MEK1/2 is a critical regulator of microtubule assembly and spindle organization during rat oocyte meiotic maturation

MEK (MAPK kinase) is an upstream protein kinase of MAPK in the MOS/MEK/MAPK/p90rsk signaling pathway. We previously reported the function and regulation of MAPK during rat oocyte maturation. In this study, we further investigated the localization and possible roles of MEK1/2. First, immunofluorescent staining revealed that p-MEK1/2 was restricted to the germinal vesicle (GV). After germinal vesicle breakdown (GVBD), p-MEK1/2 condensed in the vicinity of chromosomes and then translocated to the spindle poles at metaphase I, while spindle microtubules stained faintly. When the oocyte went through anaphase I and telophase I, p-MEK1/2 disappeared from spindle poles and became associated with the midbody. By metaphase II, p-MEK1/2 was again localized to the spindle poles. Second, p-MEK1/2 was localized to the centers of cytoplasmic microtubule asters induced by taxol. Third, p-MEK1/2 co-localized with gamma-tubulin in microtubule-organizing centers (MTOCs). Forth, treatment with U0126, a non-competitive MEK1/2 inhibitor, did not affect germinal vesicle breakdown, but caused chromosome mis-alignment in all MI oocytes examined and abnormal spindle organization as well as small cytoplasmic spindle-like structure formation in MII oocytes. Finally, U0126 reduced the number of cytoplasmic asters induced by taxol. Our data suggest that MEK1/2 has regulatory functions in microtubule assembly and spindle organization during rat oocyte meiotic maturation.     

Meeting the meiotic challenge: Specializations in mammalian oocyte spindle formation

Oocytes uniquely accumulate cytoplasmic constituents to support early embryogenesis. This unique specialization is accompanied by acquisition of a large size and by execution of asymmetric meiotic divisions that preserve precious ooplasm through the expulsion of minimal size polar bodies. While often taken for granted, these basic features of oogenesis necessitate unique specializations of the meiotic apparatus. These include a chromatin‐sourced RanGTP gradient that restricts spindle size by defining a spatial domain where meiotic spindles form, acentriolar centrosomes that rely on microtubule organizing centers to form spindle poles, and an actin‐based mechanism for asymmetric spindle positioning. Additionally, localized protein synthesis to support spindle formation is achieved in the spindle forming region, whilst protein synthesis is reduced elsewhere in the ooplasm. This is achieved through enrichment of spindle‐related mRNAs in the spindle forming region combined with local PLK1‐mediated phosphorylation and inactivation of the translational repressor EIF4EBP1. This allows PLK1 to function as an important regulatory nexus through which endogenous and exogenous signals can impact spindle formation and function, and highlights the important role that PLK1 may have in maintaining oocyte quality and fertility.     

Stage specific effects of carbendazim (MBC) on meiotic cell cycle progression in mouse oocytes

The effects of the pesticide carbendazim (MBC) on the in vitro meiotic maturation of mouse oocytes were evaluated using conventional and confocal fluorescence microscopy. The response of oocytes exposed to 0, 3, 10, or 30 μM MBC during meiotic maturation was analyzed with respect to chromosome organization, meiotic spindle microtubules, and cortical actin using fluorescent labels for each of these structures. Continuous exposure to MBC during the resumption of meiosis resulted in a dose-dependent inhibition of meiotic cell cycle progression at metaphase of meiosis-1. Drug exposure at the metaphase-anaphase transition of meiosis-1 did not interfere with cell cycle progression to metaphase-2 except at high concentrations (30 μM). At the level of spindle microtubule organization, MBC caused a loss of nonacetylated microtubules and a decrease in spindle size at 3 or 10 μM concentrations. Thirty μM MBC prevented spindle assembly when added at the beginning of meiotic maturation or caused spindle pole disruption and fragmentation when added to preformed spindles. Spindle disruption involved a loss of phosphoprotein epitopes, as monitored by MPM-2 staining, and resulted in the appearance of dispersed chromosomes that retained a metaphase-plate location on spindle fragments associated with the oocyte cortex. Polar body extrusion was impaired by MBC, and abnormal polar bodies were observed in most treated oocytes. The results suggest that MBC disrupts cell cycle progression in mouse oocytes by altering meiotic spindle microtubule stability and spindle pole integrity. Mol. Reprod. Dev. 46:351–362, 1997. © 1997 Wiley-Liss, Inc.     

