RESEARCH ARTICLE 3307
Development 134, 3307-3315 (2007) doi:10.1242/dev.005454
Vesicular traffic at the cell membrane regulates oocyte
meiotic arrest
Wassim El-Jouni, Shirley Haun, Rawad Hodeify, Azida Hosein Walker and Khaled Machaca*
Vertebrate oocytes are maintained in meiotic arrest for prolonged periods of time before undergoing oocyte maturation in
preparation for fertilization. Cyclic AMP (cAMP) signaling plays a crucial role in maintaining meiotic arrest, which is released by a
species-specific hormonal signal. Evidence in both frog and mouse argues that meiotic arrest is maintained by a constitutively active
G-protein coupled receptor (GPCR) leading to high cAMP levels. Because activated GPCRs are typically targeted for endocytosis as
part of the signal desensitization pathway, we were interested in determining the role of trafficking at the cell membrane in
maintaining meiotic arrest. Here we show that blocking exocytosis, using a dominant-negative SNAP25 mutant in Xenopus oocytes,
releases meiotic arrest independently of progesterone. Oocyte maturation in response to the exocytic block induces the MAPK and
Cdc25C signaling cascades, leading to MPF activation, germinal vesicle breakdown and arrest at metaphase of meiosis II with a
normal bipolar spindle. It thus replicates all tested aspects of physiological maturation. Furthermore, inhibiting clathrin-mediated
endocytosis hinders the effectiveness of progesterone in releasing meiotic arrest. These data show that vesicular traffic at the cell
membrane is crucial in maintaining meiotic arrest in vertebrates, and support the argument for active recycling of a constitutively
active GPCR at the cell membrane.
INTRODUCTION
Early in vertebrate oogenesis, germ cells enter the meiotic cell cycle
and arrest in prophase of meiosis I for extended periods of time, up
to 40-50 years in humans, for example (Eppig et al., 2004; Masui
and Clarke, 1979; Hassold and Hunt, 2001). During this time the
oocyte grows and accumulates crucial macromolecular components
for supporting later development (Voronina and Wessel, 2003).
Release from meiotic arrest marks the initiation of oocyte
maturation, a dramatic cellular differentiation that prepares the egg
for fertilization and early embryonic development (Eppig et al.,
2004; Bement and Capco, 1990). The molecular mechanisms
underlying oocyte meiotic arrest are not fully understood, but there
is general agreement that elevated cyclic AMP (cAMP) levels
underlie meiotic arrest in several vertebrate and invertebrate species
(Maller and Krebs, 1977; Cho et al., 1974; Stern and Wassarman,
1974; Meijer and Zarutskie, 1987; Conti et al., 2002).
Xenopus oocytes are typically matured in vitro using
progesterone, although evidence supports the argument that
androgens are the major physiological stimulus, partly because they
are the primary steroids produced in the ovary (Lutz et al., 2001).
The mechanisms by which steroids release meiotic arrest are not
fully understood, but it is clear that transcription is not required for
oocyte maturation (Masui and Markert, 1971). There is, however,
evidence supporting a role for steroid action through both a
membrane receptor and classical steroid receptors (Masui and
Markert, 1971; Bayaa et al., 2000; Tian et al., 2000; Evaul et al.,
2007). In Xenopus, progesterone-induced oocyte maturation is
associated with a rapid transient decline (20-60%) in cAMP (Maller
et al., 1979; Cicirelli and Smith, 1985), due to inhibition of adenylate
cyclase (AC) (Sadler and Maller, 1981; Sadler and Maller, 1985;
Department of Physiology and Biophysics, University of Arkansas for Medical
Sciences (UAMS), Little Rock, AR 72205, USA.
*Author for correspondence (e-mail: [email protected])
Accepted 13 July 2007
Finidori-Lepicard et al., 1981). Furthermore, interventions that
increase cAMP, either through activation of AC (Schorderet-Slatkine
and Baulieu, 1982), increasing protein kinase A (PKA) activity
(Maller and Krebs, 1977) or inhibiting cAMP phosphodiesterase
(Bravo et al., 1978; Sadler and Maller, 1987), block progesteroneinduced oocyte maturation. Supporting these results, inhibition of
PKA induces oocyte maturation in the absence of progesterone
(Maller and Krebs, 1977; Huchon et al., 1981; Sun and Machaca,
2004; Daar et al., 1993). Based on these findings, a G-protein
coupled receptor (GPCR) linked to cAMP generation has been an
attractive mechanism to explain the maintenance of oocyte meiotic
arrest. Indeed, a growing body of evidence in both mammals and
Xenopus argues that meiotic arrest is maintained by a constitutively
active GPCR. Injection of neutralizing antibodies against G␣s in
both Xenopus and mouse oocytes releases meiotic arrest (Gallo et
al., 1995; Mehlmann et al., 2002). By contrast, blocking G␣i with
pertussis toxin in Xenopus oocytes has no effect on oocyte
maturation (Sadler et al., 1984). Others have also shown that the ␤␥
subunits are also involved in maintaining oocyte meiotic arrest in
Xenopus (Sheng et al., 2001; Lutz et al., 2000). In addition, mice
with a deleted adenylate cyclase type 3 gene show defects in meiotic
arrest (Horner et al., 2003). Upstream of G␣s, a GPCR
(GPR3/GPR12) has been recently shown to be essential for
maintaining meiotic arrest in rodents (Mehlmann et al., 2004;
Freudzon et al., 2005; Ledent et al., 2005; Hinckley et al., 2005).
