The effect of taxol on first and second meiotic spindle formation was examined in oocytes of the surf clam, Spisula solidissima, by immunofluorescence staining with anti-tubulin antibody. The first meiotic spindle appeared to form as in untreated control cells. However, the spindle did not migrate toward the periphery of taxol-activated oocytes, resulting in blockage of the formation of the first polar body. In spite of inhibited microtubule depolymerization and failure of spindle disappearance, the pole separation in telophase that is typical of this material began at the same time as in untreated cells. Polymerization of the second spindle microtubules onto the spindle persisting from the first meiosis led to the formation of a triple form of spindle connected at the poles of each other. The subsequent emergence of ring-shaped microtubule-containing structures in mature activated eggs was not affected by taxol. The mechanism of meiotic spindle formation thus seemsto be different from that in mitosis, where taxol has been shown to block spindle formation completely.

Cytoplasmic microtubules are generally very labile and many biological functions associated with microtubules can be assumed to be performed through their periodic formation and breakdown within cells. Much of the evidence for the role of microtubule assembly as it relates to function has been derived from the effect of microtubule-depolymerizing drugs such as colchicine, vinblastine, griseofulvin or nocodazole (see Dustin, 1984, for a review). Taxol, on the other hand, promotes microtubule polymerization and induces extensive stabilization of microtubule structures (Schiff et al. 1979). It has therefore been possible, for the first time, to demonstrate the requirement for microtubule depolymerization in the intracellular migration of: chromosomes (Mol^-Bajer & Bajer, 1983), insulin storage granules in the pancreas (Howell et al. 1982), cholesterol to the mitochondrial site of the cholesterol side-chain cleavage enzyme (Rainey et al. 1985) or the pronucleus in fertilized sea-urchin eggs (Schatten et al. 1982).

One of the major functions of microtubules is to form the spindle during mitosis. Taxol-treated tissue culture cells can go through their cell cycle from Gz to Af (Schiff & Horwitz, 1980); however, they are incapable of forming normal bipolar spindles at mitosis. Stable bundles of microtubules formed in interphase cells are replaced by multiple asters (DeBrabander et al. 1981; Brenner & Brinkley, 1982). Taxol treatment of sea-urchin eggs results in the formation of abnormally large sperm asters that persist through several cell cycles. No mitotic spindles appear in fertilized eggs at mitosis, and the eggs undergo a cycle of nuclear breakdown and re-formation with the stabilized sperm monastral-like microtubule-containing structure (Schatten et al. 1982).

In contrast to results of extensive studies on mitosis, structural as well as physiological information on meiotic spindles is relatively scant. Using immunofluorescence staining of whole oocytes with anti-tubulin antibody, we have recently shown the formation of first and second meiotic spindles in surf clam oocytes during the process of maturation (Kuriyama et al. 1986a). Fully grown oocytes of surf clam are uniformly arrested at the germinal vesicle stage (first meiotic prophase). They undergo their germinal vesicle breakdown and two meiotic divisions when they are fertilized or parthenogenetically activated by treatment with KCL The first polar body is formed at 15–20 min after activation. Interphase between meiosis I and II is short, and the second meiotic spindle appears in the cytoplasm within 10—15 min after the first meiotic division. After the second polar body formation, fertilized eggs later form a sperm aster and mitotic spindles, whereas activated eggs form monastral/ring-shaped microtubule-containing structures, which undergo cycles of alternating formation and breakdown.

With the aim of comparing the mechanisms of spindle formation in meiosis and mitosis, we determined the effect of taxol on the formation of meiotic spindle structure in the oocytes of the surf clam, Spisula solidissima. Here we report that, although microtubules are stabilized by taxol extensively, germinal vesicle breakdown as well as the formation of meiotic spindles can occur in taxol-treated eggs as in untreated controls. The effects of taxol on meiotic spindle formation thus appear to be different from those on mitotic spindles. This difference may reflect differences in the mechanisms of mitotic and meiotic spindle formation and will be discussed from the perspective of microtubule organizing centres (MTOCs).

