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Item Open Access Estrogen receptors alpha and beta: Opposing roles in hypothalamic-pituitary-adrenal axis function and stress-related behaviors(Colorado State University. Libraries, 2008) Weiser, Michael James, author; Handa, Robert, advisorEstradiol has reported effects on mood ranging from anxiogenic to anxiolytic and depressant to anti-depressant. These opposing actions of estradiol may be explained by the existence of two distinct estrogen receptor (ER) systems, ER alpha (ERα) and ER beta (ERβ). Furthermore, there exists a sex difference in stress-related psychiatric disorders such as anxiety and depression, for which women are more susceptible than men. Common to the pathology of these disorders is a dysfunctional hypothalamic-pituitary-adrenal (HPA) axis where glucocorticoid negative feedback is impaired leading to chronically high levels of circulating glucocorticoids. The HPA axis is the main neuroendocrine axis that governs physiological responses to stressors. In rodents, basal and stress-induced activity of the HPA axis is higher in females than in males. This suggests that, if transferable to humans, the sex difference observed in HPA axis function in animal models may help explain the female predisposition for certain psychiatric disorders. The studies described in this dissertation were aimed at characterizing the distinct roles for ERα and ERβ in HPA axis activity and stress-related behaviors. The studies in Chapter 3 examine the effect of estradiol signaling through ERα or ERβ on glucocorticoid negative feedback of the HPA axis. Results indicate that estradiol impairs glucocorticoid-dependent negative feedback by activating ERα specifically at the level of the paraventricular nucleus (PVN). The studies in Chapter 4 examine the effect of estradiol signaling through ERα or ERβ on anxiety-like and depressive-like behaviors. Results indicate that selective activation of ERα is anxiogenic and depressant, whereas selective activation of ERβ is anxiolytic and antidepressant. Finally, the studies in Chapter 5 examine the effect of estradiol signaling through ERβ on behavior and HPA axis activity induced by glucocorticoid receptor (GR) activation in the central nucleus of the amygdala (CeA). Results indicate that delivery of a GR agonist to the CeA is anxiogenic and augments the HPA axis response to a stressor, and peripheral administration of an ERβ agonist blocks this effect. Collectively, these studies point to an antagonistic relationship between estradiol signaling through ERα and ERβ with respect to HPA axis activity and stress-related behaviors.Item Open Access Characterization of periattachment factor(Colorado State University. Libraries, 2008) Purcell, Scott H., author; Anthony, Russell V., advisor; Seidel, George E., advisorThe purpose of these studies was to characterize expression of periattachment factor (PF) mRNA in the developing sheep conceptus, localize PF in the sheep conceptus, determine if degrading PF mRNA in the sheep conceptus was detrimental to development, and characterize PF in human tissues and cells. Sheep conceptuses were collected on d 11, 13, 15, 16, 17, 21, and 30 post-mating. oPF mRNA exhibited a quadratic expression pattern (P<0.05). No oPF mRNA was detected in d 11 conceptuses. From d 13, oPF mRNA increased to a peak at d 16 before declining. Peak expression of oPF mRNA in the conceptus occurs during rapid elongation and initial attachment of the conceptus to the endometrium. Immunolocalization of PF in a d 15 conceptus showed predominantly nuclear staining in trophectoderm and trophendoderm. Next, embryos collected from superovulated ewes on d 8 post-mating were infected with either a lentivirus expressing a short-hairpin (sh) RNA designed to target PF mRNA for degradation, a lentivirus expressing a shRNA containing 3 mismatched nucleotides, or a control lentivirus expressing no shRNA. Following infection, blastocysts were transferred into recipient ewes and collected back on d 15 of gestation. While 94 and 88% of the control and mismatched shRNA-treated conceptuses elongated by d 15, none of the embryos treated with the lentivirus expressing shRNA against PF mRNA elongated, and most died. This indicates that oPF is required for conceptus elongation and survival. Human PF mRNA was detected in human carcinomas and in the first trimester cytotrophoblast cell line, hTR8. Immunohistochemistry showed PF in the nuclei of carcinomas and in first and second trimester cytotrophoblasts. PF was also present in the invading cytotrophoblast columns. In an in vitro invasion assay of first trimester cytotrophoblasts, hPF mRNA increased from 0, 3, to 12 h as invasion occurred. To further characterize PF in human cells, a lentiviral construct expressing an shRNA targeting the hPF mRNA sequence was developed that resulted in 63% mRNA reduction in BeWo cells.Item Open Access Characterization of mediators of cardiac and renal development in response to increased prenatal testosterone(Colorado State University. Libraries, 2008) Maresh, Ryan W., author; Anthony, Russell, advisorPreviously reported differences in cardiac and kidney weights of offspring from ewes prenatally treated with testosterone suggested the development of systemic hypertension. To determine if prenatal androgen excess influences the expression of key mediators, angiopoietin 1 (Ang1), angiopoietin 2 (Ang2), tunica interna endothelial cell kinase-2 (Tie2), angiotensin II receptor subtypes 1 and 2 (AT1 and AT2), endothelial nitric oxide synthase (eNOS), vascular endothelial growth factor (VEGF), VEGF receptor 1 (VEGF R1), VEGF receptor 2 (VEGF R2), insulin receptor β (IRβ), glucose transporter 1 (GLUT1), and glucose transporter 4 (GLUT4) were evaluated from fetal hearts, fetal kidney, left ventricle (LV), right ventricle (RV), and kidney. We hypothesized that mRNA and protein concentrations were altered and that the changes persist into adulthood. At fetal Day 90 a significant increase (p = 0.021) in IRβ mRNA concentration of was seen in the heart, whereas VEGF mRNA concentration was significantly reduced (p = 0.044) in the kidney. In 9 month RV, eNOS mRNA concentration was significantly reduced in the treatment group (p = 0.019). In the 9 month kidney, significant increases in GLUT4 mRNA concentration (p = 0.047) and eNOS protein concentration (p = 0.027) were seen in the treatment group. At 21 months of age, Ang1 mRNA concentration in the treatment group was significantly reduced (p = 0.025) in the LV. AT1 mRNA concentration was significantly decreased (p = 0.004) in the RV of the treatment group. LV eNOS concentration was significantly reduced (p = 0.032), while RV eNOS mRNA concentration was significantly decreased (p < 0.001) in the treatment group. Significant decreases in GLUT1 mRNA concentration were detected in the LV (p = 0.013) and RV (p = 0.006), and RV GLUT4 mRNA concentration was significantly decreased (p = 0.003) in the treatment group. LV IRβ concentration was significantly