human mitotic cells, generally by maintaining k-fiber minus-end clustering but also by promoting spindle pole-centrosome or centrosome-centrosome connection in specific cell types or contexts. Similar to erroneous merotelic kinetochore attachments,6 detachment of k-fiber minus ends from spindle poles evades spindle checkpoint surveillance and may therefore be a source of genomic instability in dividing cells.

Centromere defects, chromosome instability, and cGAS-STING activation in systemic sclerosis

Centromere defects in Systemic Sclerosis (SSc) have remained unexplored despite the fact that many centromere proteins were discovered in patients with SSc. Here we report that lesion skin fibroblasts from SSc patients show marked alterations in centromeric DNA. SSc fibroblasts also show DNA damage, abnormal chromosome segregation, aneuploidy (only in diffuse cutaneous (dcSSc)) and micronuclei (in all types of SSc), some of which lose centromere identity while retaining centromere DNA sequences. Strikingly, we find cytoplasmic “leaking” of centromere proteins in limited cutaneous SSc (lcSSc) fibroblasts. Cytoplasmic centromere proteins co-localize with antigen presenting MHC Class II molecules, which correlate precisely with the presence of anti-centromere antibodies. CENPA expression and micronuclei formation correlate highly with activation of the cGAS-STING/IFN-β pathway as well as markers of reactive oxygen species (ROS) and fibrosis, ultimately suggesting a link between centromere alterations, chromosome instability, SSc autoimmunity, and fibrosis.

Tumor Treating Fields (TTFields) Concomitant with Immune Checkpoint Inhibitors Are Therapeutically Effective in Non-Small Cell Lung Cancer (NSCLC) In Vivo Model.

Tumor Treating Fields (TTFields) are electric fields that exert physical forces to disrupt cellular processes critical for cancer cell viability and tumor progression. TTFields induce anti-mitotic effects through the disruption of the mitotic spindle and abnormal chromosome segregation, which trigger several forms of cell death, including immunogenic cell death (ICD). The efficacy of TTFields concomitant with anti-programmed death-1 (anti-PD-1) treatment was previously shown in vivo and is currently under clinical investigation. Here, the potential of TTFields concomitant with anti- PD-1/anti-cytotoxic T-lymphocyte-associated protein 4 (anti-CTLA-4) or anti-programmed death-ligand 1 (anti-PD-L1) immune checkpoint inhibitors (ICI) to improve therapeutic efficacy was examined in lung tumor-bearing mice. Increased circulating levels of high mobility group box 1 protein (HMGB1) and elevated intratumoral levels of phosphorylated eukaryotic translation initiation factor 2α (p-eIF2α) were found in the TTFields-treated mice, indicative of ICD induction. The concomitant application of TTFields and ICI led to a significant decrease in tumor volume as compared to all other groups. In addition, significant increases in the number of tumor-infiltrating immune cells, specifically cytotoxic T-cells, were observed in the TTFields plus anti-PD-1/anti-CTLA-4 or anti-PD-L1 groups. Correspondingly, cytotoxic T-cells isolated from these tumors showed higher levels of IFN-γ production. Collectively, these results suggest that TTFields have an immunoactivating role that may be leveraged for concomitant treatment with ICI to achieve better tumor control by enhancing antitumor immunity.

DNAR-12. COMBINING TUMOUR TREATING FIELDS WITH THERAPEUTIC DNA DAMAGE RESPONSE INHIBITORS TO INCREASE POTENCY IN HIGH-GRADE GLIOBLASTOMAS USING CLINICALLY RELEVANT EX-VIVO GLIOMA STEM CELL MODELS

