Meiotic restitution in wheat-barley hybrids

Meiotic restitution occurs in pollen mother cells (PMCs) of reciprocal F₁ hybrids between wheat and barley. In occasional PMCs, all or most of the 28 chromosomes assemble at the equatorial plate at metaphase I, but instead of undergoing anaphase I separation they reform into a mass of chromatin to form a restitution nucleus. Some of these restituted nuclei undergo a regular second division and dyads are produced among other non-restituted cells which have reached the tetrad stage of division. Other restituted nuclei fail to undergo a second division and then the PMCs appear as monads among neighbouring tetrads. Both the monads and dyads are expected to produce microsporocytes with the diploid complement of chromosomes. Chromosomes which fail to become included in the restituted nucleus form separate micronuclei and, depending on whether they undergo a regular second division or not, the PMCs containing them eventually appear as tetrads, triads or dyads. These partially restituted nuclei are expected to produce unreduced gametes, deficient for one or more chromosomes. It is postulated from these observations that restitution in wheatbarley F₁ hybrids depends on a high frequency of univalent accumulation at the equatorial plate at metaphase I and the subsequent failure of the chromosomes to undergo anaphase I separation.     

Diffuse kinetochores and holokinetic anaphase chromatin movement during mitosis in the hemipteran Agallia constricta (leafhopper) cell line AC-20

Mitosis in the hemipteran Agallia constricta (leafhopper) cell line AC-20 was examined by light microscopy of living and fixed cells. During early prometaphase the numerous small (0.30-3.0-microns) chromosomes appear as discrete units that lack a primary constriction. However, by late prometaphase the chromosomes are tightly packed at the spindle equator and are no longer clearly resolvable as individuals. When viewed from the side the metaphase chromatin appears as a 2-3-microns wide band that spans the width of the spindle; when viewed from the pole it appears as a fenestrated disk. The metaphase chromatin splits at anaphase into two sister chromatin plates, each of which exhibits holokinetic poleward movement, i.e., all parts of each plate move as a single unit with the same velocity. In many early-to-mild anaphase cells the separating sister plates are connected by chromatin-containing bridges that break as anaphase progresses. Ultrastructural analyses of serial thick and thin sections from cells fixed by conventional, OsO4/KFeCN, or high pressure rapid freezing methods, reveal that by metaphase all of the chromosomes are interconnected to form a large, irregularly shaped fenestrated disk of chromatin. Similar analyses reveal that adjacent chromatids remain interconnected throughout anaphase. Each disk of metaphase and anaphase chromatin contains numerous kinetochores recessed within its pole-facing surface. Kinetochores consist of a fine, faintly staining fibrillar material arranged along the chromatin surface as thin (0.1-0.3 micron dia.) rods varying considerably (0.15-2.3 microns) in length. From these observations we conclude that the polycentric metaphase chromatin of A. constricta, and its holokinetic behavior during anaphase, arises from the aggregation or cohesion of smaller prometaphase chromosomes, each of which contains a single, diffuse kinetochore.     

Rhodamine-labelled phalloidin stains components in the chromosomal spindle fibres of crane-fly spermatocytes and Haemanthus endosperm cells.

In crane-fly spermatocytes and Haemanthus endosperm, all metaphase and anaphase chromosomal spindle fibres were stained with rhodamine-labelled phalloidin. In crane-fly spermatocytes, each kinetochore was stained with rhodamine-labelled phalloidin at diakinesis of prophase and after colcemid caused metaphase spindles to depolymerize. Since phalloidin stains actin filaments, the distributions of rhodamine-labelled phalloidin-stained material in crane-fly spermatocytes and Haemanthus endosperm suggest that actin filaments might interact with microtubules to produce forces that move chromosomes during cell division, either directly or via an intermediate motor molecule.     

Localization and development of kinetochores and a chromatid core during meiosis in grasshoppers

Positive staining of kinetochores and a chromatid core has been achieved using a simplified silver staining method in squash preparations from meiotic chromosomes of grasshoppers. This technique permits the exact localization of kinetochores on the chromosomes whether metacentric, acrocentric or ‘telocentric’. The sister kinetochores can be observed from mid-diplotene stages but they are not differentiated during first meiotic metaphase. However they can be observed again at the onset of anaphase 1. The existence of a positively stained chromatid core in meiotic divisions is also reported. This core appears well defined inside each chromatid from diplotene to the end of the second meiotic division. The visualization of these cores in first meiotic metaphase clearly shows the points at which the chiasmata took place.     

