Maturation of porcine oocytes after cooling at the germinal vesicle stage

Maturation of porcine oocytes was examined after oocytes were cooled at the germinal vesicle stage. Cumulus-oocyte complexes (COCs) collected from medium-sized follicles were cooled at 24 degrees C or 4 degrees C for 5, 30 or 120 min in a solution with or without 1.5 M dimethylsulfoxide (DMSO). After rewarming, COCs were cultured in maturation medium at 39 degrees C, 5% CO2 in air for 44 h. Meiotic spindle organisation (by immunostaining and confocal microscopy), nuclear maturation (by orcein staining) and cytoplasmic maturation (by intracellular glutathione assay) of oocytes were examined after maturation. When COCs were cooled at 24 degrees C for various times in the medium without DMSO, a tendency to decreased spindle formation, nuclear maturation and cytoplasmic maturation was observed, but there was no statistical difference compared with controls. Addition of DMSO during cooling inhibited subsequent nuclear maturation and spindle formation. When COCs were cooled at 4 degrees C, both nuclear and cytoplasmic maturation as well as spindle formation were inhibited in most oocytes in a time-dependent manner. DMSO during cooling did not have any beneficial effect on subsequent oocyte maturation and spindle formation. These results suggest that porcine oocytes are very sensitive to a drop in the temperature before exposure to culture. Cooling oocytes before maturation inhibits their subsequent spindle organisation, nuclear and cytoplasmic maturation. Addition of DMSO to the cooling solution did not protect porcine oocytes from cooling-induced damage.     

Effects of cooling germinal vesicle-stage bovine oocytes on meiotic spindle formation following in vitro maturation.

Attempts to cryopreserve bovine oocytes result in low survival because of their sensitivity to temperatures near 0 degrees C. This study evaluates the effects of chilling germinal vesicle-stage (GV) oocytes on their formation of microtubules and the meiotic spindle. In experiment 1, five groups of GV-stage oocytes, each consisting of approximately 90 oocytes, were held at 39 degrees C as controls, or at 31 degrees C, or cooled to 24, 4 or 0 degrees C for 10 min. After being treated, all oocytes were cultured at 39 degrees C for 24 hr. Compared to the controls, holding oocytes for 10 min at 31 or 24 degrees C did not significantly alter the formation of normal spindles, but chilling them to 4 or 0 degrees C did. After 24 hr of maturation, the respective percentages of oocytes containing normal meiotic spindles observed in the controls or those held at 31 or 24 degrees C were 69.8%, 71.9%, or 69.4% (P > 0.05). In contrast, the percentages of oocytes with normal spindles after they had been cooled to 4 or 0 degrees C were 44.0% or 29.1%, respectively. In experiment 2, approximately 90 oocytes/group were cooled to 4 degrees C for various times before being warmed and cultured. Regardless of the time of exposure, cooling oocytes to 4 degrees C reduced the formation of normal spindles. The percentages of oocytes cooled to 4 degrees C for 10, 20, 30, 45, or 60 min with normal spindles were 44.0%, 38.4%, 37.5%, 34.5% and 30.9%, respectively. In experiment 3, approximately 60 oocytes per group that had been held at 31 degrees C or cooled to 24, 4 or 0 degrees C for 10 min were allowed to mature for 24 hr before being subjected to in vitro fertilization. The cleavage rates of oocytes subjected to various chilling treatments exhibited the same pattern as that of oocytes with normal spindles. That is, there were no significant differences in cleavage rates among the control oocytes and those held at 31 or 24 degrees C (70.4%, 71.8%, and 72.4%; P > 0.05). However, only 37. 0% and 30.4% of oocytes chilled to 4 or 0 degrees C cleaved after fertilization. These results suggest that: (1) chilling bovine oocytes no lower than 24 degrees C does not reduce formation of normal meiotic spindles; (2) however, chilling oocytes to 4 degrees C or lower for as little as 10 min drastically reduces the formation of normal meiotic spindles and of fertilization; (3) the rates of fertilization and cleavage of resultant zygotes mimic that of formation of normal spindles.     

