Studies of multipolar mitoses in euploid tissue cultures

Cultures of kidney epithelium and fibroblasts of 39 specimens of Microtus agrestis were investigated. In all 77 cultures multipolar mitoses were found. They were studied in living state and after pulse labelling with ³H-thymidine. The ploidy of the multipolar mitoses and of their daughter nuclei was determined by measuring the relative Feulgen-DNA content and by counting the predominantly constitutive heterochromatic sex chromosomes. Constitutive heterochromatin was demonstrated by late replication, retarded separation of the chromatids in anaphase, heteropycnosis and by the Giemsa technique of Arrighi and Hsu (1971). The latter stained also the spindle apparatus of mitoses.—In living cells, transformation of multipolar mitoses into bipolar mitoses was observed. The chromosomes of multipolar mitoses are separated into complete genomes; the daughter nuclei can be haploid, diploid, triploid or tetraploid. The chromosomes of haploid and triploid metaphases were studied with the Giemsa banding technique. The banding pattern shows an exact monosomy and trisomy, respectively, for each chromosome. Haploid nuclei are likely to be viable only in multinucleate cells, whereas triploid cells behave like diploid cells during the S period and the mitosis.Dedicated to Prof. Dr. K. Goerttler on the occasion of his 75th birthday.Supported by the Bundesministerium für Bildung und Wissenschaft of the Federal Republic of Germany.     

Die Verteilung der Chromosomen auf die Tochterzellkerne multipolarer Mitosen in euploiden Gewebekulturen von Microtus agrestis

In kidney epithelial cultures from female Microtus agrestis, 3,55% of all mitoses are multipolar, 94% of them tripolar. Feulgen photometric measurements of 21 tripolar mitoses reveal a total DNA amount corresponding to the mitotic diploid value (4c) in 5 cases, and to the tetraploid value (8c) in 16 cases, Diploid tripolar mitoses divide into one daughter nucleus with a diploid DNA value (2c) and two nuclei each with a haploid DNA value (1c). Most tetraploid tripolar mitoses divide into one daughter nucleus with a diploid DNA value (2c) and two nuclei with a triploid DNA value (3c). Also the sex chromosomes are distributed to the daughter nuclei in the relation of 2∶3∶3. This can be seen in anaphase figures as well as in interphase nuclei presumably derived from tripolar mitoses, showing chromocenters according to the number of X-chromosomes. In two cases of tripolar tetraploid mitoses the resulting nuclei have a haploid, a triploid and a tetraploid DNA value. The DNA replication pattern is always identical in the daughter nuclei of diploid and tetraploid tripolar mitoses. — Our observations suggest segregation and distribution of haploid chromosome sets or multiples of haploid sets to the daughter nuclei of multipolar mitoses. They also show a possible way of formation of haploid and triploid cells in a basically diploid tissue. Presumably triploid nuclei (with 3 chromocenters) are capable of DNA synthesis.     

Formation and division of binucleated cells in kidney cell cultures of microtus agrestis

Summary Epithelial kidney cell cultures of Microtus agrestis contain 10 to 25% binucleated cells. Observations of living cells under the phase contrast microscope showed that binucleated cells can arise by nuclear mitosis without cytoplasmic division. When binucleated cells divide the two nuclei are highly synchronized as they enter mitosis. In mitosis the chromosomes of both nuclei combine to a common metaphase plate leading to polyploid cells. In one case a tripolar spindle was seen after formation of a metaphase by the chromosomes of the two nuclei of a binucleated cell. This tripolar mitosis resulted in one binucleated and one mononucleated cell. The DNA-content (Feulgen photometry) and the distribution of heterochromatic bodies of the nuclei were corresponding to a tetraploid, a triploid and a haploid chromosome set. This suggests the possibility of somatic segregation of complete haploid sets.Supported by the Deutsche Forschungsgemeinschaft.     

