Characterization by fluorescence and electron microscopy in situ hybridization of a double Y isochromosome.

A patient with mixed gonadal dysgenesis and Y isochromosomes i(Y) is described. Lymphocyte cultures from peripheral blood contained a high proportion of 45,X cells and several other cell lines with two different marker chromosomes (mars). These markers had either a monocentric (mar1) or a dicentric appearance (mar2). Following high-resolution GTG, RBG, QFQ, and CBG bandings, five cell lines were identified; 45,X/46,X,+mar1/46,X,+mar2/47,X,+mar1x2/47,X,+mar2x 2. The percentages were 66/6/26/1/1%, respectively. Chromosome banding analyses were insufficient for characterization of the markers. In situ hybridization of specific probes for the Y centromere and its short arm showed, both in fluorescence and electron microscopy (EM), two different Y rearrangements. Mar1 is an isochromosome for the short arm i(Yp) and mar2 is a dicentric which was shown by EM to be a double isochromosome Yp, inv dup i(Yp). The breakpoint producing mar1 is within the centromere and the one producing mar2 is within one of the short arms of the Y isochromosome. The findings of different cell populations in peripheral blood lymphocytes indicate the postzygotic instability of this i(Yp).

Cytoplasmic membrane transplants in early Xenopus embryos indicate cell-cycle-specific effects.

Fragments of rough endoplasmic reticulum or Golgi complex isolated from normal adult rat liver homogenates were injected into one cell of cleaving two-cell Xenopus laevis embryos and the effects on development were monitored during early cleavage by morphological analysis. Scanning electron microscopy revealed the formation of large cells on the injected side of the embryos. Such large cells were not present in controls and thus were considered to have been formed as a consequence of delayed cleavage. Delay of cleavage was obtained with as little as 1 ng of membrane protein giving a ratio of membrane protein to embryo protein of 1:10(5). Cytological observations of microinjected embryos confirmed the occurrence of delayed cytokinesis and suggested that nuclear division became asynchronous. Since rough microsomes from proliferating tissues (i.e., livers with primary tumors and livers undergoing regeneration) showed little or no effect on cytokinesis after microinjection into early embryos, we conclude that cytoplasmic membranes may exhibit cell-cycle-specific properties important for normal development.

Detection of small, single-copy genes on protein-G-banded chromosomes by electron microscopy.

A method for the detection by electron microscopy of chromosome banding after in situ hybridization of small, nonradioactive DNA sequences is described. Typical high-resolution G-banding is produced by adding 5-bromodeoxyuridine (BrdU) during the last part of the S-phase and by applying a monoclonal antibody against the BrdU-substituted chromosome segments, followed by the addition of protein G, but no further treatment. A protocol for in situ hybridization of small, single-copy biotinylated DNA sequences and their detection by immunogold tagging on banded chromosomes is also described. This combined approach permits high-resolution mapping of small DNA sequences and should be useful in discriminating between neighboring DNA fragments.

Simultaneous visualization of chromosome bands and hybridization signal using colloidal-gold labeling in electron microscopy.

Electron microscopy (EM) is seldom used with in situ hybridization to localize DNA sequences because banding methods for chromosome identification could not be coupled to EM techniques. We have applied an immunochemical replication-banding method specific for EM to solve this problem. A thymidine synchronization/BrdUrd release protocol allows BrdUrd incorporation only into late replicating bands. A biotinylated DNA probe is hybridized in situ to its complementary sequence. The biotinylated probe and the BrdUrd-substituted DNA are simultaneously localized by different reporter/detection systems using different-sized colloidal gold particles as electron-dense tags. We demonstrate the high precision of this mapping procedure by localizing on long prophase chromosomes (greater than 1000 bands per haploid set) the pXBR-1 sequence to a small subregion of the centromeric subband Xp11.1-Xq11.1. This localization to a part of an individual prophase subband is the most precise localization ever reported on human banded mitotic chromosomes.

Detection by electron microscopy of a small subband 13q14.11 deletion in an hereditary retinoblastoma.

