Spindle dynamics in living mouse oocytes during meiotic maturation, ageing, cooling and overheating: a study by polarized light microscopy

A liquid crystal polarized light microscope (LC PolScope) was used to examine spindle dynamics in living mouse oocytes. Immature oocytes were cultured for 0-48 h and spindles were imaged with the PolScope at various time points of culture. Oocytes at metaphase I (M-I) and metaphase II (M-II) were also exposed to shifts of temperature from 25 to 41 degrees C to examine the effects of fluctuations of temperature on spindle dynamics. After examination with the PolScope, some oocytes were fixed and examined by immunocytochemical staining and confocal microscopy. After culturing for 6 h, 76% and 2% of the oocytes reached M-I and M-II stages and all oocytes had birefringent spindles. When the oocytes were cultured for 14-16 h, 88% and 6% of oocytes were at M-II and M-I stages respectively and all oocytes had birefringent spindles. However, when the oocytes were cultured for 22-48 h, the proportions of oocytes with birefringent spindles decreased as culture time was increased. Exposure of oocytes to 25 degrees C induced spindle disassembly within 10-20 min in both M-I and M-II oocytes. Most (93-100%) oocytes reassembled spindles after warming at 37 degrees C. Furthermore, exposure of oocytes at M-I stage but not at M-II stage, to 30 degrees C also induced significant microtubule disassembly. However, exposure of oocytes to 38-41 degrees C did not obviously change the quantity of microtubules in the spindles, which was measured by retardance. This study indicates that the PolScope can be used to examine spindle dynamics in living oocytes, and it has the advantage over the routine fluorescence microscope in that images can be obtained in the same individual oocyte and the quantity of microtubules can be measured by retardance in living oocytes. These results also indicate that the M-II spindle in mouse oocytes is sensitive to oocyte ageing and cooling, but not heating, and M-I spindle is more sensitive to temperature decline than M-II spindle.     

Maternal-restraint stress increases oocyte aneuploidy by impairing metaphase I spindle assembly and reducing spindle assembly checkpoint proteins in mice

Studies in both humans and animals suggest detrimental effects of psychological stress on reproduction. Although our recent study shows that maternal-restraint stress diminishes oocyte developmental potential, the mechanism behind this effect is unknown. This prompted us to study the potential role of maternal-restraint stress in the genesis of aneuploidy during meiosis I. At 24 h after equine chorionic gonadotropin injection, mice were subjected to restraint stress for 24 h. After the restraint, some mice were killed to recover immature oocytes for in vitro maturation, while others were injected with human chorionic gonadotropin to recover in vivo matured oocytes. Analysis on chromosome complements of both mature oocytes and parthenotes confirmed that maternal restraint increased aneuploidy in both in vivo and in vitro matured oocytes and that the percentage of aneuploid oocytes were three times higher in the earlier matured oocytes than in the later matured ones. Further observations indicated that maternal restraint 1) impaired metaphase I (MI) spindle assembly while inhibiting MAPK activities, 2) accelerated progression of anaphase I while down-regulating the expression of spindle assembly checkpoint (SAC) proteins, and 3) induced intraoocyte oxidative stress. The following possible model was proposed to explain the results. Maternal-restraint stress increased oocyte aneuploidy by impairing MI spindle assembly and decreasing the SAC. Whereas abnormal spindles would affect centromere attachments, a reduction in SAC would accelerate the anaphase I progression. Failure of centromere attachment, together with the hastened anaphase, would result in nondisjunction of the unattached chromosomes. Furthermore, maternal-restraint stress might also impair spindle assembly and SAC function by inducing intraoocyte oxidative stress, which would then reduce MAPK activity, a critical regulator of microtubule assembly and the establishment and maintenance of the SAC during oocyte maturation.     

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.     

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.     

