Fragmentation of polyploid nuclei in the giant cells of the rat trophoblast. I. The ultrastructure of the nuclear fragments

Electron microscope study of the nuclear fragments in the rat trophoblast has demonstrated that the division of the trophoblast giant nucleus results first in the formation of a multinuclear cell. Each nuclear fragment is covered with its own nuclear envelope made of two membranes with numerous pore complexes. The chromatin in these nuclear fragments is condenced with various degrees of condensation, which depends on the step of placenta development, cell differentiation and the degree of nuclear fragmentation. The nuclear ultrastructure in nuclear fragments also depends on the degree of nuclear fragmentation and on the level of chromatin condensation. The nucleolus has no granular component. On large fragments, with lower chromatin condensation the nucleolus is not homogenous being made of fragments of more and of less electron dense fibrilles. Small light lacunae are seen in the nucleolus where chromatin threads and strands pass on. With a high chromatin condensation in the nucleus, round small nucleoli look homogenous being made of moderately electron dense fibrilles. Products of chromosome activity have been found in the nuclear fragments: accumulations of minute granules (d = 15--20 nm), perichromatinous granules (d = 35--40 nm), and fibrillar nucleolus-like bodies. In the multinuclear cell, made as the result of fragmentation of the initially giant nucleus, all the small nuclei are first arranged very close to each other, so that the contours of the neighbouring nuclei coincide.     

Chromosome organisation in the Australian plague locust Chortoicetes terminifera

In Chortoicetes terminifera 45 independently-occurring B-chromosomes were tested and 23 distinct banding variants were detected with either G- or C-banding; six types were found more than once. In particular the Type I banding morph was detected 12 times indicating that individuals carrying this type may be under a different regime of selection compared with individuals bearing other types of banding morph; or the Type I may be subjected to a higher rate of meiotic drive in either or both sexes than other types. Also the Type I appeared to be obviously related to four other banding morphs whereas most types were not obviously related to any other banding morphs, but a few were similar in banding pattern to one or two other types. Three types of B-chromosomes were found in three or more different populations. A relatively high frequency of the Type I banding morph was found in one population, which was probably mainly composed of non-migratory individuals, and also in a laboratory-raised population. The most likely mechanisms for small changes in the banding sequence of the B-chromosomes are three-break insertions which are often indistinguishable from inversions. Rearrangements which add or delete bands, or sequences of bands, to or from B-chromosomes are probably the result of exchanges which are now known to take place in rare individuals with two B-chromosomes. The most distal region of all the banding morphs of the B-chromosome in C. terminifera, plus a short interstitial region in some types, is not late-replicating and has the banding characteristics of euchromatin. The rest of the chromatin of the B-chromosomes is heterochromatic and is the latest replicating heterochromatin in the whole genome. It consists of G-bands, which are also deeply stained with C-banding, and alternating G-interbands, which in turn are stained grey with C-banding. Both of these staining combinations are seen in heterochromatin of the normal complement. The heterochromatin of the B-chromosomes is condensed throughout 1st meiotic prophase in the male and in all somatic interphase nuclei where it can be quickly detected using the G-banding technique. The B-chromosome has a relatively constant, acrocentric morphology with a tendency to increase of length of the long arm as band numbers increase. Isochromosomes of the long arm have been seen only in laboratoryraised embryos. From egg pods with significantly fewer than expected B-chromosomes it is strongly suggested that more than one male may fertilize the eggs in a single pod.     

Intranuclear and cytoplasmic annulate lamellae in the polyploid giant cells of the trophoblast

Intranuclear and cytoplasmic annulate lamellae in polyploid giant cells of the trophoblast have been studied in rat placenta on days 12--17 of development. The annulate lamellae are present in the cytoplasm within a limited time, being visible on day 12 only. These are arranged in bundles near the nucleus to be moving then to the cytoplasm. The end parts of annulate lamellae are broadened to make cisterns of rough endoplasmic reticulum. Unlike the cytoplasmic annulate lamellae, those found within the nucleus are seen in part of the nuclei investigated throughout the whole period examined to look as single structures (not gathered in bundles), they can be branching, separating closed spaces within the nucleus (making local swellings in the loci of branching; the latter having electron dense or transparent vesicles). Association with nuclear chromatin in some regions is a peculiar feature of the intranuclear annulate lamellae. This association is especially obvious at endoprophase in the cycle ofthe polytene nucleus during the somatic conjugation--chromonemes unite in a bundle and condense. Ultrastructural changes of the annulate lamellae is noted throughout the polytene nucleus cycle and during the cell differentiation. It is supposed that in the case of temporary labile chromosome polyteny in the nuclear cycle, which is characteristic of mammalian trophoblasts, annulate lamellae can well compare, in their function, with the synaptonemal complex--these prevent from too tight associations of homologues in the course of somatic conjugation of chromosomes.     

