61 Extracellular vesicles from serum in culture media are internalized by bovine embryos produced in vitro

Abstract

Extracellular vesicles (EV) are currently considered a mechanism of cell communication. These are secreted from different cell types, including embryos, to serve as mediators of short and long distance signals. EV can be identified in vivo in different biological fluids, as well as in vitro embryo culture medium. Usually, media used for embryo in vitro culture are supplemented with serum or other protein sources that favour cell proliferation and development. Serum and protein sources contain EV, including microvesicles and exosomes that in principle can be internalized by embryonic cells. The aim of this study was to evaluate if serum-derived EV are internalized by the embryo at different stages of the early development, and if EV from the serum are required for in vitro bovine embryo development. For that, it was first evaluated if EV depleted culture media affect embryo development up to the blastocyst stage; oocytes were in vitro matured for 22 to 23 h and in vitro fertilized for 18 h. Posteriorly, presumptive zygotes were in vitro cultured in groups (25 embryos/well in 4-well plates) in SOF or SOF depleted of EV for 8 days. To evaluate EV internalization, culture media was supplemented with labelled EV and confocal imaging was performed. The EV were obtained by ultrafiltration (centrifugal filter devices 100 kDa, Amicon; Millipore, Billerica, MA, USA) for 15 min at 3000 rpm. Then, EV were stained with PKH67 dye and washed 3 times with PBS by ultrafiltration to remove excess dye. The EV labelled with PKH67 were resuspended in SOFaa depleted of EV (3 × 10⁹ particles per 500 µL) and supplemented for 24 h at the 1-cell stage (Day 1 post IVF), 16 cells (Day 4 post IVF), and early blastocyst (Day 6 post IVF) in 5% CO₂, 5% O₂, and 90% N₂. PBS with PKH67 dye was used as a control treatment. Hoechst 33343 was used to label the nuclei before washing with PBS and fixation with 0.4% paraformaldehyde. Images were acquired on a Zeiss (Zeiss, Jena, Germany) LSM 780 confocal microscope. There were no statistical differences on blastocyst rate at Day 8 between embryos cultured in SOF depleted of EV (19.5%) and control group (SOF; 22.7%; P > 0.05). We observed punctuated green fluorescence near the embryo nuclei in the 3 stages studied in embryos supplemented with EV but not in the control treatment, which indicates that EV from serum are uptaken by embryonic cells in early development. Therefore, we demonstrated uptake of EV from fetal calf serum added to culture media, although its absence does not affect embryo development.

Research was supported by FONDECYT, Chile (1170310).