Influence of Centriole Behavior on the First Spindle Formation In

Developmental Biology 208, 200–209 (1999) Article ID dbio.1998.9183, available online at http://www.idealibrary.com on

View metadata, citation and similar papers at core.ac.uk brought to you by CORE Inﬂuence of Centriole Behavior on the First Spindleprovided by Elsevier - Publisher Connector Formation in Zygotes of the Brown Alga Fucus distichus (Fucales, Phaeophyceae)

Chikako Nagasato, Taizo Motomura, and Terunobu Ichimura Institute of Algological Research, Faculty of Science, Hokkaido University, Muroran 051-0003, Japan

The influence of centrioles, derived from the sperm flagellar basal bodies, and the centrosomal material (MTOCs) on spindle formation in the brown alga Fucus distichus (oogamous) was studied by immunofluorescence microscopy using anti-centrin and anti-␤-tubulin antibodies. In contrast to a bipolar spindle, which is formed after normal fertilization, a multipolar spindle was formed in polyspermic zygote. The number of mitotic poles in polyspermic zygotes was double the number of sperm involved in fertilization. As an anti-centrin staining spot (centrioles) was located at these poles, the multipolar spindles in polyspermic zygotes were produced by the supplementary centrioles. When anucleate egg fragments were fertilized, chromosome condensation and mitosis did not occur in the sperm nucleus. Two anti-centrin staining spots could be detected, microtubules (MTs) radiated from nearby, but the mitotic spindle was never produced. When a single sperm fertilized multinucleate eggs (polygyny), abnormal spindles were also observed. In addition to two mitotic poles containing anti-centrin staining spots, extra mitotic poles without anti-centrin staining spots were also formed, and as a result multipolar spindles were formed. When karyogamy was blocked with colchicine, it became clear that the egg nucleus proceeded independently into mitosis accompanying chromosome condensation. A monoastral spindle could be frequently observed, and in rare cases a barrel-shaped spindle was formed. However, when a sperm nucleus was located near an egg nucleus, the two anti-centrin staining spots shifted to the egg nucleus from the sperm nucleus. In this case, a normal spindle was formed, the egg chromosomes arranged at the equator, and the associated MTs elongated from one pole of the egg spindle toward the sperm chromosomes which were scattered. From these results, it became clear that paternal centrioles derived from the sperm have a crucial role in spindle formation in the brown algae, such as they do during animal fertilization. However, paternal centrioles were not adequate for the functional centrosome during spindle formation. We speculated that centrosomal materials from the egg cytoplasm aggregate around the sperm centrioles and are needed for centrosomal activation. © 1999 Academic Press

INTRODUCTION 1986, 1991), the centriole, a component of the centrosome, is introduced into zygotes by the sperm. In sea urchin, the fertilizing spermatozoon contributes two distinctly sepa- Fertilization, encompassing the union of female and male rated centrioles, one associated with the axoneme and the gametes, is the key event in the life cycle of organisms other with the single sperm mitochondrion. Each of these together with meiosis. After karyogamy, the mixing of chromosomes from both gametes, a normal bipolar mitotic centrioles is converted into an active centrosome and spindle in zygotes must be accurately created to separate appears to form its own MT arrangement during fertiliza- the chromosomes into daughter cells. The centrosome, tion (Paweletz et al., 1987a,b), yet only a single sperm aster composed of a pair of centrioles and pericentriolar material is seen therein (Schatten et al., 1986). In Xenopus, each in almost all eukaryotes, has a crucial role in spindle sperm contains two closely associated centrioles (Bernar- formation, controlling the distribution and dynamics of dini et al., 1986) that give rise to a single sperm aster (Fe´lix MTs within the cell (Kimble and Kuriyama, 1992; Balczon, et al., 1994; Stearns and Kirschner, 1994). In primates 1996). (Sathananthan, 1997) and other mammals (Le Guen and In most animal fertilization except mice (Schatten et al., Crozet, 1989; Sutovsky et al., 1996), the sperm contains a

