Journal of Cell Science 109, 911-918 (1996)
Printed in Great Britain © The Company of Biologists Limited 1996
JCS3380
911
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.
912
M. G. Riparbelli and G. Callaini
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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M. G. Riparbelli and G. Callaini
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
915
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,
916
M. G. Riparbelli and G. Callaini
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).
REFERENCES
Archer, J. and Solomon, F. (1994). Deconstructing the microtubuleorganizing center. Cell 76, 589-591.
Bishop, D. K., Park, D., Xu, L. and Kleckner, N. (1992). DMC1: a meiosisspecific yeast homolog of E. coli recA required for recombination,
synaptonemal complex formation and cell cycle progression. Cell 69, 439456.
Doane, W. W. (1960). Completion of meiosis in uninseminated eggs of
Drosophila melanogaster. Science 132, 677-678.
Engebrecht, J. and Roeder, G. S. (1989). Yeast mer1 mutants display reduced
levels of meiotic recombination. Genetics 121, 237-247.
Esposito, R. E. and M. S. Esposito, M. S. (1969). The genetic control of
917
sporulation in Saccharomyces. I. The isolation of temperature-sensitive
sporulation-deficient mutants. Genetics 61, 79-89.
Félix, M., Antony, C., Wright, M. and Maro, B. (1994). Centrosome
assembly in vitro: role of γ-tubulin recruitment in Xenopus sperm aster
formation. J. Cell Biol. 124, 19-31.
Fernandez, J., Olea, N., Tellez, V. and Matte, C. (1991). Structure and
development of the egg of the Glossiphonid leech Theromyzon rude:
reorganization of the fertilized egg during completion of the first meiotic
division. Dev. Biol. 137, 142-154.
Frasch, M., Glover, D. M. and Saumweber, H. (1986). Nuclear antigens
follow different pathways into daughter nuclei during mitosis in Drosophila
embryos. J. Cell Sci. 82, 115-172.
Freeman, M., Nüsslein-Volhard, C. and Glover, D. M. (1986). The
dissociation of nuclear and centrosomal in gnu, a mutation causing giant
nuclei in Drosophila. Cell 46, 457-468.
Gard, D. L. (1992). Microtubule organization during maturation of Xenopus
oocytes: assembly and rotation of the meiotic spindles. Dev. Biol. 151, 516530.
Gard, D. L. (1993). Ectopic spindle assembly during maturation of Xenopus
oocytes: evidence for functional polarization of the oocyte cortex. Dev. Biol.
159, 298-310.
Giloh, H. and Sedat, J. W. (1982). Fluorescence microscopy: reduced
photobleaching of rhodamine and fluorescein protein conjugates by n-propyl
gallate. Science 217, 1252-1255.
Gonzalez, C., Saunders, R. D. C., Casal, J., Molina, I., Carmena, M.,
Ripoll, P. and Glover, D. M. (1990). Mutation at the asp locus of
Drosophila lead to multiple free centrosomes in syncytial embryos, but
restrict centrosome duplication in larval neuroblasts. J. Cell Sci. 96, 605-616.
Hatsumi, M and Endow, S. A. (1992). Mutants of the microtubule motor
protein, nonclaret disjunctional, affect spindle structure and chromosome
movement in meiosis and mitosis. J. Cell Sci. 101, 547-559.
Hollingsworth, N. and Byers, B. (1989). HOP1: a yeast meiotic pairing gene.
Genetics 121, 445-462.
Holy, J. and Schatten, G. (1991). Spindle pole centrosomes of sea urchin
embryos are partially composed of material recruited from maternal stores.
Dev. Biol. 147, 343-353.
Huettner, A. F. (1924). Maturation and fertilization in Drosophila
melanogaster. J. Morphol. Physiol. 39, 249-265.
Jang, J. K., Messina, L., Erdman, M. B., Arbel, T. and Hawley, R. S. (1995).
Induction of metaphase arrest in Drosophila oocytes by chiasma-based
kinetochore tension. Science 268, 1917-1919.
King, R. C. (1970). Ovarian Development in Drosophila melanogaster.
Academic Press Inc., New York.
Kuriyama, R., Borisy, G. G. and Masui, Y. (1986). Microtubule cycles in
oocytes of the surf clam, Spisula solidissima: an immunofluorescence study.
Dev. Biol. 114, 115-160.
Lutz, D. A., Hamaguchi, Y. and Inoue, S. (1988). Micromanipulation studies
of the asymmetric positioning of the maturation spindle in Chaetopterus sp.
oocytes: I. Anchorage of the spindle to the cortex and migration of a
displaced spindle. Cell Motil. Cytoskel. 11, 83-96.
Mahowald, A. P. and Strassheim, J. M. (1970). Intercellular migration of
centrioles in the germarium of Drosophila melanogaster. An electron
microscopy study. J. Cell Biol. 45, 306-320.
Mahowald, A. P. and Kambysellis, M. P. (1980). Oogenesis. In The Genetics
and Biology of Drosophila, vol. 2d (ed. M. Ashburner and T. R. F. Wright),
pp. 141-224. New York: Academic Press.
