Browsing by Author "Krisher, Rebecca L., advisor"

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Open Access
Illuminating the impact of reproductive extracellular vesicles: modeling maternal signals during preimplantation embryo development
(Colorado State University. Libraries, 2024) Menjivar, Nico Graham, author; Tesfaye, Dawit, advisor; Krisher, Rebecca L., advisor; Chicco, Adam, committee member; Hollinshead, Fiona K., committee member
Pre-implantation embryo development is a complex process beginning around the time of gametic syngamy, the process of two gametes fusing to create a zygote (the first cell of a new organism). Passively transient through the oviduct, the presumptive zygote is then characterized by a series of timely cleavage divisions, activation of the embryonic genome, compaction (morula formation), cavitation (blastocyst formation), and summing in hatching from the encapsulated zona pellucida and implantation to the uterine wall. Unfortunately, the current IVF system that occurs ex vivo, completely bypasses the critical maternal-embryonic crosstalk that would inevitably persist during the primitive stages of pre-implantation development. It is thought that the low yield of developed embryos in vitro, is in part due to the failed ability to recapitulate a suitable system that mimics the maternal environment, shunting early cleavage stage embryos for failure. However, the reservations regarding maternal signals secreted to developing embryos, the reproductively inaccessible nature of the organs, and suboptimal in vitro systems to study replicate in vivo function has limited our complex understanding of these stages. In this dissertation, I aimed to interrogate multiple aspects of preimplantation embryo development, under the primary premise of modeling maternal signal during the pre-implantation period. Utilizing the intrinsic interest in the growing field of extracellular vesicle (EV) research and their significance in intercellular signaling, particularly their communicative role in selective biological information transfer, my first exertion was developing a source of EVs from in vitro cultured granulosa cells for use during IVM (necessitating maternal signals amid the follicle microenvironment). Through the analysis of this dataset (in combination with Gebremedhn et al. 2020) together with immunofluorescence and functional experiments, we characterized diverging miRNA profiles of EVs secreted by granulosa cells subjected to polarizing thermal conditions, that are abundantly up taken by COCs and modulate key developmental events that safeguard developing embryos exposed to conditions of stress. Next, I built upon this work by generating a functional 3D organoid model to study the cellular and extracellular response of the oviduct using a multi-omics approach. Using this atlas as a guide, I characterized the functional undertakings of the oviduct during applied levels of heat stress and found its crucial role in altering the metabolic activity of maternal tissues, which likely in part functionally augment developing embryos and assume failure. Given the functional applicability of reproductive EVs acting as maternal cues, I established this suitable model as a mechanism to generate physiologically relevant EVs (in vivo-like) to offset applied stress during the initial stages of development. These EVs secreted from 3D cultured oviductal organoids were then compared with those secreted from 2D OECs and from in vivo oviductal fluid (miRNAs), and used in an IVC setting, highlighting functional maternal—embryonic crosstalk. Altogether, this dissertation highlights key functional aspects of reproductive extracellular vesicles from both the follicular microenvironment and the oviduct, highlighting the novel and incredible power of suitable in vitro systems to propagate mechanisms to understand maternal signal absent in the current in vitro systems, beginning to illuminate the 'black box' of EVs in embryo development.
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 member
Maternal 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.