Dynamics of microtubular cytoskeleton in higher plant meiosis. IV. Middle prometaphase

By means of morphological analyses of meiotic abnormalities in pollen mother cells of cereal distant hybrids, processes of cytoskeleton cycle at the middle prometaphase (chaotic stage) were studied. It was shown that elements of the bipolar spindle (central and opposite kinetochore fibres) are formed at the chaotic stage of meiotic prometaphase.     

Meiosis spindle formation and chromosome behavior in diploid potatoes with 'fused spindles' mutation

Chromosomal behaviour and spindle morphology were studied in microsporogenesis of two kinds of diploid potato clones: with normal meiosis, and with "fused spindles" (fs) occurring during the second meiotic division from prometaphase II (proMII) to telophase II (TII). For the first time, morphological effect of fs was found at the late proMII stage to be expressed as two interrelated processes: 1) abnormal chromosome movement, which resulted in joining two groups of chromosomes in the central zone of meiocytes, and 2) abnormal formation of two spindles in the direction to two division poles instead of four poles that actually led to the formation of a united bipolar spindle. Thus, it is not the fusion of two parallel spindles but the formation of united bipolar spindle that constitutes fs abnormality, while the parallel co-orientation of two spatially separated meiotic spindles is a norm in diploid potato. These primary abnormalities detected at proMII resulted in abnormalities at its subsequent meiotic stages: formation of fused spindle and united metaphase plate at MII, bipolar chromosome segration at anaphase II, formation of two telophase nuclei at TII and dyads at the tetrad stage. The results obtained evidence the polar division disturbance in diploid potato clones with fs abnormality.     

Spindle formation and dynamics of gamma-tubulin and nuclear mitotic apparatus protein distribution during meiosis in pig and mouse oocytes

This work focuses on the assembly and transformation of the spindle during the progression through the meiotic cell cycle. For this purpose, immunofluorescent confocal microscopy was used in comparative studies to determine the spatial distribution of alpha- and gamma-tubulin and nuclear mitotic apparatus protein (NuMA) from late G2 to the end of M phase in both meiosis and mitosis. In pig endothelial cells, consistent with previous reports, gamma-tubulin was localized at the centrosomes in both interphase and M phase, and NuMA was localized in the interphase nucleus and at mitotic spindle poles. During meiotic progression in pig oocytes, gamma-tubulin and NuMA were initially detected in a uniform distribution across the nucleus. In early diakinesis and just before germinal vesicle breakdown, microtubules were first detected around the periphery of the germinal vesicle and cell cortex. At late diakinesis, a mass of multi-arrayed microtubules was formed around chromosomes. In parallel, NuMA localization changed from an amorphous to a highly aggregated form in the vicinity of the chromosomes, but gamma-tubulin localization remained in an amorphous form surrounding the chromosomes. Then the NuMA foci moved away from the condensed chromosomes and aligned at both poles of a barrel-shaped metaphase I spindle while gamma-tubulin was localized along the spindle microtubules, suggesting that pig meiotic spindle poles are formed by the bundling of microtubules at the minus ends by NuMA. Interestingly, in mouse oocytes, the meiotic spindle pole was composed of several gamma-tubulin foci rather than NuMA. Further, nocodazole, an inhibitor of microtubule polymerization, induced disappearance of the pole staining of NuMA in pig metaphase II oocytes, whereas the mouse meiotic spindle pole has been reported to be resistant to the treatment. These results suggest that the nature of the meiotic spindle differs between species. The axis of the pig meiotic spindle rotated from a perpendicular to a parallel position relative to the cell surface during telophase I. Further, in contrast to the stable localization of NuMA and gamma-tubulin at the spindle poles in mitosis, NuMA and gamma-tubulin became relocalized to the spindle midzone during anaphase I and telophase I in pig oocytes. We postulate that in the centrosome-free meiotic spindle, NuMA aggregates the spindle microtubules at the midzone during anaphase and telophase and that the polarity of meiotic spindle microtubules might become inverted during spindle elongation.     