Preparation of eggs for immunofluorescence

The surf clam, Spisula solidissima, was collected at the Marine Biological Laboratory, Woods Hole, MA; starfish, Astropecten verrilli, were obtained from the Pacific Bio-Marine Laboratory Inc., Venice, CA.

Oocytes of surf clam were prepared as before and vitelline membranes were removed by protease digestion (Kuriyama et al. 1986a). Meiotic maturation was induced by addition of 0 ·25 ml of 3M-KC1 to 10ml of an oocyte suspension (Allen, 1953). Preparation and activation of starfish oocytes were performed as described (Kuriyama & Kanatani, 1981).

Eggs at appropriate stages were immunofluorescently stained with anti-tubulin antibodies as described (Kuriyama & Borisy, 1985; Kuriyama et al. 1986a). Briefly, whole eggs were first adsorbed to polylysine-coated coverslips and fixed with cold methanol in the presence of 50mM-EGTA (ethylene glycol bis(β-aminoethylether) tetraacetic acid) (Harris et al. 1980). After rehydration with phosphate-buffered saline (PBS), the coverslips were stained with monoclonal anti-yeast or-tubulin (kind gift from Dr J. V. Kilmartin) or monoclonal anti-chick brain βtubulin (Amersham) for 1 ·5 h at 37°C. The coverslips were then rinsed thoroughly with PBS, and stained with fluorescein-labelled second antibodies (P. L. Cappel Lab., Inc.). Microscopic observations were made on a Zeiss Photomicroscope III or an Olympus BH2 microscope with epifluorescence optics, and photographed with Kodak Tri-X film.

Treatment of eggs with taxol and isolation of taxol-treated meiotic spindles

Demembranated oocytes were treated with taxol at a final concentration of 5–20 μg ml−1 by addition of 1/100 to l/800vol. of 2 mg ml−1 or 4 mg ml−1 stock solution dissolved in dimethyl sulphoxide.

Taxol-treated second meiotic spindles were isolated and purified by using 1 M-glycerol, 5 mM-MES (2(7V-morpholino) ethanesulphonic acid), 1 mM-EGTA, 2mM-MgSO4, 0 ·05% Triton X-100 at pH6 ·15 as a standard isolation medium (Kuriyama & Borisy, 1983). Eggs cultured in calciumfree artificial sea water (CFSW) at room temperature were sedimented and washed twice with 1 M-dextrose to remove CFSW. The packed pellet of eggs was resuspended in 10 – 30 vol. of the isolation medium. The suspension was shaken to rupture the eggs and meiotic spindles were collected by centrifugation at 3000g for 5–10 min.

Maturation of parthenogenetically activated eggs in the presence of taxol

Removal of the vitelline membrane is the key factor for staining whole oocytes with anti-tubulin antibodies. We dissolved vitelline membranes by direct digestion with protease, permitting rapid penetration of the first and second antibodies into the cytoplasm (Kuriyama et al. 1986a). Fluorescent micrographs in the left-hand column of Fig. 1 (A,C,E) demonstrate the staining pattern of surf clam oocytes with anti-yeast a-tubulin antibody before (A) and after (C,E) activation with KC1. As reported earlier (Kuriyama et al. 1986a), no specifically differentiated microtubulecontaining structure was visible in unactivated eggs. Only a thin layer of cytoplasm surrounding the germinal vesicle was stained evenly and faintly. Shortly after activation, two bright spots appeared next to the germinal vesicle (Fig. 1C) and microtubules started to polymerize onto the dots. It eventually became possible to detect the fully grown meiotic spindle, with symmetrically elongated astral fibres in the centre of eggs (Fig. IE) within 12–15 min of activation.

Fig. 1.

Immunofluorescence micrographs of surf clam oocytes stained with monoclonal anti-tubulin antibody. Eggs were incubated with (B,D,F) or without (A,C,E) 10 μgml−1 taxol for 10 min before activation. Photographs were taken at Omin (A,B), 5 ·25 min (C), 10 min (D), 9 ·3 min (E) and 25 min (F) after activation with KC1. A, ×730; B, ×690;C, ×880; D, ×770; E, ×780; F, ×750.

