56 Nobiletin affects gene expression profiles of the ERK1/2 pathway in bovine embryos produced in vitro

Abstract

During embryo development the embryonic genome activation (EGA) is one of the most important events and in bovine embryos it occurs at the 8- to 16-cell stage. In vitro embryo production increases the levels of reactive oxygen species (ROS), which leads to the low quality of the produced blastocysts, possibly by affecting EGA. Nobiletin is an antioxidant that affects cell cycle regulation (Huang et al. 2016 Evid. Based. Complement. Alternat. Med. 2016, 2918796, https://doi.org/10.1155/2016/2918796). Therefore, we aimed to evaluate the effect of nobiletin supplementation, in two key periods of early embryo development, on blastocyst yield and expression of selected genes of the ERK1/2 pathway and oxidative stress on produced embryos. In vitro zygotes were cultured in synthetic oviductal fluid (SOF) with 5% fetal calf serum (control, C); C with 5 or 10 µM nobiletin (MedChemExpress) (N5, N10); or C with 0.03% dimethyl sulfoxide (C_DMSO; vehicle for nobiletin dilution) during the minor (21–54 h post-insemination (hpi): 2- to 8-cell; MN_EGA; 12 replicates) or major (54–96 hpi: 8- to 16-cell; MJ_EGA; 10 replicates) phase of EGA. The speed of development was considered and embryos that reached ≥8 cells at 54 hpi from MN_EGA phase and ≥16 cells at 96 hpi from MJ_EGA phase, were selected and further cultured in control medium until Day 7. Embryos at ≥8 cell (MN_EGA), ≥16 cell (MJ_EGA) stage, and Day 7 blastocysts from both periods were snap-frozen in liquid N₂ for gene expression analysis (3 pools of 10 embryos/treatment). The expression of genes related to ERK1/2 pathway (H3–3B, H3–3A, NFE2L2) and oxidative stress (GPX1) were measured by quantitative PCR; H2AFZ and ACTB were used as housekeeping genes. Statistical analysis was assessed by one-way ANOVA. At 54 hpi, irrespective of nobiletin supplementation, no differences were found in the proportion of embryos that reached the 8-cell stage between groups in both phases (≈60%). At 96 hpi, nobiletin during MJ_EGA showed a higher proportion of embryos reaching the 16-cell stage than control groups (≈70% vs. ≈60%, respectively; P < 0.001). Blastocyst yield for MN_EGA and MJ_EGA was higher (P < 0.001) for N5 (40.0 ± 0.8% and 46.7 ± 0.8%) and N10 (41.0 ± 0.9% and 54.5 ± 1.1%) compared with C (32.0 ± 0.6% and 38.4 ± 1.1%) and C_DMSO (31.2 ± 0.4% and 35.8 ± 1.0%) groups, while N10 was higher (P < 0.05) compared to N5 group in MJ_EGA. The expression of H3–3B and H3–3A were higher (P < 0.05) in 8-cell embryos from N5 and N10 groups during MN_EGA; while in 16-cell embryos, H3–3B and NFE2L2 were higher (P < 0.05) only in the N10 group compared with both controls during MJ_EGA. GPX1 was upregulated in nobiletin-supplemented groups from both phases (8- and 16-cell embryos and blastocysts) compared with controls (P < 0.05). In conclusion, nobiletin supplementation during minor or major EGA has a positive effect in pre-implantation embryo development and modifies the transcription of cell cycle and oxidative stress genes in early embryos. These benefits can be attributed to its bioactivity and indicate that it might be a tool to overcome EGA and ROS disorders in bovine in vitro-produced embryos.

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