High-resolution banding, specific for electron microscopy, was applied to chromosomes of synchronized blood lymphocytes obtained from a child with bilateral retinoblastoma. Ultrastructural analysis of the subbands in region q14.1, after synchronization and immunochemical banding, showed that the deletion in the abnormal chromosome 13 corresponds to subband 14.11, thus evidencing that the retinoblastoma gene is located within subband q14.11. This first application to a diagnostic problem of immunochemical banding suggests that, coupled with electron microscopy, this banding provides a higher resolution than that obtained with light microscopy and should be useful to pinpoint important localizations.

DNA denaturation for ultrastructural banding and the mechanism underlying the fluorochrome-photolysis-Giemsa technique studied with anti-5-bromodeoxyuridine antibodies.

G- and R-bands produced by an immunochemical approach were studied by electron microscopy (EM) to evaluate the role of DNA denaturation on banding quality. Excellent banding was observed only after adequate denaturation by HCl, NaOH and formamide, used in appropriate concentrations to provide uniform 5-bromodeoxyuridine (BrdUrd) exposure by generating single-stranded DNA. Formamide treatment resulted in less intercellular variability. High temperature and high concentrations of NaOH and HCl altered chromosomal morphology. Besides formamide, Hoechst 33258 prestaining which does not interfere with the binding of the anti-BrdUrd antibody and UV irradiation associated with formamide also produced high quality banding. On the other hand, consecutive Hoechst and UV treatment completely inhibited the immunochemical banding. The data indicate that Hoechst and UV act synergistically to disintegrate BrdUrd-substituted chromatin from which DNA is then extracted, leaving only the unsubstituted DNA stainable with Giemsa.

Electron microscopy of gold-labeled human and equine chromosomes.

We present an immunochemical technique for the detection of 5-bromo-2'-deoxyuridine (BrdU) incorporated discontinuously into the chromosomal DNA. A monoclonal anti-BrdU antibody and a protein A-gold complex were used to produce chromosome banding of human and equine chromosomes, specific for electron microscopy (EM). Well-defined bands, symmetry of sister chromatids, concordance between homologues, and band patterns similar to those observed by light microscopy facilitate chromosome identification and karyotyping. From prophase to late metaphase, chromosomes condense and bands appear to fuse. The fusion appears to be owing to chromatin reorganization. Our results underline the value of using immunogold reagents, which are ideal probes for antigen localization on chromosomes.

Dynamic G- and R-banding of human chromosomes for electron microscopy.

Synchronized human lymphocytes were exposed to 5-bromo-2'-deoxyuridine (BrdUrd) for incorporation in either G- or R-bands. The substituted bands were revealed by monoclonal anti-BrdUrd antibodies disclosed with either gold-labeled antibodies or with the protein A-gold complex. Sharp G- or R-banding, specific for electron microscopy (EM), was obtained. These banding patterns, referred to as GB-AAu (G-bands by BrdUrd using Antibodies and gold [Au]) and RB-AAu (R-bands by BrdUrd using Antibodies and gold [Au]), resemble dynamic band patterns (GBG and RBG) much more than they do morphologic band patterns (GTG and RHG). The G- and R-band patterns allow accurate chromosome identification and karyotyping. An actual karyotype of human GB-AAu-banded chromosomes at the 750 band level, photographed in the EM, is presented. The method produces excellent band separation and band contrast. Variations in band staining intensities were noted and correlated with BrdUrd enrichment. The C-band regions were positively stained after GB-AAu banding while they were negatively stained after RB-AAu banding. Telomeres appeared heterogeneous after GB-AAu banding suggesting that part of the telomeric bands might be late replicating.

Human chromosome banding specific for electron microscopy.

