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

Abstract

Background: Ovarian 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.

Methods: Follicle 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.

Results: ACVR1 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.

Conclusions: These 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.

Figure 1

Relative level of ACVR1 and ID2 in equine granulosa cells collected from mid-estrous and pre-ovulatory follicles. Values on y-axis indicate 1/normalized (Δ) Ct values relative to geometric mean of ACTB and GAPDH. * = P < 0.05 between follicular stage.

Figure 2

ACVR1 protein levels in granulosa cells isolated from mid-estrous and pre-ovulatory follicles. Values on y-axis indicate normalized values relative to ACTB.

Figure 3

Exosomal miRNAs predicted to regulate ACVR1 and ID2 transcripts. (A) MiR-27b predicted binding sites in the 3’UTR of equine ACVR1 and ID2, and miR-372 and miR-382 predicted binding sites in the 3’UTR of equine ACVR1. (B) Relative levels of miR-27b, miR-372, and miR-382 in exosomes isolated from follicular fluid of mid-estrous and pre-ovulatory follicles that are predicted to target ACVR1 and/or ID2. Values on y-axis indicate 1/normalized (Δ) Ct values relative to geometric mean of ACTB and GAPDH or miR-99b and protein ratio. * = P < 0.05 between follicular stage.

Figure 4

Levels of ACVR1 mRNA and protein and ID2 mRNA in granulosa cells following exosomal treatment. (A) Relative levels of ACVR1 and ID2 in granulosa cells following treatment with exosomes isolated from follicular fluid of mid-estrous and pre-ovulatory follicles. Values on y-axis indicate 1/normalized (Δ) Ct values and relative to geometric mean of ACTB and GAPDH. Different letters indicate P < 0.05 compared to control (no exosome treatment). (B) ACVR1 protein level in pre-ovulatory granulosa cells following treatments with follicular fluid exosomes. Replicates are composed of granulosa cells from pre-ovulatory follicles (n = 4) and exosomes isolated from follicular fluid of pre-ovulatory (n = 4) and mid-estrous follicles (n = 4). Bottom panels indicates the Western blot images with (upper band) ACVR1 and (lower band) ACTB.

Figure 5

MiRNA-27b, miR-372, and miR-382 levels in pre-ovulatory granulosa cells following exosome treatment. Values on y-axis indicate 1/normalized (Δ) Ct values relative to miR-99b. Different letters indicate P < 0.05.

Figure 6

ACVR1 mRNA and protein level in exosomes. (A) Relative levels of ACVR1 in exosomes isolated from mid-estrous and pre-ovulatory follicles. Values on y-axis indicate 1/normalized (Δ) Ct values relative to geometric mean of ACTB and GAPDH. (B) ACVR1 protein level in exosomes isolated from mid-estrous and pre-ovulatory follicles.