Browsing by Author "Tesfaye, Dawit, committee member"
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Item Embargo Assessing antimicrobial mechanisms in Langerhans cells during a Mycobacterium leprae infection(Colorado State University. Libraries, 2024) Fischbacher, Linda, author; Belisle, John, advisor; Gries, Casey, committee member; Tesfaye, Dawit, committee memberLangerhans cells are essential immune cells in the skin that maintain homeostasis and clear pathogens. Despite their importance, much is unknown about Langerhans cells, including their innate antimicrobial mechanism. Single-cell sequencing of leprosy skin lesions identified genes upregulated in Langerhans cells of reversal reaction lesions that may be associated with antimicrobial activity. CCL22, MPEG1, and IDO1 were selected to study further as potential effectors in Langerhans cells for killing Mycobacterium leprae. We hypothesized that altered metabolic processes in Langerhans cells impact antimicrobial effects on M. leprae. An in vitro model was developed to induce antimicrobial gene expression in Langerhans cell-like dendritic cells (LCDCs). IL-1β was identified as the best inducer of CCL22 and MPEG1, and IFN-γ as the best inducer of IDO1. Induction was measured by gene expression and protein production, as well as enzyme activity for IDO1 by measuring metabolites. The antimicrobial effect of IDO1 on intracellular M. leprae in LCDCs was assessed by inducing IDO1 with IFN-γ or inhibiting IDO1 activity with 1-methyltryptophan. Stimulation by this agonist or this antagonist modulated IDO1 expression and activity but did not affect M. leprae viability. The changes of intracellular tryptophan catabolites in non-stimulated and M. leprae-infected LCDCs were measured. The M. leprae infection increased two kynurenine pathway catabolites after 24 and 48 hours, 3-hydroxyanthranilic acid and quinolinic acid. These data indicate that while M. leprae did not induce IDO1 expression, it did increase IDO1 and kynurenine pathway activity. Neither metabolite has reported antimicrobial properties, but quinolinic acid may benefit M. leprae for synthesizing nicotinamide adenine dinucleotide. A different tryptophan catabolite pathway leads to serotonin production. In M. leprae-infected LCDCs, serotonin was decreased, but 5-hydroxyindoleacetic acid, a breakdown product of serotonin, was increased. The implication of these changes for an M. leprae infection of LCDCs is unknown. 5-hydroxyindoleacetic acid is also increased in Mycobacterium tuberculosis patients. This metabolite may benefit these mycobacteria as it is reported to increase PPARγ activity, which is known to support M. leprae and M. tuberculosis in macrophages. The main antimicrobial mechanism of IDO1 is depleting tryptophan from tryptophan auxotrophic pathogens. Whether the tryptophan biosynthesis pathway in M. leprae is functional was assessed using 13C-tracing, to determine if tryptophan depletion by IDO1 could kill M. leprae. In axenic media, M. leprae did not synthesize tryptophan from 13C-glucose and 13C-palmitic acid nor synthesize tryptophan from intracellular 13C-glucose. In vitro, M. leprae only synthesized tryptophan from an intermediate, anthranilic acid. Using the same method, M. tuberculosis synthesis of tryptophan from 13C-glucose was confirmed as a control. The functionality of the tryptophan biosynthesis pathway in M. leprae could not be confirmed. However, because of the homology between the M. leprae and M. tuberculosis genes for tryptophan biosynthesis, this pathway likely is functional, and M. leprae would not be killed by IDO1-mediated tryptophan depletion. These findings indicate that IDO1 is not associated with antimicrobial activity towards M. leprae in LCDCs. Instead, increased IDO1 activity induced by M. leprae infection resulted in increased tryptophan catabolites likely to benefit rather than kill M. leprae in LCDCs. M. leprae likely evades the primary killing mechanism of IDO1, tryptophan depletion, by possessing an intact pathway for tryptophan biosynthesis. Further studies to elucidate the importance of quinolinic acid and 5-hydroxyindoleactic acid for M. leprae and validate that the M. leprae tryptophan biosynthesis pathway is functional will aid in identifying essential pathways for M. leprae that can be targeted with therapeutics. Other potential antimicrobial effectors in LCDCs, including CCL22 and MPEG1, will need to be assessed to study this innate mechanism in Langerhans cells further.Item Open Access Extracellular vesicles from the equine uterus: uptake by stallion spermatozoa and effect on capacitation parameters(Colorado State University. Libraries, 2023) Granier, Shelby K., author; McCue, Patrick, advisor; Graham, James K., advisor; Hatzel, Jennifer, committee member; Tesfaye, Dawit, committee memberFertilization in mammalian species relies on the activation of spermatozoa in the female reproductive tract by a consecutive series of events termed 'capacitation'. In vivo, ejaculated equine spermatozoa are deposited directly into the uterus and eventually arrive in the ampulla of the oviduct, which is the site of fertilization. However, the roles of the uterus, oviduct, and their secretions have on equine sperm capacitation is largely unknown. Extracellular Vesicles (EVs), including microvesicles and