玻璃化冷冻对猪体外成熟卵母细胞染色体与纺锤体的影响

以冷冻环为载体,探讨玻璃化冷冻对猪体外成熟卵母细胞染色体与纺锤体影响。单用40%乙二醇(ethyleneglycol,EG)或20%EG与20%二甲基亚砜(dimethylsulphoxide,DMSO)联合作冷冻保护剂,用直投液氮或使用玻璃化冷冻仪法制冷冷冻猪体外成熟卵母细胞;解冻2h后固定并免疫荧光法染色纺锤体及染色体;挑选各试验组形态正常卵母细胞进行体外受精实验。结果表明,与单用EG以及EG和DMSO联合直投液氮方案比较,EG和DMSO联合应用并采用玻璃化冷冻仪制冷方案卵母细胞染色体正常率为30.1%,纺锤体正常率为37.2%,可明显降低卵母细胞染色体及纺锤体结构损伤(P<0.05),并明显提高卵母细胞的激活效果(P<0.05)。采用联合冷冻保护剂及玻璃化冷冻仪高速冷冻可较好维持猪卵母细胞染色体与纺锤体形态,但玻璃化冷冻明显影响猪卵母细胞体外受精后的发育能力。     

Spindle observation in living mammalian oocytes with the polarization microscope and its practical use

The meiotic spindle is crucial for normal chromosome alignment and separation of maternal chromosomes during meiosis. Conventional methods to image spindles rely on fixation and transmission electron microscope or immunofluorescence staining and fluorescence microscope, so they provide limited value to studies of spindle dynamics and human clinical in vitro fertilization. A new orientation-independent polarized light microscope, the LC Polscope, was used to examine the bi-refringent spindles in living mammalian oocytes. It was found that spindles could be imaged with the Polscope in living oocytes in all mammals so far examined, including hamster, mouse, cattle, human, and rat. The first polar body did not accurately predict the spindle location in most metaphase II oocytes. Intracytoplasmic sperm injection (ICSI) could be performed by monitoring spindle position. Studies in humans indicated that, aftr ICSI, higher fertilization and embryonic developmental rates could be achieved in oocytes with than without bi-refringent spindles. Because spindles in most mammalian oocytes are extremely sensitive to slight changes in temperature, maintenance of temperature at 37 degrees C is crucial for normal spindle function. As chromosomes#10; are usually associated with microtubule fibers in the spindles, the position of chromosomes could be indirectly located by imaging spindles. Removing spindles under the Polscope can achieve an enucleation#10; efficiency rate of 100% in mouse oocytes. The Polscope can also be used to examine the spindle dynamics, detect spindle morphology, predict chromosome misalignment, and perform spindle transfer.     

Requirement of functional telomeres for metaphase chromosome alignments and integrity of meiotic spindles

Telomerase deficiency in the mouse eventually leads to loss of telomeric repeats from chromosome ends and to end-to-end chromosome fusions, which result in defects in highly proliferative tissues. We show that telomere dysfunction resulting from telomerase deficiency leads to disruption of functional meiotic spindles and misalignment of chromosomes during meiotic division of oocytes in late-generation (G₄) mice. However, oocytes from first-generation (G₁) mice lacking telomerase showed no appreciable telomere dysfunction and exhibited chromosome alignment at the metaphase plates of meiotic spindles, in a manner similar to that of wild-type mouse oocytes. These findings suggest that telomerase does not directly influence chromosome alignment and spindle integrity. Rather, functional telomeres may be involved in mediating metaphase chromosome alignment and maintaining functional spindles during meiotic division.     

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.     

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.     

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.     

Xkid chromokinesin is required for the meiosis I to meiosis II transition in Xenopus laevis oocytes

Xkid chromokinesin is required for chromosome alignment on the metaphase plate of spindles formed in Xenopus laevis egg extracts. We have investigated the role of Xkid in Xenopus oocyte meiotic maturation, a progesterone-triggered process that reinitiates the meiotic cell cycle in oocytes arrested at the G2/M border of meiosis I. Here we show that Xkid starts to accumulate at the time of germinal vesicle breakdown and reaches its largest quantities at metaphase II in oocytes treated with progesterone. Both germinal vesicle breakdown and spindle assembly at meiosis I can occur normally in the absence of Xkid. But Xkid-depleted oocytes cannot reactivate Cdc2/cyclin B after meiosis I and, instead of proceeding to meiosis II, they enter an interphase-like state and undergo DNA replication. Expression of a Xkid mutant that lacks the DNA-binding domain allows Xkid-depleted oocytes to complete meiotic maturation. Our results show that Xkid has a role in the meiotic cell cycle that is independent from its role in metaphase chromosome alignment.     

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.     

Nuclear origin of aging-associated meiotic defects in senescence-accelerated mice

Factors of both cytoplasmic and nuclear origin regulate metaphase chromosome alignment and spindle checkpoint during mitosis. Most aneuploidies associated with maternal aging are believed to derive from nondisjunction and meiotic errors, such as aberrations in spindle formation and chromosome alignment at meiosis I. Senescence-accelerated mice (SAM) exhibit aging-associated meiotic defects, specifically chromosome misalignments at meiosis I and II that resemble those found in human female aging. How maternal aging disrupts meiosis remains largely unexplained. Using germinal vesicle nuclear transfer, we found that aging-associated misalignment of metaphase chromosomes is predominately associated with the nuclear factors in the SAM model. Cytoplasm of young hybrid B6C3F1 mouse oocytes could partly rescue aging-associated meiotic chromosome misalignment, whereas cytoplasm of young SAM was ineffective in preventing the meiotic defects of old SAM oocytes, which is indicative of a deficiency of SAM oocyte cytoplasm. Our results demonstrate that both nuclear and cytoplasmic factors contribute to the meiotic defects of the old SAM oocytes and that the nuclear compartment plays the predominant role in the etiology of aging-related meiotic defects.     

