Embryo-induced alterations in the protein profile of bovine oviductal extracellular vesicles

The oviduct provides the optimum environment for early embryonic development. Maternal-embryonic communication, which is essential for embryo quality, is mediated partly via extracellular vesicles (EVs). This study aimed to investigate the protein cargo of EVs obtained from the oviductal fluid (OF) of pregnant and cyclic heifers and their implications for maternal-embryonic communication in vivo. Oestrous cycles of crossbred beef heifers were synchronized, following which they were artificially inseminated (pregnant; n=13) or not (cyclic; n= 8) and slaughtered 3.5 days after insemination. The oviduct ipsilateral to the corpus luteum was flushed and the OF was examined to confirm the presence of a 6-8 cell embryo in pregnant animals. OF-EVs were isolated using size exclusion chromatography, concentrated by ultrafiltration, while EVs presence were characterized by flow cytometry using antibodies for specific EV markers (CD63, CD81, and CD44). Proteomic analysis was carried out using nanoLC-MS/MS with spectral counting to identify and quantify the proteins present in the EVs. Five animals from each group were used and statistical analysis was performed using ANOVA for flow cytometry data or T-test for proteomic data, both with a significance level of 5%. Bioinformatic analysis was performed with the DAVID and STRING tools. Flow cytometry analysis confirmed EV presence and no significant differences in EV markers between groups. A total of 1,101 proteins were identified: 5 unique to OF-EVs from cyclic heifers, 611 unique to pregnant heifers, and 485 in common. Among the common proteins, 93 were upregulated and 42 were downregulated in OF-EVs from the pregnant group. Functional enrichment analysis demonstrated that proteins exclusive to pregnant OF-EVs are involved in the Ras and Hippo pathways. Of note, Ras signaling is critical for mouse embryo development at the time of embryonic genome activation, which in cattle occurs in the oviduct during 8- to 16-cells transition. Furthermore, LLGL1, PATJ, and PARD6GB, members of the Hippo pathway exclusively found in pregnant OF-EVs, can regulate cell polarity and establishment of pluripotency. Additional pathways related to unique and upregulated proteins in pregnant OF-EVs include tight junction, cell adhesion molecules, and focal adhesion, which are essential for proper oviductal cell functioning and embryo development. Gene ontology analysis also revealed that upregulated proteins in pregnant OF-EVs, in comparison to EVs from cyclic animals, are associated with the immune response. In conclusion, although our model does not exclude a potential effect of sperm on the OF-EVs in the inseminated group, the study characterized a specific protein signature in OF-EVs from pregnant animals, which is likely due to the interactions established between the mother and the embryo.

Bibliographic Details

Main Authors:	Mazzarella, Rosane, Sánchez, José María, Fernández-Fuertes, Beatriz, Guisado, Sandra, Álvarez-Barrientos, A., González, Esperanza, Falcon-Perez, Juan M., Azkargorta, Mikel, Elortza, Félix, González, Encina M.
Other Authors:	Sao Paulo Research Foundation
Format:	comunicación de congreso biblioteca
Language:	English
Published:	Association of Embryo Technology in Europe 2023-09-07
Subjects:	Puerperium, Uterine involution, Uterine stiffness, Sheep,
Online Access:	http://hdl.handle.net/10261/351819
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