Chromosome Segregation Errors Research Articles

An anaphase surveillance mechanism prevents micronuclei formation from frequent chromosome segregation errors.

SummaryMicronuclei are a hallmark of cancer and several other human disorders. Recently, micronuclei were implicated in chromothripsis, a series of massive genomic rearrangements that may drive tumor evolution and progression. Here, we show that Aurora B kinase mediates a surveillance mechanism that integrates error correction during anaphase with spatial control of nuclear envelope reassembly to prevent micronuclei formation. Using high-resolution live-cell imaging of human cancer and non-cancer cells, we uncover that anaphase lagging chromosomes are more frequent than previously anticipated, yet they rarely form micronuclei. Micronuclei formation from anaphase lagging chromosomes is prevented by a midzone-based Aurora B phosphorylation gradient that stabilizes kinetochore-microtubule attachments and assists spindle forces required for anaphase error correction while delaying nuclear envelope reassembly on lagging chromosomes, independently of microtubule density. We propose that a midzone-based Aurora B phosphorylation gradient actively monitors and corrects frequent chromosome segregation errors to prevent micronuclei formation during human cell division.

Age-dependent integrity of the meiotic spindle assembly checkpoint in females requires Aurora kinase B.

A hallmark of advanced maternal age is a significant increase in meiotic chromosome segregation errors, resulting in early miscarriages and congenital disorders. These errors most frequently occur during meiosis I (MI). The spindle assembly checkpoint (SAC) prevents chromosome segregation errors by arresting the cell cycle until proper chromosome alignment is achieved. Unlike in mitosis, the SAC in oocytes is desensitized, allowing chromosome segregation in the presence of improperly aligned chromosomes. Whether SAC integrity further deteriorates with advancing maternal age, and if this decline contributes to increased segregation errors remains a fundamental question. In somatic cells, activation of the SAC depends upon Aurora kinase B (AURKB), which functions to monitor kinetochore–microtubule attachments and recruit SAC regulator proteins. In mice, oocyte‐specific deletion of AURKB (Aurkb cKO) results in an increased production of aneuploid metaphase II‐arrested eggs and premature age‐related infertility. Here, we aimed to understand the cause of the short reproductive lifespan and hypothesized that SAC integrity was compromised. In comparing oocytes from young and sexually mature Aurkb cKO females, we found that SAC integrity becomes compromised rapidly with maternal age. We show that the increased desensitization of the SAC is driven by reduced expression of MAD2, ZW10 and Securin proteins, key contributors to the SAC response pathway. The reduced expression of these proteins is the result of altered protein homeostasis, likely caused by the accumulation of reactive oxygen species. Taken together, our results demonstrate a novel function for AURKB in preserving the female reproductive lifespan possibly by protecting oocytes from oxidative stress.

Age-cumulative Effect of REC8 Reduction on Meiotic Chromosome Segregation Errors in Mice

Abstract Objective: This study aimed to explore the relationship between cohesin subunit REC8 reduction and meiosis chromosome segregation errors in the ovary. Methods: Rec8 +/− mice were generated using CRIPSR/Cas9 gene editing. The association between age and REC8 expression levels in the ovary was determined by Western blotting. Chromosome segregation errors were investigated by immunofluorescence imaging of superovulated oocytes. Wild-type and Rec8 +/− female mice at 5, 8, 20, 36, and 40 weeks were used to evaluate ovarian reserve by ovarian clearing and immunolabeling. Results: Ovary REC8 expression levels gradually decreased with age, while chromosome segregation errors increased with age. Segregation errors were more common in Rec8 +/− mice, suggesting an association with REC8 expression. The ovarian reserve capacity decreased significantly with age. The ovarian reserve in Rec8 +/− mice was inferior to that of age-matched wild-type mice, indicating important roles of age and REC8 levels in the ovarian reserve. Conclusions: REC8 reduction has an age-cumulative effect on meiotic chromosome segregation errors in mouse ovaries. Rec8 haploinsufficiency poses a major challenge in generating normal and reproductive oocytes in aging mice.

From contemplation to classification of chromosomal mosaicism in human preimplantation embryos.

