I ANDROLOGIA 30, 217-224 (1998)

Spermadhesins: A new protein family. Facts, hypotheses and perspectives

E. Topfer-Petersen', A. Romero', P. F. Varela', M. Ekhlasi-Hundrieser', Z. Dostalova', L. Sanz' and J. J. Calvete'

'Institut fur Reproduktionsmedizin, Tierarzttliche Hochschule Hannover, Germany; 'Centro de Investigaciones Biol6gicas, CSIC, Madrid, Spain

Key words. Spermadhesin-CUB domain-spermatozoa-zona pellucida-gamete recognition-capacitation

Summary. Spermadhesins are a novel family of secretory proteins expressed in the male genital tract of pig, horse and bull. They are major products of the seminal plasma and have been found to be peripherally associated to the sperm surface. The structure and function of spermadhe- sins have been thoroughly investigated in the pig, which exhibits the greatest diversity of members: AWN, AQN- 1, AQN-2, PSP-I and PSP-I1 and its glycosylated isoforms. They are multifunctional proteins showing a range of ligand-binding abili- ties, e.g. carbohydrates, sulfated glycosamino- glycans, phospholipids and protease inhibitors, suggesting that they may be involved in different steps of fertilization. Isolated porcine spermadhe- sins bind the zona pellucida glycoproteins in a cation-dependent manner with a Kd in a low micromolar range, and AWN, AQN- 1 and AQN-3 display similar binding affinity for glyoproteins containing GalP( 1-3)-GalNAc and Ga1(3( 1-4)- GlcNAc sequences in 0-linked and N-linked oligo- saccharides, respectively. During sperm passage through the epididymis AQN-3 and AWN have been shown to bind tightly to the sperm surface by interaction with the phospholipids of the mem- brane bilayer. At ejaculation the spermadhesins form a protective coat around the sensitive acro- soma1 region of the sperm head, thus possibly preventing premature acrosome reaction. During in vitro capacitation most of these aggregated sperm adhesins are lost, with the exception of phospholipid-bound spermadhesins. AWN and AQN-3 may now serve as a primary receptor for the oocyte zona pellucida, thus contributing to

Correspondence: Dr Edda Topfer-Petersen, Institut fur Reproduktionsmedizin, Tierarztliche Hochschule Hannover, Bunteweg 15, 30559 Hannover-Kirchrode, Germany.

initial binding and recognition between sperm and egg.

The amino acid sequence of spermadhesins does not show any discernible similarity with known carbohydrate recognition domains (CRD). How- ever, they belong to the superfamily of proteins with a CUB domain with a predicted all-p struc- ture. The crystal structure of the heterodimeric complex of the spermadhesins PSP-I/PSP-I1 has been solved, showing that the overall structure of both spermadhesins consists of a P-sandwich with five (parallel1 and antiparallel) P-strands. It is the first three-dimensional structure of a zona pel- lucida-binding protein and reveals the architecture of the CUB domain. The spermadhesins represent a novel class of lectins that may be involved in sequential steps of fertilization, at least in the Pig.

Introduction

The initial event in the complex, multistep process of mammalian fertilization is gamete recognition through specific complementary molecules located on the external surfaces of the spermatozoon and the oocyte. In the few species so far investigated this cell-cell adhesion mechanism appears to be mediated by a protein-carbohydrate interaction between sperm-associated lectins and glycan struc- tures of the glycoprotein network surrounding the investing egg, the zona pellucida.

A number of sperm proteins with affinity for zona pellucida glycoproteins have been identified in several mammalian species using different approaches, such as probing Western blots of sperm proteins with labeled zona pellucida pro- teins; identification of the epitopes for specific

218 E. TOPFER-PETERSEN ET AL.

polyclonal or monoclonal antisera that inhibit sperm-zona binding; and studies with crosslinking reagents. The group of zona pellucida binding molecules include enzymes (glycosyltransferases, glycosidases, and hexokinase), integral plasma membrane proteins, and peripherally associated components (Table 1 ; reviewed by Topfer-Petersen et al., 1995). However, only in a few instances have the candidate zona pellucida-binding proteins been biochemically or structurally characterized. Among those sperm proteins for which specific carbohydrate binding sites have been demon- strated, the most thoroughly characterized are mouse P1-4 galactosyltransferase, mouse sp56, rabbit Spl7, human mannose-binding protein, and porcine spermadhesins AQN- 1, AWN, AQN-3, and PSP-TI (reviewed by Topfer-Petersen & Calvete, 1996). It must be emphasized, however, that the actual participation of any of these pro- teins in gamete interaction in the in vivo situation has not been proven. Furthermore, in species such as the mouse, where several zona pellucida- binding proteins have been reported, their partici- pation in gamete interaction is an issue of vivid debate (Kalab etal . , 1994; Burks et al., 1995; Cardullo & Wolf, 1995; Gong etal., 1995; Bork,

1996; Thaler & Cardullo, 1996; Tsai & Silver, 1996).