Cytological studies on the organization of DNA in giant trophoblast nuclei of the mouse and the rat

Giant trophoblast nuclei of the mouse and the rat, known to contain hundreds, or even thousands, of times the haploid amount of DNA, have been studied by a number of cytological techniques. These nuclei appear in two morphological states:“reticulate,” in which large numbers of chromatin threads of uniform size intermingle throughout the nucleus, often radiating from clumps of heterochromatin adjacent to the nucleoli, and “homogeneous,” in which the chromatin is more evenly dispersed and individual threads are more difficult to distinguish. Intermediate morphologies are also observed. In neither case were structures resembling polytene chromosomes discernible. — Centromeric heterohromatin as revealed by the Giemsa BSG technique has been quantitatively analyzed in giant versus diploid trophoblast nuclei. Although the median number of chromocenters is slightly greater in giant than in diploid nuclei, the range is similar. In both cases, the chromocenter number is usually less than the diploid number of chromosome pairs, indicating the attraction between centromeres not only of homologous, but also of heterologous, chromosomes. By scanning microdensitometry, we have observed a constant ratio of chromocenter area: total nuclear area in giant cells. This ratio, which likely reflects the ratio of chromocenter volume: total nuclear volume, is in good agreement with that of satellite DNA: total DNA.     

Chromosome and nucleotide sequence differentiation in genomes of polyploid Triticum species

Summary The nature of genome change during polyploid evolution was studied by analysing selected species within the tribe Triticeae. The levels of genome changes examined included structural alterations (translocations, inversions), heterochromatinization, and nucleotide sequence change in the rDNA regions. These analyses provided data for evaluating models of genome evolution in polyploids in the genus Triticum, postulated on the basis of chromosome pairing at metaphase I in interspecies hybrids.The significance of structural chromosome alterations with respect to reduced MI chromosome pairing in interspecific hybrids was assayed by determining the incidence of heterozygosity for translocations and paracentric inversions in the A and B genomes of T. timopheevii ssp. araraticum (referred to as T. araraticum) represented by two lines, 1760 and 2541, and T. aestivum cv. Chinese Spring. Line 1760 differed from Chinese Spring by translocations in chromosomes 1A, 3A, 4A, 6A, 7A, 3B, 4B, 7B and possibly 2B. Line 2541 differed from Chinese Spring by translocations in chromosomes 3A, 6A, 6B and possibly 2B. Line 1760 also differed from Chinese Spring by paracentric inversions in arms 1AL and 4AL whereas line 2541 differed by inversions in 1BL and 4AL (not all chromosomes arms were assayed). The incidence of structural changes in the A and B genomes did not coincide with the more extensive differentiation of the B genomes relative to the A genomes as reflected by chromosome pairing studies.To assay changing degrees of heterochromatinization among species of the genus Triticum, all the diploid and polyploid species were C-banded. No general agreement was observed between the amount of heterochromatin and the ability of the respective chromosomes to pair with chromosomes of the ancestral species. Marked changes in the amount of heterochromatin were found to have occurred during the evolution of some of the polyploids.The analysis of the rDNA region provided evidence for rapid fixation of new repeated sequences at two levels, namely, among the 130 bp repeated sequences of the spacer and at the level of the repeated arrays of the 9 kb rDNA units. These occurred both within a given rDNA region and between rDNA regions on nonhomologous chromosomes. The levels of change in the rDNA regions provided good precedent for expecting extensive nucleotide sequence changes associated with differentiation of Triticum genomes and these processes are argued to be the principal cause of genome differentiation as revealed by chromosome pairing studies.     

Sexing the human fetus and identification of polyploid nuclei by DNA-DNA in situ hybridisation in interphase nuclei

Samples of human adult lymphocytes, fetal lymphocytes, amniotic fluid cells, and chorionic villus cells were sexed independently by cytogenetics and DNA-DNA in situ hybridisation to a tritiated Y probe. For the in situ hybridisation analysis, the presence of Y bodies (hybridisation bodies) in 100 interphase nuclei were scored after autoradiography. In all, 82/83 samples were sexed in this way (one technical failure) and 78/82 were sexed by both in situ hybridisation and cytogenetics. There was complete agreement between the two methods. There was a considerable variation (40-100%) in the percentage of interphase nuclei with a hybridisation body among the male samples, but very few nuclei from female samples showed significant hybridisation. In situ hybridisation could be used to sex the conceptus when males but not females are at risk for various X-linked genetic disorders and may also be useful for detecting 45,X/46,XY mosaicism or polyploid/diploid mosaicism. This would be particularly useful for direct preparations of chorionic villus samples, which often prove difficult to analyse cytogenetically but offer the best means of avoiding maternal contamination. Some interphase nuclei had more than one hybridisation body, and this was most commonly found among amniotic fluid cells. Comparison of sizes of nuclei with one or two hybridisation bodies strongly suggested that most of the amniotic fluid cell nuclei with two hybridisation bodies were tetraploid.     

