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Reproductive Technology |
RIKEN Bioresource Center,3 Koyadai, Tsukuba, Ibaraki 305-0074, Japan
Graduate School of Life and Environmental Sciences,4 University of Tsukuba, Tsukuba, Ibaraki 305-8572, Japan
Meiji University Graduate School,5 Kawasaki, Kanagawa 214-8571, Japan
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ABSTRACT |
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 TOP ABSTRACT INTRODUCTIONMATERIALS AND METHODSRESULTSDISCUSSIONREFERENCES |
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-tubulin antibody. Enucleation of MII oocytes caused no significant change in the number of cytoplasmic asters. The number of asters decreased after transfer of the donor nuclei into these enucleated oocytes, probably because some of the asters participated in the formation of the spindle that anchors the donor chromosomes. The cytoplasmic asters became undetectable within 2 h of oocyte activation, irrespective of the presence or absence of the donor chromosomes. After the standard NT protocol, a spindle-like structure persisted between the pseudopronuclei of these oocytes throughout the pronuclear stage. The asters reappeared shortly before the first mitosis and formed the mitotic spindle. When the donor nucleus was transferred into preactivated oocytes (delayed NT) that were devoid of free asters, the microtubules and microfilaments were distributed irregularly in the ooplasm and formed dense bundles within the cytoplasm. Thereafter, all of the delayed NT oocytes underwent fragmentation and arrested development. Treatment of these delayed NT oocytes with Taxol, which is a microtubule-assembling agent, resulted in the formation of several aster-like structures and reduced fragmentation. Some Taxol-treated oocytes completed the first cell cycle and developed further. This study demonstrates that cytoplasmic asters play a crucial role during the first cell cycle of murine NT embryos. Therefore, in mouse NT, the use of MII oocytes as recipients is essential, not only for chromatin reprogramming as previously reported, but also for normal cytoskeletal organization in reconstructed oocytes.
developmental biology, early development, embryo, gamete biology, ovum
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INTRODUCTION |
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TOPABSTRACTINTRODUCTIONMATERIALS AND METHODSRESULTSDISCUSSIONREFERENCES |
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In unfertilized murine oocytes, the microtubule-organizing centers (MTOCs), which comprise the so-called cytoplasmic asters (cytoasters), play central roles in the apposition of the male and female pronuclei and in centrosomal inheritance of cleavage stage–embryos [8]. In most animals other than the mouse, the centrosomes are inherited mainly from the fertilizing spermatozoa, from which the MTOC is organized (reviewed in [9]). Therefore, it is possible that the interactions that occur between microtubules and chromosomes during the reconstruction and first cell cycle of cloned embryos differ between mice and other animals. The present study was undertaken to determine 1) the roles of cytoplasmic asters during the first cell cycle of cloned murine embryos; and 2) the effects of NT timing on aster behavior, which may be related to the embryo fragmentation that is observed specifically in delayed NT murine oocytes.
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MATERIALS AND METHODS |
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TOPABSTRACTINTRODUCTIONMATERIALS AND METHODSRESULTSDISCUSSIONREFERENCES |
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The oocytes were cultured in bicarbonate-buffered potassium simplex optimized medium (KSOM) that was supplemented with 0.1 mg/ml polyvinyl alcohol and 3 mg/ml bovine serum albumin (BSA) [10]. Drops of the culture medium were covered with mineral oil (Nacalai, Tokyo, Japan) and maintained under 5% CO2 in air at 37°C. Oocyte handling and micromanipulation were carried out in BSA-free Hepes-buffered KSOM medium (Hepes-KSOM; pH 7.4) in air.
Preparation of Oocytes and Cumulus Cells
B6D2F1(C57BL/6 x DBA/2) female mice (SLC, Shizuoka, Japan), 8– 12 weeks of age, were used for the collection of recipient oocytes and donor cumulus cells. The mice were superovulated by the injection of 7.5 IU eCG, followed by 7.5 IU hCG about 48 h later. Oocytes were collected from the oviducts about 15 h after hCG injection and released from the cumulus cells by treatment with 0.1% bovine testicular hyaluronidase in KSOM medium. These oocytes and cumulus cells were incubated in KSOM until use. All procedures described within were reviewed and approved by the Animal Experimental Committee at the RIKEN Institute and were performed in accordance with the Guiding Principles for the Care and Use of Laboratory Animals.
