Premature Sister Chromatid Separation Research Articles

Unambiguous chromosome identification reveals the factors impacting irregular chromosome behaviors in allotriploid AAC Brassica.

The major irregular chromosome pairing and mis-segregation were detected during meiosis through unambiguous chromosome identification and found that allotriploid Brassica can undergo meiosis successfully and produce mostly viable aneuploid gametes. Triploids have played a crucial role in the evolution of species by forming polyploids and facilitating interploidy gene transfer. It is widely accepted that triploids cannot undergo meiosis normally and predominantly produce nonfunctional aneuploid gametes, which restricts their role in species evolution. In this study, we demonstrated that natural and synthetic allotriploid Brassica (AAC), produced by crossing natural and synthetic Brassica napus (AACC) with Brassica rapa (AA), exhibits basically normal chromosome pairing and segregation during meiosis. Homologous A chromosomes paired faithfully and generally segregated equally. Monosomic C chromosomes were largely retained as univalents and randomly entered daughter cells. The primary irregular meiotic behaviors included associations of homoeologs and 45S rDNA loci at diakinesis, as well as homoeologous chromosome replacement and premature sister chromatid separation at anaphase I. Preexisting homoeologous arrangements altered meiotic behaviors in both chromosome irregular pairing and mis-segregation by increasing the formation of A-genomic univalents and A-C bivalents, as well as premature sister chromatid separation and homologous chromosome nondisjunction. Meiotic behaviors depended significantly on the genetic background and heterozygous homoeologous rearrangement. AAC triploids mainly generated aneuploid gametes, most of which were viable. These results demonstrate that allotriploid Brassica containing an intact karyotype can proceed through meiosis successfully, broadening our current understanding of the inheritance and role in species evolution of allotriploid.

Requirement of Nek2a and cyclin A2 for Wapl-dependent removal of cohesin from prophase chromatin

Sister chromatid cohesion is mediated by the cohesin complex. In mitotic prophase cohesin is removed from chromosome arms in a Wapl- and phosphorylation-dependent manner. Sgo1-PP2A protects pericentromeric cohesion by dephosphorylation of cohesin and its associated Wapl antagonist sororin. However, Sgo1-PP2A relocates to inner kinetochores well before sister chromatids are separated by separase, leaving pericentromeric regions unprotected. Why deprotected cohesin is not removed by Wapl remains enigmatic. By reconstituting Wapl-dependent cohesin removal from chromatin in vitro, we discovered a requirement for Nek2a and Cdk1/2-cyclin A2. These kinases phosphorylate cohesin-bound Pds5b, thereby converting it from a sororin- to a Wapl-interactor. Replacement of endogenous Pds5b by a phosphorylation mimetic variant causes premature sister chromatid separation (PCS). Conversely, phosphorylation-resistant Pds5b impairs chromosome arm separation in prometaphase-arrested cells and suppresses PCS in the absence of Sgo1. Early mitotic degradation of Nek2a and cyclin A2 may therefore explain why only separase, but not Wapl, can trigger anaphase.

A non-canonical role of the inner kinetochore in regulating sister-chromatid cohesion at centromeres

The 16-subunit Constitutive Centromere-associated Network (CCAN)-based inner kinetochore is well-known for connecting centromeric chromatin to the spindle-binding outer kinetochore. Here, we report a non-canonical role for the inner kinetochore in directly regulating sister-chromatid cohesion at centromeres. We provide biochemical, X-ray crystal structure, and intracellular ectopic localization evidence that the inner kinetochore directly binds cohesin, a ring-shaped multi-subunit complex that holds sister chromatids together from S-phase until anaphase onset. This interaction is mediated by binding of the 5-subunit CENP-OPQUR sub-complex of CCAN to the Scc1-SA2 sub-complex of cohesin. Mutation in the CENP-U subunit of the CENP-OPQUR complex that abolishes its binding to the composite interface between Scc1 and SA2 weakens centromeric cohesion, leading to premature separation of sister chromatids during delayed metaphase. We further show that CENP-U competes with the cohesin release factor Wapl for binding the interface of Scc1-SA2, and that the cohesion-protecting role for CENP-U can be bypassed by depleting Wapl. Taken together, this study reveals an inner kinetochore-bound pool of cohesin, which strengthens centromeric sister-chromatid cohesion to resist metaphase spindle pulling forces.

