Dynamics of microtubular cytoskeleton in higher plant meiosis. V. Late prometaphase. The general scheme of anastral spindle formation

By means of morphological analyses of meiotic abnormalities in pollen mother cells (PMCs) of cereal wide hybrids, haploids and meiotic mutants, the processes involved in cytoskeleton cycle at late prometaphase (a sub-stage of transition from chaotic figure to bipolar spindle) were studied. A significance of the four processes of late prometaphase--axial orientation, lateral association, consolidation and convergance of spindle fibers--is discussed.     

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. III. Early prometaphase stage

The early prometaphase and initial stages of meiotic spindle formation in higher plant PMCs were studied by means of a new approach worked out by the authors: a morphological dissection that consists in the analysis of various abnormalities of the process under study. Wide cereal hybrids F1 were used as a source of such abnormalities: phenotypes with C-, S-shaped and combined spindle, with spindles surrounded by microtubule (MT) ring and phenotypes with chaotic circular MT system in M1. Three stages of early prometaphase not described before (disintegration of perinuclear MT band, straightening of its bundles, and their translocation throughout the cytoplasm) were revealed.     

Spo76p is a conserved chromosome morphogenesis protein that links the mitotic and meiotic programs.

Spo76p is conserved and related to the fungal proteins Pds5p and BIMD and the human AS3 prostate proliferative shutoff-associated protein. Spo76p localizes to mitotic and meiotic chromosomes, except at metaphase(s) and anaphase(s). During meiotic prophase, Spo76p assembles into strong lines in correlation with axial element formation. As inferred from spo76-1 mutant phenotypes, Spo76p is required for sister chromatid cohesiveness, chromosome axis morphogenesis, and chromatin condensation during critical transitions at mitotic prometaphase and meiotic midprophase. Spo76p is also required for meiotic interhomolog recombination, likely at postinitiation stage(s). We propose that a disruptive force coordinately promotes chromosomal axial compaction and destabilization of sister connections and that Spo76p restrains and channels the effects of this force into appropriate morphogenetic mitotic and meiotic outcomes.     

Consolidation of cytoskeleton during plant spindle formation. II. "Fused spindles"

Three mechanisms of fused spindle formation in meiosis of Solanacea have been described: 1) approach of daughter nuclei at prophase II; 2) fusion of perinuclear cytoskeleton systems at prophase II; 3) approach and fusion of prometaphase chaotic figures at prometaphase II. The process of fusion spindle formation appears to be complex and including several steps.     

“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.     

Microtubule and microfilament dynamics in porcine oocytes during meiotic maturation

Microtubule and microfilament organization in porcine oocytes during maturation in vivo and in vitro was imaged by immunocytochemistry and laser scanning confocal microscopy. At the germinal vesicle stage, microtubules were not detected in the oocyte. After germinal vesicle breakdown, a small microtubule aster was observed near the condensed chromatin. During the prometaphase stage, microtubule asters were found in association with each chromatin mass. The asters then elongated and encompassed the chromatin at the metaphase-I stage. At anaphase-I and telophase-I microtubules were detected in the meiotic spindle. Microtubules were observed only in the second meiotic spindle at the metaphase-II stage. The meiotic spindle was a symmetric, barrel-shaped structure containing anastral broad poles, located peripherally and radially oriented. Taxol, a microtubule-stabilizing agent, did not induce microtubules in oocytes at the germinal vesicle stage. After germinal vesicle breakdown, numerous cytoplasmic foci of microtubules were formed in the entire oocyte when oocytes were incubated in the presence of taxol. Microfilaments were observed as a relatively thick uniform area around the cell cortex and were also found throughout the cytoplasm of oocytes at the germinal vesicle stage. After germinal vesicle breakdown, the microfilaments were concentrated close to the female chromatin. During prometaphase, microfilaments were chromatin moved to the peripheral position. At metaphase-I, two domains, a thick and a thin microfilament area, existed in the egg cortex. Chromosomes were located in the thick microfilament domain of the cortex. In summary, these results suggest that both micro-tubules and microfilaments are closely involved with chromosomal dynamics after germinal vesicle breakdown and during meiotic maturation in porcine oocytes. © 1996 Wiley-Liss, Inc.     

Survivin is a critical regulator of spindle organization and chromosome segregation during rat oocyte meiotic maturation

Survivin is a novel member of the inhibitor of apoptosis gene family that bear baculoviral IAP repeats (BIRs), whose physiological roles in regulating meiotic cell cycle need to be determined. Confocal microscopy was employed to observe the localization of survivin in rat oocytes. At the germinal vesicle (GV) stage, survivin was mainly concentrated in the GV. At the prometaphase I (pro-MI) and metaphase I (MI) stage, survivin was mainly localized at the kinetochores, with a light staining detected on the chromosomes. After transition to anaphase I or telophase I stage, survivin migrated to the midbody, and signals on the kinetochores and chromosomes disappeared. At metaphase II (MII) stage, survivin became mainly localized at the kinetochores again. Microinjection of oocytes with anti-survivin antibodies at the beginning of the meiosis, thus blocking the normal function of survivin, resulted in abnormal spindle assembly, chromosome segregation and first polar body emission. These results suggest that survivin is involved in regulating the meiotic cell cycle in rat oocytes.     

