Fishing for alpha-L-fucosidase: Evaluating Enzyme Activity and the Reproductive Role in Zebrafish (Danio rerio)

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    Linked Agent
    Thesis advisor: Venditti, Jennifer J.
    Committee member: Coleman, William L.
    Committee member: Hare-Harris, Abby
    Degree granting institution: Bloomsburg University of Pennsylvania
    Degree name: Master of Science
    Date Created
    2020
    Note

    Although alpha-L-fucosidase has been shown to be important during reproduction in many diverse organisms, the protein’s specific role remains unclear. To our knowledge, this would be the first report of alpha-L-fucosidase in zebrafish milt. Moreover, the utilization of zebrafish will allow for an evaluation of alpha-L-fucosidase’s potential role in embryonic development. Alpha-L-fucosidase activity in zebrafish sperm was quantified using the substrate 4-methylumbelliferyl-alpha-L-fucopyranoside (4-MU-fuc) to produce a fluorometric product known as 4-methyl-umbelliferone (4-MU). Fluorometric results suggested the presence of alpha-L-fucosidase in zebrafish milt. Partitioning assays were conducted to evaluate the presence of alpha-L-fucosidase in the sperm cells versus the liquid component of milt. Although not significant, a higher amount of alpha-L-fucosidase was detected in the pellet compared to the supernatant fraction. Treatment of zebrafish samples with deoxyfuconojirimycin (DFJ), a potent and specific inhibitor of alpha-L-fucosidase, inhibited enzyme activity. Egg extracts were prepared and evaluated for the presence of alpha-L-fucosidase and revealed minimal detection. In vitro fertilization experiments were conducted to evaluate embryonic success over 72 hours in the absence and presence of DFJ. A decrease in fertilization success was noted when comparing untreated versus DFJ treated experiments 0.75-2 hours post insemination (hpi). In both untreated and DFJ treated experiments, embryos successfully developed through 72 hpi. Understanding the role of alpha-L-fucosidase in reproduction could provide an understanding that can be translated to improving infertility in humans.

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    60 leaves
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    All rights reserved. Content may be used for educational and personal use only with full attribution to the author.
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