Histone H2AFX Links Meiotic Chromosome Asynapsis to Prophase I Oocyte Loss in Mammals

Chromosome abnormalities are common in the human population, causing germ cell loss at meiotic prophase I and infertility. The mechanisms driving this loss are unknown, but persistent meiotic DNA damage and asynapsis may be triggers. Here we investigate the contribution of these lesions to oocyte elimination in mice with chromosome abnormalities, e.g. Turner syndrome (XO) and translocations. We show that asynapsed chromosomes trigger oocyte elimination at diplonema, which is linked to the presence of phosphorylated H2AFX (γH2AFX). We find that DNA double-strand break (DSB) foci disappear on asynapsed chromosomes during pachynema, excluding persistent DNA damage as a likely cause, and demonstrating the existence in mammalian oocytes of a repair pathway for asynapsis-associated DNA DSBs. Importantly, deletion or point mutation of H2afx restores oocyte numbers in XO females to wild type (XX) levels. Unexpectedly, we find that asynapsed supernumerary chromosomes do not elicit prophase I loss, despite being enriched for γH2AFX and other checkpoint proteins. These results suggest that oocyte loss cannot be explained simply by asynapsis checkpoint models, but is related to the gene content of asynapsed chromosomes. A similar mechanistic basis for oocyte loss may operate in humans with chromosome abnormalities.     

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.     

Smc1β is required for activation of SAC during mouse oocyte meiosis

Smc1β is a meiosis-specific cohesin subunit that is essential for sister chromatid cohesion and DNA recombination. Previous studies have shown that Smc1β-deficient mice in both sexes are sterile. Ablation of Smc1β during male meiosis leads to the blockage of spermatogenesis in pachytene stage, and ablation of Smc1β during female meiosis generates a highly error-prone oocyte although it could develop to metaphase II stage. However, the underlying mechanisms regarding how Smc1β maintains the correct meiotic progression in mouse oocytes have not been clearly defined. Here, we find that GFP-fused Smc1β is expressed and localized to the chromosomes from GV to MII stages during mouse oocyte meiotic maturation. Knockdown of Smc1β by microinjection of gene-specific morpholino causes the impaired spindle apparatus and chromosome alignment which are highly correlated with the defective kinetochore-microtubule attachments, consequently resulting in a prominently higher incidence of aneuploid eggs. In addition, the premature extrusion of polar bodies and escape of metaphase I arrest induced by low dose of nocodazole treatment in Smc1β-depleted oocytes indicates that Smc1β is essential for activation of spindle assembly checkpoint (SAC) activity. Collectively, we identify a novel function of Smc1β as a SAC participant beyond its role in chromosome cohesion during mouse oocyte meiosis.     

Unrepaired DNA damage facilitates elimination of uniparental chromosomes in interspecific hybrid cells

Elimination of uniparental chromosomes occurs frequently in interspecific hybrid cells. For example, human chromosomes are always eliminated during clone formation when human cells are fused with mouse cells. However, the underlying mechanisms are still elusive. Here, we show that the elimination of human chromosomes in human–mouse hybrid cells is accompanied by continued cell division at the presence of DNA damage on human chromosomes. Deficiency in DNA damage repair on human chromosomes occurs after cell fusion. Furthermore, increasing the level of DNA damage on human chromosomes by irradiation accelerates human chromosome loss in hybrid cells. Our results indicate that the elimination of human chromosomes in human–mouse hybrid cells results from unrepaired DNA damage on human chromosomes. We therefore provide a novel mechanism underlying chromosome instability which may facilitate the understanding of carcinogenesis.     

The long and the short of avian W chromosomes: no evidence for gradual W shortening

The well-established view of the evolution of sex chromosome dimorphism is of a gradual genetic and morphological degeneration of the hemizygous chromosome. Yet, no large-scale comparative analysis exists to support this view. Here, we analysed karyotypes of 200 bird species to test whether the supposed directional changes occur in bird sex chromosomes. We found no support for the view that W chromosomes gradually become smaller over evolutionary time. On the contrary, the length of the W chromosome can fluctuate over short time scales, probably involving both shortening and elongation of non-coding regions. Recent discoveries of near-identical palindromes and neo-sex chromosomes in birds may also contribute to the observed variation. Further studies are now needed to investigate how chromosome morphology relates to its gene content, and whether the changes in size were driven by selection.     

