52 Extracellular vesicles from oviduct and uterus in sequential in vitro culture affects mitochondrial activity and lipid metabolism transcripts in bovine embryos

Abstract

Oviducal fluid (OF) and uterine fluid (UF) improve the quality of embryos during in vitro culture, and their extracellular vesicles (EV) may be involved in such an effect. We aimed to evaluate the effect of EV from OF and UF in sequential in vitro culture on the development and quality of bovine embryos. Zygotes were cultured in synthetic oviduct fluid supplemented either with 3 mg mL⁻¹ BSA (n = 1584) or 5% EV-depleted fetal calf serum (dFCS, n = 1594) in absence or presence (BSA^EV, n = 1853 and dFCS^EV, n = 1473) of 3 × 10⁵ EV mL⁻¹ from OF (Day 1 to Day 4) and UF (Day 5 to Day 8), mimicking in vivo conditions. EV from oviducts (early luteal phase) and uterine horns (mid luteal phase) from slaughtered heifers were isolated by size exclusion chromatography; size and concentration were assessed by nanotracking analysis (NTA) and morphology by transmission electron microscopy (TEM). Blastocyst rate was recorded on Days 7–8 and their quality was assessed for mitochondrial activity by staining with Mitotracker Deep Red (ThermoFisher Scientific), survival rate after vitrification/warming by in vitro culture for up to 72 h, and relative mRNA abundance of lipid metabolism-related transcripts by quantitative PCR. Housekeeping genes were H2AFZ and ACTB. Data were analysed by one-way ANOVA and Tukey test. TEM confirmed the presence and morphology of EVs, and NTA indicated mode size and concentration of particles (137.2 and 151.2 nm; 2.97 × 10¹⁰ and 7.98 × 10¹⁰ particles mL⁻¹, for OF and UF, respectively). Blastocyst yield was lower (P < 0.05) in BSA groups compared with dFCS groups (BSA: 16.2 ± 1.5 and 31.0 ± 1.9; BSA^EV: 14.1 ± 1.6 and 26.2 ± 2.0% vs. dFCS: 30.5 ± 2.0 and 40.6 ± 2.4; dFCS^EV: 31.1 ± 2.5 and 39.8 ± 2.7%, Day 7 and Day 8, respectively), irrespective of EV supplementation. Blastocyst mitochondrial activity was increased (P < 0.05) by EV in dFCS^EV compared with the other groups. No differences in survival rate after vitrification/warming were found (range at 72 h: 67.1 ± 8.1 to 87.8 ± 5.7%). PPARGC1B was downregulated and ACC upregulated by EV, irrespective of protein source in medium (P < 0.05). In contrast, EV affected some transcripts depending on the protein source in the medium (CD36 upregulated in dFCS^EV, downregulated in BSA^EV; PLIN2 downregulated in dFCS^EV and ATGL downregulated in BSA^EV, P < 0.05). In conclusion, mimicking physiological conditions using EV from OF and UF in sequential IVC does not affect development but improves embryo quality by increasing blastocysts’ mitochondrial activity and favours the expression of specific lipid metabolism transcripts. Functional effects of EV may be influenced by the protein source in the medium.

This research was funded by MINECO-Spain AGL2015-70140-R, PID2019-111641RB-I00, RTI2018-093548-B-I00; YN Cajas, SENESCYT-Ecuador; CLV Leal, FAPESP-Brazil 2017/20339-3, CNPq-Brazil 304276/2018-9.