Identification and Characterization of Extracellular Vesicles and Its DNA Cargo Secreted During Murine Embryo Development

Abstract

Extracellular vesicles (EVs) are known to transport DNA, but their implications in embryonic implantation are unknown. The aim of this study was to investigate EVs production and secretion by preimplantation embryos and assess their DNA cargo. Murine oocytes and embryos were obtained from six- to eight-week-old females, cultured until E4.5 and analyzed using transmission electron microscopy to examine EVs production. EVs were isolated from E4.5-day conditioned media and quantified by nanoparticle tracking analysis, characterized by immunogold, and their DNA cargo sequenced. Multivesicular bodies were observed in murine oocytes and preimplantation embryos together with the secretion of EVs to the blastocoel cavity and blastocyst spent medium. Embryo-derived EVs showed variable electron-densities and sizes (20–500 nm) and total concentrations of 1.74 × 107 ± 2.60 × 106 particles/mL. Embryo secreted EVs were positive for CD63 and ARF6. DNA cargo sequencing demonstrated no differences in DNA between apoptotic bodies or smaller EVs, although they showed significant gene enrichment compared to control medium. The analysis of sequences uniquely mapping the murine genome revealed that DNA contained in EVs showed higher representation of embryo genome than vesicle-free DNA. Murine blastocysts secrete EVs containing genome-wide sequences of DNA to the medium, reinforcing the relevance of studying these vesicles and their cargo in the preimplantation moment, where secreted DNA may help the assessment of the embryo previous to implantation.

Extracellular Vesicles Function as Bioactive Molecular Transmitters in the Mammalian Oviduct: An Inspiration for Optimizing in Vitro Culture Systems and Improving Delivery of Exogenous Nucleic Acids during Preimplantation Embryonic Development

“…Besides these cargoes, it is now known that genomic DNA (gDNA) fragments in EVs are transportable between cells, then increase the gDNA-coding mRNA and protein expressions in the recipient cells, eventually affecting the function of recipient cells; for instance, the AT1 receptor DNA in EVs could increase AT1 receptor expression, then stimulate Na + -K + ATPase activity in recipient HEK293 cells [33]. In addition, EVs derived from embryos also contain DNA fragments that represent the full murine genome, which may be useful for embryo chromosomal noninvasive diagnosis [34].…”

Extracellular vesicles, microRNA and the preimplantation embryo: non-invasive clues of embryo well-being

“…Generally, these extracellular vesicle populations consisted of vesicles within 50-200 nm in diameter and the extracellular vesicle population being actively released from preimplantation embryos throughout early development was concluded to be largely exosomal in nature. This is supported by the observation of membrane localized, CD9+ MVBs in fixed murine cell slices from oocyte, cleavage-stage and morula-stage embryos (Simon et al, 2020), including blastocyst trophectodermal cells (Shin et al, 2017) and within trophoblast cells of bovine blastocysts (Mellisho et al, 2017). Uptake of human preimplantation embryoderived extracellular vesicles by primary human endometrial cells has been demonstrated, suggesting extracellular vesicles may be involved in the conceptus-uterus milieu that facilitates the initial attachment and growth of the extraembryonic lineages through the uterine epithelium (Giacomini et al, 2017).…”

“…Extracellular vesicles are released at all stages of human preimplantation development, including cleavage-stage embryos and zygotes (Vyas et al, 2019). Similarly, all stages of porcine (Saadeldin et al, 2014), bovine (Qu et al, 2017;Dissanayake et al, 2020) and mouse (Pallinger et al, 2018;Simon et al, 2020) preimplantation embryos have confirmed extracellular vesicle release. In all cases, the identified vesicles presented the hallmark cupped-shape, double membrane vesicle appearance and amplification of select embryonicspecific mRNA sequences (POU5F1 and NANOG [Giacomini et al, 2017]; Oct4, Sox2, Klf4, c-Myc and Nanog [Saadeldin et al, 2014]) from extracellular vesicleenriched media fractions confirmed the embryonic origin of these vesicles.…”

“…Preimplantation embryo-derived extracellular vesicles are confirmed as secretory products in spent culture media collected during human clinical studies (Abu-Halima et al, 2017;Giacomini et al, 2017;Pallinger et al, 2017;Vyas et al, 2019) in addition to those involving various animal models: the cow (Mellisho et al, 2017;Qu et al, 2017;Pavani et al, 2019;Dissanayake et al, 2020), mouse (Pallinger et al, 2018;Simon et al, 2020) and pig (Saadeldin et al, 2014). Extracellular vesicles are released at all stages of human preimplantation development, including cleavage-stage embryos and zygotes (Vyas et al, 2019).…”

“…In fact, the direct observation of exogeneous fluorescently labelled small vesicle internalization has revealed this flux is likely bidirectional (Saadeldin et al, 2014;Kim et al, 2019). Vyas et al (2019) observed extracellular vesicles within the zona pellucida itself by electron microscopy (Vyas et al, 2019), which is corroborated by Simon et al (2020). This raises the question whether the contents of the zona pellucida may provide sufficient embryo-derived material to be used as a non-invasive source of diagnostic information.…”