Chromosome no. 1 of Crepis capillaris shows defined 3D-shapes in mitotic prophase.

The shape of mitotic prophase chromosomes has been studied in root tip nuclei by confocal microscopy and 3D-image analysis. Crepis capillaris chromosome no. 1 was used as a test object. Chromosome conformation was studied in early, mid- and in late prophase. In mid- and late prophase, individual chromosomes could be distinguished on the basis of their length. Early prophase chromosomes could not be distinguished as individuals. The central axes of prophase chromosomes were traced with an automated computer procedure and then represented as a string of 3D coordinates. This representation facilitated measurement along the chromosome axis of shape parameters such as curvature (amount of bending), torsion (helical winding) and torsion sign (helical handedness). Stretches of early prophase chromosomes showed full helical turns, which could be left- or right-handed. In the later prophase stages curvature and torsion were statistically analysed. Our data on 40 midprophase chromosomes no. 1 show that they are still highly curved, but full helical turns were no longer found. Instead, an overall meandering pattern was observed. In late prophase, one central loop persisted, flanked by two preferential regions of high curvature.

The 5S rRNA gene clusters have a defined orientation toward the nucleolus in Petunia hybrida and Crepis capillaris.

The 3D localization of the 5S ribosomal RNA genes was studied in cells of the cortex zone of roots in the plant species Petunia hybrida inbred line V26 and in Crepis capillaris. The analysis was carried out on interphase nuclei (both species) and on prophase nuclei (C. capillaris). The 5S ribosomal RNA genes were detected by fluorescence in-situ hybridization and 3D images were obtained by confocal scanning laser microscopy. In both plant species, the 5S ribosomal genes were localized at the short arm of chromosome 2, which, in both plants, also possesses a satellite at its end. Statistical and visual analysis of interphase nuclei showed that: (1) there is a preference for an association of the 5S rRNA gene clusters of the two homologous chromosomes, and (2) the 5S rRNA gene clusters in both species had a preserved spatial position within the interphase nucleus and they tended to be polarized with respect to their neighbouring cells (i.e. a relic telophase orientation). Moreover, tracing of the chromosomal segment between the 5S loci and the active NOR revealed that the homologous chromosomes during early/mid prophase were aligned and that they entered the nucleolus side by side, at least for these chromosome segments. We interpret our data to mean that location of 5S rRNA near the nucleolus favours their functioning in ribosome biogenesis.

Characterisation of the nucleolar organising regions during the cell cycle in two varieties of Petunia hybrida as visualised by fluorescence in situ hybridisation and silver staining.

The cell cycle-dependent spatial position, morphology and activity of the four nucleolar organising regions (NORs) of the Petunia hybrida cultivar Mitchell and the inbred line V26 have been analysed. Application of the silver staining technique and fluorescence in situ hybridisation on fixed root-tip material revealed that these interspecific hybrids possess four NORs of which only those of chromosome 2 are active during interphase, which implies that the NOR activity is not of parental origin. However, at the end of mitosis, activity of all NOR regions could be detected, suggesting that the high demand for ribosomes at this stage of the cell cycle requires temporal activity of all NORs. Using actin DNA probes as markers in fluorescence in situ hybridisation experiments enabled the identification of the individual petunia chromosomes.

There is an upper limit of chromosome size for normal development of an organism.

A clearly definable upper tolerance limit for chromosome arm length has been found. As a rule we postulate that, for normal development of an organism, the longest chromosome arm must not exceed half of the average length of the spindle axis at telophase. Above this length, fertility and viability of the carrier individuals become severely impaired due to increasingly incomplete separation of the longest chromatids during mitosis, resulting finally in the loss of DNA. The experimental work that points to a limit in genome plasticity has been carried out on a series of field bean lines with karyotypes of considerable variation in length of individual chromosomes.

The spatial localization of 18 S rRNA genes, in relation to the descent of the cells, in the root cortex of Petunia hybrida.

