Isolation and expression of micrornas involved in both porcine reproductive and respiratory syndrome and testicular maturation

The importance of microRNAs (miRNAs) in most biological processes has been well established, though the intricacies of their post-transcriptional regulation in reproductive functions are not entirely understood. They are found both intra- and extracellularly and can be obtained by sampling bodily fluids. Aberrant miRNA expression has been reported in numerous pathophysiological conditions and due to their stability in circulating fluids, miRNAs are potential biomarkers for reproductive disease and infertility. The modern swine industry relies almost entirely on the use of artificial insemination and therefore, reproductive biomarkers would be of great utility for optimization of genetics, breeding, and pathogen detection. The overall objective of these studies was to analyze miRNAs involved in boar reproduction. The first study measured the miRNAs, let-7a and miR-15b, in immature and mature porcine testes from boars of ages 2d, 3mo, 5mo, and 18mo. Additionally, the transcripts and proteins of argonaute RISC catalytic component 2 (AGO2), high mobility group AT-hook 2 (HMGA2), and lin-28 homolog A (LIN28A) were examined using qPCR and immunofluorescence, respectively, in the immature (2d and 3mo) and mature (5mo and 18mo) testes. The second study identified known and unknown miRNAs present in sperm and seminal plasma from PRRSv-negative and PRRSv-positive boars using small RNA sequencing. Furthermore, microarrays were utilized to compare miRNA expression two days prior to (-2dpi) and six days post (6dpi) inoculation with PRRSv, in sperm, seminal plasma, and serum. No differences in let-7a expression were observed among the testes of different maturity, though a tendency was present; while, miR-15b expression was higher in the 2d age group compared to 3mo, 5mo, and 18mo. Argonaute transcript levels were lower in the sexually immature testes, whereas HMGA2 expression did not differ, with a tendency present. Transcript levels of LIN28A were decreased in 2d boars compared to 5mo and 18mo, and in 3mo boars compared to 18mo. MicroRNA sequencing resulted in 379 known and 183 potential candidate (PC) miRNAs in sperm, and 91 known and 71 PC-miRNAs in seminal plasma. Microarrays revealed differential expression on -2dpi and 6dpi of 83 sperm miRNAs, 10 seminal plasma miRNAs, and 13 serum miRNAs. Target prediction and functional enrichment analyses were performed for the differentially expressed miRNAs. Functional enrichment analysis utilized Gene Ontology (GO) and InterPro (IPR) terms and revealed enrichment for targets of 35 sperm, 9 seminal plasma, and 5 serum miRNAs. Reoccurring terms included P2X purinoreceptor, ion transport, protein-glutamine gamma-glutamyltransferase activity, and histone H4-K20 demethylation. Altogether, these studies demonstrate the involvement of miRNAs in different aspects of boar reproduction, including testicular maturation, sperm production, and influence of a viral pathogen on miRNA populations in sperm, seminal plasma, and serum.