Differential MicroRNA Expression in Sperm Cells and Seminal Plasma due to PRRSV Infection

Abstract Text:

The objectives of this study were to detect differentially expressed (DE) microRNAs (miRNAs) due to PRRSV infection in sperm cells and seminal plasma and to perform functional enrichment analyses on predicted targets of these miRNAs. All animal work was performed at the Swine Veterinary Center (Saint Peter, MN) where six terminal crossbred boars were inoculated intramuscularly with 2 mL of 2x10⁴ viral RNA copies/mL PRRSV strain 1-8-4 on day 0. Semen was collected two days prior to inoculation (-2 dpi) and six days post-inoculation (6 dpi). The cellular fraction was separated by centrifugation and both sperm cells and seminal plasma aliquots were flash frozen and stored at -80°C. RNA was isolated using the mirVana miRNA Isolation Kit and used in custom microarray analyses (LC Sciences, Houston, TX) based on our porcine sperm cell and seminal plasma sequencing data. Individual microarrays were performed using 3 biological replicates from -2 dpi and 6 dpi. Differential expression was determined significant by P < 0.05 and a fold change threshold of less than or greater than two-fold. Potential miRNA targets were predicted using miRanda 3.3a with a score threshold of 140 and energy threshold of -20 kcal/mol.  Targets were then analyzed for enrichment of Gene Ontology (GO) and InterPro (IPR) terms and were considered to be enriched if P < 0.01 using the Bonferroni correction. Microarray analyses resulted in 83 DE miRNAs in sperm and 10 DE miRNAs in seminal plasma when comparing -2 dpi and 6 dpi. Enrichment analyses revealed that the predicted targets of 35 DE sperm miRNAs and 9 DE seminal plasma miRNAs have functions and/or conserved protein domains that are significantly enriched when compared to the pig genome. Enriched terms of the sperm transcripts included lung epithelium development, cAMP response element binding protein, lactation, lung saccule development, regulation of cell size, positive regulation of cell adhesion, P2X7 purinoceptor, Peptidase S9B, Leucine-rich repeats, Peptidase C48, and phosphorylated immunoreceptor signaling. Transcripts from seminal plasma were enriched for ion channel activity, P2X purinoreceptor, Transglutaminase, endoplasmic reticulum signal peptide binding, and Signal Recognition Particles, among others. This data is the first to report differential miRNA expression in sperm and seminal plasma due to PRRSV infection. Further studies involving qPCR of predicted miRNA targets should be performed for a greater understanding of PRRSV infection at a molecular level.

Keywords:

PRRS, microRNA, semen