Altered Expression of Placental miRNAs in Ewes with Uteroplacental Insufficiency Due to Consumption of Endophyte-Infected Tall Fescue Seed.

Differential miRNA expression levels have been reported for placental tissues in cases of uteroplacental insufficiency and fetal growth restriction. Ergot alkaloids found in endophyte-infected tall fescue [Lolium arundinaceum (Schreb.) Darbysh] seed induces intrauterine growth restriction (IUGR) due to uteroplacental insufficiency. The objective of this study was to assess the impact on placental miRNA levels due to consumption of ergot alkaloids during mid to late gestation. Thirty-six Suffolk ewes (78.24 kg ± 9.5) estimated to be carrying twins at d 35 of gestation were randomly assigned to one of two treatments: endophyte-free tall fescue seed (E-; 0.0 µg ergovaline + ergovalinine/g) or endophyte-infected tall fescue seed (E+; 4.14 µg ergovaline + ergovalinine/g) from d 35 – d 85 and d 86 – d 133 of gestation creating four unique dietary treatments: E-E-, E-E+, E+E-, and E+E+. Endophyte-infected tall fescue seed was fed at a level to provide 1772 µg of ergovaline + ergovalinine/hd/d for E+ treatments while an equal weight of endophyte-free seed (0.0 µg ergovaline + ergovalinine) was fed for E- treatments. Ewes were pair-fed across treatments in order to maintain equal DMI. Fetal and maternal necropsies were performed at d 133 of gestation. Placental samples were weighed, frozen in liquid nitrogen, and stored at -80°C. Total RNA was extracted from cotyledon placental tissue using TRIzol® and a PureLink® RNA Mini Kit (ThermoFisher Scientific). Total RNA was quantified and quality was assessed using an Agilent 2100 Bioanalyzer. RIN numbers of > 7 were considered sufficient quality. A subsample (n = 4) was selected for both the E+E+ and E-E- treatment groups and total cotyledon RNA was submitted for gene expression profiling via Illumina RNA-Seq (LC Sciences). Numerous miRNA transcripts were upregulated (P < 0.01; n = 15) in E+E+ compared to E-E- and included miR-21, miR-221, miR-99a, miR-152, miR-381, miR-30a, miR-143, and miR-122. miR-21 exhibited a 9.89-fold increase in expression level for E+E+ treatment compared to E-E-. miR-21 has previously been implicated as a potential biomarker for fetal hypoxia due to upregulation in both placental and plasma/serum samples. Similar increases in miR-21 and miR-122 levels have been correlated to other pathological diseases of the placenta including placental insufficiency, IUGR, and preeclampsia.