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First published online 15 August 2007
doi: 10.1242/dev.005454

Development 134, 3307-3315 (2007)
Published by The Company of Biologists 2007
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Vesicular traffic at the cell membrane regulates oocyte meiotic arrest

Wassim El-Jouni, Shirley Haun, Rawad Hodeify, Azida Hosein Walker and Khaled Machaca*

Department of Physiology and Biophysics, University of Arkansas for Medical Sciences (UAMS), Little Rock, AR 72205, USA.

* Author for correspondence (e-mail: kamachaca{at}uams.edu)

Accepted 13 July 2007


   SUMMARY
TOP
 SUMMARY
INTRODUCTION
 MATERIALS AND METHODS
 RESULTS
 DISCUSSION
 REFERENCES
 
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.

Key words: Meiotic arrest, Oocyte maturation, Xenopus laevis, Exocytosis, Endocytosis, Clathrin, SNAP25


   INTRODUCTION
TOP
 SUMMARY
 INTRODUCTION
MATERIALS AND METHODS
 RESULTS
 DISCUSSION
 REFERENCES
 
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., 2004Go; Masui and Clarke, 1979Go; Hassold and Hunt, 2001Go). During this time the oocyte grows and accumulates crucial macromolecular components for supporting later development (Voronina and Wessel, 2003Go). 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., 2004Go; Bement and Capco, 1990Go). 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, 1977Go; Cho et al., 1974Go; Stern and Wassarman, 1974Go; Meijer and Zarutskie, 1987Go; Conti et al., 2002Go).

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., 2001Go). 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, 1971Go). There is, however, evidence supporting a role for steroid action through both a membrane receptor and classical steroid receptors (Masui and Markert, 1971Go; Bayaa et al., 2000Go; Tian et al., 2000Go; Evaul et al., 2007Go). In Xenopus, progesterone-induced oocyte maturation is associated with a rapid transient decline (20-60%) in cAMP (Maller et al., 1979Go; Cicirelli and Smith, 1985Go), due to inhibition of adenylate cyclase (AC) (Sadler and Maller, 1981Go; Sadler and Maller, 1985Go; Finidori-Lepicard et al., 1981Go). Furthermore, interventions that increase cAMP, either through activation of AC (Schorderet-Slatkine and Baulieu, 1982Go), increasing protein kinase A (PKA) activity (Maller and Krebs, 1977Go) or inhibiting cAMP phosphodiesterase (Bravo et al., 1978Go; Sadler and Maller, 1987Go), block progesterone-induced oocyte maturation. Supporting these results, inhibition of PKA induces oocyte maturation in the absence of progesterone (Maller and Krebs, 1977Go; Huchon et al., 1981Go; Sun and Machaca, 2004Go; Daar et al., 1993Go). 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{alpha}s in both Xenopus and mouse oocytes releases meiotic arrest (Gallo et al., 1995Go; Mehlmann et al., 2002Go). By contrast, blocking G{alpha}i with pertussis toxin in Xenopus oocytes has no effect on oocyte maturation (Sadler et al., 1984Go). Others have also shown that the ß{gamma} subunits are also involved in maintaining oocyte meiotic arrest in Xenopus (Sheng et al., 2001Go; Lutz et al., 2000Go). In addition, mice with a deleted adenylate cyclase type 3 gene show defects in meiotic arrest (Horner et al., 2003Go). Upstream of G{alpha}s, a GPCR (GPR3/GPR12) has been recently shown to be essential for maintaining meiotic arrest in rodents (Mehlmann et al., 2004Go; Freudzon et al., 2005Go; Ledent et al., 2005Go; Hinckley et al., 2005Go). Indeed, mice lacking the GPR3 gene exhibit spontaneous oocyte maturation and are sub-fertile (Mehlmann et al., 2004Go; Ledent et al., 2005Go). 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 ß-arrestin, which desensitize GPCRs, into Xenopus oocytes leads to progesterone-independent maturation (Wang and Liu, 2003Go).

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 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., 2006Go). 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{Delta}20) releases meiotic arrest in the absence of the physiological stimulus, progesterone. The effect of SNAP25{Delta}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., 1999Go; Quick et al., 1997Go; Awayda, 2000Go). SNAP25{Delta}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.


   MATERIALS AND METHODS
TOP
 SUMMARY
 INTRODUCTION
 MATERIALS AND METHODS
RESULTS
 DISCUSSION
 REFERENCES
 
Gamete preparation and treatments
Xenopus oocytes were obtained as previously described (Machaca and Haun, 2002Go). Oocyte maturation was induced with 5 µg/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 µM forskolin or 5x10-9 M cholera toxin (Calbiochem) for 30-60 minutes before progesterone treatment, whereas SNAP25{Delta}20-injected cells were treated with the drugs at the time of injection.