reduced (p = 0.036) in the treatment group. In the kidney, VEGF R2 mRNA concentration was significantly reduced (p = 0.024) in the treatment group. Taken together, the data supports the development of systemic hypertension and insulin resistance with age following prenatal androgen exposure.Item Open Access The use of chronic models of temporal lobe epilepsy in antiepileptic drug development(Colorado State University. Libraries, 2007) Grabenstatter, Heidi, author; Dudek, F. Edward, advisorA chronic animal model with altered ion channels, transmitter receptors and/or neural circuitry similar to temporal lobe epilepsy (TLE) may be useful in the discovery of new antiepileptic drugs (AEDs). The hypothesis was that rats with kainate-induced epilepsy are pharmacosensitive to AEDs, but high doses do not block all spontaneous seizures (i.e., these rats are "pharmacoresistant"). A repeated-measures cross-over protocol was used to show single intraperitoneal injections of topiramate, RWJ-333369, and carbamazepine reduced the frequency of spontaneous motor seizures. The same protocol with administration of 30 mg/kg and 100 mg/kg carbamazepine in specially-formulated food pellets was as effective as intraperitoneal injections, and 100 mg/kg carbamazepine administered in food three times per day completely suppressed motor seizures in 50% of the animals for a prolonged time period (i.e., 24 h) while reducing any stress to the animals. Video-EEG showed carbamazepine preferentially reduced spontaneous convulsive seizures compared to nonconvulsive seizures at 100 mg/kg, reduced seizure duration in some animals at 100 mg/kg, and caused a subtle decrease in the maximum frequency of population spikes during seizures at 30 mg/kg. These data suggest that animal models of TLE with spontaneous seizures can be used efficiently to test AEDs, and that this repeated-measures cross-over protocol is amenable to both dose-effect and time-course-of-recovery studies for the direct comparison of AEDs. This approach can provide statistical power to compensate for seizure clusters and variability across animals. These experiments also show that rats with kainate-induced epilepsy are pharmacosensitive to standard and experimental AEDs; additional studies are required to determine if this model is also pharmacoresistant.Item Open Access Status epilepticus, recurrent seizures, hippocampal damage and the estrous cycle in a model of temporal lobe epilepsy(Colorado State University. Libraries, 2008) Fawley, Jessica, author; Dudek, F. Edward, advisorTemporal lobe epilepsy is the most common form of epilepsy and is associated with hippocampal sclerosis and spontaneous recurrent seizures. These pathologies generally develop after a latent period from a precipitating brain injury, which often results in status epilepticus (SE). Sex and hormones have been reported to influence SE and mortality in both clinical and experimental settings. Temporal lobe epilepsy is also associated with an increase in reproductive disorders, which are often the result of altered pulsatile release of luteinizing hormone (LH). Gonadotropin-releasing hormone (GnRH) controls LH release; therefore, reproductive abnormalities associated with epilepsy could hypothetically involve hypothalamic disturbances, particularly to the GnRH network, resulting in altered secretion of GnRH. The aim of this dissertation was to (1) to examine the effects of SE and/or temporal lobe epilepsy on the GnRH neuronal network and (2) utilize recordings of electroencephalogram (EEG) activity to systematically quantify sex and hormone influences on SE and the subsequent recurrent seizures. I report that pilocarpine-induced SE resulted in reproductive alterations in two rodent models of temporal lobe epilepsy, which were not due to a detectable reduction in GnRH-positive neurons. There were no significant differences between the EEG parameters of SE or recurrent seizures between groups. Sex and the stage of the estrous cycle may influence pyramidal cell loss in the hippocampus at 24 h but the stage of the estrous cycle and/or sex do not seem to be predictors of long-term hippocampal damage. In summary, these data do not support the hypothesis that SE and/or temporal lobe epilepsy results in a reduction in the number of GnRH neurons or the hypothesis that sex/cycle stage influences SE, or the progression to temporal lobe epilepsy. However, these models of SE/temporal lobe epilepsy will be useful to further study temporal lobe epilepsy-associated reproductive alterations.Item Open Access The effect of aging on gene expression and mitochondrial DNA in the equine oocyte and follicle(Colorado State University. Libraries, 2009) Campos-Chillon, Lino Fernando, author; Carnevale, Elaine, advisorThe decline in fertility of aged mares is linked to declining oocyte quality. Oocyte viability is dependant on the ability of oocytes to remain in meiotic arrest until the initiation of maturation and adequate cumulus communication. We hypothesize that aging is associated with quantitative and temporal differences in meiotic arrest and resumption in oocytes, decreased oocyte secretion of paracrine factors and lower mitochondrial numbers, ultimately resulting in a dissociation of oocyte and follicular maturation. The objectives of this study were to clone and determine quantitative and temporal differences in mRNA content of the LH receptor (LHR), amphiregulin (AREG) and epiregulin (EREG) in granulosa cells; PDE4 in cumulus cells; and PDE3A, GPR3, GDF9, BMP15, and mitochondrial DNA (mtDNA) in oocytes during in vivo maturation in young (3-12 yr) and old (>20 yr) mares. Oocytes and follicular cells were collected by transvaginal follicular aspiration. Follicle maturation was induced in estrous mares with a follicle >30 mm by injection of 750 µg of recombinant equine LH. Aspirations were conducted at 0, 6, 9, and 12 h after LH administration. Total RNA was isolated from single denuded oocytes and associated lysed cumulus and granulosa cells. For each gene, mean mRNA copy number for each time point and age group were compared by ANOVA and Fisher's LSD. Regression coefficients were generated to compare oocyte mitochondrial numbers and correlations between gene expression within age groups. Expression of LHR mRNA in granulosa cells was different (p<0.05) between age groups. Young mares displayed a significant drop in LHR mRNA between 0 h and 6, 9, and 12 h; while the pattern of expression in old mares was similar (p>0.05) among times and higher (p<0.05) at 6 h than in young mares. Expression of AREG mRNA in granulosa cells peaked (p<0.05) at 9 h; however, the magnitude of expression at 6 and 9 h was higher (p<0.05) in old than young mares. Similarly, EREG expression peaked (p<0.05) at 9 h in young and old mares but was higher (p<0.05) for old mares. Expression of PDE4D peaked (p<0.05) at 6 and 12 h in old and young mares, respectively. The patterns of expression of GPR3 for oocytes of young and old mares were different and peaked (p<0.05) at 9 and 12 