We report the complete amino acid sequence of HSP-1, a major protein isolated from stallion seminal plasma or acid extracts of ejaculated spermatozoa. The protein consists of 121 amino acids organized in two types of homologous repeats arranged in the pattern AA'BB'. Each of the 13-15-residue A-type repeats contains two O-linked oligosaccharide chains. The B-type repeats span 44-47 amino acids each, are not glycosylated, and have the consensus pattern of the gelatin-binding fibronectin type-II module. This domain also occurs in the major bovine seminal plasma heparin-binding proteins PDC-109 (BSP-A1/A2) and BSP-A3. However, unlike the bovine proteins which bind quantitatively to a heparin-Sepharose column, stallion HSP-1 was recovered in both the flow-through and the heparin-bound fractions. Structural analysis showed that the two HSP-1 forms contain identical polypeptide chains which are differently glycosylated. Moreover, size-exclusion chromatography showed that heparin-bound HSP-1 associates with HSP-2, another major seminal plasma protein, into a 90 kDa product, whereas the non-heparin-bound glycoform of HSP-1 is eluted as a monomeric (14 kDa) protein. This suggests that glycosylation may have an indirect effect on the heparin-binding ability of HSP-1 through modulation of its aggregation state. On the other hand, both glycoforms of HSP-1 displayed gelatin-binding activity, indicating that the molecular determinants for binding heparin and gelatin are different.

The primary structure of HSP-7, a 14-kDa protein isolated from stallion seminal plasma, has been determined. HSP-7 belongs to the spermadhesin protein family, shares 98 76 sequence identity with the boar seminal plasma protein AWN, and, like its boar homolog, displays zona-pellucida-binding activity. Despite these conserved structural and functional features, the equine and porcine spermadhesins differ in their topography on spermatozoa.Keywords: horse seminal plasma-7 ; anti-(boar seminal plasma protein) ; zona-pellucida-binding protein ; equine spermadhesi n ; seminal plasma protein.The interaction between complementary molecules situated on the surface of capacitated spermatozoa and the extracellular glycoprotein matrix encasing the oocyte, the zona pellucida, initiates the chain of events leading to the physiological agonistinduced acrosome reaction, a necessary step of the mammalian fertilization process (Yanagimachi, 1994). However, despite the importance of gamete recognition and binding for the survival and evolution of species and the fact that the complementarymolecules-hypothesis was already postulated at the beginning of this century (Lillie, 191 3), the molecular details of sperm-zonapellucida binding remain ill defined. Many sperm-associated proteins (both integral and peripheral proteins) have been proposed as candidates for primary zona pellucida-binding molecules Calvete, 1995, 1996). However, the actual participation of most of these proteins in the in wivo process, as well as the identity of zona pellucida carbohydrate structures acting as ligands for these proteins, are still a matter of debate (Cardullo and Wolf, 1995;Litscher et al., 1995;Thall et al., 1995;Yonezawa et a]., 1995;Bork, 1996;Tsai and Silver, 1996). From in vitro studies on gamete interaction of a few mammalian species, an emerging generalization regarding primary sperm -zona-pellucida binding is the involvement of spermatozoal carbohydrate-binding proteins and oligosaccharides of the oocyte's glycoprotein vestment (Macek and Shur, 1988). This protein-carbohydrate complementarity mechanism appears to be conserved in the fertilization process throughout the whole evolutionary scale, from plants to humans (Dale, 1991).In the pig, we have characterized a family of sperm surfaceassociated 12-14-kDa lectins, the spermadhesins, some of Correspondence to Juan J. Calvete and E. Topfer-Petersen, Institut fur Reproduktionsmedizin, Tierarztliche Hochschule Hannover,

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Key words. Spermadhesin SSP-7-AWN-epitope-zona pellucida binding protein-stallion seminal plasma immunohistochemistry Summary. SSP-7 is a protein originally isolated from stallion seminal plasma. It has extensive amino acid sequence homology with boar spermadhesin AWN, and, like its porcine counterpart, SSP-7 displays zona pellucida-binding activity. Strikingly, however, immunohistochemical studies presented here show that the stallion and the boar spermadhesin homologues are secreted at different places of the male genital tract. Furthermore, indirect immunofluorescence shows that the topography of SSP-7 on the surface of stallion spermatozoa is restricted to the equatorial segment, whereas boar AWN epitopes cover the entire acrosomal cap membrane. The different cellular origin and compartimentalization of spermadhesin molecules in different species suggest that structurally related proteins could be involved in speciesspecific aspects of mammalian fertilization.