88
Nuclear envelope dynamics in oocytes: from germinal vesicle
breakdown to mitosis
PeÂter LeÂnaÂrt and Jan Ellenberg
We have recently gained new insight into the mechanisms
involved in nuclear envelope breakdown, the irreversible step
that commits a cell to the M phase. Results from mammalian cell
and star®sh oocyte studies suggest that mechanical forces of the
cytoskeleton, as well as biochemical disassembly of nuclear
envelope protein complexes, play important roles in this process.
Addresses
Gene Expression and Cell Biology/Biophysics Programmes, European
Molecular Biology Laboratory (EMBL), D-69117, Heidelberg, Germany
e-mail: jan.ellenberg@embl-heidelberg.de
Current Opinion in Cell Biology 2003, 15:88±95
This review comes from a themed issue on
Cell structure and dynamics
Edited by Michel Bornens and Laura M Machesky
0955-0674/03/$ ± see front matter
ß 2003 Elsevier Science Ltd. All rights reserved.
DOI 10.1016/S0955-0674(02)00011-X
Abbreviations
AL
annulate lamellae
ER
endoplasmic reticulum
GFP
green ¯uorescent protein
GV
germinal vesicle
GVBD GV breakdown
MPF
maturation promoting factor
NE
nuclear envelope
NEBD NE breakdown
NPC
nuclear pore complex
Introduction
The nuclear envelope (NE) has been attracting increasing attention in recent years. This structure forms the
boundary between the chromosomes and cytoplasm and
comprises two concentric nuclear membranes perforated
and joined by large aqueous channels Ð the nuclear pore
complexes (NPCs) Ð that mediate transport between
cytoplasm and nucleus [1]. In animal cells, the nuclear
membranes are stabilised by an underlying layer of intermediate ®laments, the nuclear lamina [1].
Although the biological importance of the NE has been
appreciated for a long time [2], it is only recently that a
growing list of human hereditary diseases has been attributed to defects in NE proteins, referred to as laminopathies or envelopathies [3,4]. In addition, owing to
advances in proteomics and sequencing, the very limited
number of characterised NE proteins has greatly
expanded and we are now aware of almost all the conCurrent Opinion in Cell Biology 2003, 15:88±95
stituent polypeptides [5,6]. Together with better biochemical and genetic knowledge of the NE, advanced
imaging methods have allowed greater understanding
of its dynamics in living cells. The workhorse system
for in vivo characterisation of the NE has been mammalian tissue culture cells. These have provided fundamental insights into the stable nature of the NE in interphase
[7], the intimate relationship of the NE to the endoplasmic reticulum (ER) [8,9], as well as into the mechanisms of its disassembly and reassembly during the somatic
mitotic cycle [10,11,12,13]. The results from intact
cells have brought us to signi®cantly revise the textbook
model of nuclear disassembly, according to which nuclear
membranes were thought to vesiculate after solubilisation
of the lamina (reviewed in [14,15]).
Most of our biochemical knowledge about the NE stems
from meiotic in vitro systems (Xenopus egg), nevertheless,
our understanding of NE dynamics in such cells is very
limited. Recently, studies using oocytes, which are ideally
suited to observe the dynamic changes in NE structure
during the G2/M transition in vivo, have taken place and
have revealed features that both resemble and differ from
somatic mammalian cells. In this review, we aim to
summarise ®ndings from meiotic systems, point out their
unique features and advantages and highlight basic questions of NE function that we should be able to tackle
through future work involving oocytes.
Germinal vesicle breakdown in oocytes
Why use oocytes?
Oocytes have a very different architecture to cultured
mammalian ®broblasts (Figure 1). They are large, highly
specialised cells containing stockpiles of all the cellular
components required to support rapid early embryonic
divisions in their cytoplasm. The nucleus of oocytes
arrested at the beginning of meiosis I is very large and
is termed the germinal vesicle (GV). The volumes of
star®sh and Xenopus oocyte nuclei are 100±100,000 times
larger and their envelopes contain 300±15,000 times more
NPCs than a ®broblast nucleus [16,17] (P LeÂnaÂrt, unpublished data). In contrast, the size of the chromosomes and
microtubule asters is very similar between oocytes and
somatic cells. Although the functional relevance of the
enormous GV is unclear, it may be a means of storing
spontaneously assembling NE components such as NPCs
and lamins over long periods of time.
