104 Differences in genes representation in extracellular vesicles according to bovine embryo quality

Extracellular vesicles (EVs) are membrane-enclosed nanoparticles secreted by all cells, including embryonic cells. These nanoparticles transport molecules such as proteins, lipids, small RNAs, mRNA, and DNA fragments. Recently, DNA has also been detected in EVs released by preimplantation bovine embryos. This finding is relevant because the DNA in EVs could be used as a noninvasive biopsy for embryonic genotyping. However, there is a relationship between DNA quantity in embryonic EVs, the origin of the embryo (IVF or parthenogenesis), and embryonic competence. The aim of this study was to determine if the DNA cargo (gene representation) in EVs from bovine preimplantation embryos differs in relation to the embryos’ competence to reach the blastocyst stage. Bovine embryos were produced by IVF and cultured in groups until Day 5. Then, morulae were transferred to individual culture in EV-depleted medium until Day 7. Subsequently, on Day 7, the media from blocked morulae (M-EV, n = 18) and from blastocysts (B-EV, n = 20) were individually collected for isolation of EVs by ultrafiltration using an Amicon Filter (10 kDa, 0.5 mL). Characterization of the EVs was performed by determining the presence of protein markers (CD63 and Alix) using western blot analysis, the size and concentration by nanoparticle tracking analysis, and the morphology by transmission electron microscopy. Samples were then treated with DNase I to avoid external DNA contamination. For DNA extraction, a lysis buffer from a commercial kit (Ambion) was used. DNA in EVs was quantified using a Quant-iTTM PicoGreen® dsDNA Assay Kit. Representative samples (M-EV, n = 3; B-EV, n = 3) were used for Next Generation Sequencing analysis (Novogene Co., Ltd.). Owing to the low quantity of DNA (picograms), the samples were amplified using a Whole Genome Amplification kit. The data were analyzed using FASTP software to detect the quantity and quality of the reads and for base repair (trimming). Alignment was performed using BWA software with the ARS-UCD1.2 reference genome. A Venn diagram was created for B-EV and M-EV, followed by a Gene Ontology analysis. The mean DNA concentration was 62 ± 14 ng µL⁻¹ for M-EVs and 73 ± 23 ng μL⁻¹ for B-EVs. Quality analysis showed that DNA in EVs was highly fragmented. In M-EVs, more than 80% of reads had identity with the bovine genome compared with only 20% in B-EVs. A total of 422 genes were identified in B-EVs, 195 were unique to this stage, and 107 genes were related to the term “Cytoplasm” in the cellular compartment. A total of 3709 genes were found in M-EVs, with 3482 unique to this group. Highlighted terms included “Development Process” (1180 genes), “Anatomical Structure” (1092 genes), and “Multicellular Organism Development” (905 genes), all derived from “Biological Process.” The most represented genes in M-EVs were IL17RC, SEMA5B, ANKRD55, and KIF2C, and those in B-EVs were DDAH1, ADAM12, PCDH15, and SCAF1. It was concluded that M-EVs had a higher DNA content compared with B-EVs. This higher content affects the number of genes that could be detected in embryo-derived EVs, and consequently, the use of EVs as a biopsy for genomic analysis of individual embryos.

This study was funded by Fondecyt No. 1210334.