0012-1606/99 $30.00 Copyright © 1999 by Academic Press 200 All rights of reproduction in any form reserved. First Spindle Formation in Fucus Zygotes 201 single centriole that forms a sperm aster in oocyte cyto- distichus in the following cases: (1) polyspermic zygotes, (2) plasm and subsequently duplicates, sending the new daugh- zygotes from anucleate egg fragments, (3) zygotes from ter centriole to form the opposite spindle pole. multinucleate eggs, and (4) karyogamy-blocked zygotes Contrary to the paternal inheritance of centrioles in with colchicine. From these observations, we discuss the fertilization, the most pericentriolar (centrosomal) materi- function of centrioles and centrosomal materials in forma- als including gamma-tubulin, which have true MT nucle- tion of the first spindle in the oogamous brown alga F. ating activity (Kimble and Kuriyama, 1992; Balczon, 1996), distichus. are prepared already in the egg cytoplasm. These maternal centrosomal materials gather around the sperm centrioles for activation of the centrosome on the first mitotic appa- MATERIALS AND METHODS ratus (Holy and Schatten, 1991, 1997). Therefore, the func- Culture. Receptacles of mature thalli of F. distichus subsp. tional centrosomes in zygotes are prepared by blending evanescens (C. Agardh) Powell, which is a monoecious species, sperm and egg centrosomal materials (Schatten, 1994). In were collected at Charatsunai, Muroran, Hokkaido, Japan, from vitro MT nucleation assays using Xenopus sperm and April to June of 1995–1998. Procedures for the liberation of eggs and extracts of unfertilized eggs support this idea (Stearns and sperm, fertilization, and culture were the same as those in previous Kirschner, 1994). This study shows that pericentrin and papers (Abe, 1970; Wakana and Abe, 1992; Motomura, 1994). centrin molecules are located near the sperm centrioles, but Egg fragments and polyspermy. Unfertilized eggs of F. disti- gamma-tubulin is completely lacking in sperm and is stored chus were obtained by the method of Wakana and Abe (1992). in egg cytoplasm. Briefly, oogonia and antheridia were liberated from conceptacles by Although centrosomal activation which is contributed by immersing in chilled seawater (0–4°C) containing Na2–EDTA (0.5 egg and sperm has been studied in many animals as men- mg/ml) in a petri dish. In the EDTA-containing seawater, the inner walls of the oogonia and antheridia were not ruptured. Oogonia, tioned above, this regulation of the centrosome during not antheridia, were obtained by filtering this solution through an fertilization has not been well described in other groups, 82-␮m nylon mesh. The oogonial wall was ruptured and unfertil- including plants. A distinct centrosomal structure with ized eggs were liberated within 1 h after placing oogonia in PESI centriole cannot be defined in higher plant cells and the medium (Tatewaki, 1966) containing 40 mg/liter chloramphenicol. complicated fertilization proceeds in the embryo sac within To induce polyspermy, unfertilized eggs were placed in PESI the ovule. Different from land plants, both female and male medium under continuous light overnight at 18°C and then were gametes in the brown algae, except in cases of nonflagellate inseminated with freshly liberated sperm. eggs, have anterior and posterior flagella and swim in Fragments of unfertilized eggs were