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Unequal cell division regulated by the contents of germinal vesicles
Developmental Biology 273 (2004) 76 – 86 www.elsevier.com/locate/ydbio
Unequal cell division regulated by the contents of germinal vesicles Ri-ko Matsuura a,b and Kazuyoshi Chiba a,b,* a
b
Department of Biology, Ochanomizu University, 2-1-1 Ohtsuka, Bunkyo, Tokyo 112-8610, Japan Tateyama Marine Laboratory, Ochanomizu University, 11 Kou-yatsu, Tateyama, Chiba 294-0301, Japan Received for publication 1 December 2003, revised 23 March 2004, accepted 29 April 2004 Available online 2 July 2004
Abstract Fertilization occurs during meiosis in many animals, when maternal centrosomes participate in the formation of spindles at the animal pole, which results in polar body formation. Paternal centrosomes do not participate in cell division during oocyte maturation. After meiosis, they form the spindles while the maternal centrosomes are discarded. It is unknown why paternal centrosomes do not form spindles during meiosis. Here, we show that the artificial incorporation of sperm at the animal pole of immature starfish oocytes causes unequal cell division and the formation of polar body-like fragments. The removal of germinal vesicles from the animal pole blocks the formation of polar bodylike fragments. Furthermore, translocation of germinal vesicles to the vegetal pole by centrifugation induces the extrusion of polar body-like fragments from the vegetal pole, where sperm penetration is prerequisite. After germinal vesicle breakdown, cyclin B is localized in the maternal and paternal asters and spindles near the germinal vesicle. These results suggest that germinal vesicle components such as the cdc2 – cyclin B complex interact with asters and spindles and can induce unequal cell division. During normal fertilization, paternal centrosomes are likely kept away from the germinal vesicle components, resulting in the inhibition of unequal paternal centrosome-dependent cell division. D 2004 Elsevier Inc. All rights reserved. Keywords: Polar body; Starfish; Maturation; Germinal vesicle; Centrosome; Fertilization; Cyclin B
Introduction The localization of the mitotic apparatus in polarized cells results in unequal cell division, which plays an important role during development and differentiation. Polar body formation is one of the most remarkable examples of unequal division. To produce haploid eggs without wasting stored cell components that will be used for further development, maturing oocytes release tiny polar bodies containing excessive chromosomes from the animal pole. In immature oocytes, a large nucleus called the germinal vesicle (GV) localizes at the animal pole. Translocation or removal of the GV by centrifugation results in the failure of a polar body to form. However, centrosomes, which are an important component of the mitotic apparatus or spindles, remain at the animal pole (Barakat et al., 1994; Picard et al., 1988). These results suggest that GV components may affect centrosomes or asters at the animal pole after GV breakdown * Corresponding author. Department of Biology, Ochanomizu University, 2-1-1 Ohtsuka, Bunkyo, Tokyo 112-8610, Japan. Fax: +81-3-59785370. E-mail address: [email protected] (K. Chiba). 0012-1606/$ - see front matter D 2004 Elsevier Inc. All rights reserved. doi:10.1016/j.ydbio.2004.04.038
(GVBD) and cause spindle formation and the extrusion of polar bodies. Once the spindle has been made after GVBD in the intact oocyte, the ectopic polar body is formed from the cortical region wherever the spindle containing centrosomes has been translocated (Hamaguchi, 2001; Shirai and Kanatani, 1980). Thus, GV components may irreversibly change centrosomes or asters after GVBD, resulting in spindle formation and the extrusion of polar bodies. It is unclear whether GV components can induce spindle formation or if components present at the animal pole other than GV components are also required. In starfish oocytes, GVBD is induced by 1-methyladenine (1-MA) (Kanatani et al., 1969). After GVBD, arrested oocytes at MI in the ovary are spawned (Harada et al., 2003; Oita et al., 2004) and proceed through the two meiotic cell divisions to form two polar bodies. In addition to causing GVBD, 1-MA enables oocytes to undergo normal monospermic fertilization (Chiba et al., 1990; Fujimori and Hirai, 1979). While paternal centrosomes participate in cleavage at the end of meiosis, they do not form functional mitotic apparatus during meiosis. Although the reason why sperm centrosomes do not function during polar body formation is unknown, the sperm penetration site may be different from
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the animal pole, where maternal centrosomes are presumably affected by GV components. In other words, it is possible that normal fertilization during meiosis occurs apart from the animal pole, resulting in the separation of paternal centrosomes from the GV components. If true, the artificial incorporation of sperm into the animal pole forces paternal centrosomes to interact with GV components, resulting in the formation of functional sperm centrosomes during meiosis. Thus, in this study, we tried to answer the following questions to determine whether the above hypothesis is true: 1) Do sperm (or paternal centrosomes) which penetrate the animal pole induce unequal cell division? 2) Do GV components translocated into the vegetal pole cause unequal cell division at the vegetal pole? Sperm can randomly penetrate immature starfish oocytes resulting in polyspermy when inseminated before 1-MA treatment (Chambers, 1923; Chiba and Hoshi, 1989; Clark, 1936; Longo et al., 1995; Schuetz and Longo, 1981). To answer our first question, we inseminated immature oocytes and obtained sperm-penetrating oocytes at the animal pole. Answering the second question was useful for determining whether GV components are sufficient for the induction of unequal cell division or polar body formation.
Materials and methods Materials Cytochalasin B (Sigma Chemical Co., St. Louis, MO) was dissolved in dimethyl sulfoxide (DMSO) at a concentration of 5 mg/ml, and stored at 20jC. Unfertilized and polyspermic oocytes were treated with 10 AM cytochalasin B 30 min after 1-MA addition. To enlarge polar bodies, unfertilized and polyspermic oocytes were transferred into artificial seawater (ASW) containing 2% hexylene glycol (2-methyl-2,4-pentanediol, Wako) 40 min after 1-MA addition (Saiki and Hamaguchi, 1997).
Fig. 1. PBF formation from polyspermic starfish oocytes. Morphological changes in oocytes during maturation were observed with a light microscope equipped with Nomarski differential interference contrast optics. (a – d) Unfertilized oocyte. (a) Immature unfertilized oocyte. (b) GVBD occurred 30 min after 1-MA treatment. (c) The first polar body was formed 75 min after 1-MA treatment. (d) The second polar body was formed approximately 110 min after 1-MA treatment. (e – h) Polyspermic oocytes. (e) Immature polyspermic oocytes. (f) GVBD occurred 30 min after 1-MA treatment. (g, gV) Two to three PBFs were formed at the animal pole 75 min after 1-MA treatment. (h, hV) Five more PBFs were formed about 110 min after 1-MA treatment. (gV, hV) Optical zoom at the animal pole. Arrowheads show PBFs. The following sets in parentheses show the photographs from the same oocytes: (a, b), (c, d), (e, f), (g, h), (gV, hV). Representative figures are shown. The bar represents 50 Am.
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Animal maintenance, gamete collection, and culture Starfish Asterina pectinifera were collected on the Pacific coast of Honsyu Island, Japan, and kept in laboratory aquaria supplied with circulating seawater at
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15 – 17jC. To remove follicle cells, isolated ovaries were incubated in ice-cold Ca2+-free seawater (CFSW), and released oocytes were washed twice with CFSW. Defolliculated oocytes were stored in ASW at 20jC. Oocyte maturation was induced by the addition of 1 AM 1-MA. Sperm were obtained ‘‘dry’’ by cutting the testes and keeping them on ice. Just before fertilization, sperm were diluted in ASW. Preparation of polyspermic oocytes Fig. 2. Effects of the number of penetrated sperm on PBF formation. Immature oocytes were treated with ‘‘dry’’ sperm diluted at the indicated ratio. After removal of attached sperm using protease, fertilized oocytes were treated with 1-MA. Then, the number of PBFs were counted under the microscope at 80 min (1st PBF) and 120 min (2nd PBF) after 1-MA addition corresponding to the timing of the first and second polar body formation in unfertilized oocytes, respectively. The number of sperm that entered an oocyte was counted using DAPI staining. Each bar or point represents the average of results from 20 eggs.
Defolliculated oocytes were inseminated for 10 min. To remove adhesive sperm, oocytes were treated with 0.02 % actinase-E (Kaken Pharmaceutical, Tokyo, Japan) in ASW for 5 min (Nemoto et al., 1980). Then, they were washed 5 times with CFSW to interrupt the actinase reaction. Oocytes were incubated in ASW at 20jC for 30 min.
