Presence of microtubules in isolated cortices of prophase I and metaphase II oocytes in Xenopus laevis

Summary An extensive array of microtubules has been shown to exist in the cortex of Xenopus laevis oocytes both at the prophase I and metaphase II stages. The cortical microtubules were visualized after the oocyte cortex was squashed and immunostained using anti-tubulin antibody. They are cold- and nocodazole-sensitive; their stability to both treatments decreases after meiotic maturation. Biochemical extraction of manually isolated oocyte cortices, in a microtubule-stabilizing buffer, confirms these cytological observations.     

Participation of the adhesive disc during karyokinesis in Giardia lamblia

Evidence is presented for a potential involvement of the adhesive disc on the nucleus division in Giardia lamblia. The trophozoite mitotic nucleus was studied by transmission electron microscopy, freeze-fracture, freeze-substitution and also by immunofluorescence microscopy using anti-tubulin antibodies specific to spindle microtubules and Panotic staining. Prior to cell division the nucleus elongated and a displaced disc fragment, established contact with the nucleus. A progressive nucleus indentation was coincident with the concomitant presence of a disc fragment at the constricted region. One nucleus each time progressively divided until the karyokinesis was finished and two daughter-nuclei were observed. After the first karyokinesis a second karyokinesis takes place following the same procedure. When Giardia gets the four nuclei, cytokinesis occurs. Duplicated basal bodies were seen in between the first and the second karyokinesis. Immunofluorescence microscopy, using a panel of anti-tubulin antibodies, and electron microscopy of cells processed using microtubule stabilizer buffers, or cells fast-frozen and freeze-substituted, did not reveal the presence of a typical spindle. We propose that Giardia lamblia presents an uncommon mitotic behavior where the adhesive disc, a microtubular structure, seems to participate in the karyokinesis process.     

Organisation of the cytoskeleton during in vitro maturation of horse oocytes

Meiotic maturation of mammalian oocytes is a complex process during which microfilaments and microtubules provide the framework for chromosomal reorganisation and cell division. The aim of this study was to use fluorescence and confocal laser scanning microscopy to examine changes in the distribution of these important cytoskeletal elements and their relationship to chromatin configuration during the maturation of horse oocytes in vitro. Oocytes were cultured in M199 supplemented with pFSH and eLH and, at 0, 12, 24, and 36 hr after the onset of culture, they were fixed for immunocytochemistry and stained with markers for microtubules (a monoclonal anti-alpha-tubulin antibody), microfilaments (AlexaFluor 488 Phalloidin) and DNA (TO-PRO(3)). At the germinal vesicle stage, oocyte chromatin was amorphous and poorly condensed and the microfilaments and microtubules were distributed relatively evenly throughout the ooplasm. After germinal vesicle breakdown, the microtubules were aggregated around the now condensed chromosomes and the microfilaments had become concentrated within the oocyte cortex. During metaphase I, microtubules were detected only in the meiotic spindle, as elongated asters encompassing the aligned chromosomes, and, as maturation progressed through anaphase-I and telophase-I, the spindle assumed a more eccentric position and gradually rotated to assist in the separation of the homologous chromosomes and in the subsequent formation of the first polar body. During metaphase II, the meiotic spindle was a symmetrical, barrel-shaped structure with two poles and with the chromosomes aligned along its midline. At this stage, microtubules were found intermingled with chromatin within the polar body and, although, the bulk of the microfilaments remained within the oocyte cortex, a rich domain was found overlying the spindle. Thus, during the in vitro maturation of horse oocytes both the microfilament and microtubular elements of the cytoskeleton were seen to reorganise dramatically in a fashion that appeared to enable chromosomal alignment and segregation.     