h, respectively. Magnitude of expression of PDE3A for oocytes of old mares at 6 and 9 h was higher (p<0.05) than in young mares. Expression of GDF9 and BMP15 was different (p<0.05) between ages. Mean expression of both genes in the old group was similar over time; however, in young mare oocytes maximum expression was at 6 h (p<0.05). Correlation coefficients between GDF9 and BMP15 for old and young mares were 0.94 and 0.99, respectively. Numbers of copies of oocyte mtDNA did not vary in young mares; however, there was a temporal decrease (p<0.05) of oocyte mitochondrial copy numbers in old mares. The main effect for age for mtDNA was similar for old and young mares.Item Open Access The role of interferon-tau (IFNT) in luteal gene expression, steroidogenesis, and luteal lifespan in the ewe(Colorado State University. Libraries, 2009) Bott, Rebecca Clark, author; Bruemmer, Jason, advisor; Niswender, Gordon, advisorInterferon-tau (IFNT) was evaluated for endocrine actions on the corpus luteum (CL). The hypothesis was that infusion of IFNT would increase luteal expression of interferon-stimulated gene (ISG)-15, and the length of time for ewes to return to estrus. Osmotic pumps containing 200 μg IFNT or BSA (n=12 each) were connected to the uterine vein of non-pregnant ewes 10 days post-estrus. Messenger RNA encoding ISG15 was elevated in CL from pregnant and IFNT-infused ewes (P<0.05) compared to nonpregnant and BSA-treated ewes, respectively. Luteal mRNA encoding ISG15 from ewes treated with IFNT was greater than in ewes treated with BSA (P<0.05). Serum concentrations of progesterone were not different in ewes that received infusions of BSA or IFNT. Progesterone decreased by six hours (P<0.05) in ewes that received BSA+PGF or IFNT+PGF, but did not differ in ewes that received infusions of IFNT +/- PGF at 8, 10, or 12 hours after PGF. There were no differences in prostaglandin E synthase (PGES) or prostaglandin F synthase (PGFS), or in prostaglandin dehydrogenase (PGDH), steroidogenic acute regulatory protein (StAR), peripheral type benzodiazepine receptor (PBR), cytochrome P450 side chain cleavage enzyme (CYP-11A), or 3μ-hydroxysteroid dehydrogenase (3μ-HSD). Seven day infusion of IFNT during the time frame of maternal recognition of pregnancy resulted in 20% of IFNT-treated ewes returning to estrus by d19 compared to 100% of BSA-treated ewes (P<0.01). In conclusion IFNT acts systemically, alters gene expression in the corpus luteum, and decreases the number of ewes returning to estrus by d19.Item Open Access Two types of melanopsin retinal ganglion cell in the mouse retina: the regulation of melanopsin expression(Colorado State University. Libraries, 2009) Baver, Scott Benjamin, author; Tobet, Stuart, advisorRods, cones and a subset of retinal ganglion cells (RGCs) that express the photopigment melanopsin are the sensory photoreceptors of the mammalian retina. The light-driven signals that are initiated by the photoreceptors are relayed from the retina to the brain. In addition to the role of light information in regulating the perception of colors, objects and movement, it also controls pupil size and the synchronization of daily physiological rhythms to the day/night cycle. The melanopsin-expressing RGCs, which are intrinsically photosensitive (ipRGCs), contribute especially to these two latter processes. The focus of this dissertation is the ipRGCs of the mouse retina.Item Open Access Energy substrates, metabolic regulators, and lipid accumulation during culture of in vitro-produced bovine embryos(Colorado State University. Libraries, 2007) Barceló-Fimbres, Moisés, author; Seidel, George E., Jr., advisorThe main objective of this experiment was to optimize in vitro culture conditions for bovine embryonic development, using alternative energy sources and metabolic regulators. Replacing glucose with fructose in culture medium consistently increased blastocyst production per oocyte and decreased lipid content in bovine embryos. The use of phenazine ethosulphate (PES) or fetal calf serum (FCS) supplementation did not affect embryonic development; however, PES consistently decreased, and FCS increased lipid content of embryos compared to the control. There was no effect of glucose or fructose on survival of embryos after cryopreservation by slow freezing or vitrification; however, embryos-treated with PES to reduce lipid content resulted in improved cryotolerance, and FCS decreased cryotolerance compared to the control. Transfer of embryos treated with PES during in vitro culture did not affect pregnancy rates, conceptus losses, or fetal or post-natal development in calves born normally. The sex ratio of calves born was skewed toward males. This effect likely was due to a toxic effect of glucose to female embryos cultured in vitro. Therefore, the more expanded day 7 blastocysts were mostly male embryos.Item Open Access Identification and characterization of two ovine membrane receptors for progesterone(Colorado State University. Libraries, 2007) Ashley, Ryan L., author; Nett, Terry M., advisorClassically, progesterone (P4) has been thought to act only through the well-known genomic pathway involving hormone binding to nuclear receptors (PR) and subsequent modulation of gene expression. Alternatively, there is increasing evidence for rapid, nongenomic effects of P4 in a variety of mammalian tissues; however the receptor responsible for these actions is yet to be characterized. The likelihood of a membrane P4 receptor (mPR) causing these events is quite plausible. Sheep are the experimental model often used in our laboratory, because regulation of reproductive function is very similar in ewes and women. Nongenomic actions of P4 have been described in sheep, but the receptor(s) responsible has not been identified. The objective of this dissertation was to isolate and characterize an ovine mPR distinct from the nuclear PR. A membrane protein found to bind P4 was first isolated from porcine liver and is now known as P4 receptor membrane component 1 (PGRMC1). Since PGRMC1 is expressed in a variety of species and tissues, I hypothesized that PGRMC1 was the protein responsible for the nongenomic effects of P4 in sheep. As such, the purpose of my initial studies was to determine if PGRMC1 was expressed in the sheep and further verify if PGRMC1 was present in the plasma membrane of the ovine corpus luteum (CL). A protein lacking homology to the nPRs but homologous to other reported PGRMC1 proteins was isolated from the sheep and contains a single transmembrane domain at the N-terminus. Despite the transmembrane domain, the ovine PGRMC1 displays predominant localization in the endoplasmic reticulum. During my initial studies, another putative mPR was cloned from the seatrout that displayed seven transmembrane domains, indicative of a G-protein-coupled receptor (GPCR) and upon ligand activation, also caused rapid induction of second messenger pathways. As such, I wanted to determine if this mPR was also present in the sheep. An ovine mPR distinct from the