87 Characterisation of extracellular vesicle populations secreted by bovine female reproductive tissues and embryos in vitro
B. Fernandez-Fuertes A , J. M. Sánchez A , R. Mazzarella A , A. Álvarez-Barrientos B , S. Guisado A , E. M. González C , P. Lonergan D and D. Rizos AA Department of Animal Reproduction, National Institute for Agriculture and Food Research and Technology (INIA-CSIC), Madrid, Spain
B Bioscience Applied Techniques Facility, University of Extremadura, Badajoz, Spain
C Department of Anatomy and Embryology, Veterinary Faculty, Universidad Complutense de Madrid, Madrid, Spain
D School of Agriculture and Food Science, University College Dublin, Dublin, Ireland
Reproduction, Fertility and Development 35(2) 169-170 https://doi.org/10.1071/RDv35n2Ab87
Published: 5 December 2022
© 2023 The Author(s) (or their employer(s)). Published by CSIRO Publishing on behalf of the IETS
A thorough understanding of bovine embryo-maternal interactions during the preimplantation period is crucial for the development of techniques to mitigate pregnancy loss in vivo and improve embryo development rates in vitro. Lately, the regulation of this crosstalk by maternal- and embryo-derived extracellular vesicles (EVs) has attracted significant interest; however, the localised nature of the maternal response to an embryo during the first week of pregnancy makes this communication difficult to study in vivo. Thus, the aim of this study was to determine the ability to isolate and characterise the populations of EVs secreted in vitro by (1) oviducal explants incubated in the presence or absence of eight-cell embryos (8C), or by (2) endometrial explants incubated with or without blastocysts (BL), as well as by (3) embryos alone. Cross-bred beef heifers were oestrous synchronised and slaughtered on Day 3.5 after standing oestrous to produce oviducal explants (n = 4), or on Day 7 to generate endometrial explants (n = 3). Conditioned media from the following groups was recovered after 6 h incubation: (1) oviducal explant alone, (2) oviducal explant + 8C (n = 30 per explant), (3) 8C alone (n = 100), (4) uterine explant alone, (5) uterine explant + BL (n = 10 per explant), and (6) BL alone (n = 50). Of note, two explants were recovered from each heifer to produce groups 1, 2, 4, and 5. EVs were isolated by size-exclusion chromatography, concentrated using a column filter, and characterised by flow cytometry with carboxyfluorescein succinimidyl ester and antibodies against EV surface markers (CD63, CD81, and CD44) to determine presence and differences in EV populations across groups. Data were analysed by one-way ANOVA after confirming normal distribution. Tissue region influenced the population of EVs detected, as evidenced by an increase in the percentage of total CD63+, and subpopulations CD63+CD44− and CD63+CD81− EVs in oviducal explants in comparison to uterine explants (P < 0.01). Similarly, embryo developmental stage also led to differences in EV labelling. Specifically, 8C secreted a higher percentage of CD63-CD44− EVs and a lower percentage of CD63+ and CD63+CD44+ EVs in comparison with BL (P < 0.01). Interestingly, co-culture of 8C with oviducal explants led to an increase in the percentage of CD63+CD44+ EVs in comparison with oviducal explants cultured alone (9.7 ± 1.4% vs 3.8 ± 0.7; P < 0.05). However, no differences in EV populations were detected in the conditioned media of endometrial explants incubated in the presence or absence of BL. In conclusion, both tissue region as well as embryonic stage lead to differences in the population of secreted EVs. In addition, these data suggest that embryos can induce changes in the type of EVs secreted by the oviduct. Further studies are needed to determine the cargo of these EVs and its role in the regulation of the maternal environment and early embryo development.
This work was supported by the Spanish Ministry of Science and Innovation (PID2019-111641RB-I00).