The severity of meiotic aneuploidy is associated with altered morphokinetic variables of mouse oocyte maturation.

Is there an association between morphokinetic variables of meiotic maturation and the severity of aneuploidy following in vitro maturation (IVM) in the mouse? The severity of meiotic aneuploidy correlates with an extended time to first polar body extrusion (tPB1) and duration of meiosis I (dMI). Morphokinetic variables measured using time-lapse technology allow for the non-invasive evaluation of preimplantation embryo development within clinical assisted reproductive technology (ART). We recently applied this technology to monitor meiotic progression during IVM of mouse gametes. Whether there is a relationship between morphokinetic variables of meiotic progression and aneuploidy in the resulting egg has not been systematically examined at the resolution of specific chromosomes. Next-generation sequencing (NGS) is a robust clinical tool for determining aneuploidy status and has been reverse-translated in mouse blastocysts and oocytes. Therefore, we harnessed the technologies of time-lapse imaging and NGS to determine the relationship between the morphokinetics of meiotic progression and egg aneuploidy. Cumulus-oocyte complexes were collected from large antral follicles from hyperstimulated CD-1 mice. Cumulus cells were removed, and spontaneous IVM was performed in the absence or presence of two doses of Nocodazole (25 or 50 nM) to induce a spectrum of spindle abnormalities and chromosome segregation errors during oocyte meiosis. Comprehensive chromosome screening was then performed in the resulting eggs, and morphokinetic variables and ploidy status were compared across experimental groups (control, n = 11; 25 nM Nocodazole, n = 13; 50 nM Nocodazole, n = 23). We monitored IVM in mouse oocytes using time-lapse microscopy for 16 h, and time to germinal vesicle breakdown (tGVBD), tPB1, and dMI were analyzed. Following IVM, comprehensive chromosome screening was performed on the eggs and their matched first polar bodies via adaptation of an NGS-based preimplantation genetic testing for aneuploidy (PGT-A) assay. Bioinformatics analysis was performed to align reads to the mouse genome and determine copy number-based predictions of aneuploidy. The concordance of each polar body-egg pair (reciprocal errors) was used to validate the results. Ploidy status was categorized as euploid, 1-3 chromosomal segregation errors, or ≥4 chromosomal segregation errors. Additionally, aneuploidy due to premature separation of sister chromatids (PSSC) versus non-disjunction (NDJ) was distinguished. We applied and validated state-of-the-art NGS technology to screen aneuploidy in individual mouse eggs and matched polar bodies at the chromosome-specific level. By performing IVM in the presence of different doses of Nocodazole, we induced a range of aneuploidy. No aneuploidy was observed in the absence of Nocodazole (0/11), whereas IVM in the presence of 25 and 50 nM Nocodazole resulted in an aneuploidy incidence of 7.69% (1/13) and 82.61% (19/23), respectively. Of the aneuploid eggs, 5% (1/20) was due to PSSC, 65% (13/20) to NDJ, and the remainder to a combination of both. There was no relationship between ploidy status and tGVBD, but tPB1 and the dMI were both significantly prolonged in eggs with reciprocal aneuploidy events compared to the euploid eggs, and this scaled with the severity of aneuploidy. Eggs with ≥4 aneuploid chromosomes had the longest tPB1 and dMI (P < 0.0001), whereas eggs with one to three aneuploid chromosomes exhibited intermediate lengths of time (P < 0.0001). N/A. We used Nocodazole in this study to disrupt the meiotic spindle and induce aneuploidy in mouse oocytes. Whether the association between morphokinetic variables of meiotic progression and the severity of aneuploidy occurs with other compounds that induce chromosome segregation errors remain to be investigated. In addition, unlike mouse oocytes, human IVM requires the presence of cumulus cells, which precludes visualization of morphokinetic variables of meiotic progression. Thus, our study may have limited direct clinical translatability. We validated NGS in mouse eggs to detect aneuploidy at a chromosome-specific resolution which greatly improves the utility of the mouse model. With a tractable and validated model system for characterizing meiotic aneuploidy, investigations into the molecular mechanisms and factors which may influence aneuploidy can be further elaborated. Time-lapse analyses of morphokinetic variables of meiotic progression may be a useful non-invasive predictor of aneuploidy severity. This work was supported by the Bill & Melinda Gates Foundation (INV-003385). Under the grant conditions of the Foundation, a Creative Commons Attribution 4.0 Generic License has already been assigned to the Author Accepted Manuscript version that might arise from this submission. The authors have no conflict of interest to disclose.

