Chromosome Plates Research Articles

Offspring born from chimeras reconstructed from parthenogenetic and in vitro fertilized bovine embryos.

Chimeric embryos were produced by aggregation of parthenogenetic (Japanese Red breed) and in vitro fertilized (Holstein breed) bovine embryos at the Yamaguchi Research Station in Japan and by aggregation of parthenogenetic (Red Angus breed) and in vitro fertilized (Holstein breed) embryos at the St. Gabriel Research Station in Louisiana. After embryo reconstruction, live offspring were produced at each station from transplanting these embryos. The objective of this joint study was to evaluate the developmental capacity of reconstructed parthenogenetic and in vitro fertilized bovine embryos. In experiment I, chimeric embryos were constructed: by aggregation of four 8-cell (demi-embryo) parthenogenetic and four 8-cell stage (demi-embryo) IVF-derived blastomeres (method 1) and by aggregation of a whole parthenogenetic embryo (8-cell stage) and a whole IVF-derived embryo (8-cell stage) (method 2). Similarly in experiment II, chimeric embryos were constructed by aggregating IVF-derived blastomeres with parthenogenetic blastomeres. In this experiment, three categories of chimeric embryos with different parthenogenetic IVF-derived blastomere ratios (2:6; 4:4, and 6:2) were constructed from 8-cell stage bovine embryos. In experiment III, chimeric embryos composed of four 8-cell parthenogenetic and two 4-cell IVF-derived blastomeres or eight 16-cell parthenogenetic and four 8-cell IVF-derived blastomeres were constructed. Parthenogenetic demi-embryos were aggregated with sexed (male) IVF demi-embryos to produce chimeric blastocysts (experiment IV). In the blastocyst stage, hatching and hatched embryos were karyotyped. In experiment V, chimeric embryos that developed to blastocysts (zona-free) were cryopreserved in ethylene glycol (EG) plus trehalose (T) with different concentrations of polyvinylpyrrolidone (PVP; 5%, 7.5%, and 10%). In experiment I, the aggregation rate of the reconstructed demi-embryos cultured in vitro without agar embedding was significantly lower than with agar embedding (53% for 0% agar, 93% for 1% agar, and 95% for 1.2% agar, respectively). The aggregation was also lower when the aggregation resulted from a whole parthenogenetic and IVF-derived embryos cultured without agar than when cultured with agar (70% for 0% agar, 94% for 1% agar, and 93% for 1.2% agar, respectively). The development rate to blastocysts, however, was not different among the treatments. In experiment II, the developmental rates to the morula and blastocyst stages were 81%, 89%, and 28% for the chimeric embryos with parthenogenetic:IVF blastomere ratios of 2:6, 4:4, and 6:2, respectively. In experiment III, the developmental rate to the morula and blastocyst stages was 60% and 65% for the two 4-cell and four 8-cell chimeric embryos compared with 10% for intact 8-cell parthenogenetic embryos and 15% for intact 16-cell parthenogenetic embryos. To verify participation of parthenogenetic and the cells derived from the male IVF embryos in blastocyst formation, 51 embryos (hatching and hatched) were karyotyped, resulting in 27 embryos having both XX and XY chromosome plates in the same sample, 14 embryos with XY and 10 embryos with XX. The viability and the percentage of zona-free chimeric embryos at 24 hr following cryopreservation in EG plus T with 10% PVP were significantly greater than those cryopreserved without PVP (89% vs. 56%). Pregnancies were diagnosed in both stations after the transfer of chimeric blastocysts. Twin male (stillbirths) and single chimeric calves were delivered at the Yamaguchi station, with each having both XX and XY chromosomes detected. Three pregnancies resulted from the transferred 40 chimeric embryos at the Louisiana station. Two pregnancies were lost prior to 4 months and one phenotypically-chimeric viable male calf was born. We conclude that the IVF-derived blastomeres were able to stimulate the development of bovine parthenogenetic blastomeres and that the chimeric parthenogenetic bovine embryos were developmentall

Karyotyping of human oocytes by chromosomal analysis of the second polar bodies.