obtained by stirring vigor- seawater, and a pair of centrioles exists as the flagellar basal ously a test tube containing unfertilized eggs in PESI medium using bodies (O’Kelly, 1989). Interestingly, isogamy, anisogamy, a vibro shaker. As a result, anucleate egg fragments were produced together with nonfragmented normal eggs and egg fragments con- and oogamy are all observed in brown algae while animals taining an egg nucleus. They were inseminated with freshly are only oogamous. We have clarified that the paternal liberated sperm. inheritance of centrioles is the common phenomenon, Multinucleate eggs (polygyny). Multinucleate eggs formed by independent of the type of sexual reproduction. In Colpo- irregular or no cleavage during the final stages of oogonium menia bullosa (isogamy; Motomura, 1992), Cutleria cylin- formation could be easily found, because they have a larger drica (anisogamy; Nagasato et al., 1998), and Laminaria diameter than normal eggs. These large eggs were obtained by angustata (primitive oogamy because the egg has vestigial filtering through on 82-␮m nylon mesh and were inseminated with flagella; Motomura, 1990, 1991), two pairs of centrioles sperm. derived from male and female gametes exist in zygotes after Karyogamy block. Karyogamy was blocked by colchicine by plasmogamy. However, the maternal centrioles selectively the same method used by Motomura (1995). To prevent the migration of the sperm nucleus and karyogamy with the egg disappear and the paternal centrioles remain as a part of the nucleus, zygotes were cultured in PESI medium containing 0.5 functional centrosome in these zygotes. In typical ooga- mg/ml colchicine (Wako Pure Chemical Industries) for 3 h imme- mous Fucus distichus and Pelvetia compressa (Motomura, diately after insemination. Then, they were washed five times with 1994; Bisgrove et al., 1997), the maternal centrioles do not PESI medium and cultured continuously in this medium (contain- exist in the mature eggs and the paternal centrioles from ing 40 mg/liter chloramphenicol) under conditions of 18°C and the flagellar basal bodies of sperm are introduced into the continuous light. zygotes. Immunofluorescence microscopy. Specimens were fixed with We have previously studied the paternal inheritance of 3% paraformaldehyde and 0.5% glutaraldehyde in PHEM buffer (60 centrioles in the brown algae. However, the pericentriolar mM Pipes, 25 mM Hepes, 10 mM EGTA, 2 mM MgCl2, pH 7.5) materials including gamma-tubulin, not centrioles, have containing 4% NaCl for 30 min at room temperature and they were squashed between a slide glass and a poly-L-lysine-coated cover true MTOC activity (Kimble and Kuriyama, 1992; Balczon, glass. Extruded cytoplasm was attached to the poly-L-lysine-coated 1996). Therefore, the first spindle pole formation in zygotes cover glass and was subsequently fixed for 30 min. Samples were cannot be regulated only by sperm-derived centrioles. Prob- treated with PBS containing 5% Triton X-100 for 1–2 h at room ably centrosomal materials which are already prepared in temperature to extract chlorophyll and washed with PBS several the egg cytoplasm are also necessary. In this study, we times. Then, they were incubated for 30 min in PBS containing 1 examined formation of the first spindle in the brown alga F. mg/ml NaBH4, followed by several rinsed in PBS. In some cases,

