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PubMed1977

A light- and electron-microscope study of nuclear structure throughout the cell cycle in the euglenoid Astasia longa (Jahn).

The structure of nuclei of Astasia longa in synchronized cultures was examined at the light- and electron-microscope levels. Three types of nuclei, differing mainly in chromatin conformation, were observed during interphase and were tentatively classed in the G1, S and G2-periods. The fibrillar nucleolar regions exhibited a most complex organization and appeared to consist of convoluted, coarse filaments or nucleolonemata approximately 0.15 micrometer in diameter. Chromosome condensation was evidenced first by the longer, thicker profiles of chromatin observed in late prophase. Furthermore, the nucleolus, that persists throughout mitosis, began to elongate at late prophase. Furthermore, the nucleolus, that persists thorughout mitosis, began to elongate at this stage, simultaneously with the appearance of short, unoriented profiles of intranuclear microtubules. Chromosome condensation was complete by mid-metaphase and the nucleolus was elongated into a cylindrical shape with irregular extremities. Microtubule profiles were longer than in prophase; they were now oriented parallel to the nucleolus and frequently lay closely appressed to its sides. In anaphase, the chromosomes segregated into 2 groups, one towards each extremity of the dumb-bell-shaped nucleolus. The telophase chromosomes assumed a random orientation with respect to the still intact nucleolus. Throughout the division stages the persiting nucleolus maintained its ultrastructural organization and consisted partly of conspicuous nucleolonemal profiles which tended to be oriented along the major axis of this organelle. Nucleolar separation into 2 fragments occurred late in telophase and was followed by a reformation of daughter nuclei and initiation of cell fission during cytokinesis.

Animals↗
PubMed1983

Changes in poly(a)+ RNA during male meiosis in Lilium.

Levels of poly(A)+ RNA have been investigated at each stage of male meiosis in Lilium (var. Firecracker). Two methods were employed in this work: in one extracts from labelled meiocytes were passed through oligo(dT) columns, while in the other the specific probe [3H]poly(U) was hybridized in situ with resin-embedded sections of pollen mother cells. The label contained in the eluate from the oligo(dT) columns was measured by liquid scintillation, and the quantity of [3H]-poly(U) hybridized was determined by statistical analysis of light microscopic autoradiographs. Both techniques revealed a dramatic decline in detectable poly(A)+ RNA during prophase. Lowest levels are reached in the pachytene stage, following which a gradual restoration of this species of RNA takes place in both nucleus and cytoplasm. The data presented here provide no clear indication as to whether this fall in RNA levels is caused by the action of novel enzymes specific to the meiotic prophase, by a cessation of synthesis and the activity of normal turnover processes, or by a combination of the two. Although there is some evidence from the [3H]poly(U) hybridization study that a small peak of poly(A)+ RNA synthesis may take place in leptotene, both methods indicate that there is a very low of poly(A)+ RNA synthesis throughout prophase. The presence of poly(A)+ RNA was not detected in either the accessory nucleoli or the cytoplasmic nucleoloids that characterize the nucleus and cytoplasm of these cells. These events are considered in terms of the juncture at which they occur in the plant life-cycle.

Meiosis↗
PubMed1985

Simultaneous ultrastructural localization of Ag-NOR (nucleolar organizer region) proteins and ribonucleoproteins during mitosis, in human breast cancerous tissues.

The simultaneous ultrastructural localization of the Ag-NOR proteins and ribonucleoproteins (RNP) was carried out during mitosis in human breast cancerous tissues using a one-step silver staining method followed by acetylation. Owing to the good preservation of the cells and to the high precision of the Ag staining we were able to follow the behaviour of the argyrophilic nucleolar components from prophase to late telophase. During all the phases of mitosis the Ag-NOR proteins were associated with fibrillar RNP component (without visible fibrillar centre), which originated, during prophase, from the dense fibrillar component of the nucleolus and then integrated within the periphery of some metaphasic chromosomes as irregular structures. The Ag staining of these structures was homogeneous but lower compared to those of nucleoli during prophase. The RNP sheath that covered the chromosomes was never silver stained. From anaphase to early telophase the argyrophilic RNP structures disposed within the periphery of the chromatin mass formed roundish and then spherical structures in which the Ag-NOR proteins were clustered as centrally localized spheres. These findings showed the usefulness of our method for the precise study of the spatial relationship between the Ag-NOR proteins and the chromosomes. Thus we were able to show striking segregation of both the argyrophilic structures at the periphery of the chromatin mass and of the Ag-NOR proteins within these structures. This suggests that a high degree of the structural organization in the future nucleus is already present during anaphase. This could be related to the rapid resumption of ribosomal RNA synthesis.

