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.     

Microtubule and chromatin organization during the first cell-cycle following intracytoplasmic injection of round spermatid into porcine oocytes

The objective of this study was to determine microtubule assembly and chromatin configuration in porcine oocytes during the first cell cycle following round spermatid injection into matured porcine oocytes in the presence or absence of electrical stimulation. The oocytes with two large pronuclei and two polar bodies were classified as normal fertilization at 6 to 8 h following injection. The incidence of normal fertilization following round spermatid injection with electrical stimulation was significantly higher (21/45, 47%) than that following injection alone (6/39, 15%). Although a small microtubular aster was organized near the decondensed spermatid chromatin in some oocytes (2/6, 33%, spermatid injection alone; 9/21, 29%, spermatid injection and electrical stimulation), it did not enlarge nor fill the cytoplasm. Instead, a dense network of microtubules in the cytoplasm was organized from cortex. At 12 to 15 h after injection, we classified the oocytes with closely apposed pronuclei as normal fertilization. The electrical stimulation following spermatid injection enhanced (P < 0.05) the incidence of normal fertilization (18/54, 33%) compared with spermatid injection alone (7/52, 13%). During pronuclear movement, the maternally derived microtubules filled the whole cytoplasm, which appeared to move male and female chromatin. Mitosis and two-cell division were observed at 20 to 24 h after spermatid injection with electrical stimulation (12/41, 29%). At mitotic metaphase, the microtubular spindle had focused astral poles, and chromosomes were aligned on the spindle equator. During mitosis, asters were assembled at each spindle pole, and they filled the cytoplasm. These results suggested that round spermatid nuclei of the pig can develop into a morphologically normal pronucleus in matured porcine oocytes and are competent to participate in syngamy with the ootid chromatin. In addition, functional microtubules for complete fertilization with spermatid were not associated with male-derived centrosome but were organized solely from maternal stores. Mol. Reprod. Dev. 50:221–228, 1998. © 1998 Wiley-Liss, Inc.     

Microtubule distribution during fertilization in the rabbit.

The distribution of microtubules was studied during fertilization of the rabbit oocyte by immunofluorescence microscopy after staining with an anti-alpha-tubulin antibody. In ovulated oocytes, microtubules were found exclusively in the meiotic spindle. At fertilization, the paternal centrosome generated sperm astral microtubules. During pronuclear development, the sperm aster increased in size, and microtubules extended from the male pronucleus to the egg center and towards the female pronucleus. These observations indicate that microtubules emanating from the sperm centrosome were involved in the movements leading to the union of the male and female pronuclei. At late pronuclear stage, microtubules surrounded the adjacent pronuclei. The mitotic spindle that emerged from the perinuclear microtubules contained broad anastral poles.     

The CBA mouse as a model for age-related aneuploidy in man: studies of oocyte maturation,spindle formation and chromosome alignment during meiosis

To elucidate the possible mechanism of disturbances in chromosome segregation leading to the increase in aneuploidy in oocytes of aged females we examined the meiotic spindles of CBA/Ca mice. Employing immunofluorescence with an anti-tubulin antibody, and human scleroderma serum, as well as 4-6-diamidino-2-phenylindole (DAPI) staining of chromosomes the microtubular cytoskeleton could be visualized, and the behaviour of chromosomes and centromeres of oocytes spontaneously maturing in vitro could be studied. The morphology of spindles during the first meiotic division was not conspiciously different in oocytes from young and aged mice as far as the cytoskeletal elements were concerned. Neither multipolar spindles nor pronounced cytoplasmic asters appeared in oocytes of mice approaching the end of their reproductive life (9 months and older). Oocytes of aged females also did not exhibit any sign of premature separation of parental chromosomes at prophase, obvious malorientations of bivalents, or significant lagging of chromosomes during ana and telophase. Metaphase I with all bivalents aligned at the spindle equator appeared to be a relatively brief stage in oocyte development compared with pro-and prometaphase. Therefore, already slight disturbances occuring in the timing of the developmental programme which leads to a premature anaphase transition may be responsible for the high incidence of chromosomally unbalanced gametes in aged females, rather than non-separation and lagging of chromosomes during late ana-and telophase. In a second set of experiments we compared the metaphase II spindles of spontaneously ovulated oocytes obtained from animals at different ages. Previous studies have shown that spindle length and chromosome alignment may be altered in cells predisposed to aneuploidy. To distinguish between the significance of the chronological age of the female and the physiological age of the ovaries (as indicated by the total number of oocytes remaining) we examined the spindle apparatus in young (3–4 months old) and aged (9 months and older) mice as well as CBA females which had been unilaterally ovariectomized (uni-ovx) early in adult life and were approaching the end of their reproductive life at 6–7 months of age. Measurements of the pole-to-pole distance implied that spindle length may be related to maternal age. In oocytes of aged (9 month), uni-ovx (6 month) as well as 6-month-old sham-operated controls the metaphase II spindle was significantly shorter than in oocytes of young mice. By contrast, chromosome disorder and displacement was most pronounced in the aged and uni-ovx mice whilst most oocytes from young mice and moderately aged shamtreated controls exhibited a more regular alignment of chromosomes. These results, which are consistent with recent findings in CBA mice of an increased rate of aneuploidy in females approaching the end of their reproductive life, are discussed with respect to the hypothesis that the aetiology of aneuploidy rests on the critical timing of different events in oocyte development.     

