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Microtubule Organization during Maturation of Xenopus Oocytes: Assembly and Rotation of the Meiotic Spindles DAVID L. GARD Department

of Biology,

University

of Utah, Salt Luke City, Utah 84112

Accepted December 10, 1991

Assembly of the meiotic spindles during progesterone-induced maturation of Xenopus oocytes was examined by confocal fluorescence microscopy using anti-tubulin antibodies and by time-lapse confocal microscopy of living oocytes microinjected with fluorescent tubulin. Assembly of a transient microtubule array from a disk-shaped MTOC was observed soon after germinal vesicle breakdown. This MTOC-TMA complex rapidly migrated toward the animal pole, in association with the condensing meiotic chromosomes. Four common stages were observed during the assembly of both Ml and M2 spindles: (1) formation of a compact aggregate of microtubules and chromosomes; (2) reorganization of this aggregate resulting in formation of a short bipolar spindle; (3) an anaphase-B-like elongation of the prometaphase spindle, transversely oriented with respect to the oocyte A-V axis; and (4) rotation of the spindle into alignment with the oocyte axis. The rate of spindle elongation observed in Ml (0.7 pm min-‘) was slower than that observed in M2 (1.8 pm mini). Examination of spindles by immunofluorescence with antitubulin revealed numerous interdigitating microtubules, suggesting that prometaphase elongation of meiotic spindles in Xenopus oocytes results from active sliding of antiparallel microtubules. A substantial number of maturing oocytes formed monopolar microtubule asters during Ml, nucleated by hollow spherical MTOCs. These monasters were subsequently observed to develop into bipolar Ml spindles and proceed through meiosis. The results presented define a complex pathway for assembly and rotation of the meiotic spindles during maturation of Xenopus oocytes. 0 1992 Academic Press. Inc.

Several features are known to distinguish the meiotic spindles of Xenopus oocytes from more typical mitotic In response to progesterone, stage VIXenopus oocytes and meiotic spindles in other animal cells (for reviews of are released from prophase-arrest and reenter the mei- spindle structure, see McDonald, 1989; McIntosh and otic cycle (reviewed in Gerhart, 1980). Despite recent Koonce, 1989). The meiotic spindles of Xenow oocytes progress in understanding the role of MPF and cyclins and eggs lack the characteristic centrioles and centroin resumption of the meiotic cycle (Masui and Marker& somes found at the poles of mitotic spindles in most 1971; Dunphy and Newport, 1988; Murray and animal cells (references in Gerhart, 1980; Huchon et ah, Kirschner, 1989a,b; Kobayashi et al., 1991; Minshull et 1981), exhibiting a barrel-shaped morphology and lackab, 1989,1991), little is known regarding the mechanics ing well-developed microtubule asters (Huchon et al, of spindle assembly and microtubule reorganization 1981; Karsenti et al, 1984; Dent and Klymkowsky, 1989). during maturation of Xenopus oocytes. In these respects, the meiotic spindles in Xenopus ooGerminal vesicle breakdown (GVBD) during maturacytes are similar to mitotic spindles in higher plants, tion of amphibian oocytes begins at the basal or vegetal which also lack centrioles (Bajer, 1972; McIntosh, 1983). surface of the GV (Brachet et ab, 1970; Huchon et ah, The second meiotic spindle in unfertilized Xenops 1981). Brachet et al. (1970) described a transient fibrous eggs is normally found tightly associated with the egg array that was found soon after initiation of GVBD dur- cortex, aligned parallel to the animal-vegetal (A-V) ing maturation of Xenopus oocytes. Similar arrays have axis of the egg (and therefore orthogonal to the oocyte also been observed in oocytes of other amphibian species surface) (Gerhart, 1980). In contrast, the first meiotic (Beetschen and Gautier, 1989, and references therein). spindles in maturing Xenopus oocytes have been found The fibrous arrays observed in maturing Xenops oo- in both axial and transverse orientations (Huchon et ab, cytes have subsequently been shown to consist of micro- 1981), suggesting that rotation of the meiotic spindles in Xenopus oocytes might occur subsequent to spindle astubules nucleated from a novel microtubule organizing center formed at GVBD (Huchon et al, 1981; and Jessus sembly. However, spindle rotation in Xenopus eggs has et ah, 1986). However, the role of this microtubule array not been directly demonstrated, and the mechanism by and MTOC during subsequent assembly of the meiotic which spindle position and orientation in Xenopus oocytes is determined remains largely unknown. spindles remained unclear. INTRODUCTION

0012-1606/92 $5.00 Copyright All rights

0 1992 by Academic Press, Inc. of reproduction in any form reserved.