Electron microscopy (EM) provides much higher resolution than that obtained with light microscopy (LM). Until now, however, no chromosome banding procedure specifically adapted for EM was available. To produce an easy and reproducible banding method that would allow accurate chromosome identification, we investigated the applicability of an immunochemical method. BrdU-substituted chromosomal regions can be accurately visualized by applying a monoclonal antibody against BrdU, followed by a gold-tagged secondary antibody. Since BrdU is incorporated during the last part of the S-phase, regions of darkly stained G- and C-bands are substituted. A characteristic C-banding pattern is revealed, and the G-banding obtained is sharp and allows discrimination between subbands. Its similarity with the classical G-banding observed by LM makes it easy to interpret and facilitates karyotyping.

Light and electron microscopy of Ag-NORs in domestic horse chromosomes identified after R-banding.

Silver staining shows the presence in the domestic horse of six NORs located on chromosomes 1, 26 and 31 as identified after R-banding. Following electron microscopy, the argyrophilic material was observed outside the terminal secondary constrictions (satellite stalks) on the terminal portion of the short arm of chromosome 1, outside the secondary constrictions on the proximal region of the long arms of chromosome 31, and beside the proximal region of the long arms of chromosome 26. Satellite staining applied to these chromosomes appears to reveal only the active NORs.

Easy transfer of selected mitoses from light to electron microscopy.

A method is described whereby any given chromosome spread selected by light microscopy can be transferred to a grid and studied by electron microscopy.

[Elongated mitotic cells in the neuroepithelium of Xenopus laevis and Notophtalmus viridescens]. / Cellules en mitose de forme allongée dans le neuroépithélium de Xenopus laevis et de Notophtalmus viridescens.

Most neuroepithelial cells in the embryos of Xenopus laevis and Notophtalmus viridescens become round upon entering into mitosis. However, many maintain their elongated form, or show long cytoplasmic processes, during mitosis. It therefore seems possible that, in these species, two groups of microtubules are present during mitosis: cytoplasmic microtubules and mitotic microtubules.

Peculiar pattern of interkinetic nuclear migration in the newt Notophtalmus viridescens.

The distribution of mitotic figures was studied in the neuroepithelium of Notophtalmus viridescens embryos of stages 14, 16 and 18. On the average, 34% of the mitotic figures were counted near the neurocoele (here in described as zone 1), 10% were recorded in the outer portion of the epithelium (zone 3) and 56% were found between these two regions (zone 2). It is concluded that this neuroepithelium is the site of interkinetic nuclear migration although its pattern is peculiar when compared to what occurs in the chick embryo. Also, the analysis of one micron-thick serial sections showed that the neuroepithelium in Notophtalmus viridescens is pseudo-stratified throughout neurulation.

Light and electron microscope study of osmotically induced tumor necrosis.

Necrosis of 1.5-cm Walker 256 tumors was produced by injecting a strongly hypertonic solution of glucose in and around the growths and by delaying resorption of the solution with serotonin, given s.c. at a distance. The morphological changes occurring in 13 tumors were followed by light and electron microscopic analysis of samples taken 0.25, 0.5, 1, 1.5, 3, and 5 hr after treatment. The 0.25-hr samples showed mitochondrial swelling, loss of cristae, and flocculent material within the inner compartment. Swelling of the mitochondria persisted in the 0.5-hr specimens (as it did in all subsequent samples), and it was accompanied by clumping and margination of chromatin. These changes were more pronounced at 1 hr, at which time the nuclear and plasma membranes were frequently ruptured. The endoplasmic reticulum and Golgi complex could no longer be recognized. The 3-hr material revealed ruptures of the outer mitochondrial membrane with myelin figures and discontinuous cell membranes. In the 5-hr samples, the nuclei exhibited a dark nucleoplasm and large clumps of chromatin. The perinuclear membrane was not always recognizable.

The effects of high hydrostatic pressure on microfilaments and microtubules in Xenopus laevis.