X染色体的DNA序列结构不同于6、7、8、10、11、12号染色体

雌性哺乳动物X染色体上的大部分基因均因X染色体失活作用而失去表达能力 ,X染色体长臂表现失活更明显。虽然对X染色体失活的许多方面都有所了解 ,但是仍然不清楚失活信号沿着X染色体全长扩散的机制。为了了解X染色体是否有不同于其他染色体的基因组学特征 ,这些特征是否关系到X染色体的失活扩散和维持 ,分析 6、7、8、1 0、1 1、1 2号染色体和X染色体DNA序列 7碱基 (7nt)组合水平的结构是否显示差异。从NCBI基因库(http :∥www .ncbi.nlm .nih .gov genome guide)下载 7条染色体长臂各 6 0Mb区域。将这 6 0Mb区域分为 0 5Mb (或 5 0kb)一段 ,对每一段DNA做 7nt字符串组合分析 ,如 1～ 7,2～ 8,3～ 9…… ,记录每种 7nt字符串的频率 ,A、C、G和T4个硷基的 7nt字符串共有 4 7=1 6 384种组合。根据数字差异显示的结果 (http :∥www .ncbi.nlm .nih .gov genome guide) ,选择在扁桃腺生发中心B细胞中高表达的基因 70个 ,用以计算所有内含子 (有义链 )的 7nt频率值。每个内含子被记录为一组 7nt频率值 ,求和相同基因中的所有内含子相同 7nt字符串的频率值 ,再用该和乘以该基因的表达频率得该基因 7nt字符串的频率值 ,求和 70个基因的 7nt字符串的频率值称做intron 7nt,该值试图模拟细胞中RNA小片段的总和。     

Frequency and distribution of aneuploidy in human female gametes

Summary During the past 6 years, 14 cytogenetic studies on human oocytes recovered during in vitro fertilization procedures have been published; they report contradictory results. The present survey has pooled the more than 1500 oocyte chromosome complements examined to date, in order to determine generalized trends in chromosomal abnormalities of female gametes. The overall frequency of abnormalities in mature oocytes is 24.0% with a large majority of aneuploidies (22.8%) over structural aberrations (1.2%), which could be explained by the difficulty in the detection of structural abnormalities in oocyte chromosome sets. An analysis of the distribution of non-disjunction among all chromosomes was also performed. In the A, C, D, and especially in the G groups, there is a significant difference between the observed non-disjunction and the frequencies expected from an equal partitioning of non-disjunction among all chromosomes. These data are discussed with reference to the differences obtained from cytogenetic studies on human sperm and from investigations on spontaneous abortion.     

Two new balancer chromosomes on mouse chromosome 4 to facilitate functional annotation of human chromosome 1p

To facilitate genetic screens to identify and maintain recessive mutations that map to the short arm of human chromosome 1, we have utilized chromosome engineering to generate two mouse strains that carry large inversions on the distal region of mouse chromosome 4. The inversion intervals are 16 and 22 cM in size together they cover approximately half of chromosome 4. Since recombination between the wild-type and inversion chromosomes does not occur within these inversion intervals, mutant alleles of genes mapping to this region can be identified and maintained. Therefore, these inversion chromosomes work as balancer chromosomes. These inversions have the additional advantage that they are tagged with genes encoding the visible coat color markers tyrosinase and agouti, and therefore the dosage of the inversion chromosome (+/+, Inv/+, Inv/Inv) can be visually recognized. These inversion strains will be extremely useful for mutagenesis screens that focus on functional annotation of human chromosome 1p.     