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.     

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.     

Involvement of calcium/calmodulin-dependent protein kinase kinase in meiotic maturation of pig oocytes

Calcium (Ca(2+))/calmodulin-dependent protein kinase kinase (CaMKK) is a novel member of Ca(2+)/calmodulin-dependent protein kinase (CaMK) family, whose physiological roles in regulating meiotic cell cycle needs to be determined. We showed by Western blot that CaMKK was expressed in pig oocytes at various maturation stages. Confocal microscopy was employed to observe CaMKK distribution. In oocytes at the germinal vesicle (GV) or prometaphase I (pro-MI) stage, CaMKK was distributed in the nucleus, around the condensed chromatin and the cortex of the cell. At metaphase I (MI) stage, CaMKK was concentrated in the cortex of the cell. After transition to anaphase I or telophase I stage, CaMKK was detected around the separating chromosomes and in the cortex of the cell. At metaphase II (MII) stage, CaMKK was localized to the cortex of the cell, with a thicker area near the first polar body (PB1). Treatment of pig cumulus-enclosed oocytes with STO-609, a membrane-permeable CaMKK inhibitor, resulted in the delay/inhibition of the meiotic resumption and the inhibition of first polar body emission. The correlation between CaMKK and microfilaments during meiotic maturation of pig oocytes was then studied. CaMKK and microfilaments were colocalized from MI to MII during porcine oocyte maturation. After oocytes were treated with STO-609, microfilaments were depolymerized, while in oocytes exposed to cytochalasin B (CB), a microfilament polymerization inhibitor, CaMKK became diffused evenly throughout the cell. These data suggest that CaMKK is involved in regulating the meiotic cell cycle probably by interacting with microfilaments in pig oocytes.     

BubR1 is a spindle assembly checkpoint protein regulating meiotic cell cycle progression of mouse oocyte

BubR1 (Bub1-related kinase or MAD3/Bub1b) is an essential component of the spindle assembly checkpoint (SAC) and plays an important role in kinetochore localization of other spindle checkpoint proteins in mitosis. But its roles in mammalian oocyte meiosis are unclear. In the present study, we examined the expression, localization and function of BubR1 during mouse oocyte meiotic maturation. The expression level of BubR1 increased progressively from germinal vesicle to metaphase II stages. Immunofluorescent analysis showed that BubR1 localized to kinetochores from the germinal vesicle breakdown to the prometaphase I stages, co-localizing with polo-like kinase 1, while it disappeared from the kinetochores at the metaphase I stage. Spindle disruption by nocodazole treatment caused relocation of BubR1 to kinetochores at metaphase I, anaphase I and metaphase II stages; spindle microtubules were disrupted by low temperature treatment in the BubR1-depleted oocytes in meiosis I, suggesting that BubR1 monitors kinetochore-microtubule (K-MT) attachments. Over-expression of exogenous BubR1 arrested oocyte meiosis maturation at the M I stage or earlier; in contrast, dominant-negative BubR1 and BubR1 depletion accelerated meiotic progression. In the BubR1-depleted oocytes, higher percentage of chromosome misalignment was observed and more oocytes overrode the M I stage arrest induced by low concentration of nocodazole. Our data suggest that BubR1 is a spindle assembly checkpoint protein regulating meiotic progression of oocytes.     

Cytological Observations of Microsporogenesis and Pollen Development in Triticum Sect. Trititrigia,T. aestivum and Their Hybrids

Cytological observation of microsporogenesis and pollen development of two parents, Xiaoyan 7430 and Yannong 15, and their hybrids were carried out by using squash method in this study. The results show: that microsporogenesis of the two parents, Xiaoyan 7430 and Yannong 15, is normal but their microspores usually aborted at first meiosis in pollen development. The frequency of abortion is higher in the former than the latter. However, the pollen development of which passed through the first meiosis is normal at the 2- or 3-nucleated stage. The rate of setting of both parents is basically normal. The meiosis of pollen mother cell (PMC) of two hybrids of Xiaoyan 7430 × Lumai No.1 and Xiaoyan 7430 × Yannong 15 is quite chaotic. Despite of the high frequency of univalents and multivalents at meiotic MI of PMC, and of micronucleoli in tetrad as well of the laggards at meiotic Al observed, the frequency of abortion of microspore are both at about 10%, similar to that of Xiaoyan 7430, at first meiosis of microspore in the stage of pollen development. Even though the pollen developments are basically normal in the 2- or 3-nucleated stage, but the percentage of setting of two hybrids is 28.24% and 33.82% respectively, apparently lower than that of their parents. The above-mentioned results indicate that although meiosis of PMC of F1 is chaotic, yet normal microspore could be formed and stages of pollen development cart still be carried on. The lower seed setting of F1 is not mainly due to abortion of pollen.     