Fig. 3. Hoechst staining of polar bodies and PBFs. After 1-MA treatment, unfertilized and polyspermic oocytes were stained with Hoechst. Morphological changes of living oocytes were visualized by Nomarski differential interference contrast optics (a, c, e, g, i, k, m) and fluorescent (b, d, f, h, j, l, n) microscopy. (a – f) Unfertilized oocytes. (a, b) Metaphase oocyte at 77 min after 1-MA addition. (c, d) The first polar body was formed 86 min after 1-MA addition. (e, f) The second polar body was formed 126 min after 1-MA addition. (g – n) Polyspermic oocytes. (g, h) Metaphase oocyte at 77 min after 1-MA addition. (i, j) PBFs having negative Hoechst staining were formed 86 min after 1-MA addition. (k, l) PBFs having negative Hoechst staining were formed 126 min after 1-MA addition. (m, n) One of six PBFs stained with Hoechst was extruded 126 min after 1-MA addition. (insets) An optical zoom at the animal pole. Each photo shows the representative figure of different oocytes. The bar represents 50 Am.
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Micromanipulation A micropipette was made following the procedure of Hiramoto (1974). The tapered portion near the tip of the micropipette was broken at a point where it had a diameter of about 10 Am. A micromanipulator (MO-388; NARISHIGE, Tokyo, Japan) and a microinjector (IM-9B, NARISHIGE, Tokyo, Japan) were used for the surgical manipulation. After inserting a micropipette into the unfertilized and fertilized GV-intact oocytes in injection chambers (Chiba et al., 1992), whole or partial GV components were aspirated (Miyazaki et al., 2000). Before and after aspiration, the diameter of the GV was measured to calculate the volume of the GV components. Visualization of chromosomes in living oocytes GVBD in starfish oocytes was induced by the addition of 1 AM 1-MA. Then, maturing oocytes were incubated with 10 AM Hoechst (bis-Benzimide H 33342, Sigma) in ASW for 30 min, and washed twice with ASW. They were observed using fluorescence microscopy. Immunofluorescence staining of microtubules and cyclin B Immunostaining was performed by a modification of the procedure of Miyazaki et al. (2000) and Iwao et al. (2002). Oocytes were treated with extraction buffer containing detergent (Shirai et al., 1990) and fixed with 100% methanol for 1 hour at 20jC. After fixation, they were transferred to PBS-T (phosphate-buffered saline/0.05% Tween 20) and left
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to stand for 5 min. They were then incubated with a mouse monoclonal antibody against a-tubulin (Amersham Corp., Buckinghamshire, England) for 50 min, washed with PBST, then stained with a FITC-conjugated goat anti-mouse IgG antibody (Tago, Burlingame, CA) for 40 min. After rinsing with PBS-T, they were incubated with anti-starfish cyclin B rabbit polyclonal antibody for 60 min, washed with PBS-T, then stained with a RITC-conjugated anti-rabbit IgG antibody (Jackson Immunoresearch Laboratories, Inc) for 40 min. DNA was stained with DAPI (Sigma) for 5 min, and washed with PBS-T. Centrifugation of oocytes Unfertilized oocytes were set in the injection chamber. To mark the animal pole, oocytes were sprayed with 1% Nile Blue (E. Merck, Darmstadt, Germany) – ASW (Shirai and Kanatani, 1980) using a micropipette (see also Fig. 8A, a). After local staining, oocytes were positioned with a glass needle so that their GV was located at the bottom of the chamber (see Fig. 8A, b). The chamber was inserted into an agar-tube containing 10% agar –ASW, and centrifuged at 500 g for 40 min (see Fig. 8A, c and d). Then, oocytes were inseminated and treated with 1-MA. Observation Living oocytes were observed with a Nomarski differential interference contrast or polarization contrast microscope, and images were taken by a digital camera (COOL PIX-4500, Nikon, Tokyo, Japan). Microtubules, cyclin B, and DNA
Fig. 4. Anti-a-tubulin antibody staining. At the time of the first polar body formation (75 min after 1-MA treatment), unfertilized and polyspermic oocytes were extracted with detergent, fixed with ice-cold methanol, and then stained with anti-a-tubulin antibody and DAPI to visualize tubulin (green) and chromosomes (blue). Images of tubulin and DNA were observed with a fluorescence microscope. (a) Unfertilized oocyte; (b) polyspermic oocytes; (insets) an optical zoom at the animal pole. Arrowheads indicate polar body or PBFs. The bar represents 50 Am.
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were observed with fluorescence microscopy (Zeiss, Axioskop), and images were taken by a video-enhanced microscopy, which was carried out using a video camera (C2400-08) and an image processor ARGUS-50 control system (Hamamatsu Photonics K.K.). Fluorescent intensity was measured using Image Gauge 3.3 graphic system (Fuji Photo Film, Tokyo, Japan).
Results Numerous polar bodies released from polyspermic oocytes As shown in Figs. 1a – d, immature starfish oocytes underwent GVBD after 1-MA addition and released the first and the second polar body, respectively. Because cell division of the first polar body after extrusion does not occur in starfish, the total number of the polar bodies from one oocyte was two through meiotic cell division (Fig. 1d). In a polyspermic oocyte, however, more than two polar bodies were released at the same time as the first polar body formation in unfertilized oocytes (Figs. 1g and gV). Moreover, when second polar body formation occurred in the control unfertilized oocytes (Fig. 1d), the polyspermic oocytes additionally released many polar bodies (Figs. 1h and hV). To investigate the effect of the number of sperm penetrating the oocytes on polar body formation, immature oocytes were treated with ‘‘dry’’ sperm which had been diluted from 10 3 to 10 13. As shown in Fig. 2, using 10 4 diluted ‘‘dry’’ sperm, 30 or more sperm nuclei were incorporated into an oocyte, from which over six polar bodies on average were extruded when second polar body formation occurred in the control. The number of polar bodies depended on the number of sperm incorporated into an oocyte, when 10 4 to 10 7 dilution dry sperm was added to immature oocytes. Sperm penetration was not observed by treatment with more diluted sperm (10 8 to 10 13), causing normal two polar bodies formation. These results strongly support the idea that the number of polar bodies depends on the number of sperm penetrating the animal pole. At 10 3 dilution, although oocytes seemed to try to extrude many polar bodies, cleavage of the extruding polar bodies was not sufficient, causing a reduced number of polar bodies. In this study, we called these cleaved fragments extruded from the animal pole of polyspermic oocytes polar bodylike fragments (PBFs). PBF without chromosomes In unfertilized oocytes, polar bodies contained the maternal chromosomes stained with Hoechst (Figs. 3d and f). To determine whether numerous PBFs from polyspermic oocytes contained maternal/paternal chromosome or not, living oocytes were stained with Hoechst and visualized by fluorescence microscopy. As shown in Figs. 3j and l,
Fig. 5. Enlargement of PBFs by hexylene glycol treatment. (A) The large first polar body (a) and the large second polar body (b) were released from hexylene glycol-treated unfertilized oocytes. Large PBFs were extruded by hexylene glycol treatment at the same time as the first polar body formation (c), and additional large PBFs were formed at the same time as the second polar body formation (d). (B) Oocytes treated with hexylene glycole were visualized by polarization contrast microscope optics. (a) Unfertilized oocyte; (b) polyspermic oocyte; (c) schematic representation of (b). (C) At the time of the first polar body formation (80 min after 1-MA treatment), normal fertilized and polyspermic oocytes were extracted with detergent, fixed with ice-cold methanol, and then stained with DAPI (a, d) and antia-tubulin antibody (b, e). Merge of (a) and (b) is shown in (c). Merge of (d) and (e) is shown in (f). Images of DNA (blue) and tubulin (green) were observed with a fluorescence microscope. (a – c) Oocyte was fertilized 40 min, and then added 2% hexylene glycole at 50 min after 1-MA treatment. (d – f) Polyspermic oocyte was added 2% hexylene glycole at 50 min after 1-MA treatment. The bar represents 50 Am.
