BOR - Papers in Press, published online ahead of print
June 29, 2005.
Biol Reprod 2005, 10.1095/biolreprod.105.042366
BIOLOGY OF REPRODUCTION 73, 935–941 (2005)
DOI: 10.1095/biolreprod.105.042366
© 2005 by the Society for the Study of Reproduction, Inc.
Dynamics of Microtubules and Positioning of Female Pronucleus During Bovine Parthenogenesis
Yuki Morito ,
Yukihiro Terada 1 ,
Soichi Nakamura ,
Junko Morita ,
Tomoko Yoshimoto ,
Takashi Murakami ,
Nobuo Yaegashi , and
Kunihiro Okamura
Department of Obstetrics and Gynecology, Tohoku University School of Medicine, Sendai, Miyagi 980-8574, Japan
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ABSTRACT
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The zygote centrosome, consisting of both paternal and maternal centrosomal components, is the microtubule-organizing center necessary for pronuclear migration and positioning in fertilization. Maternal centrosomal function in microtubule organization and pronuclear positioning, however, remains unclear. In the present study, we sought to elucidate the function of maternal centrosomes during bovine parthenotes in the microtubule organizational processes required to move the pronucleus to the cell center without sperm centrosomal components. Microtubule organization, pronuclear position, and distribution of gamma-tubulin, which is thought to be the major component of maternal centrosomal material, were imaged by immunocytochemistry and conventional epifluorescence microscopy. In bovine parthenotes treated with paclitaxel, a microtubule-stabilizing drug, the cytoplasmic microtubule asters became organized after chemical activation, and the microtubules radiated dynamically toward the female pronucleus. The microtubule patterns correlated well with pronuclear movement to the cell center. Microtubules aggregated at regions of gamma-tubulin concentration, but gamma-tubulin did not localize to a spot until the first interphase of bovine parthenogenesis. These findings indicate that gamma-tubulin is responsible for microtubule organization as the maternal centrosome. In bovine parthenogenesis, the maternal centrosome then organizes cytoplasmic microtubules to move the female pronucleus into the cell center. We propose that the maternal centrosome plays a role as a functional centrosome despite the lack of a sperm contribution, making this structure less competent for microtubule organization in comparison with centrosomes containing sperm centrosomal components.
fertilization, gamete biology, ovum
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INTRODUCTION
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The centrosome, which is the major microtubule-organizing center of animal cells, is important in stabilizing cell movement, establishing cell polarity, and regulating the cell cycle. This structure also plays a key role in fertilization through organization of the interphase cytoskeleton and the mitotic and meiotic spindles (for review, see [1, 2]). A typical centrosome contains a pair of centrioles surrounded by pericentriolar material, a protein complex containing gamma-tubulin (known throughout as 
-tubulin) that is directly involved in microtubule nucleation. The oocytes lose centrioles during oogenesis in humans [3, 4], rhesus monkeys [5], cows [6], and many other mammals except rodents (for review, see [1]), but they retain a stockpile of pericentriolar material. In contrast, the spermatozoa lose most centrosomal proteins but retain centrioles during spermiogenesis. In the zygote, the sperm-derived centrosome matures by acquiring maternally derived pericentriolar material components (for review, see [1, 2]). Thus, during fertilization of these animals, a functional centrosome likely is a composite structure blending both paternal and maternal centrosomal components. The sperm centrosome is required to organize the radial array of microtubules, called the sperm aster, within the inseminated oocyte of nonrodent mammals. Fertilization is only complete when the parental genomes unite; pronuclear migration and apposition require cytoplasmic dynein, a microtubule-based motor protein, and the cofactor dynactin, based on the directionality of female pronuclear movement and the dynamics of its motion along sperm aster microtubules [7–9]. The sperm aster is essential to unite the female with the male pronucleus in most nonrodent mammalian zygotes [6]. In contrast, rodents do not use a sperm aster during fertilization. Mouse spermatozoa lose centrioles during spermiogenesis [10, 11]; hence, the oocytes do not acquire centrioles after fertilization. The maternal centrosomes in this animal remain dispersed as the cytoplasmic microtubule-organizing centers because of the absence of centrioles and form multiple asters during the pronuclear stage [7, 12]. These cytoplasmic asters are attracted to the surfaces of both male and female pronuclei, and they are involved in pronuclear movement [12].