Microtubule and microfilament organization in immature, in vitro matured and in vitro fertilized prepubertal goat oocytes

The aim of our study was to analyse the cytoskeletal organization of prepubertal goat oocytes. Microtubule and microfilament organization during in vitro maturation of prepubertal and adult goat oocytes and presumptive zygotes of in vitro matured-in vitro fertilized (IVM-IVF) prepubertal goat oocytes were analysed. Oocytes were matured in M-199 with hormones and serum and inseminated with frozen-thawed sermatozoa. Oocytes and presumptive zygotes were treated with anti-alpha-tubulin antibody and fluorescein isothiocyanate (FITC)-labelled goat anti-mouse antibody to stain the microtubules. Microfilaments were localized by means of phalloidin 5 microg/ml conjugated with fluorescein isothiocyanate (FITC-phalloidin). DNA was stained with propidium iodide. Stained oocytes were observed under a confocal laser scanning microscope. At the germinal vesicle nuclear stage, microfilaments were distributed at the cortex of the oocytes. After in vitro maturation, 91.7% of metaphase II (MII) oocytes from adult goats displayed microfilaments in the cortex and within the polar body and were characterized by the presence of a microfilament thickening at the cortical region over the meiotic spindle. In prepubertal goat MII oocytes only 5.7% of oocytes displayed microfilaments at the cortex and within the polar body. After insemination, most of the zygotes displayed microfilaments distributed at the cortex. An undefined microtubular network was observed in adult and prepubertal goat oocytes at the germinal vesicle stage. After in vitro maturation, 100% of MII oocytes from adult goats displayed microtubules on the meiotic spindle and within the polar body. This pattern of distribution was observed in 71.6% of prepubertal goat oocytes. Undefined microtubule networks were present in most of the zygotes analysed. In conclusion, cytoskeletal differences were found between prepubertal and adult goat MII oocytes. Furthermore, most of the zygotes from IVM-IVF prepubertal goat oocytes displayed cytoskeletal anomalies.     

Trypanosoma cruzi: distribution of fluorescently labeled tubulin and actin in epimastigotes

Cytoskeletal components were visualized in epimastigote forms of Trypanosoma cruzi by double immunofluorescence microscopy using monospecific antibodies against tubulin and against actin. Intense staining of the flagellum and the edges of the cell body was observed when the cells were stained with anti-tubulin, reflecting the presence of the basal bodies, the flagellar axoneme and the subpellicular microtubules. A less intense staining was seen in the cell body of epimastigotes stained with anti-actin. However, an intense staining was observed with this antibody in the flagellum, in a pattern similar to that observed with anti-tubulin. It is suggested that the paraxial structure, which is formed by a complex array of 6-nm-thick microfilaments is composed, at least in part, of actin.     

Alterations to the microtubular cytoskeleton and increased disorder of chromosome alignment in spontaneously ovulated mouse oocytes aged in vivo: an immunofluorescence study

Alterations in the organization of the microtubular cytoskeleton and chromosome alignment were examined by tubulin immunofluorescence and DAPI staining during in vivo ageing of naturally ovulated, metaphase-arrested oocytes of CBA/Ca mice in the fallopian tubes. In oocytes isolated from young mice on the day of oestrus, a few hours after ovulation, when they are still tightly surrounded by cumulus, the anti-tubulin fluorescence is almost exclusively restricted to the metaphase spindle. Only some faintly staining foci are observed in the cytoplasm, which presumably represent cytoplasmic MTOC not involved in spindle formation. The spindle is usually barrel-shaped or slightly pointed at its poles and does not possess astral fibres. In oocytes aged for more than 12 h in the fallopian tubes cytoplasmic asters develop, while microtubules seem to become gradually lost from the spindle, preferentially in its central area near the chromosomes. Astral fibres are observed radiating out from the polar centrosomes into the cytoplasm. In oocytes free of cumulus, and consequently more than 24 h post-ovulation, a pronounced shrinking of the spindle is observed. The mean pole-to-pole distance becomes significantly reduced in postovulatory aged cells. At the same time astral microtubules in the cytoplasm appear to become gradually depolymerized. Age-dependent alterations in the microtubular cytoskeleton do not seem to result from a changed pattern of the post-translational detyrosylation of -tubulin in certain sets of microtubules. In freshly ovulated oocytes chromosomes in most spindles are well ordered and precisely arranged at the equatorial plane. In 11% of the cells only, there was dislocation of one or several of the chromosomes from the spindle equator. By contrast, 61.4% of bipolar spindles of postovulatory aged oocytes have chromosomes displaced from the centre of the spindle towards one of the spindle poles. The implications of the observed alterations in the microtubular cytoskeleton, shrinking of the spindle and increased disorder of chromosome alignment are discussed with regard to predisposition to aneuploidy and reduction of developmental potential of postovulatory aged oocytes.     

