154 Exosomes in Follicular Fluid Protect the Bovine Oocyte from Heat Shock

Abstract

Elevated temperature can compromise the ability of the mammalian oocyte to develop to the blastocyst stage after fertilization. The microenvironment of the oocyte is determined by the cellular and non-cellular components of the follicle including cumulus cells and follicular fluid. Here we tested whether follicular fluid contains molecules that can protect the bovine oocyte from heat shock during maturation, and if so, whether some of these protective molecules are present in exosomes. The experiments utilised ovaries from Bos taurus and admixtures of B. taurus and Bos indicus. Four separate pools of follicular fluid were prepared by aspiration of follicles from 48 to 70 slaughterhouse ovaries. Exosomes were isolated from follicular fluid by a series of centrifugation, filtration, and ultracentrifugation steps before being reconstituted in PBS. Each of the 4 exosome preparations was subject to particle size and concentration analysis. The experiments were designed as 2 × 3 factorial to test the effect of temperature and supplementation. Cumulus-oocyte complexes (COC) obtained from slaughterhouse ovaries were matured at 38.5°C for 22 h (control) or 41°C for 14 h followed by 38.5°C for 8 h (heat shock). Maturation was performed in the presence of vehicle (PBS), 10% (v/v) follicular fluid, or exosomes (16 × 10⁹ particles/mL). Data were analysed by least-squares ANOVA. Orthogonal contrasts and the mean separation test pdiff were used to compare means. Effects of treatment on cumulus cell expansion (change in diameter after maturation) were replicated 5 times using 119 to 122 COC per treatment. Effects of treatment on embryonic development after fertilization of treated COC was determined in 6 replicates using 244 to 286 embryos per replicate. Expansion was reduced by heat shock (P < 0.001), and affected by treatment (P < 0.05), with both follicular fluid and exosomes preventing the decrease in expansion caused by heat shock. Cleavage was reduced by heat shock (P < 0.001) and affected by treatment (P < 0.05) and the interaction between temperature × supplementation (P < 0.05). Although heat shock reduced the cleavage rate for vehicle-treated oocytes (77 v. 67%), there was no effect of heat shock for oocytes treated with follicular fluid FF (78 v. 74%) or exosomes (79 v. 78%; SEM = 1.4%). Heat shock also reduced the percent of cleaved embryos becoming blastocysts for the vehicle group (27 v. 17%; P < 0.05) but had no effect on percent of cleaved embryos becoming blastocysts for the follicular fluid (31% v. 26%) or exosome groups (28 v. 26%). Uptake of exosomes into isolated cumulus cells and oocytes cultured at 38.5°C for 0.5, 1, 14 and 22 h was examined using labelling of exosomes with 10 µM BODIPY® Ceramide TR (Thermo Fisher Scieintific, Waltham, MA, USA) and confocal microscopy. Exosomes were taken up by cumulus cells after culture for 1 h or later but were not taken up by oocytes. In conclusion, follicular fluid exosomes protected the oocytes from heat shock and this effect seems to be mediated by cumulus cells.

Study supported by BARD US-4719-14.