1 Laboratoire de Biologie de la Reproduction, UPRES 3410, UFR SMBH, Université Paris 13, 74, rue Marcel Cachin, 93017 Bobigny, Service dHistologie-Embryologie-Cytogénétique, Hôpital Jean Verdier (Assistance Publique-Hôpitaux de Paris), Bondy and 2 Laboratoire Entrée muqueuse du VIH et immunité muqueuse, Unité INSERM U567, Institut Cochin, 22, rue Méchain, 75014 Paris, France
3 To whom correspondence should be addressed. E-mail: jean-philippe.wolf{at}jvr.ap-hop-paris.fr
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Abstract |
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Key words: FEE/fertilization/human/integrins/RGD
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Introduction |
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Integrins constitute a large family of heterodimeric transmembrane receptors composed of covalently linked and
subunits. The major function of these cell surface molecules is to mediate cellcell and cellextracellular matrix attachment (for a review see Bowen and Hunt, 2000
). Integrin activation induces a conformational change of their outer
and
domains, inducing binding with ligands (Brakebusch and Fassler, 2003
). Ligand binding induces, in turn, integrin clustering at the cell surface, and recruitment of actin filaments and signalling proteins to the cytoplasmic domain of integrins (Giancotti and Ruoslahti, 1999
; Hynes, 2002
). Conversely, intracellular signals can induce integrins to bind to their matrix ligands (Liddington and Ginsberg, 2002
). Eighteen
and eight
subunits have been identified and form 23 known heterodimers (Zhu and Evans, 2002
). Among the integrins, some mediate preferentially adhesion to ligands such as fibronectin, vitronectin and fibrinogen by binding to the RGD motif present in the molecule. They are known as RGD-sensitive. The RGD motif has also been found in snake venom proteins that specifically inhibit integrin binding function and serve as potent integrin antagonists. The majority of these proteins interact with
1- and
3-associated integrins and their potency is at least 5002000 times higher than that of short RGD peptides (Lu et al., 2003
).
In reproductive biology, there is strong evidence that integrins and their ligands are important mediators of spermegg binding. Both spermatozoa and oocytes express a number of integrins and molecules that contain integrin recognition sites. The mouse oocyte contains integrin subunits 2,
3,
5,
6,
v,
1,
3 and
5 as detected by mRNA and/or protein analysis (Tarone et al., 1993
; Evans et al., 1995b
; Zuccotti et al., 1998
; Burns et al., 2002
). Similarly, human oocytes express integrin subunits
2,
3,
5,
6,
v,
M,
1,
2,
3,
4,
5 and
6 (Campbell et al., 1995
; Ji et al. 1998
; Sengoku et al., 2004
). Among the many different integrin subunits expressed on oocytes, a large amount of data clarifies the major role of
6
1 integrin in the process of gamete binding and/or fusion (Almeida et al., 1995
; Takahashi et al., 2000
) though it is called into question by others (Evans et al., 1997b
; Evans, 1999
; Miller et al., 2000
). However, a kinetic experiment revealed that, at an early stage of spermegg fusion, the integrin
6 subunits accumulate at the sperm-binding site. The frequency of cluster formation is closely related to that of spermegg fusion. This led Takahashi et al. to suggest that spermegg fusion actually occurs at sites where the
6
1 integrin is clustered and that this interaction occurs via direct association of integrin
6 with sperm (Takahashi et al., 2000
).
Besides oocyte integrins, their putative ligands on sperm, i.e. ADAMs such as fertilin (ADAM2) and cyritestin (ADAM3), have also been well studied as candidate genes responsible for the spermoocyte interaction. Fertilin is an
heterodimer first described on guinea pig spermatozoa as the putative ligand by which the sperm interacts with the oocyte (Primakoff et al., 1987
). Fertilin
and
subunits are the prototypes of the ADAM gene family, which are a widely distributed family of evolutionarily conserved transmembrane proteins (Blobel, 1992
; Wolfsberg, 1995
, 1996
). The disintegrin domain of ADAMs consists of 6090 amino acids and is homologous to snake venom disintegrins. The snake disintegrin domains contain RGD sequences and competitively inhibit integrin-mediated adhesion of platelets to RGD sequences in fibrinogen and other ligands (Gould et al., 1990
). Structural analysis has revealed that the RGD sequence is present at the top of an extended loop structure, similar to the location of the RGD sequence in fibronectin (Leahy et al., 1996
). However, most ADAMs do not contain an RGD sequence in their disintegrin domain. Indeed, the putative disintegrin tripeptide-binding site of fertilin presents species specificity. In the position of the RGD tripeptide, the consensus sequence in fertilin
(based on cDNA clones) is QDE in the mouse and FEE in the human (Gupta et al., 1996
; for a review see Evans, 2002
).
