Download Organisation of Xenopus oocyte and egg cortices

MICROSCOPY RESEARCH AND TECHNIQUE 44:415–429 (1999)
Organisation of Xenopus Oocyte and Egg Cortices
PATRICK CHANG, DANIEL PÉREZ-MONGIOVI, AND EVELYN HOULISTON
Unité de Biologie Cellulaire Marine (UMR. 643 CNRS -Université Paris VI), Station Zoologique 06230 Villefranche-sur-mer, France
KEY WORDS
cytoskeleton; deep etch; microtubule; actin; intermediate filament; endoplasmic
reticulum; determinant; germ plasm; mRNA localisation; cortical rotation; Surface Contraction Waves
ABSTRACT
The division of the Xenopus oocyte cortex into structurally and functionally distinct
‘‘animal’’ and ‘‘vegetal’’ regions during oogenesis provides the basis of the organisation of the early
embryo. The vegetal region of the cortex accummulates specific maternal mRNAs that specify the
development of the endoderm and mesoderm, as well as functionally-defined ‘‘determinants’’ of
dorso-anterior development, and recognisable ‘‘germ plasm’’ determinants that segregate into
primary germ cells. These localised elements on the vegetal cortex underlie both the primary
animal-vegetal polarity of the egg and the organisation of the developing embryo. The animal cortex
meanwhile becomes specialised for the events associated with fertilisation: sperm entry, calcium
release into the cytoplasm, cortical granule exocytosis, and polarised cortical contraction. Cortical
and subcortical reorganisations associated with meiotic maturation, fertilisation, cortical rotation,
and the first mitotic cleavage divisions redistribute the vegetal cortical determinants, contributing
to the specification of dorso-anterior axis and segregation of the germ line. In this article we consider
what is known about the changing organisation of the oocyte and egg cortex in relation to the
mechanisms of determinant localisation, anchorage, and redistribution, and show novel ultrastructural views of cortices isolated at different stages and processed by the rapid-freeze deep-etch
method. Cortical organisation involves interactions between the different cytoskeletal filament
systems and internal membranes. Associated proteins and cytoplasmic signals probably modulate
these interactions in stage-specific ways, leaving much to be understood. Microsc. Res. Tech.
44:415–429, 1999. r 1999 Wiley-Liss, Inc.
INTRODUCTION
Developmental and cell biologists interested in the
relationship between egg organisation and the establishment of the embryonic body plan have long appreciated
the special role of the egg cortex (for instance see
Elinson, 1980; Sardet and Chang, 1987; StewartSavage et al., 1991). In many types of embryo, developmental ‘‘determinants’’ identified by functional assays,
and specific localised maternal mRNAs with known or
suspected developmental functions tend to become associated with specific cortical regions during oogenesis
(see Gavis, 1997; Micklem, 1995). Subsequent cortical
and subcortical reorganisations associated with oocyte
maturation, fertilisation, and/or early mitotic cleavage
divisions result in the relocalisation of these localised
elements and are responsible for establishment of the
embryonic axes (Sardet et al., 1994). The nature of the
cortical elements that participate in the localisation
anchoring, release, and translocation of maternal determinants and mRNAs are not yet understood. Indeed
the composition and organisation of the cortex are still
poorly defined in most of the organisms. Xenopus offers
a good opportunity to address this problem, for practical reasons (late stage oocytes and eggs are abundant,
large, and exhibit clear external polarity) and because
of the rapidly accumulating knowledge concerning early
developmental events. In this article, we will summarise what is known about cortical organisation in
Xenopus oocytes and eggs in relation to the mechanisms of determinant localisation and relocalisation,
r 1999 WILEY-LISS, INC.
and present some ultrastructural views of replicas
prepared from isolated cortices of oocytes and eggs,
which give a novel perspective of cortical organisation.
A large number of ultrastructural and immunofluorescence studies using a variety of microscopy methods
have been published concerning the amphibian oocyte
and egg cortex. Filaments of all three cytoskeletal
systems have been identified in the cortex at various
times in various forms, essentially by immunofluorescence, while electron microscopy has revealed further
details of the organisation of these and other components, notably cortical and subcortical ER (StewartSavage et al., 1991). Actin is clearly the predominant
cortical protein, with cortical actin conferring contractility, in particular to the thickened animal cortex (Merriam and Sauterer, 1983; Merriam et al., 1983). However, despite the abundance of actin, definitive
microfilaments have proven hard to detect in the cortex, except in the cleavage furrow (Franke et al., 1976;
Larabell, 1995; Roeder and Gard, 1994). Microtubules
come and go in the subcortical region according to
spatial and temporal modulation of microtubule dynam-
Contract grant sponsor: ARC; Contract grant numbers: 6985, 9144; Contract
grant sponsor: CNRS; Contract grant sponsor: Ministère d’Education; Contract
grant number: ACC4/Al.
