Exosomes Derived from Yak Follicular Fluid Increase 2-Hydroxyestradiol Secretion by Activating Autophagy in Cumulus Cells

Abstract

Exosomes in the follicular fluid can carry and transfer regulatory molecules to recipient cells, thus influencing their biological functions. However, the specific effects of yak follicular fluid exosomes on 2-hydroxyestrodiol (2-OHE₂) secretion remain unknown. Here, we investigated whether yak follicular fluid exosomes can increase 2-OHE₂ secretion through the activation of autophagy in cumulus cells (YCCs). In vitro cultured YCCs were treated with yak follicular fluid exosomes for 6, 12, and 24 h. The effects of yak follicular fluid exosomes on autophagy and 2-OHE₂ secretion were evaluated through real-time quantitative fluorescence PCR (RT-qPCR), Western blotting (WB), transfected with RFP-GFP-LC3, immunohistochemistry, and ELISA. To further investigate whether 2-OHE₂ secretion was related to autophagy, YCCs were administered with yak follicular fluid exosomes, 3-methyladenine (3-MA), and rapamycin (RAPA). The results revealed that treatment with yak follicular fluid exosomes activated autophagy in YCCs and increased 2-OHE₂ secretion. Conversely, the inhibition of autophagy with 3-MA blocked these effects, suggesting that autophagy has an important role in 2-OHE₂ secretion in YCCs. Treatment of YCCs with rapamycin showed similar results with yak follicular fluid exosomes as there was an increase in 2-OHE2 secretion due to the activation of autophagy in the treated cumulus cells. Our results demonstrate that autophagy is enhanced by yak follicular fluid exosomes, and this is associated with an increase in 2-OHE₂ secretion in YCCs.

Keywords: 2-hydroxyestrondiol; autophagy; cumulus cells; yak follicular fluid exosomes.

Figure 1

Identification of yak follicular fluid exosomes. (A) Phase morphology of isolated YCCs. Scale bar represents 200 μm. (B) Size and shape of yak follicular fluid exosomes (white arrows) determined using transmission electron microscopy (TEM). Scale bar represents (a) 200 and (b) 100 nm. (C) Size distribution and concentration of yak follicular fluid exosomes using nanoparticle tracking analysis (NTA). (D) Western blot analysis (See Figure S1).

Figure 2

Uptake of yak follicular fluid exosomes by YCCs. Fluorescence microscopy confirmed the location of the yak follicular fluid exosomes that were tagged with PKH-26 (red fluorescence) in YCCs. Scale bar represents 50 μm.

Figure 3

Yak follicular fluid can activate autophagy in YCCs. (A) Expression of autophagy-related genes LC3 (A1), ATG5 (A2), Beclin1 (A3), ATG12 (A4) and P62 (A5) in YCCs using a qPCR assay. (B) Western blot analysis of protein expression in YCCs (C: control, E: Yak follicular fluid exosomes) (See Figure S2). (C) Quantitative results of Western blot analysis for LC3II/LC3I (C1), P62 (C2), ATG5-ATG12(C3) and Beclin1 (C4). Data are expressed as the mean ± SD. n = 6. NS means no significant difference. * p < 0.05 and ** p < 0.01 indicate significant differences between the yak follicular fluid exosome treatments and the control.

Figure 4

Enhanced autophagy flux of YFFEVs in YCCs. (A) Confocal imaging of YCCs with YFFEVs treatments after RFP-GFP-LC3 transfection. Scale bar represents 10 μm. (B) Quantitative analysis of GFP and RFP dots. Green dots represent autophagosomes, and red dots represent autophagosomes and autolysosomes. (C) The quantitative analysis of merged images with both yellow dots (autophagosomes) and red dots (autolysosomes). Data are expressed as the mean ± SD. n = 6. NS means no significant difference. ** p < 0.01 indicates a significant difference between the yak follicular fluid exosome treatments and the control.

Figure 5

Yak follicular fluid exosomes could increase 2-OHE₂ secretion in YCCs. (A) Immunofluorescence staining of CYP19A1 and CYP1A1 proteins in YCCs. Scale bar represents 50 μm. (B) Expression of 2-OHE₂ secretion-related genes CYP17A1 (B1), CYP19A1 (B2), CYP1A1 (B3) and CYP1B1 (B4) in YCCs using the qPCR assay. (C) Western blot analysis of the protein expression in YCCs (C: Control, E: Yak follicular fluid exosomes) (See Figure S2). (D) ELISA kit analyses of the concentrations of estradiol (pg/mL) in the cell supernatant. (E) Quantitative Western blot results for CYP17A1 (E1), CYP19A1 (E2), CYP1A1 (E3) and CYP1B1 (E4). Data are expressed as the mean ± SD. n = 6. NS means no significant difference. * p < 0.05 and ** p < 0.01 indicate significant differences between the yak follicular fluid exosome treatments and the control.

Figure 6

3-MA inhibits autophagy and reduces 2-OHE₂ secretion in YCCs. (A,B) Western blot analysis of protein expression in YCCs. (C) Western blot analysis of the protein expression in YCCs (See Figure S3). (D) ELISA kit analysis of the concentrations of estradiol (pg/mL) in the cell supernatant. (E) Quantitative Western blot results for CYP17A1 (E1), CYP19A1 (E2), CYP1A1 (E3) and CYP1B1 (E4). Data are expressed as the mean ± SD. n = 4. NS means no significant difference. * p < 0.05 and ** p < 0.01 indicate significant differences between the yak follicular fluid exosome treatments and the control.

Figure 7

RAPA increases 2-OHE₂ secretion by upregulating autophagy in YCCs. (A,B) Western blot analysis of the protein expression in YCCs. (C,D) Quantitative results of the Western blot (See Figure S4). (E) ELISA kit analysis of the concentrations of estradiol (pg/mL) in the cell supernatant. Data are expressed as the mean ± SD. n = 3. * p < 0.05 and ** p < 0.01 indicate significant differences between the yak follicular fluid exosome treatments and the control.