Chromosomal mosaicism is a hallmark of early human embryo development. The last decade yielded an enormous amount of information about diversity and prevalence of mosaicism in preimplantation embryos due to progress in preimplantation genetic testing of aneuploidies (PGT-A) based exclusively on molecular karyotyping of trophectoderm biopsy. However, the inner cell mass karyotype is still missing for mosaic embryos affecting the success rate of assisted reproductive medicine. Here, a classification model of chromosomal mosaicism is proposed based on the analysis of the primary zygote karyotype, the timing and types of primary and secondary chromosome segregation errors, and the distribution of mosaic cell clones between different embryonic and extraembryonic compartments of the blastocyst. Five basic principles for mosaicism analysis are introduced, namely, the estimation of the primary zygote karyotype, the investigation of additional sample point, the requirement of the second time point analysis, the delineating of reciprocity of chromosome segregation, and comprehensive chromosome screening at the single-cell level. The suggested model allows the prediction of the inner cell mass karyotype of the blastocyst and its developmental potential based on information from trophectoderm biopsy and non-invasive PGT-A using blastocoele fluid sample or spent culture medium as additional sample and time points for analysis and considering the reciprocity as a basic process in chromosome segregation errors between daughter cells in postzygotic cell divisions.

Loss of polycomb repressive complex 1 activity and chromosomal instability drive uveal melanoma progression

Chromosomal instability (CIN) and epigenetic alterations have been implicated in tumor progression and metastasis; yet how these two hallmarks of cancer are related remains poorly understood. By integrating genetic, epigenetic, and functional analyses at the single cell level, we show that progression of uveal melanoma (UM), the most common intraocular primary cancer in adults, is driven by loss of Polycomb Repressive Complex 1 (PRC1) in a subpopulation of tumor cells. This leads to transcriptional de-repression of PRC1-target genes and mitotic chromosome segregation errors. Ensuing CIN leads to the formation of rupture-prone micronuclei, exposing genomic double-stranded DNA (dsDNA) to the cytosol. This provokes tumor cell-intrinsic inflammatory signaling, mediated by aberrant activation of the cGAS-STING pathway. PRC1 inhibition promotes nuclear enlargement, induces a transcriptional response that is associated with significantly worse patient survival and clinical outcomes, and enhances migration that is rescued upon pharmacologic inhibition of CIN or STING. Thus, deregulation of PRC1 can promote tumor progression by inducing CIN and represents an opportunity for early therapeutic intervention.

Aluminum Enters Mammalian Cells and Destabilizes Chromosome Structure and Number.

Chromosome instability (CIN) consists of high rates of structural and numerical chromosome abnormalities and is a well-known hallmark of cancer. Aluminum is added to many industrial products of frequent use. Yet, it has no known physiological role and is a suspected human carcinogen. Here, we show that V79 cells, a well-established model for the evaluation of candidate chemical carcinogens in regulatory toxicology, when cultured in presence of aluminum—in the form of aluminum chloride (AlCl3) and at concentrations in the range of those measured in human tissues—incorporate the metal in a dose-dependent manner, predominantly accumulating it in the perinuclear region. Intracellular aluminum accumulation rapidly leads to a dose-dependent increase in DNA double strand breaks (DSB), in chromosome numerical abnormalities (aneuploidy) and to proliferation arrest in the G2/M phase of the cell cycle. During mitosis, V79 cells exposed to aluminum assemble abnormal multipolar mitotic spindles and appear to cluster supernumerary centrosomes, possibly explaining why they accumulate chromosome segregation errors and damage. We postulate that chronic aluminum absorption favors CIN in mammalian cells, thus promoting carcinogenesis.

Recent insights into mechanisms preventing ectopic centromere formation.

The centromere is a specialized chromosomal structure essential for chromosome segregation. Centromere dysfunction leads to chromosome segregation errors and genome instability. In most eukaryotes, centromere identity is specified epigenetically by CENP-A, a centromere-specific histone H3 variant. CENP-A replaces histone H3 in centromeres, and nucleates the assembly of the kinetochore complex. Mislocalization of CENP-A to non-centromeric regions causes ectopic assembly of CENP-A chromatin, which has a devastating impact on chromosome segregation and has been linked to a variety of human cancers. How non-centromeric regions are protected from CENP-A misincorporation in normal cells is largely unexplored. Here, we review the most recent advances on the mechanisms underlying the prevention of ectopic centromere formation, and discuss the implications in human disease.