*Correspondence to: Evelyn Houliston, Unitée de Biologie Celulaire Marine
(ERS.643 CNRS-Université Paris VI), Station Zoologique 06230 Villefrance-surmer, France. E-mail: [email protected]
Received 3 September 1998; accepted in revised form 25 November 1998
416
P. CHANG ET AL.
ics in the cell (see Houliston and Elinson, 1992) and
intermediate filaments (IFs), essentially cytokeratincontaining, form a polarised cortical network that
disassembles during meiosis (Klymkowsky, 1995). Disruption studies indicate that the actin cortex of the
oocyte provides a polarised framework with respect to
which microtubules and IFs are organised (Gard et al.,
1997). The participation of microfilaments, intermediate filaments, and microtubules in specific cortical
processes has also been investigated, and the accumulated data have been reviewed several times in recent
years (Dent and Klymkowsky, 1989; Elinson and Houliston, 1990; Elinson, 1990; Gard et al., 1995, this issue;
Klymkowsky and Karnovsky, 1994; Klymkowsky, 1995).
For this reason we have chosen not to give extensive
details of the literature again here, but rather to outline
the possible participation of the cortex in the localisation and redistribution of developmental determinants.
STEPS IN CORTICAL LOCALISATION
The sequence of localisation events in Xenopus oocytes and eggs involving the cortex is shown in Figure 1.
Specialisation of the cortex begins early during oogenesis when the oocyte is small and transparent (Stage
I–II according to Dumont, 1972), with the arrival at the
cell periphery of a curious dense structure known as the
‘‘Balbiani body’’ or ‘‘mitochondrial cloud’’ (Heasman et
al., 1984). A number of specific mRNAs and proteins, as
well as structurally defined fibrilo-granular material
that gives rise to germinal granules and eventually to
germ plasm, are associated together in the mitochondrial cloud (Al-Mukhtar and Webb, 1971; Heasman et
al., 1984). The cortical site of mitochondrial cloud
attachment, which marks the future vegetal pole of the
oocyte, does not appear to be predetermined.
The germ plasm and associated mRNAs spread out in
distinctive islands across a patch of cortex in the stage
II oocyte, marking the future vegetal pole. During
mid-oogenesis (Stage III–IV) these are joined by a
distinct set of mRNAs, which do not associate with the
germ plasm but spread out evenly over a wider cortical
region. The elaboration of the distinct animal and
vegetal cortices continues throughout oogenesis as the
oocyte accumulates yolk platelets, swelling enormously
from less than 100 µm to over 1,200 µm in diameter. In
the fully grown oocyte (Stage VI) the animal cortex is
thickened and darkly pigmented, while the vegetal
cortex is thin and transparent. On current evidence,
mRNAs specific to the vegetal hemisphere tend to be
associated with the cortex, whereas animal localised
ones tend to be cytoplasmic (Hudson et al., 1996).
Cortically-associated mRNAs and germ plasm are
released to various extents from the vegetal cortex into
the cytoplasm during meiotic maturation, and then
further redistributed by post-fertilisation reorganisations. In particular, a process of ‘‘cortical rotation’’ is
responsible for displacing dorso-anterior determinants
from the vegetal pole to be activated at the equator of
the egg. The entire cortex becomes displaced relative to
the underlying cytoplasm by about 30°C about a horizontal axis (perpendicular to the plane of the page in Fig.
1). The direction of rotation and thus the equatorial site
of dorsal determinant activation is not predetermined.
At the end of the first cell cycle, successive ‘‘Surface
Contraction Waves’’ (SCWs; Hara, 1971) that pass from
the animal to vegetal poles of the egg at each mitosis,
help to drive germ plasm islands towards the vegetal
pole (Savage and Danilchik, 1993). Subsequently, subcortical ingression associated with cleavage furrow
progression causes further redistribution of germ plasm
and perhaps of other components deeper within the
blastomeres of the early embryo (Danilchik and Denegre, 1991; Ressom and Dixon, 1988). The four cortical
processes that modulate determinant localisation: localisation to the vegetal cortex during oogenesis, determinant release during maturation, cortical rotation, and
the SCWs, will be discussed in more detail in the
following sections.
LOCALISATION OF DETERMINANTS ON THE
OOCYTE VEGETAL CORTEX
Germ Plasm
Germ plasm is clearly implicated in germ line determination in Xenopus (Ikenishi, 1986), Drosophila (Gavis,
1997), and a number of other species from a wide range
of phyla (Eddy, 1975; Wakahara, 1991; Ikenishi, 1998).