Western blots
Lysates were prepared in extraction buffer (80 mM ß-glycerophosphate, 20 mM Hepes, pH 7.5, 20 mM EGTA, 15 mM MgCl2, 1 mM sodium vanadate, 50 mM NaF, 1 mM DTT, 10 µg/ml aprotinin, 50 µg/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{Omega}. 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, 2000Go). 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 {tau} (1/Ra+Gm). {tau} 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., 1996Go).

Molecular biology
For the wild-type full-length SNAP25, mouse Snap25B in pcDNA3 (Low et al., 1998Go) 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., 1992Go). The mouse Snap25 gene was highly likely to be functional in Xenopus, as it shares 95% identity with Xenopus SNAP25. SNAP25{Delta}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, 2002Go).

Transferrin endocytosis assay
Cells were treated overnight with 332 µM 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 µg/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 (10x 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-{alpha}-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 µM 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 (20x objective).


   RESULTS
TOP
 SUMMARY
 INTRODUCTION
 MATERIALS AND METHODS
 RESULTS
DISCUSSION
 REFERENCES
 
SNAP25{Delta}20 blocks exocytosis
SNARE proteins are central to vesicular fusion events that underlie trafficking at the subcellular level (Chen and Scheller, 2001Go). SNAREs contain coiled-coil domains that interact to form a four-helix 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., 2004Go). In the case of exocytosis, two of the four helices that form the four-helix bundle are contributed by SNAP25 (Jahn, 2004Go). 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., 2004Go). 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{Delta}20) has been reported to act as a dominant-negative of exocytosis in Xenopus oocytes (Yao et al., 1999Go). Therefore, to test the role of exocytosis in maintaining meiotic arrest we constructed a SNAP25{Delta}20 clone and expressed it in oocytes (Fig. 1). By recording membrane capacitance as a direct measure of cell membrane area, we functionally confirmed that SNAP25{Delta}20 acts as a dominant-negative and effectively blocks exocytosis (Fig. 1). SNAP25{Delta}20, but not full-length wild-type SNAP25, decreased membrane area in a time-dependent fashion (Fig. 1). The expression levels of both SNAP25{Delta}20 and wild-type SNAP25 were comparable (Fig. 1).


Figure 1
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  		Fig. 1. Dominant-negative SNAP25{Delta}20 blocks exocytosis. Xenopus ocytes were injected with either wild-type or SNAP25{Delta}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{Delta}20, and no band was detected in uninjected oocytes. SNAP25{Delta}20 runs faster than wild-type full-length SNAP25 because of the deletion of the last 20 residues. Ooc, uninjected oocytes; WT, wild type.

 
SNAP25{Delta}20 induces oocyte maturation
Expression of SNAP25{Delta}20 induced progesterone-independent oocyte maturation (Fig. 2). Oocytes expressing SNAP25{Delta}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{Delta}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{Delta}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{Delta}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, 1994Go). 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., 1998Go; Nebreda and Ferby, 2000Go), and the polo-like kinase-Cdc25C cascade, which activates Cdc25C. Cdc25C is a dual-specificity phosphatase that removes the inhibitory phosphorylation at Tyr15 and Thr14 from cdc2 kinase, and constitutes the rate-limiting step in MPF activation (Perdiguero and Nebreda, 2004Go; Kumagai and Dunphy, 1991Go).

To determine whether the signaling cascade underlying oocyte maturation is activated normally in SNAP25{Delta}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{Delta}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{Delta}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{Delta}20 (Fig. 2E,F). Together, these results show that SNAP25{Delta}20 induces normal oocyte maturation at the biochemical, morphological and nuclear maturation (meiosis) levels.

Timecourse of SNAP25{Delta}20-dependent maturation
We then analyzed the timecourse of SNAP25{Delta}20-induced maturation in more detail to determine if it is equivalent to progesterone treatment. Oocytes were either injected with SNAP25{Delta}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{Delta}20 expression. After progesterone treatment, membrane area decreases gradually over time, as previously reported (Kado et al., 1981Go; Machaca and Haun, 2000Go). 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, 2000Go). 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{Delta}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{Delta}20-injected cells reaches ~135 nF when cells begin to undergo GVBD (Fig. 3A). Most interesting is the expression of SNAP25{Delta}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{Delta}20 explains the delay in GVBD kinetics (Fig. 3A). These data support the argument that once SNAP25{Delta}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{Delta}20, we tested the effects on maturation of BoNT A, a zinc-dependent protease that cleaves and inactivates SNAP25 (Sudhof, 1995Go). Injection of various amounts (100-600 nM) of the catalytically 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{Delta}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 sub-threshold 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{Delta}20-dependent exocytic block.


Figure 2
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  		Fig. 2. SNAP25{Delta}20 releases oocyte meiotic arrest in Xenopus. (A) Percentage of oocytes reaching the GVBD stage following progesterone treatment, or injection of SNAP25{Delta}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{Delta}20 ({Delta}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{Delta}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 µm for spindles and 10 µm 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{Delta}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{Delta}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{Delta}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{Delta}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).