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Fig. 6. Inhibition of PBF formation by cytochalasin B. After GVBD (30 min after 1-MA treatment), oocytes treated with cytochalasin B (10 AM at final concentration) did not form polar bodies (c, d), or PBFs (g, h). Control oocytes without cytochalasin B formed the first polar body (a) and the second polar body (b). Polyspermic oocytes without cytochalasin B formed PBFs (e, f). Photographs were taken 72 min (a, c, e, g), and 116 min (b, d, f, h) after 1-MA treatment. The bar represents 50 Am.
many PBFs without chromosomes were released. In some oocytes, a few PBFs were stained with Hoechst, while most of them were not stained (Figs. 3m and n). These results indicate that PBF did not always include sperm nuclei and maternal chromosomes. To examine the distribution of asters around the animal pole, polyspermic oocytes were fixed at the same time as PBF formation, followed by double staining with an antia-tubulin antibody and DAPI. As shown in the inset of Fig. 4b, three spots of asters and sperm nuclei were observed near the area of PBF formation, while two spots of asters and maternal chromosomes were stained in the unfertilized oocyte releasing the first polar body (Fig. 4a). Thus, PBF formation may be regulated by asters or spindles originating from sperm centrosomes at the animal pole.
bulin antibody (Fig. 5C, d – f). Instead, the size of asters in areas other than the animal pole were same as those of untreated oocytes (Fig. 4b). These results indicate that only asters and spindles present in the animal pole are sensitive to hexylene glycol. Involvement of actin in PBF release Hamaguchi et al. (2001) and Hiramoto (1982) reported that polar body formation is controlled by a decrease in actin filaments, causing a decrease in the surface force near the centrosome of the spindles or asters. To investigate the role
Effects of hexylene glycol on PBF formation Hexylene glycol enhances polymerization of tubulin in spindles or asters during polar body formation of starfish oocytes, causing extrusion of large polar bodies (Saiki and Hamaguchi, 1997; Fig. 5A, a and b). If PBF formation is also regulated by asters, the size of PBFs may be increased by hexylene glycol treatment. Indeed, as shown in Fig. 5A, c and d, large PBFs were released from polyspermic oocytes treated with hexylene glycol. Also, using polarization contrast microscopy, enlarged spindles were observed at the animal poles of polyspermic oocytes (Fig. 5B, b and c). These results support the hypothesis that PBF formation is regulated by centrosomes donated from sperm. We found that asters and spindles at the animal pole were much bigger than those distributed in other areas when we stained hexylene glycol-treated polyspermic oocytes with anti-tu-
Fig. 7. Effects of the GV components on PBF formation. Before micromanipulation, the diameter of a GV was measured. Then, the contents of a GV were partially aspirated into a micropipette inserted from the vegetal end of an oocyte. The diameter of the remaining GV was measured to calculate its % volume (remaining GV/intact GV). The volume of control oocytes is shown as 100%, which was only inserted by a micropipette without sucking the contents of the GV. The number of PBFs or polar bodies (PB) was counted under microscopy. Each symbol represents a result from one egg.
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Fig. 9. Accumulation of cyclin B in the asters and spindles at the animal pole. Normally fertilized (a – f) and polyspermic (g – l) oocytes were extracted with detergent, fixed with ice-cold methanol, and then stained with DAPI (blue), anti-a-tubulin antibody (green), and anti-cyclin B antibody (red). Images obtained by a fluorescence microscope. (a – c, g – i) Metaphase-I (60 min after 1-MA treatment); (d – f, j – l) anaphase-I (80 min after 1-MA treatment). (a – c) Accumulation of cyclin B can be seen in the meiotic spindle. (g – i) Strong staining of cyclin B can be seen clearly in the sperm-asters of the animal pole. (d – f, j – l) Cyclin B disappeared at anaphase. Representative figures are shown. The bar represents 50 Am.
of actin filaments in PBF formation, oocytes were treated with microfilament inhibitor, cytochalasin B, and then morphological changes were observed. As shown in Fig. 6, polyspermic oocytes treated with cytochalasin B did not extrude PBFs. This result indicates that polymerization or depolymerization of actin is important for PBF formation.
Effect of GV components Picard et al. (1988) showed that polar body formation is blocked when the GV components are completely removed. Similarly as shown in Fig. 7, when the GV components were sucked with a micropipette to make 30% or less GV volume, both the formation of the polar body and PBFs
Fig. 8. Translocation of the GV to the vegetal pole causing ectopic PBF. (A) To label the animal pole, oocytes in the injection chamber were sprayed with Nile Blue (a). Then, Nile Blue-stained oocytes at the animal pole were turned upside down using a glass needle (b). The chamber with stained oocytes was transferred to a centrifugation tube (c) and was centrifuged (d). (B) The GV in the stained animal pole (a) and (f) migrated to the colorless vegetal pole after centrifugation (b and g). Then, centrifuged oocytes were treated with (h) or without sperm (c), and treated with 1-MA (c, h) to induce GVBD. Ectopic PBFs were released from the colorless vegetal pole (i and j), while no polar bodies were released from unfertilized oocytes (d and e). (d and i) 80 min after 1-MA treatment. (e and j) 120 min after 1-MA treatment. The following sets in parentheses show the photographs from the same oocytes; (a – e), (f – j). The bar represents 50 Am.
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were inhibited. Instead, as in control oocytes, several PBF as well as two polar bodies were released when over 80% of the volume of the GV components remained. The number of PBFs and polar bodies were roughly dependent on the amount of GV components having 30– 80% of the original volume. Ectopic PBF formation from the vegetal pole To confirm that only the GV components in the presence of centrosomes are sufficient for the induction of unequal division, experimental translocation of the GV from the animal pole to the other cortical region in immature oocytes may be useful. Because the density of the GV components is relatively low, centrifugation of immature oocyte can shift the GV from the animal pole to the vegetal pole, although centrosomes are strictly fixed at the animal pole in immature oocytes during centrifugation (Barakat et al., 1994; Miyazaki et al., 2000; Shirai and Kanatani, 1980). Indeed, when Nile Blue-stained oocytes at the animal pole (Fig. 8A, a) were turned upside down (Fig. 8A, b) and centrifuged as shown in Fig. 8A, c and d, the GV in the stained animal pole (Fig. 8B, a and f) migrated to the colorless vegetal pole (Fig. 8B, b and g). Centrifuged oocytes were then inseminated and treated with 1-MA to induce GVBD at the vegetal pole (Fig. 8B, h). As expected, ectopic PBFs were released from the colorless vegetal pole, but not from the animal pole (Fig. 8B, i and j). These results suggest that the GV components affect the paternal centrosomes even in the vegetal pole causing unequal cell division. In control oocytes without fertilization, no polar body was released from the stained animal pole without the GV or from the vegetal pole with the GV (Fig. 8B, d and e), as reported by Barakat et al. (1994). Thus, we concluded that the GV components are sufficient for inducing unequal cell division. They may interact with paternal centrosomes or asters causing formation of spindles and ectopic PBFs. Also, formation of the big asters induced by hexylene glycol in the animal pole (Fig. 5C) may be due to the GV components. If this is the case, the GV components may localize in the asters and spindles of the animal pole. Localization of cyclin B in the asters of the animal pole Because the cdc2 – cyclin B complex accumulates in the GV just before GVBD and in spindles during metaphase in meiotic cell division (Ookata et al., 1992, 1993), it may be a candidate for GV components interacting with asters that induces unequal cell division. As shown in Figs. 9a – c, when polyspermic oocytes after GVBD were stained with anti-cyclin B antibody, asters at the animal pole more strongly reacted with the antibody than those in other areas. The average fluorescent intensity of RITC staining (anticyclin B antibody) of an aster of the animal pole was 3.5 F 0.36 (mean F SE, n = 16) times higher than that of areas other than the animal pole, while the intensity of FITC
staining (anti-tubulin antibody) of the aster at the animal pole was almost the same (1.2 F 0.08 [mean F SE, n = 16] times) as that of other areas. After metaphase, cyclin B is destroyed by the proteasome (Harada et al., 2003; Nishiyama et al., 2000; Oita et al., 2004). As expected, asters or spindles staining with anti-cyclin B antibody disappeared during the anaphase in monospermic (Figs. 9d –f) or polyspermic oocytes (Figs. 9j – l), thus confirming specificity of the staining. Although it is unknown whether the cdc2 – cyclin B complex is directly involved in the formation of functional spindles during meiotic cell division, these results support the hypothesis that GV components such as the cdc2 – cyclin complex interact with asters and spindles present near the GV after GVBD. Instead, asters apart from the GV may not receive enough GV components after GVBD, resulting in the failure of a functional mitotic apparatus to form during oocyte maturation. Thus, it is likely that GV components that interact with asters can induce unequal cell division during meiosis.