Fig. 1.

Immunofluorescence micrographs of surf clam oocytes stained with monoclonal anti-tubulin antibody. Eggs were incubated with (B,D,F) or without (A,C,E) 10 μgml−1 taxol for 10 min before activation. Photographs were taken at Omin (A,B), 5 ·25 min (C), 10 min (D), 9 ·3 min (E) and 25 min (F) after activation with KC1. A, ×730; B, ×690;C, ×880; D, ×770; E, ×780; F, ×750.

Changes in the microtubule staining pattern in taxol-treated oocytes are illustrated in the right-hand column of Fig. 1 before (B) and after (D,F) activation of eggs with KC1. In these experiments, surf clam oocytes were incubated with 10/tgml−1 taxol for 10 min before activation. During this treatment, an area intensely stained with anti-tubulin antibody appeared not only at the cortex of the cytoplasm but also at the surface of the germinal vesicle (Fig. IB). In spite of the formation of this extra immunofluorescence-positive structure, brightly stained fluorescent dots appeared as in control cells at around 10min after activation (Fig. ID). They faced each other across the germinal vesicle and appeared to push and to indent the nucleus. While the cortical ring was still brightly fluorescent, the staining intensity around the surface of the germinal vesicle faded away with progressive breakdown of the germinal vesicle. Fluorescent microtubules originating from the dots on the nucleus were more intense than in control cells, suggesting assembly of a larger number of microtubules onto the dots. Enlarging astral fibres eventually meet one another to form a bridge over the nucleus, resulting in the formation of the first polar body spindle in taxol-treated cells at around 25 min after activation (Fig. IF). Compared to control cells, taxol-treated cells seemed to require longer to form a spindle in the cytoplasm, and to have an enhanced assembly of spindle microtubules as was apparent from the more extensive fluorescence around the dots or spindle area (compare C and E with D and F, respectively). Thus taxol neither blocks the germinal vesicle breakdown nor inhibits the formation of the first meiotic spindle. This result has also been confirmed in oocytes of the starfish, Astropecten verrilli, the maturation of which was triggered by addition of 1-methyladenine (data not shown).

Since preincubation of non-activated oocytes with lO^gml−1 taxol for lOmin did not cause any blockage of first spindle formation, samples of clam oocytes in the following experiments were treated with the drug from 5 min after activation with KC1. Fig. 2 shows changes in distribution of microtubule-containing structures in taxol-treated cells at both low magnification, to show the uniformity of the staining reaction (Fig. 2B’,D’,F’,H’), and at high magnification to show the structural details (Fig. 2B,D,F,H). Untreated control cells at stages corresponding to those of taxol-treated cells (B,D,F,H) are shown in A,C,E and G, respectively. As described earlier, fluorescent ring structures were visible in the area of cortex and germinal vesicle in oocytes treated with taxol lOmin before activation (Fig. 1B,D,F). Such bright fluorescent rings were, however, not detectable in cells treated with taxol 5 min after activation. Instead, almost all microtubules visualized by anti-tubulin antibody staining were associated with the structure of the first meiotic spindle, which shows as strong fluorescence in the region of meiotic spindle (Fig. 2B). After establishment of the first meiotic spindle, this centrally located spindle started to move toward the periphery in control cells (Fig. 2A), which resulted in unequal cleavage to produce the first polar body. The spindle in taxol-treated cells, on the contrary, remained in the same central position and failed to form the polar body (Fig. 2B). This failure of spindle movement might be due to blockage by taxol of asymmetric depolymerization of astral microtubules, which may normally permit or cause the spindle to move to one side of the egg. It should be noted that the cytoplasm in taxol-treated cells (Fig. 2B,B’) shows a much cleaner immunofluorescent background as compared to the control (Fig. 2A), suggesting that the major proportion of available tubulin molecules is incorporated into the spindle.

Fig. 2.

