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State Key Laboratory of Reproductive Biology, Institute of Zoology, Chinese Academy of Sciences, Beijing 100080, China
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ABSTRACT |
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 TOP ABSTRACT INTRODUCTIONMATERIALS AND METHODSRESULTSDISCUSSIONREFERENCES |
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gamete biology, meiosis, oocyte development
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INTRODUCTION |
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TOPABSTRACTINTRODUCTIONMATERIALS AND METHODSRESULTSDISCUSSIONREFERENCES |
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The MAD1 was first identified [8] in budding yeast, and then its sequence was cloned [16]. Studies of MAD1 immunofluorescence showed that it was mainly localized in the nucleus, with a few at the spindle poles [16]. An 85-kDa protein from Xenopus egg extracts, XMAD1 is a homologue of budding yeast MAD1. The XMAD1 was localized to the nuclear envelope and the nucleus during interphase, was dissociated from the nuclear envelope at prophase, persisted at the chromosomes at prometaphase, then disappeared from chromosomes at metaphase and anaphase [17]. Additionally, MAD1 homologue was identified in human and mouse cells [18, 19]. In HeLa cells [20], human MAD1 (HMAD1) localization is similar to that in budding yeast [16] and Xenopus [17].
The functions of MAD1 have been widely studied in mitotic spindle checkpoints from yeast to mammalian cells. In budding yeast, after being treated by benomyl, MAD1-deleted cells are unable to delay the onset of anaphase and, therefore, suffer a high frequency of chromosome loss and rapid death [16]. Spindle disruption is accompanied by MAD1 phosphorylation, which requires BUB1, BUB3, and MAD2 but not BUB2 and MAD3, suggesting the possible role of MAD1 in the checkpoint [16]. Adding an anti-XMAD1 antibody to Xenopus egg extracts deactivates the checkpoint and prevents XMAD2 from localizing to unbound kinetochores [17], suggesting that XMAD1 may recruit XMAD2 to unattached kinetochores. In budding yeast, a tight complex between MAD1 and MAD2 is crucial for checkpoint function and hyperphosphorylation of MAD1 [21], which was subsequently verified by analysis of tetrameric MAD1-MAD2 crystal structure [22]. However, another study in Xenopus egg extracts showed that only a part of MAD2 formed a complex with MAD1, that the other part of MAD2 was not bound to MAD1, and that the ratio between MAD1 and MAD2 was critical for maintaining a pool of MAD1-free MAD2, which is necessary for the spindle checkpoint [17]. The MAD2 may become activated and dissociated from MAD1 at kinetochores and is replenished by the pool of MAD1-free MAD2 [23]. The HMAD1 mutation may be the origin of some cancers in humans [24, 25].
Recent studies indicate that a spindle checkpoint system also exists that monitors segregation of homologous chromosomes or chromatids during meiosis [26–28]. Some mitotic spindle checkpoint proteins, such as MAD2 [29, 30] and BUB1 [31], are present in mammalian oocytes and participate in the spindle checkpoint during meiosis I and/or meiosis II. Although MAD1 was present in Xenopus oocytes [32], its functions are still unknown during meiosis. To our knowledge, MAD1 localization and function have not been examined in mammalian meiosis. Therefore, to increase our understanding of the meiotic spindle checkpoint mechanisms during mammalian meiosis and the occurrence of aneuploidy in mammalian embryos, in the present study we used mouse oocytes as a model to study whether MAD1 is also a meiotic spindle checkpoint protein in mammals. Furthermore, we examined its detailed localization during normal meiosis and microtubule polymerization, depolymerization, and repolymerization.
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MATERIALS AND METHODS |
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TOPABSTRACTINTRODUCTIONMATERIALS AND METHODSRESULTSDISCUSSIONREFERENCES |
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Animal care and handling were conducted in accordance with policies regarding the care and use of animals promulgated by the ethical committee of the State Key Laboratory of Reproductive Biology, Chinese Academy of Sciences. The mice with color gene type of aabbcc were from an inbred strain of Kunming white mice, a native breed widely used in biological research in China [33, 34]. Prophase-stage oocytes, also called immature oocytes at the germinal vesicle (GV) stage, were collected from ovaries of 4- to 6-wk-old female mice at 48 h after eCG injection. Cumulus-enclosed GV oocytes were collected by puncturing the large antral follicles with a needle in M2 medium (Sigma Chemical Co., St. Louis, MO) supplemented with 60 µg/ml of penicillin and 50 µg/ml of streptomycin. Then, the oocytes were cultured in M2 medium at 37°C in a humidified atmosphere of 5% CO2 in air. The cumulus cell masses surrounding the oocytes were removed by treatment with 300 IU/ml of hyaluronidase (Sigma) and repeated pipetting before treatment and examination.