Indeed, mice lacking the GPR3 gene exhibit spontaneous oocyte
maturation and are sub-fertile (Mehlmann et al., 2004; Ledent et al.,
2005). Together, these data support the argument that vertebrate
oocyte meiotic arrest is maintained by a constitutively active GPCR
through the action of AC and cAMP. Consistent with this
conclusion, injection of a GPCR kinase (GRK) or ␤-arrestin, which
desensitize GPCRs, into Xenopus oocytes leads to progesteroneindependent maturation (Wang and Liu, 2003).
Given the fact that most cells have developed complex cascades
to limit and regulate GPCR signaling, it is intriguing that oocyte
meiotic arrest is dependent on extended constitutive GPCR
DEVELOPMENT
KEY WORDS: Meiotic arrest, Oocyte maturation, Xenopus laevis, Exocytosis, Endocytosis, Clathrin, SNAP25
3308 RESEARCH ARTICLE
MATERIALS AND METHODS
Gamete preparation and treatments
Xenopus oocytes were obtained as previously described (Machaca and
Haun, 2002). Oocyte maturation was induced with 5 ␮g/ml progesterone.
GVBD was detected visually by the appearance of a white spot at the animal
pole and confirmed by fixing oocytes in methanol and bisecting them in half
to visualize the germinal vesicle (see Fig. 2B). For forskolin and cholera
toxin treatments, oocytes were pre-incubated in 100 ␮M forskolin or
5⫻10–9 M cholera toxin (Calbiochem) for 30-60 minutes before
progesterone treatment, whereas SNAP25⌬20-injected cells were treated
with the drugs at the time of injection.
Western blots
Lysates were prepared in extraction buffer (80 mM ␤-glycerophosphate, 20
mM Hepes, pH 7.5, 20 mM EGTA, 15 mM MgCl2, 1 mM sodium vanadate,
50 mM NaF, 1 mM DTT, 10 ␮g/ml aprotinin, 50 ␮g/ml leupeptin, 1 mM
PMSF). Typically 1-2 oocyte equivalents were loaded per lane for Westerns.
Anti-phopho-MAPK, anti-phospho-Tyr15 of Cdc2 and anti-Cdc25C
antibodies were from Cell Signaling, and anti-SNAP25 antibody was from
Sternberger Monoclonals.
Capacitance measurement
Oocytes were voltage clamped with two microelectrodes by the use of a
GeneClamp 500 (Axon Instruments). Electrodes were filled with 3 M KCl
and had resistances of 0.5-2 M⍀. Oocytes were bathed in Ringer, in mM: 96
NaCl, 2.5 KCl, 1.8 CaCl2, 2 MgCl2, 10 HEPES, pH 7.4. Voltage stimulation
and data acquisition were controlled using pClamp8 (Axon Instruments).
Membrane capacitance (Cm) was measured using the built-in algorithm in
pClamp8 with a voltage pulse of 5 mV. This algorithm accurately
reproduced capacitance values obtained by direct calculation as previously
described (Machaca and Haun, 2000). Specifically, four steps from a –35
mV holding potential to –30 mV for 50 msec each were administered.
Capacitive current decay was averaged and fitted by a single exponential.
Membrane capacitance Cm was calculated as ␶ (1/Ra+Gm). ␶ is the time
constant obtained from the exponential fit. Ra is the access resistance and
was calculated as Vp/I0. Vp is the applied voltage pulse (5 mV) and I0 is the
instantaneous current obtained by extrapolating the experimental fit to time
0. Gm was calculated as Iss/(Vp–Ra*Iss). Iss is the steady state current
following relaxation of the capacitive transient (Takahashi et al., 1996).
Molecular biology
For the wild-type full-length SNAP25, mouse Snap25B in pcDNA3 (Low et
al., 1998) was subcloned into the BamHI-XhoI sites in the Xenopus oocyte
expression vector pSGEM, which flanks the cDNA with the 5⬘- and 3⬘UTRs from the Xenopus globin gene, thus stabilizing the resultant mRNA
in the oocyte (Liman et al., 1992). The mouse Snap25 gene was highly likely
to be functional in Xenopus, as it shares 95% identity with Xenopus
SNAP25. SNAP25⌬20 was generated from pcDNA3-SNAP25 by PCR
using a pair of primers that flanked the clone with BamHI-EcoRI sites for
subcloning into pSGEM, and that introduced a stop codon after residue 186,
thus deleting the last 20 residues. mRNAs for the SNAP25 clones in pSGEM
were produced by in vitro transcription after linearizing the vector with NheI
using the mMessage mMachine T7 kit (Ambion). The Mos clone was
previously described (Machaca and Haun, 2002).