Allocation of gamma-tubulin between oocyte cortex and meiotic spindle influences asymmetric cytokinesis in the mouse oocyte

In oocytes, asymmetric cytokinesis represents a conserved strategy for karyokinesis during meiosis to retain ooplasmic maternal factors needed after fertilization. Given the role of gamma-tubulin in cell cycle progression and microtubule dynamics, this study focused on gamma-tubulin as a key regulator of asymmetric cytokinesis in mouse oocytes. Gamma-tubulin properties were studied using multiple-label digital imaging, Western blots, quantitative RT-PCR, and microinjection strategies in mouse oocytes matured in vivo (IVO) or in vitro (IVM). Quantitative image analysis established that IVO oocytes extrude smaller first polar bodies (PBs), contain smaller spindles, and have more cytoplasmic microtubule organizing centers (MTOCs) relative to IVM oocytes. Maturation in culture was shown to alter gamma-tubulin distribution, as evidenced by incorporation throughout the meiotic spindle and within the first PB. Western blot analysis confirmed that total gamma-tubulin content remained elevated in IVM oocytes compared with IVO oocytes. Analysis of gamma-tubulin mRNA during maturation revealed fluctuations in IVO oocytes, whereas IVM oocytes maintained relatively stable at lower levels for the time points examined (0-16 h). Selective reduction of gamma-tubulin mRNA by injection of siRNA diminished both spindle and PB size, whereas overexpression of enhanced green fluorescent protein gamma-tubulin had the opposite effect. Together, these studies reinforce the notion that limiting gamma-tubulin availability during meiotic maturation ensures coordination of karyokinesis and cytokinesis and conservation of gamma-tubulin as an embryonic reserve.     

Vanadate,an inhibitor of tyrosine phosphatases,induced premature anaphase in oocytes and aneuploidy and polyploidy in mouse bone marrow cells

Protein tyrosine phosphatases are needed for activating maturation promoting factor, meiotic spindle assembly and spindle checkpoint inactivation. The protein phosphatase inhibitor vanadate was used to upset the kinase-phosphatase equilibrium during oocyte maturation (OM) and the metaphase anaphase transition (MAT) prior to cytogenetic analyses of mouse oocytes and bone marrow cells. ICR females received pregnant mare serum gonadotrophin (PMSG) and 48h later received human chorionic gonadotrophin (hCG). Vanadate doses of 0, 5, 15, and 25mg/kg were administered intraperitoneally immediately after hCG and ovulated oocytes and bone marrow cells were processed for cytogenetic analyses 18h after hCG. Data were analyzed by Chi-square and Fisher's exact tests. Vanadate induced different cytogenetic abnormalities in oocytes and in bone marrow cells. The frequencies of oocytes exhibiting premature anaphase (spontaneous activation) in vanadate exposed mice were significantly (P<0.01) elevated over controls; whereas, in bone marrow cells, the levels of tetraploidy, hyperploidy and premature centromere separation were significantly (P<0.01) increased by vanadate treatment. These results suggest that alteration of the kinase-phosphatase equilibrium during OM and the MAT leads to cytogenetic abnormalities that differ between oocytes and bone marrow cells.     

Mouse Oocyte Maturation: Meiotic Checkpoints

Mouse oocytes at different stages of maturation were fused together and the ensuing cell cycle events were analyzed with the objective of identifying checkpoints in meiosis. Fusion of maturing oocytes just undergoing germinal vesicle breakdown (GVBD) induces PCC (premature chromosome condensation) but no spindle formation in immature (GV) partner oocytes. On the other hand, fusion of metaphase I (MI) oocytes containing spindles to GV oocytes induces both PCC and spindle formation in the immature partner. Thus, while molecules required for condensation are present throughout metaphase, those involved in spindle formation are absent in early M-phase. Oocytes cultured for 6 h—early metaphase I (i.e., 2 h before the onset of anaphase I)—and then fused to anaphase-telophase I (A-TI) fusion partners block meiotic progression in the more advanced oocytes and induce chromatin dispersal on the spindle. By contrast, oocytes cultured for 8 h (late MI) before fusion to A-TI partners are driven into anaphase by signals from the more advanced oocytes and thereafter advance in synchrony to telophase I. When early (10 h) or late (12 h) metaphase II oocytes were fused to A-TI partners the signals generated from early MII oocytes block the anaphase to telophase I transition and induce a dispersal of A-TI chromosomes along the spindle. On the other hand, late MII oocytes respond to A-TI signals by exiting from the MII block and undergoing the A-TII transition. Moreover, the oocytes in late MI are not arrested in this stage and progress without any delay through A-TI to MII when fused to metaphase II partners. The signals from the less-developed partner force the MII oocyte through A-TII to MIII. In total, these studies demonstrate that the metaphase period is divided into at least three distinct phases and that a checkpoint in late metaphase controls the progress of meiosis in mammalian oocytes.     

