2 Noninvasive method for bovine embryo sexing through the analysis of DNA content in extracellular vesicles

The presence of free DNA has been demonstrated in the culture media of in vitro-produced embryos of different species. This DNA has been used for preimplantation genetic diagnosis in the human clinic, avoiding the invasive nature of embryo biopsy. However, it is difficult to discriminate embryo-derived DNA (E-DNA) from cumulus cells or sperm remnants, affecting the reliability of the results. Extracellular vesicles (EVs) are membrane-enclosed nanoparticles secreted by cells that contain DNA, among other molecules. It seems that embryonic DNA might be released within EVs. EVs can be classified as small vesicles mainly enriched in exosomes and microvesicles that are released by “healthy” cells, whereas apoptotic bodies are vesicles more heterogeneous in size that are produced following cell death. In this sense, we hypothesised that E-DNA secreted withing small vesicles could be used for bovine embryo sexing. Bovine embryos were produced by in vitro fertilisation and cultured in groups in synthetic oviducal fluid (SOF) medium up to the morula stage. Afterwards, morulae were individually cultured in 50 µL of EV-depleted medium (SOFdep) up to the blastocyst stage. SOFdep was prepared by ultrafiltration of complete SOF using centrifugal filter devices (100 kDa for 15 min at 1660 × g). Both blastocyst and its respective culture medium were individually collected and properly identified. E-EVs in culture medium were separated using a density gradient (Optiprep; Stemcell Technologies), recovering only the 1.06 g/cm³ fraction to obtain small EVs enriched in exosomes. The EV population had a mean size of 187.2 ± 5.2 nm, a concentration of 4.16e + 008 ± 5.45e + 007 particles mL⁻¹, was positive to CD9, and had a classical EV morphology. The recovered E-EVs were treated with DNase to remove bound DNA outside the EV membrane. Both embryos and E-EVs were treated with cell lysis buffer (ThermoFisher Scientific) for DNA extraction. A segment of SAT1 (a multicopy gene) and SRY (a sex ligand gene) were amplified by real-time PCR. Twelve embryos and their respective EVs were analysed. DNA from bovine tissue, female and male, was included as controls in the PCRs. SAT1 was amplified in all samples (embryo and EVs) indicating the presence of DNA in E-EVs. SRY was amplified in three of the 12 samples, which indicated three male embryos among the samples. However, SRY was amplified only in 1 of 12 EV samples, coinciding with a male embryo. Overall, 83.3% sex concordance was obtained between the embryo and its respective EV sample. No SRY amplification was obtained in EVs from two of the male embryos while SAT1 was amplified. This is most likely due to the low amount of DNA in these samples, which could be solved by including a previous step of whole-genome amplification. We concluded that bovine embryos released DNA inside small EVs, and this DNA can potentially be used for embryo sexing.

This research was supported by FONDECYT No. 11210334, Chile.