Polytene chromosomes in mouse trophoblast giant cells

Mouse trophoblast giant cells undergo successive rounds of DNA replication resulting in amplification of the genome. It has been difficult to determine whether giant cell chromosomes are polyploid as in liver cells or polytene as in Dipteran salivary glands because the chromosomes do not condense. We have examined the pattern of hybridization of mouse giant cells with a variety of in situ chromosome markers to address this question. Hemizygous markers displayed one hybridization signal per nucleus in both diploid and giant cells, while homozygous markers displayed two signals per nucleus in both cell types. These patterns are consistent with cytological evidence indicating that giant cell chromosomes are polytene rather than polyploid. However, in contrast to the situation in Dipteran salivary glands, the two homologues do not appear to be closely associated. We conclude that the mechanism of giant cell DNA amplification involves multiple rounds of DNA replication in the absence of both karyokinesis and cytokinesis, and that sister chromatids, but not homologous chromosomes, remain closely associated during this process.     

Chromosome 'speed dating' during meiosis of polyploid Brassica hybrids and haploids

Given their tremendous importance for correct chromosome segregation, the number and distribution of crossovers are tightly controlled during meiosis. In this review, we give an overview of crossover formation in polyploid Brassica hybrids and haploids that illustrates or underscores several aspects of crossover control. We first demonstrate that multiple targets for crossover formation (i.e. different but related chromosomes or duplicated regions) are sorted out during meiosis based on their level of relatedness. In euploid Brassica napus (AACC; 2n = 38), crossovers essentially occur between homologous chromosomes and only a few of them form between homeologues. The situation is different in B. napus haploids in which crossovers preferentially occur between homeologous chromosomes and a few can then form between more divergent duplicated regions. We then provide evidence that the frequency of crossovers between a given pair of chromosomes is influenced by the karyotypic and genetic composition of the plants that undergo meiosis. For instance, genetic evidence indicates that the number of crossovers between exactly the same pairs of homologous A chromosomes gets a boost in Brassica digenomic tetraploid (AACC) and triploid (AAC) hybrids. Increased autosyndesis within B. napus haploids as compared to monoploid B. rapa and B. oleracea is another illustration of this process. All these observations may suggest that polyploidization overall boosts up crossover machinery and/or that the number of crossovers is modulated through inter-bivalents or univalent-bivalent cross-talk effects. The last part of this review gives an up-to-date account of what we know about the genetic control of homologous and homeologous crossover formation among Brassica species.     

Chromosome replication in mouse intraspecific hybrids

Hybrid cells (HY SS2 and HY SS6) arising from the fusion of diploid cells of the mouse lymphosarcoma LS/BL and L cells resistant to 8-azaguanine (HGPRT-) showed slower growth and a longer generation time than the parent lines. The inter- and intrachromosomal timing and patterns of early chromosome DNA replication of parent cells was preserved in the hybrid genome and was not influenced by loss of telocentric chromosomes from LS/BL or L (HGPRT-) cells. Thus DNA chromosome replication sequences are not dependent on the presence of a complete set of chromosomes of the parent cells and do not therefore seem to be a result of interaction between chromosomes not segregated in the hybrid genome.     

Quantitative cytophotometric studies on polyploid liver cell nuclei of frog and rat

Summary Nuclei were isolated from fixed liver tissue of diploid and triploid Rana pipiens and of rat. While the frog liver nuclei present a single ploidy class on the basis of Feulgen absorption measurements, rat liver contained diploid, tetraploid, and some octoploid nuclei. Nuclear areas within single ploidy classes varied over wide ranges, especially in the frog material. The mode of this variation was dependent on ploidy. Microspectrophotometric measurements of several protein components were compared to ploidy and nuclear volume. General protein methods indicated a linear relationship to nuclear volume. Protein-bound sulfhydryl and disulfide groups were not related to nuclear volume, but could be related to ploidy. Protein tyrosine values showed a partial dependence on both factors.This investigation was supported by a grant from the U.S. Public Health Service, GM-10003-03.Post-Doctoral Trainee under U.S. Public Health Training Grant to the Department of Pathology, University of Florida, 5T1 GM 1142-03.Supported by a Career Development award from the National Institute of Child Health and Human Development, K3-DH-6176-03.