Impact of Global Transcriptional Silencing on Cell Cycle Regulation and Chromosome Segregation in Early Mammalian Embryos.

The onset of an early development is, in mammals, characterized by profound changes of multiple aspects of cellular morphology and behavior. These are including, but not limited to, fertilization and the merging of parental genomes with a subsequent transition from the meiotic into the mitotic cycle, followed by global changes of chromatin epigenetic modifications, a gradual decrease in cell size and the initiation of gene expression from the newly formed embryonic genome. Some of these important, and sometimes also dramatic, changes are executed within the period during which the gene transcription is globally silenced or not progressed, and the regulation of most cellular activities, including those mentioned above, relies on controlled translation. It is known that the blastomeres within an early embryo are prone to chromosome segregation errors, which might, when affecting a significant proportion of a cell within the embryo, compromise its further development. In this review, we discuss how the absence of transcription affects the transition from the oocyte to the embryo and what impact global transcriptional silencing might have on the basic cell cycle and chromosome segregation controlling mechanisms.

O-004 Self-correction in human preimplantation development: What do we know?

Abstract text Recent advances in preimplantation genetic testing for aneuploidy (PGT-A) and time-lapse imaging have improved our understanding of the early human embryo confirming the variable patterns of development and chromosomal status. Aneuploidy is common and increased sensitivity in PGT-A allows the non-binary reporting of euploid-aneuploid mosaicism. The PGT-A result is the inference of the biopsied embryo’s ploidy status at a point in time, by assessment of a small percentage of cells, and, whilst concordance with the rest of the embryo is high; it is not absolute. Many reports have demonstrated that, with the transfer of embryos with increasing severity and complexity of mosaicism, comes compromised implantation, reduced ongoing pregnancy rates and increased miscarriage rates. Segmental mosaic embryos have been reported to have slightly reduced implantation potential compared with euploid counterparts. However, complex mosaic embryos are widely reported to result in severely reduced implantation success, if transferred. Outside of PGT-A treatment cycles, undoubtedly fertility clinics are unwittingly transferring mosaic and aneuploid embryos daily, with variable success. The transfer of embryos in which mosaicism has been detected, although associated with lower implantation and higher miscarriage rates than euploid embryos, can lead to normal pregnancies and healthy births. We know that the placenta can harbour chromosomal aberrations which are absent from the fetus, and there are few reports of births with demonstrably high levels of mosaicism through fetal development. This raises the question as to whether correction mechanisms exist. In other words, do conceptuses become chromosomally more normal as development progresses, and what are the mechanisms, if so? PGT-A, time lapse, novel live cell imaging and in vitro model techniques have enabled a more detailed study of early embryo development and consideration of the phenomenon of self-correction. This has provided insights and hypotheses surrounding the mechanisms of development and of self-correction. The relatively lower levels of chromosome abnormality in the blastocyst, compared with cleavage stage, are well documented and indicative of some form of correction. A recent investigation reported that a large proportion of embryos initially diagnosed as mosaic were later diagnosed as euploid when assessed at day 12 of development; providing evidence of the depletion of abnormal cells throughout the early post-implantation stages. There are many time-lapse reports of anomalous ‘direct’ or multichotomous blastomere divisions being associated with aneuploidy, and leading to developmental arrest or reduced implantation potential and of temporal delays in aneuploid embryos compared with their euploid counterparts. It is possible, therefore, that errors and even attempts to repair them, in individual cells in the rapidly developing embryo; which involve complex biochemical systems, could delay karyo- and cytokinesis, resulting in these detectable delays. The embryonic mortality model suggests that there is selection against embryos based on their degree of aneuploidy, such that aneuploid cell lines are lost during implantation. We know that irregularities in blastomere cleavage can generate chromosome segregation errors but these may sporadically be confined to cells excluded, or extruded, from the morula or from the blastocyst; a possible exhibition of the clonal depletion or embryo mortality model. The trisomic/monosomic rescue model suggests that aneuploid cells can give rise to diploid cells (and possibly uniparental disomy) through mitotic chromosome losses or gains. We know that an abnormal number of pronuclei does not always produce an aneuploid blastocyst and that early embryos exhibiting multinucleation can result in healthy live births. Finally, the preferential allocation of aneuploid cells to the trophectoderm model is based on the hypothesis that euploid cells are preferentially retained in the ICM in order to achieve viability. This presentation aims to consider what we know, and discuss the theories and available evidence for self-correction.