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Spindle Assembly in Xenopus Oocytes

The scarcity of information regarding meiotic spindle assembly in Xenops oocytes stems from their size (>l mm diameter) and yolk-filled cytoplasm, attributes which render Xenopus oocytes and eggs completely opaque to conventional microscopic techniques. For this report, I have taken advantage of two features that facilitate analysis of spindle assembly during the meiotic maturation of Xenopus oocytes. First, spindles are assembled in a pigment- and yolk-free region of cytoplasm formed by GVBD (the white spot commonly used as a marker for maturation). Second, spindle assembly occurs in close proximity to the oocyte surface. These characteristics, in conjunction with the optical sectioning afforded by confocal microscopy, made possible both immunofluorescence of fixed oocytes and time-lapse studies of spindle assembly in living oocytes. The results presented define a complex pathway of spindle assembly, elongation, and rotation in Xenopus oocytes, and substantially add to our understanding of spindle mechanics in this important experimental system. MATERIALS

AND

METHODS

Chemicals were from Sigma Chemical Corporation (St. Louis, MO), unless otherwise indicated. Taxol was obtained from Dr. Matthew Suffness of the National Cancer Institute. Obtaining Oocytes and Eggs Xenopus laevis were obtained from Xenopus I (Ann Arbor, MI) and were injected with 50 units pregnant mare serum gonadotropin (Gestyl, Diosynth Inc., Chicago, IL) 3-14 days before use. Oocytes were isolated as previously described (Gard, 1991a). In some experiments, 10 mg/ml collagenase B (Boehringer Mannheim, Indianapolis, IN) was substituted for the desalted collagenase used previously. Ovulation was induced by injection of 50 units of human chorionic gonadotropin. Unfertilized eggs were dejellied with 2% cysteine (pH 8.0) prior to fixation for immunofluorescence.

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Fixation and Immunojluorescence Oocytes were fixed in formaldehyde-glutaraldehydetaxol as described previously (Gard, 1991a). Following rehydration in phosphate-buffered saline (PBS) (Gard, 1991a), oocytes were bisected with a scalpel, either laterally (parallel to the A-V axis) or equatorially. Bisected oocytes were incubated in 100 m&f NaBH, (in PBS) for 4-6 h at room temperature or overnight at 4°C. Oocytes were processed for immunofluorescence as described previously (Gard, 1991a) using DMlA, a monoclonal antibody recognizing cu-tubulin (Blose et al., 1984; ICN Biomedicals, Lisle, IL), or Tub-lA2, a monoclonal antibody recognizing tyrosinated a-tubulin (Kreis, 198’7; Sigma, St. Louis, MO). Rhodamine- and fluorescein-conjugated secondary antibodies were obtained from Orginon Cappell (Malvern, PA). Oocytes were dehydrated in methanol and cleared in benzyl-alcohol: benzyl-benzoate (1:2) (Dent and Klymkowsky, 1989; Gard, 1991a). Cleared oocytes were mounted in 0.5 mm depression slides in BA:BB. Laterally bisected oocytes were mounted cut surface up, providing a lateral cross section of the oocyte. Equatorially bisected oocytes were mounted cortical surface up, providing a polar view of the oocyte. Dual Fluorescence Microscopy of Microtubules and Chromosomes Oocytes were fixed in 100% methanol at room temperature (two changes), and were stored overnight in methanol at -20°C. Samples were processed for immunofluorescence with DMlA and fluorescein-conjugated secondary antibodies as previously described (Gard, 1991a). Oocytes were then incubated in propidium iodide (PI; 5 or 10 pg/ml; Molecular Probes, Inc. Eugene, OR) in 100% methanol (two changes for 15-30 min each), washed twice with 100% methanol (15-30 min each), cleared, and mounted as described above. While methanol is not an ideal fixative for individual microtubules (Gard, 1991a), preservation of meiotic spindles was sufficient to allow identification of the chromatin organization.

Maturation of Stage VI Oocytes Oocytes were incubated in 10 pg/ml progesterone in modified Barth’s saline H (MBSH: 88 mM NaCl, 1 mM KCl, 2.4 mMNaHCO,, 0.8 mMMgSO,, 0.3 mMCa(NO,),, 0.4 mM CaCl,, 10 mM Hepes, pH 7.4), and were monitored for the appearance of white maturation spots at the animal pole. The efficiency of maturation exceeded 90% in all experiments reported. Oocytes exhibiting WSF within 5- or lo-min intervals were collected, incubated for the indicated times in MBSH, and were fixed for immunofluorescence microscopy.

Confocal Microscopy of Fixed Oocytes Oocytes were examined on a Nikon optiphot equipped with a Bio-Rad MRC-600 laser scanning assembly using the GHS (rhodamine) filter set. 10X (NA 0.45), 40X (NA LO), and 60X (NA 1.4) objectives were used, giving optical section thicknesses of approximately 12.5, 2.6, and 1.0 pm, respectively. Images shown are Kalman averages of 6-25 successive scans (1 set/scan). A nonlinear output (provided by enhance settings of +l to +3 on the Bio-Rad MRC-600) was used during image collection to

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decrease the extreme contrast between the central spindle and individual microtubules (Gard, 1992). Dual fluorescence images of tubulin and PI were collected using the dual wavelength filter set for fluorescein and Texas red, either simultaneously or sequentially. Fluorescence crossover between the two channels was removed by subtraction. Tubulin and PI images were merged using a seven-bit alternate pixel format. Time-Lapse Confocal Microscopy of Living Oocytes Maturing oocytes were transferred to MBSH containing 5% ficoll immediately upon white spot formation. Fluorescein-conjugated bovine brain tubulin (Fl-Tb; Hyman et ak, 1990) (kindly provided by Dr. W. Sullivan, Univ. of California, Santa Cruz) was diluted to 2.5 mg/ ml in injection buffer (50 mM K glutamate, 0.5 mM MgCl,). Oocytes were microinjected with 100-200 ng of Fl-Tb within 10 min of WSF. This amount of tubulin corresponds to 20-40s of the endogenous oocyte tubulin (Gard and Kirschner, 1987). Injected oocytes were mounted in chamber slides in MBSH plus 5% ficoll and were examined on the Bio-Rad MRC-600 laser scanning confocal microscope described above, using the BHS (fluorescein) filter set. Neutral density filters were used to reduce the laser intensity to 1%. A 40X NA 1.0 plan apochromatic objective was used, providing optical sections 3-4 Frn in thickness. Linear summations of 4-8 optical sections (each a single 1-set scan) spanning 12-32 pm were collected to provide increased depth of field. Collection of a single stack of 4-8 sections required 8-16 sec. Stacks of sections were collected at approximately 2-min intervals. Spindle orientations were confirmed by serial optical sectioning at selected time intervals, using 40X or 60X objectives. Spindle lengths and diameters were measured using software supplied with the MRC-600 confocal microscope.