Identification of an Mr 60,000 polypeptide unique to the meiotic spindle of the mouse oocyte

The mouse oocyte expresses an M_r 60,000 (p60) polypeptide that is associated with the first and second meiotic spindles. Immunoreactive p60 was not detectable in the meiotic spindles of male germ cells or in mitotic spindles. P60 was identified with a polyclonal antibody whose predominant activity is directed against ankyrin. However, immunoadsorption experiments demonstrated that p60 is not an ankyrin isoform and represents a secondary activity of the polyclonal antibody. Circumstantial evidence suggest that p60 may be a micro-tubule-associated protein. Since the most obvious difference between the female meiotic spindle and other spindles is the long half-life of the former, we hypothesize that p60 may function in the maintenance of the long-lived female meiotic apparatus. © 1995 Wiley-Liss, Inc.     

Unusual cytological patterns of microsporogenesis in Brachiaria decumbens: abnormalities in spindle and defective cytokinesis causing precocious cellularization

Cytogenetic studies carried out in the tetraploid accession BRA001068 of Brachiaria decumbens, also known as cv. Basilisk, revealed an unusual pattern of microsporogenesis. The spindle in metaphase I and anaphase I became heavily stained with propionic carmine. In telophase I, the interzonal microtubules continued to be intensely stained, and during the phragmoplast formation the fibers were pushed to the cell wall, persisting until prophase II, even after cytokinesis. Due to its tetraploid condition, the accession presented many cells with precocious chromosome migration to the poles in metaphase I and laggards in anaphase I that gave rise to micronuclei in telophase I. While in other polyploid accessions of Brachiaria micronuclei remained in this condition until the second cytokinesis, the micronuclei in this accession organized their own spindle in the second division. In several microsporocytes, the micronuclei with their minispindle were divided further into microcytes by additional cytokinesis. Some curious planes of cytokinesis were found in some cells, with partitioning of cytoplasm into cells of irregular shape. The result consisted of a high frequency of abnormal products of meiosis. Quadrivalents were observed in diakinesis at low frequency, which suggests a segmental allotetraploid and the inability of both genomes to co-ordinate their activities, leading to multiple spindle and precocious cellularization. In spite of abnormal meiotic products reducing pollen fertility, seed production was normal. Enough normal pollen was available to fertilize the central-cell nucleus of the embryo sac and produce normal endosperm in this pseudogamous aposporous apomictic accession.     

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.     

Assembly of a protein sharing epitopes with sperm dynein heavy chains into meiotic spindle in the prometaphase starfish oocyte

Starfish oocyte meiosis provides a good system for studying the mechanism for prometaphase chromosome movement. Since a protein sharing epitopes with sperm dynein might be a force generator for mitosis, the contribution of such a protein was assessed in this movement. Specific antibodies to heavy chains (HCs) and intermediate chains (ICs) of dynein subunits were affinity-purified from whole antidynein serum. We confirmed that the oocytes contain several polypeptides identical to sperm dynein subunits. The anti-HCs binding to in situ antigen was examined in the oocytes permeabilized with detergent at appropriate stages of maturation with special reference to tubulin and chromosomes, and the meiotic apparatus-establishing process was described in terms of a force generator (oocyte dynein). Before resumption of maturation, dynein HCs were particularly associated with prophase chromosomes within the germinal vesicle (GV). After GV breakdown, there was a striking local accumulation of dynein HCs in the "fading GV" (nuclear matrix). When chromosomes were pulled toward the central area between 2 asters, dynein was accumulated at first at the presumptive equator and then moved to the poles, showing uneven localization on the meiotic spindle.     

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.     

MULTIPOLAR SPINDLE 1 (MPS1), a novel coiled-coil protein of Arabidopsis thaliana, is required for meiotic spindle organization

The spindle is essential for chromosome segregation during meiosis, but the molecular mechanism of meiotic spindle organization in higher plants is still not well understood. Here, we report on the identification and characterization of a plant-specific protein, MULTIPOLAR SPINDLE 1 (MPS1), which is involved in spindle organization in meiocytes of Arabidopsis thaliana . The homozygous mps1 mutant exhibits male and female sterility. Light microscopy showed that mps1 mutants produced multiple uneven spores during anther development, most of which aborted in later stages. Cytological analysis showed that chromosome segregation was abnormal in mps1 meiocytes. Immunolocalization showed unequal bipolar or multipolar spindles in mps1 meiocytes, which indicated that aberrant spindles resulted in disordered chromosome segregation. MPS1 encodes a 377-amino-acid protein with putative coiled-coil motifs. In situ hybridization analysis showed that MPS1 is strongly expressed in meiocytes.     