鲤鱼染色体组人工调控的核型证明

以囊胚细胞制备鲤鱼单倍体染色体永久片并分析了单倍体染色体的核型。用囊胚细胞及白细胞培养法制取雌核二倍体及人工三倍体的永久片。以单倍体的核型为根据对人工二倍体及三倍体的染色体配对证明,人工诱发雌核二倍体的染色体是来自两个完整的染色体组,而人工三倍体的染色体则依不同个体而有差异。有部分个体的染色体来自三套完整的染色体组,但大部分个体是非整倍体,前者的雌性个体性腺发育正常,后者是不育的。     

Unequal sister chromatid exchange in the rDNA array of Saccharomyces cerevisiae

In the yeast Saccharomyces cerevisiae the nucleolar organiser region (NOR) is located on chromosome XII. It contains 100-200 copies of rDNA--a minimum of 20 rDNA genes in tandem--and is termed the RDN locus. Yeast cells may exist in either haploid or diploid form. There are two forms of life cycle: haploid and diploid cells double by mitosis, and diploid cells are reduced to the haploid state by meiosis. Diploid cells have two homologous chromosomes for each of the 16 chromosomes. They are usually of the same size. However, in this study it is shown that homologous chromosomes XII can become different in size due to unequal sister chromatid exchange during mitosis in 'old' cells.     

Chromosome constitution of in vitro segregated haploid and diploid cells of the mouse

The composition in segregated haploid sets of paternal and maternal chromosomes has been studied in order to verify whether their composition is uniparental of mixed, fixed or variable. Primary cultures where prepared using kidneys from hybrids of strains of Mus musculus in which the parental chromosomes are distinguishable; the maternal set consists of 20 teleocentric chromosomes, the paternal set of 9 metacentric chromosomes, derived by Robertsonian fusion and 2 telocentrics. Applying Seabright's banding technique, an analysis of segregated haploid and diploid cells, which have originated spontaneously through polyploidisation-segregation processes was carried out. It was concluded that the haploid sets have a variable composition of paternal and maternal chromosomes.     

Incidence of mosaic cell lines in vivo and malsegregation of chromosome 21 in lymphocytes in vitro of trisomy 21 patients: detection by fluorescence in situ hybridization on binucleated lymphocytes

In order to detect aneuploidy in interphase human lymphocytes, both in vivo and in vitro, fluorescence in situ hybridization (FISH) was carried out on binucleated cells cytokinesis-blocked by cytochalasin B at the first mitosis after phytohemagglutinin stimulation. A pericentric chromosome-21-specific DNA probe prepared from yeast artificial chromosome clone 881D2 by the polymerase chain reaction was employed. One thousand binucleated cells per individual were scored from cultures from twelve trisomy 21 patients aged 0.01-8.9 years (mean 4.3 years) and 20 normal children of similar age. Of trisomy 21 patients, increased frequencies of disomic cells in vivo (1.690+/-1.070%) and cells containing six signals with nondisjunction (0.822+/-0.554%) were found, compared with those of monosomic 21 cells in vivo (0.265+/-0.130%) and cells containing four signals with nondisjunction in normal children (0.369+/-0.250%; P=0.000 and P=0.000, respectively). These results show that malsegregation of chromosome 21 occurs more often in trisomic 21 cells than in disomic cells from normal children. The frequency of nondisjunction was significantly higher than the loss of chromosome 21 in both cultured trisomic (0.822+/-0.554% vs 0.043+/-0.049%, P=0.000) and disomic (0.369+/-0.250% vs 0.010+/-0.30%, P=0.000) cells. Comparisons of in vivo and in vitro data on aneuploidy indicate that a cell selection mechanism may exist in vivo. All these results show that FISH, with a chromosome-specific probe, on binucleated lymphocytes is a powerful tool for simultaneously detecting mosaic cell lines in vivo and malsegregation (loss and nondisjunction) of a corresponding chromosome in vitro in the same cell population.     

Synaptonemal complex analysis in spermatocytes of diploid and triploid Chinese shrimp Fenneropenaeus chinensis

A modified surface spreading technique for synaptonemal complex (SC) analysis was tested to assess the process of chromosome synapsis in spermatocytes of diploid and induced triploid Fenneropenaeus chinensis. Spermatocytes of diploid shrimp showed typical morphological characteristics of eukaryote SC, with complete synapsis of bivalents. No recognizable bivalent associated with sex chromosomes was observed in spermatocytes of diploid shrimp. However, differences in morphology of SC, including unsynapsed univalents, bivalents, totally paired trivalents with non-homologous synapsis, partner switches and triple synapsis were identified at early pachytene stage of triploid spermatocytes. Triple synapsis was especially common at late pachytene stage in spermatocytes of triploid shrimp. The observed abnormal synapsis behavior of chromosomes in spermatocytes indicated that triploid male shrimp may find it difficult to develop normal haploid sperm.     