On the other hand, structural analyses of zona pellucida glycans have only been accomplished in the pig (Mori et al., 1991; Noguchi & Nakano, 1992; Noguchi et al,, 1992; Hirano et al., 1993; Hokke et al., 1993, 1994)) although initial studies on bovine and mouse zona pellucida oligosaccha- ride structures have been reported (Noguchi & Nakano, 1993; Katsuma et al., 1996). Again, the actual identity of the carbohydrate structures acting as ligands for sperm lectins has only been assessed in a couple of species and is still a matter of controversy. Hence, terminal a-linked galac- tose (Bleil & Wassarman, 1988) and terminal pl,4-linked N-acetylglucosamine (Miller et al., 1992; Gong et al., 1995) residues have been sug- gested to be essential for the sperm receptor activity of mouse zona pellucida. However, Thall et al. (1995) have demonstrated that genetic- engineered mice lacking Gala1,3Gal epitopes are fertile, indicating that terminal galactose residues are not strictly required for sperm adhesion to the zona pellucida. On the other hand, Litscher et al. (1 995) showed that oligosaccharide constructs bearing terminal (a- or P-linked) galactose inhibit

Table 1. Mammalian sperm proteins identified by their zona pellucida binding capability. Species code: M, mouse; Ha, hamster; Hu, human; R, rat; Ra, rabbit; P, pig; GP, guinea pig; Ho, horse; D, dog; W, wide distribution; 'GalTase, P-1,4-N- acetylglucosamine : galactose transferase; 'Integral plasma membrane protein; 3peripheral protein; 4SPI-B, serine proteinase inhibitor- binding protein; 5RTK, receptor tyrosine kinase; 6MPB, mannose-binding protein; 'Possibly GPI-anchored in the membrane; PH-20 possesses hyaluronidase activity; 'Sp 17 mRNA is present in these species; 'This carbohydrate-binding activity is deduced from the structure of the sperm protein; "SGR, sperm galactosyl receptor; "Acrosomal protein: may only play a role as secondary adhesion molecule to bind acrosome-reacted sperm to the zona pellucida

Protein Species Carbohydrate- binding activity

Reference

GalTasel,2 M terminal GlcNAc residues Miller et al., 1992 $-D-mannosidase' M,Ha,Hu,R ccl-3/al-2-Man Cornwall et al., 1991 15-20 kDa SPI-B"' M,Hu Boettger-Tong et al., 1993 ~ ~ 5 6 ~ M,Ha terminal cc-Gal Bookbinder et al., 1995 APz (52 kDa) P Peterson et al., 1991 RTK' 95 kDa M,Hu Burks et al., 1995 C-type M B P ~ Hu D-mannose Benoff, 1993 SL1Pl3 (68 kDa) W sulfogalactolipids Tanphaichitr et al., 1993 ~ ~ - 2 0 ~ 2 ~ ~ W Overstreet et al., 1995

Hunnicutt et al., 1996 p 105/45' P Hardy & Garbers, 1994, 1995 Sp17' (17 kDa) Ra (M,Hu)~ (galacto~e)~; heparin Richardson et al., 1994

Kong et al., 1995 Lea et al., 1996

Ra,Hu (galact~se)~ Mertz et al., 1995 54 kDa SGR" sperm ad he sin^^ P,Ho P-Gal in oligosaccharides Calvete et al., 1995a

Topfer-Petersen & Calvete, 1996

Urch & Patel, 1991 Richardson & O'Rand, 1996

(Pro)acrosin" W sulfated polysaccharides Jones, 1991

ANUROLOGIA 30, 2 17-224 (1998)