In addition to the data obtained in vitro, knockout mice for these genes provided evidence for the participation of the ADAMs in the spermoocyte interaction. Thus, fertilin / sperm show greatly reduced levels of binding to the oolemma, but the few that do bind are still able to fuse (Cho et al., 1998
). Moreover, some experiments suggest that integrin
6
1 mediates the binding of fertilin
and cyritestin (Chen et al., 1999
; Yuan et al., 1999; Takahashi et al., 2001
; Tomczuk et al., 2003
). Indeed, in the mouse, based on the capacity of specific monoclonal antibodies (mAbs) to inhibit fertilin
binding to the oocyte and the direct binding to this integrin of fertilin
peptides, integrin
6
1 has been suggested to be a receptor for fertilin
(Chen and Sampson, 1999
; Chen et al., 1999
; Bigler et al., 2000
).
On the other hand, members of the RGD-binding integrin subfamily (including v
1,
v
3 and
v
5) are expressed by oocytes and are implicated in fertilization by studies using RGD peptides in IVF assays (Bronson and Fusi, 1990; Ji et al., 1998
) and other work (Linfor and Berger, 2000
). Bronson and Fusi (1990)
demonstrated that RGD-dependent recognition is involved in spermoolemmal adhesion since RGD peptides inhibit the fertilization rate in the zona-free hamster egg penetration test. Furthermore, the oligopeptide GdRGDSP, specifically designed to block both fibronectin and vitronectin receptors, significantly inhibits the binding of human sperm to the oolemma of zona pellucida-free hamster oocytes (Fusi et al., 1996
). This is of importance since fibronectin and vitronectin are two glycoproteins that contain functional RGD sequences and are both present on human spermatozoa (Fusi et al., 1992
, 1994
). Integrins that recognize these ligands have been detected on spermatozoa and eggs. We similarly reported a dose-dependent and partial inhibition of the human gamete fusion by RGD peptide (Ji et al., 1998
). These results indicate that an RGD-sensitive integrin is involved in the fusion process. Biochemical analyses have implicated the
v integrin subunit on the pig oocyte in the recognition of isolated pig sperm membrane proteins (Linfor and Berger, 2000
), but direct involvement of RGD-sensitive integrins should be confirmed.
Despite this supportive evidence, a precise role for ADAMs and integrins, RGD sensitive or not, remains to be determined. Moreover, these data will also be discussed with regard to experiments showing that (i) antibody inhibition studies using different conditions did not find inhibition of spermegg fusion by an anti-6 mAb (GoH3) (Evans et al., 1997b
; Evans, 1999
; Miller et al., 2000
); (ii) oocytes from
6 knockout mice showed normal binding and fusibility with sperm (Miller et al., 2000
); and (iii) using
1 conditionally deficient females,
3 integrin null mice and anti-
3 or
v integrin function-blocking antibodies,
3
1 is not essential for spermegg binding and fusion and
1 integrin null eggs are fully functional in fertilization both in vivo and in vitro (He et al., 2003
). These results could not totally exclude the possibility of the occurrence of functional redundancy among various integrins and/or of a macromolecular complex including integrins and tetraspanins.
