Komondor, Kayla M.
(2024)
Fertilization unconventionally activates phospholipase C during the fast block to polyspermy in the African clawed frog, Xenopus laevis.
Doctoral Dissertation, University of Pittsburgh.
(Unpublished)
This is the latest version of this item.
Abstract
For most animals, fertilization by more than one sperm is lethal to a developing embryo. Accordingly, eggs use multiple mechanisms to keep additional sperm from entering the nascent zygote. One such mechanism, the fast block to polyspermy, is used by externally fertilizing animals and involves a change in the membrane potential of the egg from a negative resting potential to a positive fertilization potential. Sperm can bind to, but not enter, depolarized eggs. In frogs, fertilization opens the calcium-activated chloride channel TMEM16A, allowing an efflux of chloride ions that depolarize the egg’s membrane. Currently, the signaling pathway by which fertilization opens TMEM16A is not yet known.
Using whole-cell recordings on Xenopus laevis eggs during fertilization, I have found that inhibiting phospholipase C (PLC) using the general PLC inhibitor U73122 abolishes the fast block depolarization, and polyspermic fertilization occurred. U73122 covalently modifies PLC, which enabled me to independently treat either egg or sperm prior to fertilization to identify which gamete provides the PLC. Whole-cell fertilization recordings with U73122 pretreated sperm showed typical fast block depolarizations and monospermic development, while pretreated eggs did not exhibit a depolarization and exhibited polyspermic development, indicating that the PLC necessary for the fast block is egg-derived. X. laevis eggs have three PLC subtypes (PLCg1, PLCb1, and PLCb3). Each PLC subtype typically uses different activation methods: PLCg1 is canonically activated by tyrosine phosphorylation, and PLCb is typically activated through the Gaq/11 subunit of G-protein coupled pathways. Inhibiting these methods of canonical activation revealed typical depolarizations during whole-cell recordings and monospermic development. Together, these data reveal that the egg PLC during the fast block to polyspermy in X. laevis is activated by a novel signaling mechanism
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Details
| Item Type: |
University of Pittsburgh ETD
|
| Status: |
Unpublished |
| Creators/Authors: |
|
| ETD Committee: |
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| Date: |
13 May 2024 |
| Date Type: |
Publication |
| Defense Date: |
8 March 2024 |
| Approval Date: |
13 May 2024 |
| Submission Date: |
3 April 2024 |
| Access Restriction: |
No restriction; Release the ETD for access worldwide immediately. |
| Number of Pages: |
85 |
| 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: |
fertilization, phospholipase C, Xenopus laevis, polyspermy, TMEM16A, fast block to polyspermy, development |
| Date Deposited: |
13 May 2024 13:50 |
| Last Modified: |
13 May 2024 13:50 |
| URI: |
http://d-scholarship.pitt.edu/id/eprint/46041 |
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Fertilization unconventionally activates phospholipase C during the fast block to polyspermy in the African clawed frog, Xenopus laevis. (deposited 13 May 2024 13:50)
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