83 MicroRNA574-3p Influences Porcine Oocyte Maturation and Regulates Abundance of Proteins Critical to Early Embryo Development

Abstract

Abstract MicroRNA are small non-coding RNA involved in post-transcriptional gene regulation impacting oocyte maturation and embryo development. MIR574-3p abundance decreases during oocyte in vitro maturation (IVM) and blastocyst development. The study objective was to evaluate the role of MIR574-3p during porcine oocyte maturation and early embryo development. To assess the function of MIR574-3p during these processes, denuded GV stage oocytes injected with MIR574-3p mimic (MIR574-3p-M), MIR574-3p inhibitor (MIR574-3p-I) or negative control oligo (NC) underwent IVM for 42 hours. The number of MII arrested oocytes was decreased (P = 0.06) in the MIR574-3p-M group (67.7 ± 1.4%) as compared to the NC group (76.1 ± 1.3%), whereas maturation was not affected by MIR574-3p-I (75.6 ± 1.5%) as compared to the NC group (73.1 ± 3.6%). MII arrested oocytes were parthenogenetically activated and cultured for 7 days. Neither mimic nor inhibitor affected the cleavage or blastocyst rate. Using LC-MS/MS we evaluated changes in global protein abundance in injected oocytes after 42 hours of IVM. We identified 971 proteins in MIR574-3p-M injected oocytes, of which 57 were differentially abundant as compared to the control group. In MIR574-3p-I injected oocytes, 1007 proteins were identified, of which 107 were differentially abundant as compared to the control group. Overall, MIR574-3p-M upregulated proteins critical to membrane binding mediating sperm receptors on the zona pellucida, while it downregulated intranuclear activity such as nucleotide biosynthesis, mitotic spindle assembly and orientation; whereas MIR574-3p-I induced upregulation of proteins involved in the processes between and including protein biosynthesis and metabolism, while downregulating proteins critical to ATP, RNA, DNA and protein binding. These data suggest artificially increasing MIR574-3p abundance during IVM alters the oocyte proteome and influences meiotic progression to MII. Project was supported by Agriculture and Food Research Initiative Competitive Grant no. 2017-67015-26459 from the USDA National Institute of Food and Agriculture.