Parthenogenesis is an extraordinary process in which the activated oocyte initiates full development, resulting in a sexually mature adult without a paternal contribution. In nature, parthenogenesis is observed in many insects, crustaceans, rotifers, and reptiles. In mammals, natural parthenogenesis usually is not observed. Artificial parthenogenesis, however, can occur by activating the egg through various physical or chemical stimuli. Mammalian parthenotes serve as ideal models for studying the function of the maternal centrosome. In some nonrodent mammals, such as cows [6, 13], rabbits [14], humans [3], pigs [15], and marsupials [16], parthenotes exhibited disarrayed microtubules in the cytoplasm shortly after artificial activation, suggesting that these mammalian oocytes are able to form a functional centrosome without contributions from the sperm. A role for the maternal centrosome in microtubule organization and pronuclear movement, however, has not been fully elucidated.
The present study was conducted to test the hypothesis that nonrodent mammalian oocytes are capable of forming a functional centrosome, which is required to assemble microtubules to move the female pronucleus in the absence of sperm centrosomal components. Using bovine parthenotes in the present study, we detail the dynamics of microtubules and the distribution of -tubulin, which is assumed to be a major component of the maternal centrosome, during the first interphase of bovine parthenogenesis. Our results also identify an interaction between microtubule organization and female pronuclear movement in bovine parthenotes.
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MATERIALS AND METHODS
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Experimental design and number of oocytes examined are summarized in Figure 1. Experiments on animals were conducted according to the International Guiding Principles for Biomedical Research Involving Animals as promulgated by the Society of the Study of Reproduction.
In Vitro Maturation
Bovine ovaries were obtained after slaughter and transported to the laboratory in normal saline at 20°C. Cumulus-oocyte complexes were collected by aspiration of small follicles (diameter, 2–8 mm) using an 18-gauge needle. Oocytes with a homogenous cytoplasm, surrounded by at least three layers of cumulus cells, were selected. Oocytes matured for 22– 26 h in 80 µl of Medium 199 (M199; Gibco, Grand Island, NY) supplemented with 10% (v/v) fetal calf serum (FCS; Gibco), 50 µg/ml of gentamicin (Sigma, St. Louis, MO), 0.2 mM sodium pyruvate (Sigma), 0.12 IU/ml of FSH (Antrin; Denka Pharmaceutical, Kanagawa, Japan), and 50 ng/ml of recombinant human epidermal growth factor (Upstate, Lake Placid, NY) at 38.5°C in an atmosphere of 5% CO2 under silicone oil.
Process of Parthenogenetic Activation
Cumulus cells were separated from oocytes by a brief incubation in 2 mg/ml of hyaluronidase (Sigma) and 1 mg/ml of collagenase (Sigma) in M2 culture medium (Sigma). We then selected mature oocytes that had arrested at the second meiotic metaphase (metaphase II). A number of mature oocytes were used for the following experiments without activation. The remaining mature oocytes were activated using the method originally reported by Susko-Parrish et al. [17 and 18, 19] with the following modifications: Oocytes were exposed to 5 µM ionomycin (Sigma) for 5 min, then incubated in M199 with 10% FCS for 3 h. Next, drops of 1.9 mM 6-dimethylaminopurine (Sigma) were added for an additional 3-h incubation at 38.5°C in an atmosphere of 5% CO2 under silicone oil. The entire activation process required approximately 6 h in total. Oocytes activated by this method extruded a second polar body and, thereby, were haploid. Activated oocytes were incubated in M199 with 10% FCS until the indicated time points and then used for the following experiments.
Paclitaxel Treatment
Before zona removal, a portion of the activated oocytes were treated for 1 h with 2 µM paclitaxel (Taxol; Bristol-Myers, New York, NY) in M199 with 10% FCS. The remaining oocytes were incubated in paclitaxel-free medium (M199 plus 10% FCS) alone for 1 h. These oocytes were fixed at 2, 5, and 7 h after activation. Unactivated oocytes arrested at metaphase II also were incubated in the presence or absence of paclitaxel for 1 h before zona removal. Zonae pellucidae were removed from all oocytes by a short incubation in 5 mg/ml of pronase (Actinase-E; Kaken, Tokyo, Japan). After two washes followed by a 30-min recovery period, the zona-free oocytes were fixed as described below.
