Characterization of extracellular vesicle-coupled miRNA profiles in seminal plasma of boars with divergent semen quality status

Authors: DLAMINI, NOTSILE - Mississippi State University; NGUYEN, TINA - Mississippi State University; GAD, AHMED - Colorado State University; TESFAYE, DAWIT - Colorado State University; LIAO, SHENGFA - Mississippi State University; WILLARD, SCOTT - Mississippi State University; RYAN, PETER - Mississippi State University; FEUGANG, JEAN - Mississippi State University

Submitted to: International Journal of Molecular Sciences
Publication Type: Peer Reviewed Journal
Publication Acceptance Date: 2/1/2023
Publication Date: 2/6/2023
Citation: Dlamini, N.H., Nguyen, T., Gad, A., Tesfaye, D., Liao, S.F., Willard, S.T., Ryan, P.L., Feugang, J.M. 2023. Characterization of extracellular vesicle-coupled miRNA profiles in seminal plasma of boars with divergent semen quality status. International Journal of Molecular Sciences. 24(4):3194. https://doi.org/10.3390/ijms24043194.
DOI: https://doi.org/10.3390/ijms24043194

Interpretive Summary: Spermatozoa and the seminal plasma (SP) are the main fractions of semen whose quality significantly impacts breeding outcomes. The SP is the liquid fraction of the semen routinely used for non-invasive evaluation of semen quality without affecting sperm amount and integrity. It contains multiple molecules and structures, such as extracellular vesicles (EVs), that interact with spermatozoa to deliver their payloads, affecting sperm function. Here we explored the presence of EVs in SP of semen diagnosed as poor or good quality in boar studs; then, we evaluated their microRNA (miRNA) contents to identify a possible relationship with semen quality. Both semen groups showed comparable size distributions of EVs containing roughly similar amounts of miRNAs between poor (281) and good (271) semen quality. Both groups significantly differed in three specific miRNAs targeting several genes whose products may impair semen quality.

Technical Abstract: Sperm heterogeneity creates challenges for successful artificial insemination. Seminal plasma (SP) surrounding sperm is an excellent source for detecting reliable non-invasive biomarkers of sperm quality. Here, we isolated microRNAs (miRNAs) from SP-derived extracellular vesicles (SP-EV) of boars with divergent sperm quality statuses. Raw semen from sexually mature boars was collected for eight weeks. Sperm motility and normal morphology were analyzed, and the sperm was classified as poor- or good-quality based on standard cutoffs of 70% for the parameters measured. SP-EVs were isolated by ultracentrifugation and confirmed by electron microscopy, dynamic light scattering, and Western immunoblotting. The SP-EVs were subjected to total exosome RNA isolation, miRNA sequencing, and bioinformatics analysis. The isolated SP-EVs were round spherical structures approximately 30–400 nm in diameter expressing specific molecular markers. miRNAs were detected in both poor- (n = 281) and good (n = 271)-quality sperm, with fifteen being differentially expressed. Only three (ssc-miR-205, ssc-miR-493-5p, and ssc-miR-378b-3p) allowed gene targeting associated with cellular localization (nuclear and cytosol) and molecular functions (acetylation, Ubl conjugation, and protein kinase binding), potentially impairing sperm quality. PTEN and YWHAZ emerged as essential proteins for protein kinase binding. We conclude that SP-EV-derived miRNAs reflect boar sperm quality to enable therapeutic strategies to improve fertility.