Regulation of ACVR1 and ID2 by cell-secreted exosomes during follicle maturation in the mare.

Juliano C Da Silveira,Quinton A Winger,Gerrit J Bouma,Elaine M Carnevale

doi:10.1186/1477-7827-12-44

Juliano C Da Silveira, Quinton A Winger + Show 2 more

Open Access

https://doi.org/10.1186/1477-7827-12-44

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Abstract

BackgroundOvarian follicle growth and maturation requires extensive communication between follicular somatic cells and oocytes. Recently, intercellular cell communication was described involving cell-secreted vesicles called exosomes (50–150 nm), which contain miRNAs and protein, and have been identified in ovarian follicular fluid. The goal of this study was to identify a possible role of exosomes in follicle maturation.MethodsFollicle contents were collected from mares at mid-estrous (~35 mm, before induction of follicular maturation) and pre-ovulatory follicles (30–34 h after induction of follicular maturation). A real time PCR screen was conducted to reveal significant differences in the presence of exosomal miRNAs isolated from mid-estrous and pre-ovulatory follicles, and according to bioinformatics analysis these exosomal miRNAs are predicted to target members belonging to the TGFB superfamily, including ACVR1 and ID2. Granulosa cells from pre-ovulatory follicles were cultured and treated with exosomes isolated from follicular fluid. Changes in mRNA and protein were measured by real time PCR and Western blot.ResultsACVR1 mRNA and protein was detected in granulosa cells at mid-estrous and pre-ovulatory stages, and real time PCR analysis revealed significantly lower levels of ID2 (an ACVR1 target gene) in granulosa cells from pre-ovulatory follicles. Exposure to exosomes from follicular fluid of mid-estrous follicles decreased ID2 levels in granulosa cells. Moreover, exosomes isolated from mid-estrous and pre-ovulatory follicles contain ACVR1 and miR-27b, miR-372, and miR-382 (predicted regulators of ACVR1 and ID2) were capable of altering ID2 levels in pre-ovulatory granulosa cells.ConclusionsThese data indicate that exosomes isolated from follicular fluid can regulate members of the TGFB/BMP signaling pathway in granulosa cells, and possibly play a role in regulating follicle maturation.

Highlights

Ovarian follicle growth and maturation requires extensive communication between follicular somatic cells and oocytes
Granulosa cell treatment with exosomes isolated from follicular fluid from mid-estrous and pre-ovulatory follicles To determine if exosomes from different follicular stages can affect relative level of transforming growth factor B (TGFB)/BMP family members in granulosa cells, exosomes isolated from follicular fluid of mid-estrous and pre-ovulatory follicles were added to pre-ovulatory granulosa cells in culture
ACVR1 levels in granulosa cells were lower (P < 0.05) following treatment with exosomes isolated from follicular fluid of mid-estrous and preovulatory follicles compared to control granulosa cells (Figure 4A)

Summary

Introduction

Ovarian follicle growth and maturation requires extensive communication between follicular somatic cells and oocytes. Studies in mice reveal that transforming growth factor B (TGFB) induces miR-224 and miR-383 expression, which target Smad and Rbms thereby regulating estradiol production in response to gonadotropin stimulation [12,13]. These studies clearly indicate that miRNAs are important regulators of ovarian function by controlling various aspects of follicular growth and development, and demonstrate a role for the TGFB signaling pathway in regulating miRNA transcription, as well as miRNAs controlling TGFB family members expression and function during folliculogenesis [12]

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