A mutation in the spindle checkpoint arresting meiosis II in Brachiaria ruziziensis.

Cytological characterization of BRA005568 accession of Brachiaria ruziziensis (2n = 2x = 18) showed a totally unexpected high frequency of abnormal meiotic products, from triads to hexads, and also tetrads with micro nuclei or microcytes. Meiosis I had a low frequency of abnormalities, mainly related to the chiasma terminalization process. In meiosis II, however, frequency of abnormalities increased exceptionally. Early prophase II was normal with the chromosome set enclosed by the nuclear envelope. However, in late prophase II, owing to the breakdown of the nuclear envelope, the chromosomes were scattered in the cytoplasm. Some chromosomes did not reach the metaphase II plate and remained scattered. The behavior of sister cells was inconsistent. While in one cell the chromosomes were totally aligned at the metaphase II plate, in the other they could be found completely scattered, leading to an asynchronous cell division. Cells with scattered chromosomes were unable to progress in meiosis. Thus, anaphase II failed to occur and sister chromatids were not released. Cells with non-aligned chromosomes in the metaphase II plate did not receive the "go ahead" sign to initiate anaphase II. Consequently, the scattered chromosomes produced telophase II nuclei of different sizes in situ. The asynchronous behavior led to the formation of a wide range of meiotic products. Results suggest that the present accession contains a mutation affecting the spindle checkpoint that arrests the second meiotic division.     

Chromosome structure XIII

Summary The history of chromonemata was studied in Dissosteira Carolina L. It was found that each chromosome consists of 2 chromatids at all stages which themselves are again split into half-chromatids or chromonemata proper.Multiplication of threads is believed to occur at metaphase, the cycle being strictly analogous to that of plants with large chromosomes such as Trillium, Tradescantia, Hordeum and Secale, especially the two latter. Four threads per chromosome were observed during the last premeiotic telophase, during diaphase and diakinesis. During leptotene only 2 threads can be seen because the chromosome attenuates so much that half-chromatids can no longer be resolved. The dyad chromosome contains 8 threads which were observed in first telophase and interkinesis. The chromosome of the second anaphase contains four threads again, as the threads do not multiply during the second metaphase.The chromosome threads in Dissosteira Carolina L. are not so easily fixed as those of large plant chromosomes. The resulting images are more diffuse and less obvious, nevertheless the evidence obtainable appears cogent.Approved by the Director of the New York State Agricultural Experiment Station as Journal Paper 160, August 20, 1936.     

Confocal analysis of chromosome behavior in wheat x maize zygotes.

Herein, we profile the first embryonic mitosis in a hybrid of wheat and maize by using a whole-mount genomic in situ hybridization method and immunofluorescence staining with a tubulin-specific antibody. We have successfully captured the dynamics of each set of parental chromosomes in the first zygotic division of the hybrid embryo 24-28 h after crossing. During the first zygotic metaphase, although both sets of parental chromosomes congressed into the equatorial plate of the zygote, the maize chromosomes tended to lag in comparison with the wheat chromosomes. During anaphase, each parental chromosome separated into its sister chromosomes; however, some of the maize chromosomes lagged around the metaphase plate as segregants. The maize sister chromosomes that did move toward the pole showed delayed and asymmetric movement as compared with the wheat ones. Immunological staining of tubulin revealed a bipolar spindle structure in the first zygotic metaphase. The kinetochores of the maize chromosomes that lagged around the metaphase plate did not attach to the spindle microtubules. These results suggest that factors on the kinetochores of maize chromosomes that are required to control chromosome movement are deficient in the zygotic cell cycle.     

Identification of the Meiotic Division of Malarial Parasites

Zygotes of Plasmodium berghei were cultured 15–25 h in vitro to yield mature infective ookinetes. Samples taken in the first 5 h of culture were examined by electron microscopy. Meiotic figures were detected in the nuclei of the zygotes. Threadlike leptotene chromatids (chromosomes) condensed from attachment plaques on the nuclear envelope; chromatid pairing followed (zygotene), with synaptonemal complexes subsequently appearing (pachytene). These complexes persisted into metaphase but dissociated when the chromatids rapidly decondensed during anaphase. At telophase of the first meiotic division the kinetochores were retracted toward two small spindle complexes, which were found at widely separated poles in the nuclear envelope. The observations are consistent with a haploid genome of 8–10 chromosomes.     