Acetylation↗
PubMed2005

Shugoshin prevents dissociation of cohesin from centromeres during mitosis in vertebrate cells.

Cohesion between sister chromatids is essential for their bi-orientation on mitotic spindles. It is mediated by a multisubunit complex called cohesin. In yeast, proteolytic cleavage of cohesin's alpha kleisin subunit at the onset of anaphase removes cohesin from both centromeres and chromosome arms and thus triggers sister chromatid separation. In animal cells, most cohesin is removed from chromosome arms during prophase via a separase-independent pathway involving phosphorylation of its Scc3-SA1/2 subunits. Cohesin at centromeres is refractory to this process and persists until metaphase, whereupon its alpha kleisin subunit is cleaved by separase, which is thought to trigger anaphase. What protects centromeric cohesin from the prophase pathway? Potential candidates are proteins, known as shugoshins, that are homologous to Drosophila MEI-S332 and yeast Sgo1 proteins, which prevent removal of meiotic cohesin complexes from centromeres at the first meiotic division. A vertebrate shugoshin-like protein associates with centromeres during prophase and disappears at the onset of anaphase. Its depletion by RNA interference causes HeLa cells to arrest in mitosis. Most chromosomes bi-orient on a metaphase plate, but precocious loss of centromeric cohesin from chromosomes is accompanied by loss of all sister chromatid cohesion, the departure of individual chromatids from the metaphase plate, and a permanent cell cycle arrest, presumably due to activation of the spindle checkpoint. Remarkably, expression of a version of Scc3-SA2 whose mitotic phosphorylation sites have been mutated to alanine alleviates the precocious loss of sister chromatid cohesion and the mitotic arrest of cells lacking shugoshin. These data suggest that shugoshin prevents phosphorylation of cohesin's Scc3-SA2 subunit at centromeres during mitosis. This ensures that cohesin persists at centromeres until activation of separase causes cleavage of its alpha kleisin subunit. Centromeric cohesion is one of the hallmarks of mitotic chromosomes. Our results imply that it is not an intrinsically stable property, because it can easily be destroyed by mitotic kinases, which are kept in check by shugoshin.

Amino Acid Sequence↗
PubMed2002

Effects of proteasome inhibitor (lactacystin) and cysteine protease inhibitor (E-64-d) on processes of mitosis in Xenopus embryonic cells.

At least two different protease pathways have been implicated in the degradation that is required to control the eukaryotic cell cycle; these two pathways center on the activities of ubiquitin/proteasome and cysteine protease. The proteasome inhibitors, lactacystin and AcLLnL and the cysteine protease inhibitor E-64-d were tested for their ability to inhibit the cell cycles of Xenopus embryos. Lactacystin, AcLLnL and E-64-d all caused the complete arrest of the cell cycle. To define the specific cell cycle processes that were affected by the two inhibitors, we performed a cytological analysis. Inhibition of the cell cycle by lactacystin and E-64-d occurred during prophase and metaphase. The number of cells that arrested in prophase was 1.4-times higher in the E-64-d-treated group than in the control group and the number of arrested cells in the lactacystin-treated group was 1.4-times higher than in the E-64-d-treated group. The number of cells that arrested in metaphase was 3-to-4-times higher in the E-64-d and lactacystin groups than in the control group. These results indicate that both cysteine protease(s) and proteasomes are involved in the prophase and metaphase stages of cell division.

Acetylcysteine↗
PubMed2006

Expression patterns and action analysis of genes associated with blood coagulation responses during rat liver regeneration.

