Extracellular Vesicles Secreted by Pre-Hatching Bovine Embryos Produced In Vitro and In Vivo Alter the Expression of IFNtau-Stimulated Genes in Bovine Endometrial Cells.

The embryo-maternal interaction occurs during the early stages of embryo development and is essential for the implantation and full-term development of the embryo. In bovines, the secretion of interferon Tau (IFNT) during elongation is the main signal for pregnancy recognition, but its expression starts around the blastocyst stage. Embryos release extracellular vesicles (EVs) as an alternative mechanism of embryo-maternal communication. The aim of the study was to determine whether EVs secreted by bovine embryos during blastulation (D5-D7) could induce transcriptomic modifications, activating IFNT signaling in endometrial cells. Additionally, it aims to assess whether the EVs secreted by embryos produced in vivo (EVs-IVV) or in vitro (EVs-IVP) have different effects on the transcriptomic profiles of the endometrial cells. In vitro- and in vivo-produced bovine morulae were selected and individually cultured for 48 h to collect embryonic EVs (E-EVs) secreted during blastulation. E-EVs stained with PKH67 were added to in vitro-cultured bovine endometrial cells to assess EV internalization. The effect of EVs on the transcriptomic profile of endometrial cells was determined by RNA sequencing. EVs from both types of embryos induced several classical and non-classical IFNT-stimulated genes (ISGs) and other pathways related to endometrial function in epithelial endometrial cells. Higher numbers of differentially expressed genes (3552) were induced by EVs released by IVP embryos compared to EVs from IVV (1838). Gene ontology analysis showed that EVs-IVP/IVV induced the upregulation of the extracellular exosome pathway, the cellular response to stimulus, and the protein modification processes. This work provides evidence regarding the effect of embryo origin (in vivo or in vitro) on the early embryo-maternal interaction mediated by extracellular vesicles.

Bovine embryos release extracellular vesicles with differential miRNA signature during the compaction and blastulation stages.

Pre-implantation embryos release extracellular vesicles (EVs) to extracellular environment. In this work it is hypothesized that the EVs miRNA cargo will vary during pre-implantation development due to the constant changes in gene expression that take place through this period. The concentration, size and miRNA cargo of EVs secreted by competent bovine embryos during the period from compaction to blastulation (Day 3-7) were analyzed. For this analysis tow developmental windows were defined: W2 from 8-cells (D3) to morula (D5) and W3 from morula (D5) to blastocyst (D7). For W2, in vitro produced embryos were individually cultured in EVs-depleted medium from D3 to D5; culture media were collected and assigned to Group W2. Morulae were kept in culture up to blastocyst stage to determine the developmental competence. For W3, D5 morulae were collected and cultured individually in EVs-depleted medium up to blastocyst stage; culture media were assigned to Group W3, and blastocysts were kept in culture up to day 11 to define their competence. The mean size of EVs was similar between groups, however, EVs concentration was lower in W2. A total of 140 miRNAs were identified. From them, 79 were differentially expressed between the groups, 28 upregulated and 51 downregulated. miRNAs differentially detected between both developmental windows participate in the regulation of signaling pathways which crucial for embryonic development. It was concluded that the secretion of EVs is regulated by the developmental progress of the embryo during the pre-implantation period.

Nanoparticles from culture media are internalized by in vitro-produced bovine embryos and its depletion affect expression of pluripotency genes.

