RESEARCH ARTICLE Molecular Reproduction & Development 81:983–993 (2014) Identification of microRNAs in Exosomes Isolated from Serum and Umbilical Cord Blood, as Well as Placentomes of Gestational Day 90 Pregnant Sheep ELLANE R. CLEYS, 1 JENNIFER L. HALLERAN, 1 ERIN MCWHORTER, 1 JOANNA HERGENREDER, 2 VANESSA A. ENRIQUEZ, 1 JULIANO C. DA SILVEIRA, 1 JASON E. BRUEMMER, 2 QUINTON A. WINGER 1 , AND GERRIT J. BOUMA 1 * 1 Department of Biomedical Sciences, Colorado State University, Fort Collins, Colorado 2 Department of Animal Sciences, Colorado State University, Fort Collins, Colorado SUMMARY Despite reports that circulating levels of maternal serum exosomes increase during pregnancy and that placenta-specific microRNAs (miRNAs) have been identified in humans, little is known about exosomes and miRNAs during pregnancy in agriculture animals. In this study, we characterized the expression of 94 miRNAs in ovine placentomes at gestation day (GD) 90 by real-time PCR, and then investigated the presence of these miRNAs in exosome samples isolated from maternal jugular blood in non-pregnant ewes and at GD30 and GD90 and in umbilical blood collected at GD90. In maternal jugular exosome samples, 13 miRNAs were present in lower and 12 miRNAs were present in higher amounts at GD90 compared to non-pregnant (GD0) or GD30. Additionally, 12 miRNAs were present in higher amounts in umbilical venous exosomes compared to umbilical arterial exosomes; only miR-132 was lower in exosomes isolated from umbilical venous blood than from umbilical arterial blood. In placentome samples, miR-34c and miR135a abundance was higher in cotyledon tissue than in caruncle, while miR-183 and miR-379 amounts were higher in caruncle than cotyledon tissue. Only miR-379 was differentially expressed in all serum exosomes and placentome samples. Pathway analysis predicted that differentially expressed maternal serum exosomal miRNAs target Cellular Growth and Prolifera- tion and Organ Development pathways, while umbilical serum exosomal and pla- centomes miRNAs were predicted to target cellular development and organismal/ embryonic development. Mol. Reprod. Dev. 81: 983993, 2014. ß 2014 Wiley Periodicals, Inc. Received 21 June 2014; Accepted 27 August 2014  Corresponding author: Department of Biomedical Sciences Colorado State University Fort Collins, CO 80523. E-mail: gerrit.bouma@colostate.edu From a thesis submitted to the Academic Faculty of Colorado State University, in partial fulfilment of the requirements for the degree of PhD in Biomedical Sciences. Grant sponsor: Agriculture and Food Research Initiative; Grant number: 2010-38420-20397 Published online 30 September 2014 in Wiley Online Library (wileyonlinelibrary.com). DOI 10.1002/mrd.22420 INTRODUCTION MicroRNAs (miRNAs) are small, non-coding RNA mol- ecules that regulate gene expression by binding comple- mentary sequences in target transcripts to either promote mRNA degradation or to inhibit translation (Cai et al., 2009). Target binding to mRNAs occurs most commonly in the 3 0 untranslated region (UTR), but can also occur in the coding region itself or the 5 0 UTR. miRNAs may regulate 3080% of all genes in the human genome since a single miRNA can regulate hundreds of transcripts, while a single Abbreviations: GD#, gestation day #; HSP70, heat-shock protein 70; miRNA, microRNA. ß 2014 WILEY PERIODICALS, INC.