AIM: To study the blood coagulation response after partial hepatectomy (PH) at transcriptional level. METHODS: After PH of rats, the associated genes with blood coagulation were obtained through reference to the databases, and the gene expression changes in rat regenerating liver were analyzed by the Rat Genome 230 2.0 array. RESULTS: It was found that 107 genes were associated with liver regeneration. The initially and totally expressing gene numbers occurring in initiation phase of liver regeneration (0.5-4 h after PH), G0/G1 transition (4-6 h after PH), cell proliferation (6-66 h after PH), cell differentiation and structure-function reconstruction (66-168 h after PH) were 44, 11, 58, 7 and 44, 33, 100, 71 respectively, showing that the associated genes were mainly triggered in the forepart and prophase, and worked at different phases. According to their expression similarity, these genes were classified into 5 groups: only up-, predominantly up-, only down-, predominantly down-, up- and down-regulation, involving 44, 8, 36, 13 and 6 genes, respectively, and the total times of their up- and down-regulation expression were 342 and 253, respectively, demonstrating that the number of the up-regulated genes was more than that of the down-regulated genes. Their time relevance was classified into 15 groups, showing that the cellular physiological and biochemical activities were staggered during liver regeneration. According to gene expression patterns, they were classified into 29 types, suggesting that their protein activities were diverse and complex during liver regeneration. CONCLUSION: The blood coagulation response is enhanced mainly in the forepart, prophase and anaphase of liver regeneration, in which the response in the forepart, prophase of liver regeneration can prevent the bleeding caused by partial hepatectomy, whereas that in the anaphase contributes to the structure-function reorganization of regenerating liver. In the process, 107 genes associated with liver regeneration play an important role.

Animals↗
PubMed1976

Action of bleomycin on proliferating plant cells.

Bleomycin (10-(6) M) has been tested in Allium cepa L. meristems which are formed by a proliferating cell population growing under steady state conditions. Chromosome breaks were apparently induced by the antibiotic in cells in G2 period since anaphases with chromatid breaks were formed at a time shorter than G2 + prophase duration. Stimulation of entrance of G2 cells into mitosis is suggested both by an increase in the frequency of early prophases and by the study of waves of prophases in a synchronous subpopulation labelled by caffeine. Progression of other mitotic phases was unaffected. Nucleologenesis rate was increased by the antibiotic in a fashion resembling protein synthesis inhibitors. Protein synthesis is inhibited by 10-(6) M bleomycin to the same extent as 4 X 10(-6) M anisomycin. Both facts suggest that bleomycin has a direct inhibitory effect on protein synthesis in meristems. Given the nucleologenesis sensitivity to nucleolar RNA inhibition it is suggested that the antibiotic activity on nucleolar transcription is mediated through DNA.

Bleomycin↗
PubMed2002

[Disruption of organization of mitotic microtubules in root meristem cells of Allium cepa induced by chloral hydrate].

Data are presented on the effect of chlorahydrate on microtubule organization in the root meristem of Allium cepa. Our studies show that an incomplete preprophase band commonly appears during G2-prophase transition, yet the major effect is the lack of perinuclear microtubules, leading to inhibition of the prophase spindle formation and transition to C-mitosis. Upon chloralhydrate treatment of metaphase cells, we found cells with chromosomes regularly aligned within the metaphase plate and differently disorganized mitotic spindles. Concurrently, C-metaphase cells with remnants of kinetochore fibers were present. In addition, normal bipolar and abnormal irregular types of chromosome segregation were detected, this representing multipolar and diffuse anaphases. The major difference between them is the presence of polar microtubules during multipolar anaphase, and their lacking during diffuse anaphase. Alternatively, microtubule clusters between segregated groups of chromosomes are typical for cells with diffuse anaphase. During bipolar anaphase, excessive aster-like microtubules emanate from the spindle poles, and in telophase accessory phragmoplasts are observed at the cell periphery. The formation of incomplete phragmoplasts was observed after normal bipolar and abnormal chromosome segregation. We conclude that chloralhydrate may affect the nuclear surface capability to initiate the growth of perinuclear microtubules, thus blocking the prophase spindle formation. It also disturbs the spatial interaction between microtubules, which is crucial for the formation and functioning of various microtubular systems (preprophase band, spindle and phragmoplast).

Allium↗
PubMedDate not supplied

A topoisomerase II-dependent checkpoint in G2-phase plant cells can be bypassed by ectopic expression of mitotic cyclin B2.