Nuclear Examination of Oocytes
At various time points of culture, oocytes were fixed for examination of nuclear maturation. Nuclear stages were categorized as GV, prometaphase I (ProM-I), metaphase I (M-I), anaphase I (A-I), telophase I (T-I), and metaphase II (M-II) according to methods reported previously [35, 36].
Nocodazole and Taxol Treatment of Oocytes
Oocytes at various stages (GV, ProM-I, M-I, A-I, T-I and M-II) were treated by nocodazole or Taxol (Sigma). For nocodazole treatment, 10 mg/ ml of nocodazole in dimethyl sulfoxide (DMSO) stock (Sigma) were diluted in M2 medium to give a final concentration of 20 µg/ml, and oocytes were incubated for 10 min. For Taxol treatment, 5 mM Taxol in DMSO stock was diluted in M2 medium to give a final concentration of 10 µM, and oocytes were incubated for 45 min. After treatment, oocytes were washed thoroughly and fixed for immunofluorescence staining or were cultured for spindle recovery. In the control, oocytes were also treated in the medium with the same concentration of DMSO before examination. For spindle recovery, oocytes at the M-I (8 h of culture) and M-II (16 h of culture) stages were cultured in M2 medium for 10, 30, and 60 min after nocodazole treatment and then fixed for immunofluorescence staining.
Immunofluorescence Staining of MAD1, Microtubules, Kinetochores, and Nuclei
Rabbit anti-MAD1 antibody (0.5 mg/ml in 0.1% BSA/PHEM [60 mM PIPES, 25 mM Hepes pH 6.9, 10 mM EGTA, 8 mM MgSO4], pH 7.2) prepared against bacterially expressed Xenopus MAD1 was a kind gift from Dr. R.H. Chen (Cornell University) [17]. In a preliminary experiment, we used both rabbit serum and fluorescein isothiocyanate (FITC)-conjugated goat anti-rabbit immunoglobulin (Ig) G for the immunostaining and did not find positive MAD1 signal (image not shown); therefore, we found that this anti-XMAD1 antibody can specifically bind MAD1 in mouse oocytes.
Because we cannot depict the oocytes using three-color staining by confocal microscopy, oocytes used in the present study were double-stained for MAD1 and nuclei, microtubules and nuclei, MAD1 and microtubules, and MAD1 and kinetochores. Immunofluorescence staining was based on procedures reported previously [29–31].
For MAD1 and nuclei staining, oocytes were first treated in 0.5% Triton X-100/PHEM for 4–5 min, washed rapidly three times in PBS with 0.05% polyvinylpyrrolidone (PVP), fixed in 4% paraformaldehyde/PHEM for 20 min, and then washed three times in PBS with 0.05% PVP. After being blocked with 1% BSA/PHEM with 100 mM glycine at room temperature for 1 h, the oocytes were incubated at 4°C overnight with anti-MAD1 antibody diluted 1:100 in 1% BSA/PHEM with 100 mM glycine. After four washes in PBS with 0.05% Tween 20, the oocytes were incubated for 45 min with FITC-conjugated goat anti-rabbit IgG diluted 1:200 in 1% BSA/PHEM with 100 mM glycine. Then, the oocytes were further washed three times in PBS with 0.05% Tween 20 and stained with propidium iodide in PBS with 0.05% Tween 20 for 2–3 min. Finally, the oocytes were mounted on glass slides and examined with a TCS-4D laser scanning confocal microscope (Leica Microsystems, Bensheim, Germany).
