Abnormalities of spindle and cytokine behavior leading to the formation of meiotic restitution nuclei in intergeneric cereal hybrids

Mobile stages of meiosis have been analysed by visualizing the spindle in fertile cereal F1 hybrids. We describe four different mechanisms of the formation of restitution nuclei in meiotic division: (1) centripetal migration of telophase chromosome groups from the poles of a curved spindle at early telophase; (2) centripetal migration of the chromosome groups at late telophase when cell plate formation has failed; (3) preferable migration of univalents to one of the poles although spindle appearance is morphologically normal; and (4) in the absence of chromosome segregation where kinetochore fibers have failed to form.     

Cytological evidences of SDR-FDR mixture in the formation of 2n eggs in a potato diploid clone

Summary Macrosporogenesis and microsporogenesis were investigated in a diploid S. tuberosum x S. chacoense potato hybrid, characterized by more than 50% 2n egg formation. Fifty-five percent of dyad formation of 2n macrospores is ascribed to two meiotic abnormalities: omission of the second meiotic division, occurring at a frequency of 38%, and irregular spindle axis orientation at metaphase I at a frequency of 16%: These abnormalities give origin to a mixture of 2n eggs, composed of mostly second division restitution (SDR) and a small portion of first division restitution (FDR). Microsporogenesis showed rare dyads of 2n microspores depending on parallel spindles observed in anaphase II.Contribution no. 53 from the Center of Vegetable Breeding, CNR, Portici, Italy     

Radial spindle and the phenotype of the maize meiotic mutant, dv

The morphological phenotype of the maize meiotic mutant dv (divergent spindle) has been further analysed by visualization of the division spindle and examination of its fine structure in mother cells of pollen. Previous research showed that dv blocks convergence of spindle fibres at the poles. New observations reveal abnormalities caused by this mutation, with dv showing disturbances in nuclear envelope breakdown during vesiculation, preventing the spindle fibres from adopting a bipolar orientation (with convergence on the poles). The anomalies result in radial spindles which are similar to monoastral spindles in animal cells.     

Microtubule distribution in dv, a maize meiotic mutant defective in the prophase to metaphase transition

Microsporogenesis in Zea mays, the meiotic reduction of diploid sporocytes to haploid microspores, proceeds through a well-defined developmental sequence. The ability to generate mutants that affect the process makes this an ideal system for elucidating the role of the cytoskeleton during plant development. We have used immunofluorescence microscopy to compare microtubule distribution in wild-type and mutant microsporocytes. During normal meiosis the distribution of microtubules follows a specific temporal and spatial pattern that reflects the polar nature of microspore formation. Perinuclear microtubule staining increases and the nucleus elongates in the future spindle axis during late prophase I. Metaphase I spindles with highly focused poles align along the long axis of the anther locule. Cytokinesis occurs perpendicular to the spindle axis. The second division axis shifts 90 degrees with respect to the first division plane, thereby yielding an isobilateral tetrad of microspores. Microtubule distribution patterns during meiosis suggest that a nuclear envelope-associated microtubule organizing center (MTOC) controls the organization of cytoplasmic microtubules and contributes to spindle formation. The meiotic mutant dv is defective in the transition from a prophase microtubule array to a metaphase spindle. Instead of converging to form focused poles, the metaphase spindle poles remain diffuse as in prometaphase. This defect correlates with several abnormalities in subsequent developmental events including the formation of multinucleate daughter cells, multiple microspindles during meiosis II, multiple phragmoplasts, polyads of microspores, and cytoplasmic microtubule foci. These results suggest that dv is a mutation that affects MTOC organization.     