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.     

Functional redundancy in the maize meiotic kinetochore

Kinetochores can be thought of as having three major functions in chromosome segregation: (a) moving plateward at prometaphase; (b) participating in spindle checkpoint control; and (c) moving poleward at anaphase. Normally, kinetochores cooperate with opposed sister kinetochores (mitosis, meiosis II) or paired homologous kinetochores (meiosis I) to carry out these functions. Here we exploit three- and four-dimensional light microscopy and the maize meiotic mutant absence of first division 1 (afd1) to investigate the properties of single kinetochores. As an outcome of premature sister kinetochore separation in afd1 meiocytes, all of the chromosomes at meiosis II carry single kinetochores. Approximately 60% of the single kinetochore chromosomes align at the spindle equator during prometaphase/metaphase II, whereas acentric fragments, also generated by afd1, fail to align at the equator. Immunocytochemistry suggests that the plateward movement occurs in part because the single kinetochores separate into half kinetochore units. Single kinetochores stain positive for spindle checkpoint proteins during prometaphase, but lose their staining as tension is applied to the half kinetochores. At anaphase, approximately 6% of the kinetochores develop stable interactions with microtubules (kinetochore fibers) from both spindle poles. Our data indicate that maize meiotic kinetochores are plastic, redundant structures that can carry out each of their major functions in duplicate.     

普通小麦与瓦维洛夫山羊草属间杂种的产生及其育性的细胞学 …

通过重复授粉和７５μｇ．ｇ＾－１ＧＡ３处理,成功地获得了普通小麦（Ｔ．ａｅｓｔｉｕｍ）与瓦维洛夫山羊草（Ａｅ．ｖａｖｉｌｏｖｉｉ）ＲＭ０２８６,ＲＭ０２９１ ２个组合属间杂种的产生及其育性,期间表现出较高的可酱性,杂匀结实率分别３９．８４％和４１．２２％,杂种Ｆ１植株具有双亲的形态特征,并表现出强的生活力。通过对Ｆ１植株小孢子发生和花粉发生和花粉发育的细胞学观察发现,花粉母细胞减数分裂过程异常紊乱     

Bouquet arrest and its consequences for asynaptic meiosis in cereals--haploids and wide hybrids F1

Arrest of chromosomes at bouquet configuration at zygothene leads to meiotic restitution in some haploids and allohaploids in cereals: univalents are transported preferentially to one spindle pole in bipolar spindle because of their oriented position in the beginning of prometaphase.     

Microinjected centromere [corrected] kinetochore antibodies interfere with chromosome movement in meiotic and mitotic mouse oocytes [published erratum appears in J Cell Biol 1990 Dec;111(6 Pt 1):following 2800]

Kinetochores may perform several functions at mitosis and meiosis including: (a) directing anaphase chromosome separation, (b) regulating prometaphase alignment of the chromosomes at the spindle equator (congression), and/or (c) capturing and stabilizing microtubules. To explore these functions in vivo, autoimmune sera against the centromere/kinetochore complex are microinjected into mouse oocytes during specific phases of first or second meiosis, or first mitosis. Serum E.K. crossreacts with an 80-kD protein in mouse cells and detects the centromere/kinetochore complex in permeabilized cells or when microinjected into living oocytes. Chromosome separation at anaphase is not blocked when these antibodies are microinjected into unfertilized oocytes naturally arrested at second meiotic metaphase, into eggs at first mitotic metaphase, or into immature oocytes at first meiotic metaphase. Microtubule capture and spindle reformation occur normally in microinjected unfertilized oocytes recovering from cold or microtubule disrupting drugs; the chromosomes segregate correctly after parthenogenetic activation. Prometaphase congression is dramatically influenced when antikinetochore/centromere antibodies are introduced during interphase or in prometaphase-stage meiotic or mitotic eggs. At metaphase, these oocytes have unaligned chromosomes scattered throughout the spindle with several remaining at the poles; anaphase is aberrant and, after division, karyomeres are found in the polar body and oocyte or daughter blastomeres. Neither nonimmune sera, diffuse scleroderma sera, nor sham microinjections affect either meiosis or mitosis. These results suggest that antikinetochore/centromere antibodies produced by CREST patients interfere with chromosome congression at prometaphase in vivo.