Comparison of spontaneous and idoxuridine-induced micronuclei by chromosome painting

Fluorescence in situ hybridisation (FISH) technique with chromosome specific library (CSL) DNA probes for all human chromosomes were used to study about 9000 micronuclei (MN) in normal and idoxuridine (IUdR)-treated lymphocyte cultures of female and male donors. In addition, MN rates and structural chromosome aberrations were scored in Giemsa-stained chromosome spreads of these cultures. IUdR treatment (40 μg/ml) induced on the average a 12-fold increase of the MN rate. Metaphase analysis revealed no distinct increase of chromosome breaks but a preferential decondensation at chromosome 9q12 (28–79%) and to a lower extend at 1q12 (8–21%). Application of FISH technique with CSL probes to one male and one female untreated proband showed that all human chromosomes except chromosome 12 (and to a striking high frequency chromosomes 9, X and Y) occurred in spontaneous MN. In cultures containing IUdR, the chromosomal spectrum found in MN was reduced to 10 chromosomes in the male and 13 in the female proband. Eight chromosomes (2, 6, 12, 13, 14, 15, 17 and 18) did not occur in MN of both probands. On the contrary chromosomes 1 and especially 9 were found much more frequently in the MN of IUdR-treated cultures than in MN of control cultures. DAPI-staining revealed heterochromatin signals in most of the IUdR-induced MN. In an additional study, spontaneous and IUdR-induced MN were investigated in lymphocytes of another female donor using CSL probes only for chromosomes 1, 6, 9, 15, 16 and X. The results confirmed the previous finding that chromosomes 1 and 9 occur very often in MN after IUdR-treatment. The results indicate that decondensation of heterochromatic regions on chromosomes 1 and 9 caused by IUdR treatment strongly correlates with MN formation by these chromosomes.     

Patterns of meiotic recombination in human fetal oocytes

Abnormal patterns of meiotic recombination (i.e., crossing-over) are believed to increase the risk of chromosome nondisjunction in human oocytes. To date, information on recombination has been obtained using indirect, genetic methods. Here we use an immunocytological approach, based on detection of foci of a DNA mismatch-repair protein, MLH1, on synaptonemal complexes at prophase I of meiosis, to provide the first direct estimate of the frequency of meiotic recombination in human oocytes. At pachytene, the stage of maximum homologous chromosome pairing, we found a mean of 70.3 foci (i.e., crossovers) per oocyte, with considerable intercell variability (range 48-102 foci). This mean equates to a genetic-map length of 3,515 cM. The numbers and positions of foci were determined for chromosomes 21, 18, 13, and X. These chromosomes yielded means of 1.23 foci (61.5 cM), 2.36 foci (118 cM), 2.5 foci (125 cM), and 3.22 foci (161 cM), respectively. The foci were almost invariably located interstitially and were only occasionally located close to chromosome ends. These data confirm the large difference, in recombination frequency, between human oocytes and spermatocytes and demonstrate a clear intersex variation in distribution of crossovers. In a few cells, chromosomes 21 and 18 did not have any foci (i.e., were presumptively noncrossover); however, configurations that lacked foci were not observed for chromosomes 13 and X. For the latter two chromosome pairs, the only instances of absence of foci were observed in abnormal cells that showed chromosome-pairing errors affecting these chromosomes. We speculate that these abnormal fetal oocytes may be the source of the nonrecombinant chromosomes 13 and X suggested, by genetic studies, to be associated with maternally derived chromosome nondisjunction.     

Should Y stay or should Y go: The evolution of non‐recombining sex chromosomes

Gradual degradation seems inevitable for non‐recombining sex chromosomes. This has been supported by the observation of degenerated non‐recombining sex chromosomes in a variety of species. The human Y chromosome has also degenerated significantly during its evolution, and theories have been advanced that the Y chromosome could disappear within the next ～5 million years, if the degeneration rate it has experienced continues. However, recent studies suggest that this is unlikely. Conservative evolutionary forces such as strong purifying selection and intrachromosomal repair through gene conversion balance the degeneration tendency of the Y chromosome and maintain its integrity after an initial period of faster degeneration. We discuss the evidence both for and against the extinction of the Y chromosome. We also discuss potential insights gained on the evolution of sex‐determining chromosomes by studying simpler sex‐determining chromosomal regions of unicellular and multicellular microorganisms.     