THE QUADRIPOLAR MICROTUBULE SYSTEM AND MEIOTIC SPINDLE ONTOGENY IN HORNWORTS (BRYOPHYTA: ANTHOCEROTAE)

Ontogeny of the meiotic spindle in hornworts was studied by light microscopy of live materials, transmission electron microscopy, and indirect immunofluorescence microscopy. As in monoplastidic meiosis of mosses and Isoetes, the single plastid divides twice, and the four resultant plastids migrate into the future spore domains where they organize a quadripolar microtubule system (QMS). Additionally, a unique axial microtubule system (AMS) was found to parallel the plastid isthmus at each division in meiosis, much as in the single plastid division of mitosis. This finding is used to make a novel comparison of mitotic and meiotic spindle development. The AMS contributes directly to development of the mitotic spindle, whereas ontogeny of the meiotic spindle is more complex. Nuclear division in meiosis is delayed until after the second plastid division; the first AMS disappears without spindle formation, and the two AMSs of the second plastid division contribute to development of the QMS. Proliferation of microtubules at each plastid results in the QMS consisting of four cones of microtubules interconnecting the plastids and surrounding the nucleus. The QMS contributes to the development of a functionally bipolar spindle. The meiotic spindle is comparable to a merger of two mitotic spindles. However, the first division spindle does not terminate in what would be the poles of mitosis; instead the poles converge to orient the spindle axis midway between pairs of non-sister plastids.     

PLK1 regulates centrosome migration and spindle dynamics in male mouse meiosis

Cell division requires the regulation of karyokinesis and cytokinesis, which includes an essential role of the achromatic spindle. Although the functions of centrosomes are well characterised in somatic cells, their role during vertebrate spermatogenesis remains elusive. We have studied the dynamics of the meiotic centrosomes in male mouse during both meiotic divisions. Results show that meiotic centrosomes duplicate twice: first duplication occurs in the leptotene/zygotene transition, while the second occurs in interkinesis. The maturation of duplicated centrosomes during the early stages of prophase I and II are followed by their separation and migration to opposite poles to form bipolar spindles I and II. The study of the genetic mouse model Plk1(Δ/Δ) indicates a central role of Polo‐like kinase 1 in pericentriolar matrix assembly, in centrosome maturation and migration, and in the formation of the bipolar spindles during spermatogenesis. In addition, in vitro inhibition of Polo‐like kinase 1 and Aurora A in organotypic cultures of seminiferous tubules points out to a prominent role of both kinases in the regulation of the formation of meiotic bipolar spindles.     

HURP permits MTOC sorting for robust meiotic spindle bipolarity, similar to extra centrosome clustering in cancer cells

In contrast to somatic cells, formation of acentriolar meiotic spindles relies on the organization of microtubules (MTs) and MT-organizing centers (MTOCs) into a stable bipolar structure. The underlying mechanisms are still unknown. We show that this process is impaired in hepatoma up-regulated protein (Hurp) knockout mice, which are viable but female sterile, showing defective oocyte divisions. HURP accumulates on interpolar MTs in the vicinity of chromosomes via Kinesin-5 activity. By promoting MT stability in the spindle central domain, HURP allows efficient MTOC sorting into distinct poles, providing bipolarity establishment and maintenance. Our results support a new model for meiotic spindle assembly in which HURP ensures assembly of a central MT array, which serves as a scaffold for the genesis of a robust bipolar structure supporting efficient chromosome congression. Furthermore, HURP is also required for the clustering of extra centrosomes before division, arguing for a shared molecular requirement of MTOC sorting in mammalian meiosis and cancer cell division.     

mei-38 is required for chromosome segregation during meiosis in Drosophila females

Meiotic chromosome segregation occurs in Drosophila oocytes on an acentrosomal spindle, which raises interesting questions regarding spindle assembly and function. One is how to organize a bipolar spindle without microtubule organizing centers at the poles. Another question is how to orient the chromosomes without kinetochore capture of microtubules that grow from the poles. We have characterized the mei-38 gene in Drosophila and found it may be required for chromosome organization within the karyosome. Nondisjunction of homologous chromosomes occurs in mei-38 mutants primarily at the first meiotic division in females but not in males where centrosomes are present. Most meiotic spindles in mei-38 oocytes are bipolar but poorly organized, and the chromosomes appear disorganized at metaphase. mei-38 encodes a novel protein that is conserved in the Diptera and may be a member of a multigene family. Mei-38 was previously identified (as ssp1) due to a role in mitotic spindle assembly in a Drosophila cell line. MEI-38 protein localizes to a specific population of spindle microtubules, appearing to be excluded from the overlap of interpolar microtubules in the central spindle. We suggest MEI-38 is required for the stability of parallel microtubules, including the kinetochore microtubules.     