Copyright © 1999 by Academic Press. All rights of reproduction in any form reserved. 202 Nagasato, Motomura, and Ichimura they were treated in 0.001% trypsin in PBS for 5 min at 30°C for Cytokinesis occurred abnormally in these polyspermic zy- especially intense staining by anti-centrin antibody under immu- gotes (not shown). nofluorescence microscopy (Aruga et al., 1997). After trypsin treatment, they were rinsed for 10 min in PBS containing protease inhibitors (Complete, Boehringer-Mannheim). The samples were Anucleate Egg Fragments pretreated with blocking solution, composed of PBS containing Anucleated fragments obtained by vigorously stirring 2.5% skim milk, 5% normal goat serum, and 0.05% NaN3, for 30 min at 30°C. Then, they were incubated with polyclonal anti- unfertilized eggs can be fertilized by sperm. After plas- centrin antibody (diluted 1:250 with PBS, a gift from Dr. Melko- mogamy, the cell wall formed (Figs. 2a and 2b) and the nian, Universita¨t zu Ko¨ln, Germany) overnight at 22°C, washed sperm aster developed normally (Figs. 2d–2f). These zy- several times with PBS, and then incubated in monoclonal anti-␤- gotes, which have only a sperm nucleus and not an egg tubulin antibody (diluted 1:50, Amersham Co.) for 60 min at 30°C. nucleus, did not elongate a rhizoid nor did they undergo After rinsing with PBS, they were incubated with rhodamine-B- nuclear and cytoplasmic division (Fig. 2c). On the other conjugated goat anti-rabbit IgG (diluted 1:50, Bio Source Interna- hand, egg fragments containing an egg nucleus developed tional) and fluorescein isothiocyanate (FITC)-conjugated goat anti- normally after fertilization, although their development mouse IgG (diluted 1:50, Bio Source International) or with the was a little slower than normal zygotes (not shown). Ob- reverse combination for 60 min at 30°C. Nuclei of the samples servations made 24 h after plasmogamy indicate that the were then stained with DAPI (0.5 ␮g/ml PBS) for 10 min at room temperature, washed with PBS, and finally mounted with Mowiol sperm nucleus did not undergo any conspicuous chromatin 4-88 mounting medium (Osborn and Weber, 1982) containing 0.2% decondensation or chromosome condensation nor did it p-phenylenediamine. Observations were carried out with an Olym- divide in these zygotes. Two anti-centrin staining spots pus epifluorescence microscope (BX50-FLA), and photographs were existed around the sperm nucleus, and MTs radiated from taken using Kodak Tri-X film at ASA 1600 and developed with nearby (Figs. 2g–2i). In some cases, one of the anti-centrin Pandol (Fuji Photo Film Co. Ltd.). staining spots detached from the sperm nucleus (Figs. 2j–2l). Again, a spindle MT array was not formed and cytokinesis also did not occur in this case. RESULTS Multinucleate Eggs Polyspermy After meiosis and one mitosis, eight eggs are formed in an Polyspermy in F. distichus could be induced with high oogonium in the genus Fucus. However, in some cases the frequency when sperm were used to inseminate 1-day-old final cytoplasmic cleavage occurs abnormally or not at all, unfertilized eggs. Just after plasmogamy, numerous (sperm producing multinucleate eggs which are larger than normal. aster-like) MTs radiated from nearby sperm nuclei beneath Egg nuclei in these multinucleate eggs are located in the the plasma membrane of the zygote (Figs. 1a and 1b) as center of the cytoplasm but never fuse with each other. occurred during normal fertilization (Swope and Kropf, When sperm were used to inseminate these multinucleate 1993; Motomura, 1994; Bisgrove et al., 1997). The sperm eggs, a sperm nucleus fused with one of the egg nuclei (Figs. nuclei migrated to the egg nucleus, located in the center of 3a–3c). After karyogamy, each anti-centrin staining spot the egg cytoplasm, and fused to the egg nucleus (Figs. started to separate and migrate as it does during normal 1c–1e). Again, two anti-centrin positive spots could be fertilization (Figs. 3a–3c). In mitosis, the chromosomes observed near each of the sperm nuclei and no discrete associated with the egg nuclei, including that which fused centrin-staining structures were found around the egg with the sperm nucleus, were mixed. The spindle possessed nucleus (Fig. 1d). Therefore, we could estimate the number an eccentric form in many cases. Although each of the two of sperm that fused to an egg, although this number could anti-centrin staining spots derived from the sperm existed not be controlled. After karyogamy, centrin-staining spots at the poles of the spindle, several supplementary poles derived from the sperm separated from each other. Mitosis without anti-centrin staining spots existed (Figs. 3d–3i). In in polyspermic zygotes occurred about 16 h after plas- addition, the chromosomes were not arranged at the equa- mogamy, as it did during normal development. Character- tor (Figs. 3f and 3i). Figures 3j–3m show telophase. Clearly istically, a multipolar spindle was formed in the polysper- nuclear division had occurred abnormally, and in this case mic zygote. Figures 1f–1i show a multipolar spindle derived five daughter nuclei of irregular sizes were formed (Fig. 3m). after polyspermy. In this case, four anti-centrin positive The fertilized multinucleate eggs could elongate a rhizoid spots could be observed (Figs. 1g and 1h) and spindle MTs but cytokinesis proceeded abnormally (not shown). radiated from each of them (Fig. 1f). As a result, the chromosomes failed to arrange at the equator (Fig. 1i). Each Karyogamy-Blocked Zygotes sperm nucleus was associated with two poles during formation of the first spindle in zygote; therefore, these four poles Colchicine blocked the migration of the sperm nucleus and would be caused by two sperm fused to an egg. In telophase karyogamy in Fucus and Pelvetia (Brawley and Quatrano, (Figs. 1j–1l), each of the mitotic poles separated from one 1979; Swope and Kropf, 1993; Motomura, 1995). As men- another and the daughter nuclei migrated to each pole. tioned previously (Motomura, 1995), the concentration (0.5