Nuclear Transfer and Oocyte Activation
Nuclear transfer and oocyte activation were carried out according to the method reported previously [11, 12]. The timing of NT and oocyte activation differed according to the experimental groups, as described below. The recipient MII oocytes were enucleated, together with a small amount of the surrounding cytoplasm in Hepes-buffered KSOM that contained 7.5 µg/ml cytochalasin B (Calbiochem, Darmstadt, Germany) on a heated manipulation stage (37°C). Enucleation was performed using a Piezo-driven micromanipulator (Prime Tech, Ibaraki, Japan). The oocytes were allowed to regenerate their membranes in KSOM medium for 1–2 h. For NT, the cumulus cells were placed in 6% polyvinylpyrrolidone solution, and their nuclei were injected into enucleated oocytes in Hepes-buffered KSOM at room temperature using the Piezo-driven micromanipulator. The oocytes were activated by treatment with 3 mM SrCl2 in Ca2+-free KSOM medium for 1 h. When NT was carried out before oocyte activation (standard protocol, Group A), the oocytes were cultured for an additional 5 h in the presence of 5 µg/ml cytochalasin B to prevent the extrusion of a polar body that contained some of the donor chromosomes.
Experimental Design
This study involved five experimental groups (Fig. 1): Group A, standard NT protocol, in which the oocytes were activated 2 h after NT; Group B, simultaneous NT, in which the oocytes were activated within 5 min after NT; Group C, delayed NT, whereby NT was performed 2 h after activation; Group D, diploid parthenotes, in which the oocytes were activated without enucleation and NT; and Group E, enucleated oocytes, whereby the oocytes were enucleated and activated without NT. The distribution patterns of the microtubules, microfilaments, and chromosomes were observed at 2, 8–12, and 15 h after activation using the indirect immunofluorescence method described below.
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Some of the delayed NT oocytes were treated with Taxol, which promotes microtubule assembly, to investigate whether microtubule network reformation improved the abnormal cellular kinetics of delayed NT oocytes. In a preliminary experiment using parthenogenetic embryos, we found that Taxol treatment itself induced abnormal, uneven cleavage or mild fragmentation (consisting of two to eight fragments) of embryos. Therefore, we employed two protocols in which embryos were subjected to long- or short-term exposure to Taxol-containing medium. In the first protocol, enucleated oocytes were activated for 1 h with SrCl2 in Ca2+-free KSOM medium that contained 1.5 µM Taxol. After 1 h of treatment with Taxol in normal KSOM medium and nuclear transfer, the reconstructed oocytes were treated with Taxol for a further 1–2 h (total of 3–4 h exposure to Taxol). In the second protocol, delayed NT oocytes, which were prepared as described above (Group C), were exposed to 0.2 µM Taxol for only 10 min after nuclear transfer. In both protocols, the reconstructed oocytes were cultured in Taxol-free KSOM for 72 h, and their developmental stages were recorded.
Immunofluorescence Staining
Oocytes were fixed and immunostained for microtubules, microfilaments, and DNA using a modification of the method described by Navara et al. [9]. Briefly, zona pellucidae were removed with acidified M2 medium (pH 2.5) at 37°C. After a 30-min period of recovery at 37°C, the zona-free oocytes were attached to MAS-coated slide glasses (Matsunami Glass, Tokyo, Japan) and extracted for 2–3 min with buffer M (25% [v/ v] glycerol, 50 mM KCl, 0.5 mM MgCl2, 0.1 mM EDTA, 1 mM EGTA, 50 mM imidazole hydrochloride, and 1 mM 2-mercaptoethanol; pH 6.8) that contained 0.01%–0.2% Triton X-100. The permeabilized oocytes were transferred to dry slide glasses and fixed in 3% formaldehyde for 20 min at room temperature. Permeabilization and fixation were performed at 37°C to maintain the original microtubule configuration. Fixed oocytes were then blocked overnight with 0.1 M PBS that contained 0.1% Triton X-100 and 3 mg/ml BSA (PBS-TX-BSA) at 4°C. The oocytes were incubated with a murine monoclonal antibody against -tubulin (clone DM 1A, diluted 1:500; Sigma Chemical Co., St. Louis, MO) for 40 min at 37°C. After washing in PBS-TX-BSA, the samples were stained with FITC-conjugated goat anti-mouse IgG antibody (diluted 1:100; Sigma). They were then nuclear stained for DNA with DAPI (4,6-diamidino-2-phenylindole) or propidium iodide. For the staining of actin filaments, the oocytes were cultured with rhodamine-conjugated phalloidin (50 µg/ml; Sigma) before DNA staining. The slide glasses were mounted in antifade medium (Vectashield; Vector Labs, Burlingame, CA) and examined using a TE2000 epifluorescence microscope (Nikon, Tokyo, Japan).
