Extracellular vesicles from oviductal and uterine fluids supplementation in sequential in vitro culture improves bovine embryo quality

Abstract

Background: In vitro production of bovine embryos is a well-established technology, but the in vitro culture (IVC) system still warrants improvements, especially regarding embryo quality. This study aimed to evaluate the effect of extracellular vesicles (EVs) isolated from oviductal (OF) and uterine fluid (UF) in sequential IVC on the development and quality of bovine embryos. Zygotes were cultured in SOF supplemented with either BSA or EVs-depleted fetal calf serum (dFCS) in the presence (BSA-EV and dFCS-EV) or absence of EVs from OF (D1 to D4) and UF (D5 to D8), mimicking in vivo conditions. EVs from oviducts (early luteal phase) and uterine horns (mid-luteal phase) from slaughtered heifers were isolated by Size Exclusion Chromatography. Blastocyst rate was recorded on Days 7-8 and their quality was assessed based on lipid contents, mitochondrial activity and total cell numbers, as well as survival rate after vitrification. Relative mRNA abundance for lipid metabolism related transcripts and levels of active hormone-sensitive lipase (pHSL) proteins were also determined. Additionally, the expression levels of 383 miRNA in OF- and UF-EVs were assessed by pRT-PCR. Results: Blastocyst yield was lower (p<0.05) in BSA groups compared to dFCS groups. Survival rates after vitrification/warming were improved in dFCS-EVs compared to dFCS (p<0.05). EVs increased (p<0.05) blastocysts total cell number in dFCS-EV and BSA-EV compared to dFCS and BSA groups, while lipid content was decreased and mitochondrial activity increased in dFCS-EV (p<0.05). Lipid metabolism transcripts were affected by EVs ( PPARGC1B and ACACA , p<0.05) and protein source in the medium ( CD36, PLIN2 and ATGL , p<0.05). Levels of pHSL were lower in dFCS (p<0.05). Twenty miRNA were differentially expressed between OF- and UF-EVs and only bta-miR-148b was increased in OF-EVs (p<0.05). Conclusions: Mimicking physiological conditions using EVs from OF and UF in sequential IVC does not affect embryo development but improves blastocyst quality regarding survival rate after vitrification/warming, total cell number, lipid content, mitochondrial activity and relative changes in expression of lipid metabolism transcripts and lipase activation. Besides, EVs miRNA contents may contribute to the observed effects.

Serum-free spontaneously immortalized bovine oviduct epithelial cell conditioned medium promotes the early development of bovine <i>in vitro</i> fertilized embryos

“…Considering that no crosstalk was present between the embryos and SI-BOEC, which were used only for medium preparation, there may be some factors exerted in the conditioned medium, which were, once internalized in embryos, able to generate positive effects without further necessary interactions with oviduct cells. Extracellular vesicles derived from the bovine oviduct are known to deliver various molecules such as mRNAs, microRNAs, proteins, and lipids [8], and these molecules influence the quality [28] and yield of blastocysts [29,30]. The positive influence of the extracellular vesicles was also observed in various intra-embryo activities of the resulting embryos, such as regulation of transcription [31], modulation of phospholipid composition [32], and improvement of the TCA-cycle function [25].…”

Messenger roles of extracellular vesicles during fertilization of gametes, development and implantation: Recent advances

“…Current research indicates that O-EVs can improve embryo blastocyst formation rate, embryo survival rate, embryo hatching rate, and post-embryo transfer birth rate (Almiñana et al, 2017;Qu et al, 2019;Fang et al, 2022b;de Alcântara-Neto et al, 2022). Furthermore, O-EVs can improve embryo development after cryopreservation and survival rate after vitrification (Lopera-Vasquez et al, 2017;Leal et al, 2022;Sidrat et al, 2022). More than just O-EVs may influence embryo development in vivo.…”

“…EVs have been shown to improve the survival rate of frozen embryosLopera-Vasquez et al (2017) Bovine O-EVs can improve the re-expansion rate and hatching rate of frozen-thawed embryos, which may be related to O-EVs' ability to restore trophectoderm integrity and improve junctional cell contact and fluid fluxSidrat et al (2022) After vitrification, freezing, or heating, bovine O-EVs improved embryo survival, lipid content, and total cell numberLeal et al (2022) Pregnant mice's uterine cavity contains more I-EVs and higher levels of miRNA-21 than non-pregnant mice's uterine cavity. miRNA-21 may be involved in embryonic developmentI-EV Lv et al (2018)Cattle with endometritis have I-EVs that differ from normal I-EVs and are harmful to embryonic developmentWang et al (2019) EV secreted from the bovine blastocyst stage contains miRNA-378a-3p, which has been shown to increase the number of embryonic trophoblast cells and inner cells and improve hatching rate EO-EVPavani et al (2022) Mouse SP-EVs can improve blastocyst formation rate and ICM/TE index in sperm/oocyte mixed medium, as well as reduce embryonic cell apoptosis SP-EVMa et al (2022) Embryo implantation I-EVs are produced during pregnancy in pigs by both embryonic trophoblast cells and endometrial epithelial cells, but primarily by endometrial epithelial cells.…”