stained by both DMlA (anti cu-Tb; not shown) and TublA2 antisera (anti tyr-Tb; Fig. 1). The TMA rapidly migrated from its original site of assembly, near the basal surface of the GV, toward the animal pole of maturing oocytes (Huchon et ah, 1981). Within 10 min of WSF, the TMA in most oocytes was found within 50 pm of the animal surface (Fig. 1). Between 10 and 20 min after WSF, most oocytes contained a compact aggregate of microtubules in the animal hemisphere that was derived from the TMA. These aggregates exhibited an elongated or round profile in polar view (Fig. 2A). Often, a tenuous arc of microtubules extended from one side of the elongate MT aggregates (marked by arrows in Fig. 2A). Bipolar Ml spindles were occasionally observed as early as 20 min after WSF, but became more numerous at later times. The length of spindles observed early in Ml was quite variable, ranging from 15 to 30 pm (compare Figs. 2B and 2C). Most early spindles were barrelshaped, and often exhibited splitting of one or both spindle poles (as in Fig. 2B). Sparse microtubule asters appeared to be organized by the poles of some Ml spindles. However, it was often difficult to distinguish between microtubules of a polar aster, and microtubules penetrating from the opposite spindle pole (see Figs. 2B and 2C for examples). The majority of Ml spindles fixed between 20 and 40 min after WSF were oriented with the spindle axis transverse to the A-V axis, and thus parallel to the oocyte surface (such as the spindles in Figs. 2B and 2C). At later times, spindles were observed aligned with the AV axis, and thus orthogonal to the oocytes surface (Fig. 2D), or in intermediate orientations (not shown). The pole-to-pole length of axially oriented Ml spindles was more uniform than the early transversely oriented spindles, averaging 26 + 2 pm (SD n = 11). The average diameter of axially oriented Ml spindles was 17 t 3 pm (SD n = 35).

RESULTS

A Transient Microtubule Array Precedes Assembly of the Ml Spindle The earliest stages of germinal vesicle breakdown (GVBD) were observed in oocytes fixed prior to formation of an externally visible white maturation spot (WSF). Breakdown of the germinal vesicle was first apparent at its basal (or vegetal) surface (not shown), consistent with previous reports (Brachet et al, 1970; Huchon et al., 1981). Coincident with GVBD (prior to WSF), a transient microtubule array (TMA) was assembled from a disk-shaped MTOC formed near the basal region of the GV (Huchon et ah, 1981; Jessus et aZ.,1986). The MTOC and microtubules of the TMA were brightly

AssembZyof Monopolar Asters during Ml A substantial number of oocytes (147 oocytes from eight different females) fixed 20-60 min after WSF contained a single monopolar microtubule aster (Fig. 2E). Optical sectioning of these “monasters” (in both equatorial and lateral orientations) revealed roughly spherical shells about 10 pm in diameter, which were brightly stained with anti-tubulin (DMlA). Microtubules extending from these hollow shells formed asters approx. 30 ym in diameter, and often extended to within a few micrometers of the oocyte surface. The frequency of monasters varied substantially among different batches of oocytes. In most batches (6

DAVID L. GARD

Spindle Assembly in Xenopus Oocytes

FIG. 1. Assembly of transient microtubule array (TMA) during GVBD. (A) Lateral view of a Xenopus oocyte O-10 min after WSF. The TMA is closely apposed to the animal surface. (B) Lateral view of the TMA complex near the oocyte surface (arrows) O-5 min after WSF. Both oocytes were stained with Tub-lA2 (anti-tyrosinated tubulin). Scale bars are 125 @cmin A and 50 pm in B.

of 8), monasters were found in fewer than 10% of the oocytes fixed between 20 and 60 min after WSF. However, in two batches processed for immunofluorescence (and another used for time-lapse experiments, see below), monasters were found in 35-‘70% of the oocytes fixed during the same time period. In one batch, 88% (36 of 41) of the oocytes fixed between 20 and 30 min after WSF contained monasters. Oocytes from this batch, when fixed later in meiosis, contained normal Ml and M2 spindles, suggesting that monasters could develop into functional meiotic spindles. Cytokinesis and Assembly of the M2 Spindle The onset of cytokinesis was typically observed in oocytes fixed betwhen 80 and 120 min after WSF. The budding polar body was often found in a shallow depression in the oocyte surface (Fig. 2F). Immunofluorescence microscopy with DMlA revealed a small microtubule aster nucleated from the oocyte spindle pole during late anaphase-telophase. No evidence of an interphase nucleus was detectable in the variable interval between Ml and M2. After completion of cytokinesis, oocytes in early M2 were commonly observed to contain a disc-shaped aggregate of microtubules (Fig. 3A). These aggregates were more uniform in size and shape than those seen in early Ml, and exhibited little or no bipolar organization. Bipolar M2 spindles were found as early as 90 min after WSF in some oocytes, but became more prevalent in oocytes fixed more than 120 min after WSF. As in Ml (see above), the length of early M2 spindles varied con-