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RESULTS |
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TOPABSTRACTINTRODUCTIONMATERIALS AND METHODSRESULTSDISCUSSIONREFERENCES |
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Behavior of Cytoplasmic Asters in Nuclear-transferred MII Oocytes
We examined the behavioral patterns of the cytoplasmic asters in MII-arrested oocytes, both before and after NT. Removal of the meiotic spindles from MII oocytes did not change the number or distribution of cytoplasmic asters (14.9 ± 3.0 vs. 15.1 ± 3.8 (mean ± SD asters per oocyte; P > 0.05; Fig. 2, A and B). However, 1–2 h after transfer of the donor nucleus, the number of cytoplasmic asters decreased significantly (8.9 ± 2.9; P < 0.01), whereas a newly formed spindle enclosed the condensed donor chromosomes (Fig. 2C). The area proximal to the spindle had fewer asters than the area distal to the spindle (Fig. 2C). These findings suggest that cytoplasmic asters participate in the reformation of the metaphase spindle that anchors the donor chromosomes.
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Behavior of Microtubules in Nuclear-transferred Oocytes After Activation
To gain a better understanding of the postactivation behavior of microtubules in nuclear-transferred oocytes, we observed the temporal and spatial changes in microtubule distribution in the oocytes from three groups with different NT and activation timings (see Fig. 1 for experimental design). At 1–2 h after activation (Fig. 3, A1, B1, and C1), the oocytes proceeded to the anaphase-telophase transition. At this stage, cytoplasmic asters were undetectable in Groups A and C, whereas some asters were detected in Group B. Although the asters that had exhibited intense fluorescence disappeared, the fine microtubule network remained and was evenly distributed throughout the cytoplasm. Intense fluorescence for tubulin was restricted to the spindle between the segregating donor chromosomes in Group A (standard NT; Fig. 3A1). When the chromosomes segregated into three masses, a tridirectional spindle was formed (Fig. 3A1, inset). In Group B (simultaneous NT), dense bundles of microtubules (or assemblies of asters) often surrounded the introduced donor nucleus (Fig. 3B1).
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At 8–12 h after activation (Fig. 3, A2, B2, and C2), the oocytes were at the midpronuclear stage and formed two pseudopronuclei (Group A) or one large pseudonucleus (Group B). The spindle-shaped dense microtubule arrays were still found between the two pseudopronuclei in Group A. In Group C oocytes (delayed NT), the donor nucleus retained its original size or was slightly larger, and strong fluorescence was absent in the cytoplasm or around the nucleus. The fine microtubule network was present in the cytoplasm, although its distribution was very irregular. Many of these delayed NT oocytes had bumpy surfaces and showed irregular distributions of microfilaments and microtubules within the ooplasm (Fig. 4B). This finding is in marked contrast to the observations of oocytes from Group A, in which the microfilaments were evenly distributed only on the surface area of the cortex (Fig. 4A).
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At 15 h after activation (Fig. 3, A3 and B3), most of the oocytes in Groups A and B entered the prometaphase or metaphase, as evidenced by nuclear membrane breakdown. All of the oocytes in Group C showed complete fragmentation at this stage (see also Fig. 5). During the early prometaphase, the condensing chromosomes were surrounded by dense microtubule arrays without nucleation sites, whereas several asters reappeared in the cytoplasm (Fig. 3A3). As this stage proceeded, the asters migrated to the condensed chromosomes and gradually formed the mitotic spindle. This type of microtubule behavior was common to Groups A and B.