The maize homologue of the cell cycle checkpoint protein MAD2 reveals kinetochore substructure and contrasting mitotic and meiotic localization patterns

We have identified a maize homologue of yeast MAD2, an essential component in the spindle checkpoint pathway that ensures metaphase is complete before anaphase begins. Combined immunolocalization of MAD2 and a recently cloned maize CENPC homologue indicates that MAD2 localizes to an outer domain of the prometaphase kinetochore. MAD2 staining was primarily observed on mitotic kinetochores that lacked attached microtubules; i.e., at prometaphase or when the microtubules were depolymerized with oryzalin. In contrast, the loss of MAD2 staining in meiosis was not correlated with initial microtubule attachment but was correlated with a measure of tension: the distance between homologous or sister kinetochores (in meiosis I and II, respectively). Further, the tension-sensitive 3F3/2 phosphoepitope colocalized, and was lost concomitantly, with MAD2 staining at the meiotic kinetochore. The mechanism of spindle assembly (discussed here with respect to maize mitosis and meiosis) is likely to affect the relative contributions of attachment and tension. We support the idea that MAD2 is attachment-sensitive and that tension stabilizes microtubule attachments.     

Activated proline‐rich tyrosine kinase 2 regulates meiotic spindle assembly in the mouse oocyte

Proline‐rich tyrosine kinase 2 (PYK2), a member of the protein tyrosine kinase family, plays an important role in various cellular processes. PYK2 can be phosphorylated on tyrosine 402 by diverse stimuli at the cell surface, and recent studies have shown that this activated form of PYK2 is enriched in oocytes and required for fertilization. However, the subcellular localization and functions of activated PYK2 in oocytes remain elusive. In this study, we demonstrate that the localization of p‐PYK2 undergoes dynamic changes during in vitro maturation of mouse oocytes. The signal of p‐PYK2 is initially dispersed in the cytoplasm, but begins to decorate organized microtubules after the germinal vesicle breakdown and localizes to spindle poles at metaphase. Our data further show that p‐PYK2 colocalizes with γ‐tubulin from the germinal vesicle stage through the end of meiosis in mouse oocytes. Nocodazole treatment and washout experiments confirm that p‐PYK2 associates with the oocyte spindle and spindle poles. Moreover, pharmacological inhibition of PYK2 activity dramatically alters the morphology of the bipolar spindle and prevents oocyte maturation. Together, these data suggest that activated PYK2 may function as a component of the microtubule organizing center to regulate spindle assembly during the meiotic process of mouse oocytes.     

Naegleria’s mitotic spindles are built from unique tubulins and highlight core spindle features

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Chromatin, microtubules, and kinases activities during meiotic resumption in bitch oocytes

In contrast to the majority of mammals, canine oocytes are ovulated at immature germinal vesicle (GV) stage and complete meiotic maturation to metaphase II during 48-72 hr within the oviducts. This study aims to characterize meiotic maturation process in bitch oocytes, with both morphological and biochemical approaches. The follow-up of chromatin and microtubules during maturation was described, and MPF and MAP kinase activities were quantified at different stages of maturation. Since bitch oocyte cytoplasm is darkly pigmented, the first step was to setup an appropriate staining method for DNA. We thus compared the efficiency of two visualization techniques and demonstrated that propidium iodide coupled to confocal microscopy was a better method than Hoechst/fluorescence microscopy for nuclear stage observation (determination rates: 98.6 vs. 69.5%, respectively; P < 0.01, n = 1622 oocytes). Microtubule organization, evaluated by tubulin immunodetection, revealed subcortical and perinuclear alpha-tubulin and asters in GV oocytes and a clear network of microtubules in GVBD oocytes. In MI and MII oocytes, a symmetrical, barrel-shaped, and radially located spindle was observed. MPF and MAP kinase activities were assayed concomitantly using histone H1 and MBP as substrates. Kinase activities were detected at low levels in oocytes at GV and GVBD stages and were significantly higher at MI and MII stages. In conclusion, despite the particular pattern of meiotic resumption in canine oocytes (ovulated at GV stage), cytoskeleton/chromatin organization and kinase activities follow a similar pattern to those observed in other mammalian species.     

