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- ItemEmbargomiR-137 regulates PTP61F, affecting insulin signaling, metabolic homeostasis, and starvation resistance in Drosophila melanogaster(Colorado State University. Libraries, 2023) Saedi, Hana Ibrahim, author; Tsunoda, Susan, advisor; Hoerndli, Frederic, committee member; Amberg, Gregory, committee member; Di Pietro, Santiago, committee membermiR-137 is a highly conserved brain-enriched microRNA (miRNA) that has been associated with neuronal function and proliferation. Here, we show that Drosophila miR-137 null mutants display increased body weight with enhanced triglyceride and glucose levels and decreased locomotor activity. When challenged by nutrient deprivation, miR-137 mutants exhibit reduced motivation to feed and significantly prolonged survival. Together, these phenotypes suggest a new role for miR-137 in energy homeostasis. Genetic epistasis experiments show that the starvation resistance of miR-137 mutants involves the insulin signaling pathway, and that loss of miR-137 results in drastically reduced phosphorylation/activation of the single insulin receptor, InR, in Drosophila. We explore the possibility that the protein tyrosine phosphatase61F (PTP61F), ortholog of TC-PTP/PTP1B, known to dephosphorylate InR across species, is a potential in vivo target of miR-137. We show that loss of miR-137 results in upregulation of an endogenously tagged PTP61F protein, and that genetically increasing levels of PTP61F mimics the loss of phosphorylated InR and increased starvation resistance seen in miR-137 mutants. Finally, we show that the enhanced starvation resistance of miR-137 mutants is normalized by activation of the insulin signaling pathway in the nervous system. Our study introduces miR-137 as a new player in the regulation of central insulin signaling and metabolic homeostasis.
- ItemOpen AccessThe autism-associated loss of δ-catenin function disrupts social behavior(Colorado State University. Libraries, 2023) Mendez-Vazquez, Hadassah, author; Kim, Seonil, advisor; Tamkun, Michael, committee member; Myers, Brent, committee member; Chanda, Soham, committee memberSocial impairment is a key symptom of several neuropsychiatric disorders, including autism spectrum disorder (ASD), anxiety, depression, and schizophrenia. Despite the increasing prevalence of these disorders the physiological, cellular, and molecular factors underlying social dysfunction are still poorly understood. In humans, mutations in the δ-catenin gene have been linked to severe forms of ASD. δ-catenin is a post-synaptic scaffolding protein that is expressed in excitatory synapses and functions as an anchor for N-cadherin and the AMPA receptor (AMPAR) subunit GluA2 at the postsynaptic density. A glycine 34 to serine (G34S) mutation in the δ-catenin gene was identified in ASD patients and induces a loss of δ-catenin function, which may mediate ASD pathogenesis. The mechanism by which this G34S mutation causes loss of δ-catenin function to induce ASD remains unclear. Initial findings revealed that the G34S mutation increases glycogen synthase kinase 3β (GSK3β)-dependent δ-catenin degradation to reduce δ-catenin levels. Moreover, we found that mice possessing the G34S δ-catenin mutation have significantly reduced synaptic cortical δ-catenin and GluA2 levels. The G34S mutation was also found to differentially alter glutamatergic activity in cortical excitatory and inhibitory cells. Furthermore, G34S δ-catenin mutant mice exhibit markedly impaired social behavior, which is a characteristic feature of ASD. Most significantly, we found that inhibition of GSK3β is sufficient to reverse the G34S-induced loss of δ-catenin function in cells and mice. Altogether, our study reveals that the loss of δ-catenin function arising from the ASD-associated G34S mutation induces social dysfunction via disruptions in glutamatergic activity, and that GSK3β inhibition can reverse abnormal δ-catenin G34S-induced glutamatergic activity and social deficits.
