Giant unilamellar vesicles (GUVs) are composed of lipophilic layers and are sensitive to the action of reactive oxygen species (ROS). The use of GUVs as microcarriers of biological macromolecules is particularly interesting since ROS produced by gametes or embryos during in vitro culture can induce the opening of pores in the membrane of these vesicles and cause the release of their content. This study investigated the behavior of GUVs [composed of 2-dioleoyl-sn-glycero-3-phosphocholine and 1,2-dioleoyl-sn-glycero-3-phosphoethanolamine-N-(lissamine rhodamine B sulfonyl)] in co-culture with in vitro produced bovine embryos, as well as their embryotoxicity and effectiveness as cysteine carriers in culture medium. Embryonic developmental rates were unaffected, demonstrating the absence of toxicity of GUVs co-cultured with the embryos. No increase of intracellular ROS levels was observed in the embryos co-cultured with GUVs, indicating that the higher lipid content of the culture environment resulting from the lipid composition of the GUV membrane itself did not increase oxidative stress. Variations in the diameter and number of GUVs demonstrated their sensitivity to ROS produced by embryos cultured under conditions that generate oxidative stress. Encapsulation of cysteine in GUVs was found to be more effective in controlling the production of ROS in embryonic cells than direct dilution of this antioxidant in the medium. In conclusion, the use of GUVs in in vitro culture was found to be safe since these vesicles did not promote toxic effects nor did they increase intracellular ROS concentrations in the embryos. GUVs were sensitive to oxidative stress, which resulted in structural changes in response to the action of ROS. The possible slow release of cysteine into the culture medium by GUV rupture would therefore favor the gradual supply of cysteine, prolonging its presence in the medium. Thus, the main implication of the use of GUVs as cysteine microcarriers is the greater effectiveness in preventing the intracytoplasmic increase of ROS in in vitro produced bovine embryos.

ContentsThe epidermal growth factor receptor (EGFR) pathway is directly involved in oocyte meiotic resumption induced by a gonadotropic stimulus. Here, we used an EGFR inhibitor (AG1478) to inhibit spontaneous meiosis resumption in bovine oocytes (EGFR− group) during 8 hr prematuration and assessed the competence of such oo- cytes for embryonic development, apoptosis and gene expression in comparison withControl group which was not prematured. Data are presented as mean ± SEM.Blastocysts rate on day 7 (40.81%, averaged) and hatching rate on day 9 (77.35%, averaged) were unaffected by treatment (p > 0.05). Similarly, treatment did not affect (p > 0.05) the total cell number on day 7 (119.05, averaged) and on day 9 (189.5, averaged). Apoptosis was reduced (p < 0.05) in EGFR− group day 7-embryos compared to Control group (3.7% ± 1.0 vs. 5.2% ± 0.8). Abundance of several transcripts was upregulated (p < 0.05) in EGFR− group, including genes related to embryo development and quality (NANOG and RPLP0), epigenetic regulation (H2AFZ), apoptosis (BID) and stress response (GPX4 and HIF1A). Taken together, the results presented here demonstrated a reduction in the apoptosis index and upregulation of NANOG, H2AFZ and RPLP0 mRNA levels, which are related to embryonic development. Our data suggest that temporary meiosis blockage with EGFR inhibitor during prematuration culture of bovine oocytes may be an interesting strategy to improve embryo quality. K E Y W O R D S apoptosis, embryo development, in vitro maturation | 667 CHEDIEK DALL'ACQUA Et AL.