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Published on: July 30, 2020
Prediction of major microRNAs in follicular fluid regulating porcine oocyte development
Yuki Inoue1, Yasuhisa Munakata1, Akihisa Shinozawa2
1Department of Animal Sciences, Tokyo University of Agriculture, Kanagawa, 243-0034, Japan.
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.
Insights
Key microRNAs (miRNAs) in follicular fluid regulate pig oocyte growth. Supplementing with specific miRNAs like miR-17, -92a, and -145 improved oocyte development and blastocyst rates.
Area of Science:
- Reproductive Biology
- Molecular Biology
- Developmental Biology
Background:
- Oocyte growth and developmental potential are crucial for successful reproduction.
- MicroRNAs (miRNAs) are increasingly recognized as key regulators of cellular processes, including oocyte development.
- Follicular fluid (FF) contains various factors that influence oocyte quality, including miRNAs.
Purpose of the Study:
- To identify specific microRNAs (miRNAs) within porcine follicular fluid (FF) that play a regulatory role in oocyte growth.
- To investigate the functional impact of these identified miRNAs on in vitro oocyte development.
Main Methods:
- Small RNA sequencing was used to profile miRNAs in porcine FF exosomes.
- Ingenuity pathway analysis identified upstream regulatory miRNAs based on gene expression in granulosa cells.
- In vitro oocyte and granulosa cell cultures were performed using FF, exosome-depleted FF, and specific miRNA mimics to assess effects on oocyte growth and development.
Main Results:
- Four key miRNAs (miR-17, -27, -92a, and -145) were identified in FF exosomes.
- Supplementation with FF significantly enhanced oocyte growth, an effect dependent on exosomes.
- Specific miRNA mimics (miR-92a, miR-17b, and miR-145b) promoted antrum formation, oocyte diameter, chromatin configuration, and developmental competence to the blastocyst stage.
Conclusions:
- miR-17, -92a, and -145 are identified as major regulatory microRNAs in porcine follicular fluid influencing oocyte growth.
- Exosomes within FF are essential carriers of these regulatory miRNAs.
- These findings highlight the potential of specific miRNAs as therapeutic targets for improving oocyte quality and reproductive outcomes.
