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Bart M Gadella Faculty of Science, Department of Farm Animal Health and of Biochemistry and Cell Biology, School of Veterinary Science, University of Queensland, Gatton 4343, Queensland, Australia

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Spermatozoa interact with their immediate environment and this contact remodels the sperm surface in preparation for fertilisation. These fundamental membrane changes will be critically covered in this review with special emphasis on the very specific surface destabilisation event, capacitation. This process involves very subtle and intricate modifications of the sperm membrane including removal of suppression (decapacitation) factors and changes in the lateral organisation of the proteins and lipids of the sperm surface. Processing of sperm for assisted reproduction (storage, sex-sorting, etc.) subjects spermatozoa to numerous stressors, and it is possible that this processing overrides such delicate processes resulting in sperm instability and cell damage. To improve sperm quality, novel mechanisms must be used to stabilise the sperm surface during handling. In this review, different types of membrane stress are considered, as well as novel surface manipulation methods to improve sperm stability.

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Bart Leemans Department of Reproduction, Departments of Farm Animal Health, Biochemistry and Cell Biology, Equine Sciences, Obstetrics and Herd Health, Faculty of Veterinary Medicine, Ghent University, Salisburylaan 133, 9820 Merelbeke, Belgium

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Bart M Gadella Department of Reproduction, Departments of Farm Animal Health, Biochemistry and Cell Biology, Equine Sciences, Obstetrics and Herd Health, Faculty of Veterinary Medicine, Ghent University, Salisburylaan 133, 9820 Merelbeke, Belgium
Department of Reproduction, Departments of Farm Animal Health, Biochemistry and Cell Biology, Equine Sciences, Obstetrics and Herd Health, Faculty of Veterinary Medicine, Ghent University, Salisburylaan 133, 9820 Merelbeke, Belgium

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Tom A E Stout Department of Reproduction, Departments of Farm Animal Health, Biochemistry and Cell Biology, Equine Sciences, Obstetrics and Herd Health, Faculty of Veterinary Medicine, Ghent University, Salisburylaan 133, 9820 Merelbeke, Belgium
Department of Reproduction, Departments of Farm Animal Health, Biochemistry and Cell Biology, Equine Sciences, Obstetrics and Herd Health, Faculty of Veterinary Medicine, Ghent University, Salisburylaan 133, 9820 Merelbeke, Belgium

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Hilde Nelis Department of Reproduction, Departments of Farm Animal Health, Biochemistry and Cell Biology, Equine Sciences, Obstetrics and Herd Health, Faculty of Veterinary Medicine, Ghent University, Salisburylaan 133, 9820 Merelbeke, Belgium

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Maarten Hoogewijs Department of Reproduction, Departments of Farm Animal Health, Biochemistry and Cell Biology, Equine Sciences, Obstetrics and Herd Health, Faculty of Veterinary Medicine, Ghent University, Salisburylaan 133, 9820 Merelbeke, Belgium

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Ann Van Soom Department of Reproduction, Departments of Farm Animal Health, Biochemistry and Cell Biology, Equine Sciences, Obstetrics and Herd Health, Faculty of Veterinary Medicine, Ghent University, Salisburylaan 133, 9820 Merelbeke, Belgium

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Induction of hyperactivated motility is considered essential for triggering the release of oviduct-bound mammalian spermatozoa in preparation for fertilization. In this study, oviduct-bound stallion spermatozoa were exposed for 2 h to: i) pre-ovulatory and ii) post-ovulatory oviductal fluid; iii) 100% and iv) 10% follicular fluid (FF); v) cumulus cells, vi) mature equine oocytes, vii) capacitating and viii) non-capacitating medium. None of these triggered sperm release or hyperactivated motility. Interestingly, native FF was detrimental to sperm viability, an effect that was negated by heat inactivation, charcoal treatment and 30 kDa filtration alone or in combination. Moreover, sperm suspensions exposed to treated FF at pH 7.9 but not pH 7.4 showed Ca2+-dependent hypermotility. Fluo-4 AM staining of sperm showed elevated cytoplasmic Ca2+ in hyperactivated stallion spermatozoa exposed to treated FF at pH 7.9 compared to a modest response in defined capacitating conditions at pH 7.9 and no response in treated FF at pH 7.4. Moreover, 1 h incubation in alkaline, treated FF induced protein tyrosine phosphorylation in 20% of spermatozoa. None of the conditions tested induced widespread release of sperm pre-bound to oviduct epithelium. However, the hyperactivating conditions did induce release of 70–120 spermatozoa per oviduct explant, of which 48% showed protein tyrosine phosphorylation and all were acrosome-intact, but capable of acrosomal exocytosis in response to calcium ionophore. We conclude that, in the presence of elevated pH and extracellular Ca2+, a heat-resistant, hydrophilic, <30 kDa component of FF can trigger protein tyrosine phosphorylation, elevated cytoplasmic Ca2+ and hyperactivated motility in stallion sperm, but infrequent release of sperm pre-bound to oviduct epithelium.