FIG. 1. Spindle formation in polyspermic zygotes of Fucus distichus. (a, b) Just-fertilized polyspermic zygote. Note that MTs radiate from each of two sperm nuclei (SN). EN shows egg nucleus. (c, d, e) Karyogamy in a polyspermic zygote. Two sperm nuclei are fusing to an egg nucleus. Anti-centrin staining spots (arrows) locate on each of sperm nuclei. (f, g, h, i) Multipolar spindle in polyspermy. Four anti-centrin staining spots (arrows) can be detected, and an anti-centrin staining spot is located at each of the mitotic poles. (j, k, l) Telophase nucleus in polyspermy. A spindle with four poles formed, and chromosome masses migrated to each pole. Note that an anti-centrin staining spot is located at each pole. Microtubules (a, c, f, j), centrin (d, g, h, k), and DAPI (b, e, i, l). Scale bars, 10 ␮m (a–l).

mg/ml) and duration (for 3 h after plasmogamy) of colchicine MT arrangements could be observed around both nuclei (Figs. treatment had little effect on the subsequent development of 4d–4o). When the sperm nucleus was located close to the egg F. distichus. When karyogamy was blocked, a conspicuous nucleus, two anti-centrin staining spots were drawn toward MT array was observed near the sperm nucleus (Figs. 4a–4c). the egg nucleus from the sperm nucleus (Figs. 4d–4f). In this These MTs radiated from anti-centrin staining spots on the case, spindle MTs were normally organized around the egg sperm nucleus. On the other hand, MTs did not develop nucleus (Fig. 4d), and an anti-centrin staining spot was located prominently around the egg nucleus and a discrete MT focus at each of the spindle poles (Fig. 4e). The egg chromosomes could not be detected (Figs. 4a–4c). The previous paper were clearly arranged on the equator (Fig. 4f). Associated MTs showed that when karyogamy was blocked, the egg chromo- elongated from the one pole of the spindle toward the sperm somes condensed after DNA synthesis but the sperm chromo- chromosomes which were scattered (Fig. 4d). If only one of the somes condensed prematurely without DNA synthesis after two anti-centrin staining spots was drawn toward the egg egg chromosome condensation (Motomura, 1995). When chro- nucleus, a typical monopolar spindle was formed on the egg mosomes of both nuclei condensed, spindle or spindle-like nucleus (not shown). When the egg nucleus and the sperm

FIG. 2. Fertilization and development in anucleate egg fragments. (a, b). Fertilized egg fragments. Egg fragments (arrows) are smaller than normal eggs. Some of these egg fragments are anucleate. The cell wall was stained with 10Ϫ4% celluﬂuor in seawater. (b). The same ﬁeld as a. (c). An anucleate egg fragment could not develop 24 h after fertilization, while normal zygotes elongate rhizoids and divide (arrows). (d, e, f) Just-fertilized anucleate egg fragment. Note MTs radiating from nearby the sperm nucleus. (g, h, i) An anucleate egg fragment 24 h after fertilization. Anti-centrin staining spots near the sperm nucleus separate a little. The sperm nucleus remains in a chromatin- condensed state. MTs radiate from these anti-centrin staining structures but never form a spindle. (j, k, l) Anucleate egg fragment 24 h after fertilization. One of the anti-centrin staining spots detached from the sperm nucleus. Microtubules (d, e, f), centrin (e, h, k), and DAPI (f, i, l). Scale bars, 50 ␮m (c) and 10 ␮m (a, b, d–l).

nucleus were sufﬁciently separated (Figs. 4g–4o), anti-centrin shape and the spindle MTs did not converge at a spot on both staining spots were not drawn to the egg nucleus and re- poles (Fig. 4j). The egg chromosomes arranged at the equator mained around the sperm nucleus (Figs. 4h, 4k, and 4n). In (Fig. 4l). Numerous cytoasters frequently appeared in some these cases, a normal bipolar spindle was not formed on the zygotes after washing out colchicine (Figs. 4m–4o), but we did sperm nucleus (Figs. 4g, 4j, and 4m), even though two anti- not detect a discrete anti-centrin structure in these cytoasters centrin staining spots were located there and MTs radiated (Fig. 4n). from them. The sperm chromosomes which condensed prematurely without DNA synthesis (Motomura, 1995) were merely scattered (Figs. 4i, 4l, and 4o). By contrast, we observed DISCUSSION that a monoastral spindle was frequently formed on the egg nucleus without anti-centrin staining spots (Figs. 4g–4i) and In brown algae, the centrosome is composed of a pair of rarely a bipolar spindle (Figs. 4j–4l). The bipolar spindle on the centrioles and pericentriolar material as in animal cells (Mar- egg nucleus without anti-centrin staining spots took a barrel key and Wilce, 1975; La Claire, 1982; Katsaros et al., 1983;