DNA topoisomerase II is required for mitotic chromosome condensation and segregation. Here we characterize the effects of inhibiting DNA topoisomerase II activity in plant cells using the non-DNA damaging topoisomerase II inhibitor ICRF-193. We report that ICRF-193 abrogated chromosome condensation in cultured alfalfa (Medicago sativa L.) and tobacco (Nicotiana tabaccum L.) mitoses and led to bridged chromosomes at anaphase. Moreover, ICRF-193 treatment delayed entry into mitosis, increasing the frequency of cells having a pre-prophase band of microtubules, a marker of late G2 and prophase, and delaying the activation of cyclin-dependent kinase. These data suggest the existence of a late G2 checkpoint in plant cells that is activated in the absence of topoisomerase II activity. To determine whether the checkpoint-induced delay was a result of reduced cyclindependent kinase activity, mitotic cyclin B2 was ectopically expressed. Cyclin B2 bypassed the ICRF-193-induced delay before mitosis, and correspondingly, reduced the frequency of interphase cells with a pre-prophase band. These data provide evidence that plant cells possess a topoisomerase II-dependent G2 cell cycle checkpoint that transiently inhibits mitotic CDK activation and entry into mitosis, and that is overridden by raising the level of CDK activity through the ectopic expression of a plant mitotic cyclin.

Antineoplastic Agents↗
PubMed1978

[Effect of irradiation on early gametogenesis in Tilapia].

The 10 day old fries of Tilapia mossambica Peters were irradiated with 350 R dose. Dynamics of germ cells in control and after irradiation was studied. In 10 day old fry primordial germ cells and gonial cells were observed; in 15 days--mainly gonial cells were seen; in 20 days part of gonial cells move in the early prophase of meiosis; in 25 days gonial cells and oocytes of early prophase of meiosis are seen; in 30 days oogonial cells, oocytes of early prophase of meiosis and of previtellogenesis are observed. After irradiation of 13--14 day old fries some anomalies of mitosis were observed. But mass destruction of germ cells took place during the pachytene stage in 25 day old fries. The causes of the destruction of germ cells are discussed.

Age Factors↗
PubMed1994

Association of p34cdc2 kinase and MAP kinase with microtubules during the meiotic maturation of Xenopus oocytes.

p34cdc2 protein is found in prophase, metaphase and activated Xenopus oocytes at a similar level whereas its kinase activity oscillates within meiosis. Using an anti-PSTAIRE antibody that recognizes Xenopus p34cdc2, it was demonstrated that the major part of p34cdc2 was associated with microtubules isolated in vitro from Xenopus oocytes. Conversely, tubulin was recovered in association with p34cdc2 in p13-Sepharose pellets. The abundance of the fraction of p34cdc2 which was associated with microtubules did not oscillate during the meiotic maturation and the activation process. By contrast, the histone H1 kinase activity of p34cdc2 estimated in microtubular oocyte pellets was much higher in metaphase than in prophase oocytes. Cyclin B, which is associated in vivo with p34cdc2 in prophase and metaphase oocytes, was also present in the microtubular fractions. However, cyclin was not necessary for the binding of p34cdc2 to microtubules since p34cdc2 from activated eggs, where cyclin was missing, still copurified with microtubules. Purified MAP2, but not tubulin, was able to bind to p34cdc2, demonstrating that the association between p34cdc2 and microtubules was mediated by microtubule-associated proteins. During the meiotic maturation of Xenopus oocytes, several protein kinases were activated, among them MAP kinase. MAP kinase also associated with microtubules. It was demonstrated that both p34cdc2 kinase and MAP kinase purified from Xenopus oocytes were able to phosphorylate in vitro rat brain MAP2. However both protein kinases phosphorylated different domains of MAP2, suggesting that they might regulate microtubules in different ways.

Animals↗
PubMed1976

[Homoplastic transplantation of crayfish testes (Pontastacus leptodactylus leptodactylus (Eschscholtz, 1823)) in sexual rest into normal and destalked males and females: preliminary results].

