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Regular Article |
a Division of Reproductive Biology, Department of Gynecology and Obstetrics, Stanford University School of Medicine, Stanford, California 94305-5317
b Weizmann Institute of Science, Department of Biological Regulation, Bernhard Zondek Hormone Research Laboratory, Rehovot 76100, Israel
ABSTRACT
It is generally accepted that cyclic nucleotides are key signaling molecules in the control of oocyte meiotic resumption. Given the role of phosphodiesterases (PDEs) in cyclic nucleotide degradation, this study was undertaken to investigate the properties and regulation of PDEs expressed in rat oocytes. Cilostamide-sensitive PDE3 was the major activity detected in denuded oocytes, whereas no PDE3 activity could be detected in cumulus cells. Moreover, comparable levels of PDE3 activity were measured in cumulus-oocyte complexes (COCs) and in denuded oocytes. The oocyte PDE was recovered in the soluble fraction of the homogenate and immunoprecipitated with a specific PDE3A antibody. A significant and transient increase (P < 0.05) in PDE3 activity was measured in the oocytes after 30 min of culture (70 min after isolation) compared with immediately after collection (10 min after isolation). Conversely, no changes in activity were observed when denuded oocytes or cumulus cells were incubated for up to 130 min. Evaluation of oocyte maturation indicated that only 10% of oocytes had resumed meiosis at the peak of the PDE3 activity. A significant increase (P < 0.05) in PDE3 activity was measured in COCs when follicle-enclosed oocytes were cultured in the presence of hCG. Again, this increase preceded oocyte maturation. In conclusion, these data demonstrate that PDE3A is the major PDE form expressed in mammalian oocytes. PDE3A activity increases prior to resumption of meiosis in both spontaneous and gonadotropin-stimulated maturation. These findings strongly support the hypothesis that an increase in oocyte PDE3A activity is one of the intraoocyte mechanisms controlling resumption of meiosis in rat oocytes, at least in vitro.
cyclic adenosine monophosphate, meiosis, ovum, phosphodiesterases, signal transduction
INTRODUCTION
Mammalian oocytes enter meiosis early during embryonic life [1]. This process is arrested at the dictyate stage of the first meiotic prophase, when the nucleus assumes typical germinal vesicle (GV) form. In vivo, meiotic resumption is stimulated by the LH surge in the large preovulatory follicles. In vitro, isolated cumulus-oocyte complexes (COCs) resume meiosis spontaneously [2]. The first morphological sign of meiotic resumption is the breakdown of the nuclear membrane, a process termed germinal vesicle breakdown (GVBD).
Regulation of meiotic resumption in mammalian oocytes is not fully understood. Cyclic AMP is a key second messenger regulating oocyte maturation. Cho and colleagues [3] were the first to propose that cAMP regulates mammalian oocyte maturation. Analogues of cAMP and cyclic nucleotide phosphodiesterase (PDE) inhibitors prevent spontaneous maturation [3, 4]. In addition, forskolin, a pharmacological activator of adenylyl cyclase, transiently blocked resumption of meiosis in mouse oocytes [5]. A positive correlation was observed between the FSH concentration-blocking resumption of meiosis and cAMP levels in COCs [6]. This was later confirmed by showing that FSH transiently elevates cAMP levels both in the complex and in the oocyte [7]. By using an invasive adenylyl cyclase of Bordetella pertussis, which elevates cyclic nucleotide levels in the oocytes, it was confirmed that high cAMP levels block oocyte maturation [8].
Intracellular concentration of the second messenger cAMP is a balance between the rate of synthesis and the rate of degradation, which are regulated by adenylyl cyclases and cyclic nucleotide PDEs, respectively. The presence of adenylyl cyclase activity in mammalian oocyte sufficient to block maturation has been questioned [9–15], opening the possibility that other mechanisms control cAMP levels in the gamete. Indeed, it has been demonstrated that the oocyte is metabolically coupled to cumulus cells through gap junctions [16], opening the possibility that cAMP diffuses from granulosa cells to the oocyte [1, 17–19]. According to this view, closure of the junctions blocks the cAMP transfer from granulosa cells to the oocyte and triggers meiotic resumption. Although the breakdown of communication may occur early enough to signal oocyte maturation, a causal correlation between gap junction closure and meiotic resumption has not been conclusively established [1, 20]. In addition to a regulation of cAMP transport from the somatic compartment, we propose that the intraoocyte cAMP level is controlled through its degradation by PDEs, and an increase in PDE enzyme activity may be involved in resumption of meiosis. The nonselective PDE inhibitor isobutylmethylxanthine (IBMX) prevents oocyte maturation [4, 5] and causes an increase in oocyte cAMP levels [21]. Microinjection of a preparation of an active PDE in mouse oocyte significantly induces resumption of meiosis even in the presence of a PDE inhibitor in the culture medium [22]. Moreover, an increase in PDE activity in the Xenopus oocyte is sufficient to induce resumption of meiosis [23, 24].