FIG. 3. Spindle formation in polygyny. (a, b, c) A sperm nucleus fused with one of two egg nuclei (left nucleus). Anti-centrin staining spots had already separated. (d, e, f) A multipolar spindle in polygyny. Anti-centrin staining spots exist at two of the poles in a multipolar mitotic spindle, and other mitotic poles do not contain centrin. (g, h, i) Another multipolar spindle in polygyny. Five mitotic poles can be seen and only two have an anti-centrin positive spot. Chromosomes did not arrange at the equator. (j, k, l, m) Telophase in polygyny. Five daughter nuclei are produced in a cell containing an abnormal multipolar spindle. Two anti-centrin staining spots can be detected. Microtubules (a, d, g, j), centrin (b, e, h, k, l), and DAPI (c, f, i, m). Scale bars, 10 ␮m.

Katsaros and Garatis, 1992; Motomura and Sakai, 1985). Motomura, 1992), Cutleria cylindrica (anisogamy; Nagasato Interestingly, paternal inheritance of centrioles at fertilization et al., 1998), Laminaria angustata (primitive oogamy; Mo- also occurs in brown algae, e.g., Colpomenia bullosa (isogamy; tomura, 1990, 1991), and F. distichus (oogamy; Motomura,

FIG. 4. Spindle formation in karyogamy-blocked Fucus zygotes with colchicine. EN shows an egg nucleus and SN shows a sperm nucleus. (a, b, c) Chromosome condensation starts only in the egg nucleus. Two anti-centrin staining spots are located near the sperm nucleus, and MTs radiate from them. MTs did not develop signiﬁcantly around the egg nucleus. (d, e, f) An abnormal spindle in karyogamy-blocked zygotes after colchicine treatment. Egg chromosomes arrange at the equator but sperm chromosomes do not. A bipolar spindle is formed at the egg chromosomes and an anti-centrin staining spot is located at each pole of the bipolar spindle. MTs elongate toward the sperm chromosomes from a mitotic pole. (g, h, i) Both chromosomes in egg and sperm nuclei fully condense. Close-lying anti-centrin staining spots are located at the sperm nucleus and MTs radiate from them. Note a monopolar-like spindle in the egg nucleus. (j, k, l) Anti-centrin