The 3-D localization of transcription inactive 18 S rRNA genes was studied in interphase nuclei of Petunia hybrida root tip cells. To enable a cell type (i.e. cortex)-specific study in which also the orientation and descent of the cells could be taken into account, a method was developed to preserve the spatial organization of the root meristem. The ribosomal genes were detected by fluorescence in situ hybridization using a biotinylated cDNA probe. 3-D images of 81 nuclei, obtained by confocal scanning laser microscopy, were processed with newly developed computer software. 3-D nucleolar and nuclear dimensions, and the localization of the FISH-spots, were recorded interactively. We compared the absolute and relative position of the genes within and between files of cells of the cortex region of several roots, taking into account the genealogical relationship of the cells. Statistical analysis showed that both the relative and absolute positions of the inactive genes were random, also in more closely related cells within a file of cells. A 'relict telophase orientation' of the genes (i.e. the position of the genes in the daughter cells are mirror images of each other) could only be observed in the G0/1 phase of 'true' daughter cells; the orientation was not preserved throughout the next cell cycle.

The spatial arrangement of Allium triquetrum chain quadrivalents at metaphase I as reviewed by confocal microscopy.

We examined the three-dimensional arrangement of bivalents and, in particular, a chain of four chromosomes (chain quadrivalent) in the metaphase I spindle of pollen mother cells of Allium triquetrum by confocal microscopy. Firstly, we show by optical sectioning and three-dimensional image reconstruction that the cooriented pairs of centromeres of all seven bivalents lie virtually parallel to each other in the metaphase I spindle, parallel to the long axis of the spindle. Secondly, we likewise show that the four centromeres of the chain quadrivalent are aligned in the metaphase I spindle in, essentially, a two-dimensional array, not in a three-dimensional array, as proposed by some other authors. This two-dimensionality has its basis, we argue, in the principle that poleward directed spindle forces minimise centromere-to-pole distances and therefore align pairs of centromeres connected to opposite poles most axially (vertically) in the spindle. These distances are minimised for the quadrivalent as a whole only when it lies in two dimensions, i.e. in a plane parallel to the spindle axis.

The homing cursor: a tool for three-dimensional chromosome analysis.

When studying the three-dimensional shape of prophase chromosomes (or any other tubular structure), it is useful to represent these structures as a string of three-dimensional Cartesian coordinates along the medial axis. This procedure was automated in order to limit the number of human interactions and to improve reproducibility. In this paper the design, implementation, and validation of the automated method is presented. From the data presented it can be concluded that the cursor algorithm provides an objective and therefore reproducible method to trace the medial axes of prophase chromosomes automatically. This method could allow a more extensive understanding of the (changes in) chromosome organisation throughout the cell cycle, its relation to cell function, and the complex process of chromosome condensation.

Three-dimensional chromosome arrangement of Crepis capillaris in mitotic prophase and anaphase as studied by confocal scanning laser microscopy.

To estimate the extent of ordering of chromosomes, confocal scanning laser microscopy was used to make three-dimensional images from optical sections. For Crepis capillaris, which has 2n = 6 easily recognizable chromosomes, a statistically significant sample of 75 Feulgen-stained root tip anaphases was analysed. A comparison of the observed chromosome ordering and the expected random distribution showed a significant surplus of one of the arrangements with a juxtaposition of the two chromosomes with a nucleolus organizer region. Two of the arrangements with these chromosomes in opposite positions were never observed in our material. Another analysis of 30 mithramycin A-stained prophases and 30 meta- and anaphases showed partly different patterns of non-random chromosome distribution in the two stages of mitosis. A preference for an association of the homologues was observed for all pairs of chromosomes in prophase cells, whereas in meta- and anaphase the association only persisted for the nucleolus organizer chromosomes. This indicates that there may be some relocation of the chromosome positions during the transition from prophase to metaphase. In meta- and anaphase one of the arrangements with juxtaposed NOR chromosomes was preferred, i.e. the ordering in which chromosomes 1 and 3 occupied alternate positions. Probably, the nucleolus is an important factor in producing a non-random distribution, but there could be other factors that influence chromosome ordering as well. A comparison of the anaphase chromosome ordering in C. capillaris plants from very different localities, indicated that the observed non-random distribution was independent of the origin of the material. Existing models of chromosome disposition are not sufficient to explain the observed non-random chromosome ordering in C. capillaris.

Visualization and analysis techniques for three dimensional information acquired by confocal microscopy.