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Bart Leemans Department of Reproduction, Obstetrics and Herd Health, Faculty of Veterinary Medicine, Ghent University, Merelbeke, Belgium

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Bart M Gadella Departments of Farm Animal Health
Biochemistry and Cell Biology

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Tom A E Stout Departments of Farm Animal Health
Equine Sciences, Faculty of Veterinary Medicine, Utrecht University, Utrecht, The Netherlands

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Catharina De Schauwer Department of Reproduction, Obstetrics and Herd Health, Faculty of Veterinary Medicine, Ghent University, Merelbeke, Belgium

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Hilde Nelis Department of Reproduction, Obstetrics and Herd Health, Faculty of Veterinary Medicine, Ghent University, Merelbeke, Belgium

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Maarten Hoogewijs Department of Reproduction, Obstetrics and Herd Health, Faculty of Veterinary Medicine, Ghent University, Merelbeke, Belgium

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Ann Van Soom Department of Reproduction, Obstetrics and Herd Health, Faculty of Veterinary Medicine, Ghent University, Merelbeke, Belgium

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In contrast to man and many other mammalian species, conventional in vitro fertilization (IVF) with horse gametes is not reliably successful. The apparent inability of stallion spermatozoa to penetrate the zona pellucida in vitro is most likely due to incomplete activation of spermatozoa (capacitation) because of inadequate capacitating or fertilizing media. In vivo, the oviduct and its secretions provide a microenvironment that does reliably support and regulate interaction between the gametes. This review focuses on equine sperm–oviduct interaction. Equine sperm–oviduct binding appears to be more complex than the presumed species-specific calcium-dependent lectin binding phenomenon; unfortunately, the nature of the interaction is not understood. Various capacitation-related events are induced to regulate sperm release from the oviduct epithelium and most data suggest that exposure to oviduct secretions triggers sperm capacitation in vivo. However, only limited information is available about equine oviduct secreted factors, and few have been identified. Another aspect of equine oviduct physiology relevant to capacitation is acid–base balance. In vitro, it has been demonstrated that stallion spermatozoa show tail-associated protein tyrosine phosphorylation after binding to oviduct epithelial cells containing alkaline secretory granules. In response to alkaline follicular fluid preparations (pH 7.9), stallion spermatozoa also show tail-associated protein tyrosine phosphorylation, hyperactivated motility and (limited) release from oviduct epithelial binding. However, these ‘capacitating conditions’ are not able to induce the acrosome reaction and fertilization. In conclusion, developing a defined capacitating medium to support successful equine IVF will depend on identifying as yet uncharacterized capacitation triggers present in the oviduct.

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Bart Leemans Department of Reproduction, Obstetrics and Herd Health, Faculty of Veterinary Medicine, Ghent University, Ghent, Belgium

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Tom A E Stout Department of Equine Sciences, Faculty of Veterinary Medicine, Utrecht University, Utrecht, The Netherlands
Department of Farm Animal Health, Faculty of Veterinary Medicine, Utrecht University, Utrecht, The Netherlands

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Catharina De Schauwer Department of Reproduction, Obstetrics and Herd Health, Faculty of Veterinary Medicine, Ghent University, Ghent, Belgium

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Sonia Heras Department of Reproduction, Obstetrics and Herd Health, Faculty of Veterinary Medicine, Ghent University, Ghent, Belgium

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Hilde Nelis Department of Reproduction, Obstetrics and Herd Health, Faculty of Veterinary Medicine, Ghent University, Ghent, Belgium

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Maarten Hoogewijs Department of Reproduction, Obstetrics and Herd Health, Faculty of Veterinary Medicine, Ghent University, Ghent, Belgium

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Ann Van Soom Department of Reproduction, Obstetrics and Herd Health, Faculty of Veterinary Medicine, Ghent University, Ghent, Belgium

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Bart M Gadella Department of Farm Animal Health, Faculty of Veterinary Medicine, Utrecht University, Utrecht, The Netherlands
Department of Biochemistry and Cell Biology, Faculty of Veterinary Medicine, Utrecht University, Utrecht, The Netherlands