For microtubule and nucleus staining, oocytes were treated in 0.2% Triton X-100/PHEM for 4–5 min, washed rapidly three times in PBS with 0.05% PVP, fixed in 4% paraformaldehyde/PHEM for 20 min, and washed three times in PBS with 0.05% PVP. After being blocked in 1% BSA/ PHEM with 100 mM glycine at room temperature for 1 h, the oocytes were incubated in monoclonal anti-
-tubulin (1:16 000 in 1% BSA/PHEM with 100 mM glycine; Sigma) overnight at 4°C. After four washes in PBS with 0.05% Tween 20, the oocytes were incubated with FITC-conjugated goat anti-mouse IgG (1:200 in 1% BSA/PHEM with 100 mM glycine) for 45 min before nuclear staining according to the methods described above.
For MAD1 and microtubule costaining, oocytes were treated in 0.5% Triton X-100/PHEM for 4–5 min, washed three times in PBS with 0.05% PVP, fixed in 4% paraformaldehyde/PHEM for 20 min, and washed three times in PBS with 0.05% PVP. After being blocked in 1% BSA/PHEM with 100 mM glycine at room temperature for 1 h, the oocytes were incubated in anti-MAD1 antibody at 4°C overnight. After four washes in PBS with 0.05% Tween 20, the oocytes were incubated with FITC-conjugated goat anti-rabbit IgG (1:200 in 1% BSA/PHEM with 100 mM glycine) for 45 min. After three washes in PBS with 0.05% Tween 20, the oocytes were again blocked in 1% BSA/PHEM with 100 mM glycine at room temperature for 1 h, then stained according to the methods described above, except that the primary antibody was anti--tubulin antibody (1: 16 000 in 1% BSA/PHEM with 100 mM glycine) and the second antibody was Tetramethyl Rhodamine Isothiocyanate (TRITC)-conjugated goat anti-mouse IgG (1:200 in 1% BSA/PHEM with 100 mM glycine).
For MAD1 and kinetochore costaining, all steps were the same as those used for MAD1 and microtubule costaining, except that the primary antibody was human ANA-Centromere Autoantibody (1:500 in 1% BSA/ PHEM with 100 mM glycine; Cortex Biochem) and the second antibody was fluorescent Alexa 568-conjugated goat anti-human IgG (1:200 in 1% BSA/PHEM with 100 mM glycine; Molecular Probes, Inc., Eugene, OR).
Cooling of Oocytes and Immunostaining of MAD1 and Microtubules
To observe the relationship between MAD1 localization and partial destruction of spindle, oocytes at the M-I (8 h of culture) and M-II (16 h of culture) stages were cooled before examination. To cool the oocytes, the dishes containing the oocytes in 100-µl drops of Hepes-buffered Tyrodes with polyvinyl alcohol were kept at 37°C in advance and then placed in a refrigerator at 4°C for 10, 30, 60, and 120 min. The oocytes therefore experienced a slow cooling process, and spindles were disassembled slowly. After cooling, the oocytes were fixed for immunostaining of MAD1 and microtubules.
Microinjection of Anti-MAD1 Antibody into Immature Oocytes
To study the effects of MAD1 on oocyte nuclear maturation and chromosome alignment, anti-MAD1 antibody was injected into GV-stage oocytes. The oocytes were then cultured for 8 h (to the M-I stage) or 16 h (to the M-II stage) before confocal examination of microtubules and DNA. Rabbit anti-MAD1 antibody (0.5 mg/ml in 0.1%BSA/PHEM, pH 7.2) was injected into the cytoplasm of fully grown oocytes at the GV stage as previously reported [19–21]. Isobutylmethylxanthine was added to M2 medium to produce a final concentration of 0.2 mM to prevent GV breakdown (GVBD) during injection and treatment. A microinjection volume of 7 pl/oocyte was used in all experiments. Rabbit IgG (0.5 mg/ml in 0.1%BSA/PHEM, pH 7.2) or culture medium was also injected into other oocytes to act as controls. After microinjection, oocytes were washed and cultured in M2 medium until examination.
Statistical Analysis
All experiments were repeated three times. All percentage data were subjected to arc-sine transformation before significance analysis. Data were analyzed by ANOVA using Excel (Microsoft, Redmond, WA), and multiple-range tests were conducted with q-test. Differences at P < 0.05 were considered to be significant.