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In the non-NT oocytes (Groups D and E), the chromosomes and microtubules behaved as those in the NT groups (Groups A and C, respectively). The parthenogenetically activated oocytes in Group D (Fig. 3D) showed the same microtubule distribution pattern as those in Group A (Fig. 3A), although the chromosome composition of the (pseudo)pronuclei was different (in parthenogenetic oocytes, each pronucleus contained a haploid set of chromosomes). The oocytes in Group E were enucleated, but not nuclear-transferred (Fig. 3E). After activation, they underwent fragmentation within 15 h, in a similar fashion to the delayed NT oocytes in Group C (Fig. 3C).
In Groups A and D, the microfilament-disrupting agent cytochalasin was included in the medium for the first 6 h postactivation to prevent polar body extrusion. In preliminary experiments, we confirmed that this treatment had no significant effects on the behavioral patterns of the microtubules or cytoplasmic asters (data not shown).
Effect of Taxol Treatment on the Behavior of Delayed NT Oocytes
The behavior of microtubules and microfilaments, as described above, indicates that cytoplasmic fragmentation in delayed NT oocytes (Group C) may be attributed to the absence of cytoplasmic asters at the time of NT. To obtain further experimental evidence to support this hypothesis, we treated recipient oocytes with Taxol, which is a microtubule-assembling agent, and examined whether the enhancement of microtubule assembly improved the cellular kinetics of these delayed NT oocytes. In the first protocol, numerous aster-like structures were found in the cytoplasm 2 h after Taxol treatment (shortly before NT), irrespective of the presence or absence of oocyte chromosomes (Fig. 5A). Taxol-treated oocytes were then reconstructed with the donor nucleus that was produced by delayed NT and cultured for 15 h. As expected, the reconstructed oocytes did not show severe fragmentation that was specific for delayed NT, but did show uneven cleavage or mild fragmentation with two to eight karyoplasts/cytoplast (Fig. 5B). These oocytes showed arrested development thereafter (Table 1). In the second protocol, the oocytes were exposed to Taxol for only 10 min, immediately after nuclear transfer, to minimize the cytotoxic effect of Taxol. Although delayed NT-specific severe fragmentation was induced in some oocytes, the remaining oocytes (n = 25) survived the first cell cycle. Of the latter 25 oocytes, five (20%) developed to the morula/blastocyst stage (two morulae and three blastocysts; Fig. 5B, inset; Table 1). In contrast, all of the oocytes in the nontreated group underwent complete fragmentation (Fig. 5C).
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DISCUSSION |
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TOPABSTRACTINTRODUCTIONMATERIALS AND METHODSRESULTSDISCUSSIONREFERENCES |
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The situation described above is in sharp contrast to that pertaining to NT cloning in domestic animal species, since the MII oocytes of the latter lack cytoplasmic asters. Recent studies of microtubule configuration following somatic cell NT in cattle and pigs suggest a major contribution on the part of the donor cell centrosome (or centrosomal material, 
-tubulins) to the formation of the spindle that encloses the donor chromosomes, although newly appearing cytoplasmic asters are involved to a lesser extent [16, 17]. Interestingly, even when preactivated oocytes were used as recipients, these donor-derived centrosomes participated in the formation of spindles that were associated with the donor pronuclear-like structure and played a role in the positioning of this structure. These patterns are essentially similar to that resulting from NT using blastomere nuclei [18]. Therefore, in domestic animal species the preactivated ooplasm that receives the donor nucleus develops through several cleavages, although very few of these ooplasts reach the blastocyst stage [3, 16]. Activated bovine oocytes that are enucleated at the telophase II stage have also proven to be suitable for NT [19], which indicates a certain amount of flexibility in the timing of NT and oocyte activation in the cloning of domestic species. In contrast, murine nuclear-transferred embryos from preactivated oocytes are never cleaved, and all of them undergo severe fragmentation during the first cell cycle [4, 5]. To date, only one cloned pup has been born from embryonic stem cells using preactivated oocytes that were chemically enucleated by demecolcine [20]. It would be interesting to know whether cytoplasmic asters persisted in these experiments, since as many as 36% of the reconstructed oocytes were cleaved [20]. An interval of 1 h between oocyte activation and NT is sufficient for fragmentation, as shown in this and previous studies [5]. Thus, murine somatic cell NT appears to be relatively inflexible in terms of the timing of oocyte activation and NT, although the reason for this is unclear.