Immunocytochemistry and Conventional Fluorescence Microscopy
Microtubules, DNA, and -tubulin were stained by immunocytochemistry and examined by conventional fluorescence microscopy. Oocytes, attached to poly-L-lysine-coated coverslips in 10 mM PBS lacking Ca2+ and Mg2+, were extracted for 15 min in modified buffer M (25% [v/v] glycerol, 50 mM KCl, 0.5 mM MgCl2, 0.1 mM EDTA, 50 mM imidazole hydrochloride, 1 mM 2-mercaptoethanol, and 25 mM phenylmethylsulfonyl fluoride; pH 6.7) containing 5% (v/v) methanol and 3% (v/v) Triton X-100 at 37°C. Samples were then fixed in cold methanol for 10 min as described by Simerly and Schatten [20]. Alternatively, samples were fixed in 2% formaldehyde for 40 min at 37°C. Next, samples were permeabilized overnight in 10 mM PBS containing 0.1% (v/v) Triton X 100. After fixation and permeabilization, nonspecific antibody binding was blocked by incubation of samples in 10 mM PBS with 30 mg/ml of BSA (Sigma) and 0.1% (v/v) FCS for 1 h before incubation with primary and secondary antibodies. Primary antibodies were applied to the samples for 12 h. Samples were washed twice for 10 min each wash following addition of primary and secondary antibodies. Secondary antibodies conjugated to fluorescein isothiocyanate (FITC), tetramethyl rhodamine isothiocyanate (TRITC), or AlexaFluor-488 were applied to the samples for 1 h. The DNA was detected by labeling with 10 mg/ml of Hoechst 33342 (Dojindo, Kumamoto, Japan) for 20 min. Coverslips were mounted on glass microslides in antifade medium (Vectashield; Vector Laboratories, Burlingame, CA) to retard photobleaching, then examined using conventional epifluorescence microscopy (Optiphot-2; Nikon, Tokyo, Japan). Images were recorded digitally and archived on magnetic optical disks processed using Adobe Photoshop software (Adobe Systems, Mountain View, CA).
Antibodies
Microtubules were labeled with a mixture of monoclonal antibodies specific for ß-tubulin (clone 2-28-33, diluted 1:100; Sigma) and acetylated 
-tubulin (clone 6-11-B1, diluted 1:100; Sigma) or with an antitubulin polyclonal antibody (ATN02, diluted 1:200; Cytoskeleton, Denver, CO). A polyclonal antibody against -tubulin (diluted 1:100; Convance, Berkeley, CA) also was used. Primary antibodies were detected using FITC- or TRITC-conjugated secondary antibodies (each diluted 1:40; Zymed, San Francisco, CA) or an AlexaFluor-488-conjugated secondary antibody (diluted 1:200; Molecular Probes, Eugene, OR). Preimmune mouse IgG1 antibody (diluted 1:20; Chemicon, Temecula, CA) or rabbit IgG (diluted 1:100; Santa Cruz Biotechnology, Santa Cruz, CA) was used for control experiments.
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RESULTS
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A total of 1626 metaphase II-arrested oocytes were collected for the present study, 174 of which were examined without artificial activation. The remaining majority received artificial activation. Of the 77.4% (1124 of 1452) that extruded a second polar body, 596 were examined, excluding the oocytes that divided and fragmented. Twenty of 174 oocytes without artificial activation and 60 of 596 activated oocytes were used as controls.
Microtubule Patterns and Pronuclear Position
Seventy of 174 metaphase II-arrested oocytes did not receive paclitaxel treatment, and the rest were treated with paclitaxel. In metaphase II-arrested oocytes that did not receive artificial activation or paclitaxel treatment, microtubules were visualized in meiotic spindles, with little detectable in the cytoplasm (Fig. 2A1). The meiotic spindle was small, barrel-shaped, anastral, and tangential to the oocyte surface, with the axis in a parallel direction (Fig. 2A4). The chromosomes remained aligned on the metaphase plate (Fig. 2A2). In the metaphase II-arrested oocytes treated with paclitaxel, the meiotic spindle was enlarged and deformed (Fig. 2B4), and the chromosomes were dispersed. The chromosomes did not align on the spindle equator in any of the oocytes examined (Fig. 2B2). When mouse IgG1 was applied as primary antibody instead of anti--tubulin and anti-ß-tubulin antibodies, microtubules were not detected in metaphase II-arrested oocytes (Fig. 2, C and D).