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.     

Septin 7 is required for orderly meiosis in mouse oocytes

Septin 7 is a conserved GTP-binding protein. In this study, we examined the localization and functions of Septin 7 during mouse oocyte meiotic maturation. Immunofluorescent analysis showed that intrinsic Septin 7 localized to the spindles from the pro-MI stage to the MII stage. Knockdown of Septin 7 by siRNA microinjection caused abnormal spindles and affected extrusion of the first polar body. Septin 7 mRNA tagged with myc was injected into GV stage oocytes to overexpress Septin 7. Overexpressed Myc-Septin 7 localized to the spindle and beneath the plasma membrane displaying long filaments. Fluorescence intensity of spindle α-tubulin in myc-Septin 7-injected oocytes was weaker than that of the control group, demonstrating that Septin 7 may influence recruitment of α-tubulin to spindles. MII oocytes injected with myc-Septin 7 exhibited abnormal chromosome alignment, and parthenogenetic activation failed to allow extrusion of the second polar body, suggesting that overexpression of Septin 7 may affect extrusion of the polar body by disturbing the alignment of chromosomes and regulating α-tubulin recruitment to spindles. In summary, Septin 7 may regulate meiotic cell cycle progression by affecting microtubule cytoskeletal dynamics in mouse oocytes.     

Microtubule assembly after treatment of pig oocytes with taxol: correlation with chromosomes, gamma-tubulin, and MAP kinase.

In this study, taxol was used as a tool to study the correlation of microtubule assembly with chromosomes, gamma-tubulin and phosphorylated mitogen-activated protein (MAP) kinase in pig oocytes at different maturational stages. Taxol treatment did not affect meiotic resumption and chromosome condensation but inhibited/disrupted chromosome alignment at the metaphase plate and bipolar spindle formation and thus meiotic progression. Microtubules were co-localized with chromosomes and were found to emanate from the chromosomes in taxol-treated oocytes, suggesting that chromosomes may serve as a source of microtubule organization. In addition, the concentric emanation of microtubules within the chromosome-surrounded area in taxol-treated oocytes suggests that microtubule emanation from the chromosomes may be directed by other microtubule-organizing material. The formation of one large spindle or >/=2 spindles in oocytes after taxol removal shows that minus end microtubule-organizing material can be normally located on both sides of chromosomes only when the chromosomes are aligned on the metaphase plate. The co-localization of gamma-tubulin and phosphorylated MAP kinase with microtubule assembly in both control and taxol-treated oocytes suggests that these two proteins are associated microtubule-nucleating material in pig oocytes. However, Western blot analysis showed that neither cytoplasmic microtubule aster formation nor extensive microtubule assembly in the chromosome region induced by taxol was caused by super-activation of MAP kinase. Taxol also induced microtubule assembly depending on chromosome distribution in the first polar body. The results suggest that chromosomes are always co-localized with microtubules and that emanation of microtubules from the chromosomes may be regulated/directed by microtubule-organizing material including gamma-tubulin and phosphorylated MAP kinase in pig 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.     

MEI-1/katanin is required for translocation of the meiosis I spindle to the oocyte cortex in C elegans

In most animals, successful segregation of female meiotic chromosomes involves sequential associations of the meiosis I and meiosis II spindles with the cell cortex so that extra chromosomes can be deposited in polar bodies. The resulting reduction in chromosome number is essential to prevent the generation of polyploid embryos after fertilization. Using time-lapse imaging of living Caenorhabditis elegans oocytes containing fluorescently labeled chromosomes or microtubules, we have characterized the movements of meiotic spindles relative to the cell cortex. Spindle assembly initiated several microns from the cortex. After formation of a bipolar structure, the meiosis I spindle translocated to the cortex. When microtubules were partially depleted, translocation of the bivalent chromosomes to the cortex was blocked without affecting cell cycle timing. In oocytes depleted of the microtubule-severing enzyme, MEI-1, spindles moved to the cortex, but association with the cortex was unstable. Unlike translocation of wild-type spindles, movement of MEI-1-depleted spindles was dependent on FZY-1/CDC20, a regulator of the metaphase/anaphase transition. We observed a microtubule and FZY-1/CDC20-dependent circular cytoplasmic streaming in wild-type and mei-1 mutant embryos during meiosis. We propose that, in mei-1 mutant oocytes, this cytoplasmic streaming is sufficient to drive the spindle into the cortex. Cytoplasmic streaming is not the normal spindle translocation mechanism because translocation occurred in the absence of cytoplasmic streaming in embryos depleted of either the orbit/CLASP homolog, CLS-2, or FZY-1. These results indicate a direct role of microtubule severing in translocation of the meiotic spindle to the cortex.     

