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Journal of Cell Science 109, 911-918 (1996) Printed in Great Britain © The Company of Biologists Limited 1996 JCS3380

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Meiotic spindle organization in fertilized Drosophila oocyte: presence of centrosomal components in the meiotic apparatus Maria Giovanna Riparbelli* and Giuliano Callaini Department of Evolutionary Biology, University of Siena, Via Mattioli 4, 53100 Siena, Italy *Author for correspondence

SUMMARY We examined spindle reorganization during the completion of meiosis in fertilized and unfertilized oocytes of Drosophila using indirect immunofluorescence and laser scanning confocal microscopy. The results defined a complex pathway of spindle assembly during resumption of meiosis, and revealed a transient array of microtubules radiating from the equatorial region of the spindle towards discrete foci in the egg cortex. A monastral array of microtubules was observed between twin metaphase II spindles in fertilized and unfertilized eggs. These microtubules originated

from disk-shaped material stained with Rb188 antibody specific for an antigen associated with the centrosome of Drosophila embryos. The Drosophila egg, therefore, contains a maternal pool of centrosomal components undetectable in mature inactivated oocytes. These components nucleate microtubules in a monastral array after activation, but are unable to organize bipolar spindles.

INTRODUCTION

Drosophila melanogaster females, meiosis arrests at metaphase I and resumes irrespective of sperm entry (Doane, 1960) after passage through the oviduct (King, 1970; Mahowald and Kambysellis, 1980). Since chromosome and spindle organization during meiosis I of Drosophila oogenesis have been studied, but little information is available about meiosis II, we performed cytological studies in fertilized and unfertilized eggs with antibodies against α-tubulin. Our aim was to follow the spindle microtubule dynamic after the resumption of meiosis. Since the formation of a functional zygotic centrosome is a fundamental step during fertilization, we also tested for immunoreactive material with the Rb188 antibody which is specific for a 190 kDa protein (CP190) associated with the centrosome of the Drosophila embryo (Whitfield et al., 1988, 1995). The study of centrosomal components in the activated oocyte could help to clarify the mode of centrosome inheritance and the role of the male gamete in this process.

Although mitosis and meiosis are similar processes, they are regulated differently, and some meiosis-specific genes have been identified (Bishop et al., 1992; Engebrecht and Roeder, 1989; Esposito and Esposito, 1969; Hollingsworth and Byers, 1989). Moreover, chromosome segregation occurring in mitosis leads to the formation of genetically identical daughter cells, while the cells arising from the meiotic process contain only one member of each chromosome pair of the parental cell. Homologous chromosome segregation occurs in meiosis I, and sister chromatids segregate during meiosis II, as in mitosis. The meiotic process is arrested at specific points during oogenesis (Murray, 1992) and generally resumes after fertilization. The structure of the meiotic spindle apparatus has been extensively studied in oocytes and it has been observed that spindle assembly may differ in significant ways in meiosis and mitosis. Among these differences is the lack of astral microtubules in many meiotic systems (Rieder et al., 1993). Studies in the mouse, Xenopus and insects, have shown that the meiotic spindles do not exhibit astral microtubules and centrioles, as in mitotic spindles of higher plants (Sawin and Endow, 1993). Cytological studies of Drosophila oocytes have revealed a structurally atypical meiotic spindle. The meiotic spindle poles of Drosophila oocytes are unusual in that they are unable to nucleate astral microtubules and most of the microtubules of the spindles do not terminate at the poles (Theurkauf and Hawley, 1992; McKim et al., 1993). In female Drosophila, the chromatin plays a key role during meiotic spindle assembly, and any microtubule organizing material at the poles has not been found to be involved in this process (Hatsumi and Endow, 1992; Theurkauf and Hawley, 1992; McKim and Hawley, 1995). In

Key words: Drosophila, Meiosis, Spindle organization, Microtubule organizing center

MATERIALS AND METHODS Reagents Microtubules were detected with a monoclonal antibody against αtubulin (Amersham, Buckinghamshire, UK). Centrosomal material was detected with the Rb188 antibody (Whitfield et al., 1988) that was shown previously to be specific for a 190 kDa protein (CP190) associated with the Drosophila centrosome (Frasch et al., 1986; Oegema et al., 1995; Whitfield et al., 1995). Nuclei were visualized with the specific dye Hoechst 33258. Secondary antibodies were either goat anti-mouse- or goat anti-rabbit-conjugated IgG (Cappel, West Chester, PA) conjugated with fluorescein or rhodamine. Bovine serum albumin (BSA) was obtained from Sigma.