Electron microscopical observations on nuclear division inMicrasterias americana

Each stage of nuclear division inMicrasterias americana was investigated by electron microscopy. Some chromosomes in metaphase had two or more centromeres on them, that is, they were polycentric. The centromere was roundish, moderately dense, and partially embedded in the chromosomes. Many microtubules of the spindle fibers were attached to the centromere. Abundant granules of high electron density, derived from dictyosomes in the cytoplasm, were seen in the metaphase spindle. Only the chromosomes moved towards the poles in anaphase, while these granules remained at the equatorial plate. Many nucleoli appeared in early telophase in one or more regions in almost all chromosomes. These nucleoli fused and enlarged during telophase.     

Spoke: a 120-kD protein associated with a novel filamentous structure on or near kinetochore microtubules in the mitotic spindle

We have characterized an antiserum that recognizes a single 120-kD protein in CHO cells which is soluble and cytoplasmically localized in interphase, but which is associated with a novel filamentous structure localized on or near kinetochore microtubules in mid-mitosis. These filaments, one per sister chromatid, run from near the mitotic spindle pole to within approximately 0.3 microns of each kinetochore. In metaphase, the staining pattern shows considerable substructure at light microscopy resolution, appearing as bright nodes or striations, often with a kinked or helical appearance. This overall localization pattern is retained throughout anaphase, with the filaments shortening as the chromosomes move toward the mitotic spindle poles. Also in anaphase, a separate ring-like structure lacking a tubulin-staining component appears near the spindle poles. As cells exit mitosis, the amount of this antigen in the cell decreases seven- to tenfold. The unusual staining pattern and the specific localization of this antigen on or near kinetochore microtubules in mid-mitosis indicate that the 120-kD protein defines or is associated with an important and previously unrecognized structural element of the mitotic spindle.     

Verteilung der Mikrotubuli in Metaphase- und Anaphase-Spindeln der Spermatocyten von Pales ferruginea

One metaphase I spindle, seven anaphase I spindles of different stages, and one metaphase II spindle were sectioned in series. The ultrastructure of chromosomes was examined and microtubules (MTs) were counted. The main results of the study are summarized as follows: 1. The autosomes move at the periphery of the continuous MTs during anaphase while the sex chromosomes move more or less within this group of MTs. 2. In metaphase the antosomes have few coarse surface projections, in anaphase many, but more delicate projections of irregular shape which seem to transform into regular radial lamellae at the end of movement. 3. In metaphase continuous MTs have no contact with the chromosomal surface, while during anaphase movement continuous MTs lie closer to the chromosomes, and finally arrange themselves between the radial surface lamellae. There they show lateral filamentous connections with the chromosomal surface. 4. The MT distribution profiles of metaphase and anaphase are different. While the highest density of MTs is observed in the middle region of the spindle in metaphase, there are two density zones during autosomal movement, each in one half spindle in front of the autosomes. After the autosomes have reached the poles the distribution profile is again similar to the metaphase condition. The MT distribution in metaphase II is the same as in metaphase I. Possible explanations for these observations are discussed in detail. 5. There is an overall decrease in MT content during anaphase. 6. With the onset of anaphase MTs are seen within the spindle mantle, closely associated with mitochondria. — Several theoretical aspects of anaphase mechanism are briefly discussed.     

Dynamic localization and functional implications of Aurora-C kinase during male mouse meiosis

Aurora-C was first identified during screening for kinases expressed in mouse sperm and eggs. Herein, we report for the first time the precise subcellular localization of endogenous Aurora-C during male meiotic division. The localization of Aurora-C was analyzed by immunofluorescence staining on chromosome spreads of mouse spermatocytes or in squashed seminiferous tubules. Aurora-C was first detected at clusters of chromocenters in diplotene spermatocytes and was concentrated at centromeres in metaphase I and II. Interestingly, Aurora-C was also found along the chromosome axes, including both the regions of centromeres and the chromosome arms in diakinesis. During the anaphase I/telophase I and anaphase II/telophase II transitions, Aurora-C was relocalized to the spindle midzone and midbody. A similar distribution pattern was also observed for Aurora-B during male meiotic divisions. Surprisingly, we detected no Aurora-C in mitotic spermatogonia. Furthermore, immunoprecipitation analyses revealed that INCENP associated with Aurora-C in the male testis. We propose that INCENP recruits Aurora-C (or some other factor(s) recruit INCENP and Aurora-C) to meiotic chromosomes, while Aurora-C may either work alone or cooperate with Aurora-B to regulate chromosome segregation during male meiosis.     

