Prediction of major microRNAs in follicular fluid regulating porcine oocyte development
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REPRODUCTIVE PHYSIOLOGY AND DISEASE
Prediction of major microRNAs in follicular fluid regulating porcine oocyte development Yuki Inoue 1 & Yasuhisa Munakata 1 & Akihisa Shinozawa 2 & Ryoka Kawahara-Miki 2 & Koumei Shirasuna 1 & Hisataka Iwata 1 Received: 19 May 2020 / Accepted: 30 July 2020 # Springer Science+Business Media, LLC, part of Springer Nature 2020
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. Keywords In vitro grown oocyte . Follicular fluid . Exosome . miRNA . NGS . miR-92a
Introduction Oocyte growth is mediated via well-orchestrated communication between granulosa cells (GCs) and oocytes. The developmental competence of oocytes, including the ability to complete meiotic maturation and to develop to the blastocyst stages, increases during follicle development from small to Electronic supplementary material The online version of this article (https://doi.org/10.1007/s10815-020-01909-0) contains supplementary material, which is available to authorized users. * Hisataka Iwata [email protected] 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
large antral follicles. In addition, along with follicle development, GCs demonstrate major differential gene expression changes between the small and large antral follicles [1]. Follicular fluid (FF) is the exclusive environment that surrounds oocytes and GC complexes
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