SPERMADHESINS 219

binding of mouse sperm to unfertilized eggs in vitro, while those containing terminal GlcNac were not active. To complicate the matter, Thaler & Cardullo (1996) have found that, in mouse, sub- strates of GalTase do not interfere with sperm- zona pellucida binding, and that compounds that interact with fucosyltransferase efficiently inhibit binding of solubilized zona pellucida glycoproteins to capacitated mouse sperm. Thus it is proposed that GalTase does not possess the biochemical specificity to serve as the primary sperm surface receptor for zona pellucida and that this role could be taken over by fucosyltransferase. Thaler & Cardullo (1996) propose that the GalP 1,4-GlcNAc moiety, commonly recognized by a1,3- and al,4-fucosyltransferases, mimics all or part of the zona pellucida oligosaccharide epitope involved in sperm binding. This disaccharide sequence occurs, however, in N-linked oligosaccharides of mouse zona pellucida glycoproteins (Noguchi & Nakano, 1993; Nagdas et al., 1994), a fact that appears to be at odds with previous assignment of O-linked oligosaccharides as the sperm-binding relevant structures (Florman & Wassarman, 1985).

In the pig, contradictory results have also been reported. Yurewicz et al. (1991) showed that the O-linked glycans attached to ZPB carry the bulk of the sperm-binding activity of intact zonae pel- lucidae. The smallest of the O-linked-type oligo- saccharide bearing sperm-zona pellucida binding inhibitory activity has been recently synthesized, and has the hexasaccharide structure: GalPl,4 (HSO,-6)GlcNAc~1,3-Gal~l,4-GlcNAc~l,3-Gal Pl,3-GalNAc-ol (Spijker et al., 1996). On the other hand, Nakano and colleagues (Yonezawa et al., 1995; Nakano et al., 1996) have presented evidence that endo-P-galactosidase-treated zona pellucida (EPG-ZP) retained sperm-binding inhibitory activity, and that the elimination of N-linked carbohydrate chains from EPG-ZPB markedly reduced its inhibitory effect, whereas release of O-linked carbohydrate chains hardly reduced the inhibitory activity. The authors concluded that the tri- or tetraantennary neutral N-linked carbo- hydrate chains of porcine EPG-ZPB may play a major role in mediating binding of sperm to the zona pellucida.

From the above considerations it is clear that, although the concept that gamete interaction involves complementary molecules was put for- ward more than 80 years ago (Lillie, 1913), our understanding of the molecular basis of fertili- zation is still in its infancy. Here, the discussion will be restricted to our current knowledge on spermadhesins, a family of sperm-associated zona pellucida- and carbohydrate-binding mol- ecules. Circumstantial evidence for considering

spermadhesins as bona jide primary gamete adhesion molecules has been summarized else- where (Calvete et al., 1995a; Topfer-Petersen & Calvete, 1996a) and is beyond the scope of this review.

Spermadhesins: facts and hypotheses

Studies with isolated proteins

Spermadhesins are a group of 12- 16 kDa polypep- tides found in the seminal plasma and/or peripher- ally associated with the sperm surface of pig, bull, and horse (see Reinert et al., 1996). Boar sperm- adhesins AQN- 1, AQN-3, PSP-I, PSP-11, AWN- 1, and AWN-2 are major secretory products of the seminal vesicle epithelium; their concentrations in seminal fluid range from 0.6 to 7 mg ml-' (DostalovA et al., 1994). AWN- 1 is also synthesized by the rete testis and tubuli recti (Sinowatz et al., 1995) and is the only member of its family present on epididymal sperm where 6-7 x lo6 molecules per cell have been quantitated (Dostalova et al., 1994). After ejaculation 12-60 x lo6 molecules of each AQN-1, PSP-1, AQN-2, AQN-3, AWN-1 and AWN-2 become coated on one third of apical region of the acrosomal cap. However, most of this coating material is released during in vitro capacitation when the level of each spermadhesin reaches 5-6 x lo6 molecules per spermatozoon (Dostalova et al., 1994). A specific interaction between nonaggregated AWN- 1 and AQN-3 mol- ecules and O-phosphorylethanolamine has been reported (Dostalova et al., 1995b), suggesting that these molecules could form the first layer of the coating material by interacting with the lipid bilayer. Indeed, fractionation by affinity chroma- tography of solubilized boar sperm membrane proteins on immobilized porcine zona pellucida glycoproteins identified AWN- 1 and AQN-3 as the major zona pellucida-binding proteins (Enplin et al., 1995), indicating that these spermadhesins bind tightly to the sperm surface.