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FIG. 2. Conventional epifluorescence microscopic images of microtubules (A1 and B1), DNA (A2 and B2), and -tubulin (A3 and B3) in bovine oocytes arrested at metaphase II in the absence (A) or presence (B) of paclitaxel. Microtubules were observed in the meiotic spindles, whereas little was detected in the cytoplasm (A1 and B1). The meiotic spindle of bovine oocytes was small, barrel-shaped, anastral, and tangent to the oocyte surface, with the axis on the parallel (A4) and the chromosomes aligned along the metaphase plate (A2). In oocytes treated with paclitaxel, the meiotic spindle was enlarged and deformed (B4), and the dispersed chromosomes were not aligned on the spindle equator (B2). -Tubulin was observed in the meiotic spindle, with little detected in the cytoplasm (A3). When oocytes were treated with paclitaxel, -tubulin became concentrated in the meiotic spindles, which were enlarged and deformed (B3). When mouse IgG1 was applied as primary antibody instead of anti--tubulin and anti-ß-tubulin antibodies, microtubules were not detected in metaphase II-arrested oocytes (C and D). Bar = 15 µm
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Parthenogenetically activated oocytes formed a mesh-like network of microtubules throughout the cytoplasm during the first interphase in the absence of paclitaxel treatment (Fig. 3, A1, B1, and C1). When mouse IgG1 was applied to the oocytes as primary antibody, microtubules were not stained in parthenotes without paclitaxel treatment (Fig. 3, D–F). By 2 h after activation, the majority of oocytes formed a female pronucleus, regardless of paclitaxel treatment (192 of 194 [99%]). We then measured the approximate distances between the female pronuclear edges and the cell center (Table 1). Two hours after activation, most of the untreated parthenotes exhibited female pronuclei at distances greater than 15 µm from the cell center (63 of 94 [67%]), whereas most parthenotes displayed distances of 15 µm or less from the cell center at 7 h after activation (38 of 60 [63%]). The amount of pronuclear centering correlated well with the length of incubation after activation.
When oocytes received paclitaxel treatment after artificial activation, microtubules assembled in one of the following configurations (Table 2): 1) Microtubule asters appeared in the cytoplasm (Fig. 4A1); 2) a microtubule network was observed in regions of the cytoplasm, with connecting microtubules extending from each focus (Fig. 4B1); 3) a network of dense microtubules radiated from each focus to surround the female pronucleus throughout the cytoplasm (Fig. 4C1); or 4) a mesh-like network of microtubules was observed similar to that seen when oocytes remained untreated with paclitaxel (Fig. 4G). Table 1 explains that the frequency of each microtubule pattern observed in bovine parthenotes with paclitaxel treatment differs at each time point after activation. These microtubule patterns, with the exception of the mesh-like-network pattern, correlated well with the length of incubation after activation. Microtubule asters were observed most frequently in oocytes at 2 h after activation (44 of 98 [45%]) (Fig. 4A1), whereas a network of microtubules radiating from foci throughout the cytoplasm was observed at 7 h after activation (19 of 55 [35%]) (Fig. 4C1). Most paclitaxel-treated oocytes at 5 h after activation exhibited a microtubule network in which the connecting microtubules radiated from each focus toward the pronucleus in the partial cytoplasm (26 of 49 [53%]) (Fig. 4B1). In paclitaxel-treated oocytes at 2 h after activation, one to three large microtubule asters formed in the cortical region of the cytoplasm, not around the pronucleus. Sometimes, numerous cytoplasmic asters, similar to those detected in fertilized rodent oocytes, were observed. The application of mouse IgG1 as primary antibody instead of anti--tubulin and anti-ß-tubulin antibodies resulted in a loss of detection in parthenotes that received paclitaxel treatment (Fig. 4, D–F).