Extracellular vesicles are nanoparticles secreted by cell and have been proposed as suitable markers to identify competent embryos produced in vitro. Characterizing EVs secreted by individual embryos is challenging because culture medium itself contributes to the pool of nanoparticles that are co-isolated. To avoid this, culture medium must be depleted of nanoparticles that are present in natural protein source. The aim of this study was to evaluate if the culture medium subjected to nanoparticle depletion can support the proper in vitro development of bovine embryos. Zygotes were cultured in groups on depleted or control medium for 8 days. Nanoparticles from the medium were characterized by their morphology, size and expression of EVs surface markers. Isolated nanoparticles were labelled and added to depleted medium containing embryos at different developmental stages and evaluated after 24 hours at 2, 8-16 cells, morula and blastocyst stages. There were no statistical differences on blastocyst rate at day 7 and 8, total cell count neither blastocyst diameter between groups. However, morphological quality was better in blastocysts cultured in non-depleted medium and the expression of SOX2 was significantly lower whereas NANOG expression was significantly higher. Few nanoparticles from medium had a typical morphology of EVs but were positive to specific surface markers. Punctuated green fluorescence near the nuclei of embryonic cells was observed in embryos from all developmental stages. In summary, nanoparticles from culture medium are internalized by in vitro cultured bovine embryos and their depletion affects the capacity of medium to support the proper embryo development.

Evaluation of extracellular vesicles and gDNA from culture medium as a possible indicator of developmental competence in human embryos.

Human embryos generated in vitro have a high incidence of chromosomal abnormalities that negatively affect pregnancy rate. Embryos generated in vitro secrete extracellular vesicles (EVs) into the culture medium that could be used potentially as indicators of embryo competence. This research aimed to evaluate the concentration and size of EVs and their gDNA content as an indicator of developmental competence in human embryos. Human embryos generated by intracytoplasmic sperm injection (ICSI) were classified morphologically as of either TOP, FAIR or POOR quality. Culture medium and developmentally arrested embryos (which were not able to be used for embryo transfer) were collected. Microvesicles, exosomes (MV/Exo) and apoptotic bodies (ABs) were isolated from culture medium. Nanoparticle tracking analysis (NTA) and array comparative genomic hybridization (aCGH) analysis were performed to evaluate EVs and their gDNA content. From NTA, the diameter (mean) of MVs/Exo from TOP quality embryos was higher (112.17 nm) compared with that of FAIR (108.02) and POOR quality embryos (102.78 nm) (P < 0.05). aCGH analysis indicated that MVs/Exo and ABs carried gDNA with the presence of 23 chromosome pairs. However, when arrested embryos were compared with their respective MVs/Exo and ABs, the latter had an increased rate of chromosomal abnormalities (24.9%) compared with embryos (8.7%) (P < 0.05). In conclusion, the size of EVs from culture medium might be an alternative for evaluating competence of human embryos, however more studies are needed to validate the use of gDNA from EVs as an indicator of embryo competence.

MicroRNAs from Extracellular Vesicles Secreted by Bovine Embryos as Early Biomarkers of Developmental Competence.

During early development, embryos secrete extracellular vesicles (EVs) that participate in embryo-maternal communication. Among other molecules, EVs carry microRNAs (miRNAs) that interfere with gene expression in target cells; miRNAs participate in embryo-maternal communication. Embryo selection based on secreted miRNAs may have an impact on bovine breeding programs. This research aimed to evaluate the size, concentration, and miRNA content of EVs secreted by bovine embryos with different developmental potential, during the compaction period (days 3.5-5). Individual culture media from in vitro-produced embryos were collected at day 5, while embryos were further cultured and classified at day 7, as G1 (conditioned-culture media by embryos arrested in the 8-16-cells stage) and G2 (conditioned-culture media by embryos that reached blastocyst stages at day 7). Collected nanoparticles from embryo conditioned culture media were cataloged as EVs by their morphology and the presence of classical molecular markers. Size and concentration of EVs from G1 were higher than EVs secreted by G2. We identified 95 miRNAs; bta-miR-103, bta-miR-502a, bta-miR-100, and bta-miR-1 were upregulated in G1, whereas bta-miR-92a, bta-miR-140, bta-miR-2285a, and bta-miR-222 were downregulated. The most significant upregulated pathways were fatty acid biosynthesis and metabolism, lysine degradation, gap junction, and signaling pathways regulating pluripotency of stem cells. The characteristics of EVs secreted by bovine embryos during the compaction period vary according to embryo competence. Embryos that reach the blastocyst stage secrete fewer and smaller vesicles. Furthermore, the loading of specific miRNAs into the EVs depends on embryo developmental competence.