Mahowald, A. P., Goralski, T. J. and Caulton, T. H. (1983). In vitro
activation of Drosophila eggs. Dev. Biol. 98, 437-445.
McKim, K. S., Jang, J. K. Theurkauf, W. E. and Hawley, R. S. (1993).
Mechanical basis of meiotic metaphase arrest. Nature 362, 364-366.
McKim, K. S. and Hawley, R. S. (1995). Chromosomal control of meiotic cell
division. Science 270, 1595-1601.
Murray, A. W. (1992). Creative blocks: cell-cycle checkpoints and feedback
controls. Nature 359, 599-604.
Oegema, K., Whitfield, W. G. F. and Alberts, B. (1995). The cell cycledependent localization of the CP190 centrosomal protein is determined by
the coordinate action of two separable domains. J. Cell Biol. 131, 1261-1273.
Palazzo, R. E., Vaisberg, E., Cole, R. W. and Rieder, C. L. (1992). Centriole
duplication in lysates of Spisula solidissima oocytes. Science 256, 219-221.
Puro, J. and Nokkala, S. (1977). Meiotic segregation of chromosomes in
Drosophila melanogaster oocytes. Chromosoma 63, 273-286.
Puro, J. (1991). Differential mechanisms governing segregation of a univalent
in oocytes and spermatocytes of Drosophila melanogaster. Chromosoma
100, 305-314.
918
M. G. Riparbelli and G. Callaini
Raff, J. W., Kellogg, D. R. and Alberts, B. M. (1993). Drosophila γ-tubulin is
part of a complex containing two previously identified centrosomal MAPs. J.
Cell Biol. 121, 823-835.
Rieder, C. L., Ault, J. G., Eichenlaub-Ritter, U. and Sluder, G. (1993).
Morphogenesis of the mitotic and meiotic spindle: conclusions obtained
from one system are not necessarily applicable to the other. In Chromosome
Segregation and Aneuploidy (ed. B. K. Vig), pp. 183-197. Springer-Verlag,
Berlin.
Satoh, S. K., Oka, M. T. and Hamaguchi, Y. (1994). Asymmetry in the
mitotic spindle induced by the attachment to the cell surface during
maturation in the starfish oocyte. Dev. Growth Differ. 36, 557-565.
Sawin, K. E. and Endow, S. A. (1993). Meiosis, mitosis and microtubule
motors. BioEssays 15, 399-407.
Schatten, H., Walter, M., Biessmann, H. and Schatten, G. (1992).
Activation of maternal centrosomes in sea urchin eggs. Cell Motil. Cytoskel.
23, 61-70.
Schatten, G. (1994). The centrosome and its mode of inheritance: the reduction
of the centrosome during gametogenesis and its restoration during
fertilization. Dev. Biol. 165, 299-335.
Sluder, G., Miller, F. J., Lewis, K., Davidson, E. D. and Rieder, C. L.
(1989a). Centrosome inheritance in starfish zygotes: selective loss of
maternal centrosome after fertilization. Dev. Biol. 131, 567-579.
Sluder, G., Miller, F. J. and Rieder, C. L. (1989b). Reproductive capacity of
sea urchin centrosomes without centrioles. Cell Motil. Cytoskel. 13, 264-273.
Sonnenblick, B. P. (1950). The early embryology of Drosophila melanogaster.
In Biology of Drosophila (ed. M. Demerec), pp. 62-167. New York: Wiley
and Sons.
Stearns, T. and M. Kirschner, M. (1994). In vitro reconstitution of
centrosome assembly and function: the role of γ-tubulin. Cell 76, 623-637.
Theurkauf, W. E. and Hawley, R. S. (1992). Meiotic spindle assembly in
Drosophila females: behavior of nonexchange chromosomes and the effects
of mutations in the nod kinesin-like protein. J. Cell Biol. 116, 1167-1180.
Theurkauf, W. E., Smiley, S., Wong, M. L. and Alberts, B. M. (1992).
Reorganization of the cytoskeleton during Drosophila oogenesis:
implications for axis specification and intercellular transport. Development
115, 923-936.
Tournier, F., Cyrklaff, M., Karsenti, E. and Bornens, M. (1991).
Centrosomes competent for parthenogenesis in Xenopus eggs support
procentriole budding in cell-free extracts. Proc. Nat. Acad Sci. USA 88,
9929-9933.
Warn, R. M. and Warn, A. (1986). Microtubule arrays present during the
syncytial and cellular blastoderm stages of the early Drosophila embryo.
Exp. Cell Res. 163, 201-210.
Whitfield, W. G. F., Miller, S. E., Saumweber, H., Frash, M. and Glover, D.
M. (1988). Cloning of a gene encoding an antigen associated with the
centrosome in Drosophila. J. Cell Sci. 89, 467-480.
Whitfield, W. G. F., Chaplin, M. A., Oegema, K., Parry, H. and Glover, D.
M. (1995). The 190 kDa centrosome-associated protein of Drosophila
melanogaster contains four zinc finger motifs and binds to specific sites on
polytene chromosomes. J. Cell Sci. 108, 3377-3387.
(Received 8 December 1995 - Accepted 5 February 1996)