Ultrastructural identification of CD9 positive extracellular vesicles released from human embryos and transported through the zona pellucida.

Extracellular vesicles (EVs) are highly specific and multi-purpose vesicular structures that are released by various cell and tissue types in the body. However, the secretion of EVs from mammalian embryos, especially human, has not been well characterized. Thus, the aim of this study was to 1) identify EVs in human preimplantation embryos at different stages of their development using scanning and electron microscopy, and 2) investigate whether EVs can cross the zona pellucida (ZP) and be released from human embryos cultured in vitro. Human oocytes, zygotes, cleavage embryos and blastocysts donated for research were labeled with the tetraspanin EV marker CD9 and analyzed by scanning and transmission electron microscopy. Embryo culture conditioned media collected 3- and 5-days post fertilization were examined for the presence of EVs using electron microscopy. We detected numerous CD9 positive vesicles released from all embryos examined. They were observed on the surface of the plasma membrane, within the perivitelline space as well as throughout the zona pellucida. Interestingly, EVs were not seen in the ZP of all mature metaphase II oocytes, however, were detected just after fertilization in the ZP of zygotes and embryos. Electron microscopy using negative staining, and nanoparticle tracking analysis (NTA) of embryo conditioned culture media also showed the presence of vesicles of various sizes, which were round shaped, and had a lipid bilayer. Their size ranged from 30 to 500 nm, consistent with the sizes of exosomes and microvesicles. In conclusion, the results of the study provide evidence that human preimplantation embryos at all developmental stages secrete EVs into the perivitelline space, which then traverse through the ZP, and are then released into the surrounding culture medium. Abbreviations: EVs: extracellular vesicles; ZP: zona pellucida; CD9, CD63, and CD81: tetraspanin EV markers; NTA: nanoparticle tracking analysis; ESCRT: endosomal sorting complexes required for transport; SEM: scanning electron microscopy; TEM: transmission electron microscopy; TE: trophectoderm; ICM: inner cell mass; PVS: perivitelline space; MI: metaphase I; MII: metaphase II; GV: germinal vesicle; MVs/EXs: microvesicles/exosomes; hCG: human chorionic gonadotrophin; GnRH: gonadogrophin releasing hormone; ICSI: intracytoplasmic sperm injection; SPS: serum protein substitute; 1PN: one pronuclear zygote; 3PN: tri-pronuclear zygote; IgG: immunoglobulin G; PBS: phosphate buffer saline; ETHO: ethanol; ESED: Environmental Secondary Electron Detector; BSA: bovine serum albumin.