Micromechanical studies of mitotic chromosomes

Mitotic chromosomes respond elastically to forces in the nanonewton range, a property important to transduction of stresses used as mechanical regulatory signals during cell division. In addition to being important biologically, chromosome elasticity can be used as a tool for investigating the folding of chromatin. This paper reviews experiments studying stretching and bending stiffness of mitotic chromosomes, plus experiments where changes in chromosome elasticity resulting from chemical and enzyme treatments were used to analyse connectivity of chromatin inside chromosomes. Experiments with nucleases indicate that non-DNA elements constraining mitotic chromatin must be isolated from one another, leading to the conclusion that mitotic chromosomes have a chromatin ‘network’ or ‘gel’ organization, with stretches of chromatin strung between ‘crosslinking’ points. The as-yet unresolved questions of the identities of the putative chromatin crosslinkers and their organization inside mitotic chromosomes are discussed.     

The topology of early- and late-replicating chromatin in differentially decondensed chromosomes

In this study we used a novel technique to reveal both longitudinal and transverse differentiation within mammalian mitotic chromosomes. Structural changes in chromosomes that we term ‘differential decondensation’ were produced in cells that were first incubated in hypotonic medium (15% Hanks’ solution), then adapted to normotonic conditions and thereafter exposed to a second short hypotonic shock. Such a double hypotonic treatment (DHT) is not critical for cell viability, but considerably elongates the G2 phase of the cell cycle. Giemsa staining of differentially decondensed chromosomes corresponds to standard G-banding, but does not need the standard post-fixation treatment. Using ‘dynamic’ BrdU banding, we show that such ‘differential’ staining is a result of differential resistance of the R- and G-bands to DHT. Thus, early-replicating foci, markers of R-bands, are localized in the peripheral chromatin halo, whereas late-replicating foci, corresponding to G-bands, remain associated with the axial regions of chromatids. Remarkably, despite these major changes in the structure of the chromosomal bands, the replication foci still preserve their discrete structure.     

Meiotic chromosomes and stages of sex chromosome evolution in fish: zebrafish, platyfish and guppy

We describe SC complements and results from comparative genomic hybridization (CGH) on mitotic and meiotic chromosomes of the zebrafish Danio rerio, the platyfish Xiphophorus maculatus and the guppy Poecilia reticulata. The three fish species represent basic steps of sex chromosome differentiation: (1) the zebrafish with an all-autosome karyotype; (2) the platyfish with genetically defined sex chromosomes but no differentiation between X and Y visible in the SC or with CGH in meiotic and mitotic chromosomes; (3) the guppy with genetically and cytogenetically differentiated sex chromosomes. The acrocentric Y chromosomes of the guppy consists of a proximal homologous and a distal differential segment. The proximal segment pairs in early pachytene with the respective X chromosome segment. The differential segment is unpaired in early pachytene but synapses later in an ‘adjustment’ or ‘equalization’ process. The segment includes a postulated sex determining region and a conspicuous variable heterochromatic region whose structure depends on the particular Y chromosome line. CGH differentiates a large block of predominantly male-specific repetitive DNA and a block of common repetitive DNA in that region. This revised version was published online in July 2006 with corrections to the Cover Date.     

The telochore: A telomeric differentiation of the chromosome axis

We have analysed by means of silver staining the structure of the chromosome axis at the telomeres of meiotic chromosomes in three different grasshopper species. At metaphase I the chromatid axes run the length of the chromatids although they do not reach the chromosome ends. The axes of sister chromatids are associated and show a round differentiation at their distal ends that we have named the telochore. Telochores never contact the chromosome ends: there is always some chromatin beyond them. In late metaphase I bivalents with a distal chiasma, anaphase I and metaphase II half-bivalents and anaphase II chromatids, the axes clearly possess one telochore in each chromosome end. These results seem to indicate that telochores are differentiations of the distal ends of chromatids. We discuss the possible structural significance of telochores according to the current scaffold/radial loop model of chromatin organization of eukaryotic metaphase chromosomes. Additionally, we suggest the possible functional role of the telochore as a nucleoprotein domain forming a protective cap for telomeric DNA.     