Denuding and centrifugation of maturing bovine oocytes alters oocyte spindle integrity and the ability of cytoplasm to support parthenogenetic and nuclear transfer embryo development

The effects of cumulus cell removal and centrifugation of maturing bovine oocytes on nuclear maturation and subsequent embryo development after parthenogenetic activation and nuclear transfer were examined. Removal of cumulus cells at 4, 8, and 15 hr after in vitro maturation (IVM) or the centrifugation of denuded oocytes had no effect on maturation rates. Oocytes treated at 0 hr of IVM had a lower expulsion rate (50%) of the first polar body (PB1). The removal of cumulus cells and centrifugation affected the pattern of spindle microtubule distribution and division of chromosomes. There were almost no spindle microtubules allocated to PB1 and the spindles were swollen in anaphase I and telophase I oocytes. Approximately 20% of PB1 oocytes contained tripolar or multipolar spindles. After activation, oocytes denuded with or without centrifugation at 8 hr of IVM resulted in the lowest rate of development (3.0%). Denuded oocytes at 4, 15, and 24 hr of IVM with centrifugation or not resulted in similar blastocyst development rates (9.6%-13.2%). However, centrifugation of oocytes denuded at the beginning of IVM resulted in lower blastocyst development rate (8.1%, P < 0.05) than the noncentrifuged oocytes (17.3%). After nuclear transfer, the blastocyst development rates of oocytes denuded and centrifuged at 0, 4, and 8 hr of IVM were not different when compared to the same patch of noncentrifuged oocytes. However, oocytes denuded and centrifuged at 15 hr of IVM resulted in lower (P < 0.05) blastocyst development rates than the noncentrifuged oocytes. The results of this study suggest that removal of cumulus cells and centrifugation of denuded oocytes affect the spindle pattern. Embryo development of denuded and centrifuged oocytes may differ depending on the time of removal of cumulus cells.     

Asymmetric division of spindle microtubules and microfilaments during bovine meiosis from metaphase I to metaphase III

The kinetics of spindle and chromosomes during bovine oocyte meiosis from meiosis I to meiosis III is described. The results of this study showed that (1) oocytes began to extrude the first polar body (Pb1) at the early anaphase I stage and the Pb1 totally separated from the mother cell only when oocytes reach the MII stage; (2) the morphology of the spindle changed from barrel-shaped at the metaphase stage to cylinder-shaped at early anaphase, and then to a thin, long triangle-shaped cone at late anaphase and telophase stages; (3) chromosome morphology went from an individual visible stage at metaphase to a less defined chromatin state during anaphase and telophase stages, and then back to visible individual chromosomes at the next metaphase; (4) chromatin that connected with the floor of the cone became the polar bodies and expelled, and almost all of the microtubules (MTs) and microfilaments (MFs) composing the spindles moved towards and contributed to the polar bodies; and (5) the size of the metaphase I (MI) spindle was larger than the metaphase II (MII) and metaphase III (MIII) spindles. The MII spindle, however, is more barrel-shaped than the MI spindle. This study suggests that spindle MTs and MFs during bovine oocyte meiosis are asymmetrically divided into the polar bodies.     