Discussion In this study using starfish oocytes, we found that sperm incorporated at the animal pole caused unequal cell division making PBFs from the animal pole. The timing of PBF formation was the same as that of polar body formation in control oocytes without fertilization. The shape of PBFs was similar to that of the polar body. PBFs or polar bodies from polyspermic oocytes did not always contain chromosomes, indicating that the extrusion of normal polar bodies was inhibited during PBF formation. Thus, the incorporation of sperm in an area other than the animal pole may be important for the release of normal polar bodies without the formation of PBFs. The PBF release as well as that of polar bodies depended on GV components, because removal of the GV blocked the formation of PBFs or polar bodies. Also, translocation of the GV from the animal pole to the vegetal pole resulted in the formation of ectopic PBFs at the vegetal pole, where incorporation of sperm was prerequisite. These results indicate that unequal division after GVBD is induced by GV components. Because PBFs and polar bodies were released from the region where the GV existed, the factor present among GV components that induces unequal division most likely remains there even after GVBD. The centrosome is the major microtubule-organizing center in animal cells. It contributes to most microtubule-dependent processes, including cell polarity (Etienne-Manneville and Hall, 2003; Yamashita et al., 2003) and cell cycle progression (Hinchcliffe et al., 2001). Because polar body formation depends on maternal centrosomes (Uetake et al., 2002; Washitani-Nemoto et al., 1994), it is likely that PBF formation is also regulated by centrosomes donated from sperm. Previous studies showed that maternal centrosomes at the animal pole cannot form polar bodies if the GV is
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translocated to the vegetal pole before GVBD (Picard et al., 1988; Shirai and Kanatani, 1980). In this study, we showed that penetrated sperm with paternal centrosomes induced the formation of ectopic PBFs at the vegetal pole, where the translocation of GVBD occurred. Thus, maternal and paternal centrosomes may be able to induce the formation of functional spindles during meiosis only if affected by a factor released from the GV. Instead, the formation of asters is not likely GV-dependent, because maternal centrosomes at the animal pole remained partially functional despite the lack of GV components in centrifuged oocytes; they nucleated a lot of microtubules, but they never cooperated for spindle formation. Similarly, in polyspermic oocytes, GVindependent mitotic asters were formed from paternal centrosomes, but spindle formation did not occur at an area other than the animal pole. In starfish oocytes, cdc2 – cyclin B is activated f10 min after the application of 1-MA, followed by its accumulation in the GV (Terasaki et al., 2003). Also, even when GV migrates to the vegetal pole, cyclin B accumulates in the migrated GV (Barakat et al., 1994). Thus, the cdc2 – cyclin B complex is a possible candidate among the GV components that induce the formation of functional spindles during meiotic cell division as shown in Fig. 9. Although it remains to be determined whether cyclin B localization in asters and spindles is directly involved in spindle formation, it was reported that the cdc2 – cyclin complex phosphorylates microtubule-associated proteins (MAPs), which affect the stability of microtubules (Ookata et al., 1995; also see Cassimeris and Spittle, 2001). Such cdc2 – cyclin B complex activity may be partially responsible for the formation of functional spindles during meiosis. Because a portion of the cdc2 – cyclin B complex is present in the cytoplasm just before GVBD (Ookata et al., 1992) or in centrosomes during prophase (Jackman et al., 2003), GV-independent aster formation may be regulated by the cdc2– cyclin B complex as well. Indeed, weak staining by anti-cyclin B antibody was observed in asters present in areas other than the animal pole (Fig. 9). If this is the case, the development of spindles from asters may be dependent on the quantity or activity of the cdc2 –cyclin B complex. The families of MAPs such as XMAP310, NuMA, TPX2, and the echinoderm protein p62 are present in the nucleus during interphase and associate with mitotic spindle microtubules (see Cassimeris and Spittle, 2001). Because they are involved in the formation of functional spindles, they may be GV components regulating spindle formation at the animal pole in starfish oocytes. The identification of these maternal materials or factors used during the formation of functional asters and spindles should be further studied.
Acknowledgments We are grateful to Dr. S. Nemoto of Ochanomizu University for valuable advice. We also express our thanks to Dr. M. Tamura of Ochanomizu University for technical
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advice. We thank Dr. T. Kishimoto for providing the anticyclin B antibody. Our thanks are also due to Mr. Yasumoto for his help in the collecting starfish. This study was supported by grants from the Human Frontier Science Program, the Ministry of Education, Culture, Sports, Science and Technology of Japan, and by fund from the Cooperative Program provided by the Ocean Research Institute, University of Tokyo. References Barakat, H., Geneviere-Garrigues, A.M., Schatt, P., Picard, A., 1994. Subcellular distribution of aster-nucleated microtubule length: a more or less mitotic status of cytoplasmic areas during meiosis I of starfish oocytes. Biol. Cell 81, 205 – 213. Cassimeris, L., Spittle, C., 2001. Regulation of microtubule-associated proteins. Int. Rev. Cytol. 210, 163 – 226. Chambers, R., 1923. The mechanism of the entrance of sperm into the starfish egg. J. Gen. Phys. 5, 821 – 829. Chiba, K., Hoshi, M., 1989. Activation of starfish oocytes modifies their hormone dependent period for 1-methyladenine in meiosis reinitiation. Dev. Growth Differ. 31, 453 – 458. Chiba, K., Kado, R.T., Jaffe, L.A., 1990. Development of calcium release mechanisms during starfish oocyte maturation. Dev. Biol. 140, 300 – 306. Chiba, K., Tadenuma, H., Matsumoto, M., Takahashi, K., Katada, T., Hoshi, M., 1992. The primary structure of the alpha subunit of a starfish guanosine-nucleotide-binding regulatory protein involved in 1-methyladenineinduced oocyte maturation. Eur. J. Biochem. 207, 833 – 838. Clark, J.M., 1936. An experimental study of polyspermy. Biol. Bull. 70, 361 – 384. Etienne-Manneville, S., Hall, A., 2003. Cdc42 regulates GSK-3beta and adenomatous polyposis coli to control cell polarity. Nature 421, 753 – 756. Fujimori, T., Hirai, S., 1979. Differences in starfish oocyte susceptibility to polyspermy during the course of maturation. Biol. Bull. 157, 249 – 257. Hamaguchi, Y., 2001. Displacement of the mitotic apparatus which induces ectopic polar body formation or parthenogenetic cleavage in starfish oocytes. Dev. Biol. 239, 364 – 375. Hamaguchi, Y., Satoh, S.K., Numata, T., Hamaguchi, M.S., 2001. Response of the cortex to the mitotic apparatus during polar body formation in the starfish oocyte of Asterina pectinifera. Cell Struct. Funct. 26, 627 – 631. Harada, K., Oita, E., Chiba, K., 2003. Metaphase I arrest of starfish oocytes induced via the MAP kinase pathway is released by an increase of intracellular pH. Development 130, 4581 – 4586. Hinchcliffe, E.H., Miller, F.J., Cham, M., Khodjakov, A., Sluder, G., 2001. Requirement of a centrosomal activity for cell cycle progression through G1 into S phase. Science 291, 1547 – 1550. Hiramoto, Y., 1974. A method of microinjection. Exp. Cell Res. 87, 403 – 406. Hiramoto, Y., 1982. Rheological properties of echinoderm eggs during cell division. Biorheology 19, 71 – 78. Iwao, Y., Murakawa, T., Yamaguchi, J., Yamashita, M., 2002. Localization of gamma-tubulin and cyclin B during early cleavage in physiologically polyspermic newt eggs. Dev. Growth Differ. 44, 489 – 499. Jackman, M., Lindon, C., Nigg, E.A., Pines, J., 2003. Active cyclin B1-Cdk1 first appears on centrosomes in prophase. Nat. Cell Biol. 5, 143 – 148. Kanatani, H., Shirai, H., Nakanishi, K., Kurokawa, T., 1969. Isolation and identification of meiosis inducing substance in starfish Asterias amurensis. Nature 221, 273 – 274. Longo, F.J., Woerner, M., Chiba, K., Hoshi, M., 1995. Cortical changes in starfish (Asterina pectinifera) oocytes during 1-methyladenine-induced maturation and fertilisation/activation. Zygote 3, 225 – 239. Miyazaki, A., Kamitsubo, E., Nemoto, S., 2000. Premeiotic aster as a
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device to anchor the germinal vesicle to the cell surface of the presumptive animal pole in starfish oocytes. Dev. Biol. 218, 161 – 171. Nemoto, S., Yoneda, M., Uemura, I., 1980. Marked decrease in the rigidity of starfish oocytes by 1-methyladenine. Dev. Growth Differ. 22, 315 – 325. Nishiyama, A., Tachibana, K., Igarashi, Y., Yasuda, H., Tanahashi, N., Tanaka, K., Ohsumi, K., Kishimoto, T., 2000. A nonproteolytic function of the proteasome is required for the dissociation of Cdc2 and cyclin B at the end of M phase. Genes Dev. 14, 2344 – 2357. Oita, E., Harada, K., Chiba, K., 2004. Degradation of polyubiquitinated cyclin B is blocked by the MAPK pathway at the MI arrest in starfish oocytes. J. Biol. Chem. 279, 18633 – 18640. Ookata, K., Hisanaga, S., Okano, T., Tachibana, K., Kishimoto, T., 1992. Relocation and distinct subcellular localization of p34cdc2 – cyclin B complex at meiosis reinitiation in starfish oocytes. EMBO J. 11, 1763 – 1772. Ookata, K., Hisanaga, S., Okumura, E., Kishimoto, T., 1993. Association of p34cdc2/cyclin B complex with microtubules in starfish oocytes. J. Cell Sci. 105, 873 – 881. Ookata, K., Hisanaga, S., Bulinski, J.C., Murofushi, H., Aizawa, H., Itoh, T.J., Hotani, H., Okumura, E., Tachibana, K., Kishimoto, T., 1995. Cyclin B interaction with microtubule-associated protein 4 (MAP4) targets p34cdc2 kinase to microtubules and is a potential regulator of M-phase microtubule dynamics. J. Cell Biol. 128, 849 – 862. Picard, A., Harricane, M.C., Labbe, J.C., Doree, M., 1988. Germinal vesicle components are not required for the cell-cycle oscillator of the early starfish embryo. Dev. Biol. 128, 121 – 128.