This paper describes a method for obtaining metaphase chromosomes from human second polar bodies. The second polar body nucleus was injected into the cytoplasm of an enucleated oocyte, which is activated shortly after injection. When the polar body nucleus is transformed into a haploid pronucleus, treatment with okadaic acid was used to induce premature chromosome condensation. A total of 25 analysable chromosome plates were obtained from 38 polar bodies karyotyped using this technique. Whole chromosome painting was used to detect second polar bodies (and respectively, oocytes) with unbalanced translocations. In combination with the first polar body analysis, this technique may be useful in preimplantation genetic diagnosis for patients carrying maternal translocations.

Preparation of chromosome spreads for electron (TEM, SEM, STEM), light and confocal microscopy

In the past, ultrastructural studies on chromosome morphology have been carried out using light microscopy, scanning electron microscopy and transmission electron microscopy of whole mounted or sectioned samples. Until now, however, it has not been possible to use all of these techniques on the same specimen. In this paper we describe a specimen preparation method that allows one to study the same chromosomes by transmission, scanning-transmission and scanning electron microscopy, as well as by standard light microscopy and confocal microscopy. Chromosome plates are obtained on a carbon coated glass slide. The carbon film carrying the chromosomes is then transferred to electron microscopy grids, subjected to various treatments and observed. The results show a consistent morphological correspondence between the different methods. This method could be very useful and important because it makes possible a direct comparison between the various techniques used in chromosome studies such as banding, in situ hybridization, fluorescent probe localization, ultrastructural analysis, and colloidal gold cytochemical reactions.

Chromosomes of Pelichnibothrium speciosum (Cestoda: Tetraphyllidea)

The results obtained from the analysis of Giemsa-stained mitotic chromosome plates of P. speciosum are the first data on the karyology of Tetraphyllidea. The karyotype consists of eight pairs (2 n = 16) of chromosomes ranging from 1.62 to 7.47 μm long. The first large pair of homologues contributes 26.61% to the total chromosome length and is of intermediate meta-submetacentric structure. Chromosomes 2, 3, 5, 6, 7 and 8 are clearly acrocentric. The homologues of the fourth pair possess an intermediate submeta-subtelocentric structure. The prevailing acrocentric elements in the chromosome set indicate the rather primitive karyotypic state of P. speciosum

Ultrastructure of mitosis in Diplonema ambulator larsen and patterson (euglenozoa)

The fine structural features of mitosis are described in a small biflagellated phagotrophic protist, Diplonema ambulator. Flagella replicate early in mitosis and move to opposite sides of the nucleus where they remain lateral to the spindle poles. An array of microtubules diverge from two foci located on opposite sides of the nucleus adjacent to the nuclear envelope. At metaphase, the chromosomes compact and align to form a distinct ring around the elongating nucleolus. The chromosomal plates then separate and move towards the poles followed by elongation of the nucleus. By telophase, the nucleus and nucleolus assume a dumbbell-shape. Constriction of the nuclear envelope around the chromosomal masses cleaves the nucleolus and produces the daughter nuclei. The nuclear envelope and nucleolus persist through mitosis. A comparison of the ultrastructural features of mitosis in Diplonema with that found in the euglenoids, the group with which they are believed to be most closely related, reveals marked differences.

Micronucleus test in 2-cell embryos as a simple assay system for human sperm chromosome aberrations

A micronucleus test method to assess radiation-induced chromosomal damage in human spermatozoa is described, and its efficiency examined by comparison with that of sperm chromosome analysis. Human spermatozoa were exposed in vitro to 1.11 and 2.13 Gy of 137Cs γ-rays at a dose rate of 1.36 Gy/min. After interspecific in vitro fertilization of irradiated spermatozoa with zona-free hamster oocytes, a total of 193 monospermic eggs were examined with the micronucleus test at the 2-cell stage, and a total of 304 male pronuclear chromosome plates were analyzed according to our established method. The incidence of 2-cell embryos with micronuclei coincided well with the incidence of spermatozoa with chromosomal breaks and fragments (51.6% vs. 50.3% in the 1.11-Gy group and 82.7% vs. 79.3% in the 2.13-Gy group). A similar correlation was also found between the number of micronuclei per embryo and the number of breaks and fragments per spermatozoon (0.85 vs. 0.88 and 1.50 vs. 1.45 in the 2 dose groups, respectively). These results indicate that our micronucleus test is useful as a simple and rapid method for assessing the clastogenic effects of various environmental mutagens on human sperm chromosomes.