In humans, the fertilization mechanism is rarely studied, mainly because oocytes are rare and precious. Similarly to mouse models, ADAMs and several integrins, including the 6 and
1 subunit, have been suggested to mediate spermegg interaction (Fusi et al., 1992
; Campbell et al., 1995
; Ji et al., 1998
) but, once again, these data are called into question by other work (Sengoku et al., 2004
). To address this conundrum, we studied the human gamete interaction process using peptide mimicking the putative binding site of the disintegrin domain of human fertilin
. Effectively, since fertilin
and cyritestin genes are non-functional in human (Jury et al., 1997
; Grzmil et al., 2001
), fertilin
appears to be the best candidate for involvement in gamete interaction. As the disintegrin-binding domain of fertilin
is localized at the top of a hairpin loop, we chose to use a cyclic hexapeptide (CSFEEC). Moreover, because the RGD peptide, which inhibits spermegg fusion, does not bind to
6
1 integrin, we co-incubated the two peptides. We also propose a model of a membrane receptor in the human gamete fusion process which includes integrins and the CD9 tetraspanin.
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Materials and methods |
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For IVF, normal metaphase II (MII) stage human oocytes were inseminated and examined for the presence of pronuclei 18 h post-insemination. At day 2 post-insemination, oocytes with no signs of fertilization and apparently normal morphology were donated and included in this study.
Following oocyte collection, oocytes intended for ICSI were freed of the surrounding cumulus and corona radiata cells by brief exposure to hyaluronidase (type VIII, 80 IU/ml; Sigma) at 37°C, and gentle pipetting with a Stripper® micropipettor (135 µm diameter; Mid-Atlantic Diagnostics, Marlton, USA). They were then scored for the presence of a germinal vesicle (GV) or a polar body. In vitro matured metaphase II oocytes were obtained by incubating GV or metaphase I stage (neither GV nor polar body was present) oocytes under a 5% CO2 atmosphere at 37°C, in FertiCultTM medium (FertiPro, Beemem, Belgium) for 24 h. Zona pellucida were removed mechanically with a totally chemical and enzymatically free technique, using a pair of microdissection scissors under light Stero-microscope, in order to preserve the integrity of the membranes protein pattern (Kellom et al., 1992).
Semen from donors of proven fertility was collected after 3 days of sexual abstinence. Sperm samples were kept at 37°C until liquefaction was completed. A two-step 90/45% PureSperm® gradient (Nidacon International, Gothenburg, Sweden) was used to select motile spermatozoa (300 g for 20 min). The pellet was washed once with FertiCultTM medium by centrifugation and resuspended to a sperm concentration of 2 x 106/ml for the experiments. Semen analysis was performed according to World Health Organization criteria (World Health Organization, 1999). The sperm were kept under capacitating conditions for 2 h before insemination (Mortimer et al., 1989).
Antibodies and peptides
MAb against the human v
3 integrin (LM609) and rat mAb against the
6 integrin subunit (GoH3) were purchased from Chemicon International (London, UK). Function blocking mAb against the human
1 integrin subunit (DE9) (Bergelson, 1992) was purchased from UBI (New York, USA). Donkey anti-mouse rhodamine conjugate and a rabbit anti-ratfluorescein isothiocyanate (FITC) conjugate, mouse adsorbed (Vector Laboratories, Burlingame, CA), were used.
The sequence of the disintegrin domain of human ADAM2 is CLFMSKERMCRPSFEECDLPEYCNGSSASC (accession no. CAA67753). The FEE peptide was synthesized by Neosystem (Strasbourg, France). Its formula, CSFEEC, contained the tripeptide FEE and was cyclized by the adjunction of a cysteine at both ends. The peptide was purified by high-pressure liquid chromatography to >95% purity. The scrambled cyclic peptide CFESEC was obtained in a similar way. For immunofluorescence detection, a biotinylated and cyclized FEE peptide was obtained by adjunction of a biotinylated GlyGly chain. RGD-containing peptide (Gly-Arg-Gly-Asp-Thr-Pro) was purchased from Sigma.
