Changes in Fetal Muscle miRNA Expression from Exposure to Ergot Alkaloids in Utero.

MicroRNA are small non-coding RNA that help regulate gene expression and metabolic function. Some microRNA, called MyomiRs, are expressed predominately in skeletal muscle and regulate myogenesis. The objective of this study was to identify microRNA in skeletal muscle of fetuses exposed to ergot alkaloids in utero. Thirty-six pregnant Suffolk ewes (78.24 kg ± 9.5) were randomly assigned to dietary treatments of: endophyte-free tall fescue seed (E-; 0.0 µg ergovaline + ergovalinine/hd/d) or endophyte-infected tall fescue seed (E+; 1722 µg ergovaline + ergovalinine/hd/d) at specific stages of gestation (d35–85 or d86–133) in a 2 x 2 factorial arrangement of treatments. Fetal and maternal necropsies were performed at d133 of gestation. Semitendinosus (ST) muscle was removed from each fetus and immediately frozen in liquid nitrogen for storage at -80°C. Total cellular RNA was extracted using the mirVana miRNA Isolation Kit (Ambion, Austin, TX). Quality analysis of RNA was performed using an Agilent 2100 Bioanalyzer, with a RIN threshold of 7.0. The tcRNA from 3 fetuses per treatment from the ST was used for miRNA sequencing and data analysis (LC Sciences, Houston, TX). MicroRNA sequencing yielded 113,252,743 reads with 92,177,228 mappable to the ovine reference genome. Of the mappable reads, 27% were specific to the Ovis aries genome and 18% were specific to mammals. There were 4,242 unique miRNA identified by sequencing, which included 208 that were specific to the Ovis aries genome, and 676 that were mammalian but novel to Ovis aries. Known MyomiR (miR-1, miR133a, miR133b, miR206, miR-208b, miR-486, miR-499) in skeletal muscle were present in our samples but not (P > 0.05) differentially expressed due to treatment. miR-148b, miR-300-3p, miR-431-3p, miR-299-3p, and miR-541-5p were up-regulated (P < 0.05) in E+/E+ compared to E-/E- fetal ST muscles. miR-652, miR-628, miR-2427, miR-22-3p, miR-8118-p5, miR-376d, and miR-677 were down-regulated (P < 0.05) in E+/E+ versus E-/E-. Skeletal muscle miR-148b has been shown to reduce glucose uptake in response to insulin in humans. miR-541 promotes vascular smooth muscle cell proliferation. Exposure to ergot alkaloids in utero alters miRNA expression in fetal skeletal muscle.