- ItemOpen AccessImpact of chorionic somatomammotropin in vivo RNA interference phenotype on uteroplacental expression of the IGF axis(Colorado State University. Libraries, 2023) Hord, Taylor, author; Anthony, Russel V., advisor; Winger, Quinton A., advisor; Bruemmer, Jason, committee member; Kading, Rebekah, committee memberWhile fetal growth is dependent on many factors, optimal placental function is a prerequisite for a normal pregnancy outcome. The majority of fetal growth restricted (FGR) pregnancies result from placental insufficiency (PI). The insulin-like growth factors (IGF1 and IGF2) not only stimulate fetal growth, but also placental development and function. Previously, we demonstrated that in vivo RNA interference (RNAi) of the placental hormone, chorionic somatomammotropin (CSH), resulted in two phenotypes. One phenotype exhibits significant placental and fetal growth restriction (PI-FGR), impaired placental nutrient transport, and significant reductions in umbilical insulin and IGF1. The other phenotype does not exhibit statistically significant changes in placental or fetal growth (non-FGR). It was our objective to further characterize these two phenotypes by determining the impact of CSH RNAi on placental (maternal caruncle and fetal cotyledon) expression of the IGF axis. The trophectoderm of hatched blastocysts (9 days of gestation, dGA) were infected with a lentivirus expressing either a non-targeting sequence (NTS RNAi) control or CSH-specific shRNA (CSH RNAi) prior to embryo transfer into synchronized recipient ewes. At ≈125 dGA, pregnancies were fitted with vascular catheters to undergo steady-state metabolic studies. Nutrient uptakes were determined and tissues were harvested at necropsy. In both CSH RNAi non-FGR and PI-FGR pregnancies, uterine blood flow was significantly reduced (P≤0.05), while umbilical blood flow (P≤0.01), both uterine and umbilical glucose and oxygen uptakes (P≤0.05), and umbilical concentrations of insulin and IGF1 (P≤0.05) were reduced in CSH RNAi PI-FGR pregnancies. Fetal cotyledon IGF1 mRNA concentration was reduced (P≤0.05) in CSH RNAi PI-FGR pregnancies, whereas neither IGF1 nor IGF2 mRNA concentrations were impacted in the maternal caruncles, and either placental tissue in the non-FGR pregnancies. Fetal cotyledon IGF1R and IGF2R mRNA concentrations were not impacted for either phenotype, yet IGF2R was increased (P≤0.01) in the maternal caruncles of CSH RNAi PI-FGR pregnancies. For the IGF binding proteins (IGFBP1, IGFBP2, IGFBP3), only IGFBP2 mRNA concentrations were impacted, with elevated IGFBP2 mRNA in both the fetal cotyledon (P≤0.01) and maternal caruncle (P=0.08) of CSH RNAi non-FGR pregnancies. These data support the importance of IGF1 in placental growth and function, but may also implicate IGFBP2 in salvaging placental growth in non-FGR pregnancies.
- ItemEmbargoModeling 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.
- ItemOpen AccessInvestigation of potential advancements in bovine assisted reproductive technologies(Colorado State University. Libraries, 2023) LaVelle, Gerica Anne Helton, author; Barfield, Jennifer, advisor; Tesfaye, Dawit, advisor; Cairo, Betsy, committee member; Pinedo, Pablo, committee memberThe assisted reproductive technologies (ART) area of research within bovine encompasses a diverse group of technologies including artificial insemination, cryopreservation of gametes and embryos, in vitro fertilization, induction of multiple ovulations, sex determination of sperm and embryos, and more. These techniques have been studied thoroughly within the last forty years, yet there is a plethora of knowledge to still be discovered on a molecular basis extending to longitudinal effects of ART interference generationally. One large sector of ART research in the bovine industry is to gain information that will aide in the advancement of current protocols and practices to increase productivity and cost effectiveness. This includes improving cryopreservation of gametes, creating higher in vitro embryo production yield, reducing costs, and developing easier practices for technicians with hopes of providing a more efficient system, from both a labor and fiscal standpoint. The following dissertation contributes specifically to this research sector by providing novel information regarding cryopreservation and capacitation timing of bovine spermatozoa treated with cholesterol prior to cryopreservation, embryo production rates when such spermatozoa are used within an in vitro fertilization (IVF) system, and the use and metabolomic components of spent media from bovine embryo culture. The first chapter of this dissertation will provide background information on the complex sperm capacitation and acrosome reaction process, and methods on how to assess and modify the capacitation process via molecular processes, both pre- and post-cryopreservation. This lays the foundation for the research presented here, which focuses on the loading of bovine spermatozoa with cholesterol prior to cryopreservation, and what effect this treatment has on the cells post-thaw in relation to capacitation timing analyzed using flow cytometry within the second chapter. The first chapter also provides background to the fertilization process and in vitro production of embryos which is examined by using cholesterol-loaded spermatozoa for IVF presented in the third chapter. Additionally, contextual