Eleven different PDE families have been identified in mammals [25, 26]. In situ hybridization indicates that the mRNA of PDE4B is observed in theca cells, PDE4D in mural granulosa cells, and PDE3A in the oocyte [27]. The PDE3 family is encoded by two genes: PDE3A, which is expressed in heart [28], vascular smooth muscles [29], and in platelets [30]; and PDE3B, which is induced during adipocyte differentiation [31]. The structural organization of the proteins PDE3A and PDE3B is identical. Although the catalytic domain of PDE3A and PDE3B is very similar to other PDEs, an insert of 44 amino acids sets the PDE3 family apart from other PDE families [32]. The widely divergent N-terminal portions of PDEs contain determinants that confer specific regulatory properties [26]. PDE3 possesses a unique hydrophobic membrane-association domain with six putative transmembrane domains [32] that are believed to be important for efficient membrane association and targeting [33]. It has been shown that spontaneous resumption of meiosis is blocked by nonspecific PDE inhibitors [3, 17, 34], but specific PDE3 inhibitors are considerably more potent [27, 35], indicating an important role of the PDE3 family in regulating oocyte maturation. In addition, PDE3A has been cloned using a mouse oocyte cDNA library and the pharmacological properties of the recombinant protein correlated with the rank of potency of PDE3 inhibitors in blocking meiotic resumption [36]. The goal of the present study was to determine the properties and regulation of the PDE expressed in mammalian oocytes using different biochemical and immunochemical models.
MATERIALS AND METHODS
Animals
Sprague-Dawley rats were obtained from Simonsen Laboratories (Gilroy, CA). Animals were provided with water and rat chow ad libitum and housed in air-conditioned rooms illuminated for 14 h/day.
Culture Media and Inhibitors
Oocytes and follicle-enclosed oocytes (FEOs) were cultured in Leibovitz L-15 medium with L-glutamine 300 mg/ml (Gibco, Grand Island, NY), supplemented with 5% fetal calf serum (Gemini Bio-Products Inc., Calabasas, CA), penicillin (100 U/ml; Gibco), and streptomycin (100 mg/ml; Gibco). The PDE inhibitor 3-isobutyl-1-methylxanthine (IBMX, nonselective) was from Sigma Chemical Co. (St. Louis, MO), rolipram (PDE4-specific) was a gift from Schering-Plough Pharmaceuticals (Madison, NJ), and cilostamide (PDE3-specific) was from Dr. I. Hidaka (Nagoya University, Japan). These compounds were kept in stock solution in dimethyl sulfoxide (Sigma) and diluted in the test tubes as indicated.
Oocyte Collection and Culture
Immature female rats were injected s.c. with 10–15 IU of equine chorionic gonadotropin (eCG; Calbiochem, La Jolla, CA) between 0900 and 1000 h on Day 24–25 of age in order to enhance multiple ovarian follicular development. The animals were killed 48–50 h later by cervical dislocation. COCs were collected by puncturing the large follicles with a 25-gauge needle under a stereomicroscope. Oocytes were denuded of their cumulus cells by vortex-agitation for a few minutes in culture medium as described before [37]. Groups of a maximum of 75 COCs were cultured in organ culture dishes (Falcon, Cockeysville, MD) for 30 min to 2 h. After all treatments, the oocytes were flash frozen in liquid nitrogen and stored at -80°C.
FEO Culture
Animals were treated as described above with 10–15 IU eCG. After isolating the ovaries, large FEOs were excised under a stereomicroscope [38]. The FEOs (20–25 per dish) were cultured for 2 h in culture medium described above, with or without 5 µg/ml of hCG (CR-121 from the National Institute of Diabetes and Digestive and Kidney Diseases, Bethesda, MD). The dishes were placed in a tightly closed culture chamber (MIC-101, Billups-Rothenberg Inc., Del Mar, CA) and then flushed with O2/N2 (1:1). At the end of the culture, the oocytes were collected by puncturing the follicles.
