Follicular fluid exosomes regulate OVGP1 secretion in yak oviduct epithelial cells via autophagy in vitro.

After mammalian ovulation, oocytes enter the oviduct, causing oocyte and oviduct changes. Some studies have shown that follicular fluid exosomes (FEVs) play an important role in this regulatory process, but the specific mechanism is remains unclear. Here, we investigate the effect of FEVs on autophagy and on the synthesis and secretion of oviductal glycoprotein 1 (OVGP1) in yak oviduct epithelial cells (OECs). We added FEVs to yak OECs and collected samples at intervals. The effect of autophagy on OVGP1 synthesis and secretion was detected by manipulating the level of autophagy in OECs. The results showed that autophagy gradually increased as early as 6 h after exosome intake level increased, and the increase was most obvious 24 h after. At that time, the synthesis and secretion of OVGP1 also reached its highest levels. When the autophagy level of OECs is changed through the PI3K/AKT/mTOR pathway, OVGP1 synthesis and secretion levels also change, along with the OVGP1 levels in oviduct exosomes also change. More importantly, the addition of FEVs treatment while using 3-MA to inhibit the autophagy level in yak OECs did not change the synthesis and secretion level of OVGP1. Our results indicate that FEVs can affect the synthesis and secretion of OVGP1 by regulating the level of autophagy in OECs, and that the completion of this process may depend on the PI3K/AKT/mTOR pathway, indicating that exosomes and autophagy play important roles in the reproductive physiology of yak OECs. Our results provide new ideas in to characterizing the role of exosomes in yak reproduction.

Follicular fluid exosomes inhibit expression of BTG2 and promote glucose uptake in granulosa cells by delivering miR-21-5p.

Glucose metabolism in granulosa cells (GCs) is essential for follicle development and oocyte maturation. Porcine follicular fluid exosomes promote the proliferation of porcine GCs and the synthesis of steroid hormones. However, their role in regulating glucose uptake in GCs is unclear. The objective of this study was to elucidate the effects of porcine follicular fluid exosomes on glucose uptake in porcine GCs and the intrinsic mechanisms involved. First, transcriptome sequencing revealed that glucose metabolism-related pathways were altered in GCs treated with follicular fluid exosomes. Next, in vitro culture experiments showed that glucose uptake was increased and the IRS1/AKT signaling pathway was activated in GCs after treatment with follicular fluid exosomes. Finally, miRNA sequencing of follicular fluid exosomes revealed that miR-21-5p was the most abundant miRNA. Subsequent investigations indicated that miR-21-5p promoted glucose uptake in GCs by targeting BTG2, which activated the IRS1/AKT signaling pathway. In conclusion, the findings of this study indicate that porcine follicular fluid exosomes promote glucose uptake in porcine GCs by delivering miR-21-5p, which inhibits the expression of BTG2, activating the IRS1/AKT signaling pathway.

Follicular fluid exosome-derived miR-339-5p enhances in vitro maturation of porcine oocytes via targeting SFPQ, a regulator of the ERK1/2 pathway.

In this study, we aimed to investigate cytoplasmic maturation and miRNA expression of mature oocytes cultured in porcine follicular fluid exosomes. We also examined the effect of miR-339-5p on oocyte maturation. Twenty eight differentially expressed miRNAs were detected using miRNA-seq. We then transfected cumulus oocyte complexes with miR-339-5p mimics and inhibitor during culture. The results showed that exosomes increased endoplasmic reticulum levels and the amount of lipid droplets, and decreased ROS levels, lipid droplet size, and percentage of oocytes with abnormal cortical granule distribution. Overexpressing miR-339-5p significantly decreased cumulus expansion genes, oocyte maturation-related genes, target gene proline/glutamine-rich splicing factor (SFPQ), ERK1/2 phosphorylation levels, oocyte maturation rate, blastocyst rate, and lipid droplet number, but increased lipid droplet size and the ratio of oocytes with abnormal cortical granule distribution. Inhibiting miR-339-5p reversed the decrease observed during overexpression. Mitochondrial membrane potential and ROS levels did not differ significantly between groups. In summary, exosomes promote oocyte cytoplasmic maturation and miR-339-5p regulating ERK1/2 activity through SFPQ expression, thereby elevating oocyte maturation and blastocyst formation rate in vitro.

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

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.