nuclear PR was isolated and characterized. The ovine mPR is a 350 amino acid protein that, based on predicted structural analysis, possesses seven transmembrane domains and is expressed in the hypothalamus, pituitary, uterus, ovary and CL. The first characterization of mPR expression throughout the ovine estrous cycle in the hypothalamus, pituitary, uterus, ovary, and CL is also reported. In CHO cells that overexpress a mPR-GFP fusion protein the ovine mPR was uniquely localized to the endoplasmic reticulum and not the plasma membrane. The ovine mPR specifically binds only progestins. Progesterone, 20α-hydroxyprogesterone and 17α-hydroxyprogesterone significantly (P < 0.001) displaced binding of 3H-P4 to membrane fractions from CHO cells expressing ovine mPR. Additionally, the ovine mPR directly induces Ca2+ release from the endoplasmic reticulum upon ligand activation. Further, the ovine mPR appears to activate the MAPK pathway, specifically by phosphorylation of JNK1/2 upon ligand activation. This novel method of signaling at the endoplasmic reticulum adds to the intricacy of signaling in cells and provides a mechanistically unique method for initiating actions of P4 that may alter classical dogma regarding the mechanisms by which steroid hormones act.Item Open Access Mechanism of neuronal cell death in canine glaucoma(Colorado State University. Libraries, 2009) Alyahya, Khaleel I., author; Madl, James E., advisorGlaucoma is one of the most important causes of blindness in human and dogs. Glaucoma is characterized by a progressive loss of retinal ganglion cells that is often associated with increased intraocular pressure and decreased retinal blood flow. In previous investigations, we found that changes in glutamate distribution occur selectively in damaged areas of retinas of dogs with primary glaucoma. This glutamate redistribution is consistent with high levels of extracellular glutamate (↑GluE) contributing to excitotoxic damage to neurons. In this dissertation, we used immunohistochemical methods to test three mechanisms by which this glutamate redistribution may occur in retinas from clinical cases of canine glaucoma. First, we tested if ischemia due to microvessel loss causes the changes in glutamate distribution. We found significantly lower microvessel density in damaged regions, consistent with ischemia occurring in canine glaucoma. Second, we tested if loss of glutamine synthetase induces the glutamate redistribution. We have found significantly decreased amounts of glutamine synthetase in areas with neuronal damage and glutamate redistribution, consistence with decreased glutamate synthetase contributing to glutamate redistribution. Third, leakage of glutamate from blood vessels from inflamed areas may lead to glutamate redistribution. We found that there is albumin leakage from blood vessels in damaged regions with other inflammatory indicators in those areas. The smaller size of glutamate suggests that it should also diffuse out of blood into the extracellular fluid of the retina even more readily than albumin. Increase leakiness of blood vessels in canine glaucoma is consistent with glutamate leakage contributing to glutamate redistribution. In conclusion, our results are consistent with all three mechanisms contributing to glutamate redistribution in canine glaucoma. The dissertation includes further discussion of more refined hypotheses of the mechanisms by which glutamate redistribution and neuronal damage may occur.Item Open Access Functional organization of a cortical-medullary neural circuit mediating organismal adaptation to stress(Colorado State University. Libraries, 2023) Pace, Sebastian A., author; Myers, Brent, advisor; Hentges, Shane, advisor; Tobet, Stuart, committee member; Foster, Michelle, committee memberHindbrain regions responsible for epinephrine and norepinephrine production are critical for orchestrating stress responses, maintaining physiological equilibrium and integrating afferent information. The nuclei central to hindbrain epinephrine and norepinephrine production, create a neural network that interfaces with forebrain and spinal cord regions, facilitating the integration of neuroendocrine and autonomic functions. Despite significant strides in our comprehension of stress response systems, questions concerning the roles of sex, stress history, and circuit mechanisms endure. In this study, we unveil and characterize a prefrontal-medullary circuit crucial for the suppression of stress responses. First, anterograde and retrograde tract-tracing studies demonstrated a stress-reactive vmPFC-RVLM circuit. Activation of this vmPFC-RVLM circuit mitigates glucocorticoid stress reactivity in both males and females, by targeting non-catecholaminergic neurons. Therefore, vmPFC-RVLM circuit activation may utilize local inhibitory neurons to limit catecholaminergic activation. To better understand how chronic stress affects the medulla, we explored the impact of chronic stress on signaling machinery and revealed elevated tyrosine hydroxylase (TH) levels in both male and female rats following chronic variable stress (CVS). To understand how CVS interacts with the vmPFC-RVLM circuit, we used an intersectional TeLC (Tetanus toxin - light chain) approach to disrupt the circuit and evaluate multiple stress response systems. In males, circuit disruption and CVS largely left behavioral and cardiovascular stress reactivity unaltered, however, some neuroendocrine endpoints were affected. Conversely, females exposed to circuit disruption and chronic stress exhibited heightened stress reactivity in glycemic, corticosterone, and arterial pressure responses, coupled with avoidant-like behaviors. These findings underscore the sex-specific necessity of the vmPFC-RVLM circuit in countering chronic stress-related outcomes, emphasizing a greater protective role in females relative to males. To gain deeper insights into the role of vmPFC inputs to the RVLM in females, we once again utilized a circuit-based TeLC approach, employing in situ hybridization (ISH) coupled with immunohistochemistry (IHC) to assess TH and phenylethanolamine N-methyltransferase (PNMT) transcript density across various VLM subregions. Notably, the TeLC-induced elevation of PNMT expression in females suggests that disrupting this circuit could potentially enhance epinephrine production by RVLM neurons, potentially intensifying stress reactivity post-CVS. This comprehensive study demonstrated the critical role of the vmPFC-RVLM circuit in modulating stress responses and revealing female-specific effects in mitigating physiological, behavioral, and transcriptional outcomes after chronic stress. These findings emphasize the significance of the vmPFC-RVLM circuit in managing stress reactivity in the context of chronic stress and identify the circuit as a potential candidate for reducing stress responding.Item Embargo An investigation of synaptic vesicle docking and priming and a proposed method for quantitatively measuring both in Drosophila using electron