60 Follicular fluid extracellular vesicles: endocytosis and influence on domestic cat cumulus cells and oocytes

Extracellular vesicles (EVs, membrane-bound vesicles containing regulatory molecules secreted by all cells) have been shown to hold significant potential to improve in vitro embryo production technologies. EVs from domestic cat ovarian follicular fluid (ffEV) have been shown to be taken up by cumulus cell layers and enhance the oocyte’s ability to recover from vitrification and resume meiosis in vitro. Still, the mechanisms of EV uptake into cumulus-oocyte complexes (COCs) and the relative contributions to oocyte maturation and embryo development are not well understood. Here, we sought to (1) identify the endocytotic pathway(s) responsible for ffEV uptake in cumulus cells, and (2) elucidate the role of ffEVs on domestic cat oocyte developmental potential. First, inhibitors of clathrin-mediated endocytosis (dynasore), calveolin-mediated endocytosis (genistein, dynasore), micropinocytosis (EIPA), lipid raft-mediated endocytosis (filipin), or integrin-binding (RGD peptide) were applied to monolayer-cultured cumulus cells for 30 min at 38°C and 5% CO₂, then DiO-labelled EVs or dye-process flow through controls were co-incubated for 24 h before cells were washed and stained with Hoechst 33342 before fluorescent imaging. Area of ffEV fluorescence per cell was assessed in ImageJ (National Institutes of Health) and assessed as a value relative to ffEV-only controls for each individual animal via linear mixed model in RStudio. A reduction in ffEV uptake was observed in cells exposed to dynasore, genistein, and filipin. Next, grade I and II COCs were co-incubated with ffEV isolated from <1 mm diameter, 1–2 mm diameter and >2 mm (preovulatory) diameter antral cat follicles, compared with unsupplemented controls during IVM (protein-free medium). Following 24 h of IVM, oocytes (n = 49–52 oocytes/treatment, 11 cats) were washed and then fertilised in EV-free conditions (to eliminate influence of EVs on sperm) with 1 × 10⁶ epididymal spermatozoa mL⁻¹ for 18 h. Oocytes were pipetted to remove excess sperm, and cumulus cells were returned to their previous control versus specific-sized antral follicle EV-supplemented treatment groups for 96 h of embryo culture. Oocyte maturation rates were similar among treatment groups (mean ± s.e.m., Control: 36.5 ± 9.4%, <1 mm: 31.4 ± 10.9%, 1–2 mm: 47.1 ± 9.5%, and >2 mm: 40.2 ± 10.9%). A small but not significant (nonparametric Wilcoxon test) improvement in embryo cleavage per mature oocyte (63.6 ± 13.8%) and morula development per cleaved embryo (30.3 ± 13.8%) was observed in the >2 mm ffEV treatments compared with unsupplemented (26.1 ± 10.6 and 6.1 ± 6.1%) and <1 mm ffEV supplemented (25.4 ± 11.4 and 9.1 ± 9.1%) controls. In sum, ffEVs taken up by cumulus cells via clathrin and/or calveolin-mediated endocytosis do not significantly alter nuclear maturation rates of fresh cat oocytes. However, ffEVs may have a follicle stage-specific influence on early embryo development, which would be valuable for the improvement of in vitro embryo production technologies.