150 The influence of follicular fluid extracellular vesicles on in vitro maturation of oocytes in the domestic cat

In recent years, studies on the roles of extracellular vesicles (EVs) in reproductive processes have dramatically increased. Previously, we have shown that oviducal EVs improved sperm function and fertilising ability in the domestic cat model. However, there is still a need to understand the role of EVs during oocyte maturation. Here we aimed to investigate the effects of follicular fluid EVs (ffEVs) in cat oocyte IVM. Cat ovaries (n = 5) void of corpora lutea were obtained from local clinics after ovariohysterectomy. Follicular fluid was aspirated from antral-stage follicles (1–2 mm in diameter) and centrifuged at 2,000 × g at room temperature for 30 min to remove cells and debris. The supernatant was then mixed with 500 μL of Total Exosome Isolation Reagent (Invitrogen) and incubated overnight at 4°C. The samples were centrifuged at 10 000 × g for 1 h, and the pellet was resuspended in 50 μL of PBS. The ffEVs were then aliquoted and stored at −20°C until use. Aliquots were subjected to nanoparticle tracking analysis to determine the size and concentration of ffEVs. Cumulus–oocyte complexes (COCs) were collected by slicing the ovarian cortex from additional 23 cats (6 months to 9 years old). COCs were washed in a minimum essential medium (MEM) supplemented with 2% HEPES, 3 mg mL⁻¹ bovine serum albumin, 2 mM l-glutamine, 100 μg mL⁻¹ streptomycin, and 0.25 mM pyruvate. COCs (n = 265) were separated into grade 1 (>2 layers cumulus cells surrounding an oocyte; n = 118) and grade 2 (compact cumulus with 1 or 2 layers; n = 147) and were placed in 50-μL droplets (10–20 COCs per drop) of IVM medium consisting of 2 mM l-glutamine, 100 μg mL⁻¹ streptomycin, 2 mg mL⁻¹ polyvinyl alcohol, 0.25 mM pyruvate in MEM along with 10 μg mL⁻¹ FSH, 10 μg mL⁻¹ porcine LH, and 10 ng mL⁻¹ epidermal growth factor equilibrated under mineral oil with or without 1.5 × 10⁶ ffEVs mL⁻¹. The COCs were incubated for 24 h at 38°C in 5% CO₂, and then fixed in 4% paraformaldehyde and assessed for nuclear status and analysed using the Kruskal–Wallis test in R studio. The average particle size was 197.2 ± 8.0 nm (range, 181–217 nm) with mean concentration of 5.74 ± 5.08 × 10¹¹ (range, 5.3 × 10¹⁰ to 2.1 × 10¹²). The presence of ffEVs did not enhance the IVM rate of cat COCs. Specifically, there were no differences (P > 0.05) between ffEV-supplemented groups versus no-ffEV controls in the percentages of oocytes resuming meiosis (GVB-MII stages; grade I: −ffEVs = 92.4 ± 6.2% vs. + ffEVs = 91.8 ± 3.3%; grade 2: −ffEVs = 76.2 ± 7.8% vs. +ffEVs = 81.1 ± 4%) or completing nuclear maturation (MII stage, grade I −ffEVs = 44.6 ± 8% vs. +ffEVs = 46.8 ± 10.5%; grade II: −ffEVs = 48.9 ± 9.5% vs. +ffEVs = 41.9 ± 4.6%. In summary, the findings indicated that ffEVs did not have a beneficial effect on in vitro nuclear maturation of cat oocytes. Nevertheless, further study of the roles of ffEVs in cytoplasmic maturation of cat oocyte is warranted (Lopera-Vásquez et al. 2016 PLoS One 11, e0148083).