Microtubular spindle and centrosome structures during the cell cycle in a dinoflagellate Crypthecodinium cohnii B.: an immunocytochemical study

In order to determine if a mitotic spindle organizing center is present in dinoflagellate cells, we used a library of 12 monoclonal antibodies obtained by immunizing mice with isolated human centrosomes. When tested by immunofluorescence on cryosections of the dinoflaggelate Crypthecodinium cohnii B., a positive labeling was obtained with three of these antibodies. In interphase cells, the anti-centrosome antibodies labeled structures located either in the cell periphery, corresponding probably to both basal bodies (i.e. kinetosomes) and in the perinuclear area. In the latter case, two punctate structures were observed near the nuclear envelope. They have never been described, either in light, or in electron microscopic studies of dinoflagellates. We have designated them as centrosome-like structures. A microtubular desmose reacting positively with anti-tubulin Ab was also visible, linking kinetosomes and centrosome-like structures. During mitosis, the double punctate structures were observed at the poles of the nucleus. Double immunolabeling with tubulin and anti-centrosome Ab was also carried out and strongly suggested that in mitotic cells, centrosome-like structures, located at the poles of the mitotic spindle, were associated with microtubular bundles and probably organize and polarize them. These data indicate the existence of centrosome-like structures in C. cohnii cells and the strong conservation of some centrosomal epitopes from dinoflagellates to human. One of the antibodies (CTR 210) recognized by immunoblotting, a single protein band at 72 kDa from a total protein extract. The direct demonstration that this protein is located at the centrosome-like structures and at the kinetosomes deserves further study.     

Localization of mitotic arrest deficient 1 (MAD1) in mouse oocytes during the first meiosis and its functions as a spindle checkpoint protein

The present study was designed to investigate the localization of mitotic arrest deficient 1 (MAD1) in mouse oocytes during meiotic maturation and its relationship with kinetochores, chromosomes, and microtubules. Oocytes at various stages during the first meiosis were fixed and immunostained for MAD1, kinetochores, microtubules, and chromosomes. The stained oocytes were examined by confocal microscopy. Some oocytes were treated with nocodazole or Taxol before examination. The anti-MAD1 antibody was injected into the oocytes at the germinal vesicle (GV) stage for examination of chromosome alignment and spindle formation. It was found that MAD1 was present in the oocytes from the GV to prometaphase I stages around the nuclei. When the oocytes reached the metaphase I (M-I) to metaphase II (M-II) stages, MAD1 was mainly localized at the spindle poles. However, MAD1 relocated to the vicinity of the chromosomes when spindles were disassembled by nocodazole or cooling, and the relocated MAD1 moved back to the spindle poles during spindle recovery. Taxol treatment did not affect the MAD1 localization. Although anti-MAD1 antibody injection did not affect nuclear maturation, significantly higher proportions of injected oocytes had misaligned chromosomes when the oocytes reached the M-I to M-II stages. The results of the present study indicate that MAD1 is present in mouse oocytes at all stages during the first meiosis and that it participates in spindle checkpoint during meiosis. However, MAD1 could not check misaligned chromosomes during spindle recovery after the spindles were destroyed by drug or cooling, which caused some chromosomes to scatter in the oocytes.     