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Collection of oocytes Drosophila melanogaster (Oregon-R) flies were raised in groups of 50 males and 25 females on standard cornmeal, agar and yeast medium in 200 ml plastic containers. Eggs from 4-5-day-old flies were collected at 24°C on small agar plates supplemented with acetic acid and yeast for 30 minutes and then collected five times more for 10 minutes each. Egg precollection needs to have fertilized oocytes at the same stage, since females store fertilized eggs for different periods of time. Groups of virgin females (n=50) were also raised in plastic containers. After discarding the first eggs as above, fertilized eggs were collected again four times for 5 minutes and four times for 15 minutes. Five sets of collections were performed, at both intervals, from four groups of rapidly laying females and we obtained 572 oocytes. Unfertilized eggs were collected from virgin females ten times for 5 minutes and we obtained 55 oocytes. Fertilized and unfertilized oocytes were dechorionated in a 50% bleach solution, washed with distilled water, fixed and the vitelline envelope removed as described by Warn and Warn (1986). Briefly, dechorionated eggs were washed in distilled water, dried on filter paper and the vitelline envelope was removed by transferring the embryos to a small vial containing 3 ml n-heptane and 3 ml of a solution of 90% cold methanol in water. After shaking for 3 minutes the embryos without vitelline envelope were transferred into methanol for 7 minutes and then acetone for 5 minutes, both at −20°C. Counting the time needed to remove the chorion, the oocytes developed for a further 5 minutes before fixation. Fluorescence microscopy After fixation, the oocytes were washed three times in phosphate buffered saline (PBS) and incubated for one hour in PBS containing 0.1% bovine serum albumin (BSA). Oocytes were cut longitudinally with a razor blade and then incubated at room temperature with Rb188 antibody (dilution 1:400 in PBS/BSA). After 4-5 hours an antibody against α-tubulin (dilution 1:400 in PBS/BSA) was added for one hour. The oocytes were then rinsed three times for 10 minutes each in PBS/BSA and incubated for one hour in a mixture of secondary antibodies (dilution 1:600 in PBS/BSA). After rinsing in PBS the nuclei were stained by incubating the oocytes with 1 µg/ml Hoechst 33258 for 3-4 minutes. The oocytes were rinsed again in PBS and mounted on glass microscope slides in 90% glycerol containing 2.5% n-propyl gallate (Giloh and Sedat, 1982). Fluorescence observations were carried out with a Leitz Aristoplan microscope equipped with fluorescein, rhodamine and UV filters. Photomicrographs were taken with Kodak Tri-X 400 pro and developed in Kodak HC110 developer for 7 minutes at 20°C. Confocal microscopy The organization of microtubules in fertilized oocytes was observed using a MRC-500 laser scanning confocal microscope (Bio-Rad Microscience, Cambridge, MA) mounted on a Nikon optiphot with a ×60 Planapo. For double staining with anti-tubulin and anti-centrosome antibodies the oocytes were examined on a MRC-600 confocal microsope (Bio-Rad) with a two-laser equipment. Image collection was performed by Kalman averaging of 10-13 images to improve the signal-noise ratio. Images for pictures were contrast-enhanced in the Adobe Photoshop program in an IBM Aptiva Computer and printed on Kodak Tmax 100 ASA or Kodak Ektachrome Elite 100 ASA films using a Polaroid CI-3000 Digital Palette.

