Extracellular Vesicles from Bovine Follicular Fluid Support Cumulus Expansion

Abstract

Expansion of the cumulus complex surrounding the oocyte is critical for ovulation of a fertilizable egg. The ovulation-inducing surge of luteinizing hormone leads to an increased expression of genes such as prostaglandin-endoperoxide synthase 2 (Ptgs2), pentraxin-related protein 3 (Ptx3), and tumor necrosis factor alpha-induced protein 6 (Tnfaip6) that support cumulus expansion. Factors released by mural granulosa and cumulus granulosa cells into the follicular fluid induce paracrine signaling within the follicular compartment. The follicular fluid that separates these distinct granulosa cell types is an enriched fluid containing numerous proteins, nucleic acids, and other macromolecules. Extracellular vesicles (EVs) are also present; however, no physiologically relevant functions of follicular EVs have yet been demonstrated. In our study, the effect of follicular EVs on cumulus-oocyte complex (COC) expansion and relevant gene expression was assayed. Follicular EVs were isolated using ultracentrifugation from follicular fluid of small (3-5 mm) and large (>9 mm) antral bovine follicles, then characterized by nanoparticle tracking analysis, electron microscopy, and Western blot analysis. To test for bioactivity, mouse and bovine COCs were cultured with follicular EVs. Cumulus expansion and Ptgs2, Ptx3, and Tnfaip6 gene expression were measured following COC maturation culture. The results demonstrated that follicular EVs can support both measurable cumulus expansion and increased gene expression.

Keywords: cumulus; exosome; extracellular vesicles; follicular fluid; granulosa; oocyte.

FIG. 1

Characterization of follicular EVs from bovine follicular fluid. After centrifugation and washing, isolated follicular EVs were examined by negative staining on an electron microscope to determine the presence and characteristics of the follicular EVs. Both small (A) and large (B) antral follicles contained EVs of varying sizes, mostly spherical and some with the concave centers that are characteristic of exosomes (*). C) NTA indicated that EVs collected from small and large follicles are similar in size. NTA was repeated on three isolates of follicular EVs collected on different days, which are overlaid in this figure to graphically demonstrate the variation between these follicular EV isolates. The average diameters (nm) of particles are shown for each of the three independent preparations of small and large antral follicle EVs. D) Western blots of three collections of EVs from small and large antral follicles were probed with the CD81 antibody.

FIG. 2

Cumulus cells uptake follicular fluid EVs during COC culture. Mouse COCs were cultured 16 h with PKH67-labeled bovine follicular EVs. Individual COCs were imaged on a Zeiss Pascal confocal microscope (×40) in sections 1 μm thick and examined for internalization of follicular EVs. In the expanded COC, internalized follicular EVs are evident as green spots within cumulus cells in both the outer layers of cumulus cells (A) and inner layers adjacent (C) to the oocyte (o). As negative control to insure that the COC labeling was a result of EV uptake and not residual free-floating dye in the media, the final supernatant (wash) from the follicular EV PKH67 labeling process was added to COC cultures, and these cumulus cells were all negative for PKH67 (B). Distance between the sections in A and C is 30 μm within the same COC. A′ and C′ are the same as A and C but with DIC turned off. A″ and C″ are enlargements from the boxes identified in A′ and C′. Bar = 10 μm.

FIG. 3

Bovine follicular EVs induce mouse cumulus cell expansion. Mouse COCs were matured with follicular EVs from either small or large antral bovine follicles. COCs were imaged by DIC at the beginning (0 h) and end (16 h) of culture. Negative control COCs cultured with no FBS and with no follicular EVs did not expand (A). COCs cultured with either FBS (B; positive controls), or with follicular EVs from small (C) or large (D) antral follicles induced cumulus expansion. Cumulus expansion was quantified and the percentage change in diameter of the COCs after 16 h of in vitro maturation is shown. Bar = 75 μm. E) COCs cultured with no serum or EV exhibited very little expansion. In the absence of serum, cumulus expansion induced by follicular EVs from the large antral follicles was significantly less than that induced by small antral follicle EVs (A). F) COCs matured with follicular EVs added to media containing FBS. COCs cultured in FBS exhibited some expansion and follicular EVs increased cumulus expansion over that of whole FBS (i.e., FBS containing endogenous EVs). G) COCs matured in whole FBS, EV-free FBS (EV-FBS), with and without follicular EVs from small or large follicles. Removing serum endogenous EVs from the FBS had no effect on cumulus expansion, but addition of follicular EVs still significantly increased COC expansion (C). ^a,b,cMeans ± SEM with different superscripts (a, b, or c) are significantly different (P < 0.05).

FIG. 4

Bovine follicular EVs induce cumulus expansion of bovine COCs. Bovine COCs were matured with follicular EVs from either small or large antral bovine follicles. COCs were imaged by DIC at the beginning of culture (0 h) and following in vitro maturation (24 h). Negative control COCs cultured with no FBS and with no follicular EVs did not expand (A). COCs cultured with either small-follicle EVs (B) or large-follicle EVs (C) exhibited increased cumulus expansion. Bar = 50 μm.

FIG. 5

Follicular fluid EVs induce genes involved in cumulus expansion. COCs from mice (A) and cows (B) were matured for 16 and 24 h, respectively, followed by quantitative RT-PCR analysis of Ptgs2, Ptx3, and Tnfaip6 levels. Small-follicle EVs increased cumulus gene expression in both mouse (A) and bovine COCs (B), whereas large-follicle EVs did not. Mouse COC Tnfaip6 expression trended (P = 0.08) to increase. ^a,bMeans ± SEM with different superscripts are significantly different (P < 0.05).