Karyotype diversity and the origin of grapefruit

Grapefruit is a group of citrus of recent origin, probably resulting from a cross between pummelo and sweet-orange. Aiming to investigate this putative origin and the genetic variability among grapefruit cultivars, the karyotype of six grapefruits, two pummelos, and one tangelo cultivar (grapefruit × tangerine) were analyzed using sequential CMA/DAPI double staining and FISH with rDNA probes. The karyotypes of grapefruit ‘Duncan’ and ‘Foster’ differ from those of ‘Flame’, ‘Henderson’, ‘Marsh’ and ‘Rio Red’. The former have two chromosomes with a single CMA⁺ band in both terminal regions (C type chromosome) and six chromosomes with only one CMA⁺ terminal band (D type), whereas the latter have three C and five D type chromosomes. All accessions investigated exhibited two chromosomes with 5S rDNA but a variable number of 45S rDNA. The two former grapefruits displayed four 45S rDNA sites, whereas the remaining grapefruit cultivars had five. The two pummelos showed identical karyotypes, homozygous for CMA⁺ bands and their four rDNA sites. From each pummelo chromosome pair one chromosome seems to be present in grapefruit karyotypes. The different grapefruit karyotypes might result from independent crosses between pummelos of different karyotypic constitution and sweet-oranges. The chromosome markers found in the tangelo ‘Orlando’ and the position of their two 45S rDNA confirm the grapefruit ‘Duncan’ and the tangerine ‘Dancy’ as their parents.     

A model for chromosome structure during the mitotic and meiotic cell cycles

The chromosome scaffold model in which loops of chromatin are attached to a central, coiled chromosome core (scaffold) is the current paradigm for chromosome structure. Here we present a modified version of the chromosome scaffold model to describe chromosome structure and behavior through the mitotic and meiotic cell cycles. We suggest that a salient feature of chromosome structure is established during DNA replication when sister loops of DNA extend in opposite directions from replication sites on nuclear matrix strands. This orientation is maintained into prophase when the nuclear matrix strand is converted into two closely associated sister chromatid cores with sister DNA loops extending in opposite directions. We propose that chromatid cores are contractile and show, using a physical model, that contraction of cores during late prophase can result in coiled chromatids. Coiling accounts for the majority of chromosome shortening that is needed to separate sister chromatids within the confines of a cell. In early prophase I of meiosis, the orientation of sister DNA loops in opposite directions from axial elements assures that DNA loops interact preferentially with homologous DNA loops rather than with sister DNA loops. In this context, we propose a bar code model for homologous presynaptic chromosome alignment that involves weak paranemic interactions of homologous DNA loops. Opposite orientation of sister loops also suppresses crossing over between sister chromatids in favor of crossing over between homologous non-sister chromatids. After crossing over is completed in pachytene and the synaptonemal complex breaks down in early diplotene (= diffuse stage), new contractile cores are laid down along each chromatid. These chromatid cores are comparable to the chromatid cores in mitotic prophase chromosomes. As an aside, we propose that leptotene through early diplotene represent the missing G2 period of the premeiotic interphase. The new chromosome cores, along with sister chromatid cohesion, stabilize chiasmata. Contraction of cores in late diplotene causes chromosomes to coil in a configuration that encourages subsequent syntelic orientation of sister kinetochores and amphitelic orientation of homologous kinetochore pairs on the spindle at metaphase I.     

