Browsing by Author "Tesfaye, Dawit, committee member"
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Item Open AccessExtracellular 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 EmbargoImpact 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 EmbargoModeling 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 AccessThe 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.