Transferrin endocytosis assay
Cells were treated overnight with 332 ␮M monodansylcadaverine (MDC)
(Sigma) or the carrier control (DMSO), washed in OR2 (82.5 mM NaCl, 2.5
mM KCl, 1 mM Na2HPO4, 1 mM CaCl2, 5 mM HEPES, pH 7.5) for 5
minutes and incubated OR2 containing Alexa-fluor-633-conjugated
transferrin at a concentration of (125 ␮g/ml) for 15 minutes. Then they were
rinsed extensively in OR2, and the extent of transferrin internalization at the
vegetal pole was imaged on a Zeiss LSM510 confocal microscope (10⫻
objective). Cross sections at several planes across the oocytes were scanned
and showed consistent levels of transferrin internalization. Therefore, data
were collected from a single plane at an equivalent depth into the vegetal
hemisphere. Images were thresholded and subjected to morphometry
analysis using the MetaMorph software. This allowed quantification of the
number of early endosomes, their equivalent sphere volume, and average
and total transferrin fluorescent intensity.
Imaging spindle structure
Cells were fixed 3 hours after GVBD in 100% methanol and stored at –20°C
overnight. After rehydration in TBS:methanol (1:1) for 20 minutes, oocytes
were washed twice with TBS for 15 minutes each and blocked for 3 hours
in TBS containing 2% BSA. Oocytes were then immunolabeled with an anti␣-tubulin monoclonal antibody (DMA1, Sigma) in TBS containing 2%
BSA, followed by a Cy2-conjugated donkey anti-mouse secondary
(Jackson) for 24 hours each. The oocytes were washed five times in TBS for
24 hours and stained with 1 ␮M Sytox Orange (Molecular Probes). After
staining cells were washed in TBS for 1 hour, dehydrated in 100% methanol
for 30 minutes and cleared in benzyl alcohol/benzyl benzoate (1:2). Spindle
structure images were collected on a Zeiss LSM510 confocal microscope
(20⫻ objective).
RESULTS
SNAP25⌬20 blocks exocytosis
SNARE proteins are central to vesicular fusion events that underlie
trafficking at the subcellular level (Chen and Scheller, 2001).
SNAREs contain coiled-coil domains that interact to form a fourhelix bundle, which is important for vesicle fusion. Three helices are
contributed by Q-SNAREs present on one membrane, and the fourth
helix is contributed by an R-SNARE on the other membrane (Salaun
et al., 2004). In the case of exocytosis, two of the four helices that
form the four-helix bundle are contributed by SNAP25 (Jahn, 2004).
SNAP25 contains two coiled-coil domains at its N- and C-termini
and a central cysteine-rich domain that mediates membrane
localization through palmitoylation (Salaun et al., 2004). Because
all four SNARE helices are required for membrane fusion, deletion
of a SNAP25 coiled-coil domain is predicted to block exocytosis.
Indeed, a SNAP25 mutant that removes the last 20 residues
(SNAP25⌬20) has been reported to act as a dominant-negative of
exocytosis in Xenopus oocytes (Yao et al., 1999). Therefore, to test
the role of exocytosis in maintaining meiotic arrest we constructed
a SNAP25⌬20 clone and expressed it in oocytes (Fig. 1). By
recording membrane capacitance as a direct measure of cell
DEVELOPMENT
signaling. This raises the interesting question of the mechanisms
involved in allowing such prolonged GPCR signaling in oocytes. An
important pathway for GPCR desensitization is through endocytic
removal of the receptor from the cell membrane following GRK
phosphorylation and arrestin binding (Moore et al., 2006). If this
pathway is indeed functional in oocytes, then these cells must have
developed mechanisms to replenish active GPCRs at the cell
membrane to maintain meiotic arrest. Here we explore the role of
vesicular trafficking at the cell membrane in maintaining meiotic
arrest in Xenopus oocytes. We show that meiotic arrest requires a
functional exocytic pathway, as blocking exocytosis with a
dominant-negative SNAP25 (SNAP25⌬20) releases meiotic arrest
in the absence of the physiological stimulus, progesterone. The
effect of SNAP25⌬20 expression on trafficking at the cell membrane
was followed by measuring membrane capacitance, which provides
a direct measure of membrane area and has been shown to closely
track the trafficking pattern of several membrane proteins in
Xenopus oocytes (Peters et al., 1999; Quick et al., 1997; Awayda,
2000). SNAP25⌬20-induced maturation is normal in every aspect
tested: it induces the MAPK and Cdc25C cascades leading to MPF
activation, germinal vesicle breakdown (GVBD) and arrest at
metaphase II of meiosis with a normal bipolar spindle. Furthermore,
blocking clathrin-mediated endocytosis hinders the effectiveness of
saturating levels of progesterone in releasing oocytes from meiotic
arrest. Together, these data show that vesicular trafficking at the cell
membrane is a crucial determinant of meiotic arrest.
Development 134 (18)
Fig. 1. Dominant-negative SNAP25⌬20 blocks exocytosis. Xenopus
ocytes were injected with either wild-type or SNAP25⌬20 mRNA (5-10
ng) and membrane capacitance was measured [Cm(nF)] using two
electrode-voltage clamp over time, as indicated (mean±s.e.; n=8-20).
Western blot analysis using SNAP25 antibody shows similar levels of
expression of wild-type and SNAP25⌬20, and no band was detected in
uninjected oocytes. SNAP25⌬20 runs faster than wild-type full-length
SNAP25 because of the deletion of the last 20 residues. Ooc,
uninjected oocytes; WT, wild type.
membrane area, we functionally confirmed that SNAP25⌬20 acts as
a dominant-negative and effectively blocks exocytosis (Fig. 1).