1994). Recently, we reconfirmed the paternal inheritance of mic fertilization, all sperm nuclei fused to an egg nucleus, and centrioles in Fucus and Pelvetia fertilization using anti- the number of mitotic poles was double the number of sperm centrin antibody (Bisgrove et al., 1997) and the behavior of that were incorporated into an egg. Each pair of centrioles anti-centrin staining spots was the same as the centriole derived from the sperm would separate, migrate to the mitotic behavior observed using serial sections for electron micros- poles, generate daughter centrioles, and function as active copy (Motomura, 1994). Centrin (also known as caltractin) is mitotic poles in polyspermic zygotes similar to spindle forma- a 20-kDa, calcium-binding phosphoprotein that was first iden- tion in normal fertilization (Motomura, 1994; Bisgrove et al., tified as a major component of the basal body-associated 1997). contractile striated flagellar roots of green algae (Salisbury et Sluder and Rieder (1985) investigated spindle formation on al., 1984) and is present in eukaryotic cells including algae, both the sperm and egg nuclei in the karyogamy-blocked protozoa, fungi, animals, and higher plants (Melkonian et al., zygotes of sea urchin using colcemid. They reported that 1992; Salisbury, 1995; Schiebel and Bornens, 1995; Del Vec- when irradiance with UV light was used to inactivate photo- chio et al., 1997). In brown algae, centrin is associated with the chemically the colcemid, a functional bipolar spindle of nor- centrosome, especially around the centrioles, in vegetative mal appearance formed around the male chromosomes while cells, and basal bodies in swarmers (Katsaros et al., 1991, 1993; a monoaster or no spindle was found around the female Katsaros and Garatis, 1992; Bisgrove et al., 1997; Karyophyllis chromosomes. No centriole was located at the pole of this et al., 1997; Nagasato et al., 1998); therefore, the location of monoaster around the female nucleus. Sluder et al. (1989) centrioles in the cells can be obtained using anti-centrin further studied the spindle that formed around each of the antibody. male and female nuclei. They obtained fragments of recently Through this study, it became clear that centrioles de- fertilized eggs containing only a male nucleus, only a female rived from the sperm have a crucial role in spindle forma- nucleus, or both nuclei and observed spindle formation. Nor- tion during fertilization of the brown alga F. distichus. mal bipolar spindles formed in egg fragments containing both Centrosomal materials with MTOC activity could be male and female nuclei or only a male nucleus, but again only stored or produced de novo in the egg cytoplasm and a monoaster was formed in the fragment containing just a subsequently gathered around the sperm centrioles. Their female nucleus. In our experiments using the brown alga F. combination produced an active and functional centrosome distichus, when sperm of F. distichus fertilized an anucleate in Fucus zygotes. Therefore, the results strongly support the egg fragment, the sperm nucleus remained in the condensed idea that the functional centrosome in zygotes is a product state probably without DNA synthesis, and a normal bipolar blending maternal and paternal centrosomal components in spindle was never formed different from the case of sea urchin. animal fertilization (Holy and Schatten, 1991, 1997; Navara Further, when karyogamy was blocked using colchicine treat- et al., 1994) and that sperm centrioles function as a nucle- ment, prominent MTs developed around the egg nucleus. As ation center of these centrosomal materials. in the case of sea urchin, a monoaster was frequently observed In polyspermic animal zygotes, it is well known that a in the egg nucleus. In some cases, a bipolar, barrel-shaped multipolar spindle or supernumerary bipolar mitotic spindles spindle was also detected. However, in these cases, centrioles are formed (Holy and Schatten, 1991; Navara et al., 1994; detected as anti-centrin positive spots did not exist at the Schatten, 1994) by the incorporation of extra centrioles from poles. When the sperm nucleus was located near the egg sperm into the egg. Holy and Schatten (1991) showed that the nucleus and two centrioles migrated to the egg nucleus, a number of centrosomes in metaphase was normally two when normal bipolar spindle formed with the regular radiation of an egg received one sperm, four when it received two, six from spindle MTs at both. three, and eight from four sperm in sea urchin. Brawley (1987, In the case of polygyny in F. distichus, a multipolar spindle 1990) reported that polyspermic zygotes of F. distichus and formed as in the case of polyspermy. However, unlike the case Pelvetia fastigiata germinated normally but irregular division during polyspermy, two poles had centrioles derived from a planes occurred, and they died 1–1.5 weeks after fertilization. sperm while the others did not. In polyspermy, each of the She speculated that the lethal condition of these polyspermic poles in the multipolar spindle had centrioles. This strongly zygotes could be caused by supernumerary centrioles derived suggests that the spindle pole can form without centrioles. A from the extra sperm. In this study, we clearly show that monopolar spindle, and a bipolar one in rare cases, around the abnormal multipolar spindles form in polyspermic zygotes of egg nucleus in karyogamy-blocked zygotes would also present Fucus by using anti-␤-tubulin and anti-centrin antibodies. In the same case. Two possibilities would be considered in the the case of polyspermy in Fucus as well as in animal polysper- formation of the spindle poles without centrioles from sperm.

staining spots are located near sperm chromosomes and MTs radiate from them to the sperm chromosomes. Note that egg chromosomes are arranged at the equator and a bipolar spindle can be observed. However, the egg spindle possesses a barrel shape and both poles are broad. (m, n, o) Many cytoasters are produced by colchicine treatment, but the anti-centrin staining spot is located at the sperm chromosomes and not at these cytoasters. Normal bipolar spindles are not formed at either egg or sperm chromosomes. Microtubules (a, d, g, j, m), centrin (b, e, h, k, n), and DAPI (c, f, i, l, o). Scale bars, 10 ␮m.