Aggregated spermadhesins may serve as acro- some stabilizing factors and might constitute the subpopulation that is released during capacitation; the remaining spermadhesin molecules on capaci- tated sperm are in a good position to act as primary zona pellucida binding proteins. In line with this hypothesis, Iwamoto et al. (1995) have cloned the gene for a component of a 50 kDa boar sperm motility inhibitor (SPMI) factor composed of at least 3 noncovalently associated 14-18 kDa polypeptides. The N-terminal amino acid se- quences of the three 14- 18 kDa proteins and the complete amino acid sequence encoded by the

ANDROLOGIA 30, 217-224 (1998)

220 E. TOPFER-PETERSEN ET AL.

cDNA are identical to that of AQN-3 except for two positions (T78/G, E95/D). This suggests that aggregated AQN-3 molecules may suppress sperm motility and that the removal of this coating (decapacitating) material might be associated with the acquisition of sperm motility during swim-up and sperm capacitation (Yanagimachi, 1994).

Isolated spermadhesin AWN- 1 binds zona pel- lucida glycoproteins in a cation-independent manner with a dissociation constant (Kd) in the low micromolar range, and displays the similar binding affinity for glycoproteins containing a Gal@( 1&3)-GalNAc sequence in O-linked struc- tures (DostAlova et al., 1995a). The binding affinity of AWN-1 for Galp (1-4)-GlcNAc sequence is 50 times lower. Sialylated forms of these oligosaccha- rides appear to enhance the affinity constant by five times. Thus, the carbohydrate recognition domain (CRD) of AWN-1 may contain an essential binding site for galactose and subsites for sialic acid and N-acetylhexosamines; the N-acetylhexosamine- binding subsite of AWN- 1 may preferentially accommodate an axial-oriented (GalNAc) rather than the equatorial orientation (GlcNAc) of the 4-OH group.

The oligosaccharide- and zona pellucida- binding characteristics of spermadhesins AQN- 1 and AQN-3 have been investigated using a solid- phase assay and biotinylated glycoprotein ligands (Calvete et al., 1996b). Like AWN-1, spermadhesins AQN- 1 and AQN-3 bind glycoproteins containing Galp( 1 -4)-GlcNAc and Galp( 1-3)-GalNAc oligo- saccharide sequences with dissociation constants (Kd) of 0.08-0.8 p ~ , and to zona pellucida glyco- proteins with a Kd of 0.15-0.25 p ~ . However, 5-N-acetyl neuraminic acid ~(2-3/6)-linked to the galactose residue decreases the affinity of glycosy- lated ligands to AQN-1 three times although it did not affect oligosaccharide binding to AQN-3. In addition, AQN-3 binds preferentially to glyco- proteins with either linear or tri- and tetra- antennary carbohydrates than to those containing diantennary N-acetyllactosamine structures.

Remarkably, most of the neutral N- and O-linked carbohydrate structures of porcine zona pellucida glycoproteins contain a terminal Galp( 1-4)-GlcNAc sequence; neutral O-linked oli- gosaccharides have an internal Galp( 1 -3)-GalNAc sequence, and both disaccharide structures are also present in the anionic O-linked sugar chains. The similar but distinct oligosaccharide recog- nition capabilities of spermadhesins AQN- 1 and AQN-3 and AWN-1 suggest that, in the pig, sperm-zona pellucida binding might be mediated by lectins displaying overlapping but distinct carbohydrate-recognition abilities.

Immunohistochemical localization and m W A expression

Using indirect immunofluorescence microscopy and polyclonal monospecific anti-AWN antibodies, the immunohistochemical distribution pattern of AWN-epitopes has been investigated in tissue sec- tions of the boar genital tract (Sinowatz et al., 1995). It was found that AWN is synthesized in the rete testis and by the epithelium of the seminal vesicles. On the other hand, AWN molecules were demonstrated on in vitro capacitated spermatozoa bound to an isolated porcine zona pellucida as well as to isolated zona-encased porcine oocytes (Dostilovi et al., 1995a). Upon incubation with the oocyte for short periods (1-3 min), these in vitro capacitated spermatozoa could acrosome react and start penetration of the zona pellucida. These results indicate that AWN-1 is on the surface of fertile porcine spermatozoa. Furthermore, AWN- 1 has been immunologically shown on spermatozoa recovered from the uterotubal junction of sexual mature gilts inseminated between 8 and 12 h before surgery. In the pig, the utero-tuba1 junction is the major barrier for sperm ascent to the site of fertilization, the ampulla, and the place where spermatozoa seem to complete capacitation (Yanagimachi, 1994). Circumstantial evidence has suggested that these spermatozoa represent the subpopulation that eventually fertilize the egg (Yanagimachi, 1994).

