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BOR - Papers in Press, published online ahead of print January 11, 2006.
Biol Reprod 2006, 10.1095/biolreprod.105.044677
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BIOLOGY OF REPRODUCTION 74, 777–787 (2006)
DOI: 10.1095/biolreprod.105.044677
© 2006 by the Society for the Study of Reproduction, Inc.

Research Article

Donor Centrosome Regulation of Initial Spindle Formation in Mouse Somatic Cell Nuclear Transfer: Roles of Gamma-Tubulin and Nuclear Mitotic Apparatus Protein 11

Nguyen Van Thuan 2 3, Sayaka Wakayama 4, Satoshi Kishigami 3, and Teruhiko Wakayama 3

RIKEN Kobe Institute,3 Center for Developmental Biology, Laboratory for Genomic Reprogramming, Chuo-ku, Kobe City, Hyogo 650-0047, Japan Department of Life Science,4 Graduate School of Science and Technology, Kobe University, Kobe 657-8501, Japan

ABSTRACT

During the process of spindle-chromosome complex depletion in the oocyte, it is unclear whether both gamma-tubulin and nuclear mitotic apparatus protein 1 (NUMA1), which are required for mitotic organization and spindle assembly, are removed. The role of the donor cell centrosome and donor nuclear NUMA1 in the initial spindle morphogenesis and chromosome remodeling also remains unclear. In the present study, we show that in the mouse, the level of gamma-tubulin in the poles and around the metaphase II spindle declines significantly, whereas only approximately 10% of NUMA1 is removed during spindle-chromosome complex depletion in the recipient oocyte. This process does not impede initial spindle morphogenesis and is regulated by the centrosome of the donor cumulus cell. Retaining the donor cell centrosome establishes a monopolar spindle, whereas prior removal of the centrosome by a narrow-bore micropipette leads to bipolar spindle formation. Our data show that the centrosome of the donor cell regulates initial spindle morphogenesis and that the donor cumulus cell NUMA1 compensates for the deficiency in recipient NUMA1 during the formation of metaphase-like structures after nuclear transfer. Full-term offspring of cloned mice were obtained after injection of donor cells only with a pipette having an inner diameter of 7–8 µm, which retained the donor cell centrosome. In contrast, removing the donor cell centrosome with a small pipette impaired preimplantation development and prevented full-term development. In conclusion, the initial spindle assembly of a metaphase-like spindle is regulated by the centrosome from the donor cell in the mouse.

cloned embryos, cumulus cells, cytokines, developmental biology, donor cell centrosome, early development, gamma-tubulin, nuclear transfer, NUMA1, ovum, spindle morphogenesis

INTRODUCTION

During the artificial construction of mammalian clones by somatic cell nuclear transfer (SCNT) into spindle-chromosome complex (SCC)-depleted oocytes, early nuclear reprogramming involves morphological remodeling of the donor nucleus and its associated cytoskeletal structures. This process includes breakdown of the donor nuclear membrane, initial chromatin condensation, spindle assembly, and pronuclear formation after activation. The stage of the nuclear transfer (NT) donor cell cycle affects both the chromosome and spindle constitution [1] and subsequent embryo development [2, 3]. The timing of the breakdown of the nuclear membrane also is a main factor that affects both the karyoplast and cytoplast biology [4] and, in turn, is affected by the level of maturation-promoting factors in the recipient oocyte cytoplasm [5]. Before activation, the somatic chromosomes introduced by NT assemble on a metaphase plate that is misaligned with the spindle, or individual chromosomes may be located near the spindle poles [6–8]. Most cloned embryos observed to date exhibit a range of numerical chromosomal abnormalities [9, 10]. These deficiencies in somatic nuclear reprogramming after NT might emanate, at least in part, from failure to remodel the somatic nucleus morphologically into a functional embryonic nucleus [11].

The centrosome of the somatic cell consists of a pair of centrioles surrounded by a cloud of electron-dense material known as the pericentriolar material, which contains the gamma-tubulin (known throughout as {gamma}-tubulin) ring complexes involved in microtubule nucleation [12–14]. The centrosome does not duplicate when the cell is in the G0 or early G1 phase of the cell cycle [15–17]. In dividing vertebrate cells, centrosomes form the poles of mitotic spindles, where they direct the segregation of chromosomes and play an important role in the final stages of cell division or cytokinesis [18]. Deregulation of centrosome duplication affects their number and promotes aneuploidy, which are two characteristic features of human tumors [19]. Moreover, defective centrosomes disrupt normal cell and tissue organization and lead to the chromosomal instability found in malignant tumors [20]. In normal cells, centrosomes form bipolar spindles, but they can organize monopolar, tripolar, or multipolar mitoses in tumor cells. These abnormalities cause an unequal distribution of chromosomes during mitosis and can initiate imbalanced cell cycles in which the normal checkpoints for cell cycle control are lost [21].