An indirect test for a role of the synaptonemal complex in the establishment of sister chromatid cohesiveness

The meiotic behavior of a special maize trisome was quantitatively observed at pachytene, metaphase I, anaphase I, prophase II, metaphase II and anaphase II. The data obtained are consistent with (but do not prove) the model that sister chromatid cohesiveness at anaphase I may be established during pachytene synapsis of the chromosome regions involved. The data suggest, however, that the normal prophase II maintenance of dyad integrity by cohesiveness of sister chromatid centromere regions does not depend upon prior synapsis of these regions, although monads separated from each other on the anaphase I spindle may be delivered to the same prophase II daughter nucleus. — The strands which some of the time connect sister chromatids which are separating equationally at anaphase I show a positive Feulgen staining reaction.     

ENDOMITOSIS IN TAPETAL CELLS OF EREMURUS (LILIACEAE)

True endomitosis in the anther tapetum of the liliaceous plant Eremurus is described. The nuclear membrane does not disappear, but during metaphase the chromosomes are condensed, often considerably more than in normal mitosis. When the pollen mother cells (PMCs) go through the last premeiotic mitosis, the tapetal cells have one diploid nucleus which divides while the cell remains undivided. The two diploid nuclei may undergo an endomitosis and the resulting tetraploid nuclei a second endomitosis. An alternative pathway is an ordinary mitosis—again without cell division—instead of one of the endomitotic cycles. The cytological picture in the tapetum is further complicated by restitution in anaphase and fusion of metaphase and anaphase groups during mitosis, processes which could give rise to cells with one, two, or three nuclei, instead of the expected two or four. No sign of the so-called “inhibited” mitosis is seen in these tapetal cells. When the PMCs are in leptotene-zygotene, very few tapetal nuclei are in endomitosis. When the PMCs have reached diplotene, almost 100% of cells which are not in interphase show an endomitotic stage.     

Studies in the Mitosis of Euglena I. On the Chromosome Cycle of Euglena viridis Ehrbg.

The chromosome cycle in the vegetative division of Euglena viridis was investigated. The seeming chromatin granules in the interphase nucleus are in reality thread structures, paired and very loosely twisted. Each component of the paired threads is called a chromatid, and consists of a fine thread of even thickness, the chromonema.
In the prophase, linear contraction and thickening of the chromatids occurs by means of the spiralization of them. In the later prophase, the coiled chromonema splits into two finer strands which show the plectonemic spiral. At the metaphase, the chromosomes are arranged in the form of an equatorial ring, encircling the median portion of the elongated endosome. Nearly all of the chromosomes have a submedian or a sub-terminal and a few of them have a terminal kinetochore. In the early anaphase, separation of the sister chromosomes takes place beginning at the kinetochore. The spindle fibres in the metaphase and anaphase were not observed. The two stranded spiral in the chromosomes is separated into distinct components by the uncoiling in the later telophase, and they are transformed, in the interphase nucleus, into the paired chromatids.     

The effect of UV microirradiation of the centrosome on cell behavior. I. The destruction of the mitotic spindle and disruption of cell division during irradiation in the metaphase

A 5 second UV microirradiation of the centrosome during the early metaphase leads to a rapid (within 5 minutes) chromosome shift towards the normal pole and disorganizes the spindle. The later metaphase plate is also disorganized, chromosomes being situated chaotically in the central part of the cell. Numerous (up to 10 or more) microtubule convergence centers are observed instead of the spindle. 2-4 hours after the microirradiation some cells may enter cytotomy. The microirradiation of chromosomes and cytoplasm in similar and greater doses (exposure up to 15 seconds) did not lead to disorganization of the spindle and did not effect the normal completion of mitosis. Sometimes the 5 second microirradiation in the middle metaphase also blocked anaphase, but the microirradiation within the last 5 minutes of the metaphase always failed to block anaphase and normal completion of division.     