Implantation of Crayfish testes in sexual rest into females leads to a new onset of spermatogenetic activity characterized by a precocious appearance of spermatocytes. However, these cells degenerate: their evolution never goes further than the pachytene stage of meiosis prophase. Abortive prophases appear continuously; this also takes place in acini which develop secondarily. Implantation of testes in sexual rest into males in the same physiological shape also leads to the precocious appearance of primary spermatocytes blocked in prophase of meiosis. However, the transplant tends to reacquire a structure similar to the one of a normal resting testis. The delay of appearance of the first spermatocytes is always shortened--less than a month instead of one month and a half--if the receptive female is destalked at the time of the implantation or if the donor male has been destalked one month before the transplantation. The interpretation of the observed phenomena is discussed.

Animals↗
PubMed1997

Degeneration of spermatocytes during meiotic divisions in the golden hamster testis.

The appearance and morphology of spontaneously degenerating meiotic spermatocytes was studied in late pubertal golden hamster testes by high resolution light and electron microscopy. While degeneration of primary spermatocytes during the long prophase of meiosis I is chiefly confined to midpachytene cells in stages VII-VIII of the seminiferous cycle, the subsequent meiotic phases (and their degenerative disorders) appear side by side in stage XIII of the cycle. Degeneration regularly concerns, in decreasing order of frequency, midpachytene primary spermatocytes (prophase I), metaphases I/II, telophases I/II, prophase II, and anaphases I/II. This closely corresponds to the apparent durations of these respective phases. In all meiotic phases studied, degeneration follows the same morphological pattern, including an initial vacuolar transformation of the cytoplasm and accumulation of densely staining material along membranes and microtubules, a progressive condensation of all nuclear and cytoplasmic structures, and the final cellular disintegration. In meta- and anaphase cells, meiotic spindle fibres are often perceivable even in advanced stages of degeneration. Both the morphological uniformity of spermatocyte degeneration and the constant susceptibility of meiotic phases to degeneration throughout meiotic divisions point to a generally similar mechanism underlying the physiological loss of meiotic spermatocytes.

Animals↗
PubMed1999

In vitro binding of free cdc2 and raf kinase to membrane vesicles: a possible new regulatory mechanism for cdc2 kinase activation in Xenopus oocyte.

The G2-M transition of the cell cycle is under the control of the M-phase promoting factor (MPF) formed of cdc2 kinase and cyclin B. The Xenopus prophase-blocked oocyte contains a stockpile of cyclin B2-cdc2 complexes that are maintained inactive by a double inhibitory phosphorylation on Thr-14 and Tyr-15 of cdc2. Free cdc2 molecules that are not associated with cyclin, are present in excess as compared to cyclin B2-associated cdc2. This pool of free cdc2 is permanently recruited to associate with neosynthetized cyclin B2 in the resting prophase oocyte, to feed up the pre-MPF stockpile. During re-entry into meiosis, free cdc2 could generate with newly synthesized cyclin B a small level of active MPF, that could serve as starter to initiate the conversion of pre-MPF into MPF. It was, therefore, of high interest to investigate whether free cdc2 interacts with other proteins and what could be its intracellular localization. To address these questions, we developed an in vitro system of membrane vesicles. We demonstrate here that free cdc2 is recovered in association with the external layer of membrane vesicles, whereas cyclin B2-associated cdc2 is not. Cyclin is able to associate in vitro with cdc2-containing membrane vesicles. This association does not induce the inhibitory cdc2 phosphorylations. However, it does not lead to active complexes, suggesting that membrane vesicles prevent cdc2 activation. C-Raf1, another kinase activated during reentry into meiosis, is also totally recovered in association with the membrane vesicles.

Animals↗
PubMed1993

Dynamics of organelles in the mitotic spindles of living cells: membrane and microtubule interactions.