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RESULTS |
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TOPABSTRACTINTRODUCTIONMATERIALS AND METHODSRESULTSDISCUSSIONREFERENCES |
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As shown in Figure 1, at 0 h of culture most oocytes (95.5%) were at the GV stage. By 2–4 h of culture, 73.8– 88.4% of the oocytes underwent GVBD and proceeded to the ProM-I stage. The proportion of oocytes at the M-I stage increased to 89.7% when the oocytes were examined at 8 h, and 2.1–11.3% oocytes were at the A-I to T-I stages by 8–12 h of culture. The proportions of oocytes at the M-II stage increased as the culture time increased; accordingly, the proportions of oocytes at the M-I stage decreased. When the oocytes were cultured for 16 h, 75.2% reached the M-II stage. Some oocytes (20.8%) were still at the M-I stage at 16 h after culture. Some images of oocytes at each stage are shown in Figure 2 with nuclear (DNA) and spindle (microtubule) staining.
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Localization of MAD1 and Its Association with Chromosomes, Microtubules, and Kinetochores in Mouse Oocytes During Meiosis
Typical images of the localization of MAD1 and its association with chromosomes and microtubules in the oocytes at the GV, ProM-I, M-I, A-I, and M-II stages are shown in Figure 2 (left two columns).
GV stage Nucleus in prophase was enclosed by a nuclear membrane, and no chromatids were observed. No microtubule staining was found around the nucleus (i.e., GV). A few MAD1 spots were observed around the periphery of the GV. This typical MAD1 staining was detected in 80.4% (45/56) of oocytes. Other oocytes (19.6%) had faint or no staining.
ProM-I stage Chromosomes began to condense, but individual chromosomes could not be depicted in the oocytes at the ProM-I stage. Microtubules started to organize into a spindle, but the chromosomes still scattered in the spindle. Staining for MAD1 was found around the chromosomes. This typical MAD1 localization was detected in 85.9% (61/ 71) of the oocytes, and other oocytes (14.1%) had faint or no staining of MAD1.
M-I stage A bipolar spindle (i.e., M-I spindle) was formed, and all chromosomes were aligned at the equator of the spindles. Staining for MAD1 was found mainly at the spindle poles. However, some MAD1 was still in the cytoplasm and around the chromosomes. We found that 77.1% (91/118) of the oocytes had this typical MAD1 localization, but other oocytes (22.9%) did not have MAD1 at the spindle poles or had either faint or no MAD1 staining.
A-I to T-I stages Chromosomes started to separate in the oocytes at the A-I to T-I stages; two rows of chromosomes could be observed in the oocytes at these stages. Most MAD1 staining was found mainly at the spindle poles, but some was found in the cytoplasm. We noted that 72.4% (21/29) of the oocytes had this typical MAD1 localization. Other oocytes (27.6%) did not have MAD1 at the spindle poles or had either faint or no MAD1 staining.
M-II stage A typical M-II spindle was formed in the oocytes at the M-II stage. Staining for MAD1 was mainly localized at the spindle poles, but some was found in the cytoplasm. We noted that 86.2% (75/87) of M-II oocytes had this typical MAD1 localization, but other oocytes (13.8%) did not have MAD1 at the spindle poles or had either faint or no MAD1 staining.
Costaining of MAD1 and kinetochores As Figure 3 shows, some signals of MAD1 and kinetochores overlapped in oocytes at the GV or ProM-I stage, but most MAD1 signals did not coincide with kinetochore signals. No MAD1 signals were found at the kinetochores in oocytes at the M-I and M-II stages.
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Localization of MAD1 in Mouse Oocytes Treated with Nocodazole and Taxol
Typical MAD1 localization at each stage of oocytes is shown in Figure 2.
