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Browsing Theses and Dissertations by Author "Anthony, Russell, committee member"

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    Investigating thyroid hormone transport and regulation in chorionic somatomammotropin RNAi models of intrauterine growth restricted pregnancies
    (Colorado State University. Libraries, 2023) Donovan, Anna, author; Winger, Quint, advisor; Anthony, Russell, committee member; Cadaret, Caitlin, committee member
    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.
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    ItemOpen Access
    Physical and molecular characteristics of day 75 nuclear transfer cloned bovine conceptuses
    (Colorado State University. Libraries, 2012) De Lille, Alexandra, author; Seidel, George, advisor; Anthony, Russell, committee member; Clay, Colin, committee member; Garry, Franklyn, committee member
    This study was designed to measure fetal and placental characteristics in bovine day 75 nuclear transfer and control pregnancies. Responses included mRNA concentration of the insulin-like growth factor (IGF) system [IGF-1, IGF-2, IGF1R, IGF2R, IGFBBP-1, -2, -3] and the vascular endothelial growth factor (VEGF) system [VEGF, PlGF, VEGF1R, and VEGF2R]. Fetal attrition of the cloned pregnancies up to day 75 was high (89%, 63 out of 71 frozen embryos transferred; 8 of 16 cloned conceptuses present on day 30 survived to day 75, as did 5 of 5 controls). No significant differences in mean weights of large and medium placentomes were observed between 8 clones and 5 controls. However, the variance of mean weight of large placentomes was greater in clones than in controls; one gestation had placentomes six standard deviations larger than controls. Interestingly, the mean umbilical cord weight/length ratio was significantly greater for clones (P < 0.05). Mean fetal length, fetal weight, fetal weight/length index and mean weights for heart, brain, liver, kidneys and the mean brain/liver index did not differ between cloned and control day 75 conceptuses, but numbers per group were limited. Northern blot analysis, revealed the presence of three transcripts of 3.7kb, 2.2kb and 1.7kb for VEGF and one 1.7 kb transcript for PlGF mRNA in the cotyledons and allantochorion of day 45 cloned and control gestations. All three VEGF bands were present in both cloned and control day 75 cotyledons and caruncles, but the PlGF transcript was barely detectable, except for the cotyledons of one clone. mRNA for all of genes studied could be detected with real time PCR in day 75 cotyledons and caruncles, and fetal livers contained mRNA for all IGF's and IGFBP's evaluated. In all placentomal tissues, PlGF mRNA concentration was 100-fold less than VEGF mRNA, which seems to be the driving force for placentomal vascularization at day 75. There was a trend for a reduction by half of the PlGF mRNA concentration in caruncle of clones vs. controls (P = 0.06). VEGF2R (KDR) mRNA was abundant, but VEGF1R (Flt-1), was only present in very low concentrations; our primer set did not distinguish between soluble versus membrane bound receptor mRNA for VEGF1R. Four of the cloned conceptuses contained substantially less cotyledonary IGF1R mRNA than the other clones and controls. IGFBP-3 mRNA concentrations were very high in placentomes; IGFBP-1 and -2 mRNA concentration on the other hand was very low for clones and controls. mRNA for IGFBP-1, -2, -3, however, was abundant in day 75 fetal livers, while IGF-1 mRNA was scarce in this tissue. Fetal livers from cloned pregnancies contained 4-fold more IGF-2 mRNA than controls (P<0.01). We observed that liver IGF-2 mRNA concentration and liver weight increased with weight of the largest placentome; in clones these increases were associated with a decrease in cotyledonary IGF-2 mRNA, while the opposite occurred with controls. Interestingly, there was a trend to lower IGF2R mRNA concentrations (P = 0.09), and IGF-1 mRNA was twofold higher in cotyledons of clones (P= 0.03) compared to controls. For many measurements, means were not significantly different (P > 0.1) between clones and controls. However, unequal variances were common, and data points with statistical outlier behavior were observed for clones, which highlights the heterogeneity of the cloned population. This variation in gene expression may exacerbate abnormal placentation later in gestation, and might explain some of the increased morbidity and mortality seen in calves resulting from cloning by nuclear transfer.