A member of the tubulin superfamily, {gamma}-tubulin (48 kDa) is localized in the microtubule-organizing center and is needed for mitotic organization and spindle function [22, 23]. It is found in the poles of the spindle and in the cytoplasm at the metaphase II (MII) stage of meiosis in the mouse oocyte [24, 25]. Nuclear mitotic apparatus protein 1 (NUMA1, also known as NUMA) is an abundant, 240-kDa protein that localizes to the nucleus during interphase and accumulates at the spindle poles during mitosis [26, 27]. In addition, NUMA1 is associated with dynein, dynactin, and spindle poles in both cell extracts and cultured cells [28]. Disruption of NUMA1 function by antibody injection or immunodepletion of extracts shows that it is required for the assembly and maintenance of the spindle poles [29–33].

In primate SCNT, removal of the meiotic spindle depletes the ooplasm of NUMA1 and KIFC1 protein (also known as C1 or HSET; a member of the kinesin family), both of which are vital for mitotic spindle pole formation [34]. In cattle, the somatic centrosome is transferred during NT [35], whereas in mice, this process relies on the maternal centrosome of the oocyte [36]. Both {gamma}-tubulin and NUMA1 are thought to play important roles in the cell cycle and early reprogramming after SCNT and spindle morphogenesis, but their roles during the early spindle morphogenesis are not well understood. After injection of a somatic nucleus into a mature oocyte, the cloned construct undergoes maturation-promoting factor activation, nuclear membrane breakdown, and chromosome condensation, and a metaphase spindle-like structure forms. However, whether {gamma}-tubulin and NUMA1 of the recipient oocyte are removed during the process of SCC depletion is not known. Moreover, the roles of the somatic cell centrosome and NUMA1 during initial spindle morphogenesis, chromosome remodeling, and subsequent developmental competence of cloned mouse embryos are still not clear. To gain further insight regarding these processes, we first observed the distribution and level of {gamma}-tubulin and NUMA1 in MII oocytes before and after SCC depletion to determine whether these are removed during this process. Next, we examined the associations between the donor cumulus cell centrosome, NUMA1, and initial spindle morphogenesis. We also systematically characterized spindle morphogenesis, chromosome morphology, and both {gamma}-tubulin and NUMA1 distributions in the metaphase-like structures formed after cumulus cell nuclei had been injected into SCC-depleted mouse oocytes. Finally, we examined the effects of retention or removal of donor cell centrosomes on the development of cloned embryos.

MATERIALS AND METHODS

Collection of Oocytes and Cumulus Cells

Female B6D2F1 mice (age, 8–10 wk; Japan SCL, Inc.) were superovulated by injection of 5 IU of eCG, followed 48 h later by injection of 5 IU of hCG. All animals were handled according to the Animal Experimental Handbook at the Center for Developmental Biology, RIKEN. Oocytes were collected from the oviducts approximately 16 h after hCG injection. After collection, the cumulus cells were dispersed with 0.1% hyaluronidase (Sigma Chemical Co.) in drops of Hepes-buffered CZB medium (Hepes-CZB) [37]. The oocytes were transferred to new drops of Hepes-CZB and were denuded of almost all cumulus cells by gentle pipetting. Only denuded oocytes with a homogeneous ooplasm were selected, and these oocytes were then resuspended in new drops of KSOMaa medium (Specialty Media) containing 1% BSA (KSOM medium; Sigma), which had been covered previously with sterile paraffin oil (Nacalai Tesque). The oocytes were cultured at 37.5°C in an atmosphere of 5% CO2 in air until use.

SCC Depletion, NT, and Oocyte Activation

Cumulus cell donor NT was performed as described previously [7]. The SCC depletion was carried out using a piezo-actuated micromanipulator system (Prime Tech) and pipettes (inner diameter, 6–7 µm) in a droplet of Hepes-CZB with 5 µg/ml of cytochalasin B under mineral oil. The SCC-depleted oocytes were washed and then cultured in KSOM medium. After three washes in Hepes-CZB, collected cumulus cells were transferred into droplets of Hepes-CZB containing 12% polyvinylpyrolidone (Mr, 360 kDa; Wako). The cumulus cell membrane was removed using injection pipettes with different inner diameters (7–8, 5–6, 3–4, and 2–3 µm), depending on each experiment, by drawing in and out of the injection pipette. At the same time, each injection pipette was used to pick up several cell nuclei, and a single nucleus was injected into an intact or SCC-depleted oocyte. The oocytes were then cultured in KSOM medium, fixed at 10-min intervals until 60 min after NT (see below), and then used for immunofluorescence experiments. Another group of oocytes was cultured in the same medium for at least 1 h before artificial activation. The oocytes were activated for 1 h with 5 mM SrCl2 in Ca2+-free CZB medium with 5 µg/ml of cytochalasin B. The oocytes were then cultured for 5 h in KSOM medium supplemented with 5 µg/ml of cytochalasin B to prevent polar body extrusion. Following activation and cytochalasin B treatment, the oocytes were washed and cultured in droplets of KSOM medium under mineral oil in 5% CO2 in air at 37.5°C to permit development.