Abstract High-grade gliomas are the most common and aggressive type of primary brain cancer. Despite maximal surgical resection, followed by chemotherapy and radiotherapy, overall survival for patients remains between 10-16 months, with limited improvement in survival rates over the last 40 years. This highlights an urgent unmet clinical need to develop more effective therapeutic interventions for these devastating tumours.Tumour-Treating Fields (TTFields), which deliver low-intensity (1–3V/cm) intermediate-frequency (100–300kHz) alternating electric fields to localised tumour sites, represents an exciting new clinically-approved fourth modality for the treatment of high-grade gliomas, with randomised clinical trial evidence supporting improved overall survival (20.9 vs 16.0 months). Whilst TTFields are primarily thought to mediate their anti-cancer effects by disrupting mitotic tubulin dimer alignment leading to abnormal chromosomal segregation and mitotic cell death, recent data suggests that TTFields may also attenuate DNA damage repair efficiency and replication fork dynamics. We therefore hypothesised that combining TTFields with therapeutic DNA damage response inhibitors (DDRi), to develop next generation multimodal therapy regimes, would enhance TTFields potency in glioma cells either alone, or in combination with current standard-of-care chemoradiotherapy. To assess this, we employed clinically-relevant patient-derived primary glioma stem cell (GSC) models that represent both inter-patient and intra-tumoural heterogeneity, including unique ex-vivo models of post-surgical residual disease. We show that combining TTFields with radiation and clinically approved PARP inhibitor therapy leads to significantly increased amounts of DNA damage with concurrent decreased clonogenic survival in GSCs. Furthermore, we have shown similar impressive potency when TTFields treatments are combined with ATR inhibitors that are currently being assessed in various global clinical trials for other cancers. Overall, these exciting findings support further assessment of TTFields and DDRi combinations to underpin future clinical trials combining TTFields with clinically approved DDRi to improve outcomes for patients with currently incurable high-grade gliomas.

Co-inhibition of Aurora A and Haspin kinases enhances survivin blockage and p53 induction for mitotic catastrophe and apoptosis in human colorectal cancer

Colorectal cancer (CRC) is a leading cause and mortality worldwide. Aurora A and haspin kinases act pivotal roles in mitotic progression. However, the blockage of Aurora A and Haspin for CRC therapy is still unclear. Here we show that the Haspin and p-H3T3 protein levels were highly expressed in CRC tumor tissues of clinical patients. Overexpression of Haspin increased the protein levels of p-H3T3 and survivin in human CRC cells; conversely, the protein levels of p-H3T3 and survivin were decreased by the Haspin gene knockdown. Moreover, the gene knockdown of Aurora A induced abnormal chromosome segregation, mitotic catastrophe, and cell growth inhibition. Combined targeted by co-treatment of CHR6494, a Haspin inhibitor, and MLN8237, an Aurora A inhibitor, enhanced apoptosis and CRC tumor inhibition. MLN8237 and CHR6494 induced abnormal chromosome segregation and mitotic catastrophe. Meanwhile, MLN8237 and CHR6494 inhibited survivin protein levels but conversely induced p53 protein expression. Ectopic survivin expression by transfection with a survivin-expressed vector resisted the cell death in the MLN8237- and CHR6494-treated cells. In contrast, the existence of functional p53 increased the apoptotic levels by treatment with MLN8237 and CHR6494. Co-treatment of CHR6494 and MLN8237 enhanced the blockage of human CRC xenograft tumors in nude mice. Taken together, co-inhibition of Aurora A and Haspin enhances survivin inhibition, p53 pathway induction, mitotic catastrophe, apoptosis and tumor inhibition that may provide a potential strategy for CRC therapy.

Preimplantation Genetic Testing for Couples with Balanced Chromosomal Rearrangements.

