Gene Expression Profile of In Vitro Produced Bovine Blastocysts Exposed to Hyperglycemia During Early Cleavage Stages: Link with the Warburg Effect.

Hyperglycemia affects pre-implantation embryonic development and in vitro culture of mammalian embryos shows defined period of glucose toxicity. During this period, embryos rely primarily on oxidative phosphorylation and increased glycolysis has been correlated with developmental block at the time of embryonic genome activation. Although improvements have been made to overcome this developmental block, in vitro culture still enhances early embryo metabolism and results in poor quality blastocyst. To gain insight into the impact of in vitro culture on embryo development, we studied how enhanced metabolism affects gene expression profile of bovine embryo. In vitro produced bovine zygotes were cultured in control (0,2 mM) or high glucose (5 mM) condition during early cleavage stages then transfer to control media until reaching the blastocyst stage. We selected a 5 mM glucose exposure as hyperglycemic treatment because glucose concentration in bovine oviduct is around 2 mM and in vitro dose response experiment showed embryo-lethality at 10 mM. Microarray technology was used to compare gene expression differences between control and treated blastocysts. Glucose treatment decreases significantly the blastocyst and hatching rate compared to control (22.8% vs 37.9% and 28.8% vs 43.8%, respectively). Microarray comparison reveals a total of 104 differentially expressed probes between treated and control blastocysts (1.5 fold-change; p<0.05), targeting 64 constitutive genes, 16 3'untranslated regions, 2 splice variants and 22 uncharacterised genes. Differentially expressed genes are not related to embryo sex or hatching. Over-represented processes among differentially expressed genes in treated blastocysts are cell-adhesion, oxidative stress and glucose metabolism. Functional analysis indicts a major up-regulation of genes controlling the Warburg effect i.e emphasis to use aerobic glycolysis rather than oxidative phosphorylation. Q-PCR experiments validate genes known as markers of the Warburg effect in cancer progression (LDHA,TKTL1) as well as genes involved in metabolic stress (PPARg,TP53BP2) and extracellular matrix remodelling (ADAMTS1). These observations allowed us to conclude that hyperglycemic condition during in vitro pre-attachment development modifies subsequent expression of genes involved in metabolic control in bovine blastocyst and that these modifications could be linked to embryo quality. This research is financed by NSERC-EmbryoGENE and the Institute of Nutraceuticals and Functional Foods.

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