Investigating thyroid hormone transport and regulation in chorionic somatomammotropin RNAi models of intrauterine growth restricted pregnancies
Date
2023
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Abstract
In vivo lentiviral mediated RNA interference of chorionic somatomammotropin (CSH) results in an intrauterine growth restricted (IUGR) phenotype in sheep. Abnormal levels of thyroid hormones (THs) 3,5,3'-triiodothyronine (T3) and 3,5,3',5'-tetraiodothyronine (T4, thyroxine) and abnormal expression of placental deiodinase 2 and deiodinase 3 (DIO2/DIO3) mRNA have been implicated in IUGR. It has been reported that nutrient restricted models of IUGR pregnancies result in a decrease of thyroxine levels in maternal and fetal circulation. Transthyretin (TTR) is a TH binding molecule produced by trophoblast cells that preferentially binds T4 and may shuttle T4 through the placenta. It was our objective to better understand thyroid hormone transport during late gestation specifically in CSH RNAi models of IUGR pregnancies. We hypothesized that a reduction in placental CSH negatively impacts thyroid hormone transport from maternal circulation to fetal circulation due to perturbations in both thyroid hormone carrier protein activity and placental deiodinase enzyme expression. The trophectoderm of hatched blastocysts (9 days gestational age; dGA) was infected with lentivirus expressing either a non-targeting sequence (NTS) shRNA to create control pregnancies, or a CSH shRNA to generate a CSH knockdown model of IUGR pregnancies. Uterine vein, uterine artery, umbilical vein, and umbilical artery serum was collected via catheterization, and maternal and fetal tissue was harvested near term (~135 dGA) from growth restricted CSH RNAi pregnancies (n=4) and control pregnancies (n=4). TTR protein abundance was determined through western blot analysis, T4 and T3 levels were assessed using competitive ELISA assays, and DIO2 and DIO3 expression was measured using qRT-PCR. T4 was reduced by 16% (P ≤ 0.05) in CSH RNAi IUGR umbilical vein serum samples compared to control umbilical vein serum samples, and by 29% (P ≤ 0.05) in CSH RNAi IUGR umbilical artery serum samples compared to control umbilical artery serum samples. TTR protein was 47% (P ≤ 0.10) less abundant in CSH RNAi IUGR uterine artery serum compared to control serum and 64% (P ≤ 0.05) less abundant in CSH RNAi IUGR uterine vein serum compared to control serum. In umbilical artery serum TTR protein abundance was 47% (P ≤ 0.05) less abundant in CSH RNAi IUGR serum compared to control serum. Uterine TTR uptake tended to be reduced by 45% (P ≤ 0.10) in CSH RNAi pregnancies compared to control pregnancies, and uteroplacental utilization tended to be reduced by 48% (P ≤ 0.10) in CSH RNAi pregnancies compared to control pregnancies. Umbilical uptake was not measured in either control or CSH RNAi pregnancies. There was also no difference in TTR protein concentration in either maternal or fetal liver tissue when comparing CSH RNAi tissue to control tissue. Caruncle DIO2 mRNA expression was reduced by 60% (P ≤ 0.05) in CSH RNAi caruncle samples when compared to control caruncle samples, with no significant change in DIO2 expression between CSH RNAi samples and control samples in the cotyledon. There was no difference in DIO3 expression between CSH RNAi samples and control samples in either the caruncle or cotyledon. Our data suggests that in ovine pregnancies near term, a deficiency in CSH negatively impacts processes related to thyroid hormone transport into and throughout the placenta. Dysregulation in thyroid hormone transport could be playing a role in negatively impacting placental physiology, thus aiding in the development of IUGR.