Division polarity,development and configuration of microtubule arrays in bryophyte meiosis

Summary First and second division spindles and the three cell plates of moss meiosis are oriented in accordance with polarity established during meiotic prophase. Plastids are located at the second division poles and cytoplasmic infurrowing marks the planes along which the cytoplasm will cleave into four spores. Anaphase I spindles that terminate in two focal points of microtubules straddling opposite cleavage furrows reflect the unusual tetrahedral origin of the functionally bipolar spindle. The organelles (except for the plastids which remain in the four cytoplasmic lobes) are polarized in the first division equatorial region at the time of phragmoplast microtubule assembly and remain in a distinct band after microtubule disassembly. Prophasic spindles appear to be directly transformed into metaphase II spindles in the predetermined axes between mutually perpendicular pairs of plastids. Cell plates form by vesicle coalescence in the equatorial regions of the two sets of second division phragmoplasts at approximately the same time as a cell plate belatedly forms in the organelle band. The cytoplasmic markers (plastid migration, cytoplasmic lobing and infurrowing) that predict poles and cleavage planes in free cells lacking a preprophase band strongly strengthens the concept that division sites are capable of preserving preprogrammed signals that can be triggered later in the process of cell division.     

Chromosomal influence on meiotic spindle assembly: abnormal meiosis I in female Mlh1 mutant mice.

In mouse oocytes, the first meiotic spindle is formed through the action of multiple microtubule organizing centers rather than a pair of centrosomes. Although the chromosomes are thought to play a major role in organizing the meiotic spindle, it remains unclear how a stable bipolar spindle is established. We have studied the formation of the first meiotic spindle in murine oocytes from mice homozygous for a targeted disruption of the DNA mismatch repair gene, Mlh1. In the absence of the MLH1 protein meiotic recombination is dramatically reduced and, as a result, the vast majority of chromosomes are present as unpaired univalents at the first meiotic division. The orientation of these univalent chromosomes at prometaphase suggests that they are unable to establish stable bipolar spindle attachments, presumably due to the inability to differentiate functional kinetochore domains on individual sister chromatids. In the presence of this aberrant chromosome behavior a stable first meiotic spindle is not formed, the spindle poles continue to elongate, and the vast majority of cells never initiate anaphase. These results suggest that, in female meiotic systems in which spindle formation is based on the action of multiple microtubule organizing centers, the chromosomes not only promote microtubule polymerization and organization but their attachment to opposite spindle poles acts to stabilize the forming spindle poles.     

subito encodes a kinesin-like protein required for meiotic spindle pole formation in Drosophila melanogaster

The female meiotic spindle lacks a centrosome or microtubule-organizing center in many organisms. During cell division, these spindles are organized by the chromosomes and microtubule-associated proteins. Previous studies in Drosophila melanogaster implicated at least one kinesin motor protein, NCD, in tapering the microtubules into a bipolar spindle. We have identified a second Drosophila kinesin-like protein, SUB, that is required for meiotic spindle function. At meiosis I in males and females, sub mutations affect only the segregation of homologous chromosomes. In female meiosis, sub mutations have a similar phenotype to ncd; even though chromosomes are joined by chiasmata they fail to segregate at meiosis I. Cytological analyses have revealed that sub is required for bipolar spindle formation. In sub mutations, we observed spindles that were unipolar, multipolar, or frayed with no defined poles. On the basis of these phenotypes and the observation that sub mutations genetically interact with ncd, we propose that SUB is one member of a group of microtubule-associated proteins required for bipolar spindle assembly in the absence of the centrosomes. sub is also required for the early embryonic divisions but is otherwise dispensable for most mitotic divisions.     

Nek9 regulates spindle organization and cell cycle progression during mouse oocyte meiosis and its location in early embryo mitosis