Formation of division spindles in higher plant meiosis

Depolymerisation of the MT cytoskeleton during late prophase makes it impossible to follow the cytoskeleton cycle in centrosomeless plant meiocytes. This paper describes rearrangements of the MT cytoskeleton during plant meiotic spindle formation in normally dividing pollen mother cells in various higher plant species and forms in which the cytoskeleton does not depolymerise at prophase. In such variants of the wild-type, cytoskeleton rearrangements can be observed at late prophase/early prometaphase. Radial MT bundles coalesce in the meridian plane, reorientate tangentially, curve and give rise to a developed ring-shaped perinuclear cytoskeleton system at the meridian. During nuclear envelope breakdown this ring disintegrates and splits into a set of free MT bundles. Three sub-stages of prometaphase are indicated: early prometaphase (disintegration of perinuclear ring and invasion of MTs into the former nuclear area), middle prometaphase or chaotic stage (formation of bipolar spindle fibres), and late prometaphase (formation of bipolar spindle). Analysis of a range of abnormal phenotypes (disintegrated, multiple, polyarchal, chaotic spindles) reveals two previously unknown processes during late prometaphase: axial orientation and consolidation of the spindle fibres.     

Dynamics of cytoskeleton microtubules in higher plant meiosis. I. The perinuclear band of microtubules and the meiotic spindle formation

A planar meridional perinuclear band of microtubules was observed at the late meiotic prophase I in a range of higher plant species. A distinct high-organized structure and a long time of existence allow to consider it as a new class of MTs dependent on the cell cycle in plant meiosis. MTs of the perinuclear band convert into meiotic spindle through a complex process of spatial rearrangements.     

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.     

Formin-2 is required for spindle migration and for the late steps of cytokinesis in mouse oocytes

Female meiotic divisions in higher organisms are asymmetric and lead to the formation of a large oocyte and small polar bodies. These asymmetric divisions are due to eccentric spindle positioning which, in the mouse, requires actin filaments. Recently Formin-2, a straight actin filaments nucleator, has been proposed to control spindle positioning, chromosome segregation as well as first polar body extrusion in mouse oocytes. We reexamine here the possible role of Formin-2 during mouse meiotic maturation by live videomicroscopy. We show that Formin-2 controls first meiotic spindle migration to the cortex but not chromosome congression or segregation. We also show that the lack of first polar body extrusion in fmn2(-/-) oocytes is not due to a lack of cortical differentiation or central spindle formation but to a defect in the late steps of cytokinesis. Indeed, Survivin, a component of the passenger protein complex, is correctly localized on the central spindle at anaphase in fmn2(-/-) oocytes. We show here that attempts of cytokinesis in these oocytes abort due to phospho-myosin II mislocalization.     

PGRMC1 participates in late events of bovine granulosa cells mitosis and oocyte meiosis

Progesterone Receptor Membrane Component 1 (PGRMC1) is expressed in both oocyte and ovarian somatic cells, where it is found in multiple cellular sub-compartments including the mitotic spindle apparatus. PGRMC1 localization in the maturing bovine oocytes mirrors its localization in mitotic cells, suggesting a possible common action in mitosis and meiosis. To test the hypothesis that altering PGRMC1 activity leads to similar defects in mitosis and meiosis, PGRMC1 function was perturbed in cultured bovine granulosa cells (bGC) and maturing oocytes and the effect on mitotic and meiotic progression assessed. RNA interference-mediated PGRMC1 silencing in bGC significantly reduced cell proliferation, with a concomitant increase in the percentage of cells arrested at G2/M phase, which is consistent with an arrested or prolonged M-phase. This observation was confirmed by time-lapse imaging that revealed defects in late karyokinesis. In agreement with a role during late mitotic events, a direct interaction between PGRMC1 and Aurora Kinase B (AURKB) was observed in the central spindle at of dividing cells. Similarly, treatment with the PGRMC1 inhibitor AG205 or PGRMC1 silencing in the oocyte impaired completion of meiosis I. Specifically the ability of the oocyte to extrude the first polar body was significantly impaired while meiotic figures aberration and chromatin scattering within the ooplasm increased. Finally, analysis of PGRMC1 and AURKB localization in AG205-treated oocytes confirmed an altered localization of both proteins when meiotic errors occur. The present findings demonstrate that PGRMC1 participates in late events of both mammalian mitosis and oocyte meiosis, consistent with PGRMC1's localization at the mid-zone and mid-body of the mitotic and meiotic spindle.     

Gene expression and the control of spermatid morphogenesis in Drosophila melanogaster.