CENP-32 is required to maintain centrosomal dominance in bipolar spindle assembly

Centrosomes nucleate spindle formation, direct spindle pole positioning, and are important for proper chromosome segregation during mitosis in most animal cells. We previously reported that centromere protein 32 (CENP-32) is required for centrosome association with spindle poles during metaphase. In this study, we show that CENP-32 depletion seems to release centrosomes from bipolar spindles whose assembly they had previously initiated. Remarkably, the resulting anastral spindles function normally, aligning the chromosomes to a metaphase plate and entering anaphase without detectable interference from the free centrosomes, which appear to behave as free asters in these cells. The free asters, which contain reduced but significant levels of CDK5RAP2, show weak interactions with spindle microtubules but do not seem to make productive attachments to kinetochores. Thus CENP-32 appears to be required for centrosomes to integrate into a fully functional spindle that not only nucleates astral microtubules, but also is able to nucleate and bind to kinetochore and central spindle microtubules. Additional data suggest that NuMA tethers microtubules at the anastral spindle poles and that augmin is required for centrosome detachment after CENP-32 depletion, possibly due to an imbalance of forces within the spindle.     

Meiotic spindle assembly in Drosophila females: behavior of nonexchange chromosomes and the effects of mutations in the nod kinesin-like protein

Mature Drosophila oocytes are arrested in metaphase of the first meiotic division. We have examined microtubule and chromatin reorganization as the meiosis I spindle assembles on maturation using indirect immunofluorescence and laser scanning confocal microscopy. The results suggest that chromatin captures or nucleates microtubules, and that these subsequently form a highly tapered spindle in which the majority of microtubules do not terminate at the poles. Nonexchange homologs separate from each other and move toward opposite poles during spindle assembly. By the time of metaphase arrest, these chromosomes are positioned on opposite half spindles, between the metaphase plate and the spindle poles, with the large nonexchange X chromosomes always closer to the metaphase plate than the smaller nonexchange fourth chromosomes. Nonexchange homologs are therefore oriented on the spindle in the absence of a direct physical linkage, and the spindle position of these chromosomes appears to be determined by size. Loss-of-function mutations at the nod locus, which encodes a kinesin-like protein, cause meiotic loss and nondisjunction of nonexchange chromosomes, but have little or no effect on exchange chromosome segregation. In oocytes lacking functional nod protein, most of the nonexchange chromosomes are ejected from the main chromosomal mass shortly after the nuclear envelope breaks down and microtubules interact with the chromatin. In addition, the nonexchange chromosomes that are associated with spindles in nod/nod oocytes show excessive poleward migration. Based on these observations, and the structural similarity of the nod protein and kinesin, we propose that nonexchange chromosomes are maintained on the half spindle by opposing poleward and anti-poleward forces, and that the nod protein provides the anti-poleward force.     