Securin acetylation prevents precocious separase activation and premature sister chromatid separation

Lysine acetylation of non-histone proteins plays crucial roles in many cellular processes. In this study, we examine the role of lysine acetylation during sister chromatid separation in mitosis. We investigate the acetylation of securin at K21 by cell-cycle-dependent acetylome analysis and uncover its role in separase-triggered chromosome segregation during mitosis. Prior to the onset of anaphase, the acetylated securin via TIP60 prevents its degradation by the APC/CCDC20-mediated ubiquitin-proteasome system. This, in turn, restrains precocious activation of separase and premature separation of sister chromatids. Additionally, the acetylation-dependent stability of securin is also enhanced by its dephosphorylation. As anaphase approaches, HDAC1-mediated deacetylation of securin promotes its degradation, allowing released separase to cleave centromeric cohesin. Blocking securin deacetylation leads to longer anaphase duration and errors in chromosome segregation. Thus, this study illustrates the emerging role of securin acetylation dynamics in mitotic progression and genetic stability.

Transient excited states of the metamorphic protein Mad2 and their implications for function.

The spindle checkpoint complex is a key surveillance mechanism in cell division that prevents premature separation of sister chromatids. Mad2 is an integral component of this spindle checkpoint complex that recognizes cognate substrates such as Mad1 and Cdc20 in its closed (C-Mad2) conformation by fastening a "seatbelt" around short peptide regions that bind to the substrate recognition site. Mad2 is also a metamorphic protein that adopts not only the fold found in C-Mad2, but also a structurally distinct open conformation (O-Mad2) which is incapable of binding substrates. Here, we show using chemical exchange saturation transfer (CEST) and relaxation dispersion (CPMG) NMR experiments that Mad2 transiently populates three other higher free energy states with millisecond lifetimes, two in equilibrium with C-Mad2 (E1 and E2) and one with O-Mad2 (E3). E1 is a mimic of substrate-bound C-Mad2 in which the N-terminus of one C-Mad2 molecule inserts into the seatbelt region of a second molecule of C-Mad2, providing a potential pathway for autoinhibition of C-Mad2. E2 is the "unbuckled" conformation of C-Mad2 that facilitates the triage of molecules along competing fold-switching and substrate binding pathways. The E3 conformation that coexists with O-Mad2 shows fluctuations at a hydrophobic lock that is required for stabilizing the O-Mad2 fold and we hypothesize that E3 represents an early intermediate on-pathway towards conversion to C-Mad2. Collectively, the NMR data highlight the rugged free energy landscape of Mad2 with multiple low-lying intermediates that interlink substrate-binding and fold-switching, and also emphasize the role of molecular dynamics in its function.

Mad2 is dispensable for accurate chromosome segregation but becomes essential when oocytes are subjected to environmental stress.