Chromosome nondisjunction and instabilities in tapetal cells are affected by B chromosomes in maize

Abnormal mitosis occurs in maize tapetum, producing binucleate cells that later disintegrate, following a pattern of programmed cell death. FISH allowed us to observe chromosome nondisjunction and micronucleus formation in binucleate cells, using DNA probes specific to B chromosomes (B's), knobbed chromosomes, and the chromosome 6 (NOR) of maize. All chromosome types seem to be involved in micronucleus formation, but the B's form more micronuclei than do knobbed chromosomes and knobbed chromosomes form more than do chromosomes without knobs. Micronuclei were more frequent in 1B plants and in a genotype selected for low B transmission rate. Nondisjunction was observed in all types of FISH-labeled chromosomes. In addition, unlabeled bridges and delayed chromatids were observed in the last telophase before binucleate cell formation, suggesting that nondisjunction might occur in all chromosomes of the maize complement. B nondisjunction is known to occur in the second pollen mitosis and in the endosperm, but it was not previously reported in other tissues. This is also a new report of nondisjunction of chromosomes of the normal set (A's) in tapetal cells. Our results support the conclusion that nondisjunction and micronucleus formation are regular events in the process of the tapetal cell death program, but B's strongly increase A chromosome instability.     

Centromeric genotyping and direct analysis of nondisjunction in humans: Down syndrome

In species with chiasmate meioses, alterations in genetic recombination are an important correlate of nondisjunction. In general, these alterations fall into one of two categories: either homologous chromosomes fail to pair and/or recombine at meiosis I, or they are united by chiasmata that are suboptimally positioned. Recent studies of human nondisjunction suggest that these relationships apply to our species as well. However, methodological limitations in human genetic mapping have made it difficult to determine whether the important determinant(s) in human nondisjunction is absent recombination, altered recombination, or both. In the present report, we describe somatic cell hybrid studies of chromosome 21 nondisjunction aimed at overcoming this limitation. By using hybrids to “capture” individual chromosomes 21 of the proband and parent of origin of trisomy, it is possible to identify complementary recombinant meiotic products, and thereby to uncover crossovers that cannot be detected by conventional mapping methods. In the present report, we summarize studies of 23 cases. Our results indicate that recombination in proximal 21q is infrequent in trisomy-generating meioses and that, in a proportion of the meioses, recombination does not occur anywhere on 21q. Thus, our observations indicate that failure to recombine is responsible for a proportion of trisomy 21 cases. Received: 12 January 1997; in revised form: 16 February 1998 / Accepted: 19 February 1998     

The action of N-methyl-N-nitrosourea on non-established human cell lines in vitro. II. Non-random distribution of chromatid aberrations in diploid and Down's cells.

Chromatid gaps, breaks and aberrations involved in interchanges induced by N-methyl-N-nitrosourea (MNU) were found non-randomly distributed on individual chromosomes and chromosome segments (G bands) both in human diploid fibroblasts with trisomy 21 cultured in vitro. Aberration events were located exclusively in pale G bands. Considering cells in the first post-treatment mitosis, the pattern of aberration distribution, as revealed by the position of hot spots, varied with recovery time and was different in diploid and Down's cells. In comparison with diploid cells, the X chromosomes of Down's cells were not involved in aberrations. Despite the higher aberration frequencies of Down's cells, the number of hot spots and the proportion of aberrations located in hot spots were not increased in this cell type. Therefore, the increased chromosomal sensitivity to MNU of Down's cells does not reflect an increased sensitivity of special chromosomes or chromosome sites.     

Cytological evidence of maternal meiotic errors in a line of chickens with a high incidence of triploidy.

Direct evidence of the nature of maternal meiotic errors in a selected line of chickens with a high incidence of triploidy was obtained by using cytologically marked paternal gametes derived from a closely related avian species. Matings were made by artificial insemination of female chickens of the selection line and a control line with semen from ring-necked male pheasants. A total of five triploid, one pentaploid, and 21 diploid hybrid embryos were karyotyped. Each triploid hybrid embryo contained one set of paternal pheasant chromosomes and two sets of maternal chicken chromosomes, providing irrefutable cytological evidence that the triploids were derived from diploid ova produced by females of the selection line. The pentaploid hybrid contained one set of paternal pheasant chromosomes and four sets of maternal chicken chromosomes, indicating that it had been derived from a tetraploid ovum. Females of the selection line are thought to have a genetically mediated susceptibility to nondisjunction which is responsible for the high incidence of meiotic errors. Evidence is provided that the non-disjunction occurs at both meiosis I and meiosis II.     