SNAP25⌬20, but not full-length wild-type SNAP25, decreased
membrane area in a time-dependent fashion (Fig. 1). The expression
levels of both SNAP25⌬20 and wild-type SNAP25 were
comparable (Fig. 1).
SNAP25⌬20 induces oocyte maturation
Expression of SNAP25⌬20 induced progesterone-independent
oocyte maturation (Fig. 2). Oocytes expressing SNAP25⌬20 entered
meiosis, as marked by GVBD, with the same efficiency as
progesterone-treated oocytes (Fig. 2A). By contrast, oocytes injected
with wild-type SNAP25 mRNA did not mature (Fig. 2A).
SNAP25⌬20-dependent oocyte maturation was associated with the
appearance of a normal white spot on the animal hemisphere, and
with the expected breakdown of the nuclear envelope (Fig. 2B).
Furthermore, SNAP25⌬20-injected oocytes extruded a polar body,
showing that they completed meiosis I, and arrested at metaphase of
meiosis II with a normal metaphase II spindle (Fig. 2C).
By assessing the time required for 50% of the oocytes in the
population to reach GVBD (G50), one can obtain a measure of the
rate at which oocytes mature. SNAP25⌬20-induced maturation
occurred with significantly slower kinetics compared with
progesterone (Fig. 2D). This is not surprising given the time required
for translation of the injected mRNA before a functional block of
exocytosis can be achieved.
Oocyte maturation is ultimately dependent on the activation of
maturation-promoting factor (MPF/cdk1-cyclin B), which is the
primary activity that regulates G2/M transition in both mitosis and
meiosis, and consists of a catalytic p34cdc2 Ser/Thr kinase subunit
and a regulatory cyclin B subunit (Coleman and Dunphy, 1994). Two
signaling cascades combine to induce the dramatic activation of
MPF at GVBD: the MAPK-cascade, which leads to inhibition of the
MPF-inhibitory kinase Myt1 (Palmer et al., 1998; Nebreda and
Ferby, 2000), and the polo-like kinase-Cdc25C cascade, which
activates Cdc25C. Cdc25C is a dual-specificity phosphatase that
RESEARCH ARTICLE 3309
removes the inhibitory phosphorylation at Tyr15 and Thr14 from
cdc2 kinase, and constitutes the rate-limiting step in MPF activation
(Perdiguero and Nebreda, 2004; Kumagai and Dunphy, 1991).
To determine whether the signaling cascade underlying oocyte
maturation is activated normally in SNAP25⌬20-injected cells, we
measured the activation of MAPK and MPF (Fig. 2E). For these
experiments, oocyte lysates were prepared at different time points
during maturation: (1) when the first cells in the population reached
GVBD (GVBD); (2) when 50% of the cells reached GVBD [G50;
for this time point, lysates from oocytes with a white spot (w) and
those without (nw) were collected]; (3) when 100% of the cells in
the population reached GVBD (G100). SNAP25⌬20 induced similar
activation profiles as progesterone for both MAPK and MPF,
supporting the argument that it induces oocyte maturation using the
same pathways activated by the physiological hormone (Fig. 2E).
In a similar fashion, SNAP25⌬20 induced Cdc25C activation, as
marked by its supershift due to hyperphosphorylation (Fig. 2F). The
wild-type SNAP25 control did not activate MAPK, MPF (Fig. 2E)
or Cdc25C (Fig. 2F), although it was typically expressed at higher
levels than SNAP25⌬20 (Fig. 2E,F). Together, these results show
that SNAP25⌬20 induces normal oocyte maturation at the
biochemical, morphological and nuclear maturation (meiosis)
levels.
Timecourse of SNAP25⌬20-dependent maturation
We then analyzed the timecourse of SNAP25⌬20-induced
maturation in more detail to determine if it is equivalent to
progesterone treatment. Oocytes were either injected with
SNAP25⌬20 mRNA or treated with progesterone, and GVBD and
capacitance were measured over time. In addition, lysates were
collected for analysis of kinase activation and SNAP25⌬20
expression. After progesterone treatment, membrane area decreases
gradually over time, as previously reported (Kado et al., 1981;
Machaca and Haun, 2000). Oocytes begin to undergo GVBD when
capacitance in the population reaches ~165 nF, and capacitance
continues to decrease as maturation progresses (Fig. 3A) (Machaca
and Haun, 2000). MAPK is phosphorylated ~2.5 hours before
GVBD, and MPF activation as marked by cdc2 dephosphorylation,
does not occur until the GVBD stage (Fig. 3B). The kinetics of
kinase activation and capacitance decrease in SNAP25⌬20-injected
cells is similar (Fig. 3). MAPK activates ~1.5 hours before GVBD,
and MPF activates at the GVBD stage (Fig. 3B). Membrane
capacitance in SNAP25⌬20-injected cells reaches ~135 nF when
cells begin to undergo GVBD (Fig. 3A). Most interesting is the
expression of SNAP25⌬20 protein, which is first detectable ~2
hours post-RNA injection, and accumulates to significant levels ~5
hours after RNA injection (Fig. 3B). This 2-5 hour delay in
expression of SNAP25⌬20 explains the delay in GVBD kinetics
(Fig. 3A). These data support the argument that once SNAP25⌬20
is expressed at high enough levels to induce a significant block in
exocytosis, as measured by membrane capacitance (Fig. 3A), it is
capable of inducing oocyte maturation with similar kinetics to
progesterone. These results raise the intriguing possibility that a
block of exocytosis is a functionally important component of
progesterone-induced maturation.