tomography(Colorado State University. Libraries, 2023) Twiggs, Jasmin A., author; Reist, Noreen, advisor; Hoerndli, Frederic, committee member; Hoke, Kim, committee member; Tamkun, Michael, committee memberThe nervous system, as the body's command center, plays a crucial role in cellular communication within the brain and between the brain and other body systems. Neurons, the individual cellular units, transmit electrical information and communicate with other cells through neurotransmitter release in response to electrical stimuli. Chapter 1 introduces the foundational concepts of neuronal structure and function and delves into the mechanisms underlying neurotransmitter release. Special attention is given to the neuromuscular junction (NMJ), a well-studied chemical synapse crucial for muscle movement. The synaptic vesicle cycle is introduced, with particular emphasis on docking and priming. The significance of active zones, specialized sites for efficient signal transmission, and their associated structural components are underscored. Synaptotagmin, a pivotal protein in calcium-triggered vesicle fusion, is discussed with emphasis on its C2B polylysine motif. Throughout the chapter, the utility of Drosophila as a model system for studying synaptic processes, particularly at the NMJ, is emphasized. In sum, Chapter 1 provides the foundational knowledge essential for comprehending the intricate cellular and molecular facets of synaptic communication within the nervous system, serving as a precursor to subsequent chapters' investigations. Chapter 2 examines synaptotagmin's C2B polylysine motif and its role in synaptic vesicle docking at the Drosophila NMJ. It explores the polylysine motif's potential involvement in endocytosis, demonstrates an unaffected interaction with AP-2, and uses electron microscopy to find no significant changes in vesicle distribution. The findings suggest that the reduced neurotransmitter release in the polylysine mutant is likely due to an impairment in vesicle priming. Chapter 3 introduces a method for studying synaptic vesicle docking and priming in Drosophila, using electron tomography. I address the limitations of conventional electron microscopy and underscore the need for higher-resolution techniques to assess molecular structures that mediate physiological processes. Chapter 3 also emphasizes the significance of the contact area between docked vesicles and the presynaptic membrane as a correlate of vesicle priming. The protocol, expected results, and key considerations are discussed. The methods presented in Chapter 3 offer a promising approach for understanding synaptic processes. In Chapter 4, I discuss key considerations for when standard electron microscopy can be used for assessing vesicle docking. Then, I discuss how the electron tomography method presented in Chapter 3 could not only confirm the results found in Chapter 2, that the synaptotagmin C2B polylysine motif is not implicated in vesicle docking but could also be used to directly test the mutant's role in priming. Specific aims for future studies on the synaptotagmin polylysine mutation in Drosophila are presented, potential results and interpretations are discussed. Finally, I showcase interesting, unpublished findings from electron tomograms I have taken at the Drosophila NMJ and discuss their potential significance.Item Embargo IQGAP1 is a novel effector of gonadotropin-releasing hormone receptor signaling(Colorado State University. Libraries, 2023) Alqahtani, Huda A., author; Amberg, Gregory, advisor; Clay, Colin, committee member; Tamkun, Michael, committee member; DeLuca, Jennifer, committee memberStimulation of gonadotropin-releasing hormone (GnRH) receptors on the surface of anterior pituitary gonadotrope cells is a key signaling event for the hypothalamic-pituitary-gonadal axis. One important downstream component of GnRH receptor signaling is activation of the mitogen-activated protein kinase ERK (extracellular signal-regulated kinase), which is essential for the production of the gonadotropin luteinizing hormone. Evidence suggests that GnRH receptors reside in low-density plasma membrane domains where they participate in multiprotein signaling complexes. Here we used quantitative proteomics to identify proteins associated with low-density plasma membrane domains and to measure changes in their relative abundance in these domains in response to GnRH. Using αT3-1 gonadotropes, we identified 537 proteins in detergent-free subcellular fractions containing low-density plasma membranes. SILAC (stable isotope labeling by amino acids in cell culture) in combination with mass spectrometry demonstrated that GnRH, within 10 min, altered the association of 87 proteins with this plasma membrane fraction. Ontology analysis revealed that GnRH promoted an enrichment of actin cytoskeletal and adherens junction-related proteins including the molecular scaffold IQGAP1 and the small GTPase Rac1. Subsequent investigation revealed that the association between Rac1 and IQGAP1 increased with GnRH receptor stimulation and that GnRH increased Rac1 activity. Demonstrating functional relevance, inhibiting Rac1 reduced GnRH-dependent ERK activation. Our data reveals an upstream activation of signaling and structural molecules, including Ca2+, CDC42 and Rac1, E-cadherin, N-cadherin, and β-catenin. We also identified interactions between the scaffold protein IQGAP1 and these molecules, indicating that IQGAP1 is a fundamental regulator of GnRH-dependent signaling in gonadotropes. Furthermore, our data shows that IQGAP1 has a transcriptional regulatory role in gonadotropes treated with GnRH. In sum, these data indicate that IQGAP1 complexed with Rac1 modulates ERK activity and as such serves as an essential effector in modulating cell polarity and cell-cell contacts in gonadotropes. Altogether, our proteomics data show that acute stimulation of GnRH receptors (3 nM for 10 min) alters the PAM fraction abundance of proteins, such as IQGAP1, mechanistically linked to gonadotrope activation.Item Embargo Evaluation of feral swine as potential reservoirs, sentinels, and vectors of emerging and re-emerging zoonotic pathogens in the United States(Colorado State University. Libraries, 2023) Maison, Rachel Marie, author; Bosco-Lauth, Angela, advisor; Bowen, Richard, advisor; Borlee, Bradley, committee member; Brown, Vienna, committee member; Han, Sushan, committee memberFeral swine are an extremely adaptable and prolific invasive species present in many regions of the United States. A generalist diet, high reproductive capacity, and opportunistic nature make them a significant threat to native flora and fauna, and their destructive foraging behaviors have been attributed to substantial crop loss and property damage throughout their range. Feral swine have also been demonstrated as vectors and reservoirs for many diseases, some of which are transmissible to humans. Despite the efforts of government agencies to impede range expansion of feral swine and monitor populations