The genetic control of spermatid morphogenesis was studied by light microscopy through the analysis of meiotic and premeiotic lesions. Sperm disfunction-type male-sterile mutations were screened for novel “early effect” mutations: (1) timing mutations, in which mitochondrial aggregation occurs before instead of after meiosis; (2) mutations which affect the spindle structure, e.g., a mutant with second-division monoastral spindle; (3) mutations which cause deformations in primary spermatocyte structures. It is shown, in addition to the examples cited above, that normal meiosis may often serve as an early marker for normal differentiation, and that approximately 20% of male-sterile mutations are meiotic mutants. The role of the Y chromosome was reexamined. The interaction between Y factors and X-linked male steriles is in many cases additive, indicating that Y gene products are essential for normal development of the primary spermatocytes. Furthermore, XO males are shown to be extreme meiotic mutants. It is argued that spermatid morphogenesis is totally dependent on developmental processes in the primary spermatocyte stage. The relations among developmental processes in early spermatogenesis are discussed in terms of gene activity.     

Mouse polo-like kinase 1 associates with the acentriolar spindle poles, meiotic chromosomes and spindle midzone during oocyte maturation

We have examined the dynamics of the localisation of the polo-like kinase 1 (Plk1) during maturation of the mouse oocyte. Levels of Plk1 protein increase following germinal vesicle breakdown, at which time the enzyme begins to accumulate at discrete positions on the condensing chromosomes and, subsequently, at the poles of the meiotic spindle, which moves towards the cortex of the egg. Interestingly, at metaphase in both meiotic divisions, Plk1 shows a punctate localisation along the broad spindle poles. Moreover, the punctate distribution of Plk1 on the meiotic chromosomes appears at early anaphase to correspond to the centromeric regions. The protein relocates to the spindle midzone during late anaphase and then associates with the midbody at telophase. We have confirmed the specific pattern of immuno-localisation seen in fixed preparations by observing the distribution of Plk1 tagged with green fluorescent protein in living oocytes. We discuss the localisation of the enzyme in light of the structure of the spindle poles, which are known to lack centrioles, and the highly asymmetric nature of the meiotic divisions. Received: 8 August 1998 / Accepted: 13 September 1998     

Meiosis in the basidiomycetous fungus,Coprinus atramentarius

Summary Stages in meiosis ofCoprinus atramentarius have been studied using the electron microscope. Serial sections of late pachytene nuclei showed a diglobular spindle pole body adjacent to the nuclear membrane. After Prophase I the two globular ends of the SPB enlarged and became positioned towards the apex of the basidium and the first meiotic spindle formed across the basidium apex. The two spindles of the second meiotic division formed in the same plane but at 90° to the original spindle of the first division.     

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.     

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.     

The Microtubule-Associated Protein ASPM Regulates Spindle Assembly and Meiotic Progression in Mouse Oocytes

The microtubule-associated protein ASPM (abnormal spindle-like microcephaly-associated) plays an important role in spindle organization and cell division in mitosis and meiosis in lower animals, but its function in mouse oocyte meiosis has not been investigated. In this study, we characterized the localization and expression dynamics of ASPM during mouse oocyte meiotic maturation and analyzed the effects of the downregulation of ASPM expression on meiotic spindle assembly and meiotic progression. Immunofluorescence analysis showed that ASPM localized to the entire spindle at metaphase I (MI) and metaphase II (MII), colocalizing with the spindle microtubule protein acetylated tubulin (Ac-tubulin). In taxol-treated oocytes, ASPM colocalized with Ac-tubulin on the excessively polymerized microtubule fibers of enlarged spindles and the numerous asters in the cytoplasm. Nocodazole treatment induced the gradual disassembly of microtubule fibers, during which ASPM remained colocalized with the dynamic Ac-tubulin. The downregulation of ASPM expression by a gene-specific morpholino resulted in an abnormal meiotic spindle and inhibited meiotic progression; most of the treated oocytes were blocked in the MI stage with elongated meiotic spindles. Furthermore, coimmunoprecipitation combined with mass spectrometry and western blot analysis revealed that ASPM interacted with calmodulin in MI oocytes and that these proteins colocalized at the spindle. Our results provide strong evidence that ASPM plays a critical role in meiotic spindle assembly and meiotic progression in mouse oocytes.     