Confocal Scanning Laser Microscopy (CSLM) is particularly well suited for the acquisition of 3-dimensional data of microscopic objects. In the CSLM a specific volume in the object is sampled during the imaging process and the result is stored in a digital computer as a three-dimensional memory array. Optimal use of these data requires both the development of effective visual representations as well as analysis methods. In addition to the well known stereoscopic representation method a number of alternatives for various purposes are presented. When rendering in terms of solid-looking or semitransparent objects is required, an algorithm based on a simulated process of excitation and fluorescence is very suitable. Graphic techniques can be used to examine the 3-dimensional shape of surfaces. For (near-)real time applications a representation method should not require extensive previous data-processing or analysis. From the very extensive field of 3-D image analysis two examples are given.

Computerized analysis of chromosomal parameters in karyotype studies.

A study is presented of the possibilities and limitations of semi-automated karyotype analysis on the basis of chromosome length and centromere index. A number of computer programs have been developed for 1) quick and precise measurements of chromosome arm length with the help of a graphics tablet, 2) computing (relative) length and centromere index and statistical analyses of the data, and 3) representation of these chromosomal parameters in two-dimensional scattergrams. An ellipse representing 95% of the probability mass is drawn around the bivariate mean of each chromosome. The size and orientation of the axes are calculated from repeated measurements of the chromosomes of one metaphase plate. If there is a correlation between length and centromere index, which is often the case, the axes of the ellipse are tilted. Incorporation of such a covariance analysis proved to be of great importance for an accurate karyotype analysis. The "Computer Aided Karyotyping" package does not contain routines for an automated classification of the chromosomes. The main reason is that the variation in length and centromere index of a given chromosome in different cells is often much larger than the variation between nonhomologous chromosomes. In addition, it was our aim to develop universal karyotyping aids which can be used regardless of the species studied.

Testicular cell suspensions of the mouse in vitro.

A method is presented which allows survival and continued differentiation of male mouse germ cells for up to 12 days in culture. The system uses continuous flow perfusion at 20 degrees C in a completely chemically defined medium. To show differentiation through meiosis, mice were treated with hydroxyurea (HU) which creates a gap in the spermatogenic line. Suspensions of testicular cells of these mice, completely lacking pachytene/diplotene stages, were put into culture. After 7 or 10 days culturing, differentiation was demonstrated by the appearance of pachytene and diplotene stages and round spermatids. The velocity of the differentiation in vitro was found the same as would be expected in vivo. The number of cells which show differentiation throughout meiosis is less than would be expected in cultures of testicular cells of mice which were not treated with hydroxyurea.

The behaviour of silver-positive structures during meiotic prophase of male mice.

Air dried preparations and sections of mouse seminiferous tubules were stained with an ammoniacal silver solution to study the behaviour of silver positive structures in meiotic prophase I nuclei. The presence of RNA was investigated using specific staining techniques and RNase digestion. Pachytene nuclei showed silver precipitation at the paracentromeric region of two to six autosomal bivalents. In spermatogonia at least the chromosome nos. 12, 15, 16, 17 and 18 proved to have transcriptionally active nucleolus organizer regions. From late pachytene the Ag-positive structures migrate towards the sex vesicle. In prediffuse diplotene, when the silver spots reached the sex vesicle, a vacuole-like body appeared near the sex vesicle. At the same time significant amounts of RNA accumulate near to the sex vesicle. Finally, in diffuse diplotene a tripartite structure could be observed, composed of (a) a horseshoe-shaped structure adjacent to the sex vesicle, which contains a great deal of RNA, (b) a vacuole-like body, being enclosed by the horseshoe and (c) an Ag-positive mass, migrated from the nucleolus organizer regions. It is probable that the tripartite structure, or at least a part of it, is a large nucleolus. The significance of the structure is discussed.

Chromosome behaviour during early meiotic prophase of mouse primary spermatocytes.

A correlative light- and electron microscopical study has yielded both a description of chromosome behaviour during early meiotic prophase in mouse spermatocytes, and an accurate timing of the zygotene and early pachytene stage. The light microscopic part of the study consists of an analysis of orcein stained or C-banded, air-dried preparations, and series of separately squashed sections of seminiferous tubules. The mice used for air-dried preparations were treated with hyroxyurea and triaziquone, which reduces the spermatocyte population to a small, well-defined group of cells in meiotic interphase. The development of the restricted spermatocyte population is followed during four days. The electron microscopical study consists of an investigation of synaptonemal complexes in thin sections of seminiferous tubules in various stages of the epithelial cycle. It turned out that synaptic pairing starts about half a day after the end of the DNA replication phase. At most one and a half day later chromosome pairing is completed. In very early pachytene spermatocytes a sex vesicle is not visible. The orientation of the X and Y chromosome into a sex vesicle starts about one day after the beginning of the pachytene stage. The existence of a leptotene stage is discussed. The presence of a pre-leptotene contraction phase in male mice is excluded.

