The term ‘prostasomes’ is generally used to classify the extracellular vesicles (EVs) released into prostatic fluid by prostate epithelial cells. However, other epithelia within the male reproductive tract also release EVs that mix with ‘true’ prostasomes during semen emission or ejaculation. Prostasomes have been proposed to regulate the timing of sperm cell capacitation and induction of the acrosome reaction, as well as to stimulate sperm motility where all three are prerequisite processes for spermatozoa to attain fertilising capacity. Other proposed functions of prostasomes include interfering with the destruction of spermatozoa by immune cells within the female reproductive tract. On the other hand, it is unclear whether the distinct presumed functions are performed collectively by a single type of prostasome or by separate distinct sub-populations of EVs. Moreover, the exact molecular mechanisms through which prostasomes exert their functions have not been fully resolved. Besides their physiological functions, prostasomes produced by prostate tumour cells have been suggested to support prostate cancer spread development, and prostasomes in peripheral blood plasma may prove to be valuable biomarkers for prostate cancer.

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.

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.

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.

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.

The necessity for early interaction between the embryo and the oviductal and/or uterine environment in the horse is reflected by several striking differences between equine embryos that develop in vivo and those produced in vitro. Better understanding of the salient interactions may help to improve the efficiency of in vitro equine embryo production. In an initial experiment, cleavage-stage in vitro-produced (IVP) equine embryos were transferred into the uterus of recipient mares that had ovulated recently to determine whether premature placement in this in vivo environment would improve subsequent development. In a second experiment, an important element of the uterine environment was mimicked by adding uterocalin, a major component of the endometrial secretions during early pregnancy, to the culture medium. Intrauterine transfer of cleavage-stage IVP equine embryos yielded neither ultrasonographically detectable pregnancies nor day 7 blastocysts, indicating that the uterus is not a suitable environment for pre-compact morula stage horse embryos. By contrast, exposure to uterocalin during IVP improved capsule formation, although it did not measurably affect the development or expression of a panel of genes known to differ between in vivo and in vitro embryos. Further studies are required to evaluate whether uterocalin serves purely as a carrier protein or more directly promotes improved capsule development.