A morphologic study of unfertilized oocytes and abnormal embryos in human in vitro fertilization

The morphology of human, unfertilized oocytes and abnormal embryos cultured in vitro for 48-72 hr was examined in an attempt to learn more about oocyte maturation and reproductive failure in in vitro fertilization (IVF). About 21% of the unfertilized oocytes were totally degenerated. The majority (56%) of the remaining oocytes was arrested at the metaphase II stage. They contained coherent chromosomal plates and had extruded the first polar body with nuclear material. About 13% of oocytes underwent spontaneous activation. In most of these cases the second polar body was retained and many subnuclei or one big nucleus was formed. Five percent of metaphase II oocytes penetrated by sperm were not activated, likely as a result of oocyte immaturity. The developmental ability of abnormal embryos was poor. Several one-cell-stage zygotes were arrested at the pronuclear stage or at mitosis of the first mitotic division. Polyspermic embryos, especially those which contained four or more pronuclei, did not divide or formed uneven, multinucleated blastomeres. However, some triploid and tetraploid embryos often appeared normal morphologically despite their lethal chromosomal abnormalities.

Quantitative karyotyping of barley chromosomes by image analysis methods

Barley chromosomes were analyzed by the chromosome image analyzing system, CHIAS. Stained and unstained diploid metaphase spreads were automatically scanned and their locations on the glass slide were detected. With stained chromosomes, detection efficiency of good metaphase plates exceeded on average 90%. Three image parameters, length, area, and density volume, of each chromosome were defined and measured for 250 haploid chromosome plates. Of these parameters, total length and the arm ratio of the length were the most informative for chromosome identification. A quantitative idiogram of the N-banded barley chromosomes was established from numerical data of phase-contrast images and the image analysis of N-banded chromosomes.Key words: Hordeum vulgare, CHIAS, image analysis, automatic scanning, quantitative idiogram.

Ultrastructure of cell division in the unicellular red alga Flintiella sanguinaria

Cell division was examined with the electron microscope in the unicellular red alga Flintiella sanguinaria. Flintiella morphologically resembles Porphyridium purpureum, the only other red algal unicell that has been examined for ultra-structural details of cell division. Both genera are in the order Porphyridiales (Bangiophyceae), an unnatural assemblage of reduced forms in great need of taxonomic revision. Before mitosis, the nucleus migrates to the cell periphery. As in all red algae examined, except Porphyridium, the nucleus-associated organelle is a polar ring. At prometaphase, one or two microtubule-containing cytoplasmic invaginations penetrate the nucleus at each pole. By metaphase an intranuclear spindle is found in association with a typical metaphase chromosomal plate. The nuclear envelope is intact except for a single, large gap at each pole. Perinuclear endoplasmic reticulum is absent. An elongated anaphase interzonal midpiece is formed which breaks down at telophase. The daughter nuclei are widely separated by the chloroplast, which is constricted at the same time as daughter cell formation occurs by a cleavage furrow associated with a finely filamentous region similar to the contractile ring found in many animal cells. Because of pronounced differences in several mitotic features, it is concluded that Flintiella is not closely related to Porphyridium and instead shows closer phylogenetic ties with other macroscopic genera.

Mitosis and cytokinesis in Chroomonas africana Meyer & Pienaar (Cryptophyceae)

Mitosis and cytokinesis have been examined in a recently described species of Chroomonas , viz. Chroomonas africana Meyer & Pienaar. A comparison between this species and the three other species in which cell division has been studied ( Chroomonas salina and two Cryptomonas spp.) has revealed certain common characteristics such as an open spindle, a chromosomal plate at metaphase and similar types of spindle microtubules with indistinct kinetochores. However, the cell division process in C. africana differs from that in the above mentioned species in that (i) the spindle microtubules maintain their association with the flagellar bases throughout mitosis, (ii) the movement of the chromatin masses at anaphase is synchronous but unequal, i.e. parts of the chromatin masses move at different rates, (iii) in late anaphase and early telophase the chromatin masses arch around the flagellar bases forming cup-shaped structures and (iv) cytokinesis is accomplished by an extension of the anterior vestibular region forming a cleavage furrow which extends posteriorly. The formation of the branched vestibular region is also described.