Sororin is enriched at the central region of synapsed meiotic chromosomes

During meiotic prophase, cohesin complexes mediate cohesion between sister chromatids and promote pairing and synapsis of homologous chromosomes. Precisely how the activity of cohesin is controlled to promote these events is not fully understood. In metazoans, cohesion establishment between sister chromatids during mitotic divisions is accompanied by recruitment of the cohesion-stabilizing protein Sororin. During somatic cell division cycles, Sororin is recruited in response to DNA replication-dependent modification of the cohesin complex by ESCO acetyltransferases. How Sororin is recruited and acts in meiosis is less clear. Here, we have surveyed the chromosomal localization of Sororin and its relationship to the meiotic cohesins and other chromatin modifiers with the objective of determining how Sororin contributes to meiotic chromosome dynamics. We show that Sororin localizes to the cores of meiotic chromosomes in a manner that is dependent on synapsis and the synaptonemal complex protein SYCP1. In contrast, cohesin, with which Sororin interacts in mitotic cells, shows axial enrichment on meiotic chromosomes even in the absence of synapsis between homologs. Using high-resolution microscopy, we show that Sororin is localized to the central region of the synaptonemal complex. These results indicate that Sororin regulation during meiosis is distinct from its regulation in mitotic cells and may suggest that it interacts with a distinctly different partner to ensure proper chromosome dynamics in meiosis.     

Releasing cohesin from chromosome arms in early mitosis: opposing actions of Wapl–Pds5 and Sgo1

The cohesin complex establishes sister chromatid cohesion during S phase. In metazoan cells, most if not all cohesin dissociates from chromatin during mitotic prophase, leading to the formation of metaphase chromosomes with two cytologically discernible chromatids. This process, known as sister chromatid resolution, is believed to be a prerequisite for synchronous separation of sister chromatids in subsequent anaphase. To dissect this process at a mechanistic level, we set up an in vitro system. Sister chromatid resolution is severely impaired upon depletion of Wapl from Xenopus egg extracts. Exogenously added human Wapl can rescue these defects and, remarkably, it can do so in a very short time window of early mitosis. A similar set of observations is made for Pds5, a factor implicated previously in the stabilization of interphase cohesion. Characteristic amino acid motifs (the FGF motifs) in Wapl coordinate its physical and functional interactions with Pds5 and cohesin subunits. We propose that Wapl and Pds5 directly modulate conformational changes of cohesin to make it competent for dissociation from chromatin during prophase. Evidence is also presented that Sgo1 plays a hitherto underappreciated role in stabilizing cohesin along chromosome arms, which is antagonized by the mitotic kinases polo-like kinsase (Plk1) and aurora B.     

Fluorescent in situ hybridization in plant polytene chromosomes

Polytene chromosomes are found in specialized tissues, with high metabolic activity, of a few angiosperm genera. They differ from Diptera polytenics in several aspects, mainly because their chromatids on each chromosome are not tightly paired, nor are they so highly endoreplicated as those of Diptera. In situ hybridization with isotopic and non-isotopic probes has been successfully used in plant polytene chromosomes, mainly in Phaseolus coccineus and Vigna unguiculata, where they have been best investigated. The results reported for mitotic and polytene chromosomes of these species, and a few others, are compared aiming to ascertain the efficiency and limitations of FISH in plant polytenics. In general, polytene chromosomes either from embryo suspensor cells of P. coccineus or from anther tapetal cells of V. unguiculata proved to be quite a suitable system for localizing DNA sequences by FISH. The partially unsynapsed chromatids, typically found in plant polytenics, seem to be the most important hindrance for a precise chromosome mapping. On the other hand, the interphase polytene nucleus is a valuable system for localizing FISH signals since they conserve a spatial organization similar to that of mitotic interphase and produce much amplified signals.     

Visualization of Prekinetochore Locus on the Centromeric Region of Highly Extended Chromatin Fibers: Does Kinetochore Autoantigen CENP-C Constitute a Kinetochore Organizing Center?

