Placental TonEBP/NFAT5 Osmolyte Regulation in an Ovine Model of Intrauterine Growth Restriction Short title: TonEBP/NFAT5 and osmolyte regulation in the ovine placenta.

TonEBP/NFAT5 (the tonicity-responsive enhancer binding protein/nuclear factor of activated T cells) modulates cellular response to osmotic changes by accumulating inositol and sorbitol inside the cells. Our objective was to assess placental osmolytes, TonEBP/NFAT5 RNA and protein expression, and signaling molecules across gestation, between control and intrauterine growth restriction (IUGR) ovine pregnancies. Pregnant sheep were placed in hyperthermic (HT) conditions to induce IUGR. Placental tissues were collected at 55, 95, and 130 days gestational age (dGA) to measure inositol, sorbitol, TonEBP/NFAT5 (NFAT5), sodium-dependent myoinositol transporter (SMIT, official symbol SLC5A3), aldose reductase (AR) and NADPH, official symbol DECR1). Placental weight was reduced in IUGR compared to controls at 95 and 130 dGA. Osmolyte concentrations were similar between control and IUGR placentas, but both groups demonstrated a significant decrease in inositol concentration and an increase in sorbitol concentration with advancing gestation. Cytosolic NFAT5 protein decreased significantly from 55 to 95 dGA in both groups and nuclear NFAT5 protein increased only at 130 dGA in the IUGR group, but no differences were seen between groups for either cytosolic or nuclear NFAT5 protein concentrations. DECR1 concentrations were similar between groups and increased significantly with advancing gestational age. AR was lowest at 55dGA and SLC5A3 increased with advancing gestational age. We conclude that both placental osmolytes inositol and sorbitol (and their corresponding proteins SLC5A3 and AR) change with gestational age, and are regulated, at least in part, by NFAT5 and DECR1 (NADPH). The inverse relationship between each osmolyte across gestation (e.g. inositol higher in early gestation and sorbitol higher in late gestation) may reflect nutritional needs that change across gestation.