Yuki Inoue1, Yasuhisa Munakata1, Akihisa Shinozawa2, Ryoka Kawahara-Miki2, Koumei Shirasuna1, Hisataka Iwata3. 1. Department of Animal Sciences, Tokyo University of Agriculture, Kanagawa, 243-0034, Japan. 2. NODAI Genome Research Center, Tokyo University of Agriculture, Tokyo, 156-8502, Japan. 3. Department of Animal Sciences, Tokyo University of Agriculture, Kanagawa, 243-0034, Japan. h1iwata@nodai.ac.jp.
Abstract
PURPOSE: The aim of the present study was to identify key microRNAs (miRNAs) in porcine follicular fluid (FF) that regulate oocyte growth. METHODS: miRNAs contained in FF were determined by small RNA-seq of exosome RNA. Upstream regulator miRNA was determined by ingenuity pathway analysis using differentially expressed genes in granulosa cells (GCs) between small follicles (1-2 mm in diameter) and large follicles (3-5 mm), and between follicles containing oocytes of high developmental ability and follicles containing oocytes of low developmental ability. The candidate miRNAs overlapping among the three miRNAs group were determined. Lastly, the effect of supplementation with FF, exosome-depleted FFs, or each miRNA on in vitro oocyte growth was examined. RESULTS: The miRNAs determined were miR-17, -27, -92a, and -145. These miRNAs were found in the spent culture medium of oocytes and granulosa cells complexes and serum by small RNA sequencing. Culturing of oocytes and granulosa cells complexes collected from porcine early antral follicles (0.5-0.7 mm in diameter) with FF for 14 days improved oocyte growth; depletion of exosomes from the FFs neutralized the beneficial effect observed. miR-92a mimic increased the antrum formation and diameter, together with acetylated levels of H4K12 in oocytes. In addition, supplementation of miRNA mimics miR-17b, -92a, and -145b improved the rate of chromatin configuration, and miR-17b and -92a mimics improved the developmental ability of oocytes to the blastocyst stage. CONCLUSION: miR-17, -92a, and -145 are major miRNA candidates in follicular fluids regulating oocyte growth.
PURPOSE: The aim of the present study was to identify key microRNAs (miRNAs) in porcine follicular fluid (FF) that regulate oocyte growth. METHODS: miRNAs contained in FF were determined by small RNA-seq of exosome RNA. Upstream regulator miRNA was determined by ingenuity pathway analysis using differentially expressed genes in granulosa cells (GCs) between small follicles (1-2 mm in diameter) and large follicles (3-5 mm), and between follicles containing oocytes of high developmental ability and follicles containing oocytes of low developmental ability. The candidate miRNAs overlapping among the three miRNAs group were determined. Lastly, the effect of supplementation with FF, exosome-depleted FFs, or each miRNA on in vitro oocyte growth was examined. RESULTS: The miRNAs determined were miR-17, -27, -92a, and -145. These miRNAs were found in the spent culture medium of oocytes and granulosa cells complexes and serum by small RNA sequencing. Culturing of oocytes and granulosa cells complexes collected from porcine early antral follicles (0.5-0.7 mm in diameter) with FF for 14 days improved oocyte growth; depletion of exosomes from the FFs neutralized the beneficial effect observed. miR-92a mimic increased the antrum formation and diameter, together with acetylated levels of H4K12 in oocytes. In addition, supplementation of miRNA mimics miR-17b, -92a, and -145b improved the rate of chromatin configuration, and miR-17b and -92a mimics improved the developmental ability of oocytes to the blastocyst stage. CONCLUSION:miR-17, -92a, and -145 are major miRNA candidates in follicular fluids regulating oocyte growth.
Authors: Araceli Diez-Fraile; Tim Lammens; Kelly Tilleman; Wojciech Witkowski; Bruno Verhasselt; Petra De Sutter; Yves Benoit; Marc Espeel; Katharina D'Herde Journal: Hum Fertil (Camb) Date: 2014-03-31 Impact factor: 2.767
Authors: Massimo Mallardo; Concetta Ambrosino; Danila Cuomo; Immacolata Porreca; Michele Ceccarelli; David W Threadgill; William T Barrington; Annacristina Petriella; Fulvio D'Angelo; Gilda Cobellis; Francesca De Stefano; Maria N D'Agostino; Mario De Felice Journal: Cell Death Discov Date: 2018-12-05