76 Extracellular vesicles from oviduct and uterus in sequential culture improve the quality of bovine embryos produced in vitro

Abstract

Extracellular vesicles (EVs) are released by cells and transport cargo that affect functions of other cells. Oviductal fluid (OF) and uterine fluid (UF) have been shown to improve quality of embryos during in vitro culture (Hamdi et al. 2017 Reprod. Fertil. Dev. 30, 935-945) which may be due to their content of EV (Lopera-Vásquez et al. 2017 Reproduction 153, 461-470). Thus, the aim of this study was to evaluate the effect of EVs from OF and UF on a sequential in vitro culture system on the development and quality of bovine embryos. Zygotes were cultured in synthetic oviduct fluid (SOF) supplemented with 3 mg mL⁻¹ bovine serum albumin (BSA; n = 1228) or 5% EV-depleted fetal calf serum (dFCS, n = 1261) in the presence (BSA^EV, n = 1265 and dFCS^EV, n = 1253) or absence of 3 × 10⁵ EV mL⁻¹ from OF (Day 1 to Day 4) and UF (Day 4 to Day 9), mimicking in vivo conditions. The EVs pooled from 5 oviducts (early luteal phase) and 5 uterine horns (middle luteal phase) from slaughtered heifers were isolated by a Size Exclusion Chromatography kit (Hansa BioMed). The EV size and concentration were assessed by the nanotracking analysis system and morphology by transmission electron microscopy. Embryo development was recorded on Days 7/9. Day 7/8 blastocysts were assessed for quality by staining with (a) Hoechst 33342 (10 µg mL⁻¹, 30 min) for total cell number, (b) Bodipy 493/503 (20 µg mL⁻¹, 1 h) for lipid content (lipid droplet area in µm²), and (c) for survival rate after vitrification/warming. Data were analysed by one-way ANOVA and Tukey test. The EV concentration was 2.97 and 7.98 × 10¹⁰ particles mL⁻¹, and mode size 137.2 and 151.2 nm for OF and UF, respectively. Transmission electron microscopy confirmed EV presence and size, showing typical cup-shaped morphology. Blastocyst yield was lower (P < 0.05) on Day 7 in the BSA groups (BSA: 15.7 ± 1.9 and BSA^EV: 15.2.4%) compared with serum groups (dFCS: 28.1 ± 2.6 and dFCS^EV: 30.1 ± 2.9%) irrespective of EV supplementation; however, these differences were compensated at Days 8 and 9 (range: 30.0 ± 3.2-40.8 ± 3.9%). The EVs increased (P < 0.05) blastocyst total cell number in dFCS^EV (152.6 ± 2.9) and BSA^EV (140.5 ± 1.5) compared with dFCS (117.9 ± 2.0) and BSA groups (122.4 ± 1.1). However, lipid content was decreased (P < 0.05) in the presence of EVs only in dFCS^EV (0.231 ± 0.05 µm²) compared with BSA (0.393 ± 0.03 µm²) and BSA^EV (0.379 ± 0.03 µm²) groups. The dFCS did not differ from any group (0.371 ± 0.05 µm²; P > 0.05). Blastocyst survival after vitrification/warming was high in all groups up to 72 h (range: 80.0 ± 3.8-100%; P > 0.05). In conclusion, mimicking physiological conditions using EV from OF and UF during in vitro culture does not affect development but improves embryo quality by increasing blastocyst total cell numbers and decreasing lipid contents. These results provide evidence of the association of the reproductive tract environment and developing embryo, confirming embryo-maternal communication.

Funding was provided by MINECO-Spain AGL2015-70140-R; Y. N. Cajas, SENESCYT-Ecuador; C. L. V. Leal, FAPESP-Brazil 2017/20339-3.