Botulinum neurotoxin (BoNT) acts synergistically with
progesterone
To corroborate the results of the dominant-negative SNAP25⌬20,
we tested the effects on maturation of BoNT A, a zinc-dependent
protease that cleaves and inactivates SNAP25 (Sudhof, 1995).
Injection of various amounts (100-600 nM) of the catalytically
DEVELOPMENT
Trafficking and meiotic arrest
active light chain of BoNT A was insufficient to induce oocyte
maturation. However, when BoNT A-injected oocytes (Fig. 3C,
BoNTA 200 nM) were treated with sub-threshold levels of
progesterone (100 nM), they activated fully, with similar kinetics to
oocytes matured using supra-maximal progesterone (Fig. 3C,
Prog). By contrast, control BSA-injected oocytes exhibited low
levels of maturation at sub-threshold progesterone (Fig. 3C, BSA).
This synergistic effect of BoNT A at sub-threshold levels of
progesterone was observed in 2/4 experiments on oocytes from
different donor females, showing that BoNT A is only mildly
effective at blocking exocytosis. Indeed, in contrast to the robust
exocytic block with SNAP25⌬20, which effectively reduces
membrane capacitance (Fig. 1), no effect of BoNT A injection on
membrane capacitance could be detected (Fig. 3D). However, the
synergistic effect of BoNT A supports the argument that BoNT A
blocks exocytosis sufficiently to potentiate the effects of subthreshold levels of progesterone (Fig. 3C). This suggests that the
exocytic block is a physiological mechanism mediating
progesterone action. Therefore, the fact that BoNT A can potentiate
the ability of sub-threshold progesterone to induce maturation
supports our results with the dominant-negative SNAP25⌬20dependent exocytic block.
Development 134 (18)
Analysis of SNAP25⌬20 site of action
We were then interested in mapping the site of action of
SNAP25⌬20 in releasing meiotic arrest along the physiological
oocyte maturation cascade. Because SNAP25 acts specifically at the
cell membrane, it is expected that inhibition of early steps known to
be involved in oocyte maturation should block SNAP25⌬20mediated maturation. As discussed above, these include a block of
AC through a G-protein-dependent pathway, leading to a decrease
in cAMP levels. We therefore tested the effects of agents that
maintain cAMP levels high in the oocyte on SNAP25⌬20-mediated
maturation (Fig. 4). Forskolin activates AC and has been shown to
block progesterone-dependent oocyte maturation (SchorderetSlatkine and Baulieu, 1982). Indeed, forskolin blocks both
progesterone- and SNAP25⌬20-mediated maturation, showing that
both act upstream of AC (Fig. 4A). As expected, forskolin blocks the
activation of both MAPK and MPF compared with control cells, and
importantly forskolin does not significantly inhibit SNAP25⌬20
protein expression levels (Fig. 4A). For these analyses it is important
to confirm that factors that are known to act downstream of the step
of interest are capable of rescuing the block. This is particularly the
case for cAMP, as PKA could have multiple direct effects on
downstream effectors crucial for oocyte maturation. For example,
Fig. 2. SNAP25⌬20 releases oocyte meiotic arrest in Xenopus. (A) Percentage of oocytes reaching the GVBD stage following progesterone
treatment, or injection of SNAP25⌬20 or wild-type SNAP25 mRNA (mean±s.e.; n=4-7 experiments with >50 oocytes in each treatment). The
percentages reported are the maximal levels of GVBD reached. (B) Photographs showing the absence of a white spot on the animal hemisphere,
and the presence of the germinal vesicle (nucleus) (arrow) in untreated oocytes (Ooc) and oocytes injected with wild-type SNAP25 mRNA (WT). By
contrast, progesterone (Prog) or SNAP25⌬20 (⌬20) injection results in the appearance of a white spot on the animal pole and GVBD. Top row,
white spot; bottom row, GVBD. (C) Spindle structure (top) and polar body (bottom) in progesterone-treated and SNAP25⌬20-injected oocytes.