Fluorescence staining of human zona-free eggs
Single or double staining were performed by incubating zona-free eggs with primary antibodies for 1 h at the following concentrations: anti-6 (20 µg/ml), anti-
1 (10 µg/ml) and anti-
v
3 (10 µg/ml). These incubations were followed by washing with FertiCultTM medium prior to the staining procedure with rabbit anti-ratFITC conjugate, mouse adsorbed (10 µg/ml), and/or rhodamine-conjugated donkey anti-mouse antibody (20 µg/ml). Eggs were then fixed in 2% paraformaldehyde in phosphate-buffered saline (PBS) at room temperature for 30 min. Oocytes were mounted in Immu-mount antifade solution (Shandon, Pittsburgh, PA), and confocal analysis was performed with a TCS SP2 confocal microscope (Leica, Wetzlar, Germany), using a 63x objective. Negative controls were obtained by substituting the incubation in primary antibody by incubation in PBSbovine serum albumin (BSA) 1% alone. Non-immune rat immumnoglobulin IgG 2a was also used as control. To verify that clustering was not mediated by the antibodies, labellings were also performed after paraformaldehyde fixation and identical results were obtained.
To detect FEE binding on human eggs, zona-free oocytes were incubated for 1 h with biotinylated cyclic FEE (cFEE; 100 µmol/l). Then, they were incubated with an anti-biotin mAb (Zymed Laboratories, San Francisco, CA), a biotin-conjugated anti-mouse IgG (Chemicon International) and finally with streptavidinFITC. Detection was performed using confocal microscopy (Bio-Rad MCR1000) on a Nikon Diaphot 300.
Human spermegg fusion assay
Due to French bioethical laws, in vitro human gamete fusion assays can only be performed using zona pellucida-free oocytes. Zona pellucida-free mature eggs were pre-incubated with different concentrations of peptides for 30 min in 20 µl FertiCultTM medium drops and then inseminated with 4000 capacitated motile human spermatozoa, in the presence of the same peptides, under mineral oil. Controls were obtained by omitting the peptide or using a scrambled peptide (CFESEC). After 18 h, oocytes were washed and loaded with DNA-specific fluorochrome Hoechst 33342 (Sigma) at 5 µg/ml for 20 min. After washing, they were fixed in 4% paraformaldehyde in PBSBSA 1% for 30 min at room temperature. Eggs were mounted in slides and analysed using a Zeiss Axiophot microscope equipped with a camera and connected to an Imaging System Package (Applied Imaging, Newcastle-upon-Tyne, UK). Spermatozoa were considered as fused when their nucleus became Hoechst-stained and decondensed.
Statistical analysis
Statistical analysis of the data was determined by Statview® package. Means were compared by non-parametric Wilcoxon test. Differences were considered significant at P < 0.05.
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Results |
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Cyclic FEE peptide binds to the human oocyte membrane and increases the number of fused spermatozoa
Immunofluorescence study using the biotinylated cFEE, but not the scrambled peptide, and confocal microscopy showed that it binds to the oolemma of human zona-free oocyte and forms patches (Figure 2A and B). We therefore decided to perform a spermegg fusion assay using the cFEE peptide. According to previously reported experiments in a mouse model (Zhu and Evans, 2002) or in the zona-free hamster oocyte penetration test (Bronson et al., 1999
) which showed an inhibition of fertilization with a linear peptide, the same result was expected in a homogeneous human system. Surprisingly, while in control oocytes a mean of 19.0 ± 4.6 (mean ± SD) spermatozoa were found in the cytoplasm (Figure 3A and E), there was no inhibition of fertilization when gametes were incubated with 100 µmol/l cFEE. On the contrary, we observed an increase in the number of fused spermatozoa per oocyte (36.9 ± 11.7, P < 0.0001) (Figure 3B and E). Scrambled peptide had no effect on gamete fusion (21.4 ± 4.3, NS) (Figure 3E).
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Cyclic FEE peptide enhances the RGD-induced inhibition of human gamete fusion
As RGD peptide was known to be an inhibitor of gamete fusion, we tested the co-incubation of both cFEE and RGD peptide in the insemination medium. Co-incubation of 100 µmol/l cFEE and 100 µmol/l RGD peptide accentuated the inhibitory effect of RGD. Indeed, only 4.7 ± 3.9 fused spermatozoa per oocyte (Figure 3C and E) were observed versus 11.1 ± 3.2 (P < 0.0008) when fertilization occurs with RGD alone (Ji et al., 1998). Increasing the concentration of RGD to 200 µmol/l induced a dose-dependent decrease in the number of fused spermatozoa with a mean ± SD of 1.7 ± 2.2 (P < 0.0001) (Figure 3E). Some of these oocytes presented a complete fusion failure and only the egg metaphase could be seen (Figure 3D).