Distinct classes of lagging chromosome underpin age-related oocyte aneuploidy in mouse.

Chromosome segregation errors that cause oocyte aneuploidy increase in frequency with maternal age and are considered a major contributing factor of age-related fertility decline in females. Lagging anaphase chromosomes are a common age-associated phenomenon in oocytes, but whether anaphase laggards actually missegregate and cause aneuploidy is unclear. Here, we show that lagging chromosomes in mouse oocytes comprise two mechanistically distinct classes of chromosome motion that we refer to as "class-I" and "class-II" laggards. We use imaging approaches and mechanistic interventions to dissociate the two classes and find that whereas class-II laggards are largely benign, class-I laggards frequently directly lead to aneuploidy. Most notably, a controlled prolongation of meiosis I specifically lessens class-I lagging to prevent aneuploidy. Our data thus reveal lagging chromosomes to be a cause of age-related aneuploidy in mouse oocytes and suggest that manipulating the cell cycle could increase the yield of useful oocytes in some contexts.

Single-oocyte transcriptome analysis reveals aging-associated effects influenced by life stage and calorie restriction.

Chromosome segregation errors in oocytes lead to the production of aneuploid eggs, which are the leading cause of pregnancy loss and of several congenital diseases such as Down syndrome. The frequency of chromosome segregation errors in oocytes increases with maternal age, especially at a late stage of reproductive life. How aging at various life stages affects oocytes differently remains poorly understood. In this study, we describe aging‐associated changes in the transcriptome profile of mouse oocytes throughout reproductive life. Our single‐oocyte comprehensive RNA sequencing using RamDA‐seq revealed that oocytes undergo transcriptome changes at a late reproductive stage, whereas their surrounding cumulus cells exhibit transcriptome changes at an earlier stage. Calorie restriction, a paradigm that reportedly prevents aging‐associated egg aneuploidy, promotes a transcriptome shift in oocytes with the up‐regulation of genes involved in chromosome segregation. This shift is accompanied by the improved maintenance of chromosomal cohesin, the loss of which is a hallmark of oocyte aging and causes chromosome segregation errors. These findings have implications for understanding how oocytes undergo aging‐associated functional decline throughout their reproductive life in a context‐dependent manner.

The RAS GTPase RIT1 compromises mitotic fidelity through spindle assembly checkpoint suppression

The spindle assembly checkpoint (SAC) functions as a sensor of unattached kinetochores that delays mitotic progression into anaphase until proper chromosome segregation is guaranteed.1,2 Disruptions to this safety mechanism lead to genomic instability and aneuploidy, which serve as the genetic cause of embryonic demise, congenital birth defects, intellectual disability, and cancer.3,4 However, despite the understanding of the fundamental mechanisms that control the SAC, it remains unknown how signaling pathways directly interact with and regulate the mitotic checkpoint activity. In response to extracellular stimuli, a diverse network of signaling pathways involved in cell growth, survival, and differentiation are activated, and this process is prominently regulated by the Ras family of small guanosine triphosphatases (GTPases).5 Here we show that RIT1, a Ras-related GTPase that regulates cell survival and stress response,6 is essential for timely progression through mitosis and proper chromosome segregation. RIT1 dissociates from the plasma membrane (PM) during mitosis and interacts directly with SAC proteins MAD2 and p31comet in a process that is regulated by cyclin-dependent kinase 1 (CDK1) activity. Furthermore, pathogenic levels of RIT1 silence the SAC and accelerate transit through mitosis by sequestering MAD2 from the mitotic checkpoint complex (MCC). Moreover, SAC suppression by pathogenic RIT1 promotes chromosome segregation errors and aneuploidy. Our results highlight a unique function of RIT1 compared to other Ras GTPases and elucidate a direct link between a signaling pathway and the SAC through a novel regulatory mechanism.