First, centrosomal materials could exist around the nuclear Balczon, R. (1996). The centrosome in animal cells and its func- envelope of the egg nucleus. In polygyny, some of the centro- tional homologs in plant and yeast cells. Int. Rev. Cytol. 169, somal materials around the egg nuclear envelope would mi- 25–82. grate toward the sperm centrioles and create the two func- Bernardini, G., Stipani, R., and Melone, G. (1986). The ultrastruc- tional spindle poles, but others around egg nuclei more distant ture of Xenopus spermatozoon. J. Ultrastruct. Mol. Struct. Res. 94, 188–194. from the sperm nucleus would assemble and form supernu- Bisgrove, S. R., Nagasato, C., Motomura, T., and Kropf, D. L. (1997). merary spindle poles without centrioles. Motomura (1994), Immunolocalization of centrin during fertilization and the first using electron microscopy, observed that characteristic struc- cell cycle in Fucus distichus and Pelvetia compressa (Fucales, tures composed of fine granular masses with intermixed dense Phaeophyceae). J. Phycol. 33, 823–829. and lighter staining areas appeared around the Fucus zygotic Brawley, S. H. (1987). A sodium-dependent, fast block to nucleus 4 h after plasmogamy, and these structures migrated polyspermy occurs in eggs of fucoid algae. Dev. Biol. 124, toward the centrioles at both poles just before formation of the 390–397. first spindle and became dispersed with the radiation of MTs Brawley, S. H. (1990). Polyspermy blocks in fucoid algae and the at both poles. At present, the molecular nature of these occurrence of polyspermy in nature. In “Mechanism of Fertili- structures has not been determined, but considering observa- zation: Plant to Human” (B. Dale, Ed.), pp. 419–431, Springer- tions in this experiment and the behavior of these structures Verlag, Berlin. (Motomura 1994), they could have an important role in Brawley, S. H., and Quatrano, R. S. (1979). Effects of microtubule inhibitors on pronuclear migration and embryogenesis in Fucus spindle pole formation. Second, noncentrosomal proteins in distichus (Phaeophyta). J. Phycol. 15, 266–272. the egg nucleus and cytoplasm could interact with the spindle Compton, D. A. (1998). Focusing on spindle poles. J. Cell Sci. 111, poles without the centrosome. Recent studies using extracts 1477–1481. from Xenopus eggs and HeLa cells show that noncentrosomal Del Vecchio, A. J., Harper, J. D. I., Vaughn, K. C., Baron, A. T., proteins including NuMA (nuclear protein that associates Salisbury, J. L., and Overall, R. L. (1997). Centrin homologues in with a mitotic apparatus), dynactin, and cytoplasmic dynein higher plants are prominently associated with the developing cell are essential to organize the minus ends of MTs for spindle plate. Protoplasma 196, 224–234. formation, not centrosomal material, and the centrosome acts Fe´lix, M.-A., Antony, C., Wright, M., and Maro, B. (1994). Centro- as a microtubule donor for the developing spindle (Compton, some assembly in vitro: Role of ␥-tubulin recruitment in Xeno- 1998; Gaglio et al., 1995, 1996, 1997; Heald et al., 1996, 1997). pus sperm aster formation. J. Cell Biol. 124, 19–31. Therefore, in this experiment, the formation of the spindle in Gaglio, T., Saredi, A., and Compton, D. A. (1995). NuMA is required for the organization of microtubules into aster-like the egg nucleus in the case of karyogamy-blocked zygotes and mitotic arrays. J. Cell Biol. 131, 693–708. the extra poles of the spindle in polygyny implies the possi- Gaglio, T., Saredi, A., Bingham, J. B., Hasbani, M. J., Gill, S. R., bility that the noncentrosomal proteins such as NuMA, dyn- Schroer, T. A., and Compton, D. A. (1996). Opposing motor actin, and cytoplasmic dynein would construct these spindle activities are required for the organization of the mammalian poles. Further studies using antibodies against centrosomal mitotic spindle pole. J. Cell Biol. 135, 399–414. material such as ␥-tubulin and noncentrosomal material such Gaglio, T., Dionne, M. A., and Compton, D. A. (1997). Mitotic as NuMA would be necessary to clarify spindle formation in spindle poles are organized by structural and motor proteins in polygyny and karyogamy-blocked zygotes in Fucus. addition to centrosomes. J. Cell Biol. 138, 1055–1066. Heald, R., Tournebize, R., Blank, T., Sandaltzopoulos, R., Becker, P., Hyman, A., and Karsenti, E. (1996). Self-organization of ACKNOWLEDGMENTS microtubules into bipolar spindles around artificial chromosomes in Xenopus egg extracts. Nature 382, 420–425. 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