The absence of centrioles from spindle poles of rat kangaroo (PtK2) cells undergoing meiotic-like reduction division in vitro

Light and electron microscopy were used to study somatic cell reduction division occurring spontaneously in tetraploid populations of rat kangaroo Potorous tridactylis (PtK2) cells in vitro. Light microscopy coupled with time-lapse photography documented the pattern of reduction division which includes an anaphase-like movement of double chromatid chromosomes to opposite spindle poles followed by the organization of two separate metaphase plates and synchronous anaphase division to form four poles and four daughter nuclei. The resulting daughter cells were isolated and cloned, showing their viability, and karyotyped to determine their ploidy. Ultrastructural analysis of cells undergoing reduction consistently revealed two duplexes of centrioles (one at each of two spindle poles) and two spindle poles in each cell that lacked centrioles but with microtubules terminating in a pericentriolar-like cloud of material. These results suggest that the centriole is not essential for spindle pole formation and division and implicate the could region as a necessary component of the spindle apparatus.     

Cohesin is needed for bipolar mitosis in human cells

Multi-polar mitosis is strongly linked with aggressive cancers and it is a histological diagnostic of tumor-grade. However, factors that cause chromosomes to segregate to more than two spindle poles are not well understood. Here we show that cohesins Rad21, Smc1 and Smc3 are required for bipolar mitosis in human cells. After Rad21 depletion, chromosomes align at the metaphase plate and bipolar spindles assemble in most cases, but in anaphase the separated chromatids segregate to multiple poles. Time-lapse microscopy revealed that the spindle poles often become split in Rad21-depleted metaphase cells. Interestingly, exogenous expression of non-cleavable Rad21 results in multi-polar anaphase. Since cohesins are present at the spindle poles in mitosis, these data are consistent with a non-chromosomal function of cohesin.     

Cytology of meiosis in the triploid gynogenetic salamander Ambystoma tremblayi

Female meiosis was analyzed in the triploid gynogenetic salamander Ambystoma tremblayi to determine the mechanism by which a stable chromosome number is maintained in this unisexual species. Gross details of the reproductive cycle and the cytology of meiosis were analyzed in 20 specimens and 320 oocytes involving all stages from early diplotene to the beginning of anaphase II Ovulation apparently continues progressively involving a few oocytes at a time. Oocytes from the ovary contained chromosomes in diplotene, and diakinesis. The first metaphase was not observed since this stage occurs swiftly either immediately prior to or during ovulation. Oocytes in the most anterior region of the oviduct were in metaphase II, and those in the most posterior region were undergoing the beginning of anaphase II. Telophase II was not observed. Chromosome numbers obtained at all stages of prophase gave counts of approximately 42 bivalents, equivalent to the triploid somatic number known for this species. Similar numbers of dyads were obtained from metaphase II plates. This analysis supports earlier evidence suggesting that the triploid number of chromosomes in oocytes of A. tremblayi is doubled prior to meiosis, and the somatic number is later restored by two normal meiotic divisions.     

CENP-E, a novel human centromere-associated protein required for progression from metaphase to anaphase.

We have identified a novel human centromere-associated protein by preparing monoclonal antibodies against a fraction of HeLa chromosome scaffold proteins enriched for centromere/kinetochore components. One monoclonal antibody (mAb177) specifically stains the centromere region of mitotic human chromosomes and binds to a novel, approximately 250-300 kd chromosome scaffold associated protein named CENP-E. In cells progressing through different parts of the cell cycle, the localization of CENP-E differed markedly from that observed for the previously identified centromere proteins CENP-A, CENP-B, CENP-C and CENP-D. In contrast to these antigens, no mAb177 staining is detected during interphase, and staining first appears at the centromere region of chromosomes during prometaphase. This association with chromosomes remains throughout metaphase but is redistributed to the midplate at or just after the onset of anaphase. By telophase, the staining is localized exclusively to the midbody. Microinjection of the mAb177 into metaphase cells blocks or significantly delays progression into anaphase, although the morphology of the spindle and the configuration of the metaphase chromosomes appear normal in these metaphase arrested cells. This demonstrates that CENP-E function is required for the transition from metaphase to anaphase.