同一居群韭莲不同植株减数分裂行为差异的遗传分析

对韭莲(2n=48)小孢子母细胞减数分裂及小孢子发育进行研究。结果显示同一居群植株的减数分裂行为存在明显差异。多数韭莲植株小孢子母细胞减数分裂存在少量落后染色体、微核等现象,平均每株中具有异常分离行为的母细胞占14.02%,小孢子发育正常,但花粉无活力。并首次从减数分裂后期Ⅰ的特殊的细胞学形态证明韭莲是臂内倒位杂合体。而少数植株韭莲的小孢子母细胞减数分裂极其紊乱,后期Ⅰ出现多极分离、大量落后染色体,小孢子母细胞减数分裂总异常分离高达94.3%。四分孢子期多分孢子体高达73.4%。分析认为:前者减数分裂行为异常的原因主要由染色体结构变异所致,而后者的原因除染色体结构变异外,还可能与控制纺锤体形成的基因突变有关。     

Chromator, a novel and essential chromodomain protein interacts directly with the putative spindle matrix protein skeletor

We have used a yeast two-hybrid interaction assay to identify Chromator, a novel chromodomain containing protein that interacts directly with the putative spindle matrix protein Skeletor. Immunocytochemistry demonstrated that Chromator and Skeletor show extensive co-localization throughout the cell cycle. During interphase Chromator is localized on chromosomes to interband chromatin regions in a pattern that overlaps that of Skeletor. However, during mitosis both Chromator and Skeletor detach from the chromosomes and align together in a spindle-like structure. Deletion construct analysis in S2 cells showed that the COOH-terminal half of Chromator without the chromodomain was sufficient for both nuclear as well as spindle localization. Analysis of P-element mutations in the Chromator locus shows that Chromator is an essential protein. Furthermore, RNAi depletion of Chromator in S2 cells leads to abnormal microtubule spindle morphology and to chromosome segregation defects. These findings suggest that Chromator is a nuclear protein that plays a role in proper spindle dynamics during mitosis.     

Unravelling the mystery of female meiotic drive: where we are

Female meiotic drive is the phenomenon where a selfish genetic element alters chromosome segregation during female meiosis to segregate to the egg and transmit to the next generation more frequently than Mendelian expectation. While several examples of female meiotic drive have been known for many decades, a molecular understanding of the underlying mechanisms has been elusive. Recent advances in this area in several model species prompts a comparative re-examination of these drive systems. In this review, we compare female meiotic drive of several animal and plant species, highlighting pertinent similarities.     

The effect of low temperature on the microtubules in root meristem cells of spring and winter cultivars of wheat <Emphasis Type="Italic">Triticum aestivum L.</Emphasis>

We have studied the response of interphase and mitotic microtubule arrays in root meristem cells of spring and winter cultivars of wheat Triticum aestivum L. (Moskovskaya 35 and Moskovskaya 39) to cold stress (1 h at 0°C) and acclimation to cold (3–48 h at 0°C). We show that, in general, interphase microtubules are more resistant to cold then mitotic arrays in both cultivars. During cold stress, no changes are detected in the microtubule system of interphase cells of spring wheat, whereas the density of endoplasmic microtubules increases in interphase cells of winter wheat. During mitosis, the density of the kinetochore fibers of the spindle decreases in the cells of both cultivars, but it is prevailing in the cells of spring cultivar of wheat. During acclimation to cold, the disorganization of the cortical microtubule bundles and the enhanced growth of the endoplasmic microtubule network, which is comprised of microtubule converging centers, are observed in cells of both cultivars. However, the mitotic microtubule systems of winter and spring cultivars respond differently to cold acclimation. During prophase, a diffuse tubulin “halo,”followed by the assembly of microtubule converging centers, accumulate at the perinuclear area in the cells of winter wheat. In cells of spring cultivar, the prophase spindle is only detected during initial stages of cold acclimation. During metaphase, aberrant mitotic spindles, abnormal metaphase plates, and the excessive appearance of microtubule converging centers are observed in cells of both cultivars. Acclimation induces the disorganization of the phragmoplast and the formation of multiple microtubule converging centers during telophase in the cells of both cultivars. Microtubule converging centers are detected at the perinuclear area of daughter cells in winter wheat and in the cortical cytoplasm in spring wheat. The excessive formation of microtubule converging centers suggests the activation of microtubule assembly during prolonged exposure to low temperature. Our data also demonstrates common pathways of microtubule response to cold treatment (0°C).     

EAST interacts with Megator and localizes to the putative spindle matrix during mitosis in Drosophila

We have used immunocytochemistry to demonstrate that the EAST protein in Drosophila, which forms an expandable nuclear endoskeleton at interphase, redistributes during mitosis to colocalize with the spindle matrix proteins, Megator and Skeletor. EAST and Megator also colocalize to the intranuclear space surrounding the chromosomes at interphase. EAST is a novel protein that does not have any previously characterized motifs or functional domains. However, we show by immunoprecipitation experiments that EAST is likely to molecularly interact with Megator which has a large NH2-terminal coiled-coil domain with the capacity for self assembly. On the basis of these findings, we propose that Megator and EAST interact to form a nuclear endoskeleton and as well are important components of the putative spindle matrix complex during mitosis.