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Discussion |
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Both the composition and the cyclization of the peptide can explain the absence of an inhibitory effect. It lacked the second aspartic acid of the predicted disintegrin loop pentapeptide sequence QDECD, which has been shown to be critical for its inhibitory properties (Zhu et al., 2000). Our peptide did not contain this terminal aspartate which seems to be responsible for the inhibitory effect. Furthermore, our peptide was cyclized. It has been shown that restricting the conformational space of active peptide sequences, by using them in cyclic form, could lead to components with improved activity and receptor selectivity. This has been demonstrated for a synthetic cyclic RGD-containing peptide which was a 20- to 100-fold better inhibitor of cell adhesion to vitronectin and/or laminin fragments when compared with a linear variant (Pierschbacher and Ruoslahti, 1987
; Aumailley et al., 1991
). Such hypothesis could explain our peptides activity. Similarly, linear CAQDEC has been shown to reduce sperm binding and spermegg fusion, while the cyclic form did not have any inhibitory effect (Evans et al., 1995a
). This result may, however, be species dependent since Myles et al. (1994)
found that an equivalent cyclic peptide was an inhibitor in the guinea pig.
There is, in fact, considerable variation and inconsistency in the literature regarding the efficacy of peptide mimics used in such IVF assays. The technique of oocyte depellucidation may be critical since enzymatic techniques may modify their membrane fertilin receptors (Boldt et al., 1988, 1989
). For example, different zona removal techniques induce different distributions of the
6 integrin subunit (Tarone et al., 1993
; Zuccotti et al., 1998
; Miller et al., 2000
; Takahashi et al., 2000
). To prevent any protease digestion of membrane proteins and eventually of the receptor we are interested in, we performed a totally mechanical zona pellucida removal technique using a pair of microdissection scissors under Stero-microscope without any enzyme or chemical.
As already shown, we found a partial inhibitory effect of RGD peptide on gamete fusion (Ji et al., 1998). Interestingly, it is potentiated by the cFEE. Hence, in the presence of cFEE, the doseresponse effect of RGD achieved an almost complete inhibition for 200 µmol/l. Bronson et al. (1999)
described a similar potentiation of the inhibitory effect of RGD peptide by FEE. They used a linear FEE peptide and reported that combined SFEECDLP and G4120, a cyclic RGD-containing peptide, exhibited a strong inhibition of both adhesion and penetration at concentrations that individually had been ineffective, suggesting cooperation between the two receptor ligands during fertilization in human. They suggested that two different integrins should be involved in human gamete interaction, one of which recognizes fertilin and one which recognizes RGD-containing sperm-associated proteins such as vitronectin, and that they may cooperate (Bronson et al., 1999
). Actually, we show that the
6
1 integrin is localized on the surface of human oocyte in patches. Interestingly, RGD-sensitive
v
3 integrin is also present on the oocyte membrane and co-localizes with the
6
1 integrin, suggesting their direct cooperation within a functional complex (Figure 1F). In the mouse, Takahashi et al. (2000) also showed that
6 patches appear on the oocyte membrane at the sperm-binding site prior to fusion. We thus suggest that the functional complex to which the fertilizing sperm binds contains at least these two RGD-sensitive and RGD-independent integrins.
On the other hand, the tetraspanin CD9 is essential for spermegg fusion since female mice that have been deleted for this gene are infertile. Their infertility is related to the inability of the oolemma to fuse with the sperm (Le Naour et al., 2000; Miyado et al., 2000
). Tetraspanins are surface proteins containing four transmembrane domains and forming complexes with each other and with various membrane proteins within a network of molecular interactions called the tetraspanin web (Berditchevski, 2001
; Boucheix and Rubinstein, 2001
; Hemler, 2003
). They contribute to the formation of cell surface multimolecular complexes and thereby may participate in the functional regulation of molecules with which they associate (Berditchevski, 2001
; Boucheix and Rubinstein, 2001
; Hemler, 2003
). One hypothesis is that CD9 may be necessary for the patch formation. This could explain the inability of CD9-deleted oocytes to fuse since they are unable to form
6 integrin patches (data not shown).