It remains, however, to be established whether spermadhesins AWN- 1 and AQN-3 are actually present on the subpopulation of spermatozoa that meet the investing egg.

The expression of porcine spermadhesin mol- ecules has been also assessed by reverse trans- criptase/polymerase chain reaction (RT-PCR) amplification of mRNA extracted from different tissues (Ekhlasi-Hundrieser et al., unpubl. obs.; Fig. 1). This study confirms that the seminal ves- icles are the major place of biosynthesis of sperm- adhesins PSP-I, PSP-11, AQN-3, and AWN-1 and shows that the mRNAs coding for these proteins are, in addition, abundant in the cauda epididym- idis (Fig. 1). Preliminary experiments indicate that spermadhesin AQN- 1 follows the same expression pattern. The failure to amplify detectable amounts of AWN mRNA in the porcine rete testis might indicate that this mRNA is particularly labile (and was degraded during handling), or that very low amounts of AWN mRNA are produced by this tissue.

Spemnadhesin molecules in other species

A 14 kDa protein crossreacting with anti(porcine)- AWN antibodies has been isolated from stallion

ANDROLOGIA 30, 217-224 (1998)

SPERMADHESINS 221

Neg. Control

Testis

E. cauda Seminal vesicle Marker Neg. Control

cd m E. caput cd

U

cd Testis v,

s E. caput cd E. cauda Seminal vesicle Marker

ing gene duplication, one copy of the gene may divergently evolve under pressure dictated by the ancestral function, while the duplicate gene(s), unencumbered by a functional role, are free to search new physiological roles (Trabesinger-Ruef et al., 1996). The reason for and the consequences of the presence of multiple spermadhesin mol- ecules in porcine seminal plasma but only one member of the family in stallion seminal plasma is at present unclear.

Spermadhesins: perspectives

E. caput E. cauda Seminal vesicle

Figure 1. Electrophoretic separation of RT-PCR-amplified DNA coding for porcine spermadhesins PSP-I, PSP-11, and AWN extracted from testis segments of the epididymis: caput, cauda and seminal vesicles. Basepair (bp) markers, whose size is indicated, are shown.

seminal plasma (Calvete et al., 1994). Purified stal- lion seminal plasma AWN, like its porcine homol- ogue, can bind to isolated equine zonae pellucidae. This finding, together with the observation that stallion frozen epididymal spermatozoa possess fertilizing capacity (Barker & Gandier, 1957), sug- gests that spermadhesin AWN may be one of the factors contributing to the reproductive ability of horse epididymal sperm.

Stallion and boar AWN molecules differ only in three amino acids (Reinert etal. , 1996). Since perissodactyls, (e.g. horse) and artiodactyls, (e.g. pig) had a common ancestor over 50 million years ago, the unusually low mutational rate might suggest that the whole AWN structure is under strong selective pressure, pointing to a highly conserved conformation and, most probably, a common biological function for AWN and HSP-7 in both vertebrate species. Although the actual involvement of HSP-7 in horse fertilization has not been established, the zona pellucida-binding activity displayed by the isolated protein supports its classification as putative sperm-egg adhesion molecule.

The hypothesis that the porcine and equine AWN molecules were diverging under functional constraints, whereas the other polypeptides of the boar seminal plasma spermadhesin family were diverging more rapidly in their amino acid sequences, is in line with the proposal that, follow-

The amino acid sequence of spermadhesins does not show any discernible similarity with known carbohydrate-recognition domains, indicating that spermadhesins may belong to a novel group of animal lectins. A sequence pattern-search analysis has revealed that spermadhesins belong to a family of 16 functionally diverse proteins, many of which are known to be involved in developmental pro- cesses (Bork & Beckmann, 1993). The structure of these proteins is built by combination of several modules but all of them share the CUB domain, named after the proteins where it was first iden- tified: complement subcomponents (Clr/C 1 s), embryonic sea urchin protein (Uegf), and bone morphogenetic protein 1 (Bmpl) (Bork & Beckmann, 1993). Spermadhesins, 1 10- 133 amino acid spanning polypeptides, form a subgroup of the CUB domain family as expected by their single domain architecture.