When oocytes were treated with nocodazole, microtubules were completely destroyed in all oocytes at the M-I and M-II stages (Fig. 4); therefore, only MAD1 and chromosome staining are shown in Figure 2 (middle column). Compared to the control oocytes, the same MAD1 localization was observed in oocytes at the GV to ProM-I stages. However, significant differences were observed in oocytes from the M-I to M-II stages. In 86.7% (98/113) of oocytes at the M-I stage, MAD1 moved near the chromosomes. In 13.3% (15/113) of oocytes at the M-I stage, faint or no MAD1 signals were found. In 73.3% (22/30) of oocytes at the A-I to T-I stages, most of the MAD1 staining was close to chromosomes. However, some staining was observed at the spindle midzone, and other oocytes (26.7%) had faint or no MAD1 staining. In 82.2% (60/73) of the oocytes at the M-II stage, which is similar to the results with oocytes at the M-I stage, most MAD1 staining was localized close to chromosomes, with a few MAD1 stains scattered between the chromosomes. Other oocytes (17.8%) had faint or no MAD1 staining.
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When the oocytes were treated with Taxol (Fig. 2, right two columns), the localization of MAD1 was similar to that in the control oocytes (Fig. 2, left two columns).
MAD1 Dynamics in Response to Repolymerization of Microtubules and Its Relationship with Microtubules and Chromosomes in M-I and M-II Oocytes
In the present study, we use the phrase "misaligned chromosomes" to distinguish abnormal chromosome alignment from normal chromosome alignment. If one or more chromosomes did not align at the equator of the spindle or were not together with other chromosomes (i.e., obviously separated with others), these chromosomes were called misaligned chromosomes. We use the phrase "repolymerization of microtubules" to describe the reassembly of microtubules during spindle recovery after nocodazole treatment or cooling that depolymerized the microtubules.
As shown in Figure 4, when the oocytes were fixed soon after nocodazole treatment, microtubules were completely depolymerized in all oocytes at the M-I (n = 99) and M-II (n = 81) stages. At the same time, misaligned chromosomes were found in 93.9% (n = 93) at the M-I and 90.1% (n = 73) at the M-II satge; in these oocytes, a single or several chromosomes were not well aligned at the equator plates. Some MAD1 staining was found between the chromosomes and some close to the chromosomes in an assembling status in all oocytes at the M-I and M-II stages (Fig. 2, middle column). In the present study, to distinguish this type of MAD1 localization from typical MAD1 localization, in which MAD1 is located at the spindle poles in oocytes at the M-I and M-II stages without treatment (Fig. 2, left two columns), we named it "relocated MAD1." As shown in Figures 4 and 5, at 10 min after recovery connections between microtubules and chromosomes had been established, but intact spindles did not form. Abnormal chromosome alignment was still present in most oocytes. The MAD1 signals among the chromosomes disappeared completely in all oocytes, and most MAD1 was near the chromosomes in an assembling status. No significant differences were found between the percentages of oocytes with abnormal spindles, abnormal chromosome alignment, and MAD1 relocation (Fig. 5). As the recovery time was increased, microtubules began to repolymerize around the chromosomes, and the numbers of oocytes with abnormal spindles, abnormal chromosome alignment, and MAD1 relocation were decreased gradually (Fig. 5). After 60 min of culture, 36.4% of oocytes at the M-I and 35.2% of oocytes at the M-II stage had abnormal spindles, but 69.7% and 60.3% of oocytes at the M-I and M-II stage, respectively, still had misaligned chromosomes, suggesting that spindle recovery is faster than chromosome realignment. Furthermore, oocytes with relocated MAD1 (56.9–70.5% at the M-I and 43.3–58.2% at the M-II stage) at 30–60 min of examination were significantly (P < 0.05) fewer than those with misaligned chromosomes (69.7–83.7% at the M-I and 60.3–80.5% at the M-II stage), suggesting that in 10–13% of oocytes at the M-I and 17–22% of oocytes at M-II stage, MAD1 could not sense misaligned chromosomes. On the other hand, the numbers of oocytes with abnormal spindles (36.4–55.8% at the M-I and 35.2–44.7% at the M-II stage) were significantly (P < 0.05) fewer than those with relocated MAD1 after culture for 30 or 60 min, suggesting that in 15–20% of oocytes at the M-I and 8–14% of oocytes at the M-II stage, the connection between microtubules and chromosomes is not complete even though a normal-appearing spindle has been formed. Same results were observed in the oocytes at the M-I (Figs. 4, top, and 5A) and M-II (Figs. 4, bottom, and 5B) stages.