Immunological Procedures

After two washes in Ca2+- and Mg2+-free Dulbecco phosphate-buffered saline with 0.1% polyvinyl alcohol (PBS-PVA; Sigma), the NT oocytes or cumulus cells were fixed for 30 min in PBS-PVA with 3.5% paraformaldehyde. The fixed oocytes or cumulus cells were washed twice in PBS-PVA and then stored overnight at 4°C in PBS supplemented with 3% BSA (PBS-BSA) and 0.1% Triton X-100 (Nacalai Tesque, Inc.). To stain {gamma}-tubulin and NUMA1, the samples were washed twice in PBS-BSA and then incubated overnight at 4°C in PBS-BSA with rabbit anti-{gamma}-tubulin antibody (1:200 dilution; Sigma) or mouse anti-NUMA1 antibody (1:400 dilution; BD Biosciences). After three washes in PBS-BSA for 10 min each time, the oocytes were incubated with Alexa Fluor 488-labeled goat anti-rabbit immunoglobulin (Ig) G or goat anti-mouse IgM (1:100 dilutions; Molecular Probes, Inc.) for 60 min at room temperature. To stain {alpha}- or ß-tubulin, the samples were incubated in fluorescein isothiocyanate-conjugated anti-{alpha}-tubulin (1:100; Sigma) or anti-ß-tubulin (1:100; BD Biosciences) for 60 min. After two washes in PBS-BSA, the sample was incubated with Alexa Fluor 568-labeled (1:100 dilution; Molecular Probes). After three 10-min washes in PBS-BSA, the DNA was stained with 400 µg/ml of propidium iodide (Sigma) or with 2 µg/ml of 4',6-diamidino-2-phenylindole dihydrochloride (Molecular Probes). Following extensive washing, the oocytes or cumulus cells were mounted on slides using Vectashield mounting medium (Vector Laboratories, Inc.) and observed with a Bio-Rad Radiance 2100 confocal scanning laser microscope (Bio-Rad, Carl Zeiss, Inc.). To fix and stain the SCC-depleted pieces, they were injected into empty zona pellucidae (Fig. 1), and the fixation and staining of {gamma}-tubulin and NUMA1 were performed as described above. To retain or remove the cumulus cell centrosome, the injection pipette was reduced in internal diameter from between 7 and 8 µm to between 2 and 3 µm. The presence of the centrosome was verified by staining for {gamma}-tubulin. The state of the cumulus nuclear membrane was determined by staining with goat anti-lamin B IgG (Santa Cruz Biotechnology) and Alexa 568-labeled anti-goat antibodies.


Figure 1
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  		FIG. 1. Methods to collect SCC-depleted pieces and cumulus cells treated with injection pipettes having different inner diameters for immunological experiments. A) SCC-depleted cytoplasts. B) Four to five SCC-depleted cytoplasts were injected into an empty zona pellucida. C) The treated cumulus cell nuclei were injected into an empty zona.

Electrophoresis and Western Blot Analysis

The MII oocytes, SCC-depleted oocytes, and SCC-depleted oocytes injected with cumulus nucleus using a pipette (inner diameter, 7–8 µm) were washed twice in PBS-PVA; boiled for 5 min in 15 µl of SDS sample buffer containing 62 mM Tris (pH 6.8), 2% SDS, 5% ß-mercaptoethanol, 10% glycerol, and 0.01% bromphenol blue [38]; and then frozen at –20°C until use. The samples were run on 15% SDS-polyacrylamide gels, loaded with exactly 100 oocytes per lane, and transferred to an Immobilon-P transfer membrane (Millipore Corp.) using an ATTA semidry transfer system (ATTO Corp.) at a constant 20 V for 90 min. The membranes were blocked with 10% fetal calf serum in PBS containing 0.1% Tween 20 (PBS-Tween) for 2 h and incubated with rabbit anti-{gamma}-tubulin antibody (1:1000) or mouse anti-NUMA1 antibody (1:500) for 2 h in PBS-Tween containing 5% fetal calf serum for 4 h. After three washes in PBS-Tween, the membranes were treated with horseradish peroxidase-conjugated anti-rabbit IgG (1:1000; Amersham Biosciences) or horseradish peroxidase-conjugated goat anti-mouse IgG (1:1000; Santa Cruz Biotechnology) in PBS-Tween for 1 h at room temperature. After three 10-min washes in PBS-Tween, peroxidase activity was visualized using an ECL Plus Western Blotting Detection System (Amersham Biosciences). Treated membranes were developed on an image reader (LAS-1000 mini; Fujifilm Science Imaging), and image analysis was performed using Image Gauge V4.0 software (Fujifilm). The band intensity in intact oocytes was arbitrarily set at 100%, and other intensities were expressed relative to this value. Data are presented as the mean percentage ± SD.

Embryo Transfer

To examine the effect of retention or removal of the donor centrosome on the full-term development, cloned embryo transfer was performed at the morula and blastocyst stage (72 h after activation). From 10 to 15 morulae and blastocysts were transferred into the uterus of each surrogate mother (ICR mouse; Japan SCL) on Day 3 of pseudopregnancy following mating with vasectomized ICR males.