exosomes, are membrane enclosed nanoparticles released from most cell types that carry cargos of biologically active molecules that can affect nearby or distant recipient cells. EVs have recently been identified as playing a role in reproductive functions including sperm capacitation. The aims of the present study were: first characterize EVs collected from the uterine lumen of mares in both the estrus and diestrus phases of their reproductive cycles; and second investigate the effect these uterine EVs have on stallion sperm function. Uterine fluid from 6 mares was collected during both estrus and diestrus using a low volume uterine lavage then EVs were isolated from the fluid by ultracentrifugation, and EV concentration determined by nano-tracker analysis. The concentration of EVs obtained from estrus fluids (EEV) was 235 ± 164.029 billion EVs/mL and tended to be higher (p=0.07) than those obtained in diestrus fluids (DEV) (83.67 ± 89.328 billion EVs/mL). The average size of EVs were similar (p > 0.05) with values of 148.633 ± 11.35 nm for EEV and 146.183 ± 11.89 nm for DEV. Transmission electron microscopy delivered images of vesicles with characteristic cup-shape morphology and size consistent with NTA results. Immunoblotting confirmed the particles contained exosome markers TSG-101 and CD-63, and were negative for cytochrome C, a mitochondrial organelle marker, indicating these vesicles were indeed EVs. To determine the effect EVs have on sperm, semen from 3 Quarter Horse stallions were cryopreserved, and EVs added to samples after thawing. In the first experiment, EVs or PBS void of EVs were fluorescently labeled and incubated with frozen-thawed stallion spermatozoa for one hour and uptake was evaluated by fluorescent microscopy. Fluorescence was observed only in sperm incubated with EVs, and a greater fluorescent intensity detected in EEV treated sperm. In a second experiment, spermatozoa from each stallion were co-cultured with EEV, DEV, and PBS void of EVs (control) for 90 minutes and sperm functions associated with capacitation, including hyperactivated motility, and acrosome reactions, were evaluated using a computer assisted semen analysis unit (CASA) and flow cytometry. The percentages of hyperactively motile sperm were higher (p < 0.05) for EEV treated sperm compared to control and DEV. In addition, the percentage of acrosome reacted sperm was higher (p < 0.05) for sperm treated with EEV and DEV when compared to control. In summary, these results confirm that: 1) EVs can be isolated from uterine fluid of mares, 2) uterine derived EVs can be taken up by stallion spermatozoa, and 3) uterine derived EVs have a biological effect on stallion spermatozoa function in vitro. Consequently, it is hypothesized that EVs from the mare reproductive tract will have similar biological effects on stallion sperm function in vivo.Item Embargo Impact of testosterone on trophoblast mitochondrial function(Colorado State University. Libraries, 2022) Parsons Aubone, Agata M., author; Chicco, Adam, advisor; Tesfaye, Dawit, committee member; Tjalkens, Ron, committee memberSeveral pregnancy disorders involve placental abnormalities, including gestational diabetes mellitus (GDM) (2-10% of pregnancies), preeclampsia (PE) (6-8% of pregnancies), and polycystic ovary syndrome affects (PCOS) (6 to 15% of women in reproductive age), which not only has a negative impact on maternal health but can also lead to birth defects and postnatal health complications. These disorders commonly also present high levels of androgens in maternal blood, accompanied by placental insufficiency. The placenta in these pathologies presents morphological and physiological alterations, including in the trophoblast mitochondria. The placenta is a multifunctional, transient organ that mediates the transport of nutrients and waste to and from the fetus, gas exchange, and endocrine signaling to maintain maternal and fetal homeostasis. To facilitate these diverse and important functions and enable proper fetal growth and development, the placenta is highly metabolically active and consumes ~40% of the total oxygen. Oxygen is used for the synthesis of ATP in mitochondria, which in turn is mainly used for cholesterol transport and steroidogenesis. The placenta is well recognized as a hormone-synthesizing and secreting organ; however, studies revealed it is a target of these hormones as well and contains receptors for various steroid hormones including androgens. Placental androgen receptor (AR) is relevant in pregnancy disorders with elevated androgens such as GDM, PE, and PCOS. These are accompanied by placental pathologies that include mitochondrial adaptations that vary according to the stage of the pathology, and in advanced stages when levels of reactive oxygen species (ROS) become too high they can have detrimental effects on the placenta and can even lead to pregnancy loss. Of particular interest here is the recent observation that AR has been identified as a regulator of mitochondrial function in other tissues such as the prostate and cancer. The production of ROS and/or the decrease in antioxidant defenses are the main mechanisms underlying placental insufficiency in pathologies with elevated androgens. A better understanding of the regulation of androgen signaling in placental mitochondria will lead