Accurate chromosome segregation, monitored by the spindle assembly checkpoint (SAC), is crucial for the production of euploid cells. Previous in vitro studies by us and others showed that Mad2, a core member of the SAC, performs a checkpoint function in oocyte meiosis. Here, through an oocyte-specific knockout approach in mouse, we reconfirmed that Mad2-deficient oocytes exhibit an accelerated metaphase-to-anaphase transition caused by premature degradation of securin and cyclin B1 and subsequent activation of separase in meiosis I. However, it was surprising that the knockout mice were completely fertile and the resulting oocytes were euploid. In the absence of Mad2, other SAC proteins, including BubR1, Bub3 and Mad1, were normally recruited to the kinetochores, which likely explains the balanced chromosome separation. Further studies showed that the chromosome separation in Mad2-null oocytes was particularly sensitive to environmental changes and, when matured in vitro, showed chromosome misalignment, lagging chromosomes, and aneuploidy with premature separation of sister chromatids, which was exacerbated at a lower temperature. We reveal for the first time that Mad2 is dispensable for proper chromosome segregation but acts to mitigate environmental stress in meiotic oocytes.

Actin limits egg aneuploidies associated with female reproductive aging.

Aging-related centromeric cohesion loss underlies premature separation of sister chromatids and egg aneuploidy in reproductively older females. Here, we show that F-actin maintains chromatid association after cohesion deterioration in aged eggs. F-actin disruption in aged mouse eggs exacerbated untimely dissociation of sister chromatids, while its removal in young eggs induced extensive chromatid separation events generally only seen in advanced reproductive ages. In young eggs containing experimentally reduced cohesion, F-actin removal accelerated premature splitting and scattering of sister chromatids in a microtubule dynamics-dependent manner, suggesting that actin counteracts chromatid-pulling spindle forces. Consistently, F-actin stabilization restricted scattering of unpaired chromatids generated by complete degradation of centromeric cohesion proteins. We conclude that actin mitigates egg aneuploidies arising from age-related cohesion depletion by limiting microtubule-driven separation and dispersion of sister chromatids. This is supported by our finding that spindle-associated F-actin structures are disrupted in eggs of reproductively older females.

A multimodal genetic testing approach to a diagnosis of Roberts-SC phocomelia syndrome, an ESCO2 spectrum disorder

Abstract Diagnosis of a genetic syndrome has traditionally relied upon the identification of characteristic phenotypic findings, followed by targeted laboratory testing. Shared clinical manifestations between genetically distinct syndromes and variable expressivity of findings within a single syndrome can complicate the diagnosis. Multiple testing modalities may be necessary, even when the putative gene(s) is known. We describe the multi-pronged genetic testing applied to a patient with a suspected diagnosis of Roberts- SC phocomelia syndrome (RBS). RBS is an autosomal recessive developmental disorder (OMIM 268300) caused by pathogenic variants in the “establishment of sister chromatid cohesion N-acetyltransferase” (ESCO2 ) gene mapped to chromosome 8p21.1. The ESCO2 enzyme product is a conserved protein with acetyltransferase activity required for formation of the cohesin complex, which, among other activities, maintains sister chromatid association during S-phase of the cell cycle. Because of the defective cohesin complex, RBS joins other syndromes referred to as cohesinopathies. The patient, a newborn male, was small for gestational age, with hypertelorism, cleft lip and palate, shortened forearms, absence of the radius and thumbs, and bilateral clubbed feet. All of these findings are established characteristics of RBS. Targeted Next Generation Sequencing revealed a single variant in exon 10 of ESCO2 (c.1654C&amp;gt;T, p.Arg552*) predicted to result in a loss of function. Chromosomal microarray was also performed and showed an estimated 15 Mb region of homozygosity (ROH) extending from 8p11.21 to 8p21.2, which spans ESCO2. The finding of this ROH supported the interpretation of homozygosity for the variant, as did a family history of consanguinity. Additional cytogenetic analysis documented premature sister chromatid separation and repulsion of heterochromatin chromosomal regions characteristic of RBS. These chromosomal findings served as a functional assay supporting pathogenicity of the ESCO2 variant. Interestingly, the only report of an identical variant within exon 10 of ESCO2 was described for two brothers diagnosed with Juberg-Hayward syndrome. Juberg-Hayward was considered allelic to, but a distinct entity from RBS. In contrast, our findings support the conclusion that RBS and Juberg-Hayward are not distinct syndromes, but rather represent variability within an ESCO2 syndrome spectrum. Such unification of diagnoses based on genomic findings has influenced numerous other clinical disorders. Further, our findings demonstrate the contributions of each of the genetic testing modalities, which may be applied, selectively or collectively, based on integration with clinical and family history data.