CDK-1 inhibits meiotic spindle shortening and dynein-dependent spindle rotation in C. elegans

In animals, the female meiotic spindle is positioned at the egg cortex in a perpendicular orientation to facilitate the disposal of half of the chromosomes into a polar body. In Caenorhabditis elegans, the metaphase spindle lies parallel to the cortex, dynein is dispersed on the spindle, and the dynein activators ASPM-1 and LIN-5 are concentrated at spindle poles. Anaphase-promoting complex (APC) activation results in dynein accumulation at spindle poles and dynein-dependent rotation of one spindle pole to the cortex, resulting in perpendicular orientation. To test whether the APC initiates spindle rotation through cyclin B-CDK-1 inactivation, separase activation, or degradation of an unknown dynein inhibitor, CDK-1 was inhibited with purvalanol A in metaphase-I-arrested, APC-depleted embryos. CDK-1 inhibition resulted in the accumulation of dynein at spindle poles and dynein-dependent spindle rotation without chromosome separation. These results suggest that CDK-1 blocks rotation by inhibiting dynein association with microtubules and with LIN-5-ASPM-1 at meiotic spindle poles and that the APC promotes spindle rotation by inhibiting CDK-1.     

Intranuclear membranes and the formation of the first meiotic spindle in Xenos peckii (Acroschismus wheeleri) oocytes

The ultrastructure of spindle formation during the first meiotic division in oocytes of the Strepsipteran insect Xenos peckii Kirby (Acroschismus wheeleri Pierce) was examined in serial thick (0.25- micron) and thin sections. During late prophase the nuclear envelope became extremely convoluted and fenestrated. At this time vesicular and tubular membrane elements permeated the nucleoplasm and formed a thin fusiform sheath, 5-7 micron in length, around each of the randomly oriented and condensing tetrads. These membrane elements appeared to arise from the nuclear envelope and/or in association with annulate lamellae in the nuclear region. All of the individual tetrads and their associated fusiform sheaths became aligned within the nucleus subsequent to the breakdown of the nuclear envelope. Microtubules (MTs) were found associated with membranes of the meiotic apparatus only after the nuclear envelope had broken down. Kinetochores, with associated MTs, were first recognizable as electron-opaque patches on the chromosomes at this time. The fully formed metaphase arrested Xenos oocyte meiotic apparatus contained an abundance of membranes and had diffuse poles that lacked distinct polar MT organizing centers. From these observations we conclude that the apparent individual chromosomal spindles--seen in the light microscope to form around each Xenos tetrad during "intranuclear prometaphase" (Hughes-Schrader, S., 1924, J. Morphol. 39:157-197)--actually form during late prophase, lack MTs, and are therefore not complete miniature bipolar spindles, as had been commonly assumed. Thus, the unique mode of spindle formation in Xenos oocytes cannot be used to support the hypothesis that chromosomes (kinetochores) induce the polymerization of their associated MTs. Our observation that MTs appeared in association with and parallel to tubular membrane components of the Xenos meiotic apparatus after these membranes became oriented with respect to the tetrads, is consistent with the notion that membranes associated with the spindle determine the orientation of spindle MTs and also play a part in regulating their formation.     

Microtubule organization during maturation of Xenopus oocytes: assembly and rotation of the meiotic spindles.

Assembly of the meiotic spindles during progesterone-induced maturation of Xenopus oocytes was examined by confocal fluorescence microscopy using anti-tubulin antibodies and by time-lapse confocal microscopy of living oocytes microinjected with fluorescent tubulin. Assembly of a transient microtubule array from a disk-shaped MTOC was observed soon after germinal vesicle breakdown. This MTOC-TMA complex rapidly migrated toward the animal pole, in association with the condensing meiotic chromosomes. Four common stages were observed during the assembly of both M1 and M2 spindles: (1) formation of a compact aggregate of microtubules and chromosomes; (2) reorganization of this aggregate resulting in formation of a short bipolar spindle; (3) an anaphase-B-like elongation of the prometaphase spindle, transversely oriented with respect to the oocyte A-V axis; and (4) rotation of the spindle into alignment with the oocyte axis. The rate of spindle elongation observed in M1 (0.7 microns min-1) was slower than that observed in M2 (1.8 microns min-1). Examination of spindles by immunofluorescence with antitubulin revealed numerous interdigitating microtubules, suggesting that prometaphase elongation of meiotic spindles in Xenopus oocytes results from active sliding of antiparallel microtubules. A substantial number of maturing oocytes formed monopolar microtubule asters during M1, nucleated by hollow spherical MTOCs. These monasters were subsequently observed to develop into bipolar M1 spindles and proceed through meiosis. The results presented define a complex pathway for assembly and rotation of the meiotic spindles during maturation of Xenopus oocytes.     