The Xenopus mitochondrial cloud may be equivalent to
similarly structured ‘‘sponge bodies’’ in Drosophila oocytes that also contain RNAs involved both in patterning the embryo and in germ line determination (WilschBrauninger et al., 1997). Molecular candidates for germ
line determinant activity in Xenopus are emerging from
a subset of identified localised mRNAs that associate
with the mitochondrial cloud and subsequently with
germ plasm: Xcat-2 (Mosquera et al., 1993) codes for a
putative RNA-binding protein related to the Drosophila germ plasm component nanos, Xdaz1 is a
functional homolog of Drosophila boule (Houston et al.,
1998), and Xpat transcripts segregate with germ plasm
and then with primordial germ cells during early
development (Hudson and Woodland, 1998).
Other mRNAs in the mitochondrial cloud, notably
Xwnt11 (Ku and Melton, 1993), do not associate with
the germ plasm and may play distinct patterning roles
in the early embryo. It is possible that during metazoan
evolution, germ line determinants were the first localised informational components in eggs (Denis, 1996,
but see Dixon, 1994, for counter arguments). If germ
plasm localisation does represent the original manifestation of oocyte polarity, molecules intervening in endoderm or mesendoderm development may subsequently
have exploited the existing cortical localisation mechanisms to facilitate the organised development of associated tissues.
The mechanism by which the mitochondrial cloud
carries germinal granules and associated mRNAs to the
cortex remains enigmatic, having proven refractile to
cytoskeletal disruption (see Gard et al., 1995; Kloc et
al., 1996). At around the time the mitochondrial cloud
arrives, cortical microtubules are already present and
the cytokeratin network is beginning to develop (Gard
et al., 1997), with a loosely organised filament and ER
network attached to the cortex (Figs. 2 and 3). Following the arrival of the mitochondrial cloud at the egg
cortex, germinal granules disperse from it across the
cortex and eventually assemble into distinct, mitochondria-rich, germ-plasm islands. During subsequent
stages of oogenesis, the germ plasm material stays
closely associated with the vegetal cortex.
XENOPUS OOCYTE AND EGG CORTICES
417
Fig. 1. Summary of localisation events involving the cortex from oogenesis through to first cleavage in
Xenopus.
Dorso-Anterior Determinants
There is clear experimental evidence that determinants necessary for the development of dorso-anterior
embryonic structures are associated with the vegetal
cortex of Xenopus oocytes and eggs. Removal of vegetal
pole fragments from the egg prevents development of
dorso-anterior sructures (Kikkawa et al., 1996; Sakai,
1996), while cytoplasm taken from close to the vegetal
cortex (Fujisue et al., 1993; Holowacz and Elinson,
1993, 1995) is capable of directing the development of
ectopic dorso-anterior structures if injected into other
regions. In addition, vegetal cortices isolated from
fertilised eggs and implanted into ectopic regions of the
egg have been shown recently to harbour dorsalising
activity (Kageura, 1998), substantiating the conclusions of earlier cortical grafting experiments (Curtis,
1960), which could not be interpreted clearly (Gerhart
et al., 1981). The nature of the dorso-anterior determinants found on or close to the vegetal cortex is unresolved. They do not appear to act to induce mesoderm
formation but rather to favour the development of
dorsal structures in prospective mesoderm regions (Holowacz and Elinson, 1995), probably via the Wnt/
␤-catenin signal transduction pathway (Larabell et al.,
1997; Marikawa et al., 1997; Rowning et al., 1997).
Molecular components of dorso-anterior determinants
are likely to be found amongst the ever-growing list of
vegetally localised mRNAs, and/or the vegetal cortical
proteins to which antibodies have been raised (Denegre
et al., 1997). Certain of the known localised mRNAs
appear to function in early patterning events although
none has yet been clearly equated with dorso-anterior
determinant activity: maternal VegT, a T-box containing transcription factor (Horb and Thomsen, 1997;
Lustig et al., 1996; Stennard et al., 1996; Zhang and
King, 1996) is involved in setting up the primary
embryonic germ layers, being necessary for mesoderm
and endoderm formation in the vegetal territories of the
embryo (Horb and Thomsen, 1997; Zhang et al., 1998).
Vg1, which codes for a TGF␤ family growth factor able
to induce mesoderm (Dale et al., 1993; Thomsen and
Melton, 1993; Weeks and Melton, 1987), may be involved more specifically in formation and patterning of
dorsal mesoderm and endoderm (Joseph and Melton,
1998).
Vegetal Localised mRNAs
The localised vegetal cortical RNAs identified so far
can be divided into two sets on the basis of their time
and pathway of localisation (Forristall et al., 1995; Kloc
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P. CHANG ET AL.