Saiki, T., Hamaguchi, Y., 1997. Division of polar bodies induced by their enlargement in the starfish Asterina pectinifera. Exp. Cell Res. 237, 142 – 148. Schuetz, A.W., Longo, F.J., 1981. Hormone-cytoplasmic interactions controlling sperm nuclear decondensation and male pronuclear development in starfish oocytes. J. Exp. Zool. 215, 107 – 111. Shirai, H., Kanatani, H., 1980. Effect of local application of 1-methyladenine on the site of polar body formation in starfish oocyte. Dev. Growth Differ. 22, 555 – 560. Shirai, H., Hosoya, N., Sawada, T., Nagahama, Y., Mohri, H., 1990. Dynamics of mitotic apparatus formation and tubulin content during oocyte maturation in starfish. Dev. Growth Differ. 32, 521 – 529. Terasaki, M., Okumura, E., Hinkle, B., Kishimoto, T., 2003. Localization and dynamics of Cdc2 – cyclin B during meiotic reinitiation in starfish oocytes. Mol. Biol. Cell 14, 4685 – 4694. Uetake, Y., Kato, K.H., Washitani-Nemoto, S., Nemoto, S., 2002. Nonequivalence of maternal centrosomes/centrioles in starfish oocytes: selective casting-off of reproductive centrioles into polar bodies. Dev. Biol. 247, 149 – 164. Washitani-Nemoto, S., Saitoh, C., Nemoto, S., 1994. Artificial parthenogenesis in starfish eggs: behavior of nuclei and chromosomes resulting in tetraploidy of parthenogenotes produced by the suppression of polar body extrusion. Dev. Biol. 163, 293 – 301. Yamashita, Y.M., Jones, D.L., Fuller, M.T., 2003. Orientation of asymmetric stem cell division by the APC tumor suppressor and centrosome. Science 301, 1547 – 1550.
Unequal cell division regulated by the contents of germinal vesicles Ri-ko Matsuura a,b and Kazuyoshi Chiba a,b,* a
b
Department of Biology, Ochanomizu University, 2-1-1 Ohtsuka, Bunkyo, Tokyo 112-8610, Japan Tateyama Marine Laboratory, Ochanomizu University, 11 Kou-yatsu, Tateyama, Chiba 294-0301, Japan Received for publication 1 December 2003, revised 23 March 2004, accepted 29 April 2004 Available online 2 July 2004
Abstract Fertilization occurs during meiosis in many animals, when maternal centrosomes participate in the formation of spindles at the animal pole, which results in polar body formation. Paternal centrosomes do not participate in cell division during oocyte maturation. After meiosis, they form the spindles while the maternal centrosomes are discarded. It is unknown why paternal centrosomes do not form spindles during meiosis. Here, we show that the artificial incorporation of sperm at the animal pole of immature starfish oocytes causes unequal cell division and the formation of polar body-like fragments. The removal of germinal vesicles from the animal pole blocks the formation of polar bodylike fragments. Furthermore, translocation of germinal vesicles to the vegetal pole by centrifugation induces the extrusion of polar body-like fragments from the vegetal pole, where sperm penetration is prerequisite. After germinal vesicle breakdown, cyclin B is localized in the maternal and paternal asters and spindles near the germinal vesicle. These results suggest that germinal vesicle components such as the cdc2 – cyclin B complex interact with asters and spindles and can induce unequal cell division. During normal fertilization, paternal centrosomes are likely kept away from the germinal vesicle components, resulting in the inhibition of unequal paternal centrosome-dependent cell division. D 2004 Elsevier Inc. All rights reserved. Keywords: Polar body; Starfish; Maturation; Germinal vesicle; Centrosome; Fertilization; Cyclin B
Introduction The localization of the mitotic apparatus in polarized cells results in unequal cell division, which plays an important role during development and differentiation. Polar body formation is one of the most remarkable examples of unequal division. To produce haploid eggs without wasting stored cell components that will be used for further development, maturing oocytes release tiny polar bodies containing excessive chromosomes from the animal pole. In immature oocytes, a large nucleus called the germinal vesicle (GV) localizes at the animal pole. Translocation or removal of the GV by centrifugation results in the failure of a polar body to form. However, centrosomes, which are an important component of the mitotic apparatus or spindles, remain at the animal pole (Barakat et al., 1994; Picard et al., 1988). These results suggest that GV components may affect centrosomes or asters at the animal pole after GV breakdown * Corresponding author. Department of Biology, Ochanomizu University, 2-1-1 Ohtsuka, Bunkyo, Tokyo 112-8610, Japan. Fax: +81-3-59785370. E-mail address: [email protected] (K. Chiba). 0012-1606/$ - see front matter D 2004 Elsevier Inc. All rights reserved. doi:10.1016/j.ydbio.2004.04.038
(GVBD) and cause spindle formation and the extrusion of polar bodies. Once the spindle has been made after GVBD in the intact oocyte, the ectopic polar body is formed from the cortical region wherever the spindle containing centrosomes has been translocated (Hamaguchi, 2001; Shirai and Kanatani, 1980). Thus, GV components may irreversibly change centrosomes or asters after GVBD, resulting in spindle formation and the extrusion of polar bodies. It is unclear whether GV components can induce spindle formation or if components present at the animal pole other than GV components are also required. In starfish oocytes, GVBD is induced by 1-methyladenine (1-MA) (Kanatani et al., 1969). After GVBD, arrested oocytes at MI in the ovary are spawned (Harada et al., 2003; Oita et al., 2004) and proceed through the two meiotic cell divisions to form two polar bodies. In addition to causing GVBD, 1-MA enables oocytes to undergo normal monospermic fertilization (Chiba et al., 1990; Fujimori and Hirai, 1979). While paternal centrosomes participate in cleavage at the end of meiosis, they do not form functional mitotic apparatus during meiosis. Although the reason why sperm centrosomes do not function during polar body formation is unknown, the sperm penetration site may be different from
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the animal pole, where maternal centrosomes are presumably affected by GV components. In other words, it is possible that normal fertilization during meiosis occurs apart from the animal pole, resulting in the separation of paternal centrosomes from the GV components. If true, the artificial incorporation of sperm into the animal pole forces paternal centrosomes to interact with GV components, resulting in the formation of functional sperm centrosomes during meiosis. Thus, in this study, we tried to answer the following questions to determine whether the above hypothesis is true: 1) Do sperm (or paternal centrosomes) which penetrate the animal pole induce unequal cell division? 2) Do GV components translocated into the vegetal pole cause unequal cell division at the vegetal pole? Sperm can randomly penetrate immature starfish oocytes resulting in polyspermy when inseminated before 1-MA treatment (Chambers, 1923; Chiba and Hoshi, 1989; Clark, 1936; Longo et al., 1995; Schuetz and Longo, 1981). To answer our first question, we inseminated immature oocytes and obtained sperm-penetrating oocytes at the animal pole. Answering the second question was useful for determining whether GV components are sufficient for the induction of unequal cell division or polar body formation.
Materials and methods Materials Cytochalasin B (Sigma Chemical Co., St. Louis, MO) was dissolved in dimethyl sulfoxide (DMSO) at a concentration of 5 mg/ml, and stored at 20jC. Unfertilized and polyspermic oocytes were treated with 10 AM cytochalasin B 30 min after 1-MA addition. To enlarge polar bodies, unfertilized and polyspermic oocytes were transferred into artificial seawater (ASW) containing 2% hexylene glycol (2-methyl-2,4-pentanediol, Wako) 40 min after 1-MA addition (Saiki and Hamaguchi, 1997).
Fig. 1. PBF formation from polyspermic starfish oocytes. Morphological changes in oocytes during maturation were observed with a light microscope equipped with Nomarski differential interference contrast optics. (a – d) Unfertilized oocyte. (a) Immature unfertilized oocyte. (b) GVBD occurred 30 min after 1-MA treatment. (c) The first polar body was formed 75 min after 1-MA treatment. (d) The second polar body was formed approximately 110 min after 1-MA treatment. (e – h) Polyspermic oocytes. (e) Immature polyspermic oocytes. (f) GVBD occurred 30 min after 1-MA treatment. (g, gV) Two to three PBFs were formed at the animal pole 75 min after 1-MA treatment. (h, hV) Five more PBFs were formed about 110 min after 1-MA treatment. (gV, hV) Optical zoom at the animal pole. Arrowheads show PBFs. The following sets in parentheses show the photographs from the same oocytes: (a, b), (c, d), (e, f), (g, h), (gV, hV). Representative figures are shown. The bar represents 50 Am.