Statistical Analysis

Data were subjected to arcsine transformation in each replication. The transformed values were analyzed using one-way ANOVA. A level of P < 0.05 was considered to be significant.

RESULTS

Localization and Quantitative Analysis of {gamma}-Tubulin and NUMA1 in Mouse Oocytes before and after SCC Depletion

In intact oocytes, {gamma}-tubulin was found to be concentrated at the spindle poles at the MII stage, around the spindle, abundantly in the subspace of the spindle (Fig. 2A), and in the cytoplasm. In addition, NUMA1 was detected at the spindle poles and in the cytoplasm of oocytes (Fig. 2B). The pieces of SCC removed from oocytes showed both {gamma}-tubulin and NUMA1 at the spindle poles, and small amounts of {gamma}-tubulin and NUMA1 also were observed around the spindle and near the chromosomes (Fig. 2, C and D). The levels of {gamma}-tubulin and NUMA1 of intact oocytes, SCC-depleted oocytes, and SCNT oocytes were measured by Western blot analysis (Fig. 2, E and F). The {gamma}-tubulin level was highest in intact oocytes, significantly lower in SCC-depleted oocytes, and higher in SCNT oocytes (P < 0.05) (Fig. 2E1). The NUMA1 level was 10% lower in SCC-depleted oocytes than in intact oocytes and was slightly higher after SCNT. However, the level of NUMA1 did not differ significantly between intact oocytes, SCC-depleted oocytes, and SCNT oocytes (P > 0.05) (Fig. 2F1). We examined the localization of {gamma}-tubulin, microtubules, and NUMA1 in cloned embryos at the first mitotic interphase and metaphase stages. Both {gamma}-tubulin and microtubules were not detected in the cytoplasm of cloned embryos at the pronuclear (Fig. 3A), prometaphase (Fig. 3B), or metaphase (Fig. 3C) stage of the first mitotic cleavage. However, {gamma}-tubulin was seen as small dots in the peripheral cytoplasm associated with a dense microtubule network on the cortical embryo at the pronuclear (Fig. 3, A1 and A2), prometaphase (Fig. 3, B1 and B2), or metaphase (Fig. 3, C1 and C2) stage of the first mitotic division. In fertilized eggs, {gamma}-tubulin was highly detectable around two pronuclei at the prophase (Fig. 3D) and prometaphase (Fig. 3E) stages and was concentrated in the poles, chromosomes, and around the first mitotic metaphase spindle (Fig. 3F). In contrast, NUMA1 was detected in both the pronuclei of cloned embryos and the fertilized embryos at the pronuclear stage (Fig. 3, G and H). These results indicate that during the process of SCC depletion in oocytes, {gamma}-tubulin is partially reduced, and this may result in an abnormal redistribution of {gamma}-tubulin during the first mitotic division of cloned embryos, whereas the level of NUMA1 does not change from before to after SCC depletion.


Figure 2
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  		FIG. 2. Localization of {gamma}-tubulin and NUMA1 in metaphase II oocytes (A and B) and in species of SCC-depleted from oocytes (C and D). A) Gamma-tubulin is located in the poles and around the metaphase II spindle. B) NUMA1 is located in the poles of the spindle and in the oocyte cytoplasm. C and D) Gamma-tubulin (C) and NUMA1 (D) can be detected in the SCC-depleted cytoplasts. Arrows indicate localization of {gamma}-tubulin and NUMA1 in the metaphase II spindle before and after SCC depletion. E and E1) Western blot analyses of the {gamma}-tubulin level in intact oocytes, SCC-depleted oocytes, and SCNT oocytes. F and F1) Western blot analyses of the NUMA1 level in intact oocytes, SCC-depleted oocytes, and SCNT oocytes. The intensity of {gamma}-tubulin and NUMA1 staining was measured by densitometric analysis of the blots. Data are represented as the mean ± SD. One hundred oocytes were used per lane, and the experiment was performed three times. Values with different superscripts are significantly different (P < 0.05).


Figure 3
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  		FIG. 3. Localization of {gamma}-tubulin and NUMA1 during the first mitotic interphase and metaphase of cloned embryos and fertilized embryos. Gamma-tubulin was not detected in the cytoplasm at the pronuclear (A), prometaphase (B), or metaphase (C) stage but was seen as small dots on the peripheral cytoplasm during the first mitotic cleavage in cloned embryos (A1–C2, representing higher-magnification views of A–C, respectively). Arrows indicate localization of oocyte {gamma}-tubulin. The microtubule network is densely distributed in the peripheral embryo but is sparse in the cytoplasm. Gamma-tubulin was detected around the pronuclei at the prophase (D), prometaphase (E), and the poles and around the spindle of the first mitotic metaphase in fertilized embryos (F). The microtubule network is densely distributed in the cytoplasm, but very few microtubules can be seen in the peripheral embryo during prophase and prometaphase. NUMA1 was detected in pronuclei of both cloned embryos (G) and fertilized embryos (H).