Fig. 2. General view of a replica from a deep-etched cortex
prepared from a Stage I– II oocyte. Cortices were prepared directly on
polylysine-coated glass chips from oocytes scalpeled in half in PHEM
buffer (Schliwa et al., 1981) by a modification of the method of Elinson
et al. (1993). After 15 minutes fixation in 1% glutaraldehyde in PHEM,
the samples were fast frozen and deep-etched as described (Chang et
al., 1996). A complex system of subcortical filaments (f) is present,
interacting with frequent sheets of rough ER (rER). Large (0.7–0.8
µm) vesicular structures can be tentatively identified as cortical
granules (cg), distributed unevenly through the subcortical cytoplasm,
and the residual envelopes of yolk platelets (Y), membrane bound
packets of pinocytosed vitellogenin. The asterisk marks the position of
the stereo pairs shown in Figure 3. Magnification ⫻14,200
Fig. 3. A,B: High magnification stereo views of the Stage I-II
oocyte cortex shown in Figure 2 (from region marked with asterisk). To
appreciate fully the 3D view, inexpensive stereo viewers obtainable
from Electron Microscopy supply companies should be used. Based on
filament diameters, we can tentatively identify microtubules (mt),
intermediate filaments (if), and maybe also microfilaments (mf).
Coated pits (cp) are found at the plasma membrane (pm). Magnification ⫻71,000.
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P. CHANG ET AL.
and Etkin, 1995). A first set arrives in association with
the mitochondrial cloud. This includes all the germ
plasm associated mRNAs mentioned above along with
Xwnt11 and Xlsirt, a non-coding, repetitive sequence
RNA (Kloc and Etkin, 1994). Different transcripts take
up different positions within the cloud, and appear to
attach to the cortex in a temporal sequence so that they
take on distinct distributions within the flattened ‘‘galette’’ that forms on the vegetal cortex (Kloc and Etkin,
1995; Kloc et al., 1996). The resultant layering of the
messages (Xcat-2, then Xlsirt, then Xwnt11) is later lost
as the mRNAs disperse more widely across the cortex
(Kloc and Etkin, 1995).
The second set of localised mRNAs includes Vg1 and
VegT. These ‘‘late’’ localised mRNAs are synthesised in
the very early oocyte but only concentrate to the vegetal
cortex once the germ plasm localisation has been
established, active localisation occurring in oocytes
between stages II and IV. The transport mechanism
used by the late mRNAs does not appear to be highly
specific, since it can also function to localise inappropriate messages including the early mRNAs Xcat-2 (Zhou
and King, 1996) and Xpat (Hudson and Woodland,
1998), and tau mRNA, which is localised in axons
(Litman et al., 1996). These mRNAs all have ‘‘E2’’
motifs in the 3’UTR, which recognise the ER-associated
RNA binding protein Vera (Deshler et al., 1997, 1998).
Additional elements in the Vg1 3’UTR appear to have
overlapping roles in localisation, binding Vera and
other cytoplasmic proteins. This suggests that complexes containing multiple proteins associate with the
RNAs prior to localisation, distinct proteins mediating
specificity or localisation pathways (Gautreau et al.,
1997; Mowry, 1996). The first step in localisation of late
mRNAs is their accumulation in a wedge-shaped cytoplasmic region between the GV and the newly established germ plasm patch on the cortex. This region is
rich in Vera, apparently associating with a subpopulation of the oocyte’s ER (Deshler et al., 1997; Kloc and
Etkin, 1998). The RNAs subsequently accumulate on
the vegetal cortex, and then spread laterally to cover a
larger area than that of the early RNAs. Large flat ER
sheets and tubules are predominant features of the
cortex at this time (Fig. 4A), appearing to fuse directly
to the plasma membrane as well as being tethered by
cytokeratin-containing filaments (Fig. 4B).
Localisation of Vg1 to the vegetal pole, and perhaps
the initial steps in its cortical anchoring, depend on
microtubules (Kloc and Etkin, 1995; Yisraeli et al.,
1989). Transport may be mediated by direct binding of
RNP particles to microtubules via the Vg1 binding
protein Vg1RBP (Elisha et al., 1995) either in combination with the ER and Vera or in a parallel pathway.
Vg1RBP is highly homologous to a protein implicated in
localisation of ␤actin mRNA in fibroblasts, a process
mediated by microfilaments rather than microtubules
(Havin et al., 1998), suggestive that both cytoskeletal
systems can participate in mRNA localisation. The
polarity of the cytoplasmic microtubules between the
oocyte nucleus and the cortex is not known so it is hard
to predict which kinds of motor molecules might be
involved in message transport. Microtubule regrowth
experiments suggest that minus ends are likely to lie at
the base of the nucleus (Gard, 1991), while the specific
detection of ␥-tubulin at the vegetal cortex implicates
microtubule nucleation in the opposite direction (Gard,
1994; see also Gard, this issue).
