At fertilisation in mammals a series of Ca2+ oscillations are initiated that activate development. These Ca2+ oscillations cause the reduction of mitochondrial NAD+ and flavoproteins, suggesting that they might also stimulate changes in cytosolic ATP levels. Many events at fertilisation are triggered that require ATP; however, the changes in ATP during fertilisation are poorly defined. In this thesis intracellular C a2+ and ATP levels in individual m ouse eggs were m easured by monitoring the fluorescence of a C a 2+ dye (Oregon green bapta dextran) and lum inescence of firefly luciferase. During fertilisation of m ouse eggs it w as found that there are two phases of increase in ATP in both the cytosol and the mitochondria, during the series of sperm-induced Ca2+ oscillations. The increase in ATP is Ca2+ dependent since it did not occur when Ca2+ oscillations were prevented by BAPTA injection and, were abrogated by extracellular Ca2+ chelation. Additionally, it w as not seen when eggs were activated by cycloheximide, which does not cause a Ca2+ increase. The ATP increase is likely to be caused by oxidative phosphorylation by the mitochondria since the ATP levels in substrate free media are recovered by the addition of pyruvate. This recovery is blocked by the pyruvate uptake inhibitor ar-Cyano-4-hydroxycinnamic acid. T hese data suggest that mammalian fertilisation is associated with a sudden but transient increase in cytosolic ATP via oxidative phosphorylation, and that Ca2+ oscillations are both necessary and sufficient to cause this increase in ATP. Work in this thesis has also investigated the functionality of the sperm factor PLC?. Using luciferase tagged PLC constructs, the Ca2+ oscillation inducing ability of a series of PLC? truncated constructs, PLC5 and PLCy have been established. Results show that PLC? activation of m ouse eggs cannot be reproduced by other PLCs and that the C2, EF1 and catalytic site on the X domain are all essential for causing C a2+ oscillations.
| Identifer | oai:union.ndltd.org:bl.uk/oai:ethos.bl.uk:583999 |
| Date | January 2007 |
| Creators | Campbell, Karen |
| Publisher | Cardiff University |
| Source Sets | Ethos UK |
| Detected Language | English |
| Type | Electronic Thesis or Dissertation |
| Source | http://orca.cf.ac.uk/54106/ |
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