Kinetochore is morphologically defined as a trilaminated, highly differentiated structure at the primary constriction of mitotic chromosomes. This subcellular organella is assumed to be composed of DNA and proteins. Immunoelectron microscopy has shown that centromere autoantigens CENP-C and CENP-B localize to the kinetochore inner plate and the underlying centromeric region respectively. We previously indicated that both are DNA-binding proteins that constitute centromeric heterochromatin throughout the cell cycle. Here, we tried to elucidate how these molecules are involved in the kinetochore/centromere organization in vivo by analyzing their morphological behavior in nuclei. Using immunofluorescence microscopy, we found that CENP-C remained as round discrete dots, whereas CENP-B displayed larger surrounding materials. To examine the CENP-C-binding locus on the genome, we prepared highly extended chromatin fibers and performed simultaneous immunofluorescence and fluorescence in situ hybridization. We obsreved that centromeric alphoid DNA, targeted by CENP-B, was highly dispersed, whereas the CENP-C antigen persisted as small dots well situated on the fibers. These features reminded us of the ‘ball and cup’ structure that had been presented for ‘prekinetochore’. We propose here that CENP-C constitutes a ‘kinetochore organizing center’ tightly associating with DNA, whereas CENP-B heterochromatin offers the solid support during kinetochore maturation. This revised version was published online in August 2006 with corrections to the Cover Date.     

Ladder-like amplification of the type I interferon gene cluster in the human osteosarcoma cell line MG63

The organization of the type I interferon (IFN) gene cluster (9p21.3) was studied in a human osteosarcoma cell line (MG63). Array comparative genomic hybridization (aCGH) showed an amplification of ∼6-fold which ended at both ends of the gene cluster with a deletion that extended throughout the 9p21.3 band. Spectral karyotyping (SKY) combined with fluorescence in-situ hybridization (FISH) identified an arrangement of the gene cluster in a ladder-like array of 5–7 ‘bands’ spanning a single chromosome termed the ‘IFN chromosome’. Chromosome painting revealed that the IFN chromosome is derived from components of chromosomes 4, 8 and 9. Labelling with centromeric probes demonstrated a ladder-like amplification of centromeric 4 and 9 sequences that co-localized with each other and a similar banding pattern of chromosome 4, as well as alternating with the IFN gene clusters. In contrast, centromere 8 was not detected on the IFN chromosome. One of the amplified centromeric 9 bands was identified as the functional centromere based on its location at the chromosome constriction and immunolocalization of the CENP-C protein. A model is presented for the generation of the IFN chromosome that involves breakage–fusion–bridge events. Electronic supplementary material   The online version of this article (doi:) contains supplementary material, which is available to authorized users.     

Dependence of the structure and mechanics of metaphase chromosomes on oxidized cysteines

We have found that reagents that reduce oxidized cysteines lead to destabilization of metaphase chromosome folding, suggesting that chemically linked cysteine residues may play a structural role in mitotic chromosome organization, in accord with classical studies by Dounce et al. (J Theor Biol 42:275–285, 1973) and Sumner (J Cell Sci 70:177–188, 1984a). Human chromosomes isolated into buffer unfold when exposed to dithiothreitol (DTT) or tris(2-carboxyethyl)phosphine (TCEP). In micromanipulation experiments which allow us to examine the mechanics of individual metaphase chromosomes, we have found that the gel-like elastic stiffness of native metaphase chromosomes is dramatically suppressed by DTT and TCEP, even before the chromosomes become appreciably unfolded. We also report protein labeling experiments on human metaphase chromosomes which allow us to tag oxidized and reduction-sensitive cysteine residues. PAGE analysis using fluorescent labels shows a small number of labeled bands. Mass spectrometry analysis of similarly labeled proteins provides a list of candidates for proteins with oxidized cysteines involved in chromosome organization, notably including components of condensin I, cohesin, the nucleosome-interacting proteins RCC1 and RCC2, as well as the RNA/DNA-binding protein NONO/p54NRB.     