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FIG. 4. Microtubule organization (A–C1 and G), female pronucleus migration (A–C2), and -tubulin distribution (A–C3) in bovine parthenotes treated with paclitaxel at 2 h (A), 5 h (B), and 7 h (C) after activation. At 2 h after activation, paclitaxel-treated oocytes each exhibited one to three large microtubule asters (A1). One to three large microtubule asters formed in the cortical region of the cytoplasm, not around the pronucleus. At 5 h after activation, paclitaxel-treated oocytes contained a network of connecting microtubules radiating from each focus toward the pronucleus in the partial cytoplasm (B1). In oocytes at 7 h after activation, paclitaxel-treated oocytes possessed microtubule foci aggregated around the female pronucleus, with microtubules radially spreading from each focus to fill the entire cytoplasm (C1). Sometimes, a mesh-like network of microtubules was observed similar to that seen when oocytes remained untreated with paclitaxel at each time point (G). When oocytes were treated with paclitaxel after activation, the female pronucleus is initially observed in the cytocortical region (A2), subsequently moving to a more central position (C2). When oocytes are treated with paclitaxel, the majority of -tubulin was observed to colocalize with foci of microtubules. In paclitaxel-treated oocytes at 2 h after activation, -tubulin was primarily observed around microtubule foci, but it did not localize into a discrete spot (A3). In paclitaxel-treated oocytes at 5 h after activation, -tubulin staining correlated with aggregated microtubules, especially around microtubule networks (B3). At 7 h after activation, -tubulin was abundant throughout the cytoplasm, with increased amounts of -tubulin concentrated around the female pronucleus (C3). The application of mouse IgG1 as primary antibody instead of anti--tubulin and anti-ß-tubulin antibodies resulted in a loss of detection in bovine parthenotes received paclitaxel treatment (D–F). Bar = 15 µm
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The amount of pronuclear centering also correlated well with the length of incubation after activation when oocytes received paclitaxel treatment (Table 1). In paclitaxel-treated parthenotes at 2 h after activation, the rate at which the female pronucleus presented in the cell center was significantly lower than those at 5 and 7 h after activation (0.01 < P < 0.05 and P < 0.01, respectively). Paclitaxel does not significantly inhibit female pronuclear movement to the cell center, but paclitaxel treatment decreases the percentage of female pronuclei in a central position (Table 1). Table 3 shows that the frequency of pronuclear centering interacts with microtubule patterns formed after activation when oocytes received paclitaxel treatment, regardless of the interval to the fixation after activation. In oocytes forming cytoplasmic asters, the rate at which the female pronucleus presented in the cell center was significantly lower than that of oocytes forming another microtubule pattern. In contrast, the rate of female pronuclear centering was significantly higher when microtubules organized into a network radiating throughout the entire cytoplasm. These microtubule patterns, with the exception of the mesh-like network pattern, correlated well with the amount of pronuclear centering.
Localization of -Tubulin
In metaphase II-arrested oocytes, -tubulin, as seen for microtubules, was observed within the meiotic spindle, with little remaining in the cytoplasm (Fig. 2A3). Following treatment with paclitaxel, -tubulin concentrated in meiotic spindles, which were enlarged and deformed (Fig. 2B3).
In activated, untreated oocytes, -tubulin was detected throughout the cytoplasm in a distribution similar to that of microtubules. At 2 h after activation, increased -tubulin was found in cortical regions of the cytoplasm (Fig. 3A3). In oocytes at 5 and 7 h after activation, however, a larger proportion of cellular -tubulin was observed in the central region of the cell (Fig. 3, B3 and C3). After treatment with paclitaxel, the majority of the -tubulin in oocytes colocalized with concentrated microtubules. Two hours after activation of paclitaxel-treated oocytes, -tubulin was converged with microtubule foci, but it did not localize to clear spots (Fig. 4A3). In paclitaxel-treated oocytes at 5 h after activation, -tubulin stained microtubule aggregates, bordering on networks of microtubules (Fig. 4B3). -Tubulin was abundant in the cytoplasm at 7 h after activation, with increased amounts of -tubulin concentrated around the female pronucleus (Fig. 4C3). When rabbit IgG was applied to the oocytes as primary antibody instead of anti--tubulin antibody, -tubulin was not detected in the bovine oocytes, regardless of the artificial activation and of paclitaxel treatment (Figs. 2, C and D; 3, D–F; and 4, D–F).
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DISCUSSION
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Our results support the hypothesis that bovine oocytes can form a functional centrosome that is responsible for female pronuclear movement. After paclitaxel treatment, maternal centrosomes organized cytoplasmic microtubule asters in activated bovine oocytes at the pronuclear stage. In addition, we demonstrated using paclitaxel that cytoplasmic microtubule formation in bovine parthenotes changed with the lapse of the first interphase. Microtubule dynamics correlated with female pronuclear migration and centration. Finally, cytoplasmic microtubules aggregated at the site of -tubulin accumulation in the first interphase of bovine parthenogenesis.