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Animal maintenance, gamete collection, and culture Starfish Asterina pectinifera were collected on the Pacific coast of Honsyu Island, Japan, and kept in laboratory aquaria supplied with circulating seawater at
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15 – 17jC. To remove follicle cells, isolated ovaries were incubated in ice-cold Ca2+-free seawater (CFSW), and released oocytes were washed twice with CFSW. Defolliculated oocytes were stored in ASW at 20jC. Oocyte maturation was induced by the addition of 1 AM 1-MA. Sperm were obtained ‘‘dry’’ by cutting the testes and keeping them on ice. Just before fertilization, sperm were diluted in ASW. Preparation of polyspermic oocytes Fig. 2. Effects of the number of penetrated sperm on PBF formation. Immature oocytes were treated with ‘‘dry’’ sperm diluted at the indicated ratio. After removal of attached sperm using protease, fertilized oocytes were treated with 1-MA. Then, the number of PBFs were counted under the microscope at 80 min (1st PBF) and 120 min (2nd PBF) after 1-MA addition corresponding to the timing of the first and second polar body formation in unfertilized oocytes, respectively. The number of sperm that entered an oocyte was counted using DAPI staining. Each bar or point represents the average of results from 20 eggs.
Defolliculated oocytes were inseminated for 10 min. To remove adhesive sperm, oocytes were treated with 0.02 % actinase-E (Kaken Pharmaceutical, Tokyo, Japan) in ASW for 5 min (Nemoto et al., 1980). Then, they were washed 5 times with CFSW to interrupt the actinase reaction. Oocytes were incubated in ASW at 20jC for 30 min.
Fig. 3. Hoechst staining of polar bodies and PBFs. After 1-MA treatment, unfertilized and polyspermic oocytes were stained with Hoechst. Morphological changes of living oocytes were visualized by Nomarski differential interference contrast optics (a, c, e, g, i, k, m) and fluorescent (b, d, f, h, j, l, n) microscopy. (a – f) Unfertilized oocytes. (a, b) Metaphase oocyte at 77 min after 1-MA addition. (c, d) The first polar body was formed 86 min after 1-MA addition. (e, f) The second polar body was formed 126 min after 1-MA addition. (g – n) Polyspermic oocytes. (g, h) Metaphase oocyte at 77 min after 1-MA addition. (i, j) PBFs having negative Hoechst staining were formed 86 min after 1-MA addition. (k, l) PBFs having negative Hoechst staining were formed 126 min after 1-MA addition. (m, n) One of six PBFs stained with Hoechst was extruded 126 min after 1-MA addition. (insets) An optical zoom at the animal pole. Each photo shows the representative figure of different oocytes. The bar represents 50 Am.
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Micromanipulation A micropipette was made following the procedure of Hiramoto (1974). The tapered portion near the tip of the micropipette was broken at a point where it had a diameter of about 10 Am. A micromanipulator (MO-388; NARISHIGE, Tokyo, Japan) and a microinjector (IM-9B, NARISHIGE, Tokyo, Japan) were used for the surgical manipulation. After inserting a micropipette into the unfertilized and fertilized GV-intact oocytes in injection chambers (Chiba et al., 1992), whole or partial GV components were aspirated (Miyazaki et al., 2000). Before and after aspiration, the diameter of the GV was measured to calculate the volume of the GV components. Visualization of chromosomes in living oocytes GVBD in starfish oocytes was induced by the addition of 1 AM 1-MA. Then, maturing oocytes were incubated with 10 AM Hoechst (bis-Benzimide H 33342, Sigma) in ASW for 30 min, and washed twice with ASW. They were observed using fluorescence microscopy. Immunofluorescence staining of microtubules and cyclin B Immunostaining was performed by a modification of the procedure of Miyazaki et al. (2000) and Iwao et al. (2002). Oocytes were treated with extraction buffer containing detergent (Shirai et al., 1990) and fixed with 100% methanol for 1 hour at 20jC. After fixation, they were transferred to PBS-T (phosphate-buffered saline/0.05% Tween 20) and left
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to stand for 5 min. They were then incubated with a mouse monoclonal antibody against a-tubulin (Amersham Corp., Buckinghamshire, England) for 50 min, washed with PBST, then stained with a FITC-conjugated goat anti-mouse IgG antibody (Tago, Burlingame, CA) for 40 min. After rinsing with PBS-T, they were incubated with anti-starfish cyclin B rabbit polyclonal antibody for 60 min, washed with PBS-T, then stained with a RITC-conjugated anti-rabbit IgG antibody (Jackson Immunoresearch Laboratories, Inc) for 40 min. DNA was stained with DAPI (Sigma) for 5 min, and washed with PBS-T. Centrifugation of oocytes Unfertilized oocytes were set in the injection chamber. To mark the animal pole, oocytes were sprayed with 1% Nile Blue (E. Merck, Darmstadt, Germany) – ASW (Shirai and Kanatani, 1980) using a micropipette (see also Fig. 8A, a). After local staining, oocytes were positioned with a glass needle so that their GV was located at the bottom of the chamber (see Fig. 8A, b). The chamber was inserted into an agar-tube containing 10% agar –ASW, and centrifuged at 500 g for 40 min (see Fig. 8A, c and d). Then, oocytes were inseminated and treated with 1-MA. Observation Living oocytes were observed with a Nomarski differential interference contrast or polarization contrast microscope, and images were taken by a digital camera (COOL PIX-4500, Nikon, Tokyo, Japan). Microtubules, cyclin B, and DNA
Fig. 4. Anti-a-tubulin antibody staining. At the time of the first polar body formation (75 min after 1-MA treatment), unfertilized and polyspermic oocytes were extracted with detergent, fixed with ice-cold methanol, and then stained with anti-a-tubulin antibody and DAPI to visualize tubulin (green) and chromosomes (blue). Images of tubulin and DNA were observed with a fluorescence microscope. (a) Unfertilized oocyte; (b) polyspermic oocytes; (insets) an optical zoom at the animal pole. Arrowheads indicate polar body or PBFs. The bar represents 50 Am.
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were observed with fluorescence microscopy (Zeiss, Axioskop), and images were taken by a video-enhanced microscopy, which was carried out using a video camera (C2400-08) and an image processor ARGUS-50 control system (Hamamatsu Photonics K.K.). Fluorescent intensity was measured using Image Gauge 3.3 graphic system (Fuji Photo Film, Tokyo, Japan).
Results Numerous polar bodies released from polyspermic oocytes As shown in Figs. 1a – d, immature starfish oocytes underwent GVBD after 1-MA addition and released the first and the second polar body, respectively. Because cell division of the first polar body after extrusion does not occur in starfish, the total number of the polar bodies from one oocyte was two through meiotic cell division (Fig. 1d). In a polyspermic oocyte, however, more than two polar bodies were released at the same time as the first polar body formation in unfertilized oocytes (Figs. 1g and gV). Moreover, when second polar body formation occurred in the control unfertilized oocytes (Fig. 1d), the polyspermic oocytes additionally released many polar bodies (Figs. 1h and hV). To investigate the effect of the number of sperm penetrating the oocytes on polar body formation, immature oocytes were treated with ‘‘dry’’ sperm which had been diluted from 10 3 to 10 13. As shown in Fig. 2, using 10 4 diluted ‘‘dry’’ sperm, 30 or more sperm nuclei were incorporated into an oocyte, from which over six polar bodies on average were extruded when second polar body formation occurred in the control. The number of polar bodies depended on the number of sperm incorporated into an oocyte, when 10 4 to 10 7 dilution dry sperm was added to immature oocytes. Sperm penetration was not observed by treatment with more diluted sperm (10 8 to 10 13), causing normal two polar bodies formation. These results strongly support the idea that the number of polar bodies depends on the number of sperm penetrating the animal pole. At 10 3 dilution, although oocytes seemed to try to extrude many polar bodies, cleavage of the extruding polar bodies was not sufficient, causing a reduced number of polar bodies. In this study, we called these cleaved fragments extruded from the animal pole of polyspermic oocytes polar bodylike fragments (PBFs). PBF without chromosomes In unfertilized oocytes, polar bodies contained the maternal chromosomes stained with Hoechst (Figs. 3d and f). To determine whether numerous PBFs from polyspermic oocytes contained maternal/paternal chromosome or not, living oocytes were stained with Hoechst and visualized by fluorescence microscopy. As shown in Figs. 3j and l,
Fig. 5. Enlargement of PBFs by hexylene glycol treatment. (A) The large first polar body (a) and the large second polar body (b) were released from hexylene glycol-treated unfertilized oocytes. Large PBFs were extruded by hexylene glycol treatment at the same time as the first polar body formation (c), and additional large PBFs were formed at the same time as the second polar body formation (d). (B) Oocytes treated with hexylene glycole were visualized by polarization contrast microscope optics. (a) Unfertilized oocyte; (b) polyspermic oocyte; (c) schematic representation of (b). (C) At the time of the first polar body formation (80 min after 1-MA treatment), normal fertilized and polyspermic oocytes were extracted with detergent, fixed with ice-cold methanol, and then stained with DAPI (a, d) and antia-tubulin antibody (b, e). Merge of (a) and (b) is shown in (c). Merge of (d) and (e) is shown in (f). Images of DNA (blue) and tubulin (green) were observed with a fluorescence microscope. (a – c) Oocyte was fertilized 40 min, and then added 2% hexylene glycole at 50 min after 1-MA treatment. (d – f) Polyspermic oocyte was added 2% hexylene glycole at 50 min after 1-MA treatment. The bar represents 50 Am.