Drosophila EB1 is important for proper assembly,dynamics, and positioning of the mitotic spindle

EB1 is an evolutionarily conserved protein that localizes to the plus ends of growing microtubules. In yeast, the EB1 homologue (BIM1) has been shown to modulate microtubule dynamics and link microtubules to the cortex, but the functions of metazoan EB1 proteins remain unknown. Using a novel preparation of the Drosophila S2 cell line that promotes cell attachment and spreading, we visualized dynamics of single microtubules in real time and found that depletion of EB1 by RNA-mediated inhibition (RNAi) in interphase cells causes a dramatic increase in nondynamic microtubules (neither growing nor shrinking), but does not alter overall microtubule organization. In contrast, several defects in microtubule organization are observed in RNAi-treated mitotic cells, including a drastic reduction in astral microtubules, malformed mitotic spindles, defocused spindle poles, and mispositioning of spindles away from the cell center. Similar phenotypes were observed in mitotic spindles of Drosophila embryos that were microinjected with anti-EB1 antibodies. In addition, live cell imaging of mitosis in Drosophila embryos reveals defective spindle elongation and chromosomal segregation during anaphase after antibody injection. Our results reveal crucial roles for EB1 in mitosis, which we postulate involves its ability to promote the growth and interactions of microtubules within the central spindle and at the cell cortex.     

Distinctions in meiotic spindle structure and assembly during in vitro and in vivo maturation of mouse oocytes

To better understand the differences in cytoskeletal organization between in vivo (IVO) and in vitro (IVM) matured oocytes, we analyzed remodeling of the centrosome-microtubule complex in IVO and IVM mouse oocytes. Fluorescence imaging revealed dramatic differences in meiotic spindle assembly and organization between these two populations. Metaphase spindles at both meiosis I (M-I) and meiosis II (M-II) in IVO oocytes were compact, displayed focused spindle poles with distinct gamma-tubulin foci, and were composed of acetylated microtubules. In contrast, IVM oocytes exhibited barrel-shaped spindles with fewer acetylated microtubules and gamma-tubulin diffusely distributed throughout the spindle proper. With respect to meiotic progression, IVO oocytes were more synchronous in the rate and extent of anaphase to telophase of M-I and first polar body emission than were IVM counterparts. Furthermore, IVO oocytes showed a twofold increase in cytoplasmic microtubule organizing centers (MTOCs), and constitutive MTOC proteins (gamma-tubulin and pericentrin) were excluded from the first polar body. Inclusion of MTOC constitutive proteins in the polar body and diminished number of cytoplasmic MTOCs was observed in IVM oocytes. These findings were corroborated in IVO oocytes obtained from naturally ovulated and spontaneously cycling mice and highlight a fundamental distinction in the spatial and temporal regulation of microtubule dynamics between IVO and IVM oocytes     

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.     

The microtubular cytoskeleton and chromosomes of unfertilized human oocytes aged in vitro

Summary To detect structural alterations in human oocytes that may give rise to predisposition to aneuploidy, unfertilized human oocytes from an IVF programme were processed for indirect anti-tubulin immunofluorescence. The spindle of oocytes aged for 2 days is rather small, and bi- or multipolar. Chromosomes are no longer aligned at the spindle equator but are scattered all over the degenerating spindle. This implies that human oocytes aged for 2 days may no longer be able to develop into a chromosomally balanced, normal embryo. In oocytes aged for 3–4 days the chromosomes become more decondensed and form a restitution nucleus. Microtubules radiate out from the latter towards the cell periphery and form a network of fibres in the cytoplasm. A similar alignment of tubules is found in unfertilized, activated oocytes. Oocytes with an aberrant cytoskeleton and chromosomal array were predominantly obtained from aged females. They include two binucleated oocytes with two sets of chromosomes and two oocytes with displaced chromosomes one of which had a tripolar spindle.     

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

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

Intra-oocyte localization of MAD2 and its relationship with kinetochores, microtubules, and chromosomes in rat oocytes during meiosis