After artificial activation, we observed a mesh-like network of disarrayed microtubules in the cytoplasm of bovine oocytes at the pronuclear stage, with no formation of a cytoplasmic aster in the absence of paclitaxel treatment. These results concur with those reported by Navara et al. [6] and Shin et al. [13]. When treated with paclitaxel immediately after activation, bovine oocytes exhibited microtubule asters in the cytoplasm. Paclitaxel binds to microtubule polymers, enhancing tubulin polymerization [21, 22], stabilizing preformed microtubules, and suppressing tubulin depolymerization (for review, see [23]). These established actions could therefore account for the formation of cytoplasmic asters in paclitaxel-treated activated oocytes. Whereas mammalian oocytes contain the maternal centrosome capable of organizing microtubules in the cytoplasm [6, 15], this structure does not appear to be highly active in microtubule nucleation. Thus, cytoplasmic asters are not observed in the cytoplasm of bovine parthenotes. By promoting assembly and blocking disassembly of microtubules, the combination of paclitaxel and maternal centrosomes may induce the appearance of cytoplasmic asters.
Kim et al. [24, 25] reported that numerous microtubule foci formed in bovine oocytes after germinal vesicle breakdown following treatment with paclitaxel during oocyte maturation in vitro. We did not observe the appearance of paclitaxel-induced cytoplasmic asters in bovine oocytes arrested at metaphase II, suggesting that maternal centrosomes do not significantly organize cytoplasmic microtubules when oocytes are arrested at metaphase II. It remains possible that maternal centrosomes function during oocyte maturation, but the artificial activation of oocytes may cause maternal centrosomes to induce cytoplasmic microtubule organization.
We examined microtubule dynamics during the first interphase of bovine parthenogenesis after treatment with paclitaxel. Figure 5 shows a schematic diagram of microtubule dynamics and female pronuclear positioning during the first interphase of bovine parthenogenesis. Several microtubule asters formed in the cortical region of the cytoplasm during the early pronuclear period (Fig. 5A). At this stage, microtubules radiated from each focus toward the female pronucleus, connecting through a microtubule network (Fig. 5B). At the late pronuclear stage, microtubules extended from each focus to fill the cytoplasm, aggregating around the female pronucleus (Fig. 5C). The formation of a mesh-like network of disarrayed microtubules similar to that seen in untreated oocytes (Fig. 5D), but not the generation of microtubule asters, was observed in 21% of parthenotes at 2 h after activation. This result suggests that the ability of maternal centrosomes to organize microtubules differs from cell to cell. The centrosomal potential to nucleate microtubules might be responsible for the developmental fate of each parthenote.
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FIG. 5. Schematic diagram of microtubule dynamics and female pronuclear positioning during the first interphase of bovine parthenogenesis. Pronuclear movement from the cortical region to the cell center occurred during the first interphase of bovine parthenogenesis. The interaction between the pattern of microtubule assembly and pronuclear centration indicates that microtubule assembly is responsible for movement of the female pronucleus in bovine parthenogenesis. Several microtubule asters formed in the cortical region of the cytoplasm during the early pronuclear period (A). Then, microtubules radiated from each focus toward the female pronucleus, connecting through a microtubule network (B). At the late pronuclear stage, microtubules extended from each focus to fill the cytoplasm, aggregating around the female pronucleus (C). Low ability of maternal centrosomes to organize microtubules may result in the formation of a mesh-like network of disarrayed microtubules similar to that seen in untreated oocytes (D). Maternal centrosomes, which mainly consist of -tubulin, may move from the cytocortical region toward the female pronucleus; microtubules, radiating from each focus toward the pronucleus and cell surface, move the pronucleus to the cell center to initiate the first mitosis of the bovine parthenote
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In bovine parthenogenesis, the dynamics of microtubules following paclitaxel treatment is similar to the microtubule dynamics observed during mouse oocyte fertilization, except that no contribution is made by the sperm. Unfertilized mouse oocytes have multiple centrosomal foci. After incorporation of sperm, the microtubules radiate from each focus to surround the male and female pronuclei, eventually moving them to the cell center during fertilization in this animal [7, 12]. In nonrodent mammals, including cows, introduction of sperm centrosomal components into oocytes are required to reconstruct the complete centrosome. Zygotes form the sperm aster, which extends radially from the centrosome attached to the male pronucleus toward the female pronucleus. Astral microtubules pull the female pronucleus to the male pronucleus through the action of dynein-dynactin complexes [1, 8, 9].