Initial Spindle Morphogenesis after SCNT in Relation to SCC Depletion in Oocytes

To investigate whether SCC depletion affects initial spindle morphogenesis, cumulus cell nuclei were injected into intact and SCC-depleted oocytes. The initial tubulin morphology of the donor nuclei was determined in 365 intact and 425 SCC-depleted oocytes within 40 min after injection. Immunofluorescence analysis of the spindle formation showed that only 8% of intact and 11% of SCC-depleted oocytes initiated with a bipolar spindle (Fig. 4, A and D). In contrast, the spindle started with monopolar morphology, in which tubulin strands arose from one pole with a long tail, in 86% of intact oocytes and 85% of SCC-depleted oocytes (Fig. 4, B and E). The initial spindle morphology did not differ significantly between intact and SCC-depleted oocytes (Fig. 4G). However, 2 h after NT, 94% of the somatic chromosomes had reached a metaphase-like structure with a bipolar spindle, with several donor chromosomes not located at the equator but tending to be found at one pole of the bipolar spindles regardless of the presence or absence of the SCC (Fig. 4, C and F). Thus, initial spindle morphogenesis following SCNT appears to occur independently of the process of SCC depletion.


Figure 4
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  		FIG. 4. Spindle morphologies seen after injection of cumulus nuclei into intact (A–C) and SCC-depleted oocytes (D–F) in mice. Initial bipolar (A and D) and monopolar (B and E) spindle formation at 40 min and a bipolar metaphase spindle-like structure at 120 min after cumulus nuclei were injected into intact (C) or SCC-depleted (F) oocytes. Arrows indicate localization of several donor chromosomes asymmetrically located toward a pole of bipolar spindle. Also shown is a histogram (G) indicating the percentages of initial spindle morphologies after injection of cumulus nuclei into intact and SCC-depleted oocytes. A total of 920 oocytes (420 intact metaphase II oocytes and 500 SCC-depleted oocytes) were injected with cumulus nuclei; fixed at 10, 20, 30, or 40 min after NT; and stained with fluorescein isothiocyanate-conjugated anti-{alpha}-tubulin. The initial spindle morphology of donor nuclei injected into intact oocytes and SCC-depleted oocytes could be determined in 365 and 425 oocytes, respectively.

Initial Spindle Morphogenesis in Mouse SCNT in Relation to the Donor Cell Centrosome

To investigate why cumulus cell nuclei tend to form monopolar spindles and whether the cumulus cell centrosome contributes to the initial spindle morphogenesis after being injected into SCC-depleted oocytes, we examined the centrosomes of cumulus cells. Only 9% of cumulus cells showed two points of {gamma}-tubulin on which chromosomes condensed (Fig. 5, A and D), whereas more single centrosomes (86%) were observed in cumulus cells with decondensed chromatin and intact nuclear membranes (Fig. 5, B and D). Thus, most of the cumulus cells collected from mature oocytes were at the G0- to G1-phase transition [39]. The initial spindle morphology seen after SCNT suggested that the donor cumulus cell centrosomes might give rise to monopolar spindles. To test this hypothesis, we tried to remove the centrosomes from the cumulus cell nuclei using a fine injection pipette (inner diameter, 2–3 µm) and examined whether the loss of the donor cell centrosome affects the initial spindle morphogenesis. Using micropipettes with different inner diameters, we found many single-point centrosomes when using a normal-size (inner diameter, 7–8 µm) pipette. However, decreasing the inner diameter of the pipette to between 2 and 3 µm resulted in successful removal of 99% of the centrosomes from the donor cells (Table 1). We tested this immediately after injection into SCC-depleted oocytes and observed 78%, 20%, 10%, and 0% of single-point {gamma}-tubulin clusters using pipettes with inner diameters of 7–8, 5–6, 3–4, and 2–3 µm, respectively (Table 1 and Fig. 5, E–H). Thus, we were able to successfully remove the donor cumulus cell centrosomes.


Figure 5
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  		FIG. 5. Centrosomes of cumulus cells before and immediately after injection into SCC-depleted oocytes. Cumulus cells with double dots (A), a single dot (B), and no dot (C) of {gamma}-tubulin. Also shown is a histogram (D) indicating the percentages of centrosome numbers in cumulus cells, as are an SCC-depleted oocyte (E) and cumulus cell centrosome immediately after injection into SCC-depleted oocytes with no dot (F), a single dot (G), and double dots (H) of {gamma}-tubulin.


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  		TABLE 1. The numbers of centrosomes in cumulus cells before and immediately after injection into SCC-depleted oocytes using injection pipettes of different diameters.

Using this same system, we also examined whether these donor centrosomes affect the initial spindle morphology after mouse SCNT. We found that decreasing the inner diameter of the injection pipettes from between 7 and 8 µm to between 2 and 3 µm decreased the proportion of cells displaying the monopolar spindle morphology from 92% to 0% and increased the proportion of cells displaying the bipolar spindle morphology from 6% to 84% at 40 min after SCNT (P < 0.05) (Table 2).


