57 Proteomic analysis reveals metabolic dysregulation in in vitro-cultured bovine embryos

Abstract

In vitro culture (IVC) systems fail to completely recapitulate the in vivo environment, resulting in metabolic stress during pre-implantation development and reduced blastocyst quality. We hypothesised that IVC-induced metabolic dysregulation in bovine embryos is mediated by changes in expression and/or activity of protein biomarkers associated with key metabolic pathways. Our objectives were to determine (1) expression of enzymes involved in glycolysis (HK-2, PKM2, LDHA, B and C isoforms), entry into the tricarboxylic acid (TCA) cycle (PDH), energy sensing/fatty acid oxidation (AMPK), and the metabolic signalling pathways (AKT, ERK, STAT3, 4EBP1) at the 1-cell (1C), 8- to 16-cell (8–16 C), and Day 7 blastocyst (d7BL) stage; and (2) evaluate the functional activity of these proteins both in vivo (superovulated and flushed) and in vitro (IVM/IVF/IVC) produced embryos using capillary Western blot (Protein-Simple, JESS; n = 1 embryo/stage; n = 3 replicates). For each protein, expression was normalized with total protein abundance in the same capillary and functional activity was determine based on the ratio of phosphorylated (p) to total (t) protein abundance in each sample. Data were analysed using a two-sample t-test. Results demonstrated significantly (P < 0.05) decreased LDHB expression at 1C, decreased functional activity of PDH at 8–16 C, and a trend (P < 0.09) for decreased activity of PDH and PKM2 enzymes in 1C embryos produced in vitro. These results suggest a reduced ability of PKM2 to produce pyruvate in glycolysis, as well as reduced ability of LDHB to reversibly convert pyruvate into lactate and of PDH to convert pyruvate into acetyl-CoA for metabolism in the TCA cycle, indicating an overall slowing of aerobic metabolism. In contrast, expression of STAT3 and ERK1/2 in all stages examined, AKT in 8–16C and d7BL, and 4EBP1 in d7BL were significantly (P < 0.05) higher in IVP embryos. In addition to expression, decreased (P < 0.05) activity of ERK1/2, AKT, and 4EBP1 signalling at 1C, and a trend (P < 0.08) for decreased expression of 4EBP in 8–16C and d7BL produced in vitro was observed. Activated AKT signalling enhances glucose uptake by stimulating hexokinase. Because the activity of glycolytic enzymes (PKM2, LDHB, PDH) is reduced at the 1-cell stage in vitro, these embryos may be shifting metabolism to the pentose phosphate pathway, which might increase the ability of the embryo to protect against oxidative stress induced by the IVC environment. We observed a remarkable change in metabolic enzyme expression and activity in vitro as early as the 1C stage, suggesting that bovine embryos are highly susceptible to metabolic stress even at this early stage of development. Collectively, these results point to specific abnormalities of metabolism in IVP embryos and suggest that differentially expressed proteins and their functional activity can be used as biomarkers in optimizing culture conditions to produce high-quality embryos in vitro that more closely resemble their in vivo counterparts.