254 REGULATORY MICRORNA IN THE BIDIRECTIONAL COMMUNICATION OF BOVINE OOCYTES AND THE SURROUNDING CUMULUS CELLS

Abstract

MicroRNA (miRNA) are small molecules (˜22 nucleotide in length) that influence the expression of hundreds of genes for numerous biological processes including development. In this study we aimed to investigate the presence and role of miRNA in the bidirectional communication of oocyte and cumulus cells. For this, triplicate pools each containing 1600 immature and mature oocytes and their corresponding cumulus cells were used for miRNA isolation using miRNeasy^® Mini Kit (Qiagen, Hilden, Germany). From each oocyte and cumulus cell group, 50 ng of small RNA was used for reverse transcription using RT2 miRNA First Strand Kit (SABiosciences, Frederick, MD, USA). The resulting small RNA cDNA was used as a template to profile 88 human miRNA related to cell development and differentiation using SYBR green-based real-time PCR system. Data analysis was preformed using the comparative Ct method after normalization using endogenous control RNA (SNORD44, SNORD47, SNORD48, and U6). In addition, miR-205 and miR-210 were used for localization in pre-implantation stages of embryo using 3′digoxigenin labeled, LNA- modified in situ oligonucleotide probes (Exiqon, Vedbaek, Denmark). The result of the PCR array revealed a total of 34 and 49 miRNA to be greatly abundant in immature and mature oocyte, respectively, compared with the corresponding cumulus cells, whereas only 5 and 4 miRNA were enriched in cumulus cells compared with immature and matured oocytes, respectively. Based on expression intensity, 6 oocyte enriched (miR-205, miR-150, miR-96, miR-122, miR-146a, and miR-146b-5p) and 2 cumulus-cell enriched (miR-452 and miR-210) were selected for expression analysis in pre-implantation-stage embryos and in oocyte and cumulus cells matured with or without cumulus and oocyte factors, respectively. All oocyte-specific miRNA were found to be greatly abundant in early stages of embryo development and drop after 4-cells until the blastocyst stage, following a typical maternal transcript profile. Similar results were obtained by localization of miR-205 in pre-implantation-stage embryos, in which signals were greater until the 4-cell stage and reduced thereafter. However, miR-210 and miR-452 showed no defined profile. miR-205, miR-150, miR-122, miR-146a, miR-146b-5p, and miR-452 were found to be abundant at a greater level (P < 0.05) in oocytes matured without cumulus cells compared with those matured in the presence of cumulus cells. The expression of miR-205, miR-150, and miR-122 in cumulus cells was greater in the presence of oocyte cytoplasm during maturation, whereas 16-fold increases in relative abundance of miR-210 were observed in oocyte- optimized cumulus cells. These results evidenced that oocyte and cumulus cells have a distinct set of miRNA, which is dependent on the bidirectional communication of the oocyte and the surrounding cumulus cells. Moreover, maternal miRNA were found to persist until the major genome activation in bovine.