73 In vivo-derived bovine blastocysts secrete different population of extracellular vesicles compared to in vitro-produced counterparts

Bovine embryos secrete extracellular vesicles (EVs) that vary according to embryo competence. On the other hand, in vivo-produced embryos have better developmental potential than in vitro-produced counterparts. This work aims to evaluate the effect of embryo origin on the characteristics of EVs secreted during blastulation and hatching periods. Bovine embryos were produced by in vitro fertilisation (IVP) or collected from superovulated cows (in vivo [IVV]). The developmental windows were blastulation, Day 5–7; hatching, Day 7–9. For blastulation period, IVV and IVP morulae were cultured individually in 50 µL of EV-depleted medium (synthetic oviduct fluid supplemented with FBS and bovine serum albumin [SOFd]) until Day 7. At D7, culture media (CM) were collected from grade I blastocysts. For hatching period, IVV and IVP grade I blastocysts were cultured individually in SOFd for 48 h. At D9, CM from hatched blastocysts were collected. D7 and D9 blastocysts were kept in culture until Day 11 to assess their post-hatching survival and growth. Only CM of hatched blastocysts and those larger than 270 μm were finally used for EVs analysis. Four experimental groups were considered for analysis: IVP5–7 n = 64; IVP7–9 n = 92; IVV5–7 n = 32; IVV7–9 n = 45. Nanoparticles from individual CM were isolated and characterised as EVs by the presence of CD9, CD81, and CD63, and the morphology. EVs size and concentration were determined by nanoparticles tracking analysis (NTA). Embryos from IVV7–9 reached the largest diameter at D11 followed by IVP7–9, being smaller the embryos from the blastulation group (IVP5–7: 381.9 ± 97.5; IVV5–7: 345.7 ± 107.3; IVP7–9: 471.7 ± 77.2; IVV7–9: 508.7 ± 90.6 μm). IVV5–7 embryos released bigger EVs (EVs size: IVP5–7 124.7; IVV5–7 148.5; IVP7–9 118.1; IVV7–9 115.4 nm), while the IVP7–9 embryos released the highest quantity of EVs (EVs concentration: IVP5–7 2.9 × 10⁹; IVV5–7 2.6 × 10⁹; IVP7–9 3.8 × 10⁹; IVV7–9 2.2 × 10⁹). After NTA, EVs from individual embryos were pooled to organise at least three replicates per experimental group for miRNA analysis using RNAseq. Differential miRNA contents were identified in EVs from the experimental groups (log2FC > 1.2, FDR < 0.05). The PCA showed a greater intergroup separation between IVV and IVP groups at blastulation and less intergroup separation at hatching period. There was a higher miRNA content in EVs secreted during the blastulation compared to hatching period in either IVV or IVP embryos. However, IVP embryos secreted EVs with a higher miRNA content at both stages. The miRNAs in EVs secreted during hatching period was similar between IVV and IVP embryos but there were three miRNAs, MIR6529A, MIR6529B, and MIR30D, exclusively detected in IVV7–9. It was concluded that developmental stage and embryo origin impact EVs secretion in bovine embryos. The specific miRNAs identified in EVs secreted during hatching by IVV embryos might be a point for a mechanism of early embryo maternal communication.

This research was supported by Fondecyt 1210334 and ANID 21201280.