78 INTERCEPTION OF EXOSOMAL MESSAGES BETWEEN THE OVIDUCT AND THE EMBRYO: WHAT ARE THEY TWEETING ABOUT?

Abstract

Successful pregnancy requires an appropriate communication between the mother and the embryo(s). Recent studies indicate that exosomes, small (30–100 nm) membrane-bound vesicles of endocytotic origin, could act as intercellular vehicles in this unique communication system in the uterus. However, little is known about the role of these vesicles in the oviduct. Our study aimed at (1) demonstrating the existence of oviducal-embryo communication via exosomes, (2) deciphering the exosomal dialogue between them at the proteomic level, and (3) comparing the exosomal proteomic content to the oviducal fluid proteomic content in order to highlight the key role of exosomes in this dialogue. Cow oviducts (pool of 6 oviducts at different stages of the cycle in 4 replicates) were flushed, and exosomes were isolated by serial ultracentrifugation. Exosomes were measured by dynamic light scattering analysis, resulting in exosomes (63.25–97.03 nm) and microvesicle observations (>100 nm). Bovine embryos were produced in vitro up to the blastocyst and hatching/hatched blastocyst stages. To demonstrate the existence of the oviducal-embryo communication via exosomes, oviducal exosomes were labelled with green fluorescent dye (PKH67), filtered (0.22 µm) to remove microvesicles, and co-incubated with blastocysts and hatching/hatched (H) blastocysts for 20 h, under 5% CO₂ and 5% O₂ conditions. Subsequently, embryos were washed in exosome-free medium, fixed in 4% paraformaldehyde, and labelled with Hoechst 33342 and Actin Red Phallodin. Confocal microscopy observations confirmed that exosomes were internalized by blastocysts and H-blastocysts and located around the nucleus, demonstrating the existence of an oviducal-embryo communication via exosomes. Moreover, our results showed that the zona pellucida does not represent a barrier for exosomes and they act as natural nanoshuttles bringing oviducal signals into the embryo. Then, proteomic analysis by LC1D-nanoESI-LTQ-Orbitrap was used to decipher oviducal exosomal content, identifying 480 proteins. Gene ontology analysis revealed that a high number of these proteins were involved in metabolism (24.9%), cellular process (19.3%), and 0.8% reproductive processes. Further analysis revealed that more than 56% of exosomal proteins involved in cellular process were associated with cell-to-cell communication. Finally, exosomal proteins were compared with proteins present in oviducal fluid from a pool of samples from cows at Day 0 and Day 10 of the oestrous cycle. Comparative analysis showed that from a total of 607 proteins identified in both oviducal exosomes and fluid sources, 105 were specific to exosomes, 127 were specific to fluid, whereas 375 were common to both sources. Our findings provide the first evidence of oviducal-embryo communication via exosomes, an important first step in furthering the understanding of the oviducal environment and the role of exosomes as early mediators of embryo-maternal cross talk.

This research was supported by the EU AgreenSkills fellowship n° 267196 and EU FECUND Project n^o 312097.