Finally, the oocyte sperm receptor could be composed of at least two integrins linked by tetraspans, 6
1 and
v
3, RGD-insensitive and RGD-dependent, respectively. Effectively, both
6
1 and
3 are linked to CD9 in somatic cells.
6
1 integrin is linked to CD9 via CD151 (Serru et al., 1999
) and the
3 integrin subunit is associated with CD9 in platelets (Longhurst et al., 1999
). One can therefore speculate that the fusing spermatozoon first binds loosely to
6
1 and induces
6
1 and
v
3 patch formation. It can then bind tightly to the oocyte through the
v
3 integrin prior fusion (Figure 4). Fusi et al. (1996)
actually have described the vitronectin receptor as being the possible velcro whereby the sperm binds to the oocyte.
v
3 is, precisely, the vitronectin receptor (Fusi et al., 1996
). The fact that we detected its presence on the human oocyte surface and its co-localization with the
6
1 integrin supports this hypothesis.
|
This model allows us to propose the following interpretation of the reported data. When the two peptides are co-incubated, the cFEE peptide induces the recruitment and/or the activation of all available v
3 integrin-binding sites on which the RGD peptide binds. Increasing the RGD concentration results in fewer and fewer available binding sites. At 200 µmol/l, there are absolutely no free
v
3-binding sites where the sperm could bind and fuse. This could explain the RGD dose-dependent inhibition of the gamete fusion process in the presence of cFEE. On the contrary, when RGD peptide is incubated alone, it binds to the available
v
3 integrin. However, one can speculate that the fertilizing spermatozoon can find a free
6
1 integrin site to interact with and induce formation of a complex containing
v
3 integrin on which the sperm can bind competitively with RGD peptide. This could explain why RGD-induced inhibition of gamete fusion is always partial. When cFEE peptide is incubated alone, it may induce numerous complex binding sites on which the inseminated spermatozoa can bind and fuse easily, explaining the increase in the number of fused sperm. This model also explains how the lack of tetraspanin CD9 in CD9/ eggs prevents formation of integrin patches and consequently gamete fusion.
Our findings are in contrast to previous reports stating that integrins are not essential for spermegg fusion (He et al., 2003). However, this assumption is debatable for several reasons. First, there may be several ways in which the oocyte may fuse, some being as yet unknown (Stein et al., 2004
). However, in the physiological process, integrins are effectively present on the oolemma and obviously concerned with the fusion process according to previous reports (Fusi et al., 1992
; Almeida et al., 1995
; Evans et al., 1997a
; Takahashi et al., 2000
). Secondly, the removal of the zona pellucida may bypass a step of the fusion process rendering, for example, the
6
1 integrin useless. This would explain the fusing ability of
6 or
1 integrin gene-deleted oocytes. Thirdly, experiments with deleted genes are difficult to interpret. Indeed, studies of knockout mice have shown that frequently there are unanticipated associated defects. Hence sperm from fertilin
and cyristestin knockout mice show reduced binding to the oocyte plasma membrane as expected, but they also adhere poorly to the zona pellucida and show deficient migration from the uterus to the oviduct (Cho et al., 1998
). Analysis of the protein profile of oocytes or sperm of these knockout mice also shows multiple molecular deficiency (Nishimura et al., 2001
; He et al., 2003
; for review see Stein et al., 2004
). Finally, redundant molecules may take the place of the deleted protein and assume its functions.
In conclusion, it is the first time that a peptide with the capacity of increasing human gamete fertilizing ability is reported. Such molecule could be used for supplementing the insemination medium to improve the fertilization rate in human IVF and perhaps limit the application of ICSI in assisted reproduction. Despite contradictory data from invalidated gene models, we believe that, physiologically, integrins participate in a multimolecular complex with tetraspanin and probably other as yet undiscovered molecules.
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Acknowledgements |
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Submitted on February 2, 2005; resubmitted on May 18, 2005; accepted on July 1, 2005.
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