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.

The ability to maintain cellular volume is an important general physiological function, which is achieved by specific molecular mechanisms. Hypotonically induced swelling results in the opening of K C and Cl K ion channels, through which these ions exit with accompanying water loss. This process is known as regulatory volume decrease (RVD). The molecular mechanisms that control the opening of the ion channels in spermatozoa are as yet poorly understood. The present study investigated pathways of osmo-signalling using boar spermatozoa as a model. Spermatozoa were diluted into isotonic and hypotonic Hepes-buffered saline in the presence or absence of effector drugs, and at predetermined intervals volume measurements were performed electronically. Treatment with protein kinase C (PKC) inhibitors staurosporine, bismaleimide I and bismaleimide X led to dosedependent increases of both isotonic and hypotonic volumes (P!0.05). However, as the isotonic volume was affected more than the hypotonic volume, the kinase inhibitors appeared to improve RVD, whereas activation of PKC with phorbol dibutyrate blocked RVD. The increase in isotonic cell volume induced by bismaleimide X was observed in chloride-containing medium but not in the medium in which chloride was replaced by sulphate, implying that PKC was involved in the control of chloride channel activity, e.g. by closing the channel after volume adjustment. The protein phosphatase PP1/PP2 inhibitors calyculin and okadaic acid increased the isotonic volume only slightly but they greatly increased the relative cell volume and blocked RVD. The activation of RVD processes was found to be cAMP-dependent; incubation with forskolin and papaverine improved volume regulation. Moreover, papaverine was able to overcome the negative effect of protein phosphatase inhibitors. The mechanism of sperm RVD appears to involve (a) alterations in protein phosphorylation/dephosphorylation balance brought about by PKC and PP1 and (b) a cAMP-dependent activating pathway.

Proteins secreted along the male genital tract, are involved in the remodelling of the sperm surface which occurs during sperm transit through the male genital tract and at ejaculation. During this process, collectively called post-testicular sperm maturation, the spermatozoa acquire the ability to fertilize an egg. These proteins have been shown to contribute to early and central steps of the fertilization sequence, e.g. the establishment of the oviductal sperm reservoir, modulation of capacitation and gamete interaction. The expression of those proteins is not restricted to the epididymis, but they are also secreted in more proximal parts of the male genital tract, thus contributing to the total protein content of the seminal plasma. Their isolation from seminal plasma allows investigations of structure-function relationships of the native proteins. Major seminal plasma proteins in ungulate species belong to three protein classes, which contain widely distributed protein modules: Fn-type II proteins are characterized by two or four tandemly arranged Fn-2 modules and have been implicated in the modulation of sperm capacitation. Multiple members of the cysteine-rich secretory proteins (CRISP) have been identified in the male genital tract of a number of species. CRISP proteins have been shown to be involved in various functions related to sperm-oocyte fusion, innate host defence function and ion channel blockage. Spermadhesins occur only in ungulate species. Their carbohydrate-and zona pellucida-binding properties would suggest a role of these proteins in gamete recognition.