siderably, ranging from 15 to 30 pm (compare Figs. 3B and 3C). Most early M2 spindles (cl20 min after WSF) were observed in transverse or intermediate orientations with respect to the A-V axis, while M2 spindles in unfertilized eggs were commonly aligned with the A-V axis (Fig. 3D). However, it was not uncommon to find batches of unfertilized eggs in which a high proportion exhibited M2 spindles oriented transverse to the A-V axis (not shown). Axially oriented M2 spindles were similar in length (26 t- 3 pm; n = 15) and diameter (16 f 3 pm; n. = 21) to Ml spindles (see above). Microtubule asters were commonly observed radiating from the poles of both transverse and axially aligned M2 spindles. In the transverse spindle shown in Fig. 3C, the microtubule asters of the two poles appear approximately equal in extent, and microtubules from both poles extend to the oocyte surface. In axially aligned spindles, such as that shown in Fig. 3D, a sparse aster composed of short microtubules often extended from the interior spindle pole. Microtubules emanating from the cortical pole of axially aligned spindles contributed to a radially organized network of cortical microtubules, which merged with a less ordered microtubule network in the surrounding cortex (Gard, manuscript in preparation). Meiotic Spindles Rotate into Alignment with the A-V Axis To more firmly establish the chronology of spindle orientation with respect to the oocyte A-V axis, I examined the spindle orientation in more than 1000 oocytes

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FIG. 2. Immunofluorescence microscopy of spindle assembly during Ml. (A) Polar view of a microtubule aggregate lo-20 min after WSF (a projection of two adjacent sections). An arc of microtubules (arrows) connects the two ends of the elongated microtubule aggregate. (B, C) Polar views of transversely oriented Ml spindles 20-30 min after WSF, showing the variability of spindle length. One of the poles of the short spindle in B is split (arrow), a common feature of early Ml spindles. Microtubules extend from the poles of both spindles (see text). (D) Lateral view of an axially oriented Ml spindle at 60-70 min. A sparse aster of short microtubules (arrows) extends from the interior pole. (E) Polar view of a monaster 30-40 min after WSF. (F) Lateral view of an oocyte in late anaphase-telophase, 90-100 min after WSF. Cytokinesis of the first polar body (P) is nearly completed. A small microtubule aster is nucleated by the oocyte spindle pole. All stained with DMlA. Scale bars are 10 Wm.

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Spindle Assembly in Xenopus Oocytes

FIG. 3. Immunofluorescence microscopy of spindle assembly during M2. (A) Polar view of a microtubule aggregate preceding assembly of the M2 spindle, 80-90 min after WSF. (B) Polar view of a short M2 spindle transverse to the A-V axis, 120-150 min after WSF. (C)Lateral view of a long transversely oriented M2 spindle more than 180 min after WSF. Astral microtubules extend from both spindle poles to the cortex. (D) Lateral view of an axially aligned M2 spindle in an unfertilized egg. MT asters from the cortical pole appear to anchor the spindle to the cortex. A sparse aster of short microtubules (arrows) extends from the inner spindle pole (arrow). All stained with DMlA. Scale bars are 10 pm.

(956 Ml and 127 M2 spindles). As shown in Fig. 4, early in Ml (O-40 min after WSF) spindles were found predominantly in orientations transverse to the A-V axis (SO’90” from the A-V axis). The fraction of transverse spindles then declined, and the fraction of Ml spindles in an intermediate orientation (30”-60” from the A-V axis) peaked 50-60 min after WSF. The fraction of Ml spindles in axial alignment (within 30” of the A-V axis) rose steadily with time, and peaked immediately prior to or coincident with cytokinesis, 80-120 min after WSF. Most early M2 spindles (less than 120 min after WSF) were found in either transverse or intermediate orientation. By 180 min after WSF, nearly all M2 spindles were aligned within 30” of the A-V axis.

Chromosome Distribution Meiotic Spindles

during Assembly of the

Propidium iodide (PI) and DMlA anti-tubulin were used to examine the distribution of meiotic chromosomes during spindle assembly by dual-fluorescence confocal microscopy. PI-stained chromosomes were first apparent midway through the migration of the transient microtubule array (TMA) toward the animal pole, when the disk-shaped MTOC was loo-125 pm from the animal surface. In lateral cross section, chromosomes were observed in the basal regions of the MTOCTMA complex, often located near one edge (not shown). In polar views, oocyte chromosomes were commonly ob-

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ited metaphase chromosome organization with a welldefined metaphase plate (Figs. 51 and 5J).

