The work performed during the project is explained, according to the description of the action (DoA) originally proposed, and any deviation from the original plan has been included.
Experiment 1. Analysis of oEVs derived from bovine oviduct epithelial cells in response to the presence of absence of embryos and their embryo quality (poor vs good embryos)
In vitro co-culture of bovine oviduct epithelial cells (BOEC) with or without good or poor-quality bovine embryos was performed in this experiment. Additionally, embryos and BOEC were cultured alone. This experiment was conducted in collaboration with Dr. Rizos at the National Institute for Agriculture and Food Research and Technology (INIA), Department of Animal Reproduction, Madrid, Spain). Culture media (CM, containing the EVs) and embryos were analyzed in the laboratory at the University of Zurich. The EVs were successfully isolated from the CM and characterized using electron microcopy, Western blot, flow-cytometry, and nanoparticle tracking analysis. Subsequently, small regulatory RNAs, so-called microRNAs (miRNAs) contained in the EVs and embryos were analyzed by RNA-sequencing.
Overall, a total of 84 different miRNAs were identified in EVs and 187 in embryos. An overlap of 64 miRNAs was found between EVs and embryos, with 20 unique for EVs and 123 for embryos. The data analysis showed that the miRNA cargo of the EVs secreted by the BOEC varies depending on the embryo presence or absence. Besides, it showed changes depending on embryo quality. In addition, the miRNA profile of the early embryo is different in co-culture with BOEC or alone. Interestingly, this effect of the cells on the embryo miRNA profile is depending on the embryo quality.
Experiment 2. Bioengineered oEVs: loading EVs with regulatory RNAs to assess their ability for targeted delivery and improvement of assisted reproductive technologies
The idea of this experiment was to load in vitro derived oEVs with important regulatory RNAs and perform a transfer of these molecules via oEVs to BOEC, sperm, and embryos. We selected the miRNA miR-449a and the messenger RNA (mRNA) for OVGP1, which are known for their role in the oviduct during the passage of gametes and embryos, and which are lacking in the in vitro conditions. We developed a successful method for in vitro loading of oEVs with miR-449a and OVGP1 mRNA and showed the transfer into BOEC and sperm. The changes of the expression of selected genes (related to embryonic growth and development, oviduct metabolism, sperm-oviduct binding) in the BOEC after the oEV-mediated uptake of miR-449a indicated the potential of oEV as nanocarriers to improve ART conditions.