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Effects of Slow Cooling on MAD1 Localization and Its Relationship with Chromosome and Microtubules in M-I and M-II Oocytes
To study whether partial or minor destruction of spindles affects MAD1 localization, oocytes at the M-I and M-II stages were exposed to different degrees of cooling. As shown in Figure 6, even after 120 min of cooling, some microtubules were still attached to the chromosomes, suggesting that in our experimental conditions, slow cooling caused only partial destruction of microtubules, which is different from that in nocodazole-treated oocytes. Partial spindle disassembly was found in oocytes at both the M-I and M-II stages (Figs. 6 and 7). At 10 min after cooling, relocated MAD1 was found in 55.7% of oocytes at the M-I and 40.4% of oocytes at the M-II stage, and abnormal spindles were observed in 45.5% and 29.5% of oocytes at the M-I and M-II stage, respectively. However, no obvious chromosome misalignment was observed, suggesting that cooling for 10 min had already changed the connection between the microtubules and the chromosomes. The proportions of oocytes with abnormal (i.e., partially destroyed) spindles and "relocated" MAD1 were significantly increased as the cooling time increased. After 120 min of cooling, misaligned chromosomes were found in 31.5% of oocytes at the M-I and 29.1% of oocytes at the M-II stage, but spindles in all (100%) oocytes were completely destroyed, suggesting that chromosomes in some oocytes moved during microtubule depolymerization. Meanwhile, all oocytes at the M-I and M-II stages had relocated MAD1.
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Microinjection of MAD1 Antibody into Immature Oocytes Induced a Higher Percentage of Abnormal Chromosome Alignment
Because 89.7% of the oocytes were at the M-I stage at 8 h after culture and 75.2% at the M-II stage at 16 h after culture (Fig. 1), we chose these two time points of culture to examine the effects of microinjection of anti-MAD1 antibody on maturation and chromosome alignment in the injected oocytes. As shown in Figures 8 through 10, microinjection of antibody did not affect nuclear maturation of oocytes, and the proportions of oocytes that reached the M-I (Fig. 9) and M-II (Fig. 10) stages were the same in the injected and in the control oocytes. However, when the chromosome alignment was analyzed in oocytes at the M-I, A-I, and M-II stages, we found that significantly more oocytes (25.2% at the M-I and 78.6% at the A-I stage) had misaligned chromosomes in the antibody-injected oocytes compared to the control (6.2% and 0%, respectively) at 8 h of culture (Fig. 9). Furthermore, when the oocytes were observed at 16 h of culture, more oocytes at the M-I (60.5%) and M-II (29.6%) stages had misaligned chromosomes compared to the control (11.4% and 19%, respectively) (Fig. 10). However, no obvious spindle abnormality was observed in the injected oocytes when compared to the control oocytes (Fig. 8).
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DISCUSSION |
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TOPABSTRACTINTRODUCTIONMATERIALS AND METHODSRESULTSDISCUSSIONREFERENCES |
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Our results showed that MAD1 localization during meiosis in mouse oocytes was different from that during mitosis and meiosis in Xenopus. In budding yeast, MAD1 was distributed within the nucleus and was not found in the nuclear pores, spindle-pole bodies, or telomeres [16]. In HeLa cells, HMAD1 was not detected at centromeres during interphone but was localized to nuclear pores. When the cell cycle reached the ProM-I stage, MAD1 was detected at the kinetochores but disappeared on alignment of chromosomes at the metaphase plate [20]. In Xenopus egg extracts, XMAD1 localized to the nuclear envelope and the nucleus at the interphase. At the prophase, XMAD1 dissociated from the nuclear envelope, and a fraction of XMAD1 localized to the kinetochores and then disappeared from the kinetochores at metaphase and anaphase [17]. In the present study, we found that during normal meiosis, MAD1 was found around nuclei in the oocytes at the GV and ProM-I stages. Costaining of MAD1 and kinetochores showed that their localization did not fully overlap at the GV and ProM-I stages. Little MAD1 was found in the chromosomes at the M-I, A-I, or M-II stage. We found that most MAD1 localized at the spindle poles in the oocytes at the M-I, A-I, and M-II stages, suggesting that differences for MAD1 localization exist between mitosis and meiosis.