Division polarity and plasticity of the meiosis I spindle inCypripedium californicum (Orchidaceae)

Summary Establishment of division polarity and meiotic spindle organization in the lady's slipper orchidCypripedium californicum A. Gray was studied by immunocytochemistry, confocal and transmission electron microscopy. Prior to organization of the spindle for meiosis I, the cytoplasmic domains of the future dyad and spindle polarity are marked by: (1) constriction of the prophase nucleus into an hourglass shape; (2) reorganization of nuclear-based radial microtubules into two arrays that intersect at the constriction; and (3) redistribution of organelles into a ring at the boundary of the newly defined dyad domains. It is not certain whether the opposing microtubule arrays contribute directly to the anastral spindle which is organized in the perinuclear areas of the two hemispheres. By late prophase each half-spindle consists of a spline-like structure from which depart the kinetochore fibers. This peculiar spindle closely resembles the spline-like spindle of generative-cell mitosis in certain plants where the spindle is distorted by physical constraints of the slender pollen tube. In the microsporocyte, the elongate spindle of late prophase/metaphase is curved within the cell so that the poles are not actually opposite each other and chromosomes do not form a plate at the equator. By late telophase the poles of the shortened halfspindles lie opposite each other. Plasticity of the physically constrained plant spindle appears to be due to its construction from multiple units terminating in minipoles. Cytokinesis does not follow the first meiosis. However, the dyad domains are clearly defined by radial microtubules emanating from the two daughter nuclei and the domains themselves are separated by a disc-like band of organelles.     

Two novel meiotic restitution mechanisms in haploid maize (<Emphasis Type="Italic">Zea mays</Emphasis> L.)

Two original mechanisms of nuclear restitution related to different processes of meiotic division of pollen mother cells (PMCs) have been found in male meiosis of the lines of maize haploids no. 2903 and no. 2904. The first mechanism, which is characteristic of haploid no. 2903, consists in spindle deformation (bend) in the conventional metaphase-anaphase I. This leads to asymmetric incomplete cytokinesis with daughter cell membranes in the form of incisions on the mother cell membrane. As a result, the chromosomes of the daughter nuclei are combined into a common spindle during the second meiotic division, and a dyad of haploid microspores is formed at the tetrad stage. The frequency of this abnormality is about 50%. The second restitution mechanism, which has been observed in PMCs of haploid no. 2904, results from disturbance of the fusion of membrane vesicles (plastosomes) at the moment of formation of daughter cell membranes and completion of cytokinesis in the first meiotic division. This type of cell division yields a binuclear monad. In the second meiotic division, the chromosomes of the daughter nuclei form a common spindle, and meiosis results in a dyad of haploid microspores. The frequency of this abnormality is as high as 15%. As a result, haploid lines no. 2903 and no. 2904 partly restore fertility.     

Pairing and segregation of the sex chromosomes in X1X2-males of Dysdercus intermedius with a note on the kinetic organization of Heteropteran chromosomes

The existence of an X₁X₂-mode of sex determination is confirmed by a study of all meiotic stages in the male cotton stainer (X₁X₂ and pertinent stages in the female (X₁X₁ X₂X₂). In the male, the X-chromosomes are heterochromatic and pair end-to-end in early meiotic prophase. At diakinesis, they disjoin and align side-by-side in the center of the spindle, forming a pseudotetrad. Anaphase I is equational for the sex chromosomes. At late anaphase or telophase, X₁ and X₂ join end-to-end but form spindle fiber connections to only one of the poles of the metaphase II spindle, leading to one daughter cell without X chromosomes and one with both X₁ and X₂. An attempt is made to explain sex chromosome pairing and orientation on the basis of a telocentric organization of meiotic chromosomes. The apparent differences in the kinetic organization of mitotic and meiotic chromosomes in Heteroptera are discussed.