Mapping Proximity Associations of Core Spindle Assembly Checkpoint Proteins.

The spindle assembly checkpoint (SAC) is critical for sensing defective microtubule–kinetochore attachments and tension across the kinetochore and functions to arrest cells in prometaphase to allow time to repair any errors before proceeding into anaphase. Dysregulation of the SAC leads to chromosome segregation errors that have been linked to human diseases like cancer. Although much has been learned about the composition of the SAC and the factors that regulate its activity, the proximity associations of core SAC components have not been explored in a systematic manner. Here, we have taken a BioID2-proximity-labeling proteomic approach to define the proximity protein environment for each of the five core SAC proteins BUB1, BUB3, BUBR1, MAD1L1, and MAD2L1 in mitotic-enriched populations of cells where the SAC is active. These five protein association maps were integrated to generate a SAC proximity protein network that contains multiple layers of information related to core SAC protein complexes, protein–protein interactions, and proximity associations. Our analysis validated many known SAC complexes and protein–protein interactions. Additionally, it uncovered new protein associations, including the ELYS–MAD1L1 interaction that we have validated, which lend insight into the functioning of core SAC proteins and highlight future areas of investigation to better understand the SAC.

Chromosomal instability by mutations in the novel minor spliceosome component CENATAC

Aneuploidy is the leading cause of miscarriage and congenital birth defects, and a hallmark of cancer. Despite this strong association with human disease, the genetic causes of aneuploidy remain largely unknown. Through exome sequencing of patients with constitutional mosaic aneuploidy, we identified biallelic truncating mutations in CENATAC (CCDC84). We show that CENATAC is a novel component of the minor (U12‐dependent) spliceosome that promotes splicing of a specific, rare minor intron subtype. This subtype is characterized by AT‐AN splice sites and relatively high basal levels of intron retention. CENATAC depletion or expression of disease mutants resulted in excessive retention of AT‐AN minor introns in ˜ 100 genes enriched for nucleocytoplasmic transport and cell cycle regulators, and caused chromosome segregation errors. Our findings reveal selectivity in minor intron splicing and suggest a link between minor spliceosome defects and constitutional aneuploidy in humans.

The comprehensive variant and phenotypic spectrum of TUBB8 in female infertility.

TUBB8 is a gene that is frequently analysed in the genetic diagnosis of female infertility; 102 variants of this gene have been identified. However, the evaluation of its pathogenicity and the resulting phenotypes vary. Here, we aimed to identify novel TUBB8 variants as well as to summarize the reported variants and phenotypes in order for them to be included in genetic counselling analyses. We performed whole exome sequencing to screen for candidate variants in 100 infertile female subjects and 100 controls who were able to conceive naturally. All variants were confirmed by Sanger sequencing. The effects of the variants in oocytes/arrested embryos were assessed by morphological observations, polar body biopsies, and chromosome analysis. A molecular modelling analysis was used to evaluate the possible effects of variants on protein secondary structure. We identified 29 TUBB8 variants, of which 20 were novel and five were maternally inherited. We identified three of a total of six recurrent variants that were specific for complete cleavage failure. Moreover, we obtained evidence that TUBB8 variants with large polar bodies had chromosome segregation errors. Our study expands the spectrum of TUBB8 variants, particularly for embryonic arrest. Together with the extant knowledge of TUBB8 variants, this study provides a foundation for the genetic counselling of female infertility.

Maternal germline factors associated with aneuploid pregnancy loss: a systematic review.