Mammalian chromosomes contain cis-acting elements that control replication timing, mitotic condensation, and stability of entire chromosomes

Recent studies indicate that mammalian chromosomes contain discrete cis-acting loci that control replication timing, mitotic condensation, and stability of entire chromosomes. Disruption of the large non-coding RNA gene ASAR6 results in late replication, an under-condensed appearance during mitosis, and structural instability of human chromosome 6. Similarly, disruption of the mouse Xist gene in adult somatic cells results in a late replication and instability phenotype on the X chromosome. ASAR6 shares many characteristics with Xist, including random mono-allelic expression and asynchronous replication timing. Additional "chromosome engineering" studies indicate that certain chromosome rearrangements affecting many different chromosomes display this abnormal replication and instability phenotype. These observations suggest that all mammalian chromosomes contain "inactivation/stability centers" that control proper replication, condensation, and stability of individual chromosomes. Therefore, mammalian chromosomes contain four types of cis-acting elements, origins, telomeres, centromeres, and "inactivation/stability centers", all functioning to ensure proper replication, condensation, segregation, and stability of individual chromosomes.     

Meiotic sex chromosome inactivation in the marsupial Monodelphis domestica

In eutherian mammals, the X and Y chromosomes undergo meiotic sex chromosome inactivation (MSCI) during spermatogenesis in males. However, following fertilization, both the paternally (Xp) and maternally (Xm) inherited X chromosomes are active in the inner cell mass of the female blastocyst, and then random inactivation of one X chromosome occurs in each cell, leading to a mosaic pattern of X-chromosome activity in adult female tissues. In contrast, marsupial females show a nonrandom pattern of X chromosome activity, with repression of the Xp in all somatic tissues. Here, we show that MSCI also occurs during spermatogenesis in marsupials in a manner similar to, but more stable than that in eutherians. These findings support the suggestion that MSCI may have provided the basis for an early dosage compensation mechanism in mammals based solely on gametogenic events, and that random X-chromosome inactivation during embryogenesis may have evolved subsequently in eutherian mammals.     

A Scalable Computational Approach for Simulating Complexes of Multiple Chromosomes

Significant efforts have been recently made to obtain the three-dimensional (3D) structure of the genome with the goal of understanding how structures may affect gene regulation and expression. Chromosome conformational capture techniques such as Hi-C, have been key in uncovering the quantitative information needed to determine chromatin organization. Complementing these experimental tools, co-polymers theoretical methods are necessary to determine the ensemble of three-dimensional structures associated to the experimental data provided by Hi-C maps. Going beyond just structural information, these theoretical advances also start to provide an understanding of the underlying mechanisms governing genome assembly and function. Recent theoretical work, however, has been focused on single chromosome structures, missing the fact that, in the full nucleus, interactions between chromosomes play a central role in their organization. To overcome this limitation, MiChroM (Minimal Chromatin Model) has been modified to become capable of performing these multi-chromosome simulations. It has been upgraded into a fast and scalable software version, which is able to perform chromosome simulations using GPUs via OpenMM Python API, called Open-MiChroM. To validate the efficiency of this new version, analyses for GM12878 individual autosomes were performed and compared to earlier studies. This validation was followed by multi-chain simulations including the four largest human chromosomes (C1-C4). These simulations demonstrated the full power of this new approach. Comparison to Hi-C data shows that these multiple chromosome interactions are essential for a more accurate agreement with experimental results. Without any changes to the original MiChroM potential, it is now possible to predict experimentally observed inter-chromosome contacts. This scalability of Open-MiChroM allow for more audacious investigations, looking at interactions of multiple chains as well as moving towards higher resolution chromosomes models.     

The human cleavage stage embryo is a cradle of chromosomal rearrangements

The first cell cycles following in vitro fertilization (IVF) of human gametes are prone to chromosome instability. Many, but often not all, blastomeres of an embryo acquire a genetic makeup during cleavage that is not representative of the original zygotic genome. Whole chromosomes are missegregated, but also structural rearrangements of chromosomes do occur in human cleavage stage embryogenesis following IVF. Analysis of pre- and postnatal DNA samples indicates that the in vivo human conceptions also endure instability of chromosome number and structure during cleavage of the fertilized oocyte. This embryonic chromosome instability not necessarily undermines normal human development, but may lead to a spectrum of conditions, including loss of conception, genetic disease and genetic variation development. In this review, the structural instability of chromosomes during human cleavage stage embryogenesis is catalogued, channeled into etiologic models and linked to genomic profiles of healthy and diseased newborns.     