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Fig. 6. Inhibition of PBF formation by cytochalasin B. After GVBD (30 min after 1-MA treatment), oocytes treated with cytochalasin B (10 AM at final concentration) did not form polar bodies (c, d), or PBFs (g, h). Control oocytes without cytochalasin B formed the first polar body (a) and the second polar body (b). Polyspermic oocytes without cytochalasin B formed PBFs (e, f). Photographs were taken 72 min (a, c, e, g), and 116 min (b, d, f, h) after 1-MA treatment. The bar represents 50 Am.
many PBFs without chromosomes were released. In some oocytes, a few PBFs were stained with Hoechst, while most of them were not stained (Figs. 3m and n). These results indicate that PBF did not always include sperm nuclei and maternal chromosomes. To examine the distribution of asters around the animal pole, polyspermic oocytes were fixed at the same time as PBF formation, followed by double staining with an antia-tubulin antibody and DAPI. As shown in the inset of Fig. 4b, three spots of asters and sperm nuclei were observed near the area of PBF formation, while two spots of asters and maternal chromosomes were stained in the unfertilized oocyte releasing the first polar body (Fig. 4a). Thus, PBF formation may be regulated by asters or spindles originating from sperm centrosomes at the animal pole.
bulin antibody (Fig. 5C, d – f). Instead, the size of asters in areas other than the animal pole were same as those of untreated oocytes (Fig. 4b). These results indicate that only asters and spindles present in the animal pole are sensitive to hexylene glycol. Involvement of actin in PBF release Hamaguchi et al. (2001) and Hiramoto (1982) reported that polar body formation is controlled by a decrease in actin filaments, causing a decrease in the surface force near the centrosome of the spindles or asters. To investigate the role
Effects of hexylene glycol on PBF formation Hexylene glycol enhances polymerization of tubulin in spindles or asters during polar body formation of starfish oocytes, causing extrusion of large polar bodies (Saiki and Hamaguchi, 1997; Fig. 5A, a and b). If PBF formation is also regulated by asters, the size of PBFs may be increased by hexylene glycol treatment. Indeed, as shown in Fig. 5A, c and d, large PBFs were released from polyspermic oocytes treated with hexylene glycol. Also, using polarization contrast microscopy, enlarged spindles were observed at the animal poles of polyspermic oocytes (Fig. 5B, b and c). These results support the hypothesis that PBF formation is regulated by centrosomes donated from sperm. We found that asters and spindles at the animal pole were much bigger than those distributed in other areas when we stained hexylene glycol-treated polyspermic oocytes with anti-tu-
Fig. 7. Effects of the GV components on PBF formation. Before micromanipulation, the diameter of a GV was measured. Then, the contents of a GV were partially aspirated into a micropipette inserted from the vegetal end of an oocyte. The diameter of the remaining GV was measured to calculate its % volume (remaining GV/intact GV). The volume of control oocytes is shown as 100%, which was only inserted by a micropipette without sucking the contents of the GV. The number of PBFs or polar bodies (PB) was counted under microscopy. Each symbol represents a result from one egg.
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Fig. 9. Accumulation of cyclin B in the asters and spindles at the animal pole. Normally fertilized (a – f) and polyspermic (g – l) oocytes were extracted with detergent, fixed with ice-cold methanol, and then stained with DAPI (blue), anti-a-tubulin antibody (green), and anti-cyclin B antibody (red). Images obtained by a fluorescence microscope. (a – c, g – i) Metaphase-I (60 min after 1-MA treatment); (d – f, j – l) anaphase-I (80 min after 1-MA treatment). (a – c) Accumulation of cyclin B can be seen in the meiotic spindle. (g – i) Strong staining of cyclin B can be seen clearly in the sperm-asters of the animal pole. (d – f, j – l) Cyclin B disappeared at anaphase. Representative figures are shown. The bar represents 50 Am.
of actin filaments in PBF formation, oocytes were treated with microfilament inhibitor, cytochalasin B, and then morphological changes were observed. As shown in Fig. 6, polyspermic oocytes treated with cytochalasin B did not extrude PBFs. This result indicates that polymerization or depolymerization of actin is important for PBF formation.
Effect of GV components Picard et al. (1988) showed that polar body formation is blocked when the GV components are completely removed. Similarly as shown in Fig. 7, when the GV components were sucked with a micropipette to make 30% or less GV volume, both the formation of the polar body and PBFs
Fig. 8. Translocation of the GV to the vegetal pole causing ectopic PBF. (A) To label the animal pole, oocytes in the injection chamber were sprayed with Nile Blue (a). Then, Nile Blue-stained oocytes at the animal pole were turned upside down using a glass needle (b). The chamber with stained oocytes was transferred to a centrifugation tube (c) and was centrifuged (d). (B) The GV in the stained animal pole (a) and (f) migrated to the colorless vegetal pole after centrifugation (b and g). Then, centrifuged oocytes were treated with (h) or without sperm (c), and treated with 1-MA (c, h) to induce GVBD. Ectopic PBFs were released from the colorless vegetal pole (i and j), while no polar bodies were released from unfertilized oocytes (d and e). (d and i) 80 min after 1-MA treatment. (e and j) 120 min after 1-MA treatment. The following sets in parentheses show the photographs from the same oocytes; (a – e), (f – j). The bar represents 50 Am.
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were inhibited. Instead, as in control oocytes, several PBF as well as two polar bodies were released when over 80% of the volume of the GV components remained. The number of PBFs and polar bodies were roughly dependent on the amount of GV components having 30– 80% of the original volume. Ectopic PBF formation from the vegetal pole To confirm that only the GV components in the presence of centrosomes are sufficient for the induction of unequal division, experimental translocation of the GV from the animal pole to the other cortical region in immature oocytes may be useful. Because the density of the GV components is relatively low, centrifugation of immature oocyte can shift the GV from the animal pole to the vegetal pole, although centrosomes are strictly fixed at the animal pole in immature oocytes during centrifugation (Barakat et al., 1994; Miyazaki et al., 2000; Shirai and Kanatani, 1980). Indeed, when Nile Blue-stained oocytes at the animal pole (Fig. 8A, a) were turned upside down (Fig. 8A, b) and centrifuged as shown in Fig. 8A, c and d, the GV in the stained animal pole (Fig. 8B, a and f) migrated to the colorless vegetal pole (Fig. 8B, b and g). Centrifuged oocytes were then inseminated and treated with 1-MA to induce GVBD at the vegetal pole (Fig. 8B, h). As expected, ectopic PBFs were released from the colorless vegetal pole, but not from the animal pole (Fig. 8B, i and j). These results suggest that the GV components affect the paternal centrosomes even in the vegetal pole causing unequal cell division. In control oocytes without fertilization, no polar body was released from the stained animal pole without the GV or from the vegetal pole with the GV (Fig. 8B, d and e), as reported by Barakat et al. (1994). Thus, we concluded that the GV components are sufficient for inducing unequal cell division. They may interact with paternal centrosomes or asters causing formation of spindles and ectopic PBFs. Also, formation of the big asters induced by hexylene glycol in the animal pole (Fig. 5C) may be due to the GV components. If this is the case, the GV components may localize in the asters and spindles of the animal pole. Localization of cyclin B in the asters of the animal pole Because the cdc2 – cyclin B complex accumulates in the GV just before GVBD and in spindles during metaphase in meiotic cell division (Ookata et al., 1992, 1993), it may be a candidate for GV components interacting with asters that induces unequal cell division. As shown in Figs. 9a – c, when polyspermic oocytes after GVBD were stained with anti-cyclin B antibody, asters at the animal pole more strongly reacted with the antibody than those in other areas. The average fluorescent intensity of RITC staining (anticyclin B antibody) of an aster of the animal pole was 3.5 F 0.36 (mean F SE, n = 16) times higher than that of areas other than the animal pole, while the intensity of FITC
staining (anti-tubulin antibody) of the aster at the animal pole was almost the same (1.2 F 0.08 [mean F SE, n = 16] times) as that of other areas. After metaphase, cyclin B is destroyed by the proteasome (Harada et al., 2003; Nishiyama et al., 2000; Oita et al., 2004). As expected, asters or spindles staining with anti-cyclin B antibody disappeared during the anaphase in monospermic (Figs. 9d –f) or polyspermic oocytes (Figs. 9j – l), thus confirming specificity of the staining. Although it is unknown whether the cdc2 – cyclin B complex is directly involved in the formation of functional spindles during meiotic cell division, these results support the hypothesis that GV components such as the cdc2 – cyclin complex interact with asters and spindles present near the GV after GVBD. Instead, asters apart from the GV may not receive enough GV components after GVBD, resulting in the failure of a functional mitotic apparatus to form during oocyte maturation. Thus, it is likely that GV components that interact with asters can induce unequal cell division during meiosis.