Nek9 (also known as Nercc1), a member of the NIMA (never in mitosis A) family of protein kinases, regulates spindle formation, chromosome alignment and segregation in mitosis. Here, we showed that Nek9 protein was expressed from germinal vesicle (GV) to metaphase II (MII) stages in mouse oocytes with no detectable changes. Confocal microscopy identified that Nek9 was localized to the spindle poles at the metaphase stages and associated with the midbody at anaphase or telophase stage in both meiotic oocytes and the first mitotic embyros. Depletion of Nek9 by specific morpholino injection resulted in severely defective spindles and misaligned chromosomes with significant pro-MI/MI arrest and failure of first polar body (PB1) extrusion. Knockdown of Nek9 also impaired the spindle-pole localization of γ-tubulin and resulted in retention of the spindle assembly checkpoint protein Bub3 at the kinetochores even after 10 h of culture. Live-cell imaging analysis also confirmed that knockdown of Nek9 resulted in oocyte arrest at the pro-MI/MI stage with abnormal spindles, misaligned chromosomes and failed polar body emission. Taken together, our results suggest that Nek9 may act as a MTOC-associated protein regulating microtubule nucleation, spindle organization and, thus, cell cycle progression during mouse oocyte meiotic maturation, fertilization and early embryo cleavage.     

“Bouquet arrest”, monopolar chromosomes segregation, and correction of the abnormal spindle

According to our data, the arrest of univalents in bouquet arrangement is a widespread meiotic feature in cereal haploids and allohaploids (wide hybrids F₁). We have analyzed 83 different genotypes of cereal haploids and allohaploids with visualization of the cytoskeleton and found a bouquet arrest in 45 of them (in 30% to 100% pollen mother cells (PMCs)). The meiotic plant cell division in 26 various genotypes with a zygotene bouquet arrest was analyzed in detail. In three of them in PMCs, a very specific monopolar conic-shaped figure at early prometaphase is formed. This monopolar figure consists of mono-oriented univalents and their kinetochore fibers converging in pointed pole. Such figures are never observed at wild-type prometaphase or in asynaptic meiosis in the variants without a bouquet arrest. Later at prometaphase, the bipolar central spindle fibers join in this monopolar figure, and a bipolar spindle with all univalents connected to one pole is formed. As a result of monopolar chromosome segregation at anaphase and normal cytokinesis at telophase, a dyad with one member carrying a restitution nucleus and the other enucleated is formed. However, such phenotype has only three genotypes among 26 analyzed with a bouquet arrest. In the remaining 23 haploids and allohaploids, the course of prometaphase was altered after the conic monopolar figure formation. In these variants, the completely formed conic monopolar figure was disintegrated into a chaotic network of spindle fibers and univalents acquired a random orientation. This arrangement looks like a mid-prometaphase in the wild-type meiosis. At late prometaphase, a bipolar spindle is formed with the univalents distributed more or less equally between two poles, similar to the phenotypes without a bouquet arrest. The product of cell division is a dyad with aneuploid members. Thus, the spindle abnormality—monopolar chromosome orientation—is corrected. In some cells the correction of the prometaphase monopolus occurs by means of its splitting into two half-spindles and their rotation along the future division axis.     

{gamma}-Tubulin and microtubule organization during meiosis in the liverwort Ricciocarpus natans (Ricciaceae)

Extant liverworts are "living fossils" considered sister to all other plants and as such provide clues to the evolution of the microtubule organizing center (MTOC) in anastral cells. This report is the first on microtubule arrays and their γ-tubulin-nucleating sites during meiosis in a member of the Ricciales, a specialized, species-rich group of complex thalloid (marchantioid) liverworts. In meiotic prophase, γ-tubulin becomes concentrated at several sites adjacent to the nuclear envelope. Microtubules organized at these foci give rise to a multipolar prometaphase spindle. By metaphase I, the spindle has matured into a bipolar structure with truncated poles. In both first and second meiosis, γ-tubulin forms box-like caps at the spindle poles. γ-Tubulin moves from spindle poles to the proximal surfaces of telophase chromosomes where interzonal microtubules are nucleated. Although a phragmoplast is organized, no cell plate is deposited, and second division occurs simultaneously in the undivided sporocyte. γ-Tubulin surrounds each of the tetrad nuclei, and phragmoplasts initiated between both sister and nonsister nuclei direct simultaneous cytokinesis. The overall pattern of meiosis (unlobed polyplastidic sporocytes, nuclear envelope MTOC, multipolar spindle origin, spindles with box-like poles, and simultaneous cytokinesis) more closely resembles that of Conocephalum than other marchantiod liverworts.     