for disease, human-mediated movements as well as continued natural dispersal in some regions have made management difficult. Subsequently, this invasive species continues to threaten human and animal health throughout its current range. Reported herein are a series of laboratory and field-based investigations that seek to address the potential role(s) of feral swine in the epidemiology of a few zoonotic pathogens that are emerging or re-emerging in the United States. As pigs interact closely with soils through their natural rooting and wallowing behaviors, we were primarily interested in soil-dwelling zoonoses, and identified three of particular interest. The two bacterial species Bacillus anthracis and Burkholderia pseudomallei, as well as the fungus Coccidioides, are the causative agents of anthrax, melioidosis, and coccidioidomycosis respectively, and each is understudied as they relate to feral swine. While each of these organisms is unique in their biology and the relative threat(s) they've posed to humans and animals throughout their respective histories, they are similar in that much of their ecology remains undescribed. The often-complex ecological relationships exhibited by pathogenic organisms are vital to their continued survival and evolution and can play an important role in their dissemination to human populations. The presence of high-density populations of feral swine in many regions of the United States, as well as their ability to occupy a range of habitats, may be playing a significant role in the persistence and dissemination of these organisms. Furthermore, the interactions of feral swine with contaminated substrates within their environment, as well as high levels of interspecies interactions throughout their current range could make them a likely source of additional emerging or novel pathogens. After a summary of the literature concerning feral swine in the United States, followed by that for the causative agents of anthrax, coccidioidomycosis, melioidosis, and a few select emerging disease threats, each respective chapter within this work details investigations largely by pathogen. Within Chapter 2, we present the results of a field serosurvey in feral swine residing across regions of known anthrax endemicity in Texas, and retrospectively document bacterial exposure via enzyme-linked immunosorbent assay (ELISA). This study was performed principally to evaluate the biosentinel utility of feral swine for anthrax contaminated environments, and the ELISA utilized was developed and optimized for detecting antibodies of swine origin to B. anthracis protective antigen. We additionally report on a laboratory-based experimental infection study where which a group of juvenile feral swine were either intranasally or subcutaneously exposed to varying amounts of B. anthracis strain Sterne 34F2 spores, and seroconversion as well as bacterial shedding through the nasal passages documented over time. Through both the field-based serosurvey, as well as the experimental inoculation study, we report that feral swine serology may be used as a management strategy to indirectly identify regions contaminated with anthrax bacteria. Moreover, we report that some feral swine intranasally exposed to high amounts of B. anthracis Sterne contained detectable and viable spores within their nasal passages up to 56 days past their initial exposure event as demonstrated by bacterial culture. The presence of viable B. anthracis within the nasal mucosa well after an exposure event suggests that feral swine may be capable of spreading infectious anthrax bacteria as mechanical vectors. Chapter 3 of this work details an experimental infection study similar to that reported in the previous chapter, however, instead intranasally exposes a group of juvenile feral swine to Coccidioides posadasii strain Silveira spores. This study was performed to evaluate the pathogenesis and immune response of feral swine to one of the causative agents of coccidioidomycosis. Fungal culture of tissues collected from inoculated individuals at necropsy was performed to assess whether animals might act as reservoirs for the fungus in addition to describing the characteristics of infection. Feral swine utilized for this study did not display overt clinical signs of disease, and despite being inoculated with a large dose of fungal spores, also did not seroconvert based on the results of porcine agar gel immunodiffusion assays. Fungal culture of lung and mediastinal lymph node tissues of a subset of pigs revealed the presence of viable C. posadasii, however, examination of additional sections of the same tissues did not reveal the presence of the granulomatous lesions often associated with coccidioidomycosis. Most individuals had significant comorbidities as illustrated via histology, most notably Metastrongylus nematodes in the lungs. Despite an absence of lesions and organisms histologically, isolation of the fungus from tissues by culture indicated active infection and imply that feral swine are mildly susceptible to acute Coccidioides infection. These results suggest that in some instances, feral swine may aid in fungal dispersal on the landscape due to the presence of spherules in tissues post-mortem. Chapter 4, concerning melioidosis, reports on laboratory-based experiments evaluating an indirect ELISA for measuring antibodies to B. pseudomallei in domestic and wild swine. Described previously to test human sera, this ELISA utilized whole-cell lysate derived from B. pseudomallei strain Bp82 and was optimized and preliminarily validated by testing sera from domestic goats and domestic swine experimentally inoculated with virulent and avirulent strains of B. pseudomallei, respectively. Further evaluation of assay specificity was performed by testing sera against similar antigen preparations of the closely related bacterial species Pseudomonas aeruginosa strain PAO1. Examination of sera from laboratory animals against Bp82 and PAO1 whole-cell lysate antigens revealed that most animals seroconverted and displayed B. pseudomallei antibodies, and that little cross-reactivity existed between antigens. After preliminary assay validation, serum from wild pig populations originating from Arizona, California, and Puerto Rico were assessed using both antigens to document B. pseudomallei exposure in wild pigs residing in regions where bacterial absence or endemicity can be confidently inferred at this time. In stark contrast to the serology displayed by laboratory animals, analysis of field sera collected from these regions demonstrated high levels of cross-reactivity between Bp82 and PAO1 antigens, suggesting the assay is not suitable for use in wild pig populations. Lastly, Chapter 5 describes a general survey of feral swine removed from the Aransas National Wildlife Refuge in Texas for each of the organisms investigated in the proceeding chapters, as well as additional bacterial and viral pathogens; added pathogens that were included within this survey included Francisella