Comparative sequence analysis of a highly oncogenic but horizontal spread-defective clone of Marek’s disease virus

Marek’s disease virus (MDV) is a cell-associated alphaherpesvirus that induces rapid-onset T-cell lymphomas in poultry. MDV isolates vary greatly in pathogenicity. While some of the strains such as CVI988 are non-pathogenic and are used as vaccines, others such as RB-1B are highly oncogenic. Molecular determinants associated with differences in pathogenicity are not completely understood. Comparison of the genome sequences of phenotypically different strains could help to identify molecular determinants of pathogenicity. We have previously reported the construction of bacterial artificial chromosome (BAC) clones of RB-1B from which fully infectious viruses could be reconstituted upon DNA transfection into chicken cells. MDV reconstituted from one of these clones (pRB-1B-5) showed similar in vitro and in vivo replication kinetics and oncogenicity as the parental virus. However, unlike the parental RB-1B virus, the BAC-derived virus showed inability to spread between birds. In order to identify the unique determinants for oncogenicity and the ‘‘non-spreading phenotype’’ of MDV derived from this clone, we determined the full-length sequence of pRB-1B-5. Comparative sequence analysis with the published sequences of strains such as Md5, Md11, and CVI988 identified frameshift mutations in RLORF1, protein kinase (UL13), and glycoproteins C (UL44) and D (US6). Comparison of the sequences of these genes with the parental virus indicated that the RLORF1, UL44, and US6 mutations were also present in the parental RB-1B stock of the virus. However with regard to UL13 mutation, the parental RB-1B stock appeared to be a mixture of wild type and mutant viruses, indicating that the BAC cloning has selected a mutant clone. Although further studies are needed to evaluate the role of these genes in the horizontal-spreading defective phenotype, our data clearly indicate that mutations in these genes do not affect the oncogenicity of MDV.     

Anaphase-telophase analysis of chromosomal damage induced by chemicals

Three main aspects involved in the chemical induction of anaphase-telophase aberrations in the first mitosis after treatment were analyzed: 1) the relationship between the frequency of anaphase-telophase aberrations and the time of fixation after treatment; 2) the dose-response relationships; and 3) the proliferative rate of cells exposed to chemicals which interact with DNA by different mechanisms. Experiments were carried out using Chinese hamster ovary (CHO) cells. The compounds examined were adriamycin (ADR) and mitomycin C (MMC). The frequency of cells with chromatin bridges or with lagging chromosomes as well as the mitotic index was determined in each experiment. The results obtained showed that 1) chromatin bridges and lagging chromosomes are apparently induced during the S period of the previous interphase; 2) the increase in the cytotoxicity index (inferred from the mitotic index) and the frequency of cells with chromatin bridges and lagging chromosomes were proportional to the treatment lapse and to the dose employed; and 3) the effect of ADR on cell growth differs from the effect of MMC. While ADR decreased the mitotic activity of cells in logarithmic growth phase, MMC induced mitotic delay. In accordance with these results, the occurrence of chromatin bridges in anaphase-telophase could be explained by the induction of chromosome stickiness and, to a lesser extent, by the induction of exchange-type aberrations. On the other hand, lagging chromosomes seem to be the result of chromatid or chromosome breaks because the lagging chromosomes observed were primarily, if not all, fragments and not whole chromosomes. Our evaluation of the anaphase-telophase test indicates that it is very sensitive method for the detection of chemical clastogens, but other factors, such as mitotic depression, must be taken into account to avoid false-negative results.     

Chromatin preferences of the perichromosomal layer constituent pKi-67

The proliferation-associated nuclear protein pKi-67 relocates from the nucleolus to the chromosome surface during the G2/M transition of the cell cycle and contributes to the formation of the perichromosomal layer. We investigated the in-vivo binding preferences of pKi-67 for various chromatin blocks of the mitotic chromosomes from the human and two mouse species, Mus musculus and M. caroli. All chromosomes were decorated with pKi-67 but displayed a gap of pKi-67 decoration in the centromere and NOR regions. pKi-67 distribution in a rearranged mouse chromosome showed that the formation of the centromeric gap was controlled by the specific chromatin in that region. While most chromatin served as a substrate for direct or indirect binding of pKi-67, we identified three types of chromatin that bound less or no pKi-67. These were: (1) the centromeric heterochromatin defined by the alpha satellite DNA in the human, by the mouse minor satellite in M. musculus and the 60- and 79-bp satellites in M. caroli; (2) the pericentromeric heterochromatin in M. musculus defined by the mouse major satellite, and (3) NORs in the human and in M. musculus defined by rDNA repeats. In contrast, the conspicuous blocks of pericentromeric heterochromatin in human chromosomes 1, 9 and 16 containing the 5-bp satellite showed intense pKi-67 decoration. The centromeric gap may have a biological significance for the proper attachment of the chromosomes to the mitotic spindle. In this context, our results suggest a new role for centromeric heterochromatin: the control of the centromeric gap in the perichromosomal layer.     