Chromosomal rearrangements play an important role in infertility. Carriers of chromosomal rearrangements have a lower chance of producing normal or balanced gametes due to abnormal segregation of chromosomes at meiosis, which leads to recurrent spontaneous abortions and infertility. Preimplantation genetic testing for structural chromosome rearrangements (PGT-SR) is offered to couples who have balanced chromosomal rearrangements in order to select embryos with a balanced karyotype prior to implantation, thereby increasing the chances of pregnancy. The purpose of the current study was to assess the outcomes of PGT-SR in patients carrying various balanced chromosomal rearrangements and to assess their clinical pregnancy outcome after in vitro fertilization (IVF). In this study, infertile couples with balanced chromosomal abnormalities undergoing PGT-SR were retrospectively analyzed at a single fertility center from January 2016 to December 2019. PGT-SR was performed on 87 embryos from 22 couples in whom one partner carried a balanced translocation or an inversion. Fifty-seven (65.5%) of these embryos had unbalanced or sporadic aneuploidies, 30 (34.5%) embryos were normal or chromosomally balanced, which were then transferred in 18 couples. A higher rate of unbalanced translocations in comparison to sporadic aneuploidies was observed in couples with reciprocal translocation. The live birth rate per embryo transfer was found to be 66.6% (12/18). PGT-SR is a useful tool in selecting normal or balanced embryos for transfer in IVF, which could lead to a pregnancy by reducing the chance of miscarriages due to chromosome aneuploidy in couples with balanced chromosomal rearrangements.

Chromatin Separation Regulators Predict the Prognosis and Immune Microenvironment Estimation in Lung Adenocarcinoma.

Background: Abnormal chromosome segregation is identified to be a common hallmark of cancer. However, the specific predictive value of it in lung adenocarcinoma (LUAD) is unclear. Method: The RNA sequencing and the clinical data of LUAD were acquired from The Cancer Genome Atlas (TACG) database, and the prognosis-related genes were identified. The Kyoto Encyclopedia of Genes and Genomes (KEGG) and Gene Ontology (GO) were carried out for functional enrichment analysis of the prognosis genes. The independent prognosis signature was determined to construct the nomogram Cox model. Unsupervised clustering analysis was performed to identify the distinguishing clusters in LUAD-samples based on the expression of chromosome segregation regulators (CSRs). The differentially expressed genes (DEGs) and the enriched biological processes and pathways between different clusters were identified. The immune environment estimation, including immune cell infiltration, HLA family genes, immune checkpoint genes, and tumor immune dysfunction and exclusion (TIDE), was assessed between the clusters. The potential small-molecular chemotherapeutics for the individual treatments were predicted via the connectivity map (CMap) database. Results: A total of 2,416 genes were determined as the prognosis-related genes in LUAD. Chromosome segregation is found to be the main bioprocess enriched by the prognostic genes. A total of 48 CSRs were found to be differentially expressed in LUAD samples and were correlated with the poor outcome in LUAD. Nine CSRs were identified as the independent prognostic signatures to construct the nomogram Cox model. The LUAD-samples were divided into two distinct clusters according to the expression of the 48 CSRs. Cell cycle and chromosome segregation regulated genes were enriched in cluster 1, while metabolism regulated genes were enriched in cluster 2. Patients in cluster 2 had a higher score of immune, stroma, and HLA family components, while those in cluster 1 had higher scores of TIDES and immune checkpoint genes. According to the hub genes highly expressed in cluster 1, 74 small-molecular chemotherapeutics were predicted to be effective for the patients at high risk. Conclusion: Our results indicate that the CSRs were correlated with the poor prognosis and the possible immunotherapy resistance in LUAD.

P-694 Dosage of exogenous gonadotropins is not related to blastocyst aneuploidy or cumulative live-birth rates in PGT-A cycles