The anchorage of vegetally localised mRNAs to the
cortex is distinguishable from transport since it is not
disrupted by microtubule depolymerizing drugs (Kloc
and Etkin, 1995; Yisraeli et al., 1989, 1990). Unsurprisingly, given the major role of actin in maintaining
cortical integrity, cytochalasin treatment, was found to
release Vg1 message from the cortex into the vegetal
cytoplasm (Yisraeli et al., 1989). In another study (Kloc
and Etkin, 1995), Xlsirt, Xcat-2, and Xwnt11 as well as
Vg1 RNAs were detached from the cortex of stage III
and IV oocytes by cytochalasin treatment, but appeared
to remain associated in a disc that delaminated from
the cortex. More drastic disruption of cortical anchoring
of Vg1 mRNA has been achieved with oligonucleotides
targeted to destroy Xlsirt RNAs (Kloc and Etkin, 1994).
These repetitive sequence RNAs, which arrive with the
mitochondrial cloud (Kloc et al., 1993), are proposed to
play a structural role in the cortical anchoring of Vg1,
but not Xcat-2 mRNAs (Kloc and Etkin, 1994). Although Vg1 mRNA associates with an IF-rich cellular
residue upon detergent extraction (Pondel and King,
1988), manipulation of meiotic maturation suggests
that Vg1 mRNA is not directly linked to cortical cytokeratin filaments (Klymkowsky and Maynell, 1989;
Klymkowsky et al., 1991; see Klymkowsky, 1995). Studies of whole stage VI oocytes (Canman and Bement,
1997; Gard et al., 1997) and cortices isolated from them,
which retain the localised mRNAs (Elinson et al.,
1993), indicate that all three filament systems, as well
as mitochondria and extensive ER interact in the oocyte
cortex (Figs. 5 and 6) (Elinson et al., 1993). Microtubules and microfilaments appear to direct cytokeratin
filament organisation (Gard et al., 1997), with the IF
bundles more tightly organised in the vegetal hemisphere (Klymkowsky et al., 1987).
MODIFICATION OF DETERMINANT
AND mRNA ATTACHMENT DURING
MEIOTIC MATURATION
UV irradiation of the vegetal cortex of the immature
Stage VI oocyte, but not of the unfertilised egg, significantly reduces dorso-anterior determinant activity, suggesting that the dorso-anterior determinants are released from the cortex and/or transformed during
meiotic maturation (Elinson and Pasceri, 1989; Holowacz and Elinson, 1993). In contrast, UV irradiation
of oocyte and egg vegetal surfaces affects germ cell
formation to similar degrees (Holwill et al., 1987).
Dorso-anterior determinants may move deeper into the
cytoplasm during maturation, but are not lost from the
cortical/subcortical region since they relocate with the
vegetal cortex upon experimental 180° rotation of the
egg (Marikawa et al., 1997). Vg1 mRNA is released into
the vegetal cytoplasm from its tight cortical association
early during meiotic maturation, whereas Xcat-2 remains associated with the cortical germ plasm throughout meiosis (Mosquera et al., 1993; Forristall et al.,
1995; Weeks and Melton, 1987). The small islands of
mitochondria and germinal granules present beneath
the vegetal oocyte surface (Heasman et al., 1984;
Savage and Danilchik, 1993) coalesce during oocyte
maturation to form more clearly distinct germ plasm
Fig. 4. Deep-etched cortex prepared from a Stage III oocyte as
described in Figure 1. Immunolabeling of IFs with anticytokeratin
monoclonal 1h5 (Klymkowsky et al., 1987) was performed as described
(Chang et al., 1996). A: General view, magnification ⫻28,500. B: High
magnification stereo view from a different area of the same cortex.
Magnification ⫻99,500. A large tubular patch of rough ER (rER) is
connected to the plasma membrane (pm) both directly (large arrowhead) and via filaments lableled with the 1h5 antibody (arrows
indicate gold-liganded secondary antibody). Annotations as in Figures
2 and 3. Note the rER lumen (lumen), and the wall (w) of rER tethered
by filaments (f) to the plasma membrane.
Fig. 5. General view of a deep-etched cortex prepared from the animal hemisphere of a Stage VI
oocyte. Abundant intermediate-sized filaments run in wide loose bundles (large arrows) across the
cytoplasmic side of a complex thick layer containing cortical granules (cg). Magnification ⫻14,200.