Direct and indirect non-disjunction in the origin of trisomy in cultured human lymphocytes

The aim of the present work was to investigate the processes involved in the origin of trisomic karyotypes, i.e. co-migration of sister chromatids (mitotic non-disjunction, MND) and recovery of micronuclei (MN) originating from lagging chromosomes/chromatids at anaphase (mitotic indirect non-disjunction, MIND), and to evaluate their relative contribution to aneuploidy in human lymphocytes mitotically activated in vitro. Therefore, phytohaemagglutinin-stimulated human lymphocytes from one donor were treated with 10 and 25 nM colchicine and analysed through two cell cycles by means of both molecular (FISH with centromeric DNA probes specific for chromosomes 7 and 11) and classical cytogenetic techniques. The following events were analysed: (i) chromosome/chromatid loss (a MN-generating event) in M(1) bipolar ana-telophases; (ii) MN recovery in M(2+) prophases; (iii) non-disjunction and loss of chromosomes 7 and 11 by FISH analysis in cytochalasin B-induced binucleate cells; (iv) spontaneous frequency of trisomic cells by chromosome counting and FISH analysis in M(1) c-metaphases; (v) induced frequency of trisomic cells by chromosome counting and FISH analysis in M(2) c-metaphases. Our results indicate that MND plays a major role compared with MIND in the origin of trisomic karyotypes, being approximately 4- to 5-fold higher in colchicine-treated cells. Moreover, remarkable reductions in the observed frequencies of trisomic cells were recorded in comparison with the expected ones, with an observed/expected frequency ratio of trisomic M(2) c-metaphases ranging between 1/3 and 1/6.     

ICRF‐193, an anticancer topoisomerase II inhibitor,induces arched telophase spindles that snap,leading to a ploidy increase in fission yeast

ICRF‐193 [meso‐4,4‐(2,3‐butanediyl)‐bis(2,6‐piperazinedione)] is a complex‐stabilizing inhibitor of DNA topoisomerase II (topo II) that is used as an effective anticancer drug. ICRF‐193 inhibits topo II catalytic activity in vitro and blocks nuclear division in vivo. Here, we examined the effects of ICRF‐193 treatment on chromatin behavior and spindle dynamics using detailed live mitotic cell analysis in the fission yeast, Schizosaccharomyces pombe. Time‐lapse movie analysis showed that ICRF‐193 treatment leads to an elongation of presumed chromatin fibers connected to kinetochores during mid‐mitosis. Anaphase spindles begin to arch, and eventually spindle poles come together abruptly, as if the spindle snapped at the point of spindle microtubule overlap in telophase. Segregating chromosomes appeared as elastic clumps and subsequently pulled back and merged. The snapped spindle phenotype was abolished by microtubule destabilization after thiabendazole treatment, accompanied by unequal chromosome segregation or severe defects in spindle extension. Thus, we conclude that ICRF‐193‐treated, unseparated sister chromatids pulling toward opposite spindle poles produce the arched and snapped telophase spindle. ICRF‐193 treatment increased DNA content, suggesting that the failure of sister chromatids to separate properly in anaphase, causes the spindle to break in telophase, resulting in polyploidization.     

