Novel and differentially abundant microRNAs in sperm cells, seminal plasma, and serum of boars due to porcine reproduction and respiratory syndrome virus infection.

The objectives of this study were to identify and determine relative abundance of miRNAs in boar sperm, seminal plasma (SP), and serum pre- and post-viral infection. Functional enrichment analyses on predicted targets of miRNAs of interest were performed. Boars (n = 6) were inoculated with porcine reproductive and respiratory syndrome virus (PRRSv) strain 1-8-4 (Day 0). Semen and serum were collected on Day -2 and 6. Sperm and SP were separated and aliquots were flash frozen and stored at -80 °C. Serum was frozen and stored at -80 °C. Total RNA was isolated from sperm and SP samples and subjected to RNA sequencing. Microarray analysis was performed using the Day -2 and 6 RNA samples from serum, sperm and SP. Potential miRNA targets were predicted using miRanda 3.3a and targets were then analyzed for enrichment of Gene Ontology) and InterPro terms and were considered to be enriched if P < 0.01 using the Bonferroni correction. Microarray analyses resulted in 83, 13, and 10 miRNAs with differences in abundances in sperm, serum, and SP, respectively, when comparing Day -2 and 6. Results from enrichment analyses indicated that the predicted targets of 35, nine, and five miRNAs with differences in abundances for sperm, SP, and serum, respectively, that have functions and/or conserved protein domains that are enriched when compared to the pig genome. Enriched terms for P2X purinoceptors were identified for sperm, SP and serum. Enriched terms for cell adhesion were identified for sperm and serum transcripts. Enriched terms for cell signaling were identified for sperm and SP transcripts.

The bull sperm microRNAome and the effect of fescue toxicosis on sperm microRNA expression.

Tall fescue [Schedonorus phoenix (Scop.) Holub] accounts for nearly 16 million hectares of pasture in the Southeastern and Mid-Atlantic U.S. due to its heat, drought, and pest resistance, conferred to the plant by its symbiotic relationship with the endophyte Neotyphodium coenophialum. The endophyte produces ergot alkaloids that have negative effects on the growth and reproduction of animals, resulting in the syndrome known as fescue toxicosis. The objectives of our study were to identify microRNA (miRNA) present in bovine sperm and to evaluate the effects of fescue toxicosis on sperm miRNA expression. Angus bulls were assigned to treatments of either toxic or non-toxic fescue seed diets. Semen was collected and subjected to RNA isolation. Three samples from each treatment group were chosen and pooled for deep sequencing. To compare miRNA expression between treatment groups, a microarray was designed and conducted. For each of the top ten expressed miRNA, target prediction analysis was conducted using TargetScan. Gene ontology enrichment was assessed using the Database for Annotation, Visualization and Integrated Discovery. Sequencing results elucidated the presence of 1,582 unique small RNA present in sperm. Of those sequences, 382 were known Bos taurus miRNA, 22 were known but novel to Bos taurus, and 816 were predicted candidate miRNA that did not map to any currently reported miRNA. Of the sequences chosen for microarray, twenty-two showed significant differential expression between treatment groups. Gene pathways of interest included: regulation of transcription, embryonic development (including blastocyst formation), Wnt and Hedgehog signaling, oocyte meiosis, and kinase and phosphatase activity. MicroRNA present in mature sperm appears to not only be left over from spermatogenic processes, but may actually serve important regulatory roles in fertilization and early developmental processes. Further, our results indicate the possibility that environmental changes may impact the expression of specific miRNA.

Cloning and expression of porcine Dicer and the impact of developmental stage and culture conditions on MicroRNA expression in porcine embryos.

MicroRNA (miRNA) is a class of small, single-stranded ribonucleic acids that regulate gene expression post-transcriptionally and are involved in somatic cell, germ cell, and embryonic development. As the enzyme responsible for producing mature miRNA, Dicer is crucial to miRNA production. Characterization of Dicer and its expression at the nucleotide level, as well as the identification of miRNA expression in reproductive tissues, have yet to be reported for the domestic pig (Sus scrofa), a species important for disease modeling, biomedical research, and food production. In this study we determined the primary cDNA sequence of porcine Dicer (pDicer), confirmed its expression in porcine oocytes and early stage embryos, and evaluated the expression of specific miRNA during early embryonic development and between in vivo (IVO) and in vitro (IVF) produced embryos. Total cellular RNA (tcRNA) was isolated and subjected to end point RT-PCR, subcloning, and sequencing. The pDicer coding sequence was found to be highly conserved, and phylogenetic analysis showed that pDicer is more highly conserved to human Dicer (hDicer) than the mouse homolog. Expression of pDicer mRNA was detected in oocytes and in IVO produced blastocyst embryos. Two RT-PCR procedures were conducted to identify and quantitate miRNA expressed in metaphase II oocytes (MII) and embryos. RT-PCR array was conducted using primers designed for human miRNA, and 86 putative porcine miRNA in MII and early embryos were detected. Fewer miRNAs were detected in 8-cell (8C) embryos compared to MII and blastocysts (B) (P=0.026 and P<0.0001, respectively). Twenty-one miRNA (of 88 examined) were differentially expressed between MII and 8C, 8C and B, or MII and B. Transcripts targeted by the differentially expressed miRNA were enriched in gene ontology (GO) categories associated with cellular development and differentiation. Further, we evaluated the effects of IVF culture on the expression of specific miRNA at the blastocyst stage. Quantitative RT-PCR was conducted on blastocyst tcRNA isolated from individual IVO and IVF produced embryos for miR-18a, -21, and -24. Only the expression level of miR-24 differed due to culture conditions, with lower levels detected in the IVO embryos. These data show that pDicer and miRNA are present in porcine oocytes and embryos. In addition, specific miRNA levels are altered due to stage of embryonic development and, in the case of miR-24, due to culture conditions, making this miRNA a candidate for screening of embryo quality. Additional studies characterizing Dicer and miRNA expression during early embryonic development from IVO and IVF sources are required to further examine and evaluate the use of miRNA as a marker for embryo quality.