Germline Missense Variants in CDC20 Result in Aberrant Mitotic Progression and Familial Cancer.

Two germline CDC20 missense variants that segregate with cancer in two families compromise the spindle assembly checkpoint and lead to aberrant mitotic progression, which could predispose cells to transformation. See related commentary by Villarroya-Beltri and Malumbres, p. 3432.

O-162 Emerging mechanisms of oocyte genome integrity

Abstract Ageing is accompanied by aneuploidy in mammalian eggs, which underlies common pregnancy failures in reproductively older females. This is consistent with gradual, ageing-related depletion of centromeric cohesion proteins leading to premature separation of sister chromatids. However, such progressive cohesion loss does not satisfactorily explain the sharp rise in egg aneuploidy near the end of female reproductive life. Here we show that F-actin helps to keep most sister chromatids together after centromeric cohesion has deteriorated in ageing mammalian eggs. By combining targeted protein degradation with advanced microscopy of chromosomal dynamics in eggs of young and aged females, we demonstrate that actin mitigates premature sister chromatid separation arising from centromeric cohesion loss by limiting microtubule-dependent chromatid disengagement. We propose that impairment of this function underlies ageing-related exponential rise in egg aneuploidy.

Oocyte aneuploidy rates in river and swamp buffalo types (Bubalus bubalis) determined by Multi-color Fluorescence In Situ Hybridization (M-FISH)

Aneuploidy is one of the main causes of fetal and embryonic mortality in mammals. Nonetheless, its incidence in domestic ruminants has been investigated little. Indeed, no incidence data have ever been reported for water buffalo. To establish the incidence of aneuploidy in this species, we analysed in vitro matured metaphase II (MII) oocytes with corresponding first polar bodies (I PB) of the river (2n = 50) and swamp (2n = 48) buffaloes. For the first time, six river type probes (corresponding to chromosomes 1–5 and heterosome X), were tested on swamp buffalo metaphases using Multicolor-Fluorescent In Situ Hybridization (M-FISH) before their use on oocytes MII metaphases. Of the 120 total Cumulus Oocyte Complexes (COCs, 60 for each buffalo type) subjected to in vitro maturation, 104 reached the MII stage and were analysed by M-FISH. Haploid chromosome arrangement and visible I PB were observed in 89 of the oocytes (45 in river and 44 in swamp type). In the river type, the analysis revealed one oocyte was disomic for the chromosome X (2.22%). In the swamp type, one oocyte was found to be nullisomic for chromosome X (2.27%); another was found to be nullisomic for chromosome 5 (2.27%). We also observed one oocyte affected by a premature separation of sister chromatids (PSSC) on the chromosome X (2.27%). In both buffalo types, no abnormalities were detected in other investigated chromosomes. Based on merged data, the overall aneuploidy rate for the species was 3.37%. Oocytes with unreduced chromosomes averaged 1.92% across the two types, with 1.96% in river and 1.88% in swamp. The interspecies comparison between these data and cattle and pig published data revealed substantial difference in both total aneuploidy and diploidy rates. Reducing the negative impact of the meiotic segregation errors on the fertility is key to more sustainable breeding, an efficient embryo transfer industry and ex-situ bio-conservation. In this respect, additional M-FISH studies are needed on oocytes of domestic species using larger sets of probes and/or applying next generation sequencing technologies.