Euploid segregation through multipolar mitosis in mammalian cell cultures

Cultures were made of kidney cells of male Rhesus monkey and a karyological analysis of these cells was carried out at various times after plating using the chromosome banding method of Seabringht (1972), in order to study the segregation phenomena which take place in cell cultures in vitro through multipolar mitoses and to identify segregating cells. — Several segregating cells were found: haploid cells, diploid cells with two X chromosomes and triploid cells which were strictly euploid. Polyploidization-segregation cycles in the cell cultures and their mechanism and chronological sequence were analyzed. — On the basis of these results it was suggested that the genome is organized in haploid sets capable of segregating as units and of producing strictly euploid segregating daughter cells.     

Meiotic hybridogenesis in triploid Misgurnus loach derived from a clonal lineage

Triploid loaches Misgurnus anguillicaudatus are derived from unreduced diploid gametes produced by an asexual clonal lineage that normally undergoes gynogenetic reproduction. Here, we have investigated the reproductive system of two types of triploids: the first type carried maternally inherited clonal diploid genomes and a paternally inherited haploid genome from the same population; the second type had the same clonal diploid genomes but a haploid genome from another, genetically divergent population. The germinal vesicles of oocytes from triploid females (3n=75) contained only 25 bivalents, that is, 50 chromosomes. Flow cytometry revealed that the majority of the progeny resulting from fertilization of eggs from triploid females with normal haploid sperm were diploid. This indicates that triploid females mainly produced haploid eggs. Microsatellite analyses of the diploid progeny of triploid females showed that one allele of the clonal genotype was not transmitted to haploid eggs. Moreover, the identity of the eliminated allele differed between the two types of triploids. Our results demonstrate that there is preferential pairing of homologous chromosomes as well as the elimination of unmatched chromosomes in the course of haploid egg formation, that is, meiotic hybridogenesis. Two distinct genomes in the clone suggest its hybrid origin.     

Simultaneous formation of haploid, diploid and triploid eggs in diploid–triploid mosaic loaches

In the loach Misgurnus anguillicaudatus , very few diploid–triploid mosaic individuals, which are generated by accidental incorporation of the sperm nucleus into diploid eggs produced by clonal diploid loach, occur in nature. Ploidy examination of gynogenetic progeny induced by activation with ultraviolet-irradiated goldfish sperm indicated that diploid–triploid mosaic females laid haploid, diploid and triploid eggs, simultaneously. In addition, triploid eggs exhibited larger egg sizes. Microsatellite genotyping of diploid–triploid mosaics revealed that triploid genotypes of mosaic mothers possessed two alleles specific to the clonal diploid and one allele from normal diploid male. Diploid eggs from a mosaic mother had genotypes absolutely identical to the diploid clone. Most genotypes of triploid eggs were identical to the mosaic mother, and one of the three alleles of the mosaic mother was transmitted to haploid eggs. These results suggested that diploid germ cells, which had a clonal genome, were differentiated into clonal diploid eggs, and triploid and haploid eggs were produced from triploid germ cells in the same ovary of mosaic individuals.     

Meiotic Nondisjunction and Recombination of Chromosome III and Homologous Fragments in Saccharomyces Cerevisiae

A yeast strain, in which nondisjunction of chromosome III at the first-meiotic division could be assayed, was constructed. Using chromosome fragmentation plasmids, chromosomal fragments (CFs) were derived in isogenic strains from six sites along chromosome III and one site on chromosome VII. Whereas the presence of the CFs derived from chromosome III increased considerably the meiosis I nondisjunction of that chromosome, the CF derived from chromosome VII had no effect on chromosome III segregation. The effects of the chromosome III-derived fragments were not linearly related to fragment length. Two regions, one of 12 kb in size located at the left end of the chromosome, and the other of 5 kb, located at the center of the right arm, were found to have profound effects on chromosome III nondisjunction. Most disomics arising from meioses in strains containing chromosome III CFs did not contain the CF; thus it appears that the two chromosome III homologs had segregated away from the CF. Among the disomics, recombination between the homologous chromosomes III was lower than expected from the genetic distance, while recombination between one of the chromosomes III and the fragment was frequent. We suggest that there are sites along the chromosome that are more involved than others in the pairing of homologous chromosomes and that the pairing between fragment and homologs involves recombination among these latter elements.     