RESULTS There are two main difficulties in studying female meiosis in Drosophila: many yolk granules make immunofluorescence analysis of the internal features of whole mounts of oocytes very difficult, and females lay eggs at different stages of development, because they retain fertilized eggs in the uterus prior

Table 1. Classification of the figures scored Time of development* 10 minutes n (%)

20 minutes n (%)

Meiosis I Anaphase Telophase

63 (19.8) 55 (17.3)

Meiosis II Prophase Metaphase Anaphase Telophase

31 (9.7) 81 (25.5) 34 (10.7) 17 (5.3)

28 (11) 119 (46.8) 21 (8.3)

First cleavage Multinuclear stages

26 (8.2) 11 (3.5)

50 (19.7) 36 (14.2)

318

254

Total†

*Oocytes (left column) were collected for 5 minutes and fixed after 5 minutes. Oocytes (right column) were collected for 15 minutes and fixed after 5 minutes. †Oocytes were fixed and stained with anti-tubulin antibody and Hoechst dye to score meiotic and mitotic figures

to oviposition. To facilitate observation of the meiotic spindles we took advantage of their localization in yolk free cytoplasmic areas at the anterior end of the oocyte. By tilting the specimens under the microscope, we were able to observe spindle orientation and organization. To avoid difficulties caused by the fact that females retain fertilized oocytes for different periods of time we allowed flies to lay eggs several times before collecting eggs for our study (see Materials and Methods). Unfortunately, as reported in Table 1, we were Fig. 1. Immunofluorescence staining with anti-tubulin (A1-E1) and anti-centrosome (A2-E2) antibodies, and Hoechst dye (A3-E3) of fertilized oocytes during anaphase (A) and telophase (B) of meiosis I, metaphase (C), anaphase (D), and telophase (E) of meiosis II. (A1) The anaphase I spindle is orientated radially to the oocyte surface. (A2) The centrosomal material is not visible in the midzone of the spindle (arrows), and unspecific chromosome staining is observed. (A3, and inset) Dot-like chromosomes 4 (arrowheads) move precociously to the poles. (B1) The spindle elongates in telophase and microtubues radiate from the midzone (arrowhead). (B2) The centrosomal material is still undistinguishable (arrows). (B3) Chromosomes form two distinct masses at the poles. (C1) During metaphase II a monastral array of microtubules (arrowhead) is found between twin spindles aligned in tandem. (C2) A discrete cluster of centrosomal material (arrow) is detected at this time in form of a ring-like structure (inset) at the center of the monaster. (C3) Chromosomes are disposed in two distinct masses in the midzone of the spindles. (D1) At anaphase the monaster of microtubules is separated by a small gap from the twin spindles; inset, detail of a focus for subcortical microtubules. (D2) The centrosomal material (arrow) shows a typical complex substructure (inset). (D3) Haploid complements are aligned in tandem. (E1) Telophase II spindles still show tandem alignment. (E2) The centrosomal material (arrow) is labeled in a discontinuous manner (inset). (E3) The putative female pronucleus (arrowhead) is found deeper than the other chromosomes in the egg cytoplasm. Insets in B3, C3, and E3 show merged images of the centrosomal material (arrow) and chromosomes (arrowheads). Insets in the middle column shows high magnification images of the immunoreactive material detected by Rb188 antibody. Open arrows indicate the sperm aster in C1 and E1, and the sperm head in C3 and E3. Bar: 30 µm for panels; 20 µm for insets in A2, A3, B2, B3, C3, D1, D3, E3; 5 µm for insets in C2, D2, E2.

Meiotic spindle in fertilized Drosophila oocyte

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unable to obtain fully synchronized oocytes. Development had reached the first cleavage division in 13.3% and later stages in 8.2% of the eggs. However, we observed a large number of meiotic figures (78.5%), ranging from anaphase of the first division to telophase of the second division. Fertilized oocytes were easily identified by the sperm head and aster, which were visible in the oocyte throughout the meiotic process (Fig. 1). Spindle organization after resumption of meiosis in Drosophila oocyte Meiosis in Drosophila eggs is arrested at metaphase of the first meiotic division (King, 1970) and nonexchange chromosomes are typically positioned between the spindle plate and the poles (Puro and Nokkala, 1977; Puro, 1991) of highly tapered metaphase spindles without astral microtubules (Theurkauf and Hawley, 1992). After the oocyte passes through the oviduct (Mahowald and Kambysellis, 1980) or following artificial activation (Mahowald et al., 1983), the meiotic divisions resume. We found that the anaphase I spindle differed considerably from the tapered metaphase I spindle, since its midzone was very expanded (Fig. 1A1). At this stage, the spindle was always orientated at right angles to the anterior-posterior axis of the oocyte, with one pole very close to the oocyte surface. The anaphase I spindle was formed by two arrays of microtubules (Fig. 2A): (i) parallel microtubules that extended from one pole to the other, forming the main axis of the spindle; (ii) peripheral microtubules that originated from the poles and moved away from the longitudinal axis of the spindle giving a top-like shape to the meiotic apparatus. Peripheral microtubules met in the equatorial region of the spindle. Daughter chromosomes were clearly separated and the dot-like chromosome 4 was the first