We have crystallized (Romero et al., 1996) and solved the crystal structure of a noncovalent heter- odimeric complex of spermadhesins PSP-I and PSP-I1 (Romero et al., 1997). PSP-I/PSP-I1 is the first zona pellucida-binding protein to have its three-dimensional structure, and reveals, in addition, the architecture of the CUB domain. The overall structure of the PSP-I and PSP-I1 subunits consists of a P-sandwich made up by two sheets each containing five (parallel and antiparal- lel) P-strands (Fig. 2). The heterodimer PSP- I/PSP-I1 displays a zona pellucida-binding site located in the PSP-I1 subunit (Calvete et al., 1995b)) although its carbohydrate-binding speci- ficity remains to be established. Furthermore, PSP- I/PSP-I1 does not bind to the sperm surface, clearly indicating that this spermadhesin complex will not play a role in gamete interaction. Nevertheless, due to the large amino acid sequence identity displayed by any pair of spermadhesins, the conformation of the CUB domain is expected to be highly conserved in other spermadhesin molecules.

Structure-function correlations are best under-

ANDROLOGIA 30, 2 17-224 (1998)

222 E. TOPFER-PETERSEN ET AL.

I a I

I I I I

I I

PSP-I b

Asn

i 3 PSP-II

Figure 2. a. Schematic representation of the secondary structural elements of a spermadhesin molecule (PSP-I). The position of the two conserved disulphide bridges (Cys-S-S-Cys) is depicted. The discontinuous line marks the border between the two p-sheets of the p-sandwich. N and C, N- and C-terminus, respectively. b. Stereo representation of the quaternary structure of PSP-I/PSP-11. The relative position of the glycosylation sites of PSP-I (Am5') and PSP-I1 (Amg8), and the putative carbohydrate recognition domain PSP-I1 are shown. The latter, based on studies on other spermadhesins (see above), is located around the Am5' residue. Tyr4', an aromatic residue in a position often present in the carbohydrate recognition domain of animal and vegetal lectins is shown.

stood in the case of spermadhesin AWN-1. The carbohydrate-binding site of spermadhesin AWN- 1 and AQN-3 has been localized around a conserved asparagine residue at position 50 (Calvete et al., 1993a, b). A phosphorylethanolamine-binding region has been mapped to a discontinuous region of AWN- 1 comprising residues 6- 12 and 104- 108 (Enplin et al., 1995), and a conformational heparin- binding surface, which partly overlaps with the phosphorylethanolamine-binding region, resides in an opposite location to the carbohydrate-binding region of spermadhesin AWN-1 (Calvete et al., 1996a). Molecular replacement and modelling studies based on the three-dimensional structure of PSP-I/PSP-I1 (Fig. 2), in conjunction with stra- tegies based on molecular biology protocols, (i.e. site-directed mutagenesis), will aid the elucidation of the structural basis of the ligand (car- bohydrates, phosphorylethanolamine, heparin)-

binding capabilities of AWN- 1 and other members of the spermadhesin protein family. On the other hand, future structure elucidation of the sperm- adhesin-ligand complexes will aid in establishing the molecular determinants of the spermadhesin- zona pellucida glycoconjugate interaction.

Spermadhesins are peripherally associated sperm proteins. Their attachment to the sperm surface is particularly dependent on the physico- chemical conditions of the medium surrounding spermatozoa within the female genital tract. The in uitro situation may or may not mimic the environment found by spermatozoa after insemi- nation. Therefore, a critical point in unambigu- ously assigning the proposed biological role to spermadhesin molecules is whether spermadhesins actually reach the site of fertilization.

Acknowledgements

The work of the authors' laboratories has been financed by grants from the Deutsche Forschungsgemeinschaft (To 1 14/ 3- 1 and Ca 209/ 1 - 1) and the Bundesministerium fur Forschung, Bildung und Technologie (0 1KY9503), Bonn, Germany, and from the Direccion General de Investigation Cientifica y Tkcnica (PB95-0077 and PB93-0 1 ZO), Madrid, Spain.

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