Miscarriage describes the spontaneous loss of pregnancy before the threshold of viability; the vast majority occur before 12 weeks of gestation. Miscarriage affects one in four couples and is the most common complication of pregnancy. Chromosomal abnormalities of the embryo are identified in ∼50% of first trimester miscarriages; aneuploidy accounts for 86% of these cases. The majority of trisomic miscarriages are of maternal origin with errors occurring during meiotic division of the oocytes. Chromosome segregation errors in oocytes may be sporadic events secondary to advancing maternal age; however, there is increasing evidence to suggest possible maternal germline contributions to this. The objective of this review was to appraise critically the existing evidence relating to maternal germline factors associated with pregnancy loss secondary to embryo aneuploidy, identify limitations in the current evidence base and establish areas requiring further research. The initial literature search was performed in September 2019 and updated in January 2021 using the electronic databases OVID MEDLINE, EMBASE and the Cochrane Library. No time or language restrictions were applied to the searches and only primary research was included. Participants were women who had suffered pregnancy loss secondary to numerical chromosomal abnormalities of the embryo. Study identification and subsequent data extraction were performed by two authors independently. The Newcastle-Ottawa Scale was used to judge the quality of the included studies. The results were synthesized narratively. The literature search identified 2198 titles once duplicates were removed, of which 21 were eligible for inclusion in this systematic review. They reported on maternal germline factors having variable degrees of association with pregnancy loss of aneuploid origin. The Online Mendelian Inheritance in Man (OMIM) gene ontology database was used as a reference to establish the functional role currently attributed to the genes reported. The majority of the cases reported and included were secondary to the inheritance of maternal structural factors such as Robertsonian translocations, deletions and insertions. Germline factors with a plausible role in aneuploid pregnancy loss of maternal origin included skewed X-inactivation and CGG repeats in the fragile X mental retardation (FMR1) gene. Studies that reported the association of single gene mutations with aneuploid pregnancy loss were conflicting. Single gene mutations with an uncertain or no role in aneuploid pregnancy loss included mutations in synaptonemal complex protein 3 (SYCP3), mitotic polo-like kinase 4 (PLK4) and meiotic stromal antigen 3 (STAG3) spindle integrity variants and 5,10-methylenetetrahydrofolate reductase (MTHFR). Identifying maternal genetic factors associated with an increased risk of aneuploidy will expand our understanding of cell division, non-disjunction and miscarriage secondary to embryo aneuploidy. The candidate germline factors identified may be incorporated in a screening panel for women suffering miscarriage of aneuploidy aetiology to facilitate counselling for subsequent pregnancies.

Compromised MPS1 Activity Induces Multipolar Spindle Formation in Oocytes From Aged Mares: Establishing the Horse as a Natural Animal Model to Study Age-Induced Oocyte Meiotic Spindle Instability.

Aneuploidy originating during meiosis in oocytes is the major cause of reduced fertility, implantation failure and miscarriage in women beyond their mid-thirties. Loss of chromosome cohesion, and defective microtubule dynamics and spindle assembly are, in turn, the major contributors to the error-prone nature of chromosome segregation in the oocytes of older women. However, the underlying molecular defects are not well understood. Altered function of MPS1 and AURKC have been shown to induce multipolar spindle phenotypes in murine oocytes and cancer cells, however, their role in reproductive aging associated oocyte aneuploidy is not known. Although age-related gamete and embryonic aneuploidy has been studied in female rodents, the horse may be a more appropriate animal model. Similar to women, aged mares suffer from reduced fertility and an increased incidence of oocyte aneuploidy. Moreover, mares show a long interval (decades) to reproductive senescence and, unlike rodents but similar to women, horse oocytes assemble the meiotic spindle in a slow and unstable manner, independent of microtubule organizing centers. In this study we found that oocytes from aged mares have lower expression of mRNA for Mps1, Spc25 and AurkC than oocytes from young mares while gene expression for other meiosis regulators did not differ. To assess the ability of horse oocytes to correctly form a bipolar spindle, in vitro matured MII oocytes were allowed to re-form their spindle after nocodazole-induced microtubule depolymerization. To investigate the importance of MPS1 and AURKC function in spindle (re)assembly, various concentrations of a MPS1 inhibitor (MPS1i, Compound 5) or an AURK inhibitor (AURKi, ZM447439) were included after nocodazole washout. MII oocytes from aged mares showed a higher incidence of spindle abnormalities after exposure to MPS1i. In contrast, Aurora kinase inhibition severely impaired microtubule organization and spindle formation in all oocytes, irrespective of mare age. In conclusion, gene expression for the kinases Mps1, Spc25, and AurkC is reduced in oocytes from aged mares. Moreover, spindle (re)assembly in aged mares’ oocytes is more unstable when Mps1 is inhibited. Overall, this suggests that compromised Mps1 activity predisposes to meiotic spindle instability in aged mare oocytes. This spindle instability could predispose to chromosome segregation errors.