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  		TABLE 2. Initial spindle morphologies 40 min after cumulus cell nuclear transfer into SCC-depleted oocytes using injection pipettes of different diameter.

We next examined the morphogenesis of the monopolar spindles after injecting cumulus cells with an intact centrosome (inner diameter of pipette, 7–8 µm) into SCC-depleted oocytes, and the results are shown in Figure 6. At 20 min after SCNT, microtubules arose from the cumulus cell centrosomes, when numerous asters were found in the oocyte cytoplasm (Fig. 6, A and A1). At 30 min after SCNT, the cumulus cell centrosomes with pairs of centrioles were recruiting microtubulin continuously, and a monopolar spindle was established in which microtubules extended around the condensing somatic chromosomes (Fig. 6, B and B1). By 40 min after SCNT, the microtubule strands had extended and enclosed the cumulus chromosomes, and a pair of donor {gamma}-tubulin dots could be seen clearly at the poles of the monospindle (Fig. 6, C and 6C1). Some very small dots of oocyte {gamma}-tubulin also were seen in the oocyte cytoplasm (Fig. 6C, white arrows). Finally, a bipolar spindle with misaligned chromosomes at its equator was formed, although {gamma}-tubulin dots were observed at only one pole of the spindle (Fig. 6D). We also found that several donor chromosomes were always in close contact with the pole of the spindle with {gamma}-tubulin. When the cumulus nuclei were injected using pipettes with an inner diameter of 2–3 µm, which effectively removed the centrosome as described above, 84% of the injected oocytes had formed a bipolar spindle 40 min later (Table 2), but no {gamma}-tubulin dots were observed at the poles of the bipolar spindle at 120 min after NT (Fig. 6E). However, most of the metaphase-like chromosomes are located at the equator of the spindle.


Figure 6
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  		FIG. 6. Initial monopolar spindle morphogenesis after injection of cumulus cell nuclei with intact centrosomes into SCC-depleted oocytes. A) At 20 min after SCNT, a pair of centrioles ({gamma}-tubulin) is seen on the surface of cumulus cell nucleus, and {gamma}-tubulin also is detected in the oocyte cytoplasm, which contains some cytoplasmic asters. B and C) By 30 min after SCNT, microtubulin is being recruited at the cumulus cell centrosome (B) and formed a clear monopolar spindle at 40 min after SCNT (C). A1, B1, and C1) Higher-magnification views of A, B, and C, respectively. Arrows indicate localization of oocyte {gamma}-tubulin. D) Only one dot of {gamma}-tubulin is seen in the pole of the bipolar spindle at the metaphase-like structure 120 min after injection of a cumulus nuclei with a retentive centrosome into SCC-depleted oocyte. E) No {gamma}-tubulin dot is seen in the poles of the bipolar spindle at the metaphase-like structure when the SCC oocyte was injected with a cumulus nucleus with its centrosome removed.

Initial Spindle Morphogenesis in Mouse SCNT in Relation to Donor Cell NUMA1l

In previous experiments, we found that NUMA1 of oocytes was not reduced by SCC depletion. However, the relationship between the donor cell NUMA1 and the initial spindle morphogenesis in mouse SCNT is still unclear. Because NUMA1 plays an essential role in organizing the microtubule "minus" ends at the spindle poles in vertebrate cells [40], NUMA1 localizes to the interphase nucleus and concentrates at the polar ends of the spindle during mitosis [26, 27]. In the cumulus cells studied here, NUMA1 was spread diffusely throughout the nucleus during interphase (Fig. 7A). Twenty minutes after NT, the cumulus NUMA1 began to form aggregates (Fig. 7B). By 30 min after NT, these aggregates became larger and had various morphologies, including multiple dots (Fig. 7C), double dots (Fig. 7D), and a single dot in the donor nucleus (Fig. 7E). Decreasing the inner diameter of the injection pipette to between 2 and 3 µm caused faster aggregation of NUMA1 than an inner diameter of 7–8 µm or 5–6 µm and a higher frequency of double dots detected at 30 min after NT (P < 0.05) (Fig. 7H). The narrow injection pipettes disrupt most of the cumulus nuclear membrane (unpublished data), probably making the nucleus leaky to cytoplasmic factors that might enhance the aggregation of NUMA1 to double dots. By 60 min after NT, a high percentage of double-dot NUMA1 aggregates was observed regardless of the diameter of the injection pipette at 60 min after NT (Fig. 7H). Eighty-six percent of the 50 oocytes with a metaphase-like cumulus chromosome observed at 2 h after NT had double dots of NUMA1, one at each pole of the spindle (Fig. 7F). In contrast, only 9% of chromosomes had single dots of NUMA1 at one pole of the metaphase spindle-like structure (Fig. 7G). However, the initial spindle formation was always observed before the condensation of NUMA1 following SCNT. Although we have no direct proof, comparing the results of the timing of the initial spindle formation, donor NUMA1 aggregation, and the numbers of NUMA1 clusters in the donor cell after NT and on the poles of spindles suggests that the donor cell NUMA1 does not determine the early spindle morphology but, instead, participates in the spindle poles in the metaphase spindle-like structure. Based on these results, we propose a model of initial spindle morphogenesis in SCNT, the details of which are shown in Figure 8.