M2 Spindles

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Time-Lapse Confocal Microscopy of Spindle Assembly in Living Oocytes

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Time-lapse confocal microscopy of living Xenopus oocytes microinjected with fluorescein-conjugated tubulin 30 (FLTb; Hyman et ak, 1990) was used to confirm the sequence of spindle assembly observed in fixed oocytes by immunofluorescence microscopy. Modified (fluorescent or biotinylated) tubulins have been widely used to exam40 50 60 70 60 > 60 cl20 150 160 ine the dynamics and organization of microtubules in Time after WSF (min) vitro and in vivo. While exhibiting some impairment of q Tram spindles 4 Inter spindles 0 Axial spindles assembly rates (Belmont et al, 1990; Sawin and Mitchison, 1991), modified tubulins have been found to accuFIG. 4. Spindle orientations during Ml and M2. The percentage of spindles in transverse (stippled), intermediate (black), or axial rately reflect the organization and dynamics of unmodi(white) orientations was determined at the end of the indicated interfied or endogenous tubulins (Salmon et al., 1984; Saxton vals (10 min in Ml, n = 956; 30 min in M2, ?z= 127). et al., 1984; Saxton and McIntosh, 1987; Sammak and Borisy, 1988; Belmont et al, 1990; Sawin and Mitchison, 1991). served clustered toward one side of the MTOC-TMA Time-lapse sequences of spindle assembly (Ml, M2, or complex, and were surrounded by a region of increased both) were obtained from more than 25 oocytes (from a microtubule density (Fig. 5A). Condensed chromosomes total of 10 different females) injected with Fl-Tb. More were typically found embedded within the variably than 20 additional oocytes were injected with Fl-Tb and shaped microtubule aggregates of oocytes fixed O-20 examined by serial optical sectioning at one or more min after WSF (Fig. 5B), and were evident both stages of meiosis. No evidence of laser-induced photosurrounding the monasters in oocytes fixed 20-60 min damage was apparent in Fl-Tb-injected oocytes folafter WSF (Fig. 5C), as well as within the hollow MTOC lowed by time-lapse confocal microscopy. Spindles obof the monasters (not shown). served in living oocytes by incorporation of Fl-Tb inTransversely oriented Ml spindles of all sizes exhib- jected with FI-Tb and followed in time lapse (in some ited chromosome configurations consistent with prome- instances for more than 2 hr), were similar in size, appearance, and chronology to those observed in fixed ootaphase (Figs. 5D and 5E). The chromosome distributions in spindles with intermediate orientation were cytes. The lack of apparent photodamage may result also consistent with prometaphase (not shown). In con- from the large size and tubulin content of Xenopus ootrast, well-defined metaphase chromosome configura- cytes (Gard and Kirschner, 1987). Photobleaching of flutions were commonly found in axially oriented Ml spin- orescent spindles was apparent during extensive optical dles (Fig. 5F), and later stage Ml spindles (anaphase sectioning at single time points. However, spindle fluoand telophase) were almost invariably in an axial orien- rescence rapidly recovered in the interval between images, due to the rapid turnover of spindle microtutation. Unlike the chromatin of the polar body, which formed bules (Salmon et al., 1984; Saxton et ak, 1984). The correa highly compacted mass during late telophase of Ml, spondence between the results obtained by immunofluorescence microscopy and those obtained by time-lapse individual oocyte chromosomes remained distinguishable within a highly compacted microtubule aggregate confocal microscopy of living oocytes suggests that both (Fig. 5G). No evidence of an interphase nucleus was techniques accurately depict the sequence of meiotic found in oocytes fixed in the interval between comple- spindle assembly. Within 20-30 min after WSF (lo-20 min after injection of Ml and assembly of the M2 spindle. In this regard, meiotic “interphase” in Xenopus oocytes was simi- tion), Fl-Tb was incorporated into a brightly fluorescent lar to that in eggs of clams and starfish (Doree et aZ., microtubule aggregate near the animal pole (Fig. 6A at 28 min). These fluorescent microtubule aggregates were 1983; Meijer and Guerrier, 1984). Transversely oriented M2 spindles exhibited chromo- variable in shape and were similar in overall size and some distributions consistent with prometaphase (Fig. appearance to the aggregates of microtubules and chro5H). Axially aligned spindles of oocytes matured in vi- mosomes observed in oocytes examined by immunofluotro, and in ovulated unfertilized eggs, commonly exhib- rescence. In most instances, these microtubule aggre-

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FIG. 5. Dual fluorescence microscopy of chromosome distribution and spindle assembly during meiosis. (A) Polar view of the basal region of the TMA complex at O-10 min after WSF. Chromosomes (arrows) are surrounded by a region of increased microtubule density (sum of three sections). (B) Polar view of a microtubule aggregate 5-15 min after WSF (sum of two sections). Condensed chromosomes are embedded within the elongate microtubule aggregate. (C) Condensed chromosomes surround this monaster, 35-45 min after WSF (polar view, sum of two sections). (D, E) Transversely oriented Ml spindles at 20-30 min after WSF have dispersed chromosomes, consistent with prometaphase. (F) Lateral view of an axially aligned Ml spindle with distinct metaphase plate, 70-80 min after WSF. (G) Chromatin in the polar body(P) forms a compact mass just after completion of cytokinesis. Oocyte chromosomes are embedded in a dense microtubule aggregate surrounding the oocyte spindle pole (projection of four sections). (H) Polar view of an early prometaphase M2 spindle at 90-100 min after WSF (projection of three sections). (I) Lateral view of an axial M2 spindle with well-defined metaphase plate. The polar body is evident to the upper left (P). (J) Polar view of an M2 metaphase plate in an unfertilized egg. Scale bars are 10 pm in A-I and 5 pm in J.