Immunofluorescence micrographs of activated surf clam oocytes stained with monoclonal anti-tubulin antibody. Eggs were incubated with (B,B’,D,D’,F,F’,H,H’) or without (A,C,E,G) 5–10 μgml−1 taxol from 5 min after activation with KCI. Photographs were taken at 16min (A), 25min (B), 27 ·5min (B’), 30min (D), 32min (C), 36min (D’), 39min (E), 40min (F,F’) and 80min A,B, ×620; C, ×550;D, ×460; B’,D’, ×300; E, ×640; F, ×600; G, ×560; H, ×520; F,H, ×320.

Fig. 2.

Immunofluorescence micrographs of activated surf clam oocytes stained with monoclonal anti-tubulin antibody. Eggs were incubated with (B,B’,D,D’,F,F’,H,H’) or without (A,C,E,G) 5–10 μgml−1 taxol from 5 min after activation with KCI. Photographs were taken at 16min (A), 25min (B), 27 ·5min (B’), 30min (D), 32min (C), 36min (D’), 39min (E), 40min (F,F’) and 80min A,B, ×620; C, ×550;D, ×460; B’,D’, ×300; E, ×640; F, ×600; G, ×560; H, ×520; F,H, ×320.

After first polar body formation, the remaining microtubules in both control egg and polar body disintegrated, as indicated by the increased fluorescence in the cytoplasm in response to anti-tubulin antibodies (Fig. 2C). The meiotic poles divided immediately after polar body formation and structures for the second meiosis appeared in the polar body as well as in the egg soon after complete polar separation (Fig. 2E). In contrast to control cells, meiotic microtubules were stabilized extensively (Fig. 2D) and did not depolymerize in taxol-treated eggs. The fluorescent background in the cytoplasm of taxol-treated eggs (Fig. 2D,D’) was still low, as in cells shown in Fig. 2B. Although the first meiotic spindle did not disappear in the presence of taxol, spindle poles started to separate normally as in control cells, resulting in elongated forms of the poles (Fig. 2D). Polymerization of the second spindle microtubules onto the spindle persisting from the first meiosis led to the formation of triple-formed spindles connected to each other’s poles (Fig. 2F,F’; the configuration is more clearly seen in Fig. 3A).

Fig. 3.

Light micrographs of triple-formed spindles. A,C,C’, immunofluorescence; BB’, phase-contrast. Triple-formed spindles induced within activated oocytes (A) by taxol were isolated in a glycerol-containing medium (B,B’,C,C’). A, ×740; B, ×1050;B ×1240; B’, ×420; C’, ×430.

Fig. 3.

Light micrographs of triple-formed spindles. A,C,C’, immunofluorescence; BB’, phase-contrast. Triple-formed spindles induced within activated oocytes (A) by taxol were isolated in a glycerol-containing medium (B,B’,C,C’). A, ×740; B, ×1050;B ×1240; B’, ×420; C’, ×430.

As reported previously, parthenogenetically activated surf clam eggs, having completed meiosis, fail to proceed to mitosis (Kuriyama et al. 1986a). Instead they form microtubule-containing structures different from mitotic spindles. They sometimes resemble monasters, or sometimes appear as brightly stained ring or oval structures with anti-tubulin antibody staining (Fig. 2G). As is evident in Fig. triple-formed spindles were gradually replaced by these ring-shaped microtubule-containing structures in taxol-treated mature unfertilized surf clam eggs. Compared to the structure in control cells (Fig. 2G), however, this structure in taxol-treated eggs was much larger and showed brighter fluorescence (Fig. 2H,H’). The fluorescence background in the cytoplasm of taxol-treated eggs was also much less intense than in controls.