The cytoskeletal dynamics observed during NT in this study indicate that the inability of murine preactivated oocytes to behave as appropriate recipients may be because of the lack of asters (or microtubule-nucleating activity) in the ooplasm at the time of NT. The cytoplasmic asters disappeared rapidly (within 2 h) after oocyte activation in all of the experimental groups, except for Group B, in which some asters remained for reasons unknown. Aster disappearance occurred irrespective of the presence or absence of the MII spindle (i.e., the oocytes in all the experimental groups, including the parthenogenetic group, had exactly the same pattern of aster disappearance). This phenomenon has also been noted with normally fertilized oocytes that were activated by spermatozoa [21, 22]. The disappearance of cytoplasmic asters from activated murine oocytes may reflect the loss of microtubule-nucleating activity. In the fertilized oocytes at S phase, a few -tubulin spots remained in the vicinity of the pronuclei, although well-developed cytoplasmic asters were not discernible.
The patterns of distribution of the fine microtubule networks in the cytoplasm, from which asters disappeared following activation, differed significantly among the experimental groups. The networks in the oocytes of the standard NT group (delayed activation) were distributed uniformly throughout the cytoplasm, whereas those in the delayed NT group showed irregular, patchy, or band distributions. The latter situation was also observed for enucleated oocytes that were activated without NT (Group E). In addition to these abnormal distributions of microtubules, we detected phalloidin-reactive actin filaments, which normally show a distinctive intense cortical band. It is known that contractile ring formation by actin filaments promotes cell division [23]. It seems unlikely that apoptosis is involved in this cascade, since there was no morphological evidence for apoptotic changes, such as nuclear splitting and chromatin aggregation, in the nuclei (data not shown). Therefore, the abnormal recruitment of actin filaments in delayed NT oocytes may lead directly to cytoplasmic fragmentation. However, the reason why abnormal distributions of microtubules and actin filaments occur in delayed NT oocytes remains unclear. To address this question, we treated delayed-NT oocytes with Taxol. This drug induces the formation of aster-like structures in activated oocytes. Therefore, the donor nucleus was introduced into oocyte cytoplasm that contained asters, as in standard NT. Interestingly, severe cytoplasmic fragmentation, which is inevitable in delayed NT oocytes, was not found in oocytes that were treated with Taxol for 3–4 h. However, Taxol induced abnormal cleavage or mild fragmentation (two to eight fragments) of oocytes, and we could not assess the developmental ability of the delayed-NT oocytes that were rescued by Taxol treatment. Therefore, in the second protocol, we treated the oocytes with Taxol for only 10 min to minimize the cytotoxicity of Taxol. As expected, fragmentation occurred in some oocytes owing to delayed NT, whereas the remaining oocytes escaped both fragmentation and Taxol toxicity and developed further. These findings clearly indicate that the presence of asters, or related microtubule nucleation activity, in the recipient ooplasm at the time of NT is crucial for the establishment of the normal nuclear-cytoskeletal configuration in nuclear-transferred oocytes. Thus, the use of MII oocytes is essential for successful mouse somatic cell cloning by NT, at least with regard to cytoskeletal dynamics.
Previously, it was reported that in the case of murine nuclear-transferred oocytes, normal chromatin reprogramming events, which include the silencing of donor nucleus transcription, the accumulation of TATA box-binding proteins, and increased DNase I sensitivity, occur only when the donor nucleus is introduced into MII-arrested recipient oocytes [6]. Chromatin reprogramming leads to zygotic gene activation at the appropriate time point after oocyte activation, and thereafter to normal embryo development. Taken together, our results and those of previous studies suggest that the use of MII oocytes as recipients in mouse cloning experiments is critical, not only for chromatin reprogramming but also for normal cytoskeletal organization in the reconstructed oocytes. This strict requirement is unique to murine somatic cell cloning.
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ACKNOWLEDGMENTS |
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FOOTNOTES |
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2 Correspondence: A. Ogura, RIKEN Bioresource Center, 3-1-1, Koyadai, Tsukuba, Ibaraki 305-0074, Japan. FAX: 81 29 836 9172; ogura{at}rtc.riken.go.jp
Received: 1 May 2004.
First decision: 4 June 2004.
Accepted: 11 August 2004.
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REFERENCES |
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TOPABSTRACTINTRODUCTIONMATERIALS AND METHODSRESULTSDISCUSSIONREFERENCES |
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