Automated human chromosome segmentation and feature extraction: Current trends and prospects

Automated human chromosome segmentation and feature extraction aim to improve the overall quality of genetic disorder diagnosis by addressing the limitations of tedious manual processes such as expertise dependence, time-inefficiency, observer variability and fatigue errors. Nevertheless, significant differences caused by staining methods, chromosome damage which may occur during imaging, cell and staining debris, inhomogeneity, weak boundaries, morphological variations, premature sister chromatid separation, as well as the presence of overlapping, touching, di-centric and bent chromosomes pose challenges in automated human chromosome segmentation and feature extraction. This review paper extensively discusses how the approaches presented in literature have addressed these challenges, and their strengths and limitations. Human chromosome segmentation algorithms are presented under four broad categories; thresholding, clustering, active contours and convex-concave points-based methods. Chromosome feature extraction methods are discussed under two main categories based on banding-pattern and geometry. In addition, new insights for the improvement of fully automated karyotyping are provided.

Meiosis-specific cohesin complexes display essential and distinct roles in mitotic embryonic stem cell chromosomes

BackgroundCohesin is a chromosome-associated SMC–kleisin complex that mediates sister chromatid cohesion, recombination, and most chromosomal processes during mitosis and meiosis. However, it remains unclear whether meiosis-specific cohesin complexes are functionally active in mitotic chromosomes.ResultsThrough high-resolution 3D-structured illumination microscopy (3D-SIM) and functional analyses, we report multiple biological processes associated with the meiosis-specific cohesin components, α-kleisin REC8 and STAG3, and the distinct loss of function of meiotic cohesin during the cell cycle of embryonic stem cells (ESCs). First, we show that STAG3 is required for the efficient localization of REC8 to the nucleus by interacting with REC8. REC8-STAG3-containing cohesin regulates topological properties of chromosomes and maintains sister chromatid cohesion. Second, REC8-cohesin has additional sister chromatid cohesion roles in concert with mitotic RAD21-cohesin on ESC chromosomes. SIM imaging of REC8 and RAD21 co-staining revealed that the two types of α-kleisin subunits exhibited distinct loading patterns along ESC chromosomes. Third, knockdown of REC8 or RAD21-cohesin not only leads to higher rates of premature sister chromatid separation and delayed replication fork progression, which can cause proliferation and developmental defects, but also enhances chromosome compaction by hyperloading of retinoblastoma protein–condensin complexes from the prophase onward.ConclusionsOur findings indicate that the delicate balance between mitotic and meiotic cohesins may regulate ESC-specific chromosomal organization and the mitotic program.

Cohesin-Mediated Chromatin Interactions and Autoimmunity.

Proper physiological functioning of any cell type requires ordered chromatin organization. In this context, cohesin complex performs important functions preventing premature separation of sister chromatids after DNA replication. In partnership with CCCTC-binding factor, it ensures insulator activity to organize enhancers and promoters within regulatory chromatin. Homozygous mutations and dysfunction of individual cohesin proteins are embryonically lethal in humans and mice, which limits in vivo research work to embryonic stem cells and progenitors. Conditional alleles of cohesin complex proteins have been generated to investigate their functional roles in greater detail at later developmental stages. Thus, genome regulation enabled by action of cohesin proteins is potentially crucial in lineage cell development, including immune homeostasis. In this review, we provide current knowledge on the role of cohesin complex in leukocyte maturation and adaptive immunity. Conditional knockout and shRNA-mediated inhibition of individual cohesin proteins in mice demonstrated their importance in haematopoiesis, adipogenesis and inflammation. Notably, these effects occur rather through changes in transcriptional gene regulation than through expected cell cycle defects. This positions cohesin at the crossroad of immune pathways including NF-kB, IL-6, and IFNγ signaling. Cohesin proteins emerged as vital regulators at early developmental stages of thymocytes and B cells and after antigen challenge. Human genome-wide association studies are remarkably concordant with these findings and present associations between cohesin and rheumatoid arthritis, multiple sclerosis and HLA-B27 related chronic inflammatory conditions. Furthermore, bioinformatic prediction based on protein-protein interactions reveal a tight connection between the cohesin complex and immune relevant processes supporting the notion that cohesin will unearth new clues in regulation of autoimmunity.

