162 Differences in microRNA cargo of low-fertility bull spermatozoa before and after incorporation of extracellular vesicles isolated from proven fertilty bulls’ seminal plasma

Some studies support the role of extracellular vesicles (EVs) present in seminal plasma (SP) in assisting sperm to reach functional maturity. We hypothesise that the RNA, microRNA (miRNA), proteins, and other molecules in these EVs are able to influence the biological function of spermatozoa. In this context, it is conceivable that EV cargo of SP from bulls of proven fertility could improve the semen quality of low-fertility bulls. In a previous study on semen provided by a bull centre with respective progeny data, the incorporation of EVs derived from SP (SP-EVs) of proven fertility bulls into low-fertility bulls’ spermatozoa was found to improve the rate of in vitro embryo production (Lange-Consiglio et al. 2022 Reprod. Fertil. 3, 313–327). Assuming that this change is due to the transfer of miRNAs contained in EVs, the present study investigated (1) the differences in miRNA cargo between SP-EVs of proven and low-fertility bulls and (2) the miRNA cargo of low-fertility spermatozoa before and after treatment with SP-EVs of proven fertility bulls. At first, SP-EVs of 3 proven and 3 low-fertility bulls were isolated (by ultracentrifugation at 100 000g for 1 h at +4°C), and NanoSight analysis did not detect any differences in terms of concentration. Then, RNA from SP-EVs of proven and low-fertility bulls was extracted to prepare small RNA libraries, which were sequenced by the Illumina HisEqn 2500 System. In the second step, EVs isolated from SP of 3 proven fertility bulls were labelled with PKH-26 and co-incubated with low fertility spermatozoa (400 × 10⁶ EVs in 1 mL with 5 × 10⁶ sperm) and their presence in the middle piece of spermatozoa was detected by confocal and transmission electron microscope after 3 h. RNA extraction was carried out on spermatozoa of 3 low-fertility bulls, before and after EV-treatment to create libraries. A general linear model was used in the Bioconductor EdgeR package to generate lists of miRNAs with statistically significant different expression between sample groups. From the sequencing analysis, 82 differently expressed (DE) miRNAs between SP-EVs of proven and low-fertility bulls were detected, with miR-2284x most present in the SP-EVs of proven fertility bulls. In low-fertility spermatozoa before and after their treatment with EVs, 48 DE miRNAs were identified. Among them, miR-2284x, miR-2284y, and miR-101 were found to be more present in spermatozoa incubated with EVs than in non-treated ones. These results show that miR-2284x present in the EVs of proven fertility bulls could be transferred into the spermatozoon of low-fertility ones, improving their fertility in vitro (Kropp et al. 2017 BMC Genomics 18, 280). This miRNA targets the TFB2M gene, which is more highly expressed in embryos derived in vitro from higher fertility bulls (Kropp et al. 2017 BMC Genomics 18, 280). Our data support the hypothesis that alterations of EV molecule delivery in vivo could influence mechanisms regulating sperm functioning, and thus fertility.