Fig. 2. Gallery of meiotic figures showing microtubule organization in Drosophila fertilized oocytes. (A) Anaphase I; (B) telophase I; (C) prophase II; (D) metaphase II; (E) anaphase II; (F) telophase II. Arrows point to subcortical foci. Each image is a linear projection of 10 optical sections taken at 0.5 µm intervals. Bar, 10 µm.

to move towards the poles (Fig. 1A3). As the chromosomes approached the poles during telophase (Fig. 1B3) the spindle axis became more visible and the peripheral microtubules organized in discrete bundles that stretched from the spindle midzone towards small foci in the subcortical cytoplasm (Figs 1B1, 2B). The prophase II spindle had a thick core of parallel microtubules with a narrow midzone and peripheral microtubules mostly organized in short bundles (Fig. 2C). These bundles were no longer visible, or only slightly, in later meiotic stages. The second meiotic division led to the formation of a pair of twin spindles orientated radially with respect to the oocyte surface (Fig. 1C1). The spindle poles were typically anastral, but a monastral array of microtubules was observed between the spindles (Fig. 2D). The chromosomes were all massed at the metaphase plate, without precociously moving nonexchange chromosomes (Fig. 1C3). The monastral array of microtubules persisted between twin spindles throughout anaphase (Figs 1D1, 2E) and telophase (Figs 1E1, 2F). Thin bundles of microtubules linked the monaster and subcortical foci, from which microtubules radiated towards the oocyte surface (Fig. 1D1, inset). Towards the end of meiosis, the spindles became thinner and shorter and a large gap separated the monastral array of microtubules from the spindle poles (Fig. 2F). The innermost haploid complement, the putative female pronucleus, moved away from the monaster (Fig. 1D3,E3). After the male and female pronuclei fused together, the remnant haploid complements were found at the oocyte surface. The proximal spindle persisted for a short time (not shown) but the monastral array of microtubules was no longer visible. Meiotic spindle reorganization in activated oocytes is schematized in Fig. 3.

Meiotic spindle in fertilized Drosophila oocyte

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Fig. 3. Schematic representation of meiotic spindle organization in fertilized Drosophila oocytes. (A) Anaphase I; (B) telophase I; (C) prophase II; (D) metaphase II; (E) anaphase II; (F) telophase II.

Centrosomal material was a component of the meiotic apparatus in fertilized oocytes When fertilized oocytes were incubated with Rb188 antibody (Whitfield et al., 1988), that was shown previously to be specific for the CP190 centrosomal component of Drosophila embryo (Oegema et al., 1995; Whitfield et al., 1995), a stagespecific staining pattern was observed. Rb188 did not recognize distinct structures associated with anaphase and telophase I spindles (Fig. 1A2,B2). A distict ring was instead visible during metaphase II at the center of the monastral array of microtubules in the region between the twin spindles (Fig. 1C2). During anaphase II, the centrosomal material assumed a more complex staining pattern and internal radial substructures with a cartwheel-like disposition could be recognized (Fig. 1D2). During telophase the centrosomal material appeared as a thick, unevenly stained disc (Fig. 1E2). Immunoreactive

Fig. 4. Metaphase (A) and anaphase (B) spindles during the second meiotic division of fertilized eggs double stained for microtubules (green) and centrosomal material (orange) and analyzed by confocal microscopy. Note the association of the centrosomal material with the aster of microtubules. Bar, 10 µm.

