Browsing by Subject "Danio rerio"

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    Biophysics of Zebrafish (Danio rerio) Sperm
    (Elsevier, 2009-02) Hagedorn, M.; Ricker, J.; McCarthy, M.; Meyers, S.A.; Tiersch, T.R.; Varga, Z. M.; Kleinhans, F.W.; Physics, School of Science
    In the past two decades, laboratories around the world have produced thousands of mutant, transgenic, and wild-type zebrafish lines for biomedical research. Although slow-freezing cryopreservation of zebrafish sperm has been available for 30 years, current protocols lack standardization and yield inconsistent post-thaw fertilization rates. Cell cryopreservation cannot be improved without basic physiological knowledge, which was lacking for zebrafish sperm. The first goal was to define basic cryobiological values for wild-type zebrafish sperm and to evaluate how modern physiological methods could aid in developing improved cryopreservation protocols. Coulter counting methods measured an osmotically inactive water fraction (Vb) of 0.37 ± 0.02 (SEM), an isosmotic cell volume (Vo) of 12.1 ± 0.2 μm3 (SEM), a water permeability (Lp) in 10% dimethyl sulfoxide of 0.021 ± 0.001(SEM) um/min/atm, and a cryoprotectant permeability (Ps) of 0.10 +/− 0.01 (SEM) × 10−3 cm/min. Fourier transform infrared spectroscopy indicated that sperm membranes frozen without cryoprotectant showed damage and lipid reorganization, while those exposed to 10% glycerol demonstrated decreased lipid phase transition temperatures, which would stabilize the cells during cooling. The second goal was to determine the practicality and viability of shipping cooled zebrafish sperm overnight through the mail. Flow cytometry demonstrated that chilled fresh sperm can be maintained at 92% viability for 24 h at 0°C, suggesting that it can be shipped and exchanged between laboratories. Additional methods will be necessary to analyze and improve cryopreservation techniques and post-thaw fertility of zebrafish sperm. The present study is a first step to explore such techniques.
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    The Roles of Danio Rerio Nrf2 Paralogs in Response to Oxidative Stress in the Pancreatic Beta Cell
    (2020-06) Doszpoly, Agnes; Linnemann, Amelia; Anderson, Ryan; Wek, Ronald
    Oxidative stress can disrupt cellular homeostasis, leading to cellular dysfunction and apoptosis. The Nrf2 transcription factor regulates the antioxidant response in cells by binding to antioxidant response elements (ARE) in DNA and activating genes of enzymes that combat oxidative stress. During the pathogenesis of diabetes mellitus (DM), β-cells are exposed to increased amounts of reactive oxygen species (ROS) that cause oxidative stress. Zebrafish (ZF) are excellent models for studying the dynamic mechanisms associated with DM pathogenesis, and we recently developed a ZF model of β-cell apoptosis caused by ROS. Two paralogs of Nrf2 have been identified in ZF, Nrf2a and Nrf2b, but their roles in pancreas development and/or β-cell survival are unknown. To investigate their roles, Nrf2a and Nrf2b antisense morpholinos (MO) were injected into Day 0 ZF embryos and analyzed over time. While Nrf2a MO showed no obvious phenotypes compared to WT, Nrf2b MO exhibited reduced pancreas size and islets with disrupted morphology. Ins:NTR Nrf2a MO showed reduced β-cell loss upon exposure to Metronidazole (MTZ) under generation of ROS compared to WT. Sequence analysis of ZF nrf2b in 3-day post-fertilization (dpf) embryos revealed a novel splice variant containing an additional exon that has not been described. Further investigation of Nrf2a and Nrf2b is likely to yield additional insights regarding the function and regulation of the NRF2-signaling pathway and their roles in β-cell protection under oxidative stress.