Relative abundance of extra-cellular miRNAs in bovine follicular fluid: Implication for cell–cell communication during oocyte growth

Abstract

s from the 4th Mammalian Embryo Genomics meeting Relative abundance of extra-cellular miRNAs in bovine follicular fluid: Implication for cell–cell communication during oocyte growth M.M.H. Sohel, S. Seifi Noferesti, D. Salilew-Wondim, M. Hoelker, F. Rings, E. Tholen, J. Udin, C. Looft, K. Schellander, D. Tesfaye ∗ Animal Breeding and Husbandry Group, Institute of Animal Science, University of Bonn, Germany Here we aimed to investigate the expression pattern of the circulating extra-cellular miRNAs in exosome and non-exosomal fraction of follicular fluid consisted of fully grown or growing oocytes and to validate exosome mediated cell–cell communication between follicular cells. For this, follicles of 5–8mm diameter (n=120) were isolated and individual COCs were subjected to brilliant cresyl blue (BCB) staining and classified as BCB+ (fully grown, n=60) and BCB− (growing, n=60) groups. The corresponding follicular fluid, granulosa cells and theca cells were used for further molecular analysis. miRNAs isolated from exosomal and nonexosomal portion of follicular fluid from the two categories was used for cDNA synthesis and subsequent analysis using a humanmiRNA PCR array (with 745miRNA). Results revealed that 25miRNAs (16 up and 9 down regulated) in exosomes and 30miRNAs (21 up and 9 down regulated) in non-exosomal portion of follicular fluid were differentially expressed (fold change≥2 and p<0.05) between growing vs. fully grown oocyte group. Among these miRNAs, miR-654-5p and miR-640 were found to be enriched in exosomal portion of follicular fluid containing growing oocytes, while miR-526b* and miR-373 were highly abundant in exosomal portion of follicular fluid This paper is part of a special issue entitled: 4th Mammalian Embryo Genomicsmeeting, Guest Edited byMarc-Andre Sirard, ClaudeRobert and Julie Nieminen. containing fully grown oocytes. In-silico analysis of miRNAs enriched in follicular fluid containing growing oocytes revealed that genes are involved indifferent signalingpathways like ubiquitin mediated proteolysis, focal adhesion, oocyte meiosis, MAPK signaling pathways to be potential targets, which are crucial for follicular development. Co-culture of granulosa cells with PKH67 dye labeled exosomes showedsuccessfuluptakeof exosomesby thosecells and subsequently elevated the endogenous miRNAs level in 2.5–5.5 fold. In conclusion, the present study highlighted the oocyte growth status dependent differences of circulatingmiRNAprofiles in follicularfluidandexosomemediated uptake of thosemolecules by surrounding cells in follicular microenvironment. http://dx.doi.org/10.1016/j.anireprosci.2014.06.011 Global transcriptome analysis of bovine blastocysts developed under alternative vivo/vitro culture conditions during specifics stages of development A. Gad1,4,∗, U. Besenfelder2, V. Havlicek2, M. Holker1, F. Rings1, I. Dufort3, M.A. Sirard3, K. Schellander1, D. Tesfaye1 1 Institute of Animal Science, University of Bonn, Bonn, Germany 2 Institute of Animal Breeding and Genetics, University of Veterinary Medicine, Vienna, Austria 3 Centre de recherche en biologie de la reproduction, Universite Laval, Quebec, Canada 4 Department of Animal Production, Faculty of Agriculture, Cairo University, Giza, Egypt During the period frommaturation until blastocyst formation, several critical events occur in embryos which are regulated by a harmonized expression of genes. However, the exact influence of in vitro culture conditions 0378-4320/$ – see front matter