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 spermadhesins have been thoroughly investigated in the pig, which exhibits the greatest diversity of members: AWN, AQN‐1, AQN‐2, PSP‐I and PSP‐II and its glycosylated isoforms. They are multifunctional proteins showing a range of ligand‐binding abilities, e.g. carbohydrates, sulfated glycosamino‐glycans, phospholipids and protease inhibitors, suggesting that they may be involved in different steps of fertilization. Isolated porcine spermadhesins 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 Galβ(1–3)‐GalNAc and Galβ(1–4)‐GlcNAc sequences in O‐linked and N‐linked oligosaccharides, 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 membrane bilayer. At ejaculation the spermadhesins form a protective coat around the sensitive acrosomal 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 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). However, they belong to the superfamily of proteins with a CUB domain with a predicted all‐β structure. The crystal structure of the heterodimeric complex of the spermadhesins PSP‐I/PSP‐II has been solved, showing that the overall structure of both spermadhesins consists of a β‐sandwich with five (parallell and antiparallel) β‐strands. It is the first three‐dimensional structure of a zona pellucida‐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.

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Sperm are stored in the isthmic region of the oviduct under conditions that maintain viability and suppress early capacitation steps until ovulation occurs. The initial contact between sperm and oviductal epithelium is mediated by carbohydrate-protein interactions. In the pig, the carbohydrate recognition system has been shown to involve oligomannosyl structures. The spermadhesins AWN and AQN1 are the dominant porcine carbohydrate-binding sperm proteins. The objective of this study was to demonstrate that AQN1 contributes to sperm binding to the oviductal epithelium. AQN1 showed a broad carbohydrate-binding pattern as it recognizes both alpha- and beta-linked galactose as well as Manalpha1-3(Manalpha1-6)Man structures, whereas AWN bound only the galactose species. Binding of ejaculated sperm to oviductal epithelium was inhibited by addition of AQN1 but not by AWN. Mannose-binding sites were localized over the rostral region of the sperm head. Flow cytometry showed that, under capacitating conditions, the population of live sperm was shifted within 30 min toward an increase in the proportion of cells with low mannose- and high galactose-binding. The loss of mannose-binding sites was accompanied by the loss of AQN1 in sperm extracts and the significant reduction in the sperm-oviduct binding. The oviductal epithelium was shown by GNA-lectin histochemistry and by SDS-PAGE and lectin blotting of the apical membrane fraction to express mannose components that could be recognized by AQN1. These results demonstrate that the sperm lectin AQN1 fulfils the criteria for an oviduct receptor in the pig and may play a role in the formation of the oviductal sperm reservoir.

By adopting internal fertilization, the meeting of both gametes -the sperm and the egg -and thus the highly coordinated sequence of interactions leading to fertilization, occur in the female reproductive tract. In mammals, the oviduct has been shown to translate the requirements of the female, coordinating sperm activation (capacitation) and sperm transport with the arrival of the ovulated egg. A hierarchy of carbohydrate-based interactions accompanies these events ranging from the binding of uncapacitated sperm to the oviductal epithelium (establishment of the female sperm reservoir), to the primary and secondary binding processes contributing to gamete recognition and sperm penetration of the oocyte zona pellucida. The current perspective will focus on the carbohydrate-recognition systems in the binding events during fertilization in the pig. The roles of the major carbohydrate-binding proteins, the spermadhesins and the acrosomal serine proteinase, pro/acrosin are discussed under consideration of recent structural data. The glycans and the glycoproteins of the porcine oviduct with a focus on the candidate sperm receptors as well as the zona pellucida N-glycans of prepuberal pigs have been characterized by a mass spectrometric approach. Furthermore, some preliminary data supporting the hypothesis that the zona pellucida has to undergo a maturation process during oocyte development are presented. KEY WORDS: gamete interaction, spermadhesin, acrosin, zona pellucida, glycoprotein, glycan A short review of mammalian fertilizationThe fertilization of an egg by a sperm is the fundamental event in life, as it culminates in the creation of a new individual. In mammals, the meeting of the fertilizing competent sperm and the ovulated egg is the beginning of a highly coordinated sequence of cellular interactions between the haploid gametes, which leads to the formation of the diploid zygote and initiates embryonic development.The first contact between sperm and egg takes place at the outer surface of the surrounding extracellular matrix of the egg, the zona pellucida (ZP). By binding to distinct oligosaccharides of the ZP glycoproteins, the fertilizing sperm recognizes the egg. Upon anchoring the sperm to the egg through the ZP carbohydrates the signaling cascade leading to acrosomal exocytosis of the sperm is activated allowing the sperm to pass through the zona pellucida. The acrosome-reacted sperm in the end interacts and fuses with the egg plasma membrane, which in turn enables the egg to complete meiosis, to initiate the mechanisms to prevent Int.

Sperm are stored in the isthmic region of the oviduct under conditions that maintain sperm viability and suppress motility. This region is also the site in which essential steps of the capacitation process are coordinated with the appearance of the ovulated egg. The influx of Ca(2+) and phosphorylation of sperm proteins are features of the ongoing capacitation process. Using a cell-culture system of oviductal epithelial cells, it was found that sperm bound to the epithelial cells showed a reduced Ca(2+) uptake and almost no tyrosine phosphorylation as shown by indirect immunofluorescence. Furthermore, sperm viability, measured as membrane integrity with propidium iodide, is significantly prolonged as compared to sperm in suspension. The formation of the sperm reservoir appears to be mediated by carbohydrate-protein interaction. In the pig, it has been found that mannosyl-oligosaccharides exposed by the epithelial cells are high-affinity ligands for sperm-associated lectins. Ovalbumin and mannopentaose are effective inhibitors of sperm binding to explants of oviductal epithelium. Spermadhesins, a new class of animal lectins and the major secretory products of the porcine seminal vesicle, associate with the sperm surface at ejaculation and are candidate molecules for the receptors of the epithelial carbohydrates.