The present study was designed to investigate subcellular localization of MAD2 in rat oocytes during meiotic maturation and its relationship with kinetochores, chromosomes, and microtubules. Oocytes at germinal vesicle (GV), prometaphase I (ProM-I), metaphase I (M-I), anaphase I (A-I), telophase I (T-I), and metaphase II (M-II) were fixed and immunostained for MAD2, kinetochores, microtubules and chromosomes. The stained oocytes were examined by confocal microscopy. Some oocytes from GV to M-II stages were treated by a microtubule disassembly drug, nocodazole, or treated by a microtubule stabilizer, Taxol, before examination. Anti-MAD2 antibody was also injected into the oocytes at GV stage and the injected oocytes were cultured for 6 h for examination of chromosome alignment and spindle formation. It was found that MAD2 was at the kinetochores in the oocytes at GV and ProM-I stages. Once the oocytes reached M-I stage in which an intact spindle was formed and all chromosomes were aligned at the equator of the spindle, MAD2 disappeared. However, when oocytes from GV to M-II stages were treated by nocodazole, spindles were destroyed and MAD2 was observed in all treated oocytes. When nocodazole-treated oocytes at M-I and M-II stages were washed and cultured for spindle recovery, it was found that, once the relationship between microtubules and chromosomes was established, MAD2 disappeared in the oocytes even though some chromosomes were not aligned at the equator of the spindle. On the other hand, when oocytes were treated with Taxol, MAD2 localization was not changed and was the same as that in the control. However, immunoblotting of MAD2 indicated that MAD2 was present in the oocytes at all stages; nocodazole and Taxol treatment did not influence the quantity of MAD2 in the cytoplasm. Significantly higher proportions of anti-MAD2 antibody-injected oocytes proceeded to premature A-I stage and more oocytes had misaligned chromosomes in the spindles. The present study indicates that MAD2 is a spindle checkpoint protein in rat oocytes during meiosis. When the spindle was destroyed by nocodazole, MAD2 was reactivated in the oocytes to overlook the attachment between chromosomes and microtubules. However, in this case, MAD2 could not check unaligned chromosomes in the recovered spindles, suggesting that a normal chromosome alignment is maintained only in the oocytes without any microtubule damages during maturation.     

Positioning and elongation of the fission yeast spindle by microtubule-based pushing

In eukaryotic cells, proper position of the mitotic spindle is necessary for successful cell division and development. We explored the nature of forces governing the positioning and elongation of the mitotic spindle in Schizosaccharomyces pombe. We hypothesized that astral microtubules exert mechanical force on the S. pombe spindle and thus help align the spindle with the major axis of the cell. Microtubules were tagged with green fluorescent protein (GFP) and visualized by two-photon microscopy. Forces were inferred both from time-lapse imaging of mitotic cells and, more directly, from mechanical perturbations induced by laser dissection of the spindle and astral microtubules. We found that astral microtubules push on the spindle poles in S. pombe, in contrast to the pulling forces observed in a number of other cell types. Further, laser dissection of the spindle midzone induced spindle collapse inward. This offers direct evidence in support of the hypothesis that spindle elongation is driven by the sliding apart of antiparallel microtubules in the spindle midzone. Broken spindles recovered and mitosis completed as usual. We propose a model of spindle centering and elongation by microtubule-based pushing forces.     

Microinjected carboxylated beads move predominantly poleward in sea urchin eggs

Observations on living mitotic cells have suggested that material in the spindle moves poleward during mitosis. In order to investigate this movement, sea urchin eggs have been microinjected with 0.25-micron diameter carboxylated fluorescent beads. When fluorescent beads were injected into unfertilized Lytechinus variegatus eggs, no motility was detected. When injected into mitotic cells, beads moved to the spindle poles. Individual beads moved rapidly, in a saltatory fashion, and followed generally linear paths. Beads appeared to move along astral fibers, were generally excluded from the spindle proper, and accumulated at the spindle poles. Some dispersion of the beads away from the pole was observed as cells completed mitosis, but the majority of beads retained a polar location. After depolymerization of spindle microtubules with nocodazole, some dispersion of beads into the cytoplasm was also observed. Beads moved along taxol-induced astral microtubules and accumulated at astral centers. These observations reveal that negatively charged beads accumulate rapidly at mitotic centers, moving toward the minus end of the microtubules. Neither the bidirectional motility of similar beads in interphase cells nor the plus-end-directed bead motility seen in axons was observed in these mitotic cells.     

The chromosomal basis of meiotic acentrosomal spindle assembly and function in oocytes

Several aspects of meiosis are impacted by the absence of centrosomes in oocytes. Here, we review four aspects of meiosis I that are significantly affected by the absence of centrosomes in oocyte spindles. One, microtubules tend to assemble around the chromosomes. Two, the organization of these microtubules into a bipolar spindle is directed by the chromosomes. Three, chromosome bi-orientation and attachment to microtubules from the correct pole require modification of the mechanisms used in mitotic cells. Four, chromosome movement to the poles at anaphase cannot rely on polar anchoring of spindle microtubules by centrosomes. Overall, the chromosomes are more active participants during acentrosomal spindle assembly in oocytes, compared to mitotic and male meiotic divisions where centrosomes are present. The chromosomes are endowed with information that can direct the meiotic divisions and dictate their own behavior in oocytes. Processes beyond those known from mitosis appear to be required for their bi-orientation at meiosis I. As mitosis occurs without centrosomes in many systems other than oocytes, including all plants, the concepts discussed here may not be limited to oocytes. The study of meiosis in oocytes has revealed mechanisms that are operating in mitosis and will probably continue to do so.     