Abstract Study question Does dosage of gonadotropins affect aneuploidy rate rates or cumulative live birth rates (CLBRs) after the transfer of euploid embryos? Summary answer We did not find evidence that dosage gonadotropin influenced significantly the rate of aneuploidy and CLBRs after transfer of euploid embryos. What is known already Aneuploidy rates increase steadily with age, reaching >80% in women >42 years old. The goal of controlled ovarian stimulation obtaining more embryos to biopsy through the recruitment of several follicles, reaching more euploid embryos. However, several studies have suggested that a high response to stimulation might be embryotoxic and/or increase aneuploidy rates by enhancing the abnormal segregation of chromosomes during meiosis. Furthermore, a study on young donors showed differences in the euploidy rates, suggesting an iatrogenic etiology resulting from different stimulation methods. Study design, size, duration A multicenter retrospective study, with the preimplantation genetic test for aneuploidy (PGT-A) between January 2013 and January 2020 collected from clinics IVIRMA in Europe. 6832 cycles of in vitro fertilization (IVF) with PGT-A were included in the study Participants/materials, setting, methods A total of 62131 embryos were analyzed for ploidy status. Embryos that were subjected to chromosomal analysis were performed blastomere by aCGH (array Comparative Genomic Hybridization) or NGS (Next-Generation Sequencing) analysis. The embryos were biopsied on days 5 and 6. Women were divided into two age groups (<37 and ≥37 years old). Outcomes were compared between different total gonadotropin dosages (<3000 and ≥3000 IU), the dosage of hMG, and numbers of oocytes retrieved. Main results and the role of chance The total number of aneuploid embryos was 28336 of the 62131 embryos genetically screened using PGT-A. The embryonic aneuploidy rate was 59.4% (95%CI 58.5-60.3). In the group of women with gonadotropins dosage <3000 IU the embryonic aneuploidy rate was 59.7% (95%CI 58.7-60.8) and in the group with gonadotropins dosage ≥3000 IU was 58.7% (95%CI 57.1-60.3) with no significant difference between them (P = 0.279). In the group of younger patients (<37 y.o) the aneuploidy rate demonstrated no significant differences between patients who received low gonadotropin dosages and patients with high gonadotropin dosages (P = 0.06). Aneuploidy rates in the group of older patients (≥37 y.o) were similar regardless of gonadotropin dosing (P = 0.21). Dividing the according to the number of oocytes retrieved (10, 11-15, >15), the analysis of aneuploidy rate revealed no association between the gonadotropin dosages administrated depending on the total number of retrieved oocytes and age. The aneuploidy rate was also analyzed considering the administration of hMG, there was no statistical difference in the aneuploidy rate with low or high hMG dosages also when it was stratified by age. The cumulative live-birth rate in the groups of different gonadotropins dosage and different ages of patients was similar across the group. Limitations, reasons for caution The retrospective nature is the major limitation of this study, also patients included in the study received PGT-A using NGS and CGH arrays technology. The aCGH analysis used is less sensitive than more recent NGS technology. Wider implications of the findings Our study demonstrated that gonadotropin dosage did not affect aneuploidy rate or cumulative live-birth rates suggesting that the high doses of gonadotropins used in ART cycles may be safe. Trial registration number not applicable

Micronucleus formation during early cleavage division is a potential hallmark of preimplantation embryonic loss in cattle

In assisted reproductive technology (ART)-derived embryos of non-rodent mammals, including humans and cattle, chromosome segregation errors are highly likely to occur during early cleavage division, resulting in aneuploidy, including mosaicism. However, the relationship between chromosomal segregation errors during early cleavage and subsequent embryonic development has not been detailed in these mammals. In the present study, we developed non-invasive live-cell imaging of chromosome segregation dynamics using a histone H2B-mCherry mRNA probe in bovine preimplantation embryos. Chromosome segregation errors in early cleavage affected blastocyst formation. Especially, embryos that underwent abnormal chromosome segregation (ACS) with multiple or large micronucleus formation rarely developed into blastocysts. Embryos with the severe ACS had prolonged cell cycle duration. After transfer of blastocysts with live-cell imaging of chromosome segregation to ten cows, six became pregnant and four of them gave full-term offspring. Interestingly, two of them were derived from blastocysts with ACS. Hence, chromosomal segregation errors with micronucleus formation during early cleavage can be a fatal hallmark of preimplantation embryogenesis in cattle. This technique has shown potential for understanding the relationship between chromosome segregation error and subsequent embryo development, and for selecting viable ART-derived embryos for medical and livestock production.