Widespread distribution of the major polypeptide component of MAP 1 (microtubule-associated protein 1) in the nervous system

We prepared a monoclonal antibody to microtubule-associated protein 1 (MAP 1), one of the two major high molecular weight MAP found in microtubules isolated from brain tissue. We found that MAP 1 can be resolved by SDS PAGE into three electrophoretic bands, which we have designated MAP 1A, MAP 1B, and MAP 1C in order of increasing electrophoretic mobility. Our antibody recognized exclusively MAP 1A, the most abundant and largest MAP 1 polypeptide. To determine the distribution of MAP 1A in nervous system tissues and cells, we examined tissue sections from rat brain and spinal cord, as well as primary cultures of newborn rat brain by immunofluorescence microscopy. Anti-MAP 1A stained white matter and gray matter regions, while a polyclonal anti-MAP 2 antibody previously prepared in this laboratory stained only gray matter. This confirmed our earlier biochemical results, which indicated that MAP 1 is more uniformly distributed in brain tissue than MAP 2 (Vallee, R.B., 1982, J. Cell Biol., 92:435-442). To determine the identity of cells and cellular processes immunoreactive with anti-MAP 1A, we examined a variety of brain and spinal cord regions. Fibrous staining of white matter by anti-MAP 1A was generally observed. This was due in part to immunoreactivity of axons, as judged by examination of axonal fiber tracts in the cerebral cortex and of large myelinated axons in the spinal cord and in spinal nerve roots. Cells with the morphology of oligodendrocytes were brightly labeled in white matter. Intense staining of Purkinje cell dendrites in the cerebellar cortex and of the apical dendrites of pyramidal cells in the cerebral cortex was observed. By double-labeling with antibodies to MAP 1A and MAP 2, the presence of both MAP in identical dendrites and neuronal perikarya was found. In primary brain cell cultures anti-MAP 2 stained predominantly cells of neuronal morphology. In contrast, anti-MAP 1A stained nearly all cells. Included among these were neurons, oligodendrocytes and astrocytes as determined by double-labeling with anti-MAP 1A in combination with antibody to MAP 2, myelin basic protein or glial fibrillary acidic protein, respectively. These results indicate that in contrast to MAP 2, which is specifically enriched in dendrites and perikarya of neurons, MAP 1A is widely distributed in the nervous system.     

DOC1R: a MAP kinase substrate that control microtubule organization of metaphase II mouse oocytes

For the success of fertilization, spindles of vertebrate oocytes must remain stable and correctly organized during the arrest in metaphase II of meiosis. Using a two-hybrid screen with MAPK as a bait, we have recently identified MISS (MAPK interacting and spindle stabilizing) which controls mouse oocyte metaphase II spindle stability. Using the same screen, we identify another MAPK partner, DOC1R (Deleted in oral cancer one related), a murine homologue of a potential human tumor suppressor gene. We characterize DOC1R during mouse oocyte meiosis resumption. DOC1R is regulated by phosphorylation during meiotic maturation by MPF (M-phase promoting factor) and by the MOS/./MAPK pathway. DOC1R and a DOC1R-GFP fusion localize to microtubules during meiotic maturation. Consistent with this microtubular localization, we show, by antisense and double-stranded RNA injection, that depletion of DOC1R induces microtubule defects in metaphase II oocytes. These defects are rescued by overexpressing a Xenopus DOC1R, showing that they are specific to DOC1R. Thus, the discovery of DOC1R, a substrate of MAPK that regulates microtubule organization of metaphase II mouse oocytes, reinforces the importance of this pathway in the control of spindle stability during the metaphase II arrest.     