Annotations as in Figures 2 and 3.
Fig. 6. General view of a deep-etched cortex prepared from the vegetal hemisphere of the same St VI
oocyte as shown in Figure 5. The IF bundles (large arrows) are more compact and appear to run between
the cortical granules (cg), closer to the plasma membrane (pm) than in the animal half. Magnification
⫻14,200. Annotations as in Figures 2 and 3.
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P. CHANG ET AL.
islands (Czolowska, 1969) but remain close to the
vegetal surface.
Determinant/localised mRNA dispersal during oocyte maturation accompanies extensive remodelling of
the whole egg, including the cortex. Some structural
changes reflect the change in cell cycle state from
interphasic (prophase of meiosis I) to mitotic (metaphase of meiosis II), while others are related to the
change from the stable organisation of the stage VI
oocyte, which may remained unchanged for months in
the ovary, to one that is unstable and poised for
activation by the fertilising sperm. The most striking
change to the cortex during maturation is the setting
up of a specialised cortical endoplasmic reticulum (cER),
most highly developed in the animal hemisphere and
implicated in the fertilisation response (Campanella et
al., 1984; Charbonneau and Grey, 1984; Dersch et al.,
1991; Stewart-Savage et al., 1991). This extensive cER
network is established very close to, and connecting
with, the plasma membrane (Gardiner and Grey, 1983),
now enveloping cortical granules that reorganise from a
deep to a regular array very close to the plasma
membrane (Campanella et al., 1984). Remodelling of
the actin cortex occurs in parallel with the setting up of
the cortical granule-associated cER, manifest as the
development of contractability, inducible by fertilisation or calcium ionophore. The robust contraction of the
animal cortex of the unfertilised egg cannot be prevented with cytochalasins (Merriam and Sauterer,
1983), suggesting that an extensive actomyosin filament system has been set up below the animal surface
(Merriam et al., 1983; see Larabell, 1995). Vinculin is
also recruited into the cortex during the period of oocyte
maturation and oviposition (Evans et al., 1990).
Cortical cytokeratin filaments throughout the egg
are also extensively remodelled during meiotic maturation (Klymkowsky, 1995). In the animal hemisphere,
there is a brief assembly of IF cable network, less
developed in this region of the Stage VI oocyte, then a
disassembly to phosphorylated oligomers induced indirectly by activation of the universal mitotic kinase MPF
(Klymkowsky and Maynell, 1989; Klymkowsky et al.,
1991). In the vegetal hemisphere, the preformed cytokeratin cable network disintegrates completely. The
release of Vg1 mRNA from the cortex precedes this IF
disassembly and can be distinguished from it experimentally (Klymkowsky et al., 1991). Thus, Vg1 mRNA
anchoring to the vegetal cortex appears not to be simply
dependent on any one of the cytoskeletal systems, but
probably on a combination of these, and/or other as yet
unanalysed structural components.
CORTICAL ROTATION
Cortical rotation is necessary for the development of
dorso-anterior structures in the frog embryo (Gerhart
et al., 1989). During the cortical rotation, dorsoanterior determinants relocate from around the vegetal
pole to a broad area spreading toward the equator in
the direction of cortical movement (Fujisue et al., 1993;
Kikkawa et al., 1996; Sakai, 1996). This relocation is
necessary to allow the determinants to activate certain
downstream targets in prospective mesoderm regions
that stimulate ‘‘organiser’’ activity (Darras et al., 1997).
Whether determinant substances are carried with the
cortex or translocated independently along aligned
subcortical microtubules on vesicles or particles (Rowning et al., 1997) is not known.
The mechanism of cortical rotation depends on interaction between subcortical microtubules and elements
anchored to the cortex proper. Prior to the cortical
rotation, microtubules tend to grow outwards from the
center of the egg (Houliston and Elinson, 1991b; Elinson and Palacek, 1993). The interaction of these microtubules with the vegetal cortex, the region where the
rotation movement is likely to be generated (see Houliston and Elinson, 1992), is different from that at the
animal cortex (Houliston and Elinson, 1991b; Schroeder
and Gard, 1992). In the animal hemisphere, outwardgrowing microtubules abut the thickened, pigmented
cortex abruptly with minimal bending. This behaviour,
and/or the relative fluidity of the animal cytoplasm,
may be important to allow sperm aster expansion to
drive pronuclear migration earlier in the cell cycle (see
Reinsch and Gönczy, 1998). In contrast, microtubules
that cross the dense vegetal yolk mass turn in the
yolk-poor subcortical vegetal cytoplasm and continue to
polymerise parallel to the vegetal cortex. Differences in
the interactions of microtubules with the animal and
vegetal cortices have also been demonstrated by observing ectopic meiotic spindle formation and rotation
(Gard, 1993).
