Regulation of transthyretin by human placenta

Thyroid hormones (TH), in particular thyroxine (T4), are essential in ensuring the fetus develops correctly during pregnancy. However, during the first trimester of gestation, the fetus relies solely on a maternal supply of T4, which must traverse the placenta before entering the fetal circulation. The exact mechanisms of transplacental delivery of T4 however are yet unknown. TH cell membrane transporters have been found within the placenta and at different stages of gestation. Our group had recently described the placental production and secretion of a TH binding protein, transthyretin (TTR), and postulated that placental TTR plays a role in T4 transfer to the fetus. Therefore, the aims of my thesis were (i) to determine the regulation of TTR expression in human placenta using both placental cells lines (JEG-3 and primary trophoblasts) and human tissue; and (ii) to measure the ontogeny of TTR expression in placentas collected from early and term pregnancies. JEG-3 and trophoblast cells were cultured at different oxygen concentrations (1%, 3%, 8%, 21%) to mimic those present in vivo during placental development. A hypoxia mimetic drug, desferrioxamine (DFO), was also used at a concentration of 200 μM. Both TTR mRNA and protein expression increased significantly in cells cultured under hypoxic (1%, 3% and DFO) conditions in comparison to 8% and 21% O2. Significant increases in TTR uptake of 125I-TTR and Alexa 594-TTR (both in the presence of 10 μM T4) by JEG-3 and trophoblast cells were also observed when cells were cultured at 1% and 3% O2 or treated with DFO in comparison to 8% and 21% O2. The ontological changes in TTR expression were then measured using human placental tissue collected from early (6-17 weeks gestation) and term (38-39 weeks gestation) pregnancies. Contrary to observations from JEG-3 and primary trophoblast in vitro cell cultures, TTR mRNA and protein expression increased linearly in placental tissue from 6 to 13 weeks gestational age, the period when placental oxygen concentrations rise from 1 to 8% O2. Thereafter, TTR expression remained constant in comparison to term (38-39 weeks gestation) placenta, where oxygen concentrations range from 8% to 10% O2. To demonstrate further that oxygen is a regulator of TTR, JEG-3 cells were transfected with TTR promoter reporter constructs and luciferase activity was measured. JEG-3 cells cultured at 1% and 3% O2 and treated with DFO showed significantly increased TTR promoter activity in comparison to cells cultured at 8% and 21% O2. My thesis demonstrates for the first time that oxygen has the capacity to regulate placental TTR expression in an in vitro cell culture setting. From this it was postulated that early in gestation TTR concentrations would be at its highest before decreasing as oxygen within the placenta increased. However, the ontogenic changes in TTR expression measured in human placental tissue collected at different stages of gestation did not reflect these findings. Therefore, it can be concluded that although oxygen may be one such regulatory factor of TTR in the placenta, other unknown factors may affect the expression of placental TTR during different stages of gestation. There is still much to be understood of the role that TTR plays in the transplacental delivery of T4 during gestation alongside the other known TH transporters. It is clear that many complex physiological parameters are involved in this process, which may lead to altered TH delivery across placenta to the developing fetus. Keywords placenta, oxygen, thyroxine, transthyretin, pregnancy, fetus Australian and New Zealand Standard Research Classifications (ANZSRC) 110306 Endocrinology – 40% 111401 Fetal Development and Medicine – 30% 111402 Obstetrics and Gynaecology – 30