87 Pharmacological inhibition of the AKT pathway during the late stages of bovine embryo culture: Underlying mechanisms of actions

The PI3K-AKT pathway is a regulator associated with numerous biological processes crucial for cell death and growth. SH6 is a specific AKT inhibitor that has been reported to negatively affect embryo viability; however, the underlying molecular mechanisms of AKT during embryogenesis remain unclear. The current study aimed to demonstrate the impact of SH6 on embryo development, oxidative stress, and epigenetic regulation when treatment occurred at late embryo culture stages (Day 6 post-fertilization). We first checked the impact of SH6 administration on the in vitro production and hatching rates of bovine embryos at Day 8 post-fertilization. Also, the underlying mechanisms of the AKT inhibition on apoptosis, autophagy, DNA methylation, and developmental indicators were assessed at both transcriptional and translational levels using RT-qPCR and immunofluorescence assay, respectively. The differences between experimental groups (n = 15–20 blastocysts) were analyzed using ANOVA test, and P-values less than 0.05 were considered statistically significant. Results indicated that embryo exposure to SH6 at different concentrations (20, 40, and 60 µM) remarkably retarded blastocyst production by 19.4 ± 1.78, 14 ± 1.38, and 8 ± 1.05, respectively, as compared with 31.8 ± 1.77 in control embryos. The exposure also decreased their hatching ratios in a dose-dependent manner (26.6 ± 4.63, 12.2 ± 3.72, and 0 ± 0) for the 20, 40, and 60 µM SH6 treatments compared with the control (53.2 ± 3.22). RT-qPCR revealed an increase in the mRNA levels of caspase-3, LC3B, ATG7, and beclin-1 apoptosis and autophagy markers, and the DNA methyltransferase 1 (DNMT1) gene in the 40 µM SH6-embryos. Downregulation was observed for DNA methyltransferase 3 α (DNMT3A) and β (DNMT3B) genes and for phosphofructokinase 1 (PFK1), GATA-binding protein 6 (GATA6), NADH dehydrogenase subunit 2 (ND2), and occludin (OCC) blastocyst development markers in the 20 µM SH6 group. Based on immunofluorescence, the ROS and LC3B levels were greatly increased, while the SOX2 and Brdu proliferation markers were downregulated in 20 µM SH6-embryos, unlike the untreated blastocysts. In conclusion, we elucidated the impact of SH6 administration during late embryo culture and the critical role of AKT signaling in preserving embryo viability via the direct impact on blastocyst development, proliferation, survival, and epigenetics.