Outward-growing vegetal microtubules, together with
ones nucleated locally in the vegetal cortical region,
form a striking array of aligned microtubules covering
the whole vegetal surface of the egg (Elinson and
Rowning, 1988). This vegetal subcortical microtubule
array is strongly implicated in the process of cortical
rotation, and the cortical rotation movement and the
alignment of the microtubules mutually reinforce one
another (see Houliston and Elinson, 1992; Gerhart et
al., 1989). The aligned vegetal subcortical microtubules
stay attached to experimentally isolated cortices and
can thus be visualised in deep etch replicas, where they
are seen to be sandwiched in layers between extensive
sheets of rough ER (Fig. 7; Chang et al., 1996). This
observation reinforces the hypothesis that ER could
provide the cortical substrate for microtubule motor
attachment to create the cortical rotation movement.
Interactions generating the rotation movement could
also occur directly between subcortical microtubules
and cortical actin, or between microtubules and associated cytokeratin filaments, although the latter possibility seems less likely since cytokeratin filaments are
sparse at this time and are mainly located towards the
cytoplasmic side of the subcortical microtubule layer
(Houliston and Elinson, 1991a). Various observations
suggest that microtubule motor(s) cause the cortical
rotation by moving cortically attached elements towards the plus-ends of microtubules: the uniform polarity of the microtubules in the direction of cortical
movement (Houliston and Elinson, 1991a), the observed displacement of the majority of vegetal subcortical microtubules relative to the cortex (Houliston, 1994;
Larabell et al., 1996), and the independence of cortical
rotation from continuing microtubule polymerisation
(Houliston, 1994). Immunofluorescence studies on isolated cortices indicate that the architypical plus-end
directed motor kinesin is associated with the aligned
subcortical vegetal microtubules and ER, and so could
XENOPUS OOCYTE AND EGG CORTICES
contribute to the cortical movement by generating
movement between these structures (Houliston and
Elinson, 1991a). In contrast, immunogold localisation
of the abundant plus-end directed motor protein Eg5 on
replicas of cortices isolated during the cortical rotation
revealed an exclusive association with microtubules
and microtubule bundles (Chang et al., 1996), despite
indications from whole mount immunofluorescence that
Eg5 might also be associated with the ER (Houliston et
al., 1994). Thus, if Eg5 contributes to the rotation
movement, it must do so by some sort of microtubulemicrotubule sliding mechanism (Larabell et al., 1996).
High-resolution localisation of kinesin and the many
other kinesin-like proteins present in Xenopus eggs has
yet to be accomplished. To understand the mechanism
of cortical rotation it will be necessary to determine
which microtubule subpopulations are involved in force
generation, as well as to identify the motor molecules(s)
utilized.
SURFACE CONTRACTION WAVES AND
CORTICAL CONTRACTIONS
Germ plasm loses its tight cortical association following fertilisation but remains in a subcortical band
25–30 µm thick (Ressom and Dixon, 1988). This moves
with the vegetal cortex if it is displaced animally by
180° rotation, disrupting subsequent formation and
migration of primary germ cells (Cleine and Dixon,
1985). Another indication that the germ plasm remains
under the influence of the cortex, or at least of organisational changes in the subcortical cytoplasm, is that the
SCWs of successive mitotic cycles participate in moving
the germ plasm islands toward the vegetal pole (Savage
and Danilchik, 1993).
The SCWs traverse the egg from animal to vegetal
poles at the time of each mitosis (Hara, 1971; Yoneda et
al., 1982; Sawai, 1982). There are two distinct SCWs in
each cell cycle. The first appears to be a wave of general
relaxation of the egg rather than a contraction. It
coincides with the activation of MPF at the onset of
mitosis. MPF activation is initiated in the animal
cytoplasm and propagates by a post-translational autocatalytic activation mechanism to the vegetal pole
(Pérez-Mongiovi et al., 1998; Rankin and Kirschner,
1997). The first SCW appears, at least in part, to reflect
depolymerisation of subcortical microtubules as a consequence of the wave of MPF activation (Pérez-Mongiovi
et al., 1998; Schroeder and Gard, 1992). MPF activation
in the vegetal hemisphere coincides with the end of the
cortical rotation, which stops precisely as the first SCW
passes. Microtubule depolymerisation provoked by the
MPF activation wave would explain the influence of the
first SCW on the germ plasm, since many aspects of
germ plasm redistribution following fertilisation are
sensitive to microtubule depolymerisation (Ressom and
Dixon, 1988; Savage and Danilchik, 1993). Germ plasm
aggregates are known to associate with subcortical
microtubules via the kinesin-like motor protein Xklp1
(Robb et al., 1996). It has been proposed that the first
SCW may, like the second SCW (see below), depend on a
propagated wave of calcium release (Jaffe, 1999). Although calcium transients are implicated in both activation and inactivation of MPF in Xenopus egg cytoplasm
(Lindsay et al., 1995), no calcium waves have yet been
visualised in the cortex concurrent with the first SCW.