tularensis, and SARS-CoV-2, the causative agents of tularemia and COVID-19, respectively. A suite of samples, including external parasites (e.g., ticks), nasal swabs, blood, and a series of tissues were collected from each feral swine in the field and analyzed by bacterial and fungal culture, serology, and viral assays to determine if any pigs were actively infected or previously exposed to a range of pathogens. General and selective culture of tissue samples did not reveal active infection with B. anthracis, Coccidioides spp., B. pseudomallei, or other pathogenic bacteria, however, serology illustrated that a subset of pigs were previously exposed to B. anthracis. Further examination of tissues histologically illustrated a high degree of parasitic infection in the majority of pigs, particularly with Metastrongylus nematodes. Finally, three ixodid tick species, all adults, were collected off of the majority of feral swine sampled, none of which appeared to be carrying infectious virus based on the results of general cytopathic effects assays. Results generated for this study further confirm that feral swine are hosts for a range of ixodid ticks present in Texas and include species that are known to pose a risk to the health of livestock, wildlife, and humans. Moreover, serological results indicating previous exposure to anthrax-causing bacteria agree with past ecological modeling studies that have suggested environmental suitability for B. anthracis in the region of Aransas National Wildlife Refuge. Taken together, these investigations demonstrate that feral swine in the United States very likely are contributing to the ecology of anthrax and coccidioidomycosis by being mechanical vectors and possible reservoirs of B. anthracis and Coccidioides, respectively. Serological data demonstrated from field and laboratory studies additionally support the use of feral swine as biosentinels within landscapes undergoing invasive species management and that may be contaminated with B. anthracis. While the serological data generated through the B. pseudomallei investigations reported here currently do not support use of a crude whole-cell lysate-based ELISA to evaluate samples collected from the field, we hypothesize that feral swine may be reservoirs and/or vectors of melioidosis-causing bacteria. Future investigations should continue to examine additional antigens for serologic testing, as laboratory-infected swine demonstrated a measurable antibody response after intranasal exposure with Bp82. Additional follow-up studies should also be conducted to further describe the relative role(s) that feral swine may play in the ecology and epidemiology of B. anthracis and Coccidioides, particularly if regions with high incidence of animal or human disease correspond with high densities and activity of feral swine. Future research should continue to screen feral swine as well as the ectoparasites that feed upon them for emerging or re-emerging pathogens, especially in areas with high biodiversity or species of conservation concern, as well as in areas with high levels of human activity.Item Open Access Oocyte metabolism – a potential link between mare conditions and impaired fertility(Colorado State University. Libraries, 2023) Di Donato Catandi, Giovana, author; Carnevale, Elaine M., advisor; Krisher, Rebecca L., advisor; Chicco, Adam J., committee member; Chen, Thomas W., committee memberMaternal advanced aging and obesity are known for negatively affecting reproductive outcomes by directly impacting the oocyte and the follicular environment, where the oocyte develops and matures. Success of early embryonic development relies on appropriate ability of the oocyte to produce energy. Whether maternal conditions of the mare impact oocyte metabolic function had not been previously determined. In the studies described throughout this dissertation, novel microsensors were utilized to quantify aerobic and anaerobic metabolism of single equine oocytes. Additional and complementing end points were obtained through high- resolution respirometry of granulosa cells and metabolomic profiling of oocytes and cumulus cells. The overarching hypothesis of this dissertation is that mare conditions known to impair fertility, namely advanced age and obesity, affect oocyte metabolism, ultimately impairing oocyte developmental potential. It was additionally hypothesized that dietary supplementation to old or obese mares would reach and affect the ovarian follicular environment and the oocyte, improving its metabolic function and quality. To test these hypotheses, a series of three projects were conducted to: 1) Investigate effects of mare advanced aging on oocyte metabolism; 2) Determine the potential of diet supplementation to old mares to improve oocyte metabolism; 3) Investigate effects of mare obesity on oocyte metabolism and the potential of diet supplementation on normalizing metabolic alterations. Findings from these projects revealed that mare advanced aging impairs oocyte aerobic and anaerobic metabolic function, contributing to limited embryonic metabolism and development after intracytoplasmic sperm injection (ICSI). Short-term dietary supplementation to old mares with feed additives, specifically formulated to improve mitochondrial metabolism and overall equine health, was able to improve mitochondrial metabolism of granulosa cells and oocytes, promoting greater embryonic rates after ICSI in comparison to a control grain supplementation. Additionally, the findings here reported demonstrate that mare obesity promotes several alterations in the ovarian follicle, including excess of reactive oxygen species production by granulosa cells, lipid accumulation in cumulus cells and oocytes, and excessive oocyte aerobic and anaerobic metabolism. Dietary supplementation to obese mares with similar feed components mitigated many of the obesity-associated follicular changes, likely contributing to oocyte quality. Collectively, these novel discoveries contribute to knowledge and understanding of the direct effects of maternal conditions of the mare on the ovarian follicle and oocyte, elucidating cellular mechanisms by which advanced aging and obesity disturb fertility. Furthermore, these findings reveal the benefits of dietary interventions in improving oocyte metabolism and quality. Dietary supplementation represents a non-invasive and feasible approach to tackle female subfertility. Assuredly the results presented throughout this dissertation will contribute to the equine reproduction industry, with potential to have a translational impact on the human fertility industry, by not only elucidating direct effects of maternal conditions on oocyte metabolism, but also by providing a practical method for rescuing it in vivo.Item Open Access Gas6/AXL signaling contributes to GnRH-dependent activation of pituitary gonadotropes(Colorado State University. Libraries, 2023) Mohammad Zadeh, Pardis, author; Amberg, Gregory C., advisor; Hoerndli, Frederic, committee member; Stasevich, Timothy, committee