Microscopic observation of isolated triple-formed spindles

Triple-formed spindles induced by treatment of activated oocytes with taxol (Figs 2F,F’, 3A) were isolated in the medium used for isolation of mitotic apparatus or cytasters from fertilized or unfertilized sea-urchin eggs (Kuriyama & Borisy, 1983). Mass-isolated triple-formed spindles revealed by phase-contrast and fluorescence microscopy are presented in Fig. 3B’,C’, respectively. Observation at high magnification with phase-contrast (Fig. 3B) and fluorescence microscopy after staining with anti-tubulin antibody (Fig. 3C) has revealed the detailed structure of these peculiar spindles. Each pole split from the first meiotic spindle established the second spindle linked with the original one. Condensed chromosomes were still positioned at the metaphase plate in the first meiotic spindle. This indicates that microtubule organization in the second meiotic spindles occurs independently of the chromosome condensation for the second meiosis.

The microtubule-stabilizing drug, taxol, shows different effects on spindle formation during mitosis and meiosis. It enables somatic cells and zygotes to enter mitosis, but mitotic cells do not form any normal spindles (DeBrabander et al. 1981; Brenner & Brinkley, 1982). On the other hand, the process of first meiotic spindle formation is not affected by taxol at all and the meiotic poles can separate normally as in control cells. Second meiotic spindles, although they are of unusual shape, being connected with the persistent first meiotic spindle, are formed in the taxol-treated eggs at almost the same time as in untreated cells. The microtubule drugs, iso-pyropyl-.’V-carbamate (Crozet & Szollosi, 1979) and taxol (Albertini, 1981), have been reported to inhibit germinal vesicle breakdown in mammalian oocytes. Further studies will be required to clarify this apparent discrepancy concerning the effect of taxol on germinal vesicle breakdown in oocytes.

In animal cells, a pair of centrioles is present at each mitotic pole. Precisely controlled reproduction of centrioles is believed to be essential for regulating the total number of mitotic poles. We have already shown that taxol specifically blocks the duplication of centrioles in cultured mammalian cells (Kuriyama et al. 1986b). The abnormal composition of centrioles at each spindle pole in taxol-treated cells might cause the abnormal shape of mitotic spindles. The fact that splitting of meiotic poles is independent of the taxol treatment seems to imply that either taxol does not block centriole duplication in Spisula oocytes or centrioles are not duplicated from first to second meiosis even in the absence of taxol. Longo & Anderson (1969) mentioned that each aster of the meiotic spindle in Mytilus edulis oocytes contained only one centriole in the second meiotic spindle pole. If the same is true in Spisula oocytes, centriole duplication may not occur between first and second meiosis, which makes the cells insensitive to taxol.

Otto & Schroeder (1984) have reported the presence of microtubule arrays in the cortex of immature starfish oocytes. These structures could be visualized only in the isolated cortex and not in whole oocytes because of background fluorescence diffused throughout the cytoplasm. Since taxol enhances microtubule organization, strong fluorescence detected at the cortical region in taxol-treated immature oocytes of the surf clam (Fig. IB) might correspond to an exaggerated form of the cortical microtubules described by Otto & Schroeder (1984). Besides the cortical ring of microtubules, we could visualize a microtubule-containing structure around the germinal vesicle following taxol treatment. Therefore, it might also be reasonable to expect the presence of microtubules around the nucleus in untreated oocytes. Further examination with the electron microscope will be required to demonstrate the presence of such microtubule-containing structures.

It is well-established that microtubules are involved in the intracellular movement of various kinds of organelles (Girbardt, 1968; Aronson, 1971; Wolff & Williams, 1973; Murphy & Tilney, 1974; Malaisse et al. 1975; Smith et al. 1975; Poon & Day, 1976; Hyams & Stebbings, 1977; Wolf, 1978; Heath & Heath, 1978; Taylor & Fuller, 1980; Mori et al. 1982). Recent genetic analyses of the cytoskeleton provide evidence supporting direct participation of both a and /J-tubulins in the mechanism of nuclear movement of the fungi, Aspergillus nidulans (Oakley & Morris, 1980, 1981). The results of the present investigation suggest that translocation of the first meiotic spindle in the clam ooplasm is also microtubule-related; more specifically, it is a microtubule depolymerization-dependent process. The mechanism by which the disassembly of microtubules contributes to spindle migration towards the animal pole is not known. The most reasonable explanation would be that astral microtubules radiating from one side of the pole keep on polymerizing, whereas the astral microtubules from the other side continue to depolymerize. This coordination of assembly/disassembly to microtubules at each pole may result in asymmetric pushing of the spindle to one side of the egg. In fact, evidence has already been provided by Ishizaka (1969) and Kawamura (1977) in separate experiments that asymmetric growth of the asters causes the spindle shift in grasshopper spermatocytes. Taxol induces equal amounts of tubulin polymerization at both poles and then stabilizes microtubules and blocks their depolymerization. The equal size of the two asters causes the spindle to stay at the centre of the cell.