Despite its size, the GV breaks down (a process referred to as GV breakdown, or GVBD) when oocytes
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Nuclear envelope dynamics in oocytes LeÂnaÂrt and Ellenberg 89
Figure 1
The specialised architecture of oocytes. (a) Fibroblast from rat kidney (NRK) cells. (b) Mouse oocyte. (c) Starfish oocyte. (d) Xenopus oocyte. (e)
Nucleus isolated from a Xenopus oocyte. The schemes in the upper panel are drawn to the same scale to illustrate size differences.
re-enter meiosis, a process termed maturation. This process can be triggered experimentally by simple addition
of a maturation hormone (e.g. progesterone for Xenopus or
1-methyladenine for star®sh oocytes), which induces a
precisely timed sequence of events. The large size of the
nucleus, the accurate timing of maturation and the autonomous development and transparency of the cells found
in many marine oocytes constitute great advantages for in
vivo studies of nuclear dynamics by confocal imaging. A
detailed characterisation of NE dynamics in maturing
star®sh oocytes was carried out recently and has led to
the proposal of a new model of GVBD [18,19].
the nuclear boundary is completely disrupted. This was
con®rmed by the observation that green ¯uorescent protein (GFP)-tagged NPC-associated proteins dissociate
simultaneously with the dextran entry [19] and led to
the suggestion that gradual disassembly of the NPC is the
®rst event in NEBD. Ultrastructural studies in early
Drosophila embryos undergoing mitosis further supported
this hypothesis by showing partially disassembled intermediates of NPCs in a largely intact NE during prometaphase [20].
A new model of germinal vesicle breakdown
The change in NE permeability before the clearly visible
rupture of the NE correlates well with the nuclear accumulation of maturation promoting factor (MPF) in star®sh
oocytes (M Terasaki, personal communication). Nuclear
entry of MPF during early prophase was observed in
®broblasts and sea urchin eggs using a cyclin-B±GFP
fusion protein [21,22], and the same was seen using
biochemical methods in star®sh [23], Xenopus [24] and
mouse [25] oocytes. It has been suggested that MPF
accumulation in the nucleus is related to its activation
[26]. Active MPF would thus be likely to phosphorylate
substrates during and immediately after nuclear entry.
Such substrates include several NE proteins, namely
nucleoporins, inner nuclear membrane proteins and
lamins [27±31]. Phosphorylation of NE proteins would
lead to their dissociation from the nuclear periphery and
A convenient way to assay GVBD is to introduce inert
¯uorescent markers into the cytoplasm and follow the
mixing of cytoplasm and nucleoplasm during oocyte
maturation. Fluorescent 70 kDa dextran injected into
the cytoplasm of immature star®sh oocytes is excluded
from the nucleus, and only enters during GVBD
[18,19]. According to the classical de®nition, NE
breakdown (NEBD) begins when the sharp boundary
between cytoplasm and nucleus visible using transmitted
light microscopy disappears and the cytoplasmic yolk
starts to mix with the nucleoplasm (Figure 2). Dextran
entry, however, starts around 10 minutes before these
obvious signs of NE disruption [19]. The increased
permeability of the NE, marked by the dextran entry,
indicates that the NE disassembly processes begin before
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Does maturation promoting factor trigger germinal
vesicle breakdown?
Current Opinion in Cell Biology 2003, 15:88±95
90 Cell structure and dynamics
Figure 2
would explain the increase in NE permeability that
accompanies MPF entry. Early dextran entry can also
be observed in ®broblasts and sea urchin embryos
(J Ellenberg, unpublished data). It is therefore likely that
these early events of NEBD are as conserved through
evolution as nuclear MPF accumulation. We should,
therefore, revise our de®nition of NEBD to begin with
NPC disassembly detectable by changes in NE permeability. Notably, the simultaneously altered properties of
the nucleocytoplasmic transport machinery may play a
role in the regulation of the G2/M transition, since nuclear
accumulation of MPF together with its activators is
believed to be involved in the auto-ampli®cation of
MPF [26,32].
Rupturing the nuclear envelope: the permeabilisation
wave
Dextran entry into the nucleus in maturing starfish oocytes: the two
phases of NEBD. Tetramethyl-rhodamine-labelled 70 kDa dextran
was injected into the cytoplasm of the oocyte. Before any change
could be seen on the differential interference contrast (DIC) image,
the dextran slowly starts to enter the nucleus (frames 8:00±11:00),
reflecting the beginning of the disassembly of the pore complex. The
slow entry is then followed by a rapid wave of dextran entry (frame
12:00), coinciding with the disappearance of the sharp
nucleocytoplasmic boundary on the transmitted light image
(arrowheads). Time is given as minutes:seconds. Bar ˆ 10 mm. The
scheme illustrates the `top view' of the NE. Model is adapted
from [19].