information is explained regarding the type of culture systems within IVF, where embryos are individually cultured or in groups. This information is required for the fourth chapter which includes experimentation with spent culture media and the study of the metabolomic components of media exposed to group embryo culture. Pre-loading bovine sperm with cholesterol prior to freezing is known to increase cryosurvival, though the timing of capacitation in these sperm has not yet been evaluated. The objective of the study performed in chapter two was to determine if there is a potential delay in capacitation timing in these sperm due to the increased cholesterol content. Flow cytometric evaluation was utilized to assess viability, and stain technology to assess acrosome intactness (Propidium Iodide/FITC-PNA), intracellular calcium levels (Propidium Iodide/FLUO 3-AM) and membrane fluidity (Merocyanine 540/YO-PRO-1). Through this investigation it was determined that treatment of sperm with cholesterol-loaded cyclodextrin (CLC) (2 mg/mL) increased cryosurvival and viability over time, though longer time to capacitate was required compared with non-treated sperm. Treatment with CLC also significantly decreased membrane fluidity in sperm (P<.05). by 1 hour, resulting in increased intracellular calcium and increased acrosome reaction, and consequently viability loss by 3 hours. Taken together, these results determined sperm pre-loaded with cholesterol display significantly improved post-thaw viability but require longer to capacitate which may hinder fertilization capacity and/or require adjustments to timing of in vitro fertilization. Given the delayed capacitation timing of sperm treated with CLC, it was then investigated if CLC-treated sperm could be successful in IVF to determine if the additional retained cholesterol hindered fertilization ability and consequently, embryo production. The research summarized in chapter three examines the fertilization ability of CLC-treated sperm and subsequent in vitro embryo development by offsetting sperm preparation timing in order to adjust for delayed capacitation due to the increased cholesterol content of CLC-treated sperm. Additionally, treatment with methyl-β-cyclodextrin (MβCD) was utilized as a capacitation inducing agent by causing an efflux of cholesterol from the sperm plasma membranes during the sperm preparation incubation period. Bovine sperm pre-loaded with cholesterol via CLC prior to cryopreservation did not result in significant difference in cleavage or blastocyst formation (P>0.05) when sperm preparations were completed 90 minutes prior to co-incubation to adjust for delayed capacitation timing. There was significant decline (P<0.05) in embryo cleavage percentage in both control and CLC-treated sperm groups when MβCD was used as a capacitation inducer at 2 mg/ml. The findings presented in chapter three demonstrate that bovine sperm treated with CLC resulted in a nonsignificant difference in embryo production when used in IVF, even when delayed capacitation timing is adjusted for in sperm preparation. This concludes that the fertilization ability of the spermatozoa treated with CLC was not impaired, and that the most important application of CLC-treated sperm is not within in vitro fertilization examination, but rather, within in vivo models utilizing AI synchronization timing adjusted for delayed capacitation. Here the benefit of improved cryosurvival rates can be best utilized, potentially resulting in the ultimate goal of increased fertility. One of the most widely pursued research avenues to improve embryo development is advancements in culture media and conditions. Many attempts have been made to optimize in vitro production of embryos by component supplementation and recipe adjustment. An additional culture condition with a dramatic impact on bovine preimplantation embryogenesis is whether embryos are cultured individually or in groups, where increased embryo production is observed in group culture conditions. These items together, have created an active area of research which analyzes culture media after exposure to embryo culture to examine the artifacts secreted by embryos including various amino acids, proteins, and other metabolites that are considered to be embryotrophic, or positive signals from companion embryos. Chapter four experiments include culturing embryos both individually and in groups with spent culture media from a group culture donor cycle with embryo production analyzed via cleavage and blastocyst formation with time-lapse imaging. Similar cleavage percentages between control and spent media were observed, however, an increase in blastocyst formation was observed in grouped embryos compared with single embryos. No difference (P>.05) was observed in the timing of developmental events in embryos cultured in control or spent media within individual culture. Analysis of the metabolomic profile of spent culture media with gas chromatography-mass spectrometry (GC-MS) was also completed, suggesting potential embryotrophic factors in the spent media. These findings conclude that bovine embryos progress in a more timely and developmentally differentiated degree when cultured within groups, and artifacts left within the spent culture media were not sufficient to result in significant increase in embryo production within spent media culture.