Int J Mol Sci. 2018 Dec 21;20(1). pii: E38. doi: 10.3390/ijms20010038.

Isolation and Characterization of Functionally Active Extracellular Vesicles from Culture Medium Conditioned by Bovine Embryos In Vitro.

Pavani KC¹, Hendrix A^2,³, Van Den Broeck W⁴, Couck L⁵, Szymanska K⁶, Lin X⁷, De Koster J⁸, Van Soom A⁹, Leemans B¹⁰.

Author information

1: Department of Reproduction, Obstetrics and Herd Health, Faculty of Veterinary Medicine, Ghent University, Salisburylaan 133, B-9820 Merelbeke, Belgium. KrishnaChaitanya.Pavani@UGent.be.
2: Laboratory of Experimental Cancer Research, Department of Radiation Oncology and Experimental Cancer Research, Ghent University, C. Heymanslaan 10, B-9000 Ghent, Belgium. An.Hendrix@UGent.be.
3: Cancer Research Institute Ghent (CRIG), C. Heymanslaan 10, B-9000 Ghent, Belgium. An.Hendrix@UGent.be.
4: Department of Morphology-Faculty of Veterinary Medicine, Ghent University, Salisburylaan 133, B-9820 Merelbeke, Belgium. Wim.VandenBroeck@UGent.be.
5: Department of Morphology-Faculty of Veterinary Medicine, Ghent University, Salisburylaan 133, B-9820 Merelbeke, Belgium. liesbeth.couck@UGent.be.
6: Department of Basic Medical Sciences-Physiology group, Faculty of Medicine and Health Sciences, Ghent University, De Pintelaan 185, B-9000 Ghent, Belgium. Katarzyna.Szymanska@UGent.be.
7: Department of Nutrition, Genetics, and Ethology, Faculty of Veterinary Medicine, Ghent University, B-9820 Merelbeke, Belgium. linxiaoyuan23@163.com.
8: Department of Reproduction, Obstetrics and Herd Health, Faculty of Veterinary Medicine, Ghent University, Salisburylaan 133, B-9820 Merelbeke, Belgium. Jenne.DeKoster@UGent.be.
9: Department of Reproduction, Obstetrics and Herd Health, Faculty of Veterinary Medicine, Ghent University, Salisburylaan 133, B-9820 Merelbeke, Belgium. Ann.VanSoom@UGent.be.
10: Department of Reproduction, Obstetrics and Herd Health, Faculty of Veterinary Medicine, Ghent University, Salisburylaan 133, B-9820 Merelbeke, Belgium. baleeman.leemans@UGent.be.

Abstract

Extracellular vesicles (EVs) play a possible role in cell⁻cell communication and are found in various body fluids and cell conditioned culture media. The aim of this study was to isolate and characterize EVs in culture medium conditioned by bovine embryos in group and to verify if these EVs are functionally active. Initially, ultracentrifuged bovine serum albumin (BSA) containing medium was selected as suitable EV-free embryo culture medium. Next, EVs were isolated from embryo conditioned culture medium by OptiPrep^TM density gradient ultracentrifugation. Isolated EVs were characterized by nanoparticle tracking analysis, western blotting, transmission, and immunoelectron microscopy. Bovine embryo-derived EVs were sizing between 25⁻230 nm with an average concentration of 236.5 ± 1.27 × 10⁸ particles/mL. Moreover, PKH67 EV pre-labeling showed that embryo-secreted EVs were uptaken by zona-intact bovine embryos. Since BSA did not appear to be a contaminating EV source in culture medium, EV functionality was tested in BSA containing medium. Individual embryo culture in BSA medium enriched with EVs derived from conditioned embryo culture medium showed significantly higher blastocyst rates at day 7 and 8 together with a significantly lower apoptotic cell ratio. In conclusion, our study shows that EVs play an important role in inter embryo communication during bovine embryo culture in group.

KEYWORDS:

OptiprepTM density gradient ultracentrifugation; bovine; embryo culture; embryo–embryo communication; extracellular vesicles; uptake of embryo-secreted EVs