PDE Assay
The oocytes were homogenized in an isotonic buffer (IPA) containing 10 mM sodium phosphate buffer pH 7.2, 50 mM NaF, 150 mM NaCl, 2 mM EDTA, 5 mM 2-mercaptoethanol, 30 mM sodium pyrophosphate, 3 mM benzamidine, 5 µg/ml leupeptin, 20 µg/ml pepstatin, 2 mM phenylmethylsulfonyl fluoride, and 1 µM microcystin (all from Sigma). In some experiments, Triton X-100 (0.5%) (Sigma) was used as a detergent. The homogenate was centrifuged for 30 min at 14 000 x g to obtain a soluble fraction. PDE activity was assayed using 1 µM cAMP (Sigma) as substrate according to the method of Thompson [39]. Samples were assayed at 34°C in a final volume of 200 µl; the solution consisting of 40 mM Tris-HCl pH 8.0, 10 mM MgCl2, 5 mM 2-mercaptoethanol, 1 mg/ml BSA (Fraction V), and 1 µM cAMP (all from Sigma) and 15 nM [3H]cAMP (Perkin-Elmer Life Sciences, Boston, MA) (approximately 0.1 x 106 cpm/tube; 30 Ci/mmol). To determine the contribution of different PDEs to the overall PDE activity, specific inhibitors (cilostamide for PDE3 and rolipram for PDE4) were added to the incubation mixture at 10 µM. The activity measured in the presence of the specific inhibitor was subtracted from the total activity and the difference was referred to as PDE3 or PDE4 activity. This pharmacological quantitation of different PDEs has been validated using recombinant proteins [30, 31, 40, 41] or PDE-deficient mice [42].
Immunoprecipitation
COCs were homogenized in IPA buffer and centrifuged as described above. The soluble fraction was incubated with 20 µl of anti-PDE3A [36] preadsorbed on Protein A-Sepharose (Sigma). The homogenate was immunoprecipitated overnight at 4°C by gentle agitation. A negative control was performed by using comparable amounts of preimmune serum. At the end of incubation, samples were centrifuged at 1000 x g for 5 min. The pellets were washed three times with PBS and 0.1% BSA. After washing, the pellets were resuspended in Tris-BSA (40 mM Tris-HCl pH 8.0, 1 mg/ml BSA) before measuring PDE activity.
Analysis of Meiotic Maturation
Oocytes were collected at different times of incubation and the presence of GV was monitored by Hoffman interference microscopy. Those oocytes in which no clear GV was present were scored as GVBD. In some experiments, the presence of a nucleus was detected by orcein staining. The percentage of GVBD oocytes in at least three experiments was averaged and reported as the mean ± SEM.
Analysis of Results
Values are expressed as the mean of the activity measured ± SEM. Results were analyzed by a two-way ANOVA. When the ANOVA indicated a significant difference (P < 0.05), the results were compared by the Fisher protected least significant difference test.
RESULTS
The PDE assay was optimized to measure the activity in rat COCs. When increasing numbers of COCs were used under these conditions, the hydrolysis of cAMP increased in a linear fashion and was proportional to the number of COCs (Fig. 1). All subsequent assays were then performed on extracts of 10–15 COCs.
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PDE Activity in COCs Is Recovered in the Soluble Fraction
In different tissues, PDE3s have been recovered in both the supernatant fraction and the particulate fraction [32], whereas PDE3A is recovered primarily in the supernatant fraction in both platelets [43] and placenta [44]. This experiment was undertaken to assess the subcellular localization of the cilostamide-sensitive PDE activity in COC homogenate. The PDE activity was recovered mostly in the soluble fraction regardless of the presence of Triton X-100 (Fig. 2). The cilostamide-sensitive PDE activity can be estimated by subtracting from the total activity the activity measured in presence of the inhibitor. Similar amounts of cilostamide-sensitive PDE activity were measured in presence of detergent, whereas some increase in total PDE activity was instead observed when Triton X-100 was used in the homogenization buffer. This may be due to direct activation or solubilization of PDEs other than PDE3s.
