207 Anti-aging effect of follicular fluid exosome-like extracellular vesicles on bovine oocytes matured in vitro

Aging of mature metaphase-II (MII) bovine oocytes by prolonged IVM reduces their quality and embryo development capacity (Jiang et al. 2019 J. Reprod. Dev. 65, 499–506). The aim of the present work was to study the effects of follicular fluid (FF) extracellular vesicle complementation during IVM on deleterious alterations in aged mature oocytes caused by prolonged in vitro culture. The FF from 3–8 mm follicles was subjected to differential centrifugations up to 100 000g, and final preparations were enriched in exosome-like nanovesicles (ffExo), as reported earlier (Uzbekova et al. 2020 Front Vet Sci. 7, 584948). In vitro maturation of slaughterhouse-derived cumulus–oocyte complexes (COCs) was performed for 24 h in TCM-199 with 0.5 mM sodium pyruvate, 100 ng mL⁻¹ epidermal growth factor and 3 mg mL⁻¹ BSA, used as a control. Experimental groups were supplemented with ffExo either equivalent to FF (Group 1, ffExo extracted from 1 mL of FF were added to 1 mL of IVM medium), or twice concentrated (Group 2), as these concentrations were beneficial for oocyte competence (Singina et al. 2022 Reprod. Fertil. Dev. 34, 305–306). Oocyte aging was induced by post-IVM culture for 12 h or 24 h in TCM-199 with 10% FCS, since these times were earlier determined (Lebedeva et al. 2015 Front. Genet. 6, 274; Singina et al. 2021 Pharmaceuticals 14, 684). After 12 h of aging, IVF was performed during 16 h in BO-IVF medium, followed by in vitro embryo development in BO-IVC medium (IVF Bioscience, England) until Day 7 (n = 4, 116–121 oocytes per treatment). After 24 h of aging, rate of MII-oocytes, chromosome quality and apoptosis rate were evaluated (n = 3, 75–76 oocytes per treatment). Nuclei in oocytes and blastocysts were analysed using 4′,6-diamidino-2-phenylindole (DAPI) and TUNEL staining. Analysis of variance and Tukey test were applied for statistics. Oocyte maturation rate was similar in 24-h aging groups with and without ffExo (ranging from 81.3% to 86.7%), whereas ffExo in both concentrations reduced the frequency of chromosome abnormalities (decondensation, adherence, clumping) in the MII-oocytes from 44.3 ± 0.7% (control) to 29.2 ± 1.3% in Group 1 (P < 0.001) and 35.5 ± 1.4% in Group 2 (P < 0.01). Also, ffExo supplementation during IVM decreased MII-oocyte apoptosis rate to 13.8 ± 0.2% in Group 1 (P < 0.001) and 19.4 ± 0.3% in Group 2 (P < 0.01) compared with control (26.2 ± %) after 24 h of aging. After IVF, cleavage rate of 12-h aged control oocytes was 53.5 ± 2.9 and blastocyst rate was 17.3 ± 1.6%, and ffExo supplementation significantly increased cleavage and blastocyst rate in Group 1 (63.8 ± 2.9% and 26.5 ± 0.7%, respectively; P < 0.05). No significant effect on blastocyst cell number was observed in both ffExo groups compared with control; however, percentages of apoptotic nuclei in the blastocysts were significantly lower in both Group 1 and Group 2 (4.2 ± 0.08% and 4.4 ± 0.17%, respectively) compared with control (7.1 ± 0.14%; P < 0.001). In conclusion, ffExo supplemented during IVM at equivalent to FF concentration can exert the long-term anti-aging effect on bovine oocytes.

Supported by the Russian Science Foundation (project No 19–16–00115-n).