Bull spermatozoa uptake of extracellular vesicles from bovine seminal plasma

Extracellular vesicles (EVs) are important mediators of intercellular communication by transferring microRNAs (miRNAs) that are able to repress translation of messenger RNA. Their presence in seminal plasma suggests a role on sperm fertility. It is already known that bull seminal plasma contains fertility-associated proteins that are predictive of high and low fertility [1]. In addition, in bull, a difference in miRNA content between high and low spermatozoa motility has been observed, which highlight the importance of the role played by EVs on fertility [2]. We hypothesize that co-incubation of sperm of low fertility bulls with EVs isolated from the seminal plasma of high fertility bulls could improve their fertility. Before testing this hypothesis, a preliminary work was carried out with the aim to investigate the presence and type of EVs in bovine seminal plasma and their interaction with spermatozoa. Ejaculates of eight Holstein bulls (4\u20136 years age) were collected weekly by artificial vagina. The ejaculates were centrifuged at 1600 g for 10 min to pellet spermatozoa and then centrifuged again at 2400 g for 30 min to eliminate cell debris and large vesicles. After centrifugation, the supernatants were collected, filtered twice (0.45 and 0.22 \ub5m) and stored at -80\ub0C. A double ultra-centrifugation at 100.000 g for 1 h was performed. Finally, the pellet was re-suspended in a small volume of TRIS buffer and kept at -80\ub0C until used. Three ejaculates of the same animal were pooled to detect concentration and size of EVs by Nanosight Instruments. To trace interaction with spermatozoa by fluorescence microscopy, EVs were labeled with PKH26 dye and a dose\u2013response curve in three replicates was performed. A suspension of 1x106 sp/ml was co-incubated with 200 or 400 x106 EVs labelled with pKH26 for 30, 60, 90, 120, 150 and 180 minutes at 38.5\ub0C. The end point of incubation was at 24h. Internalization of EVs was assessed by confocal microscopy. Our results showed that the size of EVs of all samples ranged from 145.1 to 187.7 nm, with an average of 166\ub129 nm. For all seminal plasma samples, the number of EVs ranged from 3.62 to 6.08x1013 particles/ml, with an average of 4.37\ub10.61x1013. Based on size, these EVs can be categorized as shedding vesicles. Confocal microscopy was set to take fluorescent images at different plans scanned every 0.12 \ub5m from top to bottom of the spermatozoa. Our results showed that no fluorescence signal was detectable after coincubation with 200x106 EVs. At the concentration of 400 x106 EVs, up to 60 minutes no signal was detectable while at 90\u2019 spermatozoa showed a fine granular fluorescent pattern within the intermediate portion. At 120\u2019 signal was within the acrosome and at 180\u2019 spermatozoa were stained for the whole length supposing a distribution of incorporated EVs throughout all the cell. At 24 h, the fluorescence signal decreased. In conclusion, this is the first report demonstrating that bull spermatozoa incorporate EVs from bull semen. We hypothesize a transfer of molecules, like miRNAs and other non coding RNA molecules, from EVs to spermatozoa probably involved in sperm fertility. [1] Killian et al. Biol Reprod 1993,49:1202. [2] Capra et al. BMC Genomics 2017,18:14