Porcine nuclei in early growing stage do not possess meiotic competence in matured oocytes

To determine whether the nuclei of early growing stage porcine oocytes can mature to the MII stage, we examined meiotic competence of nuclei that had been fused with enucleated GV oocytes using the nuclear transfer method. In vitro matured oocytes were enucleated and then fused with early growing oocytes (30-40 μm in diameter) from 5 to 7-wk-old piglets using the hemagglutinating virus of Japan (HVJ). Reconstructed oocytes were cultured for 24 h to the MII stage. Although these oocytes extruded the first polar body, they did not contain normal haploid chromosomes, and the spindles were misaligned or absent at the metaphase II (MII) stage. Furthermore, maturation promoting factor (MPF) activity levels were low in oocytes reconstructed with early growing oocytes at metaphase I (MI) and MII. In contrast, mitogen-activated protein kinase (MAPK) activity was detected between the MI and MII stages, although at slightly lower levels. In conclusion, the nuclei of early growing oocytes did not accomplish normal meiotic division in matured oocytes due to misaligned or absent spindle formation.     

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.     

Nek9 regulates spindle organization and cell cycle progression during mouse oocyte meiosis and its location in early embryo mitosis

Nek9 (also known as Nercc1), a member of the NIMA (never in mitosis A) family of protein kinases, regulates spindle formation, chromosome alignment and segregation in mitosis. Here, we showed that Nek9 protein was expressed from germinal vesicle (GV) to metaphase II (MII) stages in mouse oocytes with no detectable changes. Confocal microscopy identified that Nek9 was localized to the spindle poles at the metaphase stages and associated with the midbody at anaphase or telophase stage in both meiotic oocytes and the first mitotic embyros. Depletion of Nek9 by specific morpholino injection resulted in severely defective spindles and misaligned chromosomes with significant pro-MI/MI arrest and failure of first polar body (PB1) extrusion. Knockdown of Nek9 also impaired the spindle-pole localization of γ-tubulin and resulted in retention of the spindle assembly checkpoint protein Bub3 at the kinetochores even after 10 h of culture. Live-cell imaging analysis also confirmed that knockdown of Nek9 resulted in oocyte arrest at the pro-MI/MI stage with abnormal spindles, misaligned chromosomes and failed polar body emission. Taken together, our results suggest that Nek9 may act as a MTOC-associated protein regulating microtubule nucleation, spindle organization and, thus, cell cycle progression during mouse oocyte meiotic maturation, fertilization and early embryo cleavage.     

Transient exposure to the Eg5 kinesin inhibitor monastrol leads to syntelic orientation of chromosomes and aneuploidy in mouse oocytes

Aneuploidy may result from abnormalities in the biochemical pathways and cellular organelles associated with chromosome segregation. Monastrol is a reversible, cell-permeable, non-tubulin interacting inhibitor of the mitotic kinesin Eg5 motor protein which is required for assembling and maintaining the mitotic spindle. Monastrol can also impair centrosome separation and induce monoastral spindles in mammalian somatic cells. The ability of monastrol to alter kinesin Eg5 and centrosome activities and spindle geometry may lead to abnormal chromosome segregation. Mouse oocytes were exposed to 0 (control), 15, 30, and 45 microg/ml monastrol in vitro for 6 h during meiosis I and subsequently cultured for 17 h in monastrol-free media prior to cytogenetic analysis of metaphase II oocytes. A subset of oocytes was cultured for 5 h prior to processing cells for meiotic I spindle analysis. Monastrol retarded oocyte maturation by significantly (P < 0.05) decreasing germinal vesicle breakdown and increasing the frequencies of arrested metaphase I oocytes. Also, significant (P < 0.05) increases in the frequencies of monoastral spindles and chromosome displacement from the metaphase plate were found in oocytes during meiosis I. In metaphase II oocytes, monastrol significantly (P < 0.05) increased the frequencies of premature centromere separation and aneuploidy. These findings suggest that abnormal meiotic spindle geometry predisposes oocytes to aneuploidy.     