Cytology of Encephalartos hildebrandtii A. Br. & Bouche

The chromosomes of Encephalartos hildebrandtii A. Br. &Bouche were studied from root tip cells (2n–27), and theplant was found to be a triploid on the basic chromosome number9. Karyotype analysis has shown that the somatic complementconsists of two sets of chromosomes, a diploid set of 18 chromosomesin nine paris, and a haploid set of 9, and therefore it is suggestedthat this is an allotriploid. This is the first report of a polyploid species in the genusEncephalartos, and also in the whole of the Cycadales.     

Haploid Meiosis in Arabidopsis: Double-Strand Breaks Are Formed and Repaired but Without Synapsis and Crossovers

Two hallmark features of meiosis are i) the formation of crossovers (COs) between homologs and ii) the production of genetically-unique haploid spores that will fuse to restore the somatic ploidy level upon fertilization. In this study we analysed meiosis in haploid Arabidopsis thaliana plants and a range of haploid mutants to understand how meiosis progresses without a homolog. Extremely low chiasma frequency and very limited synapsis occurred in wild-type haploids. The resulting univalents segregated in two uneven groups at the first division, and sister chromatids segregated to opposite poles at the second division, leading to the production of unbalanced spores. DNA double-strand breaks that initiate meiotic recombination were formed, but in half the number compared to diploid meiosis. They were repaired in a RAD51- and REC8-dependent manner, but independently of DMC1, presumably using the sister chromatid as a template. Additionally, turning meiosis into mitosis (MiMe genotype) in haploids resulted in the production of balanced haploid gametes and restoration of fertility. The variability of the effect on meiosis of the absence of homologous chromosomes in different organisms is then discussed.     

Genetic Relationships between Haploid and Triploid Targionia (Targioniaceae, Hepaticae)

The species complex Targionia hypophylla-Targionia lorbeeriana includes a haploid and a triploid cytotype in Europe and the Atlantic islands. As shown by isozyme analysis, high levels of fixed heterozygosity and duplication of one of the chromosome sets characterize the triploids that are fertile. Hybridization between two haploids, followed by chromosome doubling and meiotic nondisjunction can explain triploidy. Both allopolyploidy and autopolyploidy are invoked for the origin of the triploid. The duplicated chromosome set most likely comes from the existing haploid that could be one of the putative parents. The morphological intergradation between the two cytotypes, their identical ecological requirements and sympatric distribution can be understood in the light of these data. A haploid Texan colony analyzed for comparison can be discarded as the other putative parent, which remains unknown. Moreover, three triploid African and Australasian colonies do not share the same duplicated genome as the triploids from Europe and the Atlantic islands. Electrophoretic data indicate independent origins of the European-Atlantic and African-Australasian polyploids.     

Diploid,but not haploid,human embryonic stem cells can be derived from microsurgically repaired tripronuclear human zygotes

Human embryonic stem cells have shown tremendous potential in regenerative medicine, and the recent progress in haploid embryonic stem cells provides new insights for future applications of embryonic stem cells. Disruption of normal fertilized embryos remains controversial; thus, the development of a new source for human embryonic stem cells is important for their usefulness. Here, we investigated the feasibility of haploid and diploid embryo reconstruction and embryonic stem cell derivation using microsurgically repaired tripronuclear human zygotes. Diploid and haploid zygotes were successfully reconstructed, but a large proportion of them still had a tripolar spindle assembly. The reconstructed embryos developed to the blastocyst stage, although the loss of chromosomes was observed in these zygotes. Finally, triploid and diploid human embryonic stem cells were derived from tripronuclear and reconstructed zygotes (from which only one pronucleus was removed), but haploid human embryonic stem cells were not successfully derived from the reconstructed zygotes when two pronuclei were removed. Both triploid and diploid human embryonic stem cells showed the general characteristics of human embryonic stem cells. These results indicate that the lower embryo quality resulting from abnormal spindle assembly contributed to the failure of the haploid embryonic stem cell derivation. However, the successful derivation of diploid embryonic stem cells demonstrated that microsurgical tripronuclear zygotes are an alternative source of human embryonic stem cells. In the future, improving spindle assembly will facilitate the application of triploid zygotes to the field of haploid embryonic stem cells.