148 Bull spermatozoa uptake of extracellular vesicles from bovine seminal plasma

Extracellular vesicles (EV) are important mediators of intercellular communication because they transfer microRNA (miRNA) that are able to repress translation of mRNA. Their presence in seminal plasma suggests a role in sperm fertility. It is known that bull seminal plasma contains fertility-associated proteins that are predictive of high and low fertility (Killian et al. 1993 Biol. Reprod. 49, 1202-1207). In addition, a difference in miRNA content between high and low spermatozoa motility has been observed in bulls, highlighting a potential role of EV on fertility (Capra et al. 2017 BMC Genom. 18, 14). We hypothesised that co-incubation of sperm of low-fertility bulls with EV isolated from the seminal plasma of high-fertility bulls could improve their fertility. Before testing this hypothesis, a preliminary study was carried out to investigate the presence and type of EV in bovine seminal plasma and their interaction with spermatozoa. Ejaculates of eight Holstein bulls collected weekly by artificial vagina were centrifuged at 1600×g for 10min to pellet spermatozoa and then centrifuged again at 2400×g for 30min to eliminate cell debris and large vesicles. After centrifugation, supernatants were collected and filtered twice (0.45 and 0.22µm) and stored at −80°C. A double ultracentrifugation at 100 000×g for 1h was performed, and pellets resuspended in a small volume of Tris buffer were kept at −80°C until used. Three ejaculates of the same bull were pooled to detect the concentration and size of EV by Nanosight Instruments. To trace the interaction with spermatozoa by fluorescence microscopy, EV were labeled with PKH26 dye and a dose-response curve in three replicates was performed. A suspension of 1×106 spermmL−1 was co-incubated with 200 or 400×106 EV labelled with pKH26 for 30, 60, 90, 120, 150, and 180min at 38.5°C. The end point of incubation was at 24h. Internalisation of EV was assessed using confocal microscopy. Our results showed that the size of EV ranged from 145.1 to 187.7nm, with an average of 166±29nm. For all seminal plasma samples, the number of EV ranged from 3.62 to 6.08×1013 particlesmL−1, with an average of 4.37±0.61×1013. Based on size, these EV can be categorised as shedding vesicles. Confocal microscopy was set to take fluorescent images at different planes scanned every 0.12µm from top to bottom of the spermatozoa. Our results showed that no fluorescence signal was detectable after co-incubation with 200×106 EV. At the concentration of 400×106 EV, up to 60min no signal was detectable, whereas at 90min spermatozoa showed a fine granular fluorescent pattern within the intermediate portion. At 120min, the signal was within the acrosome, and at 180min the spermatozoa were stained for the whole length, supposing a distribution of incorporated EV throughout all the cell. At 24h, the fluorescence signal decreased. In conclusion, this is the first study to demonstrate that bull spermatozoa incorporate EV from bull semen. We hypothesise that a transfer of molecules, such as miRNA and other noncoding RNA molecules, from EV to spermatozoa is probably involved in sperm fertility.