member; Winger, Quinton, committee memberGonadotropin-releasing hormone (GnRH) receptor plays a fundamental role in reproduction and is prevalent in various urogenital, reproductive, and non-reproductive cancers. Beyond the conventional G protein-coupled receptor signaling, GnRH receptors interact functionally with multiple receptor tyrosine kinases. AXL, a receptor tyrosine kinase found in various tissues and numerous tumors, is the focus of this dissertation to discover its impact, along with its endogenous ligand Gas6, on GnRH receptor signaling. In this study, clonal murine pituitary αT3-1 and LβT2 gonadotrope cell lines were utilized to evaluate the effect of AXL activation on GnRH receptor-dependent signaling pathways. A combination of ELISA and immunofluorescence techniques was employed to analyze AXL and GnRH receptor expression in αT3-1 and LβT2 cells, as well as in murine and human pituitary sections. Additionally, ELISA was used to quantify alterations in ERK phosphorylation, pro-MMP9 production, and the release of LHβ. The abundance of Egr-1 transcripts was measured using digital droplet PCR. To assess αT3-1 and LβT2 cell migration responses to GnRH and AXL, trans-well migration assay was used. Results showed the presence of AXL, alongside GnRH receptors, in αT3-1 and LβT2 gonadotrope cell lines, as well as in murine and human pituitary sections. In line with AXL's potentiating role, Gas6 enhanced GnRH-dependent ERK phosphorylation in αT3-1 and LβT2 cells. Furthermore, Gas6 increased the abundance of Egr-1 transcripts, suggesting enhanced post-transcriptional GnRH receptor responses. Notably, in LβT2 cells, Gas6/AXL signaling not only stimulated LHβ production but also enhanced GnRH receptor dependent pro-MMP9 protein generation and promoted cell migration, underlining its functional significance. In summary, our findings unveil a hitherto undiscovered role for AXL as a modulator of GnRH receptor signaling.Item Embargo Evaluating learning resource selection to support gross anatomy education(Colorado State University. Libraries, 2023) Martin, Jason, author; Magee, Christianne, advisor; West, Andrew, committee member; Meyer, Carolyn, committee member; Kaiser, Leann, committee member; Winger, Quinton, committee memberAnatomy educators are tasked with developing, maintaining, and revising comprehensive curricula that strengthen the foundation of a growing body of scientific knowledge necessary to be successful medical professionals. This work sought to evaluate the impact of instructional timing, spatial ability, self-efficacy, and resource preference in the animal anatomy classroom for undergraduate and graduate students at Colorado State University. It identified factors relevant to optimizing student performance on animal gross anatomy examinations. The first chapter provides a background on anatomy learning resources in the context of self-efficacy and spatial ability research. Chapter two found that following a transition to remote instruction, students value resources that assist with navigating their learning ecology and assist with content mastery. Chapter three was a five-year retrospective study that identified a correlation between prior examination experience and dissection examination scores. Chapter four compared two measures of visuospatial ability with atlas- and specimen-based animal anatomy assessments following experimental single-resource and real-world multi-resource instruction. Chapter five was a mixed-methods analysis that developed an experimental framework used to describe the relationship between learner self-efficacy and animal gross anatomy assessment scores. Using this framework, increased time attending in-person didactic lectures and teaching assistants mediated open laboratories was found to be beneficial for low self-efficacy students while independent exploration of open-laboratory was beneficial for high self-efficacy students. An experimental study reported in chapter five failed to find a relationship between resource preference and performance on experimental anatomy assessments. When taken together, the research provides suggestions for anatomy educators seeking to use the selective distribution of learning resources to improve the instruction of animal anatomy students.Item Open Access A retinal contribution to chronic opioid-induced sleep/wake dysfunction(Colorado State University. Libraries, 2023) Bergum, Nikolas, author; Vigh, Jozsef, advisor; Myers, Brent, committee member; Hentges, Shane, committee member; Chanda, Soham, committee memberLight is among the most important environmental factors that regulate mammalian sleep/circadian behaviors. Melanopsin-expressing intrinsically photosensitive retinal ganglion cells (ipRGCs) transmit environmental light information to key sleep/circadian centers in the brain through a process known as photoentrainment. Interestingly, past studies have revealed that ipRGCs express µ-opioid receptors (MORs), the primary molecular target for opioid analgesics. Furthermore, MOR agonists can directly inhibit ipRGC firing. Therefore, we hypothesize that opioid drugs acting on MORs expressed by ipRGCs could disrupt ipRGC-mediated regulation sleep/wake rhythms in response to environmental light/dark cycles. To test this idea, we need to confirm that morphine reaches the mouse retina following systemic delivery. To accomplish this, tissue (retina, brain and serum) was collected from mice following intraperitoneal morphine administration. Importantly, results from this study show that systemically administered morphine selectively accumulates in the mouse retina, but not the serum or the brain. To test the role that MORs expressed by ipRGCs play in opioid-induced dysregulation of sleep/circadian behaviors, we used mini-telemetry devices to assess how chronic morphine alters their sleep/wake behavior in mice. Importantly, we performed these experiments in wildtype mice along with mice lacking MORs exclusively in ipRGCs (McKO). Results from these studies reveal that McKO animals exhibit decreased morphine-induced locomotion compared to controls, which implicates MORs expressed by ipRGCs as a mediator of opioid-induced sleep-wake alterations. Finally, we tested whether ipRGCs developed cellular tolerance to MOR agonists following chronic exposure to morphine. The lack of cellular tolerance development at the level of solitary ipRGCs provides a potential cellular correlate for the persistent sleep/wake dysfunction commonly attributed to chronic opioid exposure. Taken together, these findings support the idea that opioid accumulation in the eye persistently activate MORs on ipRGCs, continuously altering the ability of ipRGCs to transmit light information to the brain's sleep/wake circuitry. This alteration in photic input to the brain could underlie some of the sleep/wake problems associated with long-term opioid use.Item Open Access 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 memberIn 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.