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FIG. 6. Time-lapse confocal microscopy of spindle assembly and rotation during Ml. (A) An example of Ml spindle assembly from a compact aggregate of microtubules, seen by time-lapse confocal microscopy. The approximate elapsed times (since WSF) are indicated. Small arrows at 28 min indicate an arc of microtubules similar to those seen in fixed oocytes. A plot of spindle length versus time is also shown in Fig. 8A (solid circles). (B) Selected images from a time-lapse sequence showing rotation of a bipolar Ml spindle from transverse (46 min) to axial orientation (101 min). At 64 min the spindle appears foreshortened, due to perspective. Scalloping along the edges of the axially oriented spindle at 101 min (arrows; just prior to onset of anaphase) represents the positions of metaphase chromosomes. The polar body (arrows) and M2 spindle (oriented intermediate to the section plane) are shown at 186 minutes (the M2 spindle appears foreshortened due to perspective). Bars are 10 pm.

gates underwent a variable amount of compaction (Fig. 6A at 28-36 min) before rapidly elongating into a bipolar spindle-oriented transversely to the A-V axis of the oocyte (Fig. 6A at 40-56 min). The axis of spindle elongation bore no consistent relationship to that of the preceding condensation or compaction. Rotation of an Ml spindle, from its initial orientation transverse to the oocyte axis, into alignment with the A-V axis is shown in Fig. 6B. The timing, rate, and extent of spindle rotation varied considerably among oocytes. In five oocytes followed by time lapse, rotation of the Ml spindles was observed to begin 35-50 min after WSF, and spindles reached axial orientations 65-85 min after WSF.

Anaphase in Fl-Tb-injected oocytes was observed 95110 minutes after WSF. Formation of a recognizable polar body, observed in 10 of 15 oocytes followed through late Ml (see Figs. 6B, 7A, 7B, and 9), occurred between 110 and 155 min after WSF. The spindle poles retained by oocytes after completion of cytokinesis appeared as compact fluorescent foci approximately 3-5 grn in diameter (Fig. 7A at 116 min.). These microtubule foci gradually increased in both apparent brightness and diameter (Fig. 7A, 116-128 min), before rapidly elongating into bipolar M2 spindles (Fig. 7A, 132-148 min). In all cases examined to date (complete time-lapse sequences of M2 in more than 10 oocytes, and several partial sequences), elongation of the

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FIG. 7. Time-lapse confocal microscopy of spindle assembly and rotation during M2. (A) A time-lapse sequence showing elongation of an M2 spindle. The polar body (P; outlined with small arrows) is apparent at 116 min (since WSF), along with the oocyte spindle pole (large arrow). The M2 spindle rapidly elongates transverse to the A-V axis (132-148 min.). A plot of spindle length versus time is also shown in Fig. 8B (solid circles). (B) A transverse M2 spindle rotates into axial alignment. The polar body (P in the 150-min panel) is apparent. Note the migration of the spindle from its initial location (an arrow at 178 min marks the initial position of the upper spindle pole). Scale bars are 25 pm.

M2 spindle occurred transverse to the A-V axis of the oocyte. Rotation of the M2 spindle, from its original transverse orientation into alignment with the A-V axis, was

followed by time-lapse microscopy in five oocytes (Fig. 7B). Spindle rotation was observed between 120 and 240 min after WSF, agreeing well with the results of immunofluorescence microscopy. In several examples, such as

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filled circles correspond to the oocyte shown in Fig. 7A). The mean maximum rate of M2 elongation was 1.8 pm min-’ (kO.3; n = 7), occurring over a span of 4-8 min. The entire phase of elongation lasted lo-15 min, during which time the maximum length increased from approx. 7-10 pm to 25-30 pm. The maximum lengths of the M2 spindles observed in oocytes injected with Fl-Tb were similar to those observed in fixed oocytes by immunofluorescence microscopy (see above). The differences between the rates of spindle elongation in Ml and M2 were probably not due to variability between oocytes, since two of the oocytes for which data are presented in Fig. 8 were followed in both Ml and M2 (open diamonds and open squares in 8A and 8B).

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Time (5 minute intervals)

FIG. 8. Rates of spindle elongation differ during Ml and M2. (A) Lengths of three Ml spindles plotted versus elapsed time. Spindles are plotted against an arbitrary time scale, to more clearly show the individual changes in length. The actual elapsed times (since WSF) at the start of each plot were 28 min (filled circles, shown also in Fig. 6A), 21 min (open diamonds), and 26 min (open squares). (B) The lengths of five M2 spindles are plotted against time. The actual elapsed starting times (since WSF) were 168 min (open circles), 161 min (open triangles), 124 min (filled circles, also shown in Fig. ‘7A), 132 min (open diamonds, from the same oocyte as the Ml spindle plotted with open diamonds in Fig. 8A), and 121 min (open squares, from the same oocyte as the Ml spindle plotted with open squares in Fig. 8A).

Monaster Formation in Living Oocytes Six oocytes (from two females) injected with Fl-Tb developed round or slightly elongate fluorescent microtubule aggregates which appeared hollow in optical sections (Fig. 9 at 19-39 min). These hollow microtubule aggregates were similar in size and appearance to the monasters observed in fixed oocytes by immunofluorescence (compare Figs. 2C and 9). Two oocytes containing monasters were followed by time-lapse microscopy. In these oocytes, monasters persisted for about 30 min (from 25 to 50 min after WSF in the example shown in Fig. 9), and then rapidly developed into bipolar spindles (Fig. 8 at 56-64 min), rotated into axial alignment (see below), and proceeded through anaphase (completion of cytokinesis is shown at 124 min in Fig. 9). Five of the six Fl-TB-injected oocytes containing monasters in early Ml completed anaphase of Ml (the sixth oocyte lysed prior to reexamination), and at least four assembled M2 spindles.