Single nucleotide polymorphism array and cytogenetic analyses of ovarian teratomas in children.

Teratomas are the most common tumors in the ovary during childhood. Previous studies suggested that they may be derived from germ cells at any developmental stage from premeiotic oogonia through meiotic oocytes to post-meiotic ova. The majority of mature teratomas reveal normal karyotypes and immature teratomas show higher frequency of chromosomal abnormalities. We analyzed fresh tissue samples from 25 primary ovarian teratomas and three extraovarian deposits using whole genome single nucleotide polymorphism (SNP) array and karyotype. SNP array detected five patterns of copy neutral loss of heterozygosity (CN-LOH): failure of meiosis I (type I) in 12 tumors, failure of meiosis II (type II) in six tumors, endoreduplication of a haploid ovum (type III) in two tumors, premeiotic error (type IV) in four tumors, and both meiotic I and meiotic II errors in one tumor (type V). Three tumors with type I error had a single chromosome showing meiotic II error, and two tumors with type II error had a single chromosome showing premature sister-chromatid separation in meiosis I. Lack of recombination in multiple chromosomes in meiosis I were common, chromosomes 17, 7, 8, 21, and 22 were most commonly involved. Abnormal karyotypes were observed in four teratomas including +3, del(3q), +7, +8, +12, and i(18q). The extraovarian deposits revealed the same CN-LOH pattern as the primary teratoma. In summary, SNP array reveals the origin of ovarian teratoma and we propose a new mechanism that consecutive meiotic I and II errors occur frequently in ovarian teratomas.

Spindle assembly checkpoint competence in aneuploid canine malignant melanoma cell lines

The spindle assembly checkpoint (SAC) is a surveillance mechanism that prevents unequal segregation of chromosomes during mitosis. Abnormalities in the SAC are associated with chromosome instability and resultant aneuploidy. This study was performed to evaluate the SAC competence in canine malignant melanoma (CMM) using four aneuploid cell lines (CMeC1, CMeC2, KMeC, and LMeC). After treatment with nocodazole, a microtubule disrupting agent, CMeC1, KMeC, and LMeC cells were arrested in M phase, whereas CMeC2 cells were not arrested, and progressed into the next cell cycle phase without cytokinesis. Chromosome spread analysis revealed a significantly increased rate of premature sister chromatid separation in CMeC2 cells. Expression of the phosphorylated form of the SAC regulator, monopolar spindle 1 (Mps1), was lower in CMeC2 cells than in the other CMM cell lines. These results indicate that the SAC is defective in CMeC2 cells, which may partially explain aneuploidy in CMM. Thus, CMeC2 cells may be useful for further studies of the SAC mechanism in CMM and in determining the relationship between SAC incompetence and aneuploidy.

Mathematical modeling of human oocyte aneuploidy

Aneuploidy is the leading contributor to pregnancy loss, congenital anomalies, and in vitro fertilization (IVF) failure in humans. Although most aneuploid conceptions are thought to originate from meiotic division errors in the female germline, quantitative studies that link the observed phenotypes to underlying error mechanisms are lacking. In this study, we developed a mathematical modeling framework to quantify the contribution of different mechanisms of erroneous chromosome segregation to the production of aneuploid eggs. Our model considers the probabilities of all possible chromosome gain/loss outcomes that arise from meiotic errors, such as nondisjunction (NDJ) in meiosis I and meiosis II, and premature separation of sister chromatids (PSSC) and reverse segregation (RS) in meiosis I. To understand the contributions of different meiotic errors, we fit our model to aneuploidy data from 11,157 blastocyst-stage embryos. Our best-fitting model captures several known features of female meiosis, for instance, the maternal age effect on PSSC. More importantly, our model reveals previously undescribed patterns, including an increased frequency of meiosis II errors among eggs affected by errors in meiosis I. This observation suggests that the occurrence of NDJ in meiosis II is associated with the ploidy status of an egg. We further demonstrate that the model can be used to identify IVF patients who produce an extreme number of aneuploid embryos. The dynamic nature of our mathematical model makes it a powerful tool both for understanding the relative contributions of mechanisms of chromosome missegregation in human female meiosis and for predicting the outcomes of assisted reproduction.