FINE STRUCTURAL STUDIES ON THE INTERPHASE AND DIVIDING APICAL CELLS OF SPHACELARIA TRIBULOIDES (PHAEOPHYTA)1

ABSTRACTThe apical cells of Sphacelaria tribuloides Menegh. are larger than other thallus cells, contain more organelles and appear polarized. Their tip portion, where they grow, contains a well developed Golgi apparatus, abundant endoplasmic reticulum (ER) membranes, mitochondria, chloroplasts and a large number of small vacuoles. It seems likely that a continuous flow of membranous material from the ER membranes to the dictyosomes and from the latter to the plasmalemma of the extending tip portion takes place. In contrast, the basal pole possesses fewer organelles and is occupied mainly by large‐sized, sometimes central vacuoles. The apical cells undergo two distinct types of highly asymmetrical differential divisions giving rise to cells of the thallus and hair initials. During the early stages of mitosis the nuclear envelope remains intact, except for fenestrated poles. Microtubules pass through the fenestrae into the nucleoplasm. During meta‐phase, a typical chromosome plate is organized. The sites of attachment of spindle microtubules to the chromosomes are structurally different from the rest of the chromosomes. At late anaphase, the nuclear envelope breaks down completely. During telophase, a new membrane encloses the chromosomes which are decondensed and the nucleoli are reorganized. Cytokinesis proceeds long after mitosis at a stage in which the nuclei have increased in size and have moved farther apart. A membranous furrow develops centripetally, without the participation of microtubules. However, microtubules traverse the thin cytoplasmic strands which, in both interphase and cytokinetic cells, meander among the vacuoles of the basal pole of the cell and the internuclear space. Dictyosomes appear to be involved in the subsequent wall deposition.

Mitosis in the freshwater red algaBatrachospermum ectocarpum

Mitotic ultrastructure was observed in meristematic cells of carposporophyte generations of the freshwater red algaBatrachospermum ectocarpum. Prior to nuclear division, cell elongation occurs resulting in the nucleus being located at the proximal end of the cell and separated by a large central vacuole from a distal “empty region” which the daughter nucleus eventually occupies. In late prophase, nuclear envelope-attached polar rings are situated adjacent to shallow nuclear invaginations. At prometaphase the nuclear invaginations extend deeply into the nucleus forming continuous cytoplasmic channels containing microtubules. Perinuclear ER and a typical equatorial chromosomal plate are formed by metaphase at which time the nuclear envelope lining the cytoplasmic channels has dispersed. Chromosomal and non-chromosomal microtubules converge at the poles where a single, large gap is seen in the otherwise intact nuclear envelope. Polar rings were not detected in the few mitotic cells observed beyond prometaphase but are thought to be present. During anaphase an interzonal midpiece is formed and the distal-most incipient daughter nucleus moves laterally past the central vacuole into the apical “empty region”. Features of mitosis inBatrachospermum are believed to be intermediate between those exhibited by the lower and higher orders of red algae, this being consistent with the taxonomic placement of the genus in theNemaliales, the least advanced order of the classFlorideophyceae.

The Dynamics of Micronuclear Mitosis in the Living Ciliate Spirostomum teres as Revealed by Polarization Microscopy1

