Sperm binding to oviduct epithelial spheroids varies among males and ejaculates but not among females in pigs.

The elimination of ejaculates and males with low fertility despite good sperm motility and morphology is crucial to maintain high pregnancy rates after artificial insemination (AI) in farm animals. The ability of sperm to survive in the female tract is particularly crucial in pigs due to the large variation in the timing between AI and ovulation and the high number of oocytes to fertilise. The objective of this study was to characterise a new in vitro model of oviduct sperm reservoir using porcine oviduct epithelial spheroids (OES) and to assess the variability in sperm binding to OES among gilts, boars and their ejaculates. Isthmic mucosa fragments were collected from gilt oviducts at a slaughterhouse, and after 48 h of culture, the OES that had spontaneously formed were sorted according to their vesicle shape and size (150-200 µm in diameter) for characterisation and sperm binding assays. The OES contained viable, cytokeratin-positive and vimentin-negative cells, of which 36.4 ± 2.0% were multiciliated. The average proportion of multiciliated cells per OES did not change among culture replicates. After co-incubation with boar fresh semen, only sperm of normal morphology were found to bind, by their head, to cilia of OES. The density of sperm bound to the OES surface increased linearly with sperm concentration. The bound sperm density on OES was used to assess the binding capacity of fresh ejaculates collected from Pietrain boars. For a given ejaculate, the bound sperm density did not vary among pools of OES female donors. The analysis of five successive ejaculates from nine boars indicated significant differences in bound sperm densities on the OES among individual boars and their ejaculates (P < 0.01). There was no correlation between the sperm bound density and sperm parameters measured by computer-assisted sperm analysis or the initial dilution of the ejaculate. In conclusion, the OES characterised in this study offered physiological conditions to study sperm binding to the isthmic reservoir and evidenced that sperm from different ejaculates and different boars vary in their ability to bind to these oviduct spheroids despite homogeneous motility and morphology.

Porcine oviductal extracellular vesicles interact with gametes and regulate sperm motility and survival.

Once in the female reproductive tract, spermatozoa undergo several modifications to acquire their complete fertilizing ability. Interactions between the oviductal fluid (OF) and gametes contribute to a successful fertilization. Recently, oviductal extracellular vesicles have been identified as an important part of the OF but their interactions with gametes are not fully understood. In the present study, we aim at determining the patterns of interactions between porcine oviductal extracellular vesicles (poEVs) and gametes (spermatozoa and oocytes). Moreover, we evaluate the effect of poEVs on sperm survival and motility to better understand the mechanisms by which poEVs modulate the processes leading to fertilization. Evaluation of poEVs uptake by spermatozoa showed that poEVs bind to spermatozoa in a time and dose dependent manner. Co-incubation of spermatozoa with poEVs (0.2 µg/µL) increased fresh and frozen sperm survival after 6 and 17 h, respectively. By contrast, poEVs supplementation reduced the total and progressive sperm motility after 2 h. Additionally, we demonstrated that poEVs interacted with the cumulus cells, zona pellucida (ZP) and oocyte, being able to cross the ZP. Besides, we showed that poEVs delivered their cargo into the oocyte, by the transfer of OVGP1 protein. In conclusion, our results demonstrated that poEVs are able to interact with both gametes. Besides, the findings from the present study showed that poEVs may participate in maintaining sperm viability and reducing motility, functions associated with the oviduct sperm reservoir. Although further investigations are needed, our results indicate that poEVs can be a potential tool to improve sperm life span during sperm handling and enhance IVF outcomes.