Genome‑wide DNA hypomethylation drives a more invasive pancreatic cancer phenotype and has predictive occult distant metastasis and prognosis potential.

Genome-wide DNA hypomethylation is the most common molecular feature in human cancers associated with chromosomal instability (CIN), which is involved in the mechanisms that regulate pancreatic cancer (PC) metastasis. It was investigated whether genome-wide DNA hypomethylation affects the phenotype in PC via CIN in vitro, and its significance on the biological behavior of PC was verified. The relative demethylation level (RDL) of long interspersed nucleotide element-1 (LINE-1) in human PC cell lines was used to characterize DNA hypomethylation using methylation-specific quantitative (q)PCR. CIN was estimated by changes in chromosomal copy number using comparative genomic hybridization analysis. Abnormal segregation of chromosomes was assessed by immunocytochemistry, and the DNA damage response was evaluated using the number of anti-γH2AX positive cells. Invasion ability was assessed using a Matrigel invasion assay. Clinical specimens from 49 patients with PC who underwent curative surgery were evaluated for a correlation of DNA hypomethylation with clinical outcome. Successful induction of genome-wide DNA hypomethylation in PC cells led to copy number changes in specific chromosomal regions. The number of cells with abnormal segregation of chromosomes significantly increased with the number of anti-γH2AX positive cells. The invasive potential of these cells also significantly increased. The occurrence of occult distant metastasis in the clinical specimens and receiver operating characteristic analysis clearly identified those who were and were not likely to have occult distant metastasis, with high LINE-1 RDL significantly correlated with the presence of occult distant metastasis (P=0.035) and poor prognosis (P=0.048). The significance of genome-wide DNA hypomethylation on the biological behavior of PC, which promotes a more invasive phenotype via CIN in vitro and predicts the susceptibility to occult distant metastasis and poor prognosis in patients with PC was revealed.

Preimplantation chromosomal mosaics, chimaeras and confined placental mosaicism.

Some human preimplantation embryos are chromosomally mosaic. For technical reasons, estimates of the overall frequency vary widely from <15 to >90% and the true frequency remains unknown. Aneuploid/diploid and aneuploid/aneuploid mosaics typically arise during early cleavage stages before the embryonic genome is fully activated and when cell cycle checkpoints are not operating normally. Other mosaics include chaotic aneuploid mosaics and mixoploids, some of which arise by abnormal chromosome segregation at the first cleavage division. Chimaeras are similar to mosaics, in having two genetically distinct cell populations, but they arise from more than one zygote and occur less often. After implantation, the frequency of mosaic embryos declines to about 2% and most are trisomic/diploid mosaics, with trisomic cells confined to the placenta. Thus, few babies are born with chromosomal mosaicism. This review discusses the origin of different types of chromosomal mosaics and chimaeras; their fate and the relationship between preimplantation chromosomal mosaicism and confined placental mosaicism in human conceptuses and animal models. Abnormal cells in mosaic embryos may be depleted by cell death, other types of cell selection or cell correction but the most severely affected mosaic embryos probably die. Trisomic cells could become restricted to placental lineages if cell selection or correction is less effective in placental lineages and/or they are preferentially allocated to a placental lineage. However, the relationship between preimplantation mosaicism and confined placental mosaicism may be complex because the specific chromosome(s) involved will influence whether chromosomally abnormal cells survive predominately in the placental trophoblast and/or placental mesenchyme.Lay summaryHuman cells normally have 23 pairs of chromosomes, which carry the genes. During the first few days of development, some human embryos are chromosomal mosaics. These mosaic embryos have both normal cells and cells with an abnormal number of chromosomes, which arise from the same fertilised egg. (More rarely, the different cell populations arise from more than one fertilised egg and these embryos are called chimaeras.) If chromosomally abnormal cells survive to term, they could cause birth defects. However, few abnormal cells survive and those that do are usually confined to the placenta, where they are less likely to cause harm. It is not yet understood how this restriction occurs but the type of chromosomal abnormality influences which placental tissues are affected. This review discusses the origin of different types of chromosomally abnormal cells, their fate and how they might become confined to the placenta in humans and animal models.