Ultrastructural localization of microtubule-associated proteins (MAP) 1 in cilia of the respiratory tract

The presence and localization of high molecular weight microtubule-associated proteins of the MAP 1 class in ciliated cells of porcine and rat respiratory tract was studied by immunoblotting and immunoelectron microscopy. Ciliary shafts of the porcine tracheal epithelium were isolated using a method that minimizes contamination of the preparation by other cellular fragments and fat. Immunoblotting with rabbit antibodies to bulk MAP 1 from hog brain clearly revealed the presence of anti-MAP 1-immunoreactive high molecular weight proteins of the MAP 1 size in these preparations. To localize MAP 1 proteins at the ultrastructural level, rat and porcine tracheal epithelia were embedded in LR White and subjected to immunogold electron microscopy. Anti-MAP 1-immunoreactive material was found at ciliary shafts and basal bodies, but not at basal feet or ciliary rootlets. Interestingly, the necklace region between the shaft and the basal body of the cilium was hardly reactive with anti-MAP 1 antibodies. This may indicate a reduced stability of ciliary microtubules in this region and could be an explanation why ciliary shafts in general break more easily there than elsewhere.     

Cytoskeletal architecture and immunocytochemical localization of microtubule-associated proteins in regions of axons associated with rapid axonal transport: the beta,beta''-iminodipropionitrile-intoxicated axon as a model system

Axons from rats treated with the neurotoxic agent beta,beta'-iminodipropionitrile (IDPN) were examined by quick-freeze, deep-etch electron microscopy. Microtubules formed bundles in the central region of the axons, whereas neurofilaments were segregated to the periphery. Most membrane-bounded organelles, presumably including those involved in rapid axonal transport, were associated with the microtubule domain. The high resolution provided by quick-freeze, deep-etch electron microscopy revealed that the microtubules were coated with an extensive network of fine strands that served both to cross-link the microtubules and to interconnect them with the membrane-bounded organelles. The strands were decorated with granular materials and were irregular in dimension. They appeared either singly or as an extensive anastomosing network in fresh axons. The microtubule-associated strands were observed in fresh, saponin-extracted, or aldehyde-fixed tissue. To explore further the identity of the microtubule-associated strands, microtubules purified from brain tissue and containing the high molecular weight microtubule-associated proteins MAP 1 and MAP 2 were examined by quick-freeze, deep-etch electron microscopy. The purified microtubules were connected by a network of strands quite similar in appearance to those observed in the IDPN axons. Control microtubule preparations consisting only of tubulin and lacking the MAPs were devoid of associated strands. To learn which of the MAPs were present in the microtubule bundles in the axon, sections of axons from IDPN-treated rats were examined by immunofluorescence microscopy using antibodies to MAP 1A, MAP 1B, MAP 2, and tubulin. Anti-MAP 2 staining was only marginally detectable in the IDPN-treated axons, consistent with earlier observations. Anti-MAP 1A and anti-MAP 1B brightly stained the IDPN-treated axons, with the staining exclusively limited to the microtubule domains. Furthermore, thin section-immunoelectron microscopy using colloidal gold-labeled second antibodies revealed that both anti-MAP 1A and anti-MAP 1B stained fuzzy filamentous structures between microtubules. In view of earlier work indicating that rapid transport is associated with the microtubule domain in the IDPN-treated axon, it now appears that MAP 1A and MAP 1B may play a role in this process. We believe that MAP 1A and MAP 1B are major components of the microtubule-associated fibrillar matrix in the axon.     

Widespread cellular distribution of MAP-1A (microtubule-associated protein 1A) in the mitotic spindle and on interphase microtubules

In the accompanying paper (Bloom, G.S., T.A. Schoenfeld, and R.B. Vallee, 1983, J. Cell Biol. 98:320-330), we reported that microtubule-associated protein 1 (MAP 1) from brain comprises multiple protein species, and that the principal component, MAP 1A, can be detected in both neuronal and glial cells by immunofluorescence microscopy using a monoclonal antibody. In the present study, we sought to determine the cellular and subcellular distribution of MAP 1A in commonly used cultured cell systems. For this purpose we used immunofluorescence microscopy and immunoblot analysis with anti-MAP 1A to examine 18 types of mammalian cell cultures. MAP 1A was detected in every culture system examined. Included among these were cells of mouse, rat, Chinese hamster, Syrian hamster, Potoroo (marsupial), and human origin derived from a broad variety of tissues and organs. Anti-MAP 1A consistently labeled mitotic spindles and stained cytoplasmic fibers during interphase in most of the cultures. These fibers were identified as microtubules by co-localization with tubulin in double-labeling experiments, by their disappearance in response to colchicine or vinblastine, and by their reorganization in response to taxol. The anti-MAP 1A stained microtubules in a punctate manner, raising the possibility that MAP 1A is located along microtubules at discrete foci that might represent sites of interaction between microtubules and other organelles. Verification that MAP 1A was, indeed, the reactive material in immunofluorescence microscopy was obtained from immunoblots. Anti-MAP 1A stained a band at the position of MAP 1A in all cultures examined. These results establish that MAP 1A, a major MAP from brain, is widely distributed among cultured mammalian cells both within and outside of the nervous system.     