Overheating is detrimental to meiotic spindles within in vitro matured human oocytes

The present study was designed to examine the effects of overheating on meiotic spindle morphology within in vitro matured human oocytes using a polarized light microscope (Polscope). Immature human oocytes at either germinal vesicle or metaphase I stage were cultured in vitro for 24-36 h until they reached metaphase II (M-II) stage. After maturation, oocytes at M-II stage were imaged in the living state with the Polscope at 37, 38, 39 and 40 degrees C for up to 20 min. After heating, oocytes were returned to 37 degrees C and then imaged for another 20 min at 37 degrees C. The microtubules in the spindles were quantified by their maximum retardance, which represents the amount of microtubules. Spindles were intact at 37 degrees C during 40 min of examination and their maximum retardance (1.72-1.79) did not change significantly during imaging. More microtubules were formed in the spindles heated to 38 degrees C and the maximum retardance was increased from 1.77 before heating to 1.95 at 20 min after heating. By contrast, spindles started to disassemble when the temperature was increased to 39 degrees C for 10 min (maximum retardance was reduced from 1.76 to 1.65) or 40 degrees C for 1 min (maximum retardance was reduced from 1.75 to 1.5). At the end of heating (20 min), fewer microtubules were present in the spindles and the maximum retardance was reduced to 0.8 and 0.78 in the oocytes heated to 39 degrees C and 40 degrees C, respectively. Heating to 40 degrees C also induced spindles to relocate in the cytoplasm in some oocytes. After the temperature was returned to 37 degrees C, microtubules were repolymerized to form spindles, but the spindles were not reconstituted completely compared with the spindles imaged before heating. These results indicate that spindles in human eggs are sensitive to high temperature. Moreover, maintenance of an in vitro manipulation temperature of 37 degrees C is crucial for normal spindle morphology.     

Septin 7 is required for orderly meiosis in mouse oocytes

Septin 7 is a conserved GTP-binding protein. In this study, we examined the localization and functions of Septin 7 during mouse oocyte meiotic maturation. Immunofluorescent analysis showed that intrinsic Septin 7 localized to the spindles from the pro-MI stage to the MII stage. Knockdown of Septin 7 by siRNA microinjection caused abnormal spindles and affected extrusion of the first polar body. Septin 7 mRNA tagged with myc was injected into GV stage oocytes to overexpress Septin 7. Overexpressed Myc-Septin 7 localized to the spindle and beneath the plasma membrane displaying long filaments. Fluorescence intensity of spindle α-tubulin in myc-Septin 7-injected oocytes was weaker than that of the control group, demonstrating that Septin 7 may influence recruitment of α-tubulin to spindles. MII oocytes injected with myc-Septin 7 exhibited abnormal chromosome alignment, and parthenogenetic activation failed to allow extrusion of the second polar body, suggesting that overexpression of Septin 7 may affect extrusion of the polar body by disturbing the alignment of chromosomes and regulating α-tubulin recruitment to spindles. In summary, Septin 7 may regulate meiotic cell cycle progression by affecting microtubule cytoskeletal dynamics in mouse 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.     

Thiol Protease Inhibitor,E-64-d,Prevents Spindle Formation during Mouse Oocyte Maturation1

E-64-d, a membrane permeant derivative of E-64, the thiol protease inhibitor, was found to prevent meiotic maturation of mouse oocytes in a dose dependent manner. When immature mouse oocytes were incubated with E-64-d for up to 14 hr, first polar body emission was blocked to 36% at 200 μg/ml and 6% at 400 μg/ml, but germinal vesicle breakdown occurred normally. Cytological analysis revealed that meiotic spindles were not formed, while chromosome condensation occurred. Thus, E-64-d prevents oocytes from progressing to the first meiotic metaphase. When exposed to E-64-d after 8 hr of incubation without E-64-d, one-fourth of oocytes completed the first meiotic division but never progressed to the second metaphase. In three-fourth of the oocytes inhibited to emit the first polar body, spindles disappeared after incubation with E-64-d. The results suggest that E-64-d promotes disassembly of meiotic spindles resulting in inhibition of meiotic maturation. We propose that thiol protease is involved in spindle formation in mouse meiotic maturation.