215 Fetal calf serum-derived extracellular vesicles’ effects on oocyte lipid accumulation during in vitro maturation in cattle

In vitro maturation (IVM) can affect oocyte competence and metabolism, exacerbating lipid accumulation in the cumulus-oocyte complex (COC). The fetal calf serum (FCS) is a major factor driving lipid accumulation during IVM. The role of FCS subcomponents, like extracellular vesicles (EVs), is still uncertain. Herein, we aimed to investigate the effect of FCS-derived EVs on oocyte lipid accumulation during IVM in cattle. For this, COCs (n = 273, six repetitions) retrieved from 3–6 mm follicles from abattoir ovaries were IVM under different medium conditions: (i) FCS (n = 56), consisting of base medium (TCM199 Earle’s salt, L-glutamine, 2.2 g/L sodium bicarbonate, 50 µg/mL gentamicin, 0.2 mM sodium pyruvate, 0.5 µg/mL FSH, and 50 mg/mL hCG) supplemented with 10% fetal calf serum; (ii) EVs-depleted FCS (FCS − EV, n = 56), consisting of base medium supplemented with 10% EVs-free FCS); (iii) EVs-depleted FCS supplemented with EVs from FCS (FCS − EV + EV, n = 55) consisting of base medium supplemented with 10% of EVs-free FCS, in which the same proportion of FCS-derived EVs was added; (iv) BSA (n = 50), consisting of base medium supplemented with 0.8 mg/mL of bovine albumin; and (v) BSA supplemented with FCS-derived EVs (BSA + EV, n = 56), consisting of base medium supplemented with 0.8 mg/mL of BSA and FCS-derived EVs corresponding to 10% (v/v). The COCs were kept in IVM for 9 h and then were denuded, fixed with paraformaldehyde 4% for 12 min, and incubated in 0.1% saponin for 1 h, followed by 1 h incubation in 20 µg/mL of BODIPY 493/503. Slides were mounted with 20 µL de ProLongTM Gold antifade reagent. Lipid droplets (LDs) from oocytes’ central slice were captured in Confocal Leica TCS SP5 Microscope and analysed through the Nucleus Counter tool of ImageJ software (National Institutes of Health). The ratio between the LDs area and the oocyte area (LD/OO ratio) was calculated. Results were submitted to ANOVA and means were compared by Tukey’s test with 5% significance level. No significant differences were found (P = 0.7592). LD/OO ratios were: FCS = 0.147 ± 0.0605; FCS − EV 0.147 ± 0.0685; FCS − EV + EV = 0.150 ± 0.0787; BSA = 0.130 ± 0.0617; BSA + EV = 0.142 ± 0.0756. These data indicate that, for this experiment, the EVs from FCS were not a major factor driving oocyte lipid accumulation during the first 9 h of IVM and other factors may be involved. The literature has shown different lipid accumulation in experimental groups using FCS and BSA in the IVM medium; however, this has been demonstrated after 24 h of IVM.

This research was supported by CAPES finance code 001; CNPq 407223/2021-5; and FAPESP 2021/09123-4.