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.
Highlights
Embryo-endometrial communication is mediated by different mechanisms including extracellular vesicles (EVs) with a plethora of molecules released to this interface
Ultrastructural visualization of murine oocytes using TEM identified the existence of multivesicular bodies (MVBs) in the cytoplasm
The presence of MVBs was observed in the blastomeres at different embryo developmental stages (E2.5 and E3.5), migrating from the cytoplasm to the plasma membrane where their content was secreted outwards through the zona pellucida (Figure 1B)
Summary
Embryo-endometrial communication is mediated by different mechanisms including extracellular vesicles (EVs) with a plethora of molecules released to this interface. EXOs originate from the endosomal pathway as late endosomes that evolve into multivesicular bodies and migrate from the perinuclear region to the cell surface by fusion [20,21] Their sizes range between 30–150 nm and are identified by classical molecular markers such as tetraspanins (CD63, CD9, CD81), flotillin-1, or heat shock 70-kDa proteins, recent reports considered that other EVs might share these markers [22]. Their cargo portrays their functionality in regulating communication by means of proteins, cytokines, lipids, RNA, or DNA [23]
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