to new insights and opportunities to understand and treat disorders of pregnancy that affect a significant number of pregnant women. Studying the human placenta in vivo presents several complications, so it is necessary to use in-vitro models. There are several human trophoblast cell lines available, but none are perfect replacements for the original organ, rather each one has qualities that allow investigators to choose one best suited for their study. The overall goal of our studies is to investigate the role of AR signaling in trophoblast cell mitochondrial respiration. Our hypothesis is that AR signaling regulates mitochondrial oxygen consumption and ROS production. The first chapter will provide an overview on the role of androgens in placental physiology and pregnancy, and the role of mitochondria in trophoblast cell function. In the second chapter, we present studies aimed at characterizing mitochondrial respiration in existing placental cell lines and elucidating a possible role for AR signaling in mitochondria. Specifically, we first demonstrated the presence of AR in placental mitochondria. Next mitochondrial oxygen consumption and ROS production are characterized and compared using an Oroboros O2K oxygen respirometer in three well-known human (ACH-3P, BeWo, and Swan-71) and one immortalized ovine trophoblast cell (iOTR) line. Finally, ACH-3P cells are selected to test mitochondrial responses to testosterone, mimicking placental pathologies seen in GDM, PE, and PCOS. Our results revealed that both human ACH-3P and Swan-71 cells, as well as the sheep iOTR cells, demonstrated normal oxygen consumption and ROS production following the addition of selected complex protein substrates. Chronic testosterone treatment led to significant increased ROS production in ACH-3P cells, which correlates with what has been observed in term placentas of women with placental hyperandrogenism. In conclusion, the ACH-3P cell line is a good in vitro model to study placental mitochondrial respiration. Ultimately, the presented data provide new information regarding the possible role of AR signaling in placental mitochondria and will pave the way for future studies aimed at uncovering the mechanism of AR regulation of mitochondrial function in normal and abnormal pregnancies (discussed in Chapter 3).Item Embargo Metabolic support of preimplantation embryo growth and viability(Colorado State University. Libraries, 2024) Fresa, Kyle Joseph, author; Carnevale, Elaine, advisor; Chicco, Adam, advisor; Tesfaye, Dawit, committee member; Krisher, Rebecca, committee member; Gentile, Christopher, committee memberEarly embryo metabolism involves essential and dynamic biological reactions that support viability, growth, and pregnancy establishment. Embryo metabolism not only serves to provide energy through ATP synthesis, but also facilitates the production of macromolecules such as proteins, nucleotides, and lipids. The ways in which embryos balance catabolic and anabolic activity during the preimplantation stage are not well understood; however, understanding these processes may lead to improved fertility treatments, embryo culture, and pregnancy outcomes. The studies described in this dissertation utilize innovative methods, such as stable isotope tracer analysis to track carbon and nitrogen flux through various pathways, oxygen microsensors to determine individual embryo respiration under various conditions, and proteomic analysis to determine the impacts of metabolic disturbances on embryo viability. The overarching hypothesis of this dissertation is that embryo viability is dependent on efficient and tightly regulated metabolic activity, and disturbances to metabolic function ultimately lead to reduced developmental potential. To test this hypothesis, a series of projects were conducted to 1) evaluate the importance of phosphoenolpyruvate carboxykinase (PEPCK) during early development, 2) uncover the function of PEPCK to support catabolic and anabolic activity in early embryos, and 3) determine the impacts of delayed embryo development on embryo metabolism and pathway regulation. These projects revealed important insights into the impact of embryo metabolism on development, including the discovery of a novel, PEPCK-mediated pathway that embryos utilize to balance energy production and biosynthesis. Furthermore, the impact of delayed embryo development was demonstrated to alter embryo metabolic activity and pathway regulation, including increased aerobic activity and altered protein expression. These findings improve our understanding of metabolic activity and regulation during preimplantation development, highlighting the impact of metabolic activity to promote ATP production, biosynthesis, developmental kinetics, and ultimately survival. The experimental outcomes presented in this dissertation provide a foundation for targeted approaches to improve embryo development and reproductive success.Item Open Access Modeling human trophoblast development during the peri-implantation period using extended embryo culture(Colorado State University. Libraries, 2023) Logsdon, Deirdre Maria, author; Winger, Quinton, advisor; Krisher, Rebecca, advisor; Yuan, Ye, committee member; Tesfaye, Dawit, committee member; DeLuca, Jennifer, committee memberDuring the peri-implantation period, a