212 Supplementation of follicular fluid-extracellular vesicles during bovine oocyte maturation and its effect on embryo development in a serum-free group and individual culture system
N. Azari-Dolatabad A B , D. Eshghi Chaharborj A C , C. Benedetti A , A. Fernandez Montoro A , J. Leroy B , A. Van Soom A , K. Pavani A D and O. Bogado Pascottini A BA Department of Internal Medicine, Reproduction and Population Medicine, Faculty of Veterinary Medicine, Ghent University, Merelbeke, Ghent, Belgium
B Department of Veterinary Sciences, Gamete Research Center, Veterinary Physiology and Biochemistry, University of Antwerp, Wilrijk, Antwerp, Belgium
C Department of Clinical Sciences, School of Veterinary Medicine, Shiraz University, Shiraz, Iran
D Department for Reproductive Medicine, Ghent University Hospital, Ghent, Belgium
Reproduction, Fertility and Development 35(2) 235-235 https://doi.org/10.1071/RDv35n2Ab212
Published: 5 December 2022
© 2023 The Author(s) (or their employer(s)). Published by CSIRO Publishing on behalf of the IETS
In in vivo conditions, oocytes mature in the presence of follicular fluid (FF). Intercellular communication within the ovarian follicle is crucial to coordinate the development of a competent oocyte that is capable of being fertilised and undergoing embryogenesis. It has been described how extracellular vesicles (EVs) play a crucial role in follicle-oocyte communication. Therefore, we evaluated, in two experiments, whether supplementation of different concentrations of EVs isolated from FF of the nulliparous heifer during bovine oocyte maturation would yield extra benefits for embryo developmental competence in a group or individual culture system. The FF samples were collected from nulliparous heifer using ovum pickup. The EVs were isolated from FF of the nulliparous heifer, using OptiPrep™ density gradient ultracentrifugation. First, we confirmed the presence of EVs in the FF by transmission electron microscopy, followed by Western blotting and nanoparticle tracking analysis (NTA). To determine the particle size and concentration of EVs, NTA was performed with a NanoSight LM10 microscope. The protein concentration of the EV samples was measured by Nanodrop™. Cumulus-oocyte complexes (COCs) were aspirated from follicles between 4 and 8 mm in diameter, and only oocytes with uniformly granulated cytoplasm and surrounded by more than three compact layers of cumulus cells were selected for in vitro maturation-fertilisation-culture in the group (500 µL) and individual (20-µL droplets under paraffin oil) culture systems. All statistical analyses were performed using R-core. In the first experiment, different concentrations of FF-EVs (control, 5, 10, 25, and 50 µg protein/mL) were supplemented to the maturation medium in the group culture system. We could not demonstrate a positive effect of EVs in group culture on BR, and higher concentrations of EVs (25 and 50 µg/mL) even significantly (P < 0.05) decreased the developmental competence of oocytes. Next, we supplemented three different concentrations of FF-EVs (control, 6.5, 12.5, and 25 µg protein/mL) to the maturation medium in an individual culture system. Here, with the lowest tested concentration (6.5 µg/mL) of EVs, we could significantly (P < 0.05) improve the BR (37.6 ± 2.4%) compared with individual control (22.2 ± 2.5%) and the other concentrations (12.5 µg/mL [15.8 ± 3.1%] and 25 µg/mL [21 ± 2.2%]) as well. Interestingly, the lowest concentration of EVs (6.5 µg/mL) in the individual culture system improved BR to the same level as the control in the group culture system (37.6 ± 2.4% vs 38.4 ± 2.1%). In conclusion, we presume that any positive effect of the FF-EVs during oocyte maturation got lost in the group culture system since the oocytes and embryos produce their own embryotropins, probably also contained within EVs. However, individual culture systems, where inter-oocyte or inter-embryo cooperation is lost, the positive effect of FF-EVs during oocyte maturation was visible in the increased embryo competence.