Spindle structure was visualized by indirect immunofluorescence using an anti-tubulin antibody and chromosomes were stained with Sytox Orange
(scale bars: 5 ␮m for spindles and 10 ␮m for polar body). (D) Time required for 50% of the oocytes in the population to reach the GVBD stage of
maturation (GVBD50) following progesterone treatment or SNAP25⌬20 mRNA injection (mean±s.e.; n=7 experiments from different female
donors). (E) Western blot analysis of cells treated with progesterone or injected with either SNAP25⌬20 or SNAP25 wild-type mRNA. Lysates were
prepared at different time points during oocyte maturation: (1) untreated oocytes; (2) when oocytes first reached the GVBD stage (GVBD); (3) when
50% of the cells reach GVBD (G50). In this case, lysates were prepared from cells with (w) and without (nw) a white spot; (4) when 100% of the
cells reached the GVBD stage (G100). Because oocytes injected with wild-type SNAP25 mRNA do not undergo GVBD, lysates were prepared at the
same time points when SNAP25⌬20-injected cells reached the G50 and G100 milestones, indicated as G50eq and G100eq, respectively. Blots were
probed with anti-phospho-MAPK, anti-phospho-cdc2 and anti-SNAP25 antibodies. (F) Lysates from oocytes that have undergone GVBD at the G50
time point were prepared and western blot analysis performed to assess Cdc25C activation and SNAP25 expression. Cdc25C activation was
detected as a supershift on the gel due to hyperphosphorylation (arrowheads) from the basal state (arrow) observed in oocytes. In contrast to
progesterone or SNAP25⌬20, no shift is detected in oocytes or SNAP25 wild-type injected cells. The middle band is a non-specific band. Blots were
stripped and re-probed with anti-SNAP25 antibody (lower panel).
DEVELOPMENT
3310 RESEARCH ARTICLE
cdc25 has been identified as a target for PKA, leading to its
inhibition (Duckworth et al., 2002). Therefore, it is possible that high
levels of PKA block maturation at later steps along the oocyte
maturation cascade, such as cdc25. To rule out this possibility we
confirmed that the forskolin block could be rescued by Mos injection
to directly activate the MAPK cascade (Fig. 4C). These results show
that SNAP25⌬20 acts upstream of AC to release oocyte meiotic
arrest.
We also tested the effect of cholera toxin, which ADP-ribosylates
G␣s and activates it, leading to increased activation of AC and a rise
in cAMP levels (Gill and Meren, 1978). Similarly to forskolin,
cholera toxin blocks both progesterone- and SNAP25⌬20-mediated
maturation, without dramatically affecting SNAP25⌬20 protein
expression levels (Fig. 4B). However, in contrast to forskolin, Mos
RNA injection was ineffective at rescuing the cholera toxinmediated inhibition (Fig. 4C), supporting the argument that cholera
toxin – which is likely to be a more potent activator of AC, as it
catalytically activates G␣s – inhibits maturation by acting both at the
early steps and later steps downstream of Mos during maturation.
Nonetheless, the forskolin results confirm that SNAP25⌬20 acts
upstream of AC, consistent with the functional role of SNAP25 at
the cell membrane.
RESEARCH ARTICLE 3311
Role of endocytosis in maintaining meiotic arrest
If as predicted by the exocytosis block data, the residence time of a
constitutive G-protein coupled receptor at the plasma membrane
ultimately modulates meiotic arrest, a block of endocytosis is
expected to negatively modulate oocyte maturation. Specifically,
clathrin-dependent endocytosis would be of primary interest,
because it is the predominant internalization pathway for activated
GPCRs (Moore et al., 2006). To test the role of endocytosis in
meiotic arrest, we inhibited both constitutive and clathrin-mediated
endocytosis (Fig. 5). Constitutive endocytosis in Xenopus oocytes,
the housekeeping pathway that maintains plasma membrane
homeostasis, can be inhibited using Clostridium botulinum C3
exoenzyme, which ADP-ribosylates and inactivates RhoA
(Schmalzing et al., 1995). Indeed, injection of oocytes with C3
exoenzyme results in an increase in membrane capacitance
consistent with an endocytic block (Fig. 5A). However, blocking
constitutive endocytosis did not affect the rate or extent of oocyte
maturation (Fig. 5B), arguing that the constitutive endocytic
pathway is not involved in regulating meiotic arrest.
We next tested the effect of inhibition of the clathrin mediated
endocytic pathway using monodansylcadaverine (MDC) (Schlegel
et al., 1982). We first developed a functional assay to confirm the
Fig. 3. Timecourse analysis of
SNAP25⌬20-induced oocyte maturation
in Xenopus. (A) Extent of GVBD (black)
and membrane capacitance (blue) in
progesterone- (left panel) and SNAP25⌬20(right panel) treated cells. The inset (below
left) shows no change in capacitance or
maturation in untreated oocytes over a
similar timecourse. (B) MAPK and MPF
activation and SNAP25 expression over the
maturation timecourse following
SNAP25⌬20 mRNA injection (left panel)
and progesterone treatment (right panel).
(C) Oocytes were either left untreated and
matured with maximal levels of
progesterone (16 ␮M), or injected with the
light chain of BoNT A (200 nM) or BSA as
the carrier control and matured with subthreshold levels of progesterone (100 nM).
The maturation timecourses for the three
treatments show that BoNT A injection
potentiates the effects of sub-threshold
levels of progesterone on maturation. This
experiment was repeated four times on
oocytes from different donor females, and
the potentiation effects of BoNT A were
observed in 2/4 experiments.
(D) Membrane capacitance (Cm)
measurements at different times, as
indicated, after BSA or BoNT A (200 nM)
injection, showing that BoNT A is
ineffective at reducing Cm.
DEVELOPMENT
Trafficking and meiotic arrest
3312 RESEARCH ARTICLE
Development 134 (18)
Fig. 4. Activation of adenylate cyclase inhibits
SNAP25⌬20-induced maturation. (A,B) Extent of
maturation of Xenopus oocytes treated with progesterone or
injected with SNAP25⌬20 mRNA in the presence or absence
of 100 ␮M forskolin (A) or 5⫻10–9 M cholera toxin (B).