Overexpression of cyclin A1 promotes meiotic resumption but induces premature chromosome separation in mouse oocyte.

Mammalian cyclin A1 is prominently expressed in testis and essential for meiosis in the male mouse, however, it shows weak expression in ovary, especially during oocyte maturation. To understand why cyclin A1 behaves in this way in the oocyte, we investigated the effect of cyclin A1 overexpression on mouse oocyte meiotic maturation. Our results revealed that cyclin A1 overexpression triggered meiotic resumption even in the presence of germinal vesicle breakdown inhibitor, milrinone. Nevertheless, the cyclin A1-overexpressed oocytes failed to extrude the first polar body but were completely arrested at metaphase I. Consequently, cyclin A1 overexpression destroyed the spindle morphology and chromosome alignment by inducing premature separation of chromosomes and sister chromatids. Therefore, cyclin A1 overexpression will prevent oocyte maturation although it can promote meiotic resumption. All these results show that decreased expression of cyclin A1 in oocytes may have an evolutional significance to keep long-lasting prophase arrest and orderly chromosome separation during oocyte meiotic maturation.

36. TWO HEALTHY BABIES BORN AFTER IMPLANTATION OF MULTIPLE CHROMOSOMALLY-RESCUED OOCYTES

Introduction Meiotic rescue is a well-known phenomenon but little is known about live births after oocyte rescue, because of the shortage of data on PB biopsy. PGT-A has been performed for many years to increase the chance of pregnancy, especially in older women. Embryo aneuploidy is a major cause of pregnancy failure and is largely of maternal meiotic origin (>95%), with the risk increasing exponentially from approximately 35 years of age. Molecular analysis of polar bodies provides indirect assessment of an oocyte's chromosomal status by determining whether the chromosomes correctly segregated during meiosis I and II. In about one third of cases, missegregation of one or two chromosomes in meiosis I can be rescued in meiosis II, leading to a euploid oocyte. We present two cases of healthy babies born after single and double chromosome-rescued oocytes Material and methods A 42- (case 1) and a 44-year old woman (case 2) underwent ART treatment with PGT-A PB, after histories of 3 and 2 prior ART failures respectively. Both couples underwent ICSI with first and second PB biopsy and comprehensive chromosome analysis by Array-CGH. The mechanisms leading to missegregation in PB1 and PB2 (nondisjunction or premature separation of sister chromatids (PSSC)) were analysed using chromosome-specific STRs. Results and conclusions Case 1: a single euploid fertilized oocyte was transferred, after double rescue of chromosomes 3 and 18 (PB1 -3, -18; PB2: +3, +18) at day 2, resulting in a singleton pregnancy. Case 2: a single euploid fertilized oocyte was transferred after rescue of chromosome 16 (PB1: -16; PB2:+16) at day 3. In case 1, the aneuploidy of chromosomes 3 and 18 in the first PB was caused by PSSC followed by the extrusion of 2 homologous chromosomes in the second PB. In case 2, chromosome 16 underwent PSSC and extrusion of a single chromatid in PB1, followed by nondisjunction in meiosis II and extrusion of two sister chromatids in PB2. In both cases, the rescued oocyte was the only euploid oocyte out of 12 retrieved and they were competent for generating a pregnancy. The two women gave birth at term to healthy babies (a girl of 3280 g and a boy of 3500 g). To our knowledge, this is the first report of healthy births after a multiple chromosomally-rescued oocyte. PGT-A of PBs is a valid method with the limitation of detecting only maternal aneuploidies and so is appropriate for screening of older women. Analysis of the 2 polar bodies by aCGH is valuable not only for aneuploidy screening in ART for older women, but also to understand the mechanisms leading to missegregation and rescue in oocytes.