Parental genome unification is highly error-prone in mammalian embryos

SummaryMost human embryos are aneuploid. Aneuploidy frequently arises during the early mitotic divisions of the embryo, but its origin remains elusive. Human zygotes that cluster their nucleoli at the pronuclear interface are thought to be more likely to develop into healthy euploid embryos. Here, we show that the parental genomes cluster with nucleoli in each pronucleus within human and bovine zygotes, and clustering is required for the reliable unification of the parental genomes after fertilization. During migration of intact pronuclei, the parental genomes polarize toward each other in a process driven by centrosomes, dynein, microtubules, and nuclear pore complexes. The maternal and paternal chromosomes eventually cluster at the pronuclear interface, in direct proximity to each other, yet separated. Parental genome clustering ensures the rapid unification of the parental genomes on nuclear envelope breakdown. However, clustering often fails, leading to chromosome segregation errors and micronuclei, incompatible with healthy embryo development.

Dysregulated Gene Expression of Imprinted and X-Linked Genes: A Link to Poor Development of Bovine Haploid Androgenetic Embryos.

Mammalian uniparental embryos are efficient models for genome imprinting research and allow studies on the contribution of the paternal and maternal genomes to early embryonic development. In this study, we analyzed different methods for production of bovine haploid androgenetic embryos (hAE) to elucidate the causes behind their poor developmental potential. Results indicate that hAE can be efficiently generated by using intracytoplasmic sperm injection and oocyte enucleation at telophase II. Although androgenetic haploidy does not disturb early development up to around the 8-cell stage, androgenetic development is disturbed after the time of zygote genome activation and hAE that reach the morula stage are less capable to reach the blastocyst stage of development. Karyotypic comparisons to parthenogenetic- and ICSI-derived embryos excluded chromosomal segregation errors as causes of the developmental constraints of hAE. However, analysis of gene expression indicated abnormal levels of transcripts for key long non-coding RNAs involved in X chromosome inactivation and genomic imprinting of the KCNQ1 locus, suggesting an association with X chromosome and some imprinted loci. Moreover, transcript levels of methyltransferase 3B were significantly downregulated, suggesting potential anomalies in hAE establishing de novo methylation. Finally, the methylation status of imprinted control regions for XIST and KCNQ1OT1 genes remained hypomethylated in hAE at the morula and blastocyst stages, confirming their origin from spermatozoa. Thus, our results exclude micromanipulation and chromosomal abnormalities as major factors disturbing the normal development of bovine haploid androgenotes. In addition, although the cause of the arrest remains unclear, we have shown that the inefficient development of haploid androgenetic bovine embryos to develop to the blastocyst stage is associated with abnormal expression of key factors involved in X chromosome activity and genomic imprinting.

Uniparental disomy: Origin, frequency, and clinical significance.

Uniparental disomy (UPD) is defined as two copies of a whole chromosome derived from the same parent. There can be multiple mechanisms that lead to UPD; these are reviewed in the context of contemporary views on the mechanism leading to aneuploidy. Recent studies indicate that UPD is rare in an apparently healthy population and also rare in spontaneous abortion tissues. The most common type of UPD is a maternal heterodisomy (both maternal allele sets present). Isodisomy (a duplicated single set of alleles) or segmental loss of heterozygosity is sometimes encountered in SNP-based microarray referrals. Decisions regarding the most appropriate follow-up testing should consider the possibility of consanguinity (that will generally involve multiple regions), an imprinted gene disorder (chromosomes 6, 7, 11, 14, 15, 20), expression of an autosomal recessive disorder, and an occult aneuploid cell line that may be confined to the placenta. Upd(16)mat, per se, does not appear to be associated with an abnormal phenotype. UPD provides an insight into the history of early chromosome segregation error and understanding the rates and fate of these events are of key importance in the provision of fertility management and prenatal healthcare.