Immunolocalization of tubulin isoforms and post-translational modifications in the protists Tritrichomonas foetus and Trichomonas vaginalis

In the present report we show the distribution of multiple tubulin isoforms in Trichomonas vaginalis and Tritrichomonas foetus, flagellated parasitic protists of the urogenital tracts of human and cattle, respectively, using immunofluorescence and immunoelectron microscopy. We used several monoclonal and polyclonal anti-tubulin antibodies from different sources and recognizing variant tubulin isoforms. Our results demonstrate that: (1) there is a heterogeneous distribution of the different tubulin isoforms in the main microtubular cell structures, such as axostyle, flagella, basal bodies, and mitotic spindle, (2) the axostyle-pelta junction is a structure with high affinity for glutamylated tubulin antibodies in T. foetus, (3) the spindle labeling is positive to anti-glutamylated tubulin and anti-alpha-tubulin (TAT1 and purchased from Amersham) antibodies in T. vaginalis but it is negative in T. foetus, (4) the nuclear matrix and the cytosol presented positive reaction using glutamylated and TAT1 (anti-alpha-tubulin) antibodies only in T. vaginalis, and (5) the Golgi complex exhibited staining using the glutamylated tubulin antibody. The present data corroborate with the idea of the existence of a heterogeneous population of microtubules in these protists and of a subset of intracytoplasmic microtubules. Microtubule diversity may reflect distinct tubulins, diverse microtubule-associated proteins, or a combination of both.     

Cell cycle-dependent behavior of microtubules in hybrids of mouse oocytes and blastomeres.

The behavior of microtubules was studied in hybrids formed between mouse oocytes arrested in metaphase II or activated parthenogenetically and mouse embryo interphase blastomeres. In all cases the interphase blastomere's network of microtubules disassembles rapidly after fusion with oocytes. Introduction of interphase cytoplasm and nuclei to metaphase oocytes during fusion induces the polymerization of new microtubules in the cytoplasm and in the meiotic spindle. The degree and the duration of this facilitated polymerization of microtubules was positively correlated with the volume of blastomeres used for fusion. The blastomere nuclei induce the formation of microtubular frames, which become more evident when the chromatin undergoes premature condensation. Finally, spindle-like structures are formed around the prematurely condensed chromosomes. In hybrids activated around the time of fusion, the blastomere nuclei undergo pronuclear-like transformation. These hybrids develop an interphase network of microtubules typical for activated oocytes. These results are discussed with regards to the cell cycle control of microtubule behavior.     

Change in the heterogeneous distribution of tubulin isotypes in mitotic microtubules of the sea urchin egg by treatment with microtubule depolymerizing or stabilizing drugs.

In the mitotic sea urchin egg, the spindle microtubules were composed of different tubulin isotypes from those of astral microtubules using monoclonal antibodies [Oka et al. (1990) Cell Motil. Cytoskeleton, 16, 239-250]. Three of the antibodies, D2D6, DM1B, and YL1/2, were specific for spindle microtubules, astral microtubules and reactive with both microtubules, respectively. The mitotic sea urchin egg was treated with microtubule depolymerizing (colcemid and nocodazole) and stabilizing (hexylene glycol) drugs and change in the heterogeneous distribution of the tubulin isotypes was investigated by the immunofluorescence procedure using these three monoclonal anti-tubulin antibodies. We observed that: (1) the microtubule depolymerizing drugs caused quick depolymerization of most mitotic microtubules, and a small number of spindle microtubules remaining were stained with all three antibodies; (2) hexylene glycol induced many microtubules in the mitotic apparatus, which was stained with D2D6 but was not stained with DM1B; (3) hexylene glycol also induced a great number of miniasters in the cytoplasm, and they were stained with three antibodies. These results suggest that these drugs altered the distribution of tubulin isotypes in the mitotic microtubules during depolymerization or polymerization within a short time.     