that shown in Fig. 7B, M2 spindles were observed to migrate a substantial distance during or just prior to rotating into axial alignment. In time lapse, spindle migration appeared to be a continuous movement, often resulting in positioning of the M2 spindle up to 50 pm from the first polar body. Quantitative analysis of the elongation of three Ml and five M2 spindles is shown in Fig. 8. A period of comDISCUSSION paction is evident in two of the three Ml spindles shown A Transient Microtubule Array Precedes Assembly of (Fig. 8A; filled circles correspond to the spindle shown the Ml Spindle in Fig. 6A). During this period, the maximum length of the nascent spindle decreased at a rate of l-2 pm min-‘. GVBD in amphibian oocytes begins at the vegetal, or The third spindle was first observed at the onset of elon- basal, surface of the GV (Brachet et ak, 1970; Huchon et gation. All three examples of Ml spindles exhibited an al, 1981; this report). The onset of GVBD is followed by elongation phase lasting lo-15 min, during which the the assembly of a TMA from a disk-shaped MTOC assopole-to-pole spindle lengths increased at an average ciated with the basal region of the GV (Brachet et ak, rate of 0.7 pm min-l (+O.l; n = 3). The extent of Ml 1970; Huchon et al., 1981; Jessus et al., 1986; Beetschen elongation ranged from 6 to 13 pm in the oocytes shown. and Gautier, 1989; this report). This unique MTOC-miThe maximum lengths of Ml spindles observed in Fl- crotubule array then rapidly moves toward the animal Tb-injected oocytes ranged from approximately 24 to 30 pole, through the nucleoplasm released by breakdown of pm, and were thus similar to those observed in fixed the germinal vesicle. The close association between the oocytes examined by immunofluorescence microscopy condensed meiotic chromosomes and the TMA (Huchon (see above). et al., 1981; Jessus et al., 1986; Beetschen and Gautier, Elongation of M2 spindles was consistently more 1989; this report), suggests that this microtubule array rapid and extensive than that observed in Ml (Fig. 8B, functions to gather and transport the oocyte chromo-

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Spindle Assembly in Xenopus Oocytes

527

FIG. 9. Time-lapse confocal microscopy of monaster and spindle assembly. A round microtubule aggregate (at 19 min after WSF) developed into a hollow monaster (29 and 39 min) which persisted for about 30 min, before rapidly elongating to form a bipolar spindle (56 and 64 min; spindle appears foreshortened due to perspective). The oocyte then completed Ml and cytokinesis, forming the first polar body (arrows denote margins of polar body; M denotes the midbody). Scale bar (shown at 19 min) is 10 pm at 19-64 min., and 25 pm at 124 min.

somes to the animal pole, as well as serving as the immediate precursor of the Ml spindle. The mechanism underlying the pole-ward migration of the TMA during oocyte maturation, at rates of lo-20 pm min-l, has yet to be conclusively established. Ryabova et al. (1986) have suggested that microfilaments, and not microtubules, are responsible for migration and anchoring of the meiotic spindles in maturing Xenopus oocytes. In contrast, Beetschen and Gautier (1989) reported that colcemid and nocodazole inhibit nuclear migration in maturing oocytes of axolotl. We are currently reevaluating the role of f-actin and microtubules in the assembly and migration of this developmentally important microtubule array during oocyte maturation. Spindle Assembly, Elongation, and Rotation during Oocyte Maturation Immunofluorescence microscopy and time-lapse confocal microscopy of living oocytes injected with fluorescein-tubulin revealed four stages common to the assembly of the first (Ml) and second (M2) meiotic spindles during maturation of Xenopus oocytes: (1) Formation of a compact aggregate of microtubules associated with the condensed meiotic chromosomes;

(2) Reorganization of the microtubules-chromosome aggregate and formation of a short bipolar spindle; (3) Rapid elongation of the spindle during prometaphase, in an orientation transverse to the oocyte A-V axis; (4) Rotation of the spindle into alignment with the A-V axis. Both meiotic spindles were assembled from compact aggregates of microtubules immediately surrounding the condensed meiotic chromosomes. During Ml, the nascent spindle formed from the transient microtubule array assembled at GVBD, and was variable in shape and extent of compaction. In contrast, the microtubulechromosome aggregate which precedes the M2 spindle was more homogeneous in size and shape, a possible consequence of its assembly from the oocyte spindle pole remaining after Ml. Formation of these microtubule-chromosome aggregates suggests that chromatin might play an important role in the early stages of spindle assembly and organization in Xenopus oocytes, as has been previously proposed for Xenops eggs and other cell types (Karsenti et ah, 1984; Church et al., 1986; Steffen et al., 1986; Sawin and Mitchison, 1991; Theurkauf and Hawley, 1992; see