425
The second SCW, which immediately precedes the
advancing cleavage furrow, is a true cortical contraction
wave. Its progress coincides with the passage of MPF
inactivation across the egg (Pérez-Mongiovi et al., 1998;
Rankin and Kirschner, 1997). The mechanism of the
second SCW is clearly likely to be closely tied in with
the actin-based contraction that drives the cleavage
furrow. Increased contractility of actin in the cortex at
the end of mitosis is thought to involve actomyosin
interactions favoured by phosphorylation of myosin
light chain kinase upon MPF inactivation (Satterwhite
et al., 1992). Intracellular calcium release appears
likely to play a role in cleavage furrow progression and
the second SCW, since localised calcium release has
been detected at the time of the second SCW, associated
with the furrow (Keating et al., 1994; Muto et al., 1996).
A more experimentally tractable actin-based cortical
contraction induced by cytoplasmic calcium release
occurs following fertilisation in amphibian eggs (Merriam and Sauterer, 1983). This violent isotropic contraction of the actin-thickened animal cortex, which drags
the entering sperm nucleus toward the animal pole, has
been shown to represent a cortical response to the
massive IP3-mediated wave of cytoplasmic calcium
release triggered by the fertilising sperm (Kubota et al.,
1987; Larabell and Nuccitelli, 1992; see Elinson, 1980;
Larabell, 1995). The wave of calcium release from the
cER following fertilisation also promotes exocytosis of
cortical granules (Andreuccetti et al., 1984). Like the
second SCW, the fertlisation-triggered cortical contraction accompanies MPF inactivation, in this case the
release from meiotic metaphase arrest. Waves of peristaltic cortical contraction accompanied by calcium
transients can also be triggered artificially during
interphase by microinjection of IP3 (Muto and Mikoshiba, 1998).
Germ plasm movement accompanying the second
SCW is less distinct than at the first SCW and difficult
to discriminate from cleavage-associated movements. The
participation of the cortex in determinant localisation can
be considered to be essentially complete by the time of first
cleavage, as germ plasm and probably the dorso-anterior
determinants move into subcortical and deeper cytoplasm, eventually reaching nuclei of different embryonic blastomeres to have their developmental effects.
PERSPECTIVES
Despite the wealth of descriptions and functional
studies concerning the organisation of different components of the Xenopus egg cortex at different times, there
remain large gaps in our knowledge. It is difficult to
integrate the different pieces of published information
and to form a full or clear picture of the structure of this
dynamic region at the times when determinants are
translocated, anchored or released. With the growing
realisation that different cytoskeletal systems do not
function independently but interact intimately (Canman and Bement, 1997; Gard et al., 1997), we must
consider that the distribution of motor molecules, membrane systems, and determinants is likely to depend on
all different types of filaments as well as the regulation
of their interaction by changing cytoplasmic conditions,
for instance during cell cycle transitions. Further structural studies of the cortex, as well as, of course, the
molecular characterisation of the dorso-anterior and
Fig. 7. View of a deep-etched cortex isolated manually from a
fertilised egg during cortical rotation and immunolabelled with antitubulin antibody YL1/2 (Kilmartin et al., 1982) and subsequent goat
anti-rat liganded with 5-nm gold particles prior to freezing and replica
preparation (methods described in Chang et al., 1996). Bundled
microtubules (arrowheads) and microtubules (small arrows) can be
seen. Magnification ⫻28,500. Annotations as in Figures 2 and 3.
XENOPUS OOCYTE AND EGG CORTICES
germ line determinants and the analysis of their localisation at the ultrastructural level, will be required to
unravel this problem.
ACKNOWLEDGMENTS
Anti-cytokeratin monoclonal 1h5 developed by M.
Klymkowsky was obtained from the Developmental
Studies Hybridoma Bank maintained by the University
of Iowa, Department of Biological Sciences, Iowa City,
IA 52242 under contract N01-HD-7–3263 from the
NICHD. We thank Clare Hudson (Marseille) for useful
hints about mRNA localisation sequences and Christian Sardet for support. The original research described
was funded by ARC grants 6985 and 9144 to E.H, and
by financement from the CNRS and Ministère
d’Education (ACC4/ AI ‘‘Biocell’’).
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