194 Extracellular vesicles derived from ampullary oviductal fluid improve developmental competence of bovine oocytes

Abstract

In vivo, oocytes mature in the presence of follicular fluid (FF) and ampullary oviductal fluid (AOF), starting from premature to final maturation stages (3-6 h postovulation). Extracellular vesicles (EVs) have been identified as important mediators of gamete/embryo-maternal interactions. They carry regulatory molecules, such as microRNAs and mRNAs. So far, the functionality of EVs derived from FF and AOF has not yet been determined. The aim of this study was to evaluate the effect of EVs derived from FF and AOF during in vitro oocyte maturation (IVM) on the development and quality of resulting bovine embryos. Follicular fluid of a preovulatory follicle was collected by ovum pickup, and AOF was collected from the oviducts of slaughtered cows in early luteal phase. Then, EVs from FF and AOF were isolated by size exclusion chromatography and confirmed by western blot. The concentration of EVs was determined by NanoDrop and Nanoparticle tracking analysis. Integrity and size of EVs were assessed by electron microscopy. Bovine oocytes (n = 1331) were allocated at random to five groups. In the control group (C), oocytes (n = 347) were cultured for 22.5 h in maturation medium (TCM-199 and epidermal growth factor 20 ng mL⁻¹) (MM). In the negative control group (NC), oocytes (n = 331) were cultured for 18 h in MM, and then transferred into fresh MM for 4.5 h. In the FF group, oocytes (n = 162) were cultured in MM supplemented with FF EVs (12.5 µg mL⁻¹) for 18 h, and then transferred to MM for 4.5 h. In the AOF group, oocytes (n = 328) were cultured for 18 h in EV-free MM, and then transferred to MM supplemented with AOF EVs (1.7 µg mL⁻¹) for 4.5 h. In the FF+AOF group, oocytes (n = 163) were cultured in MM supplemented with FF EVs for 18 h, and then transferred into MM supplemented with AOF EVs for 4.5 h. At 22.5 h post-incubation, mature oocytes were fertilized in vitro. At 21 h post-insemination, presumptive zygotes were denuded and cultured in synthetic oviductal fluid with insulin-transferrin-selenium for 8 days. Cleavage rate and blastocyst rate were recorded at 48 h and 8 days post-insemination, respectively. Blastocyst quality was assessed by differential apoptotic staining. The data were analysed using one-way ANOVA. Cleavage rate was not affected by the treatment, whereas blastocyst yield in the AOF group (48.75%) was significantly higher than that in the C (42.91%) and NC (37.72%) groups. In addition, embryos produced in the AOF group showed the highest number of trophectoderm cells and inner cell mass cells, and lowest number of apoptotic cells (P < 0.05). Trophectoderm and inner cell mass cells were higher and apoptotic cells were lower in FF and FF+AOF compared with the C and NC groups. In conclusion, adding AOF EVs to oocyte MM in the last 4.5 h of IVM improves quantity and quality of blastocysts. In addition, although FF EVs showed no effect on blastocyst yield, they improved blastocyst quality by increasing cell number.