Nanoparticles from culture media are internalized by in vitro-produced bovine embryos and its depletion affect expression of pluripotency genes

Abstract

Extracellular vesicles are nanoparticles secreted by cell and have been proposed as suitable markers to identify competent embryos produced in vitro. Characterizing EVs secreted by individual embryos is challenging because culture medium itself contributes to the pool of nanoparticles that are co-isolated. To avoid this, culture medium must be depleted of nanoparticles that are present in natural protein source. The aim of this study was to evaluate if the culture medium subjected to nanoparticle depletion can support the proper in vitro development of bovine embryos. Zygotes were cultured in groups on depleted or control medium for 8 days. Nanoparticles from the medium were characterized by their morphology, size and expression of EVs surface markers. Isolated nanoparticles were labelled and added to depleted medium containing embryos at different developmental stages and evaluated after 24 hours at 2, 8-16 cells, morula and blastocyst stages. There were no statistical differences on blastocyst rate at day 7 and 8, total cell count neither blastocyst diameter between groups. However, morphological quality was better in blastocysts cultured in non-depleted medium and the expression of SOX2 was significantly lower whereas NANOG expression was significantly higher. Few nanoparticles from medium had a typical morphology of EVs but were positive to specific surface markers. Punctuated green fluorescence near the nuclei of embryonic cells was observed in embryos from all developmental stages. In summary, nanoparticles from culture medium are internalized by in vitro cultured bovine embryos and their depletion affects the capacity of medium to support the proper embryo development.

Extracellular Vesicles Secreted by Pre-Hatching Bovine Embryos Produced In Vitro and In Vivo Alter the Expression of IFNtau-Stimulated Genes in Bovine Endometrial Cells

“…Then, cells were trypsinized and seeded in 35 mm-diameter culture dishes at a density of 0.3 × 10 6 until they reached the 60% confluence. At this time, the culture medium was replaced by an EV-depleted medium which was produced by ultrafiltration (centrifugal filter devices 100 kDa, Amicon, Miami, FL, USA) of culture medium (B911-500, Merck, Rahway, NJ, USA) for 15 min at 1660 g [ 93 ]. The EVs derived from in vivo- or in vitro-produced embryos were labeled with PKH67 (Sigma-Aldrich) fluorescent dye according to the manufacturer’s instructions to track the internalization.…”