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Prediction of major microRNAs in follicular fluid regulating porcine oocyte development

  • Reproductive Physiology and Disease
  • Published:
Journal of Assisted Reproduction and Genetics Aims and scope Submit manuscript

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.

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Data availability

All data sets about RNA-seq and small RNA-seq data are registered in DDJB as described in text.

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Acknowledgments

We thank Rumi Ohtake and Naoko Kosuge (Tokyo University of Agriculture) for technical support.

Funding

This study was supported by the Science Research Promotion Fund of the Promotion and Mutual Aid Corporation for Private Schools of Japan.

Author information

Authors and Affiliations

  1. Department of Animal Sciences, Tokyo University of Agriculture, Kanagawa, 243-0034, Japan

    Yuki Inoue, Yasuhisa Munakata, Koumei Shirasuna & Hisataka Iwata

  2. NODAI Genome Research Center, Tokyo University of Agriculture, Tokyo, 156-8502, Japan

    Akihisa Shinozawa & Ryoka Kawahara-Miki

Authors
  1. Yuki Inoue
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  2. Yasuhisa Munakata
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  3. Akihisa Shinozawa
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  4. Ryoka Kawahara-Miki
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  5. Koumei Shirasuna
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  6. Hisataka Iwata
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Contributions

I.Y, and M.Y conducted culture experiment; S.A, M.R, and M.Y conducted RNA-seq and small RNA-seq; H.I designed this experiment; H.I, K.S, M.Y, and I.Y wrote this paper.

Corresponding author

Correspondence to Hisataka Iwata.

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Conflict of interest

The authors declare that they have no conflict of interest.

Ethics approval

Based on the policy of the animal ethics committee of Tokyo University of Agriculture, the use of oocytes collected from porcine ovaries was approved, because all ovary samples were collected from a slaughterhouse where ovaries were discarded due to a lack of edible use.

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All authors have approved the manuscript and agree to its resubmission.

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Inoue, Y., Munakata, Y., Shinozawa, A. et al. Prediction of major microRNAs in follicular fluid regulating porcine oocyte development. J Assist Reprod Genet 37, 2569–2579 (2020). https://doi.org/10.1007/s10815-020-01909-0

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  • DOI: https://doi.org/10.1007/s10815-020-01909-0

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