Assays for mitotic chromosome condensation in live yeast and mammalian cells

The dynamic reorganization of chromatin into rigid and compact mitotic chromosomes is of fundamental importance for faithful chromosome segregation. Owing to the difficulty of investigating this process under physiological conditions, the exact morphological transitions and the molecular machinery driving chromosome condensation remain poorly defined. Here, we review how imaging-based methods can be used to quantitate chromosome condensation in vivo, focusing on yeast and animal tissue culture cells as widely used model systems. We discuss approaches how to address structural dynamics of condensing chromosomes and chromosome segments, as well as to probe for mechanical properties of mitotic chromosomes. Application of such methods to systematic perturbation studies will provide a means to reveal the molecular networks underlying the regulation of mitotic chromosome condensation.     

Fertilization and early embryology: Cytogenetic and fluorescent in-situ hybridization chromosomal studies on in-vitro fertilized and intracytoplasmic sperm injected 'failed-fertilized' human oocytes

This study was undertaken to establish baseline data on thechromosomal status of ‘failed-fertilized’ oocytesderived from in-vitro fertilization (IVF) or intracytoplasmicsperm injection (ICSI) procedures. A cytogenetic analysis wasundertaken on 162 IVF and 51 ICSI oocytes. In all, 82.1% (133/162)of the IVF and 78.4% (40/51) of the ICSI oocytes had metaphaseII (Mil) plates, of which 50.4% of the IVF and 47.5% of theICSI oocytes were analysed further. Chromosomes of the G-group(21–22) were identified with the majority of the anomalies.No overall significant difference in the aneuploidy rate wasfound for the IVF (37.3%) or ICSI (31.6%) oocytes, or with maternalage. However, chromosome anomalies, e.g. diploidy, fragmentedand broken chromatids, single sperm and oocyte chromatids, werefound in oocytes from IVF patients aged >36 years and inthe ICSI oocytes throughout the maternal age range (31–38years). The status of the polar body chromatin indicated thatthere was no overall significant difference in the maturationof the IVF and ICSI oocytes. Evidence of successful sperm deliverywas found in 72.5% (37/51) of the ICSI failed-fertilized oocytes.In this group there was a significant increase in the incidenceof premature chromosome condensation: 19.6% (10/51) containedsperm chromosomes, 7.8% (4/51) had swollen sperm heads, andthe remaining 45.0% had condensed sperm heads. The presenceof both sperm and Mil oocyte chromosomes was found in 19.6%(10/51) of the ICSI and 8.6% (14/162) of the IVF failed-fertilizedoocytes. Specific fluorescent in-situ hybridization DNA probeswere used to re-analyse the chromosomes of karyotyped ‘failed-fertilized’IVF oocytes and, for the first time, applied to the karyotypedchromosomes of failed-fertilized ICSI oocytes. The hybridizationefficiency was 86–95% for the centromere probe and 100%for probes 21 and 18.     

The control of chromosome segregation during mitosis in epithelial cells by substrate elasticity

Materials of defined elasticity, including synthetic material scaffolds and tissue-derived matrices, can regulate biological responses of cells and in particular adhesion, migration, growth and differentiation which are essential parameters for tissue integration. These responses have been extensively investigated in interphase cells, but little is known whether and how material elasticity affects mitotic cells. We used polyelectrolyte multilayer films as model substrates with elastic modulus ranging from Eap = 0 up to Eap = 500 kPa and mitotic PtK2 epithelial cells to address these important questions. Soft substrates (Eap < 50 kPa) led to abnormal morphology in chromosome segregation, materialized by chromatin bridges and chromosome lagging. Frequency of these damages increased with decreasing substrate stiffness and was correlated with a pro-apoptotic phenotype. Mitotic spindle was not observed on soft substrates where formation of chromatin damages is due to low β1-integrin engagement and decrease of Rac1 activities. This work constitutes the first evidence that soft substrates hinder epithelial cell division. In perspective, our findings emphasize the prime incidence of the material elasticity on the fate of the phenotype, especially of stem cells in the mitotic phase.