EP363: A review of characteristic features identifying balanced chromosomal translocations with increased potential for 3:1 meiotic segregation

A carrier of a balanced translocation is at risk for abnormal chromosome segregation during the meiotic events of gamete formation. During meiosis all four chromosomes, both derivatives and their normal homologs, pair together forming a four-chromosome structure or quadrivalent. Segregation of the quadrivalent occurs in several iterations. Most typically, the outcome of this segregation results in 2 of the quadrivalent chromosomes per gamete in either an alternate, adjacent-1 or adjacent-2 configuration.

Production of offspring from vacuum-dried mouse spermatozoa and assessing the effect of drying conditions on sperm DNA and embryo development.

Freeze-dried sperm (FD sperm) are of great value because they can be stored at room temperature for long periods of time, However, the birth rate of offspring derived from FD sperm is low and the step in the freeze-drying process particularly responsible for low offspring production remains unknown. In this study, we determined whether the drying process was responsible for the low success rate of offspring by producing vacuum-dried sperm (VD sperm), using mouse spermatozoa dried in a vacuum without being frozen. Transfer of embryos fertilized with VD sperm to recipients resulted in the production of several successful offspring. However, the success rate was slightly lower than that of FD sperm. The volume, temperature, and viscosity of the medium were optimized to improve the birth rate. The results obtained from a comet assay indicated that decreasing the drying rate reduced the extent of DNA damage in VD sperm. Furthermore, even though the rate of blastocyst formation increased upon fertilization with VD sperm, full-term development was not improved. Analysis of chromosomal damage at the two-cell stage through an abnormal chromosome segregation (ACS) assay revealed that reduction in the drying rate failed to prevent chromosomal damage. These results indicate that the lower birth rate of offspring from FD sperm may result from the drying process rather than the freezing process.

In vitro and in silico assessment of cytotoxicity and chromosome instability induced by saxitoxin in human derived neural cell line

In freshwater, saxitoxins (STX) are produced by different cyanobacteria genera, including Raphidiopsis. Data regarding cytogenotoxicity effects of STX on human cells are scarse, this merit further studies of its toxicology. This study assessed the cytotoxicity and the chromosome instability of STX on SHSY-5Y human cell line. The CBMN assay allows the detection of chromosome breaks and abnormal chromosomal segregation. Additionally, in silico systems biology approach, used to search for known and predicted interaction networks, was applied to study the interactions between STX and SHSY-5Y cellular components. The results of the CBMN assay demonstrated that STX concentrations of 2.5 - 10 µg/L induced cytostasis and chromosome instability in a dose-response relationship. Apoptosis was detected after exposure of SHSY-5Y cultured cells to STX concentration of 10 µg/L. The results of the systems biology analysis revealed the interaction of STX with proteins related with acetylcoline pathway, cell cycle regulation and apoptosis. Furthermore, combining the in vitro and in silico approachs, it was possible to suggest a mechanism of action of STX in SHSY-5Y cells. Overall, the data demonstrated the cytotoxicity and mutagenicity of environmentally relevant concentrations of STX. These results should be considered when setting up guidelines for monitoring STX in water supply.

The Inactivation of Arabidopsis UBC22 Results in Abnormal Chromosome Segregation in Female Meiosis, but Not in Male Meiosis.

