Isolation and Characterization of Functionally Active Extracellular Vesicles from Culture Medium Conditioned by Bovine Embryos In Vitro

Abstract

Extracellular vesicles (EVs) play a possible role in cell–cell communication and are found in various body fluids and cell conditioned culture media. The aim of this study was to isolate and characterize EVs in culture medium conditioned by bovine embryos in group and to verify if these EVs are functionally active. Initially, ultracentrifuged bovine serum albumin (BSA) containing medium was selected as suitable EV-free embryo culture medium. Next, EVs were isolated from embryo conditioned culture medium by OptiPrepTM density gradient ultracentrifugation. Isolated EVs were characterized by nanoparticle tracking analysis, western blotting, transmission, and immunoelectron microscopy. Bovine embryo-derived EVs were sizing between 25–230 nm with an average concentration of 236.5 ± 1.27 × 108 particles/mL. Moreover, PKH67 EV pre-labeling showed that embryo-secreted EVs were uptaken by zona-intact bovine embryos. Since BSA did not appear to be a contaminating EV source in culture medium, EV functionality was tested in BSA containing medium. Individual embryo culture in BSA medium enriched with EVs derived from conditioned embryo culture medium showed significantly higher blastocyst rates at day 7 and 8 together with a significantly lower apoptotic cell ratio. In conclusion, our study shows that EVs play an important role in inter embryo communication during bovine embryo culture in group.

The Separation and Characterization of Extracellular Vesicles from Medium Conditioned by Bovine Embryos

“…Earlier, Mellisho et al [21] have demonstrated that 500 µL of medium conditioned by bovine blastocysts was sufficient for EV isolation, but apparently, in this study, no standardized EV isolation method was applied. In our previous study, initially, we used 500 µL of embryo-conditioned medium, but this was not sufficient for either characterization or quantification of EVs [6]. In conclusion, the separation of EVs with high specificity from 1 mL of embryo culture medium was addressed in the current study.…”

“…Routine in vitro methods were used for bovine embryo production, as described previously by Pavani et al [6]. Briefly, at the local abattoir, cow ovaries were obtained and processed within 2 h after collection.…”

“…On top of the SEC column, 1 mL of embryo-conditioned medium was loaded and fractions of 1-mL eluate were collected in 16 fractions. Resulting fractions were concentrated with Amicon Ultra-2 10 k centrifugal filters (UFC201024, Merck Millipore, Billerica, MA, USA) as previously described by Pavani et al [6] and the eluates of each fraction were retrieved from the flow-through reservoir and stored at −80 • C for further EV characterization.…”

“…Recently, our group has demonstrated that bovine embryos cultured in a group can release functionally active EVs with sizes ranging from 25 to 250 nm. Embryo-derived EVs are possible autocrine embryotropins playing a crucial role in embryo development [6]. Recently, Giacomini et al [7] have also shown that conditioned media from human embryos cultured in vitro for 3 days or up to the blastocyst stage contained EVs with a diameter of 50 to 200 nm.…”

Sexual dimorphic expression and release of transcription factors in bovine embryos exposed to oxidative stress

“…It is not only the number of EVs released into extracellular space but also the molecular cargo of those EVs, which was differed between male and female exposed to OS. As shown in Figure , EVs from male embryos at higher oxygen level contained higher level of NFE2L2 and SOD1 transcripts compared to those cultured under low oxygen level, which might be beneficial of development of embryos (Pavani et al, ). The role of EVs is not only in cell–cell communication (Saeed‐Zidane et al, ) but also in the removal of undesirable cellular molecules (Takahashi et al, ).…”