Figure 7
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  		FIG. 7. The distribution of NUMA1 in cumulus cell nuclei before and after injection into SCC-depleted oocytes. Distribution of NUMA1 in the cumulus cell nucleus before injection (A), and 20 min (B) and 30–60 min (C–E) after SCNT. Double dots (F) and one dot (G) of NUMA1 are seen in the poles of the bipolar spindle at the metaphase-like structure 120 min after SCNT. Also shown is a histogram (H) indicating the morphological changes among cumulus cell NUMA1 distributions 30 and 60 min after injection into oocytes.


Figure 8
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  		FIG. 8. Our proposed model of initial spindle morphogenesis after injection of cumulus cell nuclei with or without centrosomes into SCC-depleted mouse oocytes. When a cumulus cell nucleus with single centrosome is injected into an SCC-depleted oocyte, it nucleates oocyte cytoplasmic microtubulin to form a monopolar spindle at the early stage (monopolar pathway). When a cumulus cell nucleus lacking a centrosome is injected, the cumulus chromatin spontaneously recruits microtubules to form an early bipolar spindle (bipolar pathway). However, at 120 min after SCNT, a bipolar metaphase spindle-like structure is formed regardless of whether the monopolar or bipolar pathway occurs. For the monopolar spindle pathway, donor centrosome is detected at one pole, whereas in the bipolar spindle pathway, no donor centrosome is detected at the poles of the bipolar spindle. Donor NUMA1 is detected at both poles at the metaphase spindle-like structure.

Impairment of the First Cleavage and Preimplantation Development of Cloned Embryos by Removal of the Donor Cell Centrosome with Small Pipettes

Table 3 shows the effects of retention or removal of the donor cell centrosome on the development of preimplantation and full-term cloned embryos. Decreasing the inner diameter of the injection pipette (i.e., removing the donor cell centrosome) did not affect the pronuclear formation at 6 h after activation, but it significantly impaired the competence of 1-cell cloned embryos passing the first mitotic cleavage. With an inner diameter of 2–3 µm, only 34% of SCNT oocytes reached 2-cell embryos; in contrast, 91% of SCNT oocytes using pipettes with an inner diameter of 7–8 µm and 88% of SCNT oocytes using pipettes with an inner diameter of 5–6 µm reached 2-cell embryos (P < 0.05). Most of the blocked 1-cell cytoplast embryos produced by small pipettes (inner diameters, 3–4 and 2–3 µm) were fragmented, whereas cytoplast embryos of the blocked 1-cell cloned embryos produced by pipettes with an inner diameter of 7–8 µm were not fragmented at 24 h. At 72 h after SCNT, 48% of donor cell centrosomes injected using a pipette with an inner diameter of 7–8 µm reached the morula and blastocyst stage. In contrast, the percentages of cloned embryos that developed to the morula and blastocyst stage were 31% for pipettes with an inner diameter of 5–6 µm, 15% for those with an inner diameter of 3–4 µm, and 6% for those with an inner diameter of 2–3 µm (P < 0.05) (Table 3). Finally, cloned mice (1.6%) were obtained after embryo transfer using pipettes with an inner diameter of 7–8 µm but not after embryo transfer using pipettes with inner diameters of 5–6, 3–4, or 2–3 µm.


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  		TABLE 3. Effects on preimplantation development and full-term offspring using injection pipettes of different diameters.*

DISCUSSION

The centrosome plays an important role in the assembly of the bipolar spindle, which is needed for the correct segregation of chromosomes during cell division and directs many of the microtubule-based processes within the cell [41, 42]. In the zygote of sheep [43, 44], humans [45], rhesus monkeys [46], rabbits [47], and cattle [35], the centrosome typically is introduced by the fertilizing spermatozoon. In contrast, in the mouse zygote, the centrosome in the first mitotic metaphase spindle arises from the oocyte's (maternal) centrosome [36]. During SCNT, removal of the SCC depletes the ooplasm of NUMA1 and KIFC1, both of which are vital for mitotic spindle pole formation [34]. In cattle, the somatic cell centrosome is transferred during NT [35]. However, the association between SCC depletion and donor cell centrosomes and the role of initial spindle morphogenesis after SCNT is not well understood.

Our immunofluorescence observations showed that both {gamma}-tubulin and NUMA1 in the poles and around the MII spindle were removed during SCC depletion of the recipient oocyte. However, Western blot analysis of oocytes before and after SCC depletion showed that the level of {gamma}-tubulin decreased significantly, whereas the level of NUMA1 did not differ significantly from that in the intact oocyte. Taken together, our results indicate that the process of SCC depletion in the mouse oocyte removes {gamma}-tubulin but not NUMA1.