The distribution and dynamics of the membranous organelles in two cell types were investigated during cell division. Live cells (either PtK2 or LLC-PK1) labeled with the vital dye 3,3'-dihexyloxacarbocyanine iodide [DiOC6(3)] were observed via serial optical sectioning with the laser-scanning confocal microscope. Z-series of labeled, dividing cells were collected every 1-2 minutes throughout mitosis, beginning at prophase and extending to the spreading of the daughter cells. Membrane distribution began to change from the onset of prophase in both cell types. When the mitotic spindle formed in prometaphase, fine tubular membranes, similar to those extending out to the edges of interphase cells aligned along the kinetochore spindle fibers. The lacy polygonal network typical of interphase cells persisted beneath the spindle, and a membrane network was also associated with the dorsal layer of the cell. As PtK2 cells reached metaphase, their spindles were nearly devoid of membrane staining, whereas the spindles of LLC-PK1 cells contained many tubular and small vesicular membranous structures. X-Z series of the LLC-PK1 metaphase spindle revealed a small cone of membranes that was separated from the rest of the cytoplasm by kinetochore MTs. In both cell types, as chromosome separation proceeded, the interzone remained nearly devoid of membranes until the onset of anaphase B. At this time the elongating interzonal microtubules were closely associated with the polygonal network of endoplasmic reticulum. Cytokinesis caused a compression, and then an exclusion of organelles from the midbody. Immunofluorescence staining with anti-tubulin antibodies suggested that spindle membranes were associated with microtubules throughout mitosis. In addition, taxol induced a dense and extensive collection of small vesicles to collect at the spindle poles of both cell types. Nocodazole treatment induced a distinct loss of organization of the membranous components of the spindles. Together these results suggest that microtubules organize the membrane distribution in mitotic cells, and that this organization may vary in different cell types depending on the quantity of microtubules within the spindle.

Anaphase↗
PubMed1993

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.

Algorithms↗
PubMed1995

Culture of pachytene spermatocytes for analysis of meiosis.

An impediment to the investigation of mammalian spermatogenic meiosis has been the lack of an appropriate system for experimental manipulation of meiotic prophase cells. We report here the use of a simple system for the short-term culture of pachytene spermatocytes. We have assayed parameters of cell function pertinent to meiotic prophase, namely chromosome pairing and synapsis. During the culture period of 24-48 hr, cells maintained typical pachytene morphology, chromatin condensation patterns, and chromosome pairing, as assessed by light and electron microscopy. Uridine incorporation, monitored by autoradiography, reflected the chromosomal distribution found in vivo in that the autosomal chromosomes were transcriptionally active, while the sex chromosomes were not. Thus features of chromosome pairing and sex chromatin inactivation are maintained in these cultures. We have conducted experiments to demonstrate that cultured pachytene spermatocytes can be useful for the analysis of agents, some of which may be suspected mutagens, that might affect chromosome structure and function during meiosis. Treatment of cells with actinomycin D revealed a differential effect on chromatin condensation in the autosomes versus the sex chromosomes. Camptothecin, a topoisomerase inhibitor, induced desynapsis of paired chromosomes. Okadaic acid, a phosphatase inhibitor, induced premature metaphase-I condensation of pachytene chromosomes. This last experiment suggests that these cultured cells may be useful for analysis of meiotic cell cycle controls. Taken together, these results demonstrate a culture system that can be useful for analysis of meiotic events as well as in screening for potential mutagenic agents that might affect meiotic chromosome structure and function.

Animals↗
PubMed1982

The prolongation of mitotic stages in SV40-transformed vs nontransformed human fibroblast cells.

Early studies on the duration of mitotic stages and on metaphase-to-prophase ratios in a number of normal and neoplastic cells indicated that the process of mitosis becomes altered during the course of oncogenesis. However, the nature of these changes and their effects on each of the mitotic stages are still unclear. With the use of time lapse cinemicrography, we have compared the durations of mitotic stages of SV40/WI-38 and SV40/WI-26 cells to those of their normal counterparts and to other nontransformed human fibroblasts. We also examined the relative frequencies of the individual mitotic stages in fixed preparations of WI-38 and SV40/WI-38 cells. The data show that metaphase durations are increased in the transformed cells and as much as 3-4.7-fold in SV40/WI-38 cells compared to WI-38 and other nontransformed cells. Other stages are also prolonged though to lesser degrees. These findings suggest that increased metaphase/prophase ratios observed in many tumors are due to increases in duration of metaphase rather than to shorter prophases, and that increased mitotic indices commonly observed in malignant tumors and sometimes used as an index of growth rate are at least in part due to the prolongation of mitotic stages.

Anaphase↗