Timecourses of maturation were carried out and the plateau
levels of GVBD at the end of the experiments are reported
(above). The activation of MAPK and MPF and the expression
level of SNAP25⌬20 are also shown (below). Lysates for
western blot analysis were prepared from cells at the GVBD50
time point. (C) Percent GVBD of Mos mRNA- (10 ng) injected
oocytes in the presence or absence of 100 ␮M forskolin or
5⫻10–9 M cholera toxin. The inset (below) shows the time to
GVBD50 for Mos-injected cells±forskolin. CT, cholera toxin;
Forsk, forskolin.
5C). We quantified the levels of transferrin uptake using the number
and equivalent volume of these vesicular structures in a crosssectional confocal image (Fig. 5C). MDC effectively blocked
transferrin endocytosis, as the number of the presumed early
endosomes detected in MDC-treated cells was dramatically reduced
(34.3±11.7% of control) (Fig. 5C). Although the average volume of
labeled endosomes tended to be smaller in MDC-treated cells, the
Fig. 5. Inhibition of clathrin-mediated endocytosis negatively regulates oocyte maturation. (A) Membrane capacitance [Cm(nF)] of control
Xenopus oocytes and oocytes injected with C3 exoenzyme (1 ng/oocyte) 2 hours earlier. (B) GVBD timecourse from a representative experiment
after C3 exoenzyme injection. Cells were injected with C3 exoenzyme 1 hour before progesterone treatment or left untreated. The histograms
indicate percent GVBD and normalized time to GVBD50 (mean±s.e.; n=3). GVBD levels are those reached at the end of the experiment, typically no
longer than 18 hours. (C) Transferrin endocytosis assay. Examples of confocal cross-sectional images through oocytes used to quantify Alexa-633labeled transferrin internalization after overnight incubation with either the carrier control DMSO or 332 ␮M MDC. The number of labeled vesicular
structures and their equivalent sphere volumes were quantified, as detailed in Materials and methods. (D) Membrane capacitance of oocytes
incubated in the DMSO carrier control and oocytes treated with 332 ␮M monodansyl cadaverine (MDC) overnight. (E) GVBD time course from a
representative experiment after MDC treatment. Cells were incubated with MDC overnight or left untreated before progesterone treatment. The
histograms indicate percent GVBD and normalized time to GVBD50 (mean±s.e.; n=5). Scale bar: 100 ␮m. Con, control oocytes.
DEVELOPMENT
ability of MDC to inhibit clathrin-mediated endocytosis in Xenopus
oocytes. We used fluorescently labeled transferrin as a classical
marker for clathrin-mediated endocytosis (Dautry-Varsat, 1986),
especially because Xenopus oocytes possess functional transferrin
receptors (Lund et al., 1990). Although transferrin readily labels
small endocytic vesicles, the most reliable signal was from large
vesicular structures, which are potentially early endosomes (Fig.
data did not reach statistical significance (Fig. 5C). These results
show that MDC blocks clathrin-mediated endocytosis in Xenopus
oocytes and can be used to test the role of this pathway on meiotic
arrest.
In contrast to the C3 exoenzyme treatment (Fig. 5A), MDC did
not increase membrane capacitance (Fig. 5D), arguing that clathrindependent endocytosis has a much smaller contribution to
membrane area homeostasis compared with constitutive
endocytosis. However, blocking clathrin-dependent endocytosis
with MDC slows down the rate and inhibits the extent of
progesterone-mediated maturation (Fig. 5E). This shows that
inhibition of clathrin-mediated endocytosis negatively regulates the
ability of progesterone to relieve meiotic arrest. Similar results were
obtained with SNAP25⌬20-mediated maturation (not shown).
Therefore, the endocytic blockade experiments support the exocytic
block data, as they both show that vesicular recycling at the cell
membrane is a crucial determinant of oocyte meiotic arrest.
DISCUSSION
The prolonged arrest of oocytes in meiotic prophase awaiting oocyte
maturation is a fascinating biological problem that has been tackled
for several decades, yet the mechanisms involved remain obscure.
Recent data support the argument that GPCR signaling is crucial in
maintaining meiotic arrest (Mehlmann, 2005). In this study we have
addressed the role of vesicular trafficking at the cell membrane in
maintaining meiotic arrest. We have shown that blocking exocytosis
induces hormone-independent oocyte maturation that is normal in
every aspect tested. We used a dominant-negative SNAP25 to inhibit
exocytosis, because SNAP25 localizes to the cell membrane
(Gonzalo and Linder, 1998) and is thus likely to block trafficking
specifically at this subcellular compartment. The fact that an
exocytic block is sufficient to induce oocyte maturation is consistent
with the idea that continuous insertion of membrane proteins,
including possibly a constitutively active GPCR, is required for
meiotic arrest. Alternatively, the exocytic block data would also be
consistent with a potential autocrine mechanism for maintaining
meiotic arrest, where the oocyte secretes signals that act on cellsurface receptors to maintain meiotic arrest.