Caenorhabditis elegans Aurora A kinase is required for the formation of spindle microtubules in female meiosis

In many animals, female meiotic spindles are assembled in the absence of centrosomes, the major microtubule (MT)-organizing centers. How MTs are formed and organized into meiotic spindles is poorly understood. Here we report that, in Caenorhabditis elegans, Aurora A kinase/AIR-1 is required for the formation of spindle microtubules during female meiosis. When AIR-1 was depleted or its kinase activity was inhibited in C. elegans oocytes, although MTs were formed around chromosomes at germinal vesicle breakdown (GVBD), they were decreased during meiotic prometaphase and failed to form a bipolar spindle, and chromosomes were not separated into two masses. Whereas AIR-1 protein was detected on and around meiotic spindles, its kinase-active form was concentrated on chromosomes at prometaphase and on interchromosomal MTs during late anaphase and telophase. We also found that AIR-1 is involved in the assembly of short, dynamic MTs in the meiotic cytoplasm, and these short MTs were actively incorporated into meiotic spindles. Collectively our results suggest that, after GVBD, the kinase activity of AIR-1 is continuously required for the assembly and/or stabilization of female meiotic spindle MTs.     

Nek9 regulates spindle organization and cell cycle progression during mouse oocyte meiosis and its location in early embryo mitosis

Nek9 (also known as Nercc1), a member of the NIMA (never in mitosis A) family of protein kinases, regulates spindle formation, chromosome alignment and segregation in mitosis. Here, we showed that Nek9 protein was expressed from germinal vesicle (GV) to metaphase II (MII) stages in mouse oocytes with no detectable changes. Confocal microscopy identified that Nek9 was localized to the spindle poles at the metaphase stages and associated with the midbody at anaphase or telophase stage in both meiotic oocytes and the first mitotic embyros. Depletion of Nek9 by specific morpholino injection resulted in severely defective spindles and misaligned chromosomes with significant pro-MI/MI arrest and failure of first polar body (PB1) extrusion. Knockdown of Nek9 also impaired the spindle-pole localization of γ-tubulin and resulted in retention of the spindle assembly checkpoint protein Bub3 at the kinetochores even after 10 h of culture. Live-cell imaging analysis also confirmed that knockdown of Nek9 resulted in oocyte arrest at the pro-MI/MI stage with abnormal spindles, misaligned chromosomes and failed polar body emission. Taken together, our results suggest that Nek9 may act as a MTOC-associated protein regulating microtubule nucleation, spindle organization and, thus, cell cycle progression during mouse oocyte meiotic maturation, fertilization and early embryo cleavage.     

fumble encodes a pantothenate kinase homolog required for proper mitosis and meiosis in Drosophila melanogaster

A number of fundamental processes comprise the cell division cycle, including spindle formation, chromosome segregation, and cytokinesis. Our current understanding of these processes has benefited from the isolation and analysis of mutants, with the meiotic divisions in the male germline of Drosophila being particularly well suited to the identification of the required genes. We show here that the fumble (fbl) gene is required for cell division in Drosophila. We find that dividing cells in fbl-deficient testes exhibit abnormalities in bipolar spindle organization, chromosome segregation, and contractile ring formation. Cytological analysis of larval neuroblasts from null mutants reveals a reduced mitotic index and the presence of polyploid cells. Molecular analysis demonstrates that fbl encodes three protein isoforms, all of which contain a domain with high similarity to the pantothenate kinases of A. nidulans and mouse. The largest Fumble isoform is dispersed in the cytoplasm during interphase, concentrates around the spindle at metaphase, and localizes to the spindle midbody at telophase. During early embryonic development, the protein localizes to areas of membrane deposition and/or rearrangement, such as the metaphase and cellularization furrows. Given the role of pantothenate kinase in production of Coenzyme A and in phospholipid biosynthesis, this pattern of localization is suggestive of a role for fbl in membrane synthesis. We propose that abnormalities in synthesis and redistribution of membranous structures during the cell division cycle underlie the cell division defects in fbl mutant cells.     