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Phosphodiesterase Activity in COCs and Denuded Oocytes
Considerable variation was observed in the total PDE activity measured in different groups of COCs (Fig. 3A). The total PDE activity was almost completely inhibited by IBMX (90%, 1 mM), a nonspecific PDE inhibitor, whereas the activity was partially inhibited by both cilostamide (32%, 10 µM), a specific PDE3 inhibitor, and rolipram (25%, 10 µM), a specific PDE4 inhibitor. The activity measured in denuded oocytes (DOs) is only a fraction of the total activity measured in COCs (Fig. 3B). However, this activity was inhibited almost completely by both cilostamide and IBMX, whereas rolipram had no effect (Fig. 3B). Cilostamide-sensitive PDE activity measured in COCs (3.5 ± 0.3 fmol/min per COC) and DOs (2.8 ± 0.5 fmol/min per DO, Fig. 3C) were similar, supporting the notion that the PDE3 measured in COCs is entirely derived from the oocyte. In agreement with this conclusion, no significant PDE3 activity could be measured in cumulus cells (see below). Because the cilostamide-sensitive PDE3 activity was constant in these measurements, the variation in total PDE activity reflects the varying numbers of cumulus cells that were recovered in different COC preparations.
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Immunoprecipitation of PDE3A Activity in COCs
Two different PDE3s have been identified, PDE3A and PDE3B [32]. Because in situ hybridization has shown a PDE3A transcript to be present in the oocyte [27], and PDE3A has been cloned from a mouse oocyte cDNA library [36], this experiment was undertaken to determine whether the oocyte PDE is recognized by a PDE3A-specific antibody. The soluble fraction of COC homogenate was subjected to immunoprecipitation using an anti-PDE3A (see Materials and Methods). A preimmune serum from the same rabbit was used to control for the specificity of the immunoprecipitation. When the PDE3A-specific antibody was used, the PDE activity was quantitatively recovered in the immunoprecipitation pellet and was entirely inhibited by cilostamide (Fig. 4A). The immunoprecipitation (IP) was specific because negligible cilostamide-sensitive activity was recovered after precipitation with the preimmune serum (Fig. 4B).
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Increase in PDE3 Activity During Spontaneous Maturation of COCs
To determine whether PDE activity changes during oocyte maturation, cilostamide-sensitive PDE activity was monitored at different time points during spontaneous maturation. A significant increase (P < 0.05) in cilostamide-sensitive PDE activity was measured 70 min after COC isolation (Fig. 5A). This twofold increase was transient and the activity returned to the basal level within 30 min. Evaluation of meiotic resumption (i.e., GVBD) was performed on 200 oocytes processed for each time point. At the peak of PDE activation (70 min after isolation where oocytes have been cultured for 30 min), 8.5% ± 6.5% of oocytes had resumed meiosis (insert, Fig. 5A). At 130 min after COC isolation, 92.5% ± 2.5% had resumed meiosis. This is a first indication that the cilostamide-sensitive PDE activity in rodent oocytes is activated prior to spontaneous resumption of meiosis. When the PDE activity was measured in denuded oocytes incubated for the same length of time, no change in PDE3 activity could be detected (Fig. 5B). No change in PDE3 activity was detectable when COCs were denuded after incubation (data not shown). Under these assay conditions, no PDE3 activity was detected in cumulus cells at any time of incubation (Fig. 5C).
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Increase in PDE3 Activity in COCs Isolated from hCG-Stimulated FEOs
Because the mechanism of spontaneous resumption of meiosis is not fully understood, the cilostamide-sensitive PDE activity was measured in a hormone-stimulated system. FEOs were cultured in the absence or presence of hCG for 2 h. Total PDE activity in COCs was increased by hCG, but the significance was not reached because of the variation between experiments (data not shown). However, the cilostamide-sensitive PDE activity was significantly stimulated (P < 0.05) by the hCG treatment (Fig. 6). In our experimental conditions, the oocytes did not resume meiosis within the time frame of 2 h (data not shown). Previous studies have also shown that the stimulation of meiosis by hCG/LH results in resumption of meiosis by 50% of the oocytes after 3 h and 90% after 4 h [45].
