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Shedova E.N., Singina G.N., Uzbekova S. Uzbekov R., Lukanina V.A., Tsyndrina E.V. Effect of extracellular vesicles of follicular origin during in vitro maturation and ageing of bovine oocytes on embryo development after in vitro fertilization. Sel'skokhozyaistvennaya Biologiya [Agricultural Biology], 2022, Vol. 57, № 6, p. 1178-1187.
(how to cite this article)

doi: 10.15389/agrobiology.2022.6.1178eng

UDC: 636.2:591.39

Acknowledgements:
Supported financially by the Russian Science Foundation (project No. 19-16-00115-П)

 

EFFECT OF EXTRACELLULAR VESICLES OF FOLLICULAR ORIGIN DURING in vitro MATURATION AND AGEING OF BOVINE OOCYTES ON EMBRYO DEVELOPMENT AFTER in vitro FERTILIZATION

E.N. Shedova1, G.N. Singina1 ✉, S. Uzbekova2, R. Uzbekov3, 4,
V.A. Lukanina1, E.V. Tsyndrina1

1Ernst Federal Research Center for Animal Husbandry, 60, pos. Dubrovitsy, Podolsk District, Moscow Province, 142132 Russia, e-mail shedvek@yandex.ru, g_singina@mail.ru (✉ corresponding author), kristybatle@gmail.com,
vip.krilochkina@mail.ru;
2Physiology of Reproduction and Behavior unit, INRAE, CNRS, IFCE, Tours University, 37380 Nouzilly, France, e-mail svetlana.uzbekova@inrae.fr;
3Laboratory of Cell Biology and Electron Microscopy, Faculty of Medicine, University of Tours, 10 bd Tonnelle, Tours, 37032, France, e-mail rustem.uzbekov@univ-tours.fr;
4Faculty of Bioengineering and Bioinformatics, Lomonosov Moscow State University, 1-73, Leninskiye Gory, GSP-1, Moscow, 19234 Russia

ORCID:
Shedova E.N. orcid.org/0000-0002-9642-2384
Uzbekov R. orcid.org/0000-0002-9336-5484
Singina G.N. orcid.org/0000-0003-0198-9757
Lukanina V.A. orcid.org/0000-0003-4744-7873
Uzbekova S. orcid.org/0000-0002-6989-6904
Tsyndrina E.V. orcid.org/0000-0002-3263-2358

Received September 12, 2022

Extracellular vesicles (EVs) isolated from ovarian follicular fluid (FF) are involved in vivo in the regulation of meiosis in female gametes. Recent studies suggested follicular EVs as potential regulators of oocyte quality capable to increase the efficiency of embryo production technologies in vitro (in vitro embryo production, IVP). In this work for the first time, we have analyzed embryo development competence of bovine oocytes after in vitro maturation (IVM) in the presence of EVs and ageing before in vitro fertilization. The aim of the study was to determine the effects of these conditions to oocyte ageing-related transformations during IVM in terms of their ability to develop blastocysts, as well as the quality of IVP embryos. The EVs were separated from FF by serial centrifugations and final ultracentrifugation at 100,000g. The isolated EV fraction contained 37.5 mg of total protein per ml of FF. Isolated vesicular fractions were analyzed using transmission electron microscopy that confirmed their enrichment in exosome-like EVs. For functional experiments, cumulus-oocyte complexes (COCs) were in vitro maturated in TC-199 medium containing 3 mg/ml of bovine serum albumin, 0.5 mM sodium pyruvate and 100 ng/ml EGF in the absence (control) or presence of EVs. Vesicles were added to IVM culture at the physiological concentration (EVs isolated from 1 ml of FF were added to 1 ml of IVM medium). After 24 hours of maturation, COCs were transferred to an ageing medium, cultured for further 12 hours, then in vitro fertilized and cultured for embryo development. At day 3 after in vitro fertilization, morphological evaluation of cleaved oocytes was carried out, and at day 7, the number of embryos developed to blastocyst stage and their quality were evaluated. The number of nuclei per embryo, calculated using the cytological method, served as an assessment of the quality of the embryo. In four independent experiments performed, the number of COCs in each group varied from 116 to 121. The cleavage rate of control oocytes was lower than that in the experimental EVs group (53.5 2.9 vs. 63.8 2.9 %, respectively, p < 0.05). In addition, EVs had a positive effect on embryo development up to the blastocyst stage after IVM and aging of the oocytes. In control, blastocyst rate was 17.3±1.6 %, and the presence of EVs during IVM increased this rate to 20.2±2.5 % (p < 0.05), whereas quality of produced embryos did not change. According to the reported data, EVs from follicular fluid added during IVM may increase the resistance of bovine oocytes to age transformations and, consequently, improve oocyte competence to embryo development after aging in vitro. Therefore, EVs can improve extracorporeal oocyte maturation and the efficiency of in vitro embryo production techniques in cattle.

Keywords: extracellular vesicles, bovine follicular fluid, bovine oocytes, in vitro maturation, oocyte aging, embryo development.

 

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