material was no longer visible after telophase and disappeared when the haploid complements reached the surface of the oocyte (not shown). Double exposure revealed that the centrosomal material was located between the chromosome clusters at the end of telophase I and in metaphase II (Fig. 1B3,C3, insets), but very close to sister complements of tandem spindles in telophase II (Fig. 1E3, inset). Computer generated images of fertilized oocytes double labeled for tubulin and Rb188 showed the overlap between centrosomal material and the focus for aster microubules (Fig. 4A,B). Despite the shape changes during the second meiotic division, the diameter of the centrosomal structures did not show appreciable variations throughout meiosis, thickness increasing slightly from metaphase to telophase of the second meiotic division. No staining was found inside the subcortical foci, around which short microtubules were organized (Fig. 1B2,D2). However,

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Fig. 5. Anaphase II unfertilized egg stained with anti-tubulin (A) and anticentrosome (B) antibodies, and Hoechst dye (C). Arrow in B indicates centrosomal material. Bar, 15 µm.

due to the small size of these structures and the background of the yolk region, we cannot completely exclude the possibility that a feeble punctate labeling existed. Meiotic progression followed the same pathway in fertilized and unfertilized eggs Since the centrosomal material was found far from the sperm head, any participation of the male gamete in the assembly and behavior of this structure can presumably be excluded. However, to fully eliminate this eventuality and the possibility that the correct meiotic spindle assembly depended on sperm entry into the egg, we investigated the dynamics of the meiotic apparatus in unfertilized eggs. It is known that unfertilized eggs also complete the meiotic process after laying (Doane, 1960) and our data confirmed that the ability of the meiotic apparatus to assemble through meiosis was not affected by the absence of the male gamete. When we stained the microtubules and DNA in 55 newly laid unfertilized eggs, we observed spindle organization and chromosome configurations as described in fertilized oocytes. Centrosomal material was also found in these eggs. The meiotic spindles in unfertilized eggs were indistinguishable from corresponding spindles of fertilized eggs. Fig. 5 shows a typical anaphase II spindle, composed of twin spindles aligned in tandem, orientated at right angles to the oocyte surface and separated by a monastral array of microtubules. DISCUSSION In the Drosophila oocyte, meiosis is arrested in metaphase of the first division (King, 1970), when a tapered spindle aligned parallel to the egg surface, forms (Theurkauf and Hawley, 1992). The chromosomes are therefore located in the cortical region near the anterior pole, whereas fusion of parental complements occurs in the inner ooplasm. How does the female pronucleus reach the interior of the egg? In his pioneering work on the maturation and fertilization of the Drosophila oocyte, Huettner (1924) reported that the second meiotic spindles are arranged in tandem and disposed perpendicularly to the longitudinal axis of the egg with the innermost spindle carrying the female pronucleus. These observations were confirmed by Sonnenblick (1950) and more recently by Hatsumi and Endow (1992). This pattern of spindle organization is probably involved in the migration of the female pronucleus deeper into the egg near the cytoplasmic domain of the male pronucleus. Since the oocytes were fixed at least 5 minutes after laying, which was the time required to remove the chorion, we were unable to determine precisely when the meiotic spindle of Drosophila changed orientation. However, spindle rotation

