LAUSR

Maternal obesity can impair offspring metabolic health, however the precise mechanism underpinning programming is unknown. Ten-Eleven translocase (TET) enzymes demethylate DNA using the TCA cycle intermediary α-ketoglutarate and may be involved in programming offspring health. Whether TETs are disrupted by maternal obesity is unknown. 5-6 week old C57Bl/6 female mice were fed a control diet (CD; 6% fat, n=175) or a high-fat diet (HFD; 21% fat, n=158) for six weeks. After superovulation oocytes were collected for metabolic assessment, or females were mated and zygotes cultured for embryo development, foetal growth, and assessment of global DNA methylation (5mC, 5hmC, 5fC and 5caC) in the 2-cell embryo. Zygotes collected from superovulated CBAF1 females were cultured in media containing α-ketoglutarate (0mM, 1.4mM, 3.5mM, or 14.0mM) or with 2-hydroxyglutarate (2HG) (0mM or 20mM), a competitive inhibitor of α-ketoglutarate, with methylation and blastocyst differentiation assessed. After HFD, oocytes showed increased pyruvate oxidation and intracellular ROS, with no changes in Tet3 expression, while 2-cell embryo global 5hmC DNA methylation was reduced and 5fC increased. Embryos cultured with 1.4 mM α-ketoglutarate had decreased 2-cell 5mC, whilst 14.0mM α-ketoglutarate increased the 5hmC:5mC ratio. In contrast supplementation with 20 mM 2HG increased 5mC and decreased the 5fC:5mC and 5caC:5mC ratios. α-ketoglutarate up to 3.5mM did not alter embryo development, whilst culturing in 14.0mM α-ketoglutarate blocked development at the 2-cell. Culture with 2HG delayed embryo development past the 4-cell and decreased blastocyst total cell number. In conclusion, disruptions in metabolic intermediates in the preimplantation embryo may provide a link between maternal obesity and programming offspring for ill health.