Xenopus laevis embryos of stages 14-20 were subjected, for periods of 5-330 min, to hydrostatic pressures ranging from 500 to 10 000 psi. The specimens were fixed under corresponding pressures and their neuroepithelium was studied under light and electron microscopy. A pressure of 3000 psi, maintained for as long as 180 min, did not inhibit neurulation though it induced slight deformities of the neuroepithelium. A pressure of 4000 psi, applied for 180 min, disrupted the apical ring of microfilaments and blocked neurulation. The cells lost their dissymmetry. The effect was reversible. Lengthening the duration of treatment to 330 min caused the neuroepithelial cells to loose their microtubules and to become round. This situation was not reversible. Our results indicated that microfilaments are more sensitive than microtubules, that both organelles became increasingly sensitive as the exerted pressure was increased and that microtubules of older embryos exhibited a better resistance. Finally, we showed a correlation between the presence of microfilaments and the constricted state of the cellular apices and a relationship between the presence of microtubules and cell elongation.

Microtubules, interkinetic nuclear migration and neurulation.

The hypotheses dealing with mechanisms of neurulation are reviewed briefly. The phenomenon of interkinetic nuclear migration is thought to be an important factor to be considered in the invagination of the neuroepithelium in the chick embryo. Evidence is presented that implicates cytoplasmic microtubules in this phenomenon. It is suggested that microtubules not only participate in cell elongation but also that they are involved, through interkinetic nuclear migration, in the broadening of the basal region of the cells; this widening progressively creates the strain that ensures the invagination of the neuroepithelium.

The effect of LSD on the surface of neuroepithelial cells.

The neuroepithelial cells of 10 control chick embryos and of 22 exposed to lysergic acid diethylamide (LSD) (50 microgram/ml) were examined in scanning electron microscopy (SEM). In specimens exposed to LSD, the cells are swollen and their surface loses its cytoplasmic projections. Labelling techniques applied in transmission electron microscopy (TEM) show that ruthenium red attaches to the surface of the neuroepithelial cells in the form of a continuous dark line in both controls and treated specimens. However, when cationized ferritin or lanthanum is used, the label appears in the form of a continuous line in the controls whereas it is discontinuous in specimens exposed to LSD. These observations suggest that LSD alters the components of the neuroepithelial cell surface in the young chick embryo.

A stereological approach to the study of neural organogenesis in Xenopus laevis.

Stereological methods are applied to the study of structural changes undergone, during neurulation, by the neuroepithelium of early embryos of Xenopus laevis. In light microscopy, we evaluate: the section area of the neuroepithelium, the nucleocytoplasmic ratio, the volume fraction of the intercellular spaces, of the nuclei and of the cytoplasm. In electron microscopy, the volumetric density and the surface ratio of mitochondria as well as the surface density of the endoplasmic reticulum are measured. Occasionally, the data found for Xenopus are compared to those found earlier in a similar study of the chicken embryo.

[Review of the effects of cytochalasin B on cell division, cell movemnts, cell exchanges, and cell morphology]. / Revue des effets de la cytochalasine B sur la division, les mouvements, les échanges et la morphologie cellulaire.

Cytochalasin B is known to induce quiescence in cells. We present here a comprehensive view dealing with the effects of cytochalasin B on cell division, movements, exchanges and morphology. The controversial mode of action of the drug is also considered.

The effect of LSD on the histology and ultrastructure of the neuroepithelium of young chick embryos: a stereological study.

Five control chick embryos and fifteen exposed to LSD at concentrations of 2.5, 12.5 and 50 mug/ml were examined. The morphometric analysis performed at the light microscope level shows that (1) the section area of the neural tube increases with the two highest doses of lysergic acid diethylamide (LDS) employed, (2) the volume fraction of the intercellular spaces decreases with the two highest doses of LSD, (3) the volume fraction of the nuclei in the neural tube is not modified with any concentrations of LSD employed, (4) the volume fraction of the cytoplasm in the neural tube increases with all three concentrations, and (5) the nucleo-cytoplasmic ratio decreases with all three doses employed. Moreover, at the ultrastructural level, it was found that (1) the volume fraction of mitochondria in the cytoplasm decreases at doses of 12.5 and 50 mug/ml (2) the surface to volume ratio of the mitochondria is unchanged with any of the concentrations of LSD employed and (3) the surface density of the endoplasmic reticulum in the cytoplasm increases only with a dose of 2.5 mug/ml of LSD.