After nocodazole treatment, which completely depolymerized microtubules, the connection between microtubules and chromosomes was disrupted, and the oocytes were arrested at metaphase [16, 29]. In the present study, we found that localization of MAD1 in oocytes at the GV and ProM-I stages did not change after nocodazole treatment. However, MAD1 in oocytes at the M-I to M-II stages relocated after nocodazole treatment: Some moved near the chromosomes in an assembling status, and others moved to chromosomes and attached to kinetochores in a scattering status. This relocation in MAD1 during spindle disruption obviously is different from that in mitosis [20], in which MAD1 was at the kinetochores when the spindle was disrupted by nocodazole. This may be caused by totally different localizations of MAD1 between mouse oocytes (present study) and mitotic cells [20].
Nocodazole-induced spindle disassembly is reversible [29, 30]. As shown in the present study, after 10 min of recovery most microtubules were repolymerized, and it appeared that chromosomes had connected to the chromosomes even though intact spindles had not formed in the oocytes. However, MAD1 that had scattered in the chromosomes completely disappeared in all oocytes, and most MAD1 started to move toward the spindle poles in an assembling status while misaligned chromosomes still existed in most oocytes. These results indicate that once the connection between microtubules and chromosomes is established, MAD1 moves away from this site. As the recovery time was increased, the proportions of oocytes with normal chromosome alignment, intact spindle formation, and MAD1 localization were increased. However, even after 60 min of recovery, the numbers of oocytes with misaligned chromosomes were still very high compared to oocytes with abnormal spindles and relocalized MAD1, suggesting that in some oocytes, spindles and MAD1 localization return to normal status but that the chromosomes do not. These results indicate that some chromosomes move during spindle disruption and may not be able to realign at the metaphase plate after spindle recovery. These results also indicate that MAD1 merely senses the connection between chromosomes and microtubules but, for unknown reasons, cannot sense the misaligned chromosomes.
On the other hand, during slow cooling, which partially destroyed spindles, the proportions of oocytes with misaligned chromosomes were dramatically lower than those of oocytes with misaligned chromosomes induced by nocodazole treatment. These results indicate that complete and fast spindle disruption causes chromosomes to move but that partial spindle disruption does not significantly induce chromosome to move. However, even after 10 min of slow cooling, MAD1 in some oocytes started to relocate, suggesting that the connection between microtubules and chromosomes is affected even though no obvious spindle abnormality and misaligned chromosomes were observed. As the cooling time increased, spindles in most oocytes changed. However, microtubules were still found around the chromosomes, and these microtubules may hold the chromosomes. This may be the reason why the proportions of oocytes with misaligned chromosomes did not change during the 10–120 min of cooling. However, at each time point of examination, the percentages of oocytes with relocated MAD1 and abnormal spindles were always higher than the percentages of those with misaligned chromosomes, suggesting that in some oocytes, MAD1 relocates after sensing minor alteration between microtubules and chromosomes and moves to the location where the microtubules and chromosomes disconnect. These results indicate that minor changes in the microtubule-chromosome connection can be detected by MAD1 relocation. On the other hand, we found that even in some cooled oocytes, relocated MAD1 and abnormal spindles were observed but chromosome alignment seemed to be normal. This may be caused by some microtubules still being connected with chromosomes; hence, the location or alignment of chromosomes is not affected by partial microtubule depolymerization. However, this partial disconnection between microtubules and chromosomes activates the MAD1 so that MAD1 relocates after these changes. Taken together, these results suggest that MAD1 can sense minor defects in the spindles and that MAD1 staining may be a more sensitive index than spindle morphology [37] in monitoring the relationship between microtubules and chromosomes.
After Taxol treatment, which stabilized microtubules and reduced the tension between microtubules and kinetochores [38, 39], MAD1 localization was not changed in all oocytes regardless of their stage. These results indicate that MAD1 merely senses attachment of chromosomes to microtubules but not the tension between microtubules and chromosomes.
