Identification and characteristics of extracellular vesicles from bovine blastocysts produced in vitro

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.

Effect of BMP15 and/or AMH during in vitro maturation of oocytes from involuntarily culled dairy cows

“…In addition, these blastocysts showed arrested (slower) development (80% in early blastocyst from involuntarily culled dairy cows versus 32% in beef cattle). This is indicative of reduced embryo quality (Muthukumar et al, ; Shapiro, Daneshmand, Garner, Aguirre, & Thomas, ; Sugimura et al, ; Mellisho et al, ). Developmental speed of embryos has been considered a criterion to predict embryo competence; in fact, embryos must follow the developmental program of their species to have a normal development (Rooke et al, ; Sugimura et al, ; Van Soom, Ysebaert, & de Kruif, ).…”

Embryonic extracellular vesicles as informers to the immune cells at the maternal–fetal interface

“…The presence of the EVs has been demonstrated in the embryo culture medium from different species, such as bovines [28], porcines [29], mice [30] and humans [7]. The diameter of EVs found in the embryo spent media is quite different among these species, as shown in Table 1.…”

Symposium review: Embryo survival—A genomic perspective of the other side of fertility

“…MicroRNA are stable in fluids because they are short, able to bind proteins (Wahid et al, 2010), and can be packaged within extracellular vesicles (EV), which are membranous transport bodies that serve as shuttles for molecules such as miRNA, mRNA, DNA, and proteins . Bovine embryos can secrete EV to culture medium (Mellisho et al, 2017). They have been the focus of several recent studies because they have the ability to travel greater distances throughout the body.…”