 
Analysis of SNAP25{Delta}20 site of action
We were then interested in mapping the site of action of SNAP25{Delta}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{Delta}20-mediated 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{Delta}20-mediated maturation (Fig. 4). Forskolin activates AC and has been shown to block progesterone-dependent oocyte maturation (Schorderet-Slatkine and Baulieu, 1982Go). Indeed, forskolin blocks both progesterone- and SNAP25{Delta}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{Delta}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, cdc25 has been identified as a target for PKA, leading to its inhibition (Duckworth et al., 2002Go). 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{Delta}20 acts upstream of AC to release oocyte meiotic arrest.


Figure 3
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  		Fig. 3. Timecourse analysis of SNAP25{Delta}20-induced oocyte maturation in Xenopus. (A) Extent of GVBD (black) and membrane capacitance (blue) in progesterone- (left panel) and SNAP25{Delta}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{Delta}20 mRNA injection (left panel) and progesterone treatment (right panel). (C) Oocytes were either left untreated and matured with maximal levels of progesterone (16 µM), or injected with the light chain of BoNT A (200 nM) or BSA as the carrier control and matured with sub-threshold 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.

 
We also tested the effect of cholera toxin, which ADP-ribosylates G{alpha}s and activates it, leading to increased activation of AC and a rise in cAMP levels (Gill and Meren, 1978Go). Similarly to forskolin, cholera toxin blocks both progesterone- and SNAP25{Delta}20-mediated maturation, without dramatically affecting SNAP25{Delta}20 protein expression levels (Fig. 4B). However, in contrast to forskolin, Mos RNA injection was ineffective at rescuing the cholera toxin-mediated 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{alpha}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{Delta}20 acts upstream of AC, consistent with the functional role of SNAP25 at the cell membrane.

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., 2006Go). 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., 1995Go). 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.


Figure 4
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  		Fig. 4. Activation of adenylate cyclase inhibits SNAP25{Delta}20-induced maturation. (A,B) Extent of maturation of Xenopus oocytes treated with progesterone or injected with SNAP25{Delta}20 mRNA in the presence or absence of 100 µM forskolin (A) or 5x10-9 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{Delta}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 µM forskolin or 5x10-9 M cholera toxin. The inset (below) shows the time to GVBD50 for Mos-injected cells±forskolin. CT, cholera toxin; Forsk, forskolin.

 


Figure 5
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  		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-633-labeled transferrin internalization after overnight incubation with either the carrier control DMSO or 332 µM 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 µM 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 µm. Con, control oocytes.

 
We next tested the effect of inhibition of the clathrin mediated endocytic pathway using monodansylcadaverine (MDC) (Schlegel et al., 1982Go). We first developed a functional assay to confirm the 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, 1986Go), especially because Xenopus oocytes possess functional transferrin receptors (Lund et al., 1990Go). Although transferrin readily labels small endocytic vesicles, the most reliable signal was from large vesicular structures, which are potentially early endosomes (Fig. 5C). We quantified the levels of transferrin uptake using the number and equivalent volume of these vesicular structures in a cross-sectional 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 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 clathrin-dependent 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{Delta}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
TOP
 SUMMARY
 INTRODUCTION
 MATERIALS AND METHODS
 RESULTS
 DISCUSSION
REFERENCES
 
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, 2005Go). 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, 1998Go) 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 cell-surface receptors to maintain meiotic arrest.

The SNAP25{Delta}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 sub-threshold 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., 1995Go). Although the efficiency of brefeldin at inducing meiotic arrest is poor compared with progesterone and SNAP25{Delta}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 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., 2005Go; Freudzon et al., 2005Go). 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, 2001Go). Recently this receptor was cloned and demonstrated to induce oocyte maturation through positive induction (Zhu et al., 2003Go; Ben Yehoshua et al., 2006Go). 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., 1981Go; Machaca and Haun, 2000Go) and is due to an early block of exocytosis, which is crucial for the formation of the fluid-filled blastocoele cavity during embryogenesis (Muller, 2001Go). The blastocoele is formed due to polarized vectorial transport of Na+ ions into the intercellular space by an epithelium that surrounds the embryo (Muller, 2001Go). 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, 1995Go; Muller, 2001Go). 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., 1984Go; Gardiner and Grey, 1983Go). Protein secretion is blocked early on in maturation, specifically between the trans-Golgi network and the plasma membrane (Colman et al., 1985Go; Leaf et al., 1990Go), while other intracellular trafficking events (such as ER to Golgi) are unaffected (Leaf et al., 1990Go; Ceriotti and Colman, 1989Go). 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., 1991Go; Gawantka et al., 1992Go). 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.


   ACKNOWLEDGMENTS
 
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 GrantP20RR16460 (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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