Identification and Functional Analysis of miRNAs in Seminal Plasma Exosomes of Landrace Boar (Sus scrofa domestica)
WU Zhi-Sheng1, CHEN Hui-Fang1, LIU Jun-Jie1, LIU Kai2, BAI Yin-Shan1,2
1 School of Life Science and Engineering, Foshan University, Foshan 528231, China; 2 Shanwei Animal Husbandry Research Institute, Shanwei 516600, China
Abstract：Exosomes (EXs) are a kind of small cellular vesicles with a diameter of 30~150 nm, which mediate the material transport between cells and play a significant regulatory role in development and maturation of gamete. Some studies also have shown that seminal plasma EXs (spEXs) played an important role in preservation and fertilization of sperm. However, the research on the expression regulation of spEXs miRNAs have not been reported at present. In this study, the expression of porcine spEXs miRNAs and their potential regulatory effect on gene expression in sperm fertilization were explored. The semen of elite Landrace boar (Sus scrofa domestica) was used to extract the spEXs, which were identified by electron microscopy, analysis of particle size and marker protein expression. Then the spEXs were extracted and used to sequence the miRNAs. Next, the highly expressed miRNAs were analyzed and their target genes were predicted. Finally, enrichment analysis of Gene Ontology (GO) and KEGG pathways were conducted. A total of 617 miRNAs were identified and 110 new miRNAs were discovered. The functional analysis of miRNAs with high expression showed that they had potential regulatory effects on sperm motility, metabolism and capacity. Among these miRNAs, the highly expressed miR-10 family (ssc-miR-10a-5p and ssc-miR-10b) could potentially regulate the expression of phosphodiesterase (Pde), inhibited the degradation of the second messengers (cAMP or cGMP) in sperm. Then the activation of cAMP/(protein kinase A) PKA signaling pathway and the phosphorylation of seminal a kinase anchoring proteins (AKAP) promoted the maintenance of sperm fertility. Sperm mobility inhibitor (SPMI) could inhibit sperm movement by inhibiting the motility of flagellum ATPase, which was a potential target gene of ssc-miR-204. And the high abundant ssc-miR-28-5p could target downstream regulatory N Myc downstream regulator 2 (Ndrg2), which inhibited the production of key enzymes in glycolysis. The enrichment analysis showed that target regulation of ssc-miR-28-5p could promote glycolysis and increase the amount of lactic acid in sperm, which was consistent with the study that sperm contained a lot of lactic acid. The found miRNAs found will provide basic data for preservation of porcine semen and development of semen in this study, which has a significant research value in the study of sperm fertilization mechanism.
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