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DISCUSSION
The present study provides evidence that PDE3A is activated prior to resumption of meiosis in rat oocytes. An increase in PDE activity was observed during both spontaneous and LH-induced maturation in vitro, indicating that this activation is a common pathway for oocyte meiotic resumption regardless of the stimulus applied. Because a decrease in cAMP is required for resumption of meiosis, we propose that PDE3A is an essential component of the signaling pathway controlling oocyte maturation.
It has long been known that in vitro oocyte maturation in rodents is blocked by a nonspecific PDE inhibitor, IBMX [17, 21, 34], or by hypoxanthine, which most probably acts through inhibition of PDE activity [18, 46]. More recently, it has also been shown that pharmacological inhibition with selective PDE3 inhibitors, but not PDE4 inhibitors, blocks oocyte maturation in vitro [27] and in vivo [35], supporting an important role of the oocyte PDE3 in regulating resumption of meiosis. In addition, these observations are consistent with the findings that nonselective PDE inhibitors [3, 4, 46, 47] or cAMP analogues [17, 48] that activate protein kinase A (PKA), block resumption of meiosis. Cumulatively, these data are consistent with the idea that PDE3 has a permissive role in the control of oocyte cAMP levels allowing resumption of meiosis.
The use of PDE inhibitors, however, does not allow distinction between the impact of constitutive versus regulated PDE activity. The present data add a new dimension to the understanding of the role of PDE3A in oocyte maturation. A significant increase in PDE3A activity was observed during both spontaneous and LH-induced in vitro resumption of meiosis. Inhibition of PDE4s did not abolish PDE activation (data not shown), suggesting that the activated PDE is not PDE4. In addition, only a small fraction of the PDE activity may be attributable to PDE1, because 90% of PDE activity is cilostamide-sensitive and most of the activity is immunoprecipitated using a specific PDE3A antibody. More importantly, this activation occurs prior to meiotic resumption in both experimental models.
In our experimental model, we detected an activation of PDE3 activity only when the interactions between oocytes and cumulus cells are maintained. In line with this hypothesis, no increase in PDE activity was detectable in cultured denuded oocytes. In addition, cumulus cells do not express significant PDE3 activity and no changes were detected during culture. Although it is very unlikely, we cannot formally exclude that PDE3 activity is absent in cumulus cells under basal conditions, and that it is induced during culture.
The activation of PDE3A in the oocyte may be one of the required steps allowing reentry of the dictyate oocyte into the M-phase of the cell cycle. Moreover, PDE3A may play an active role by transducing signals necessary for resumption of meiosis. An increase in cAMP degradation, together with a decrease in permeability of the gap junctions, may cause a drop in cAMP concentration in the oocyte. This in turn causes inactivation of PKA and changes in phosphorylation of cell cycle regulators that control the activity of the M-phase promoting factor. The involvement of this signaling pathway in meiotic resumption is inferred by our data and by the observation that cAMP-dependent protein kinase inhibitors or other antagonists of cAMP binding to PKA, block resumption of meiosis [27]. Proof that PDE3 activation is, per se, sufficient to trigger resumption of meiosis could not be obtained due to the spontaneous resumption of meiosis in vitro, which is an intrinsic property of mammalian oocytes. However, two observations support this possibility. First, it has been shown that injection of a crude PDE preparation in mouse oocyte causes partial meiotic resumption [22]. Although the interpretation of these data requires some caution because oocytes were cultured in the presence of the nonselective PDE inhibitor IBMX, which blocks maturation, it suggests that an increase in PDE activity is sufficient to signal resumption of meiosis. Second, in the Xenopus oocyte when maturation occurs only after progesterone or IGF-1 stimulation, injection of a PDE mRNA resulted in resumption of meiosis, Mos translation, and mitogen-activated protein kinase activation [23]. The activity of this expressed PDE is necessary because inhibitors specific for the injected PDE blocked oocyte maturation. Insulin/insulin-like growth factor-1 has also been shown to stimulate PDE activity and resumption of meiosis in Xenopus oocytes [49]. Along the same line, preincubation with a PDE3 inhibitor blocks insulin-stimulated oocyte maturation [50, 51]. These findings suggest that, at least in amphibians, an increase in PDE activity is a necessary and sufficient signal to promote resumption of meiosis. Our finding that hCG stimulation of FEOs cultured in vitro results in an increase in oocyte PDE3 activity strongly supports the notion that in vivo resumption of meiosis involves PDE3 activation.
