135 Effect of different concentrations of follicular fluid exosome-like extracellular vesicles on in vitro oocyte maturation and embryo development in cattle

Follicular fluid (FF) extracellular vesicles (ffEVs) affect different functions of follicular cells and can improve quality of enclosed oocytes due to ffEV cargo of different proteins, RNAs and lipids; thus, ffEVs can be used to optimize assisted reproductive technologies in cattle. The aim of the study was to determine the optimal concentration of ffEV preparations in IVM medium that is beneficial to blastocyst development and quality. Differential centrifugation of FF from 3- to 8-mm follicles was used for extraction of ffEVs. Transmission electron microscopy demonstrated that the mean diameter of ffEV was 48 ± 17 nm, indicating that ffEV preparations correspond mainly to exosomes, which were precipitated with protein globules. Western blot analysis of ffEVs revealed the presence of the exosome-specific markers CD81 and CD63. Penetration of ffEVs to cumulus cells and oocytes during IVM was demonstrated using PHK67 labelling and confocal microscopy. IVM medium (TCM199 with 100 ng mL⁻¹ epidermal growth factor and 3 mg mL⁻¹ bovine serum albumin used as a control) was supplemented with ffEVs in concentration either equivalent to FF (ffEV extracted from 1 mL of FF was added to 1 mL of IVM medium, 1:1), or twice concentrated (2:1), or twice diluted (1:2). Cumulus–oocyte complexes from the same size follicles underwent IVM during 24 h, followed by 16 h of IVF in BO-IVF medium, and embryo development in BO-IVC medium (IVF Bioscience), until Day 5, and then in the same medium supplemented with 5% fetal calf serum up to Day 7, at 38.5°C and 5% CO₂. Blastocyst total cell number and apoptosis rate were determined using 4′,6-diamidino-2-phenylindole (DAPI) and terminal deoxynucleotidyl transferase dUTP nick-end labeling (TUNEL) staining. The data from 3 to 4 independent experiments were analysed by ANOVA and Tukey test, and results are shown in Table 1. Maturation rate (determined during post-IVF oocyte stripping) and cleavage rate did not differ between conditions, whereas blastocyst rate was higher in the groups supplemented with FF-equivalent, or concentrated ffEVs. Moreover, corresponding blastocysts showed lower apoptotic rate, compared to the control group. The most significant effect in term of embryo cell number was observed with 2-fold concentrated ffEV preparations, suggesting more efficient ffEV uptake by the oocytes at this concentration. In conclusion, simple preparation of ffEVs by differential centrifugation is enriched in exosome-like vesicles, and may be used during IVM, at concentrations at least equivalent or superior to FF, to obtain higher quality blastocysts.

Table 1.  Results of IVM following supplementation of IVM with no follicular fluid extracellular vesicles (ffEVs) (control) and medium supplementaed with ffEVs at ratios of 1:1, 2:1, and 1:2

This research was supported by the Russian Science Foundation (project No 19-16-00115).