Pronuclear movement from the cortical region to the cell center occurred in the first interphase of bovine parthenogenesis. The interaction between the pattern of microtubule assembly and pronuclear centration leads us to believe that microtubule assembly is responsible for movement of the female pronucleus in bovine parthenogenesis. This correlation also suggests that microtubule foci move from the cytocortical region toward the female pronucleus; microtubules, radiating from each focus toward the pronucleus and cell surface, move the pronucleus to the cell center to initiate the first mitosis of the bovine parthenote (Fig. 5).
During the fertilization of many animals, including sea urchins [26], rabbits [27], and cows [6], pronuclear migration and positioning involves the organization of microtubules by the centrosome. In porcine fertilization, pronuclear movement and apposition do not proceed when microtubule assembly is inhibited [28]. Whereas treatment of activated oocytes for 1 h with paclitaxel decreased the rate of female pronuclear centration, the differences between these cells and untreated oocytes were not statistically significant. During fertilization, pronuclear migration and centration require regulated microtubule growth and shrinkage from the centrosome mediated by tubulin assembly and disassembly. Paclitaxel, by inhibiting these processes, inhibits pronuclear movement [26]. Reduction of the rate of female pronuclear centering in paclitaxel-treated oocytes is consistent with the concept that movement of the female pronucleus in parthenogenesis requires microtubule dynamic instability, as is required during fertilization.
The colocalization of cytoplasmic microtubules with -tubulin suggests that -tubulin nucleates cytoplasmic microtubules during the first interphase of bovine parthenogenesis. -Tubulin was observed throughout the cytoplasm of bovine parthenotes in the absence of paclitaxel treatment, but increasing amounts of -tubulin were found in cortical regions in the early stage of interphase and surrounding the female pronucleus at later stages. These findings agree with the observations that microtubule foci move from the cell cortex to the center in paclitaxel-treated parthenotes. In the present study, -tubulin did not localize to this region during the first interphase of bovine parthenogenesis. Shin et al. [13] reported that a -tubulin focus was observed in parthenogenetically activated bovine oocytes at the 8-cell stage. Those authors concluded that a functional centrosome, which could retain the ability to duplicate and organize a microtubule aster, may organize during the third cell cycle of bovine parthenogenesis by de novo synthesis. We propose that a maternal centrosome, which consists of centrosomal material, including -tubulin, assembles microtubules and, with paclitaxel, organizes microtubule asters that are capable of moving the female pronucleus and forming the mitotic spindle. The maternal centrosome alone, however, is incomplete as a functional centrosome, because it lacks the ability to replicate itself or form a spindle pole.
In conclusion, the present results support the theory that the maternal centrosome has the ability to fulfill a role as a functional centrosome despite impairment in the nucleation of microtubules and a lack of ability to self-replicate or form spindle poles without sperm centrosomal contributions. After activation, the maternal centrosomes of bovine oocytes have the ability to organize cytoplasmic microtubule asters using paclitaxel. In bovine parthenogenesis, microtubules dynamically assemble and disassemble, moving the female pronucleus to the cell center during the interphase before the first mitosis. The present study also indicates that -tubulin is the major component of the maternal centrosome organizing microtubules to function in the migration and centration of the female pronucleus. Movement of the female pronucleus in parthenogenesis requires maternal centrosomes and microtubule dynamics, as required during fertilization.
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ACKNOWLEDGMENTS
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We thank Dr. Gerald Schatten (Pittsburgh Development Center, University of Pittsburgh School of Medicine, Pittsburgh, PA) for helpful comments and encouragement. We also appreciate the advice and expertise of Dr. Christopher Payne (Pittsburgh Development Center, University of Pittsburgh School of Medicine) and Dr. Toshitaka Horiuchi (Hiroshima Prefectural University, Hiroshima, Japan).
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FOOTNOTES
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1 Correspondence: Yukihiro Terada, Department of Obstetrics and Gynecology, Tohoku University School of Medicine, 1-1, Seiryo-cho, Aoba-ku, Sendai, Miyagi 980-8574, Japan. FAX: 81 22 717 7258; terada{at}mail.tains.tohoku.ac.jp 
Received: 30 March 2005.
First decision: 24 April 2005.
Accepted: 29 June 2005.
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ABSTRACT
INTRODUCTION