It should be noted that the astral microtubules in the first meiotic spindle are prominent only in the migrating spindles of control cells, but not in those of taxol-treated cells (see Fig. 2A,B). Since meiotic spindles are generally of anastral shape (Wilson, 1928), astral microtubules detected in the first meiotic spindles of the clam oocytes (Fig. 2A) might have developed only for shifting the spindle toward the animal pole by the mechanism discussed above. In contrast, stabilization of microtubules and promotion of microtubule assembly by taxol is striking only in the spindle region and not in the astral portion (Fig. 2B). This results in the formation of a typical meiotic spindle in taxol-treated oocytes without any visible asters (Fig. 2B). Assuming that no astral microtubules are present in taxol-treated cells, lack of association between the spindle and the inner side of the egg surface could make movement of the spindle towards one side of the egg impossible. Detailed electron-microscopic analysis is required to determine whether centrally positioned meiotic spindles contain astral microtubules in taxol-treated eggs.

Polar body formation can be considered as an extreme case of unequal division. In sea-urchin embryos, the four vegetal cells at the 8-cell stage cleave unequally to form four micromeres and four macromeres. Dan and his coworkers examined the unequal division of sea-urchin eggs extensively and concluded that the eccentric position of the mitotic spindles resulted in the unequal division of the four vegetal cells (Dan, 1979; Dan et al. 1983). In contrast to the surf clam oocytes, in sea-urchin eggs the asymmetric orientation of the mitotic apparatus is due to the migration of the restingnucleus, rather than of the spindle (Dan, 1979). Electron-microscopic examination has revealed the presence of a granule-free area in the vegetal pole in sea-urchin embryos to which the eccentric spindle is attached (Tanaka, 1981; Dan et al. 1983). When the cortical differentiation is destroyed by treatment with detergent such as sodium lauryl sulphate (SLS), connection between the cortex and nucleus is disturbed and no further migration of the resting nucleus to the periphery can be seen (Tanaka, 1976). This results in an equal division of the vegetal quartet to produce identical sized blastomeres. The same kind of a granule-free area is also found at the animal pole of oocytes in S. solidissima (Dan & Ito, 1984). However, we failed to block the migration of the first meiotic spindle to the periphery by treatment of surf clam oocytes with 1 ×10−3 to 5 ×10−3% SLS or 20 μM-cytochalasin B (Kuriyama, unpublished), which was reported to be more than enough to inhibit micromere formation (Tanaka, 1976, 1981). Therefore, it is not clear whether microfilaments or interaction with the cortex is directly involved in the spindle movement in oocytes of the surf clam. The mechanism of nuclear movement for micromere formation in sea-urchin eggs and spindle migration for polar body formation in surf clam oocytes might be different from each other.

The author thanks Dr G. G. Borisy of the University of Wisconsin, Dr J. V. Kilmartin of the MRC Laboratory of Molecular Biology, Cambridge, England, and Mr Tim Knotek of the University of Minnesota for their kind support, providing monoclonal anti-tubulin antibody and photographic assistance, respectively. Special thanks are due to Professor K. Dan for his valuable discussions. This research was supported by a Steps Toward Independence Fellowship from Marine Biological Laboratory at Woods Hole, a University of Minnesota Graduate School Grantin-Aid, an ACS Institutional Research Grant (1N-13-X-8) and the Minnesota Medical Foundation (CRF-69-85).

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