Current Opinion in Cell Biology 2003, 15:88±95
In star®sh oocytes, the slow entry of dextrans that is
accompanied by nuclear accumulation of MPF is followed by a second phase of GVBD, demonstrated by a
rapid, dramatic wave of dextran entry when the NE has
become completely permeable [19] (Figure 2). This
wave coincides with the ®rst signs of NE disruption that
are visible with the transmitted light microscope [19].
The wave of dextran ¯ow into the nucleus is somewhat
reminiscent of the mechanical rupture observed in mammalian cells, where the dextrans enter through large holes
in the NE that are torn open by spindle microtubules
[10]. In star®sh oocytes, however, large discontinuities
are not observed in the nuclear membrane during complete permeabilisation using ¯uorescent lipid dyes ([18];
P LeÂnaÂrt, unpublished data) and the lamina still forms a
continuous mesh at the ultrastructural level at this time
[33]. It is, therefore, likely that permeabilisation starts by
a local fenestration of the membrane caused by complete
removal of the NPCs. This permeabilisation would then
be propagated from the initial site in a wave across the
surface of the NE. Computer simulations of such an
extended permeabilisation zone precisely explained the
crescent shape of the entering wave front [19]. The
lamina is then depolymerised several minutes after the
mixing of the nucleus and the cytoplasm is complete.
Only at this time do the nuclear membranes detach from
the lamina and become absorbed into the ER, without
obvious signs of vesiculation ([18]; P LeÂnaÂrt, unpublished data).
Fibroblasts versus oocytes
A simple explanation for the differences between microtubule-mediated lamina tearing in mammalian ®broblasts
[10] and the fenestration wave observed in star®sh
oocytes [19] is that, in oocytes, microtubules are simply
unable to generate the force necessary to tear the lamina
of the nucleus, due to its sheer size. Forces generated by
chromosome condensation in ®broblasts [10] are also
unlikely to lay a role in tearing the lamina of the oocyte
nucleus because the chromosomes are also relatively
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Nuclear envelope dynamics in oocytes LeÂnaÂrt and Ellenberg 91
very small and are already partially condensed in the
G2-arrested cell [34]. Furthermore, it has been shown
that GVBD in star®sh proceeds without delay and morphological changes in the absence of microtubules [35],
whereas in ®broblasts microtubule depolymerisation
causes a delay of NEBD and leads to nuclear permeabilisation similar in appearance to that observed in star®sh
oocytes [10,11].
The nuclear envelope from ®rst meiotic
division to embryonic mitosis
After GVBD is complete, chromosomes progress to the
metaphase plate of meiosis I, rapidly enter anaphase and
telophase and then the ®rst polar body is extruded. The
remaining chromosomes promptly align again in the
second meiotic spindle. The oocytes of most vertebrates
arrest at this stage (metaphase II) and only enter anaphase
II upon fertilisation. In contrast, most echinoderms complete meiosis before fertilisation and form the second
polar body and the female pronucleus before sperm entry.
During meiosis I and II, oocytes are in M phase, with high
MPF activity [36]. Therefore, NE proteins such as lamins
and nucleoporins are phosphorylated, preventing lamin
polymerisation and NPC assembly. Interestingly,
although MPF activity drops between the two meiotic
divisions, the NE does not reform around the chromosomes [36,37]. Preventing NE formation is probably an
important prerequisite to inhibiting DNA replication in
the reducing division.
A change of coats: assembly of the male pronucleus
after fertilisation
While the female pronucleus contains pore complexes
and a lamina and is competent for nucleocytoplasmic
transport, the sperm nucleus has a specialised and highly
compacted structure. Sperm chromatin is tightly condensed because somatic histones are replaced with protamines Ð sperm-speci®c basic proteins in vertebrates
[38] Ð or with sperm-speci®c histones in sea urchins
[39,40]. The NE is reduced, contains only a limited
number of specialised INM (inner nuclear membrane)
proteins [41], and completely lacks NPCs [42,43]. The
result is a nucleus in which the sperm chromatin is
hermetically sealed by an uninterrupted double membrane. Reports on sperm lamina are somewhat controversial, but it seems that sea urchin sperm contains some
patches of lamina [39,44] whereas lamins are even more
reduced in mouse sperm [41,44].
Immediately after the sperm enters the oocyte at fertilisation, its pore-less NE is rapidly replaced by the male
pronuclear envelope, which is similar in composition to
that of the female pronucleus [42]. The `change of coats'
begins with the disassembly of the double membrane,
thereby exposing the sperm chromatin to the egg cytoplasm [42,45]. This allows the rapid incorporation of
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maternal histones and other chromatin proteins and is
re¯ected by the extensive swelling of the sperm [39,45].