Protein kinase B (PKB/Akt) is required for the completion of meiosis in mouse oocytes

Akt, also known as protein kinase B, is implicated in many cellular processes. Akt is phosphorylated at two residues, Thr308 and Ser473. Thr308-phosphorylated Akt is present in pericentriolar materials, while localization of Ser473-phosphorylated Akt was similar to that of microtubules in metaphase oocytes. Spindles were shorter and aberrant in oocytes injected with Thr308- or Ser473-phosphorylated Akt antibodies. Specifically, Thr308- and Ser473-phosphorylated Akts function individually and are both necessary to assemble the metaphase II (MII) spindle. Moreover, the functions of Thr308- and Ser473-phosphorylated Akts differ in MII oocytes. Although oocytes exhibited second polar body (PB2) emission after the injection of a peptide for Thr308, the chromosomal alignment and microtubular organization were aberrant. In contrast, the injection of a peptide for Ser473 caused a failure of PB2 emission. These results suggest that Thr308- and Ser473-phosphorylated Akts are individually involved in fertilization to complete meiosis, including different roles (i.e., Ser473-phosphorylated Akts are involved in PB2 emission, whereas Thr308-phosphorylated Akts regulate the organization of microtubules).     

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.     

Cytoskeletal organization of rat oocytes during metaphase II arrest and following abortive activation: A study by confocal laser scanning microscopy

In metaphase II arrested rat oocytes (M il), microtubles were found in the taper-shaped meiotic spindle and in the cytoplasm as asters and free microtubules. Whereas spindle microtubules were acetylated, those located in the cytoplasm were not. Cytoplasmic microtubules were also labile as assessed by mild cooling. In contast to mouse oocytes, rat microtubule organizing centers (MTOCs) did not react with MPM-2 antibody by immunofluorescence despite the fact that this antibody reacts with several proteins as shown by immunoblot. However, cytoplasmic MTOCs in M II-arrested rat oocytes could be detected by their nucleating capacity in the presence of taxol, a drug that induced the formation of numerous cytoplasmic asters. In addition, taxol caused a change in the spindle shape and the formation of astral microtubules at the spindle poles. Meiotic spindles (as well as chromosomes devoid of microtubules after nocodazoletreatment) were overlaid by an actin-rich domain. Spontaneous abortive activation led to the extrusion of the second polar body followed by another metaphase arrest— metaphase III; however, normal spindles did not form and dispersed chromosomes surrounded by microtubles were observed. Electron microscopic studies confirmed these observations and revealed that the kinetochores are located deep within the chromosomes in contrast to mouse kinetochores, and this might be responsible for the absence of a metaphase III spindle in the rat oocyte. Induced activation caused transition to interphase with the formation of a characteristic microtubule network. This study shows that there are several significant differences in the cytoskeletal organization of rat and mouse oocytes. © 1993 Wiley-Liss, Inc.     

Post-translational modifications of tubulin and the dynamics of microtubules in mouse oocytes and zygotes

The distribution of post-translationally modified forms of tubulin has been studied in mouse oocytes arrested in meiotic metaphase II and in interphase eggs after fertilisation. Tyrosinated and acetylated microtubules are present in the meiotic spindle but detyrosinated ones are not. Acetylation only occurs in the most stable subpopulation of microtubules in the spindles ("pole to kinetochore"). After fertilisation, many microtubules of the interphase array become acetylated, but detyrosination occurs only at a very low level.