Spindle-view系统在猪卵母细胞去核中的应用

应用Spindle-view对体外成熟培养36、42、44和48h的猪体外成熟卵母细胞减数分裂纺锤体进行去核操作,并与传统去核方法(McGrath-Solter去核法,挤压去核法)相比较,结果表明:①在42~48h之间利用Spindle-view得到的猪卵纺锤体影像与极体的相对位置没有明显的变化;②Spindle-view适合用于猪体外成熟卵母细胞减数分裂纺锤体的观察及去核;去核效率与其他两种方法相比差异极显著(95.5%,42.1%,74.2%,P<0.01);③纺锤体成像是否清晰可用于猪卵母细胞的质量监控。     

The critical role of the MAP kinase pathway in meiosis II in Xenopus oocytes is mediated by p90(Rsk)

BACKGROUND: During oocyte maturation in Xenopus, progesterone induces entry into meiosis I, and the M phases of meiosis I and II occur consecutively without an intervening S phase. The mitogen-activated protein (MAP) kinase is activated during meiotic entry, and it has been suggested that the linkage of M phases reflects activation of the MAP kinase pathway and the failure to fully degrade cyclin B during anaphase I. To analyze the function of the MAP kinase pathway in oocyte maturation, we used U0126, a potent inhibitor of MAP kinase kinase, and a constitutively active mutant of the protein kinase p90(Rsk), a MAP kinase target. RESULTS: Even with complete inhibition of the MAP kinase pathway by U0126, up to 90% of oocytes were able to enter meiosis I after progesterone treatment, most likely through activation of the phosphatase Cdc25C by the polo-like kinase Plx1. Subsequently, however, U0126-treated oocytes failed to form metaphase I spindles, failed to reaccumulate cyclin B to a high level and failed to hyperphosphorylate Cdc27, a component of the anaphase-promoting complex (APC) that controls cyclin B degradation. Such oocytes entered S phase rather than meiosis II. U0126-treated oocytes expressing a constitutively active form of p90(Rsk) were able to reaccumulate cyclin B, hyperphosphorylate Cdc27 and form metaphase spindles in the absence of detectable MAP kinase activity. CONCLUSIONS: The MAP kinase pathway is not essential for entry into meiosis I in Xenopus but is required during the onset of meiosis II to suppress entry into S phase, to regulate the APC so as to support cyclin B accumulation, and to support spindle formation. Moreover, one substrate of MAP kinase, p90(Rsk), is sufficient to mediate these effects during oocyte maturation.     

Nek9 regulates spindle organization and cell cycle progression during mouse oocyte meiosis and its location in early embryo mitosis

Nek9 (also known as Nercc1), a member of the NIMA (never in mitosis A) family of protein kinases, regulates spindle formation, chromosome alignment and segregation in mitosis. Here, we showed that Nek9 protein was expressed from germinal vesicle (GV) to metaphase II (MII) stages in mouse oocytes with no detectable changes. Confocal microscopy identified that Nek9 was localized to the spindle poles at the metaphase stages and associated with the midbody at anaphase or telophase stage in both meiotic oocytes and the first mitotic embyros. Depletion of Nek9 by specific morpholino injection resulted in severely defective spindles and misaligned chromosomes with significant pro-MI/MI arrest and failure of first polar body (PB1) extrusion. Knockdown of Nek9 also impaired the spindle-pole localization of γ-tubulin and resulted in retention of the spindle assembly checkpoint protein Bub3 at the kinetochores even after 10 h of culture. Live-cell imaging analysis also confirmed that knockdown of Nek9 resulted in oocyte arrest at the pro-MI/MI stage with abnormal spindles, misaligned chromosomes and failed polar body emission. Taken together, our results suggest that Nek9 may act as a MTOC-associated protein regulating microtubule nucleation, spindle organization and, thus, cell cycle progression during mouse oocyte meiotic maturation, fertilization and early embryo cleavage.     

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.     