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In contrast to various other mammalian species, conventional in vitro fertilization (IVF) with horse gametes is not reliably successful. In particular, stallion spermatozoa fails to penetrate the zona pellucida, most likely due to incomplete activation of stallion spermatozoa (capacitation) under in vitro conditions. In other mammalian species, specific capacitation triggers have been described; unfortunately, none of these is able to induce full capacitation in stallion spermatozoa. Nevertheless, knowledge of capacitation pathways and their molecular triggers might improve our understanding of capacitation-related events observed in stallion sperm. When sperm cells are exposed to appropriate capacitation triggers, several molecular and biochemical changes should be induced in the sperm plasma membrane and cytoplasm. At the level of the sperm plasma membrane, (1) an increase in membrane fluidity, (2) cholesterol depletion and (3) lipid raft aggregation should occur consecutively; the cytoplasmic changes consist of protein tyrosine phosphorylation and elevated pH, cAMP and Ca2+ concentrations. These capacitation-related events enable the switch from progressive to hyperactivated motility of the sperm cells, and the induction of the acrosome reaction. These final capacitation triggers are indispensable for sperm cells to migrate through the viscous oviductal environment, penetrate the cumulus cells and zona pellucida and, finally, fuse with the oolemma. This review will focus on molecular aspects of sperm capacitation and known triggers in various mammalian species. Similarities and differences with the horse will be highlighted to improve our understanding of equine sperm capacitation/fertilizing events.

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Bart Leemans Department of Reproduction, Departments of Farm Animal Health, Biochemistry and Cell Biology, Equine Sciences, Obstetrics and Herd Health, Faculty of Veterinary Medicine, Ghent University, Belgium

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Bart M Gadella Department of Reproduction, Departments of Farm Animal Health, Biochemistry and Cell Biology, Equine Sciences, Obstetrics and Herd Health, Faculty of Veterinary Medicine, Ghent University, Belgium
Department of Reproduction, Departments of Farm Animal Health, Biochemistry and Cell Biology, Equine Sciences, Obstetrics and Herd Health, Faculty of Veterinary Medicine, Ghent University, Belgium

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Tom A E Stout Department of Reproduction, Departments of Farm Animal Health, Biochemistry and Cell Biology, Equine Sciences, Obstetrics and Herd Health, Faculty of Veterinary Medicine, Ghent University, Belgium
Department of Reproduction, Departments of Farm Animal Health, Biochemistry and Cell Biology, Equine Sciences, Obstetrics and Herd Health, Faculty of Veterinary Medicine, Ghent University, Belgium

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Edita Sostaric Department of Reproduction, Departments of Farm Animal Health, Biochemistry and Cell Biology, Equine Sciences, Obstetrics and Herd Health, Faculty of Veterinary Medicine, Ghent University, Belgium

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Catharina De Schauwer Department of Reproduction, Departments of Farm Animal Health, Biochemistry and Cell Biology, Equine Sciences, Obstetrics and Herd Health, Faculty of Veterinary Medicine, Ghent University, Belgium

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Hilde Nelis Department of Reproduction, Departments of Farm Animal Health, Biochemistry and Cell Biology, Equine Sciences, Obstetrics and Herd Health, Faculty of Veterinary Medicine, Ghent University, Belgium

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Maarten Hoogewijs Department of Reproduction, Departments of Farm Animal Health, Biochemistry and Cell Biology, Equine Sciences, Obstetrics and Herd Health, Faculty of Veterinary Medicine, Ghent University, Belgium

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Ann Van Soom Department of Reproduction, Departments of Farm Animal Health, Biochemistry and Cell Biology, Equine Sciences, Obstetrics and Herd Health, Faculty of Veterinary Medicine, Ghent University, Belgium

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In many species, sperm binding to oviduct epithelium is believed to be an essential step in generating a highly fertile capacitated sperm population primed for fertilization. In several mammalian species, this interaction is based on carbohydrate-lectin recognition. d-galactose has previously been characterized as a key molecule that facilitates sperm–oviduct binding in the horse. We used oviduct explant and oviduct apical plasma membrane (APM) assays to investigate the effects of various carbohydrates; glycosaminoglycans; lectins; S-S reductants; and the capacitating factors albumin, Ca2+ and HCO3 on sperm–oviduct binding in the horse. Carbohydrate-specific lectin staining indicated that N-acetylgalactosamine, N-acetylneuraminic acid (sialic acid) and d-mannose or d-glucose were the most abundant carbohydrates on equine oviduct epithelia, whereas d-galactose moieties were not detected. However, in a competitive binding assay, sperm–oviduct binding density was not influenced by any tested carbohydrates, glycosaminoglycans, lectins or d-penicillamine, nor did the glycosaminoglycans induce sperm tail-associated protein tyrosine phosphorylation. Furthermore, N-glycosidase F (PNGase) pretreatment of oviduct explants and APM did not alter sperm–oviduct binding density. By contrast, a combination of the sperm-capacitating factors albumin and HCO3 severely reduced (>10-fold) equine sperm–oviduct binding density by inducing rapid head-to-head agglutination, both of which events were independent of Ca2+ and an elevated pH (7.9). Conversely, neither albumin and HCO3 nor any other capacitating factor could induce release of oviduct-bound sperm. In conclusion, a combination of albumin and HCO3 markedly induced sperm head-to-head agglutination which physically prevented stallion sperm to bind to oviduct epithelium.

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