Abstract
Extracellular vesicles (EVs) have been identified within different body fluids and cell culture media. However, there is very little information on the secretion of these vesicles during early embryonic development. The aims of this work were first to demonstrate the secretion of extracellular vesicles by pre-implantation bovine embryos and second to identify and characterize the population of EVs secreted by bovine blastocysts during the period from day seven to nine of embryo culture and its correlation with further embryo development up to day 11. Bovine embryos were produced by in vitro fertilization (IVF) or parthenogenetic activation (PA) and cultured until blastocyst stage. Blastocyst selection was performed at day 7 post IVF/PA considering two variables: stage of development and quality of embryos. Selected blastocysts were cultured in vitro for 48 hours in groups (exp. 1) or individually (exp. 2) in SOF media depleted of exosomes. At day 9 post IVF/PA the media was collected and EVs isolated by ultracentrifugation. Transmission electron microscopy revealed the presence of heterogeneous vesicles of different sizes and population: microvesicles (MVs) and exosomes (EXs) of rounded shape, enclosed by a lipid bi-layer and ranging from 30 to 385 nm of diameter. Flow cytometry analysis allowed identifying CD63 and CD9 proteins as exosome markers. Nanoparticle tracking analysis generated a large number of variables, which required the use of multivariate statistics. The results indicated that the concentration of vesicles is higher in those blastocysts with arrested development from day 9 up to day 11 of in vitro development (6.7 x 108 particles/ml) derived from IVF (p <0.05), compared to PA blastocysts (4.7 x 108 particles/ml). Likewise, the profile (concentration and diameter) of particles secreted by embryos derived from IVF were different from those secreted by PA embryos. In conclusion, we demonstrated that bovine blastocysts secrete MVs/EXs to the culture media. Data suggest that characteristics of the population of EVs vary depending on embryo competence.
Highlights
There is increasing evidence that Extracellular vesicles (EVs) secreted by cells play important roles in cell-cell communication and they can be identified in vivo in different biological fluids, including blood, urine, as well as in vitro in cell culture media [1,2]
The first experiment was aimed to identify the presence of EVs in the culture media from pre-implantation bovine embryos produced by in vitro fertilization (IVF) and parthenogenetic activation (PA) and to identify structures associated with EVs biogenesis in the intracellular compartments of the blastomeres
The rates of blastocysts at day 7 and of expanded blastocysts at day 9 are shown in Table 1 and S2 Fig. No statistical differences in the blastocyst rate, embryo size and expanding rate at day 7 were observed between the two groups (PA and IVF)
Summary
There is increasing evidence that EVs secreted by cells play important roles in cell-cell communication and they can be identified in vivo in different biological fluids, including blood, urine, as well as in vitro in cell culture media [1,2]. EVs are related to biological events including tumorigenesis, metabolism, coagulation, intercellular communication and the immune system [3] and they have multiple applications in diagnosis and therapies of different pathologies [4]. There are three groups of EVs that can be identified by their size and shape: microvesicles (MV) are the biggest vesicles (diameter: 100–1000 nm) followed by apoptotic bodies (AB: 50– 500 nm) and, exosomes (EXs), the smallest vesicles with a range size of 30–120 nm [5]. Morphological identification and characterization requires several technologies such as transmission electronic microcopy (TEM), flow cytometry (FACS) and nanoparticle tracking analysis (NTA) [1,9]
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