Effect of extracellular vesicles of follicular origin during in vitro maturation and ageing of bovine oocytes on embryo development after in vitro fertilization

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 function show_eabstract() { $('#eabstract1').hide(); $('#eabstract2').show(); $('#eabstract_expand').hide(); }