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references therein for additional discussion of the role of chromatin in spindle assembly). Sawin and Mitchison (1991) have suggested that bipolar spindle morphology is established and maintained by a hierarchy of interactions between (1) chromosomes and microtubules, and (2) anti-parallel microtubules emanating from the spindle poles. While the general outline of meiotic spindle assembly in Xenopus oocytes is consistent with this model, the mechanism by which the initial bipolarity of acentrosomal meiotic spindles is established remains unclear. The rapid elongation of meiotic spindles observed in livingxenopus oocytes is consistent with the wide range of lengths observed for transversely oriented spindles in early Ml and M2. The numerous interdigitated microtubules apparent in immunofluorescence micrographs of elongating spindles (especially Figs. 2D-2E and 5B) suggests that active sliding of anti-parallel microtubules powers spindle elongation inXenopusoocytes. Similar mechanisms have been postulated for anaphase B elongation of mitotic spindles (Leslie and PickettHeaps, 1983; Saxton and McIntosh, 1987; Masuda et al., 1988). The rates of spindle elongation in Xenow oocytes, 0.7 pm mind1 in Ml and 1.8 pm min? in M2, are comparable to those reported for anaphase B in other cell types (reviewed in Heath, 1980; Baskin and Cande, 1988). Somewhat surprisingly, elongating meiotic spindles in Xenopus oocytes exhibited dispersed chromosome distributions consistent with prometaphase. The aggregation-elongation sequence of assembly distinguishes meiotic spindles in Xenoms oocytes from the mitotic spindles observed during the rapid cleavage stages of early Xenom embryos (Gard et ah, 1990) or in Xenopus egg extracts in vitro (Sawin and Mitchison, 1991). In most mitotic cells, including those of Xenoms blastulae (Gard et ab, 1990), duplication and separation of the spindle poles occurs prior to mitosis (reviewed in McIntosh, 1983; Mazia, 1987; Vandre and Borisy, 1989; McIntosh and Koonce, 1990; see Heath, 1980 for a discussion of variations of mitosis in lower eukaryotes). While meiotic spindles in amphibians lack the morphologically distinguishable centrioles or centrosomes associated with the spindle poles of other animal cells (Gerhart, 1980; Huchon et aZ.,1981), several studies suggest that Xenopus eggs and embryos contain a maternal pool of centrosome components (Jessus et ah, 1986; Gard et al., 1990; Verde et al., 1991; Stearns et al., 1991). The aggregation-elongation pathway of meiotic spindle assembly observed in Xenopus oocytes could serve to recruit and partition maternal centrosome components to the poles of the meiotic spindles. Similar patterns of spindle organization and elongation have been observed during meiosis in Drosophila (Theurkauf and Hawley, 1992) and mouse (Wassarman and Fujiwara, 1978) oo-

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cytes, which also lack centrioles (Theurkauf and Hawley, 1992; Szollosi et aZ.,1972). Thus, the observed pathway of microtubule aggregation, spindle organization, and elongation may be a common feature in the assembly of acentrosomal meiotic spindles. Both meiotic spindles in Xenopus oocytes were initially observed transverse to the A-V axis, and subsequently rotated into axial alignment during prometaphase. These results clarify previous observations that meiotic spindles of Xenopus oocytes were found in both transverse and axial orientations (Huchon et a& 1981). Rotation of spindles or MTOC axes with respect to a developmental axis has been described in eggs or embryos of both plants and animals (Hyman and White, 1987; Fernandez et aZ.,1990; Kropf et aZ.,1990; Beetschen and Gautier, 1989) and is normally associated with asymmetric or determinative cell divisions. While the mechanisms underlying spindle rotation have not been established, microtubules have been implicated in rotation of the spindle axis in embryos of C. elegans (Hyman, 1989) and Pelvetia (Kropf et a& 1990), and in leech oocytes (Fernandez et al, 1990). The observation of astral microtubules extending from the spindle poles to the cortex of Xenopus oocytes (as in Fig. 5D) is consistent with the involvement of microtubules in spindle rotation. Monaster Assembly during Maturation Oocytes

of Xenopus

The functional and physiological significance of the monasters observed during maturation of Xenms oocytes remains unclear. These monasters, which were similar in appearance to those observed in fragmented echinoderm eggs (Sluder et al, 1989), were found in a minority of oocytes (-40%) from most frogs. However, up to 88% of the maturing oocytes from some females formed monasters. Both immunofluorescence and timelapse microscopy suggested that after persisting in oocytes for 30-60 min, monasters could continue through meiosis. We cannot at this time distinguish between the possibilities that monasters represent a second pathway for assembly of the first meiotic spindle or a temporarily arrested stage in the more commonly observed pathway of spindle assembly. Assembly of the meiotic spindles is but one example of the substantial morphological and physiological changes accompanying oocyte maturation (Gerhart, 1980; Bement and Capco, 1990). The dramatic reorganization of the cytoplasmic microtubule network which accompanies maturation of Xenopus oocytes (Gard, 1991b) will be described in a subsequent report. In summary, confocal immunofluorescence and timelapse microscopy revealed a complex pathway for assem-

DAVID L. GARD

Spindle Assemblyin XenopusOocytes

bly and rotation of the meiotic spindles during maturation of Xenop oocytes. Given the expanding use of Xenoms egg extracts for studying cell cycle regulation and microtubule dynamics, maturing Xenopw oocytes provide an interesting and tractable model for examining the regulation of spindle assembly, dynamics, and localization during the meiotic cell cycle in V&O. The author thanks members of my lab, Drs. M. Beckerle, D. Kropf, and E. King, for helpful discussions during the course of these~experi’ ments and preparation of this manuscript. Alison Friend, Valerie Danielson, and Amy Roeder assisted with some of the experiments. Thanks to Dr. W. Sullivan for generously providing the fluoresceintubulin used in these experiments and Dr. M. Suffness for supplying taxol. Special thanks to Dr. Ed King for his invaluable assistance with the confocal microscope facility.

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