A Highlights from MBoC Selection: Cell shape impacts on the positioning of the mitotic spindle with respect to the substratum

All known mechanisms of mitotic spindle orientation rely on astral microtubules. We report that even in the absence of astral microtubules, metaphase spindles in MDCK and HeLa cells are not randomly positioned along their x-z dimension, but preferentially adopt shallow β angles between spindle pole axis and substratum. The nonrandom spindle positioning is due to constraints imposed by the cell cortex in flat cells that drive spindles that are longer and/or wider than the cell''s height into a tilted, quasidiagonal x-z position. In rounder cells, which are taller, fewer cortical constraints make the x-z spindle position more random. Reestablishment of astral microtubule–mediated forces align the spindle poles with cortical cues parallel to the substratum in all cells. However, in flat cells, they frequently cause spindle deformations. Similar deformations are apparent when confined spindles rotate from tilted to parallel positions while MDCK cells progress from prometaphase to metaphase. The spindle disruptions cause the engagement of the spindle assembly checkpoint. We propose that cell rounding serves to maintain spindle integrity during its positioning.     

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.     

Effects of cooling on meiotic spindle structure and chromosome alignment within in vitro matured porcine oocytes

Meiotic spindle structure and chromosome alignment were examined after porcine oocytes were cooled at metaphase II (M II) stage. Cumulus-oocyte complexes (COCs) collected from medium size follicles were cultured in an oocyte maturation medium at 39 degrees C, 5% CO(2) in air for 44 hr. At the end of culture, oocytes were removed from cumulus cells and cooled to 24 or 4 degrees C for 5, 30, or 120 min in a solution with or without 1.5 M dimethyl sulfoxide (DMSO). After being cooled, oocytes were either fixed immediately for examination of the meiotic spindle and chromosome alignment or returned to maturation medium at 39 degrees C for 2 hr for examination of spindle recovery. Most oocytes (65-71%) cooled to 24 degrees C showed partially depolymerized spindles but 81-92% of oocytes cooled at 4 degrees C did not have a spindle after cooling for 120 min. Quicker disassembly of spindles in the oocytes was observed at 4 degrees C than at 24 degrees C. Cooling also induced chromosome abnormality, which was indicated by dispersed chromosomes in the cytoplasm. Limited spindle recovery was observed in the oocytes cooled to both 4 and 24 degrees C regardless of cooling time. The effect of cooling on the spindle organization and chromosome alignment was not influenced by the presence of DMSO. These results indicate that the meiotic spindles in porcine M II oocytes are very sensitive to a drop in the temperature. Both spindle and chromosomes were damaged during cooling, and such damage was not reversible by incubating the oocytes after they had been cooled.     

Ultrastructure of bovine oocytes exposed to Taxol prior to OPS vitrification

Our objective was to document potential subcellular consequences of treatment with the microtubule stabilizer Taxol with or without subsequent vitrification of cow and calf oocytes by the open pulled straw (OPS) method. Oocytes were divided into four experimental groups for cows and four groups for calves: (1) a control group fixed immediately after maturation; (2) an OPS group cryopreserved by conventional OPS; (3) a Taxol/CPA group exposed to 1 microM Taxol and cryoprotective agents (CPAs); and (4) a Taxol/OPS group vitrified by OPS including 1 microM Taxol to the vitrification solution. All oocytes were processed for light and transmission electron microscopy. The main injuries were observed on the metaphase plate and the spindle. In control oocytes, the metaphase appeared as condensed chromosomes arranged in a well-organized metaphase plate and the spindle showed well organized microtubules in both cow and calf oocytes. However, in cow OPS oocytes, the metaphase plate was disorganized into scattered chromosomes or the chromosomes were condensed into a single block of chromatin. In addition, microtubules were not organized as typical spindles. In contrast, cow Taxol/OPS oocytes as well as both cow and calf Taxol/CPAs oocytes showed well-organized metaphase plates and normal spindle morphology. All calf OPS and calf Taxol/OPS oocytes displayed a single block of chromatin and no microtubules could be observed around the chromosomes. In conclusion, treatment with 1 microM Taxol before and during vitrification did not induce adverse changes in the oocyte cytoplasm or metaphase spindles in adult bovine oocytes, but stabilized the metaphase and spindle morphology.