KLP-18, a Klp2 kinesin,is required for assembly of acentrosomal meiotic spindles in Caenorhabditis elegans

The proper segregation of chromosomes during meiosis or mitosis requires the assembly of well organized spindles. In many organisms, meiotic spindles lack centrosomes. The formation of such acentrosomal spindles seems to involve first assembly or capture of microtubules (MTs) in a random pattern around the meiotic chromosomes and then parallel bundling and bipolar organization by the action of MT motors and other proteins. Here, we describe the structure, distribution, and function of KLP-18, a Caenorhabditis elegans Klp2 kinesin. Previous reports of Klp2 kinesins agree that it concentrates in spindles, but do not provide a clear view of its function. During prometaphase, metaphase, and anaphase, KLP-18 concentrates toward the poles in both meiotic and mitotic spindles. Depletion of KLP-18 by RNA-mediated interference prevents parallel bundling/bipolar organization of the MTs that accumulate around female meiotic chromosomes. Hence, meiotic chromosome segregation fails, leading to haploid or aneuploid embryos. Subsequent assembly and function of centrosomal mitotic spindles is normal except when aberrant maternal chromatin is present. This suggests that although KLP-18 is critical for organizing chromosome-derived MTs into a parallel bipolar spindle, the order inherent in centrosome-derived astral MT arrays greatly reduces or eliminates the need for KLP-18 organizing activity in mitotic spindles.     

Defects in cytoskeletal microtubule deployment of microsporocytes contribute to fertility loss in genic male-sterile Chinese cabbage

The microtubular cytoskeleton of male-sterile Chinese cabbage was examined to characterize cytoplasmically based defects during microsporogenesis of fertile and sterile microsporocytes. At the onset of meiosis, microtubules (MTs) in fertile microsporocytes were short and anisotropically oriented in the microsporocyte cytoplasm. As the microsporocytes entered metaphase I, the MTs constructed a bisymmetrical spindle characterized by conspicuous kinetochore fibers closely associated with chromosomes in the medial plane. During anaphase I, interzonal MTs become conspicuous between the two sets of chromosomes and the polar regions become more distant as spindle MTs are depleted, essentially disappearing at telophase I. Radially distributed MTs increased and the microsporocyte entered meiosis II, producing two spindles at angles to one another within the wall of the microsporocyte. Indicative of the completion of anaphase II is the formation of a field of aligned MTs between two non-sister nuclei, after which the cytoplasm produced centripetal furrows, meeting in the center of the cell and dividing it into four microspores at the completion of cytokinesis. In sterile microsporocytes, however, an abnormal arrangement of MTs occurred at the conclusion of anaphase II. Although two spindles formed, the angle and the boundary between the spindles were not maintained. At the onset of telophase II, the two spindles migrated to a central region and laterally fused in irregular orientations in which the decondensing chromatin of the non-sister nuclei may form separate or merged nuclei, followed by irregular cytokinesis. The result of meiosis was 41.8 % two binuclear products, and 58.2 % one diploid and one binuclear sterile products.     

Localization of the mei-1 gene product of Caenorhaditis elegans, a meiotic-specific spindle component

Genetic evidence suggests that the product of the mei-1 gene of Caenorhabditis elegans is specifically required for meiosis in the female germline. Loss-of-function mei-1 mutations block meiotic spindle formation while a gain-of-function allele instead results in spindle defects during the early mitotic cleavages. In this report, we use immunocytochemistry to examine the localization of the mei-1 product in wild-type and mutant embryos. During metaphase of meiosis I in wild- type embryos, mei-1 protein was found throughout the spindle but was more concentrated toward the poles. At telophase I, mei-1 product colocalized with the chromatin at the spindle poles. The pattern was repeated during meiosis II but no mei-1 product was visible during the subsequent mitotic cleavages. The mei-1 gain-of-function allele resulted in ectopic mei-1 staining in the centers of the microtubule- organizing centers during interphase and in the spindles during the early cleavages. This aberrant localization is probably responsible for the poorly formed and misoriented cleavage spindles characteristic of the mutation. We also examined the localization of mei-1(+) product in the presence of mutations of genes that genetically interact with mei-1 alleles. mei-2 is apparently required to localize mei-1 product to the spindle during meiosis while mel-26 acts as a postmeiotic inhibitor. We conclude that mei-1 encodes a novel spindle component, one that is specialized for the acentriolar meiotic spindles unique to female meiosis. The genes mei-2 and mel-26 are part of a regulatory network that confines mei-1 activity to meiosis.