Discussion In this study using starfish oocytes, we found that sperm incorporated at the animal pole caused unequal cell division making PBFs from the animal pole. The timing of PBF formation was the same as that of polar body formation in control oocytes without fertilization. The shape of PBFs was similar to that of the polar body. PBFs or polar bodies from polyspermic oocytes did not always contain chromosomes, indicating that the extrusion of normal polar bodies was inhibited during PBF formation. Thus, the incorporation of sperm in an area other than the animal pole may be important for the release of normal polar bodies without the formation of PBFs. The PBF release as well as that of polar bodies depended on GV components, because removal of the GV blocked the formation of PBFs or polar bodies. Also, translocation of the GV from the animal pole to the vegetal pole resulted in the formation of ectopic PBFs at the vegetal pole, where incorporation of sperm was prerequisite. These results indicate that unequal division after GVBD is induced by GV components. Because PBFs and polar bodies were released from the region where the GV existed, the factor present among GV components that induces unequal division most likely remains there even after GVBD. The centrosome is the major microtubule-organizing center in animal cells. It contributes to most microtubule-dependent processes, including cell polarity (Etienne-Manneville and Hall, 2003; Yamashita et al., 2003) and cell cycle progression (Hinchcliffe et al., 2001). Because polar body formation depends on maternal centrosomes (Uetake et al., 2002; Washitani-Nemoto et al., 1994), it is likely that PBF formation is also regulated by centrosomes donated from sperm. Previous studies showed that maternal centrosomes at the animal pole cannot form polar bodies if the GV is
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translocated to the vegetal pole before GVBD (Picard et al., 1988; Shirai and Kanatani, 1980). In this study, we showed that penetrated sperm with paternal centrosomes induced the formation of ectopic PBFs at the vegetal pole, where the translocation of GVBD occurred. Thus, maternal and paternal centrosomes may be able to induce the formation of functional spindles during meiosis only if affected by a factor released from the GV. Instead, the formation of asters is not likely GV-dependent, because maternal centrosomes at the animal pole remained partially functional despite the lack of GV components in centrifuged oocytes; they nucleated a lot of microtubules, but they never cooperated for spindle formation. Similarly, in polyspermic oocytes, GVindependent mitotic asters were formed from paternal centrosomes, but spindle formation did not occur at an area other than the animal pole. In starfish oocytes, cdc2 – cyclin B is activated f10 min after the application of 1-MA, followed by its accumulation in the GV (Terasaki et al., 2003). Also, even when GV migrates to the vegetal pole, cyclin B accumulates in the migrated GV (Barakat et al., 1994). Thus, the cdc2 – cyclin B complex is a possible candidate among the GV components that induce the formation of functional spindles during meiotic cell division as shown in Fig. 9. Although it remains to be determined whether cyclin B localization in asters and spindles is directly involved in spindle formation, it was reported that the cdc2 – cyclin complex phosphorylates microtubule-associated proteins (MAPs), which affect the stability of microtubules (Ookata et al., 1995; also see Cassimeris and Spittle, 2001). Such cdc2 – cyclin B complex activity may be partially responsible for the formation of functional spindles during meiosis. Because a portion of the cdc2 – cyclin B complex is present in the cytoplasm just before GVBD (Ookata et al., 1992) or in centrosomes during prophase (Jackman et al., 2003), GV-independent aster formation may be regulated by the cdc2– cyclin B complex as well. Indeed, weak staining by anti-cyclin B antibody was observed in asters present in areas other than the animal pole (Fig. 9). If this is the case, the development of spindles from asters may be dependent on the quantity or activity of the cdc2 –cyclin B complex. The families of MAPs such as XMAP310, NuMA, TPX2, and the echinoderm protein p62 are present in the nucleus during interphase and associate with mitotic spindle microtubules (see Cassimeris and Spittle, 2001). Because they are involved in the formation of functional spindles, they may be GV components regulating spindle formation at the animal pole in starfish oocytes. The identification of these maternal materials or factors used during the formation of functional asters and spindles should be further studied.
Acknowledgments We are grateful to Dr. S. Nemoto of Ochanomizu University for valuable advice. We also express our thanks to Dr. M. Tamura of Ochanomizu University for technical
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advice. We thank Dr. T. Kishimoto for providing the anticyclin B antibody. Our thanks are also due to Mr. Yasumoto for his help in the collecting starfish. This study was supported by grants from the Human Frontier Science Program, the Ministry of Education, Culture, Sports, Science and Technology of Japan, and by fund from the Cooperative Program provided by the Ocean Research Institute, University of Tokyo. References Barakat, H., Geneviere-Garrigues, A.M., Schatt, P., Picard, A., 1994. Subcellular distribution of aster-nucleated microtubule length: a more or less mitotic status of cytoplasmic areas during meiosis I of starfish oocytes. Biol. Cell 81, 205 – 213. Cassimeris, L., Spittle, C., 2001. Regulation of microtubule-associated proteins. Int. Rev. Cytol. 210, 163 – 226. Chambers, R., 1923. The mechanism of the entrance of sperm into the starfish egg. J. Gen. Phys. 5, 821 – 829. Chiba, K., Hoshi, M., 1989. Activation of starfish oocytes modifies their hormone dependent period for 1-methyladenine in meiosis reinitiation. Dev. Growth Differ. 31, 453 – 458. Chiba, K., Kado, R.T., Jaffe, L.A., 1990. Development of calcium release mechanisms during starfish oocyte maturation. Dev. Biol. 140, 300 – 306. Chiba, K., Tadenuma, H., Matsumoto, M., Takahashi, K., Katada, T., Hoshi, M., 1992. The primary structure of the alpha subunit of a starfish guanosine-nucleotide-binding regulatory protein involved in 1-methyladenineinduced oocyte maturation. Eur. J. Biochem. 207, 833 – 838. Clark, J.M., 1936. An experimental study of polyspermy. Biol. Bull. 70, 361 – 384. Etienne-Manneville, S., Hall, A., 2003. Cdc42 regulates GSK-3beta and adenomatous polyposis coli to control cell polarity. Nature 421, 753 – 756. Fujimori, T., Hirai, S., 1979. Differences in starfish oocyte susceptibility to polyspermy during the course of maturation. Biol. Bull. 157, 249 – 257. Hamaguchi, Y., 2001. Displacement of the mitotic apparatus which induces ectopic polar body formation or parthenogenetic cleavage in starfish oocytes. Dev. Biol. 239, 364 – 375. Hamaguchi, Y., Satoh, S.K., Numata, T., Hamaguchi, M.S., 2001. Response of the cortex to the mitotic apparatus during polar body formation in the starfish oocyte of Asterina pectinifera. Cell Struct. Funct. 26, 627 – 631. Harada, K., Oita, E., Chiba, K., 2003. Metaphase I arrest of starfish oocytes induced via the MAP kinase pathway is released by an increase of intracellular pH. Development 130, 4581 – 4586. Hinchcliffe, E.H., Miller, F.J., Cham, M., Khodjakov, A., Sluder, G., 2001. Requirement of a centrosomal activity for cell cycle progression through G1 into S phase. Science 291, 1547 – 1550. Hiramoto, Y., 1974. A method of microinjection. Exp. Cell Res. 87, 403 – 406. Hiramoto, Y., 1982. Rheological properties of echinoderm eggs during cell division. Biorheology 19, 71 – 78. Iwao, Y., Murakawa, T., Yamaguchi, J., Yamashita, M., 2002. Localization of gamma-tubulin and cyclin B during early cleavage in physiologically polyspermic newt eggs. Dev. Growth Differ. 44, 489 – 499. Jackman, M., Lindon, C., Nigg, E.A., Pines, J., 2003. Active cyclin B1-Cdk1 first appears on centrosomes in prophase. Nat. Cell Biol. 5, 143 – 148. Kanatani, H., Shirai, H., Nakanishi, K., Kurokawa, T., 1969. Isolation and identification of meiosis inducing substance in starfish Asterias amurensis. Nature 221, 273 – 274. Longo, F.J., Woerner, M., Chiba, K., Hoshi, M., 1995. Cortical changes in starfish (Asterina pectinifera) oocytes during 1-methyladenine-induced maturation and fertilisation/activation. Zygote 3, 225 – 239. Miyazaki, A., Kamitsubo, E., Nemoto, S., 2000. Premeiotic aster as a
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