Shortly after swelling, the new NE starts to assemble
around the chromatin to form the male pronucleus
[42,45]. While most of the pronuclear envelope originates from the ER of the egg [42,45], specialised areas
of the original sperm shell are retained at the tip and at the
centrosome-associated basis of the nucleus. These structures are believed to be important for the formation of the
new NE [42,45,46].
Remarkably, in sea urchins and other species, pronuclear
assembly occurs in an interphase cytoplasm in which the
female pronucleus is already present [42] and does not
require synthesis of new protein [46]. Annulate lamellae
(AL) are the obvious candidates for the source of NE
material in this case. The AL are specialised areas of the
ER, packed with pre-assembled NPCs and are found in
abundance in most oocytes [47]. AL are believed to be
depots of NPCs to be used during rapid early embryonic
divisions [48]. In M phase, AL pore complexes are disassembled, making the soluble nuclear pore proteins
available for NE assembly in anaphase and telophase.
In contrast, in species where fertilisation occurs after
meiosis II in interphase, AL are intact, as are the NPCs
of the female pronucleus. Therefore, a different assembly
mechanism must function to form the male pronucleus. A
simple model would be that the AL attach to the chromatin surface as pre-assembled nuclear membrane building blocks. This model is indirectly supported by the fact
that AL move towards the male chromatin along the
sperm aster (J Ellenberg, unpublished data) and that
nocodazole blocks pronuclear development, presumably
by preventing AL clustering [49,50]. Indeed, upon depolymerisation of the microtubules, AL remain scattered
throughout the cytoplasm and sperm chromatin is
wrapped in pore-less membranes which fail to fuse with
the female pronucleus [50]. Similarly, in Xenopus egg
extracts, NPC insertion into the NE that is assembled
around sperm chromatin can be blocked by nocodazole
[51].
Mutual attraction: pronuclear movement
After AL clustering, the growing sperm aster captures the
female pronucleus and the two pronuclei rapidly move
towards each other in a dynein-mediated process [52,53].
A similar mechanism appears to be responsible for the
dynein-dependent attachment of the microtubules to
nuclei during NEBD in ®broblasts [10,11] and the
migration of the centrosomes in Drosophila [54]. Candidates for mediation of the microtubule NE/AL interaction are NPCs, because they are the only known common
components of both AL and NE. Moreover, only these
structures are known to connect the outer nuclear membrane to the lamina, thus providing suf®cient mechanical
stability to move the whole nucleus via microtubule
motors. In contrast, simply attaching microtubules to
Current Opinion in Cell Biology 2003, 15:88±95
92 Cell structure and dynamics
Figure 3
the outer nuclear membrane would probably only result
in the formation of an ER tubule [53] unless this attachment is mediated by a complex spanning the perinuclear
space ([55]; see also Update). It will be important in the
future to identify the key molecules involved in this
interaction, with the sea urchin egg possibly becoming
one of the model systems of choice in this area due to its
easily assayed pronuclear fusion and the progress that has
been made in the sequencing of its genome.
Pronuclear fusion: how to merge two complete nuclear
envelopes
Once the pronuclei of sea urchin eggs are in close proximity, their NEs fuse by a poorly understood mechanism.
Although pronuclear fusion is not universal to all oocytes
(e.g. mouse pronuclei only appose and the male and
female chromosomes congress in a common mitotic spindle upon ®rst mitotic cleavage), similar fusions occur in
many vertebrate species after mitosis, during early
embryonic development in the process of karyomere
fusion. Karyomeres are mini-nuclei that form around each
chromosome during anaphase (see also Update). Their
NEs contain pores and support nucleocytoplasmic transport and DNA replication [56]. Only later in replication
do these karyomeres fuse to form one common nucleus
containing all chromosomes. The reason for karyomere
formation remains unclear, but may re¯ect a need for a
prompt entry into S phase in these rapidly dividing
blastomeres.