from a position parallel to the egg surface to a radial orientation presumably occurs during or shortly after the oocyte passes through the oviduct, since several authors report early anaphase I spindles perpendicular to the oocyte surface (Huettner, 1924; McKim et al., 1993; Jang et al., 1995). Spindle rotation has been described during maturation of Xenopus oocytes. In this species the meiotic spindle was observed to move from its initial orientation parallel to the cortex into alignment with the animal-vegetal axis (Gard, 1992). How spindle orientation is achieved and maintained during meiosis is an intriguing question. Microtubules linking spindle poles to the oocyte surface have been implicated in rotation and anchoring of the meiotic apparatus in Xenopus oocytes (Gard, 1993) and in other organisms (Fernandez et al., 1991; Lutz et al., 1988; Satoh et al., 1994), but this does not seem to be the case in the Drosophila oocyte, since the meiotic spindles lack astral microtubules (Theurkauf and Hawley, 1992). However, the observation that a transient array of microtubules linked the meiotic apparatus to discrete subcortical foci suggests that in Drosophila also the orientation of the meiotic spindle requires a functional interaction between the spindle and the oocyte cortex. We cannot exclude the possibility that this transient microtubular array may be an artifact of fixation even if these microtubules have been always observed in our preparations. The microtubule array observed between twin spindles at metaphase, anaphase and telophase of the second meiotic division was presumably organized by discrete material stained by the Rb188 antibody and might be an intermediate between the anastral poles of the meiotic I spindles and the astral poles of the mitotic spindles in early embryos. This is an interesting finding, because microtubule organizing centers found during stages 1-6 of oogenesis in Drosophila were no longer detectable in older oocytes (Theurkauf et al., 1992). Though at least two centrosome-associated proteins, CP190 (Frasch et al., 1986; Whitfield et al., 1988, 1995), and γ-tubulin (Raff et al., 1993), are believed to be of maternal origin in Drosophila, microtubule organizing centers have not yet been detected in oocytes before the resumption of meiosis (Theurkauf and Hawley, 1992), perhaps because the centrosomal material occurs in an undetectable dispersed form. Microtubule nucleation resumed from discrete cytoplasmic foci after the Drosophila oocyte passed through the oviduct, suggesting that the centrosomal material undergoes functional changes correlated to oocyte maturation. This agrees with studies showing that centrosomes are undetectable in unfertilized sea urchin and surf clam oocytes, whereas microtubule assembly regains after artificial activation or parthenogenesis (Kuriyama et al., 1986; Palazzo et al., 1992; Schatten et al., 1992). These observations raise intriguing questions on the mechanism by which centro-

Meiotic spindle in fertilized Drosophila oocyte somal proteins are maintained in a quiescent state in the inactivated oocyte and the formation of a functional microtubule organizing center is triggered following activation. Since wildtype unfertilized Drosophila eggs (present data) and unfertilized eggs laid by females homozygous for the mutations gnu (Freeman et al., 1986) and asp (Gonzalez et al., 1990) also have functional microtubule organizing centers, the paternal contribution is not required for the activation of maternal centrosomal material, at least in this species. This material stimulates microtubule assembly in a monastral array after the Drosophila oocyte passed through the oviduct, but it is usually unable to direct the formation of a normal bipolar spindle. This is consistent with the finding of maternal centrosomes with intact microtubule nucleating properties, but incapable of reproducing or doubling, in starfish eggs (Sluder et al., 1989a). Ultrastructural studies indicate that the reproductive properties of the centrosomes are associated with centrioles in mature sea urchin eggs (Sluder et al., 1989b). Since centrioles have been detected only in the early stages of Drosophila oogenesis (Mahowald and Strassheim, 1970), the centrosomal material, visible after resumption of meiosis, is presumably devoid of such organelles, and is therefore incapable of reproducing in a normal fashion. The fact that a centrosomal antigen is conserved from the egg to the embryo indicates that the maternal contribution is essential for a fully functional centrosome in Drosophila. This agrees with the suggestion that the zygotic centrosome is assembled from paternal and maternal components (Holy and Schatten 1991; Félix et al., 1994; Stearns and Kirschner, 1994; Schatten, 1994; Archer and Solomon, 1994). These observations raise questions on the centrosome inheritance in certain parthenogenetic insects, such as aphids and hymenopterans, in which both fertilized and unfertilized eggs usually develop. In this case, the unfertilized egg is able to assemble functional centrosomes without paternal contribution. Since studies on Xenopus (Tournier et al., 1991) and surf clam oocytes (Palazzo et al., 1992) have suggested that all the necessary components for centrosome formation and reproduction are present in the unfertilized egg, specific mechanisms must exist to inactivate or activate maternal components during fertilization and parthenogenetic development, respectively. We thank Dr William G. F. Whitfield for the generous gift of the Rb188 antibody. We are also thankful to A. Minacci for his help with laser scanning confocal microscopy and L. Gamberucci for computer processing techniques. This work was partially supported by grants from Murst (40% and 60%), C.N.R., and the Human Capital and Mobility Program of the European Community (CHRX-CT94-0642).

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(Received 8 December 1995 - Accepted 5 February 1996)