The SNAP25⌬20 mutant induces a very efficient exocytic block,
which leads to a dramatic decrease in membrane surface area. By
contrast, when we used other interventions, such as injection of
tetanus toxin or BoNT A to inhibit exocytosis, we were not able to
induce a significant decrease in membrane capacitance or oocyte
maturation. Nonetheless, BoNT A potentiates the ability of subthreshold levels of progesterone to induce maturation, arguing that
even mild inhibition of exocytosis has significant functional
consequences in terms of inducing maturation. This supports the
argument that a robust block of exocytosis is required to release the
oocyte from meiotic arrest. Consistent with this conclusion, blocking
ER-to-Golgi transport with brefeldin A, which ultimately leads to
disruption of the Golgi and inhibition of exocytosis, was reported to
induce oocyte maturation in Xenopus (Mulner-Lorillon et al., 1995).
Although the efficiency of brefeldin at inducing meiotic arrest is
poor compared with progesterone and SNAP25⌬20, it illustrates the
point that other interventions that significantly inhibit exocytosis can
release meiotic arrest.
Consistent with a crucial role for the exocytic pathway in
maintaining meiotic arrest, blocking clathrin-mediated but not
constitutive endocytosis negatively regulates the ability of
progesterone to release meiotic arrest. One interpretation of these
data is that because activated GPCRs are internalized through a
clathrin-mediated pathway, inhibition of this desensitization route
RESEARCH ARTICLE 3313
will increase the number of active GPCRs at the cell membrane, thus
countering the effects of progesterone. However, direct evidence for
this hypothesis will have to await identification of the constitutively
active GPCR responsible for maintaining meiotic arrest in Xenopus
oocytes.
Recent mouse and rat data show that constitutively active GPCRs
(GPR3/12) are required for meiotic arrest (Hinckley et al., 2005;
Freudzon et al., 2005). A similar mechanism could be functional in
Xenopus, especially because frog oocytes maintain meiotic arrest
after removal of the surrounding follicular cells, arguing for an
oocyte-autonomous mechanism for meiotic arrest. A membrane
progesterone receptor has been hypothesized for a long time in
Xenopus to induce meiotic maturation (Maller, 2001). Recently this
receptor was cloned and demonstrated to induce oocyte maturation
through positive induction (Zhu et al., 2003; Ben Yehoshua et al.,
2006). This shows that it is not functioning as the constitutively
active GPCR to maintain high cAMP levels. Rather, it supports the
argument that signaling through this membrane progesterone
receptor antagonizes a putative constitutively active GPCR pathway
that maintains meiotic arrest.
Physiological relevance of the exocytosis block
A gradual decrease in membrane surface area during Xenopus
oocyte maturation is well documented (Kado et al., 1981; Machaca
and Haun, 2000) and is due to an early block of exocytosis, which is
crucial for the formation of the fluid-filled blastocoele cavity during
embryogenesis (Muller, 2001). The blastocoele is formed due to
polarized vectorial transport of Na+ ions into the intercellular space
by an epithelium that surrounds the embryo (Muller, 2001). In
Xenopus the biogenesis of this polarized epithelium can be traced
back to the exocytosis block during oocyte maturation, which leads
to sequestration of most ionic transporters into an intracellular
vesicular pool (Muller and Hausen, 1995; Muller, 2001). As the
early blastomeres rapidly divide, they incorporate these intracellular
vesicles containing ionic channels and transporters into their
basolateral membranes. The apical membrane of this epithelium is
formed by the oocyte cell membrane, which is devoid of most
transporters, thus forming a polarized epithelium around the
embryo.
Additional morphological and functional evidence supports the
early exocytosis block during Xenopus oocyte maturation. At the
ultrastructural level, the decrease in surface area during oocyte
maturation is illustrated by the disappearance of microvilli, which
are enriched in oocytes but practically absent in eggs (Campanella
et al., 1984; Gardiner and Grey, 1983). Protein secretion is blocked
early on in maturation, specifically between the trans-Golgi network
and the plasma membrane (Colman et al., 1985; Leaf et al., 1990),
while other intracellular trafficking events (such as ER to Golgi) are
unaffected (Leaf et al., 1990; Ceriotti and Colman, 1989). Therefore,
an early exocytic block while endocytosis stays functional leads to
a dramatic decrease in membrane surface area during Xenopus
oocyte maturation. It is believed that oocytes employ this
mechanism to stock membranes and membrane proteins internally
in preparation for the rapid cell divisions in embryogenesis (Angres
et al., 1991; Gawantka et al., 1992). The mechanisms by which
progesterone blocks exocytosis are unknown, but it is clear that
vesicular trafficking at the cell membrane is crucial not only for
maintaining meiotic arrest but also for early embryogenesis.
We are grateful to Michael Hollmann for the gift of the Xenopus oocyte
expression vector pSGEM, and to Paul Roche for the mouse Snap25B clone.
We also acknowledge the use of the Confocal Microscopy Laboratory at the
University of Arkansas for Medical Sciences, which is supported by NIH Grant
DEVELOPMENT
Trafficking and meiotic arrest
P20RR16460 (PI: Larry Cornett, INBRE, Partnerships for Biomedical Research in
Arkansas) and NIH/NCRR Grant S10RR19395 (PI: Richard Kurten, “Zeiss LSM
510 META Confocal Microscope System“). This work was funded by grant
GM61829 from the NIH.
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