Topologically, the fusion of two complete nuclei poses
several problems. The crosslinked NE structure with two
nuclear membranes, NPCs and the lamina is very stable
during interphase [7]. Therefore, nuclear fusion most
likely requires local (or complete) disassembly of the
lamina, removal of NPCs from the fusion site, speci®c
fusion of the outer membranes and speci®c fusion of the
inner membranes (Figure 3). Dissolving the lamina is
likely to be the ®rst step, because this would allow the
otherwise anchored NPCs to diffuse laterally [7]. An
example of local lamina disassembly was recently provided for the case of viral egress from the nucleus (see
[57]). Once the NPCs are cleared from the fusion site,
the outer membranes may fuse, forming an intermediate
state with the two inner membranes lying close together
NE dynamics during fertilisation in echinoderms. (i) As the sperm
enters the oocyte, it is still surrounded by the pore-less sperm NE. (ii)
The sperm envelope disassembles and the nucleus swells as a result
of chromatin reorganisation. (iii) The pronuclear envelope forms,
utilising the NPC reserves stored in the AL, which are moved along the
sperm aster microtubules. (iv±vii) Pronuclei then move towards each
other and fuse. Insert: A possible model for pronuclear fusion. (a,b)
NPCs have to be removed from the fusion site, which presumably also
requires local disassembly of lamina. (c) Outer membranes then fuse.
(d,e) The inner membranes then follow suit. Microtubules and
centrosomes, red; chromatin, blue; lamina, green; NE, yellow; NPCs,
dark yellow.
Current Opinion in Cell Biology 2003, 15:88±95
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Nuclear envelope dynamics in oocytes LeÂnaÂrt and Ellenberg 93
[45,58]. Similar intermediate states can be seen on
electron micrographs, suggesting that this structure exists
for prolonged periods of time [45,58]. Later, the inner
membranes also fuse, mixing the content of the two
nuclei. Initially, only a narrow channel connects the
two pronuclei, but this then slowly increases in size
[59], indicating a constraint on the spread of the fusion
site, possibly by the remnants of the lamina±NPC network. Considering the complex structure of the NE, the
multiple intermediates that may form and the slow
kinetics of membrane fusions, pronuclear fusion is probably a complex, multistep process. It is likely that pronuclear fusion requires more elaborate machinery than
that beginning to be characterised for nuclear assembly
[60]. It may also share features with remodelling of
interphase nuclei seen during replication [61] and viral
infection [62].
Conclusions
Egg extracts are well-established systems for the study of
NE biochemical remodelling and have been used for
more than 20 years [42,63]. As this review has highlighted, the transparent oocytes available in many species
are also ideally suited to the analysis of meiosis and
embryonic mitosis in the intact cell. In echinoderms
especially, M-phase NE dynamics can be analysed with
excellent spatial and temporal resolution by advanced
imaging techniques and are easily manipulated by microinjection. Oocyte systems provide a very different cellular
context for imaging M-phase processes compared with
the commonly used cultured mammalian somatic cells.
This is particularly apparent in the case of NEBD, which
we now know, from the study of star®sh oocytes, is most
likely to start with the gradual disassembly of the nuclear
pore, coinciding with the accumulation of MPF in the
nucleus. This is much more dif®cult to appreciate in
mammalian cells where mechanical events involving
the mitotic spindle dominate entry into mitosis. Other
fundamental nuclear processes are just beginning to be
examined in intact cells and, again, oocytes will be an
invaluable investigational aid. Pronuclear migration provides an excellent model for the interactions of nuclei
with microtubules that occur in many cells with functions
as diverse as nuclear positioning, NEBD and centrosome
separation. Pronuclear and karyomere fusion exemplify
the dazzling topological problem of merging two entire
nuclei. Finally, pronuclear assembly after meiosis may be
an effective model for the study of interphase NE rearrangements. With genome sequences of the sea urchin in
the pipeline, there are many insights still to come from
oocytes, systems that have proven to be both classical and
state-of-the-art tools of cell biology.
Update
Recent work has provided further evidence for protein
interactions spanning the lumen of the NE. The protein
ANC-1 might bridge between the actin cytoskeleton and
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the INM protein UNC-84, thereby tethering the nucleus
to the cytoskeleton [64]. Hinkle et al. [65] determined
the localisation and dynamics of the small GTPase Ran by
injecting ¯uorescently labelled recombinant protein into
living cells. They found it to be localised to the NE and to
chromosomes in mouse, Xenopus and star®sh eggs, as well
as in somatic mammalian cells. Time series of activated
Xenopus eggs show an excellent example of karyomere
formation during anaphase, with Ran immediately localised to the envelope of the re-forming mininuclei around
each chromosome [65].
Acknowledgements
The authors would like to thank Mark Terasaki for sharing unpublished data,
Lisa Mehlmann for the picture of the mouse oocyte (Figure 1) and Gustavo
Gutierrez for the Xenopus oocyte (Figure 1). We are grateful to Mark Terasaki
and Philippe Collas for critically reading the manuscript. We apologise to
those whose work we have not cited owing to space limitations.
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Current Opinion in Cell Biology 2003, 15:88±95
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Current Opinion in Cell Biology 2003, 15:88±95