Cell division of binuclear cells induced by caffeine: Spindle organization and determination of division plane

Synchronously dividing binuclear cells were induced in root tips ofTriticum turgidum by caffeine treatment. Spindle and other microtubular configurations of such cells were studied using tubulin immunofluorescence and electron microscopy. The binuclear cells developed one, two or three preprophase microtubule bands longitudinally, transversely or rarely in a cross configuration. During the mitotic entry binuclear cells formed prophase spindles separately around each nucleus. When the nuclei were located fairly apart, their spindle structures developed independently throughout all mitotic phases. But when the nuclei were located closely together their metaphase and anaphase spindles shared a common polar region. However, the two spindles in such cells retained their functional autonomy. They display structurally independent minipoles in the common polar region. After anaphase the neighbouring nonsister chromosome groups of nuclei divided by a common polar region come to lie close together and in telophase, become enclosed by a common nuclear envelope. During cytokinesis of binuclear cells cell plates were formed only between sister nuclei. These cell plates may develop normally or may curve or branch giving rise to aberrant daughter cell walls. The peculiar mode of spindle and spindle polar region organization of binuclear cells and determination of the division plane in them are discussed.     

In vitro development of non-enucleated rat oocytes following microinjection of a cumulus nucleus and chemical activation

The present study examined in vitro development and the cytological status of non-enucleated rat oocytes after microinjection of cumulus nuclei and chemical activation. Oocyte-cumulus complexes were collected from gonadotropin-treated prepubertal female Wistar rats 14 h after human chorionic gonadotropin (hCG) injection. Cumulus nuclei were injected into ovulated oocytes and then stimulated in the presence of 5 mM SrCl2 for 20 min at various time points (0-3.5 h) after injection. Some of the reconstituted eggs were cultured to observe the pronuclear formation, cleavage, and blastocyst formation. The incidences of eggs forming at least one pronucleus or containing two pronuclei were not significantly different among the periods (82.4-83.5% and 43.4-51.9%, respectively). Nor did the incidences of eggs cleaving (86.7-97.7%) and developing to the blastocyst stage (0-3.5%) differ depending on when, after injection, stimulation began. When some of the reconstituted eggs were observed for cytological morphology 1-1.5 h after injection, 71.7% of the eggs caused premature chromatin condensation, but only 46.2% of them formed two spindles around each of maternal and somatic chromatins. However, the morphology of the somatic spindles differed from that of the spindles, which formed around the oocyte chromatins. Only 7.5% of the eggs contained the normal chromosomal number. In many reconstituted oocytes, before activation, an abnormal spindle formation was observed in the somatic chromatins. In conclusion, these results show that non-enucleated rat oocytes injected with cumulus nuclei can form pronuclei and cleave following chemical activation, whereas blastocyst formation is very limited, probably caused by abnormalities in the spindle formation and distribution of somatic chromatids.     

The kinesinlike protein Subito contributes to central spindle assembly and organization of the meiotic spindle in Drosophila oocytes

In the oocytes of many species, bipolar spindles form in the absence of centrosomes. Drosophila melanogaster oocyte chromosomes have a major role in nucleating microtubules, which precedes the bundling and assembly of these microtubules into a bipolar spindle. Here we present evidence that a region similar to the anaphase central spindle functions to organize acentrosomal spindles. Subito mutants are characterized by the formation of tripolar or monopolar spindles and nondisjunction of homologous chromosomes at meiosis I. Subito encodes a kinesinlike protein and associates with the meiotic central spindle, consistent with its classification in the Kinesin 6/MKLP1 family. This class of proteins is known to be required for cytokinesis, but our results suggest a new function in spindle formation. The meiotic central spindle appears during prometaphase and includes passenger complex proteins such as AurB and Incenp. Unlike mitotic cells, the passenger proteins do not associate with centromeres before anaphase. In the absence of Subito, central spindle formation is defective and AurB and Incenp fail to properly localize. We propose that Subito is required for establishing and/or maintaining the central spindle in Drosophila oocytes, and this substitutes for the role of centrosomes in organizing the bipolar spindle.