95 Culture medium with reduced nutrient concentrations alters the mTORC1 and AMPK pathway activities in bovine blastocysts

Our previous studies demonstrated improved blastocyst formation when bovine embryos were cultured in the in-house prepared culture medium with nutrient concentrations diluted to 6.25% of standard medium and supplemented with exogenous lipid and l-carnitine. Single-cell transcriptomic analysis revealed that the improved blastocyst formation in reduced nutrient culture condition is correlated with less active cellular metabolism and biosynthesis processes, increased autophagy, and transmembrane transport activities in these embryos. The current study aims to investigate the metabolism differences between the embryos derived in the standard culture medium (IVC) and the medium with reduced nutrients (IVR). The abattoir-derived COCs were matured in BO-IVM for 22 h, fertilized in BO-IVF for 20 h, and at least 100 presumptive zygotes were randomly assigned to IVC or IVR medium for in vitro culture until Day 8 under 6% CO₂ and 5% O₂ condition. Expanded and hatched blastocysts were collected for the downstream analysis. All the experiments were conducted in triplicates. Data were presented as mean ± standard error of the mean and analysed by Student’s test. The reactive oxygen species (ROS) live cell imaging was conducted by staining blastocysts with DCFDA. Images were taken using confocal fluorescent microscopy, and fluorescent intensities were analysed using ImageJ software. The ROS level was significantly higher in IVC-blastocysts compared to the IVR-blastocysts, suggesting elevated metabolic activities and oxidative stress in the IVC-blastocysts (1.00 ± 0.34 in IVR-blastocysts vs 6.40 ± 1.76 in IVC-blastocysts, P < 0.01). The mTORC1 and AMPK signaling activities were examined by measuring the ratio of phosphorylated and total proteins of P70S6, a mTORC1 downstream target, and AMPK, using the capillary JESS western blot. The IVR-blastocysts had 2.41 ± 0.40 (P < 0.05) times AMPK activity and 0.37 ± 0.07 (P < 0.01) times mTORC1 activity compared to the IVC-blastocysts. The significantly lower mTORC1 activity may explain the reduced metabolic and biosynthetic processes in the IVR-blastocysts, while the higher AMPK activity in IVR-blastocysts is related to a more active hydrolysis and autophagy processes identified in the single-cell transcriptomic analysis. The correlation between the pathway activities and single-cell transcriptomic analysis was further validated by examining genes related to mTORC1 (INSIG1, CD81, IDI1, GAMT, GSTP1) and AMPK (CLU, GAA, PPARGC1, and TGM2) signaling pathways, in which mTORC1 signaling related genes were up-regulated in the IVC-blastocysts, and AMPK signaling related genes were up-regulated in the IVR-blastocysts. In conclusion, the current study provided functional evidence to support our single-cell transcriptomic analysis. Altered mTORC1 and AMPK signaling pathways may play an essential role in embryo metabolic changes and contribute to the improved embryo development in medium with reduced nutrients.