We tested the hypothesis that SCC depletion from the oocyte affects the spindle morphogenesis, and we followed the development in somatic NT in the mouse. We rejected our initial hypothesis based on our previous report [8] and the serial observations of the early spindle formation in the present study, in which most of the initial morphology was monopolar regardless of whether the recipient oocyte was intact or SCC depleted. However, most somatic chromosomes reached the bipolar metaphase spindle by 2 h after NT regardless of whether the process started with a monopolar or a bipolar spindle. These results raise the question of whether the donor cell centrosome defines the initial spindle morphogenesis in SCNT. The number of centrosomes determines the number of spindle poles formed [48], and monopolar spindles form in the presence of a single centrosome or when separation of centrosomes is blocked [49–51]. During the normal cell cycle, the centrosome does not duplicate in the G0 phase but, rather, initiates duplication either at the end of the G1 phase or during the S phase [15, 16, 52]. In the present study, we demonstrated that most of the cumulus cells collected from mature oocytes were at the G0 to G1 phase, with a single centrosome [39], and that only a single centrosome was obtained at only one pole of the monopolar spindle (20–40 min after NT) or of the bipolar spindle at the metaphase-like structure (60–120 min after NT). Thus, the centrosome does not duplicate in the metaphase condition [53, 54].

Interestingly, we found that the donor cell centrosome determines the initial morphology of the spindle following mouse SCNT. Retention of the donor cell centrosome resulted in the formation of a monopolar spindle, whereas removal of the donor cell centrosome induced the early formation of a bipolar spindle. Our observations suggest that the presence of a cumulus cell single centrosome following NT provides a dominant focal microtubule site that can overrule the natural tendency of microtubules to self-organize into a bipolar array around chromosomes [51]. We found that although some oocyte {gamma}-tubulin foci were detectable around the cumulus nucleus, the microtubules were, however, already nucleated in the centrosome of the cumulus nucleus to form a monopolar spindle. This suggests that the functions of {gamma}-tubulin in the oocyte cytoplasm and donor centrioles differ for the recruitment of microtubules. In contrast, removing the donor centrosome appears to cause the cumulus chromatin to recruit microtubules to form spontaneously an early bipolar spindle. This result is supported by previous reports showing that bulk chromatin can nucleate microtubules to form spindles in the absence of centrosomes [51, 52]. Moreover, mammalian somatic cells can use a centrosome-independent pathway for spindle formation, which normally is masked by the presence of the centrosome [55, 56]. The observation of NUMA1 of the donor nucleus before and after injection into SCC-depleted oocytes suggests that donor cell NUMA1 does not determine the initial spindle morphology but may contribute to the organization of the poles of the metaphase spindle-like structure 1–2 h after NT. Our results are similar to those of Khodjakov et al. [56], who found that both poles in such spindles are well focused and contain NUMA1 but that the centrosomal pole lacks {gamma}-tubulin.

A bipolar spindle formed at 2 h after NT regardless of the existence of the centrosome. Only one dot of {gamma}-tubulin was found at the pole of the bipolar spindle when the donor centrosome was retained. In contrast, removing the donor centrosome resulted in a lack of {gamma}-tubulin dots at both poles of the metaphase spindle-like structure. Retention of the donor cumulus cell resulted in the formation of the monopolar spindle at the early stage. However, both in vitro culture for development and embryo transfer were associated with increased rates of embryo development to the blastocyst stage and offspring. Although we have no direct proof, our results suggest that the donor cell centrosome may be needed for normal development of the cloned embryo, or the destruction of the nuclear envelope by centrosome removal, mentioned in passing in Results, may have caused some other kind of catastrophic damage. We propose a distinct order of microtubule nucleation in SCNT of mice, which involves first {gamma}-tubulin of the donor cell centrosome and then deposition of cumulus nuclear NUMA1 at the poles of the bipolar spindle at the metaphase-like structure. In contrast, in the absence of the donor cell centrosomes, assembly of the bipolar spindle seems to occur through the self-organization of microtubules around donor chromatin.

In conclusion, we found that during the process of SCC depletion, the main part of {gamma}-tubulin, which is needed for correct mitotic spindle formation, is removed, whereas NUMA1 is not significantly affected. Our findings demonstrate that in the mouse, the initial spindle assembly of a metaphase-like spindle is regulated by the centrosome from the donor cell. Our data also show that donor cell NUMA1 is not affected by this process but contributes to the organic poles of the bipolar spindle.

FOOTNOTES

1 Supported by grants-in-aid for Creative Scientific Research (13GS0008) and for Young Scientists (A) (15681014) to T. W. from MEXT, Japan. Back

2 Correspondence: Nguyen Van Thuan, RIKEN Kobe Institute, Center for Developmental Biology, Laboratory for Genomic Reprogramming, 2–2–3 Minatojima-minamimachi, Chuo-ku, Kobe City, Hyogo 650-0047, Japan. FAX: 81 78 306 3095; nvthuan{at}cdb.riken.jp Back

Received: 14 June 2005.

First decision: 7 July 2005.

Accepted: 9 January 2006.

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