ABSTRACT. Micronuclear mitosis in living Spirostomum teres has been studied by sensitive polarization microscopy, and the dynamic aspects of micronuclear division are described. The small, spherical, interphase micronuclei lie in form‐fitting depressions in the macronuclear surface. Nuclear division begins with the rounding and slight swelling of the macronucleus and, coincidentally, the micronuclei move out of the depressions and away from the macronucleus, increase in size, and become weakly birefringent. As mitosis proceeds, the micronuclei increase in uniaxial birefringence and elongate to form irregular ovoids that convert to angular structures displaying principal axes of positive birefringence so divergent as to appear oriented at a right angle to one another. Micronuclei maintain this appearance for as long as 60 min and then abruptly change to rectangular‐shaped structures, increase in uniaxial birefringence, and begin anaphase elongation. The somewhat dumbbell‐shaped micronuclei lengthen at the constant rate of 2.0 μm/min to reach lengths >70 μm. It appears that little half‐spindle shortening occurs during spindle elongation. Accompanying the changes in micronuclear spindle length are changes in birefringence. Just before elongation begins, presumably metaphase, the micronucleus is uniformly and intensely birefringent. At the magnifications employed, a chromosome plate is not clearly visible as a region of reduced birefringence. As elongation begins, the putative half‐spindles are more birefringent than is the interzone, a condition that is maintained until the spindles have achieved ∼30% elongation, at which time a region of increased birefringence develops at the center of the interzone. This pattern persists for a very short time, then gives way to a uniform birefringence of the entire separation spindle that is maintained until elongation is completed. The rate of micronuclear spindle elongation, changes in micronuclear dimensions, and corresponding changes in birefringence are discussed with respect to possible mechanisms of mitosis.

MITOSIS IN THE OCTAFLAGELLATE PRASINOPHYTE,PYRAMIMONAS AMYLIFERA(CHLOROPHYTA)1

ABSTRACTCell division is described in the octaflagellate prasinophytePyramimonas amyliferaConrad and is compared in related genera. Basal bodies replicate at preprophase and move toward the poles. Cells remain motile throughout division. The nuclear envelope disperses and chromosomes begin to condense at prophase. Pairs of multilayered kinetochores are evident on the chromosomes of the metaphase plate. Spindle microtubules extending from the region of the basal bodies and rhizoplasts attach to the kinetochores or extend from pole to pole. Numerous vesicles and ribosomes have entered the nuclear region and the incipient cleavage furrow invaginates. The chromosomes move toward the poles at anaphase leaving a broad interzonal spindle between the two chromosomal plates. The nuclear envelope reforms first around the chromatin on the side adjacent to the spindle poles and later on the interzonal side. The cleavage furrow progresses into the interzonal spindle at telophase. By late telophase the nucleoli have reformed and the chromosomes have decondensed. The interzonal spindle has not been observed late in telophase. As the cleavage furrow nears completion the cells begin to twist and contort, ultimately separating the two cells.

Influence of chew on swallow: Robert J. NelsonJ.A.M.A. 241:4, Jan. 19, 1979

Lysyl oxidases (LOXs) are enzymes that permit the covalent crosslinking of the component chains of collagen and elastin. These enzymes are present inside the nuclei of certain mammalian cells. Previous studies have proposed LOX binding to histone H1 in vitro, and histone H1 is known to control global chromatin compaction and mitotic chromosome architecture. Therefore, in the present study, we analyzed chromatin supraorganizational changes, mitotic abnormalities, mitotic indices and cell death ratios in COS-7 and NRK-49F cells with high and low lox expression levels, respectively. The objective was to support biochemical data of LOX–H1 interaction, by providing evidence of chromatin remodeling in vivo, under different lox expressions. Chromatin decondensation assessed by image analysis was observed in COS-7 cells with increased lox expression. This decondensation is suggested to be promoted by LOX actions on histone H1, which loosens the DNA–H1 complex. In NRK-49F cells transfected with antisense lox or subjected to treatment with beta-aminopropionitrile (BAPN), chromatin condensation and nuclear phenotypic variability were found, which may be due to reduced LOX–H1 interaction. When lox expression was increased in COS-7 cells, the frequency of irregular chromosome plates was not affected, but cell proliferation decreased and “cell death preceded by multinucleation” increased. In NRK-49F cells there was accelerated proliferation induced by transfection with the antisense lox, and confirmed when cells were treated with BAPN. Apoptosis increased in NRK-49F cells only with BAPN treatment whereas cell death preceded by multinucleation increased only after antisense lox transfection. The data presented herein regarding chromatin remodeling indirectly support the hypothesis that LOX binds to histone H1 in vivo. Cell proliferation in COS-7 and NRK-49F cells and cell death at least in COS-7 cells agree with predicted effects of LOX interference in these processes.