Protein ubiquitination is important for the regulation of meiosis in eukaryotes, including plants. However, little is known about the involvement of E2 ubiquitin-conjugating enzymes in plant meiosis. Arabidopsis UBC22 is a unique E2 enzyme, able to catalyze the formation of ubiquitin dimers through lysine 11 (K11). Previous work has shown that ubc22 mutants are defective in megasporogenesis, with most ovules having no or abnormally functioning megaspores; furthermore, some mutant plants show distinct phenotypes in vegetative growth. In this study, we showed that chromosome segregation and callose deposition were abnormal in mutant female meiosis while male meiosis was not affected. The meiotic recombinase DMC1, required for homologous chromosome recombination, showed a dispersed distribution in mutant female meiocytes compared to the presence of strong foci in WT female meiocytes. Based on an analysis of F1 plants produced from crosses using a mutant as the female parent, about 24% of female mutant gametes had an abnormal content of DNA, resulting in frequent aneuploids among the mutant plants. These results show that UBC22 is critical for normal chromosome segregation in female meiosis but not for male meiosis, and they provide important leads for studying the role of UBC22 and K11-linked ubiquitination.

Genome-Wide Allele-Specific Expression in Obligately Asexual Daphnia pulex and the Implications for the Genetic Basis of Asexuality.

Although obligately asexual lineages are thought to experience selective disadvantages associated with reduced efficiency of fixing beneficial mutations and purging deleterious mutations, such lineages are phylogenetically and geographically widespread. However, despite several genome-wide association studies, little is known about the genetic elements underlying the origin of obligate asexuality and how they spread. Because many obligately asexual lineages have hybrid origins, it has been suggested that asexuality is caused by the unbalanced expression of alleles from the hybridizing species. Here, we investigate this idea by identifying genes with allele-specific expression (ASE) in a Daphnia pulex population, in which obligate parthenogens (OP) and cyclical parthenogens (CP) coexist, with the OP clones having been originally derived from hybridization between CP D. pulex and its sister species, Daphnia pulicaria. OP D. pulex have significantly more ASE genes (ASEGs) than do CP D. pulex. Whole-genomic comparison of OP and CP clones revealed ∼15,000 OP-specific markers and 42 consistent ASEGs enriched in marker-defined regions. Ten of the 42 ASEGs have alleles coding for different protein sequences, suggesting functional differences between the products of the two parental alleles. At least three of these ten genes appear to be directly involved in meiosis-related processes, for example, RanBP2 can cause abnormal chromosome segregation in anaphase I, and the presence of Wee1 in immature oocytes leads to failure to enter meiosis II. These results provide a guide for future molecular resolution of the genetic basis of the transition to ameiotic parthenogenesis.

Enhanced UV-B radiation affects AUR1 regulation of mitotic spindle morphology leading to aberrant mitosis

Enhanced UV-B radiation can lead to a variety of stress responses, including effects on cell cycle regulation and mitosis. Aurora kinases are part of the serine/threonine kinase family and play important roles in cell cycle regulation and mitosis. We hypothesize that there may be a connection between these two processes.In this study, the dynamics of chromosomal (H2B-YFP) and AUR1-GFP changes after enhanced UV-B radiation were observed using confocal microscopy, and gene and protein expression patterns under UV-B stress were quantified using RT-qPCR and Western blotting techniques. We analyzed the responses of the AUR1 overexpression to UV-B stress. We measured maximum quantum yield of photosystem Ⅱ as a proxy for UV-B stress. The recovery capacity of AUR1 overexpression strains was analyzed.In our research, we observed that enhanced UV-B radiation affects the subcellular positioning of AUR1, resulting in abnormalities in the positioning and location of the spindle at the poles, which ultimately affects the separation of chromosomes, resulting in “partition-bundle division” and the incorrect direction of division. At the same time, our results also indicated that low-dose UV-B can induce the expression of AUR1, and this overexpression of AUR1 can alleviate the damage caused by UV-B radiation.In summary, the results of our study show that enhanced UV-B radiation can change the activity and expression of AUR1, which is one of the causes of abnormal chromosome segregation. AUR1 participates in the response to UV-B stress, and, to a certain extent, can improve the UV-B tolerance of plants.