A sequential analysis of meiosis in the male mouse using a restricted spermatocyte population obtained by a hydroxyurea/triaziquone treatment.

A method is described to restrict the spermatocyte population in mice and other rodents using hydroxyurea (HU) and triaziquone (T). HU affects cells in S-phase, whereas T is an agent especially active on spermatogonia and not on spermatocytes. An application of three i.p. HU injections with 12 h intervals, followed about nine days later by one i.p. T injection creates two large gaps in the spermatogenic line. The two gaps enclose a small, well-defined group of primary spermatocytes in meiotic interphase. - The development of the restricted spermatocyte population is followed day by day. The analysis of meiosis in male mice has revealed the correct sequence of meiotic, and especially prophase I stages. On account of clearly visible differences in chromosome morphology the diplotene stage could be divided into three periods. It is suggested to use the following nomenclature: pre-diffuse diplotene, diffuse diplotene and post-difuse diplotene. The experiment was also informative about the timing of the stages in spermatocyte development by correlating the days at which the successive stages were observed with the corresponding stage of the epithelial cycle. The calculation of the position and duration of the diffuse diplotene, enables us to put forward a proposal about the significance of the diffuse diplotene. - A combination of the HU/T method with cell separation techniques provides good perspectives for detailed biochemical studies on processes taking place during meiosis.

A sequential screening of the cytogenetic damage induced by triaziquone.

A sequence is described of test procedures for a screening in vivo of the clastogenic potential of the alkylating agent triaziquone (Trenimon). Two intraperitoneal injections of 0.125 mg/kg body weight caused a considerable increase in the number of aberrations in both the micronucleus test and the bone-marrow metaphase test, but not in the spermatocyte translocation test or the spermatogonial metaphase test. With the latter test a severe cell-killing effect was detected. An analysis of whole mounts of seminiferous tubules showed that 0.125 mg/kg was a lethal dose for all B- and intermediate-type spermatogonia and partly killed A-type spermatogonia. A single administration of the same dose caused stable chromosomal rearrangements in spermatids that could be demonstrated with the F1 translocation test, and gave rise to dominant lethality of fetuses originating from post-meiotic sperm. The comparative triaziquone study has provided arguments in favor of the micronucleus test as a reliable screening method for chromosomal aberrations. The analysis of seminiferous tubules is a recommendable method for studying lethal effects of a compound on germ cells, whereas the F1 translocation test gives important information about viable aberrations and their effect on the fertility of the progeny.

Spermatogenesis in the Chinese hamster.

In this article a description of the process of spermatogenesis in the Chinese hamster is given. Spermiogenesis could be divided into 16 steps. The cycle of the seminferous epithelium was divided into 12 stages, coinciding with the first 12 steps of spermiogenesis. The relative and absolute duration of the stages was determined. The duration of the cycle of the seminiferous epithelium was found to be 17.0 days. The morphology of the spermatogonia was studied in seminiferous tubules mounted "in toto." Four classes of spermatogonia could be discerned; undifferentiated A spermatogonia (Ais, Apr, Aal), differentiated A spermatogonia (A1, A2, A3). In spermatogonia and B spermatogonia (B1, B2). It is interesting to note that the last generation of spermatogonia (B2) arises at the beginning of stage 7 and divides to give rise to primary spermatocytes in the second half of this stage; in most other species the last generation of spermatogonia arises in stage 4, giving rise to primary spermatocytes in stage 6. The unidifferentiated A spermatogonia were counted in six stages of the cycle, together with the differentiated A, In or B spermatogonia present in the same stages. The results of these cell counts are discussed in detail. One of the conclusions that could be drawn about the behaviour of the Ais and Apr spermatogonia during the cycle of the epithelium was that there is mitotic activity in these cells in several stages of the cycle. It is suggested that this mitotic activity serves to generate the Aal spermatogonia, which after one or more divisions transform into Al spermatogonia between stages 2 and 8.