Visualization of centriole-bodies using silver stain

Duplicate staining of human metaphase chromosomes, first with Giemsa followed by silver, revealed the presence of two small silver-stained bodies not seen in the Giemsa stained metaphases. Similar bodies were subsequently found in the metaphases of several animal groups. The size, structure, spatial relationships to the nucleus, behavior throughout the cell cycle, and apparent universal presence of these bodies suggest that they are either centrioles or associated centriolar structures. These centriole-bodies can be seen throughout the cell cycle, even in hypotonically spread C-metaphase chromosome plates. The silver stain procedure allows enough resolution to distinguish parent and daughter centriole-bodies at interphase, thus permitting visualization of the replication, maturation and separation stages of these bodies with the light microscope.

Malorientation of mitotic figures in the early lens rudiment of aphakia mouse embryos.

In early embryonic epithelia most cell divisions are oriented with their cleavage furrows perpendicular to the free surface of the tissue. This results in the generation of two daughter cells, which are equivalent with regard to their apical surface. Mitotic figures, in which the long axis through the centers of the chromosome plates (chromatin masses) is perpendicular to the surface, are seen only occasionally. The lens placode and early lens cup of mouse embryos homozygous for the gene aphakia show significantly greater numbers of maloriented mitoses than normal control lenses. The axis connecting the centers of the chromosomal plates is frequently perpendicular or oblique to the surface. This may result in the production of daughter cells, which are unequal as far as their position at the tissue surface is concerned. Later, the lumen of the mutant lens vesicle fills up with cells, which are apparently released from the lens epithelium. Further development of the lens and the eye is grossly disturbed. The release of these cells may be a consequence of the malorientation of their divisions, or both may be the result of a defect in the spatial organization of the lens tissue, possibly due to abnormal cellular adhesion.

Spermatogonial Budding in Fleabeetles

SUMMARYIn the subtribe Oedionychina of the fleabeetles, there are only four spermatocytes I per cyst, and the same number of spermatogonia per cyst just before meiosis. Observation of living spermatogonia shows that (at least) the last spermatogonial mitosis is unequal, resulting in budding of small cytoplasmic bodies from each cell. When the anaphasic spindle elongates, one of the chromosome plates does not move much from the equator, whereas the opposite one reaches the cell membrane and buds out with a small amount of cytoplasm. It is believed that cytoplasmic polarization causes the budding: at the budding pole, polar gelification seems to be lacking, rendering that pole little resistant against the pushing action of the elongating spindle, whereas at the opposite pole, the cytoplasm seems to be particularly dense and resistant.Evolution may have utilized the budding to limit reduction of cell size that otherwise occurs in succesive cell cycles of definitive gonia. Giant sex chromosomes that arrange t...

Anaphase structure in mitotic cells typified by spindle elongation

Cells that are typified by anaphase spindle elongation have been chosen from five species and examined by electron microscopy. Filament morphology including diameter of individual filaments is constant throughout metaphase and anaphase within the limitations of the method employed. Filaments have been observed in the interzone in numerous anaphase mitotic figures, although the number of filaments there is less than on the poleward sides of the chromosome plates. During late anaphase in these cells, filaments disappear poleward first and then progressively toward the cleavage plane, and a disarrangement of filaments may be observed near their ends. Filaments are seen characteristically in parallel arrays, but definite perpendicular orientations have been observed in several cases. Golgi bodies and mitochondria may persist with largely unaltered morphology throughout division.

The behaviour of certain nuclear antigens in mitosis

Human sera containing auto-antibodies against three distinct components of cell nuclei have been used with the fluorescent antibody technique to study the fate of their respective antigens during mitosis in He La cells. The distribution of deoxyribonucleoprotein is shown to follow the chromosomes, whereas a soluble nuclear protein and an unidentified constituent of the nucleolus do not take part in the formation of the chromosomes and are found as a halo surrounding the chromosomal plate. These antigens return to the nucleus during telophase.