Bainbridge, Rachel E
(2023)
Signaling Mechanisms of Polyspermy Blocks.
Doctoral Dissertation, University of Pittsburgh.
(Unpublished)
Abstract
Fertilization is a tightly regulated process requiring numerous signaling processes. My dissertation research focused on two: PLC activation in the egg following fertilization, and zinc effects on gamete fertility.
Fertilization initiates polyspermy blocks that stop fertilization by multiple sperm. Eggs from most animals use the slow block to polyspermy, which occurs minutes after fertilization and involves the release of cortical granules to create a barrier that surrounds the nascent zygote. In external fertilizers, eggs also use the fast block, which occurs seconds after fertilization, and involves an electrical depolarization of the egg plasma membrane. In eggs of the African clawed frog, Xenopus laevis, the fast block depolarization is mediated by the calcium-activated chloride channel TMEM16A following a phospholipase C (PLC)- dependent calcium increase. PLC-y1 is the most abundant PLC isozyme in the egg, typically activated by phosphorylation of a conserved tyrosine at the enzyme’s active site. I used tyrosine kinase inhibitors during whole cell recording to observe the fast block during X. laevis fertilization. I found that blocking tyrosine phosphorylation did not affect the fast block depolarization, suggesting that another pathway activates the fast block to polyspermy in X. laevis eggs.
In contrast to the fast block, the slow block is used by eggs from most sexually reproducing animals. During the slow block, mammalian eggs have been shown to release zinc into the extracellular milieu. Here we demonstrated that X. laevis eggs also release zinc post-fertilization, and that at physiologic concentrations, extracellular zinc stopped fertilization in diverse external fertilizers. By independently treating X. laevis sperm or eggs with zinc prior to fertilization, we demonstrated that the zinc targets both gametes to stop fertilization.
In addition to high extracellular zinc surrounding already fertilized eggs, mammalian sperm encounter high zinc concentrations as they are mixed with the seminal fluid at mating. I established a fluorometry assay using the zinc indicator FluoZin-3 to monitor cytoplasmic zinc in mammalian sperm. Because mammalian sperm both encounter varying concentrations of extracellular zinc and must undergo changes post-mating to gain the ability to fertilize an egg, we believe that zinc may alter sperm physiology.
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Details
| Item Type: |
University of Pittsburgh ETD
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| Status: |
Unpublished |
| Creators/Authors: |
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| ETD Committee: |
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| Date: |
25 January 2023 |
| Date Type: |
Publication |
| Defense Date: |
1 December 2022 |
| Approval Date: |
25 January 2023 |
| Submission Date: |
8 December 2022 |
| Access Restriction: |
No restriction; Release the ETD for access worldwide immediately. |
| Number of Pages: |
146 |
| Institution: |
University of Pittsburgh |
| Schools and Programs: |
Dietrich School of Arts and Sciences > Biological Sciences |
| Degree: |
PhD - Doctor of Philosophy |
| Thesis Type: |
Doctoral Dissertation |
| Refereed: |
Yes |
| Uncontrolled Keywords: |
polyspermy block, fertilization, embryonic development, zinc, copper, slow block to polyspermy, fast block to polyspermy, phospholipase c |
| Date Deposited: |
25 Jan 2023 13:59 |
| Last Modified: |
25 Jan 2023 13:59 |
| URI: |
http://d-scholarship.pitt.edu/id/eprint/43961 |
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