To further check functions of MAD1 in the oocytes during meiosis, anti-MAD1 antibody was injected into oocytes at the GV stage to reduce the amount of MAD1. When the injected oocytes were cultured for examination of nuclear maturation and chromosome alignment, we found that reducing MAD1 in the oocytes did not affect oocyte nuclear maturation and spindle formation. These results indicate that MAD1 does not participate in nuclear maturation and spindle formation. However, a significantly higher number of injected oocytes had abnormal chromosome alignment compaerd to the control oocytes. These misaligned chromosomes were present in oocytes at the M-I, A-I, and M-II stages, and their occurrence was especially higher in oocytes during the transition from the M-I to A-I stages, suggesting that MAD1 antibody reduced the amount of MAD1 in the oocytes and affected the function of MAD1, which in turn caused chromosome misalignment in the oocytes. These results were consistent with those in mitosis, in which MAD1 mutants failed to delay the metaphase-to-anaphase transition in response to microtubule depolymerization [16]. However, whether the oocytes with abnormal chromosome alignment have chromosome abnormalities, such as the gaining and losing of chromosomes after meiosis, cannot be determined at this point and needs further investigation by chromosome analysis.
It has been suggested that mitotic spindle checkpoint proteins, such as MAD1, MAD2, MAD3 (BUBR1), BUB1, and BUB3, lie in the same checkpoint pathway that controls the onset of anaphase [7, 11–15]. However, exact checkpoint pathway and signal transduction during anaphase-metaphase are still unknown, especially during mammalian meiosis. Recently, it has been found that MAD2 [29, 30, 40] and BUB1 [31] also participate in the meiotic spindle checkpoint in mouse [29–31] and rat [40] oocytes. However, the localizations of these checkpoint proteins are different. For example, BUB1 was present in the mouse oocytes from the GVBD to M-I stages, disappeared at the late A-I stage, and reappeared at the M-II stage [31]. Costaining of kinetochores and BUB1 indicated that all BUB1 was present at the kinetochores [31]. When MAD2 was examined in mouse [30] and rat [40] oocytes, it was found that MAD2 was localized around the chromosomes after the GVBD and ProM-I stages. When the oocytes progressed to the M-I stage, in which all chromosomes had aligned at the spindle equator, MAD2 disappeared. No MAD2 could be detected in the oocytes from the M-I to M-II stages. However, when the oocytes from the M-I to M-II stages were treated with nocodazole to destroy the spindles, MAD2 was reactivated in the oocytes at all stages. Costaining of kinetochores and MAD2 indicated that all MAD2 was present at the kinetochores [31, 40]. However, in the present study, we found that MAD1 was present in the oocytes at all stages; however, its location was changed during oocyte nuclear progression (from near chromosomes to the spindle poles). Nocodazole treatment caused MAD1 relocation to the chromosomes, but spindle recovery allowed MAD1 to return to the spindle poles. These results indicate that these spindle checkpoint proteins may participate in a same spindle checkpoint pathway but that their locations and expressions are different. They may cooperate through the same signal transduction pathway, or they may work independently. Further studies are necessary to address their relationships and detailed functions of each protein.
In conclusion, this is, to our knowledge, the first detailed examination of MAD1 localization and its function as a spindle checkpoint protein in mouse oocytes, as a mammalian model, during meiosis. Our results clearly indicate that mitotic spindle checkpoint protein MAD1 is also a meiotic spindle checkpoint protein in mammalian oocytes. The present results also indicate that MAD1 is activated in the oocytes at all stages of the cell cycle during meiosis and participates in the spindle checkpoint during normal meiosis. When normal meiosis is disrupted artificially at any point of the cell cycle, MAD1 cannot accurately check the misaligned chromosomes. The MAD1 transiently moves around the nucleus and the metaphase plate to check the connection between microtubules and chromosomes. Minor changes between this connection cause relocation of MAD1 in the metaphase plate. Examination of MAD1 in the oocytes might be a more sensitive marker than spindle morphology for monitoring environmental changes during in vitro manipulation of mammalian oocytes.
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ACKNOWLEDGMENTS |
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FOOTNOTES |
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2 Correspondence: Wei-Hua Wang, State Key Laboratory of Reproductive Biology, Institute of Zoology, Chinese Academy of Sciences, 25 BeiSiHuanXi Road, Haidian, Beijing 100080, China. FAX: 86 106 265 0042; wangweihua11{at}yahoo.com
Received: 8 June 2004.
First decision: 7 July 2004.
Accepted: 23 August 2004.
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