Chromosome painting in Arabidopsis thaliana

Chromosome painting, that is visualisation of chromosome segments or whole chromosomes based on fluorescence in situ hybridization (FISH) with chromosome-specific DNA probes is widely used for chromosome studies in mammals, birds, reptiles and insects. Attempts to establish chromosome painting in euploid plants have failed so far. Here, we report on chromosome painting in Arabidopsis thaliana (n = 5, 125 Mb C(-1)). Pools of contiguous 113-139 BAC clones spanning 2.6 and 13.3 Mb of the short and the long arm of chromosome 4 (17.5 Mb) were used to paint this entire chromosome during mitotic and meiotic divisions as well as in interphase nuclei. The possibility of identifying any particular chromosome region on pachytene chromosomes and within interphase nuclei using selected BACs is demonstrated by differential labelling. This approach allows us, for the first time, to paint an entire autosome of an euploid plant to study chromosome rearrangements, homologue association, interphase chromosome territories, as well as to identify homeologous chromosomes of related species.     

Meiotic drive and sex chromosome cycling

Sex-linked meiotic drive is found in a broad variety of taxa, including insects, birds, and mammals. In populations of some species, we see four types of sex chromosomes segregating: normal and driving X chromosomes and susceptible and resistant Y chromosomes. A theoretical analysis shows that a stable four-chromosome equilibria is a more common outcome in these systems than previously recognized. Cycling of sex chromosome frequencies and associated changes in the sex ratio are other predicted outcomes. The absence of cycling in nature may be due to migration among populations.     

Extraordinarily conserved chromosomal synteny of Citrus species revealed by chromosome‐specific painting

Reliable identification of individual chromosomes in eukaryotic species is the foundation for comparative chromosome synteny and evolutionary studies. Unfortunately, chromosome identification has been a major challenge for plants with small chromosomes, such as the Citrus species. We developed oligonucleotide‐based chromosome painting probes for all nine chromosomes in Citrus maxima (Pummelo). We were able to identify all C. maxima chromosomes in the same metaphase cells using multiple rounds of sequential fluorescence in situ hybridization with the painting probes. We conducted comparative chromosome painting analysis in six different Citrus and related species. We found that each painting probe hybridized to only a single chromosome in all other five species, suggesting that the six species have maintained a complete chromosomal synteny after more than 9 million years of divergence. No interchromosomal rearrangement was identified in any species. These results support the hypothesis that karyotypes of woody species are more stable than herbaceous plants because woody plants need a longer period to fix chromosome structural variants in natural populations.     

Chromosome arrangement and nuclear architecture but not centromeric sequences are conserved between Arabidopsis thaliana and Arabidopsis lyrata

In contrast to the situation described for mammals and Drosophila, chromosome territory (CT) arrangement and somatic homologous pairing in interphase nuclei of Arabidopsis thaliana (n = 5) are predominantly random except for a more frequent association of the chromosomes bearing a homologous nucleolus organizer region. To find out whether this chromosome arrangement is also characteristic for other species of the genus Arabidopsis, we investigated Arabidopsis lyrata ssp. lyrata (n = 8), one of the closest relatives of A. thaliana. First, we determined the size of each chromosome and chromosome arm, the sequence type of centromeric repeats and their distribution between individual centromeres and the position of the 5S/45S rDNA arrays in A. lyrata. Then we demonstrated that CT arrangement, homologous pairing and sister chromatid alignment of distinct euchromatic and/or heterochromatic regions within A. lyrata interphase nuclei are similar to that in A. thaliana nuclei. Thus, the arrangement of interphase chromosomes appears to be conserved between both taxa that diverged about 5 million years ago. Since the chromosomes of A. lyrata resemble those of the presumed ancestral karyotype, a similar arrangement of interphase chromosomes is also to be expected for other closely related diploid species of the Brassicaceae family.