At present, the exact mechanism bringing about PDE activation in oocytes is unknown. This activation is transient and can be detected only in COCs, suggesting that the communication between cumulus cells and oocytes is necessary for this activation. Undoubtedly, phosphorylation by different kinases plays an important role in PDE3A activation. Consistent with the results of PDE3B in adipose tissue [32], PKA and protein kinase B (PKB) can phosphorylate and activate the recombinant PDE3A [36]. Considering that PKA is necessary to maintain meiotic arrest, the oocyte PKA should be already active in oocytes at the GV stage. Therefore, it is difficult to conceive a PKA-dependent phosphorylation of PDE3A in oocyte maturation, unless compartmentalization of signaling is present. It is possible, however, that after the LH surge, an additional increase in cAMP above the concentration required for maintaining the meiotic blockade in the oocyte. This increase could then be translated into an activation of PDE3 and may contribute, together with a decrease in gap junction permeability, to a decrease in cAMP and meiotic resumption. Further studies are necessary to validate this hypothesis.
PKB is distal to PI-3 kinase and the phosphoinositide-dependent kinase that are activated by several growth factors. PKB is expressed in Xenopus oocytes and is a component of the pathway that controls resumption of meiosis [23]. Interestingly, epidermal growth factor (EGF) has been shown to activate meiotic resumption in rodents [52]. Because the PI-3 kinase-PKB pathway plays an important role in EGF signaling, it is possible that EGF causes an activation of PDE3A. Consistent with this view, an EGF-receptor Erb3 has recently been identified in human oocytes [53]. PKB is also a key molecule of the signaling pathway used by the c-kit receptor. Although c-kit is essential for fertility [54], it produces a delay rather than a stimulation in oocyte maturation [55].
A previous study on mouse oocytes has suggested that the oocyte PDE is involved in the decrease in cAMP associated with resumption of meiosis [5]. However, no increase in PDE activity during spontaneous oocyte maturation was detected. The reason for these apparent discrepancies with our results may be twofold. In the model used by Bornslaeger et al. [5] the oocytes were cultured without cumulus cells. According to our data, it is possible that somatic cells are necessary to convey a signal for PDE activation through the gap junctions and this signal is lost in denuded oocytes. Because changes in total PDE activity are minor under our experimental conditions, a PDE activation may remain undetected or be masked by variable number of cumulus cell. Finally, oocytes were maintained in IBMX prior to maturation [5], and this may have had an effect either on PDE activation or on PDE measurement. Further experiments are necessary to resolve these discrepancies.
Bornslaeger et al. [5] reported that cGMP inhibited the cAMP hydrolysis of oocyte extracts by approximately 50%, indicating, interestingly, that a cGMP-inhibited PDE (PDE3) is present in the oocyte. The presence may explain the observation that microinjection of cGMP causes a delay in meiotic resumption [56]. In vitro, dibutyryl-cGMP has a slight inhibitory effect on oocyte maturation, but the inhibition is much higher and persistent for more than 20 h in the presence of dibutyryl-cAMP [57]. Although cGMP can be hydrolyzed by PDE3A, the hydrolysis is sufficiently slow so that cGMP may function essentially as a competitive inhibitor in intact cells [32]. This has been demonstrated in platelets, in which an increase in cGMP is associated with an increase in cAMP due to the inhibition of PDE3A [32]. Whether this mode of regulation is physiologically relevant in the oocyte is unknown. Genetic disruption of endothelial nitric oxide synthase has an effect on oocyte maturation and ovulation [58].
In conclusion, the present study demonstrates that PDE3A activation precedes in vitro resumption of meiosis. This activation may be essential in the control of the cAMP level that leads to meiotic resumption. Understanding how this enzyme is regulated may therefore provide important insight into the signals and mechanisms controlling resumption of meiosis.
ACKNOWLEDGMENTS
Special thanks to Linda Lan and Kathleen Conti for help with the PDE assays, Carsten Bo Andersen for the generation of the PDE3A antibody and numerous fruitful discussions, and Caren Spencer for editorial assistance.
FOOTNOTES
First decision: 5 January 2001.
1 This research is supported by National Institutes of Health grant HD31544 to M.C. F.J.R. is supported by a fellowship from NSERC of Canada. 
2 Correspondence. FAX: 650 725 7102; marco.conti{at}stanford.edu
Accepted: June 26, 2001.
Received: December 4, 2000.
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