human embryo must transition from a pre-implantation stage blastocyst to a gastrulating embryonic disc surrounding by the primitive placenta. The primitive placenta at this time establishes contact, proliferates, invades, modulates the maternal immune system, and provides a primitive form of nutrients to the implanting embryo proper. Insights into this period have been largely stunted due to the ethical and technical challenges that accompany human embryo research. Studies using donated human embryos following fertility treatment are complicated by confounding infertility diagnoses and limited sample sizes. The development of the extended culture system has provided an avenue to functionally study the peri-implantation period. Further, by using a variety of models including mouse embryos, human embryos, and stem cell-derived blastoids in the extended culture system, researchers are finally able to begin to piece together the puzzle of the peri- implantation period. Here, our objectives were to demonstrate the utility of mouse models in modeling human trophoblast during peri-implantation extended culture, examine and summarize human development during peri-implantation in the context of confounding fertility diagnoses, compare human trophoblast in extended culture to other widely available regenerative trophoblast models, and determine to what extent blastoids are able to reflect human peri-implantation development and maternal-fetal crosstalk in extended culture. Further, we show that estrogen signaling in trophectoderm may be conserved between mouse and human embryos, aged embryos exhibit hindered growth in extended culture, peri-implantation trophoblast cells have unique transcriptional priorities, and the presence of endometrial stromal cells encourage fusion of syncytiotrophoblasts. Our studies both reinforce the significance of the extended culture system and lay the groundwork for future studies on early trophoblast and embryo development during peri-implantation.Item Open Access The impact of placental SLC2A3 (GLUT-3) RNA interference on fetal growth and physiology at mid-gestation in sheep(Colorado State University. Libraries, 2022) Lynch, Cameron S., author; Anthony, Russell V., advisor; Tesfaye, Dawit, committee member; Engle, Terry, committee memberGlucose is the primary energy substrate for fetal oxidative processes and growth. In order for glucose to be transported from maternal to fetal circulation in the ruminant placenta, it must be sequentially transported by SLC2A1 (GLUT-1) on the maternal-fetal syncytial layer, then by SLC2A3 (GLUT-3) on the apical trophoblast membrane, and again by SLC2A1 on the basolateral trophoblast membrane. SLC2A1 is the most abundant placental facilitative glucose transporter, and as such, is believed to be the primary glucose transporter in human and sheep placenta. However, SLC2A3 exhibits a five-fold greater affinity and transport capacity for glucose. As such, in addition to its location on the apical trophoblast membrane, any deficiency in SLC2A3 could impact trophoblast glucose uptake and placental transfer of glucose to the fetus, thus potentially altering placental development and setting the stage for fetal hypoglycemia and intrauterine growth restriction (IUGR). It was our objective to use placenta-specific RNA interference (RNAi) to diminish SLC2A3, and determine the impact at mid- gestation (75 dGA) in sheep. The resulting pregnancies underwent a terminal surgery at 75 dGA. SLC2A3 RNAi resulted in a 37% reduction (p ≤ 0.05) in placental SLC2A3 concentration. SLC2A3-deficiency resulted in decreased fetal growth as evident by reduced fetal weight (p ≤ 0.10), head circumference (p ≤ 0.05), femur length (p ≤ 0.05), and tibia length (p ≤ 0.05). While there were no significant reductions in maternal glucose or insulin concentrations, the SLC2A3 RNAi pregnancies had decreased umbilical vein (p ≤ 0.05) and umbilical artery (p ≤ 0.05) glucose concentrations, as well as reduced umbilical artery insulin (p ≤ 0.10). Additionally, apparent attempts at compensation for SLC2A3-deficiency, by increasing SLC2A1, CSH, and IGF-2, were unable to prevent fetal hypoglycemia and the impacts on fetal development. Placental SLC2A1 concentration were increased (p ≤ 0.10), however this increase in expression was unable to prevent fetal hypoglycemia. The significant increase in umbilical vein CSH concentrations (p ≤ 0.05) appeared to preserve fetal liver weight and circulating umbilical concentrations of IGF-1, both of which are commonly decreased in IUGR pregnancies. SLC2A3-deficiency also resulted in a significant increase in IGF-2 (p ≤ 0.05), IGF1R (p ≤ 0.05), and IGF2R (p ≤ 0.05) expression. This suggests an apparent attempt to increase placental growth via IGF-2 acting through IGF1R, while IGF2R, which primarily acts to sequester and degrade IGF-2, doesn't allow placental growth to be overstimulated. While it has been suggested that SLC2A3 is predominantly important in late gestation, our data indicate that SLC2A3 is important for normal fetal development and appears to be a rate limiting glucose transporter during the first-half of gestation. A deficiency in SLC2A3 impacts trophoblast glucose uptake and subsequently glucose transfer to the fetus, and appears to set the stage during early gestation for the development of IUGR.