Long lasting calcium (Ca2+) oscillations are necessary and sufficient for mammalian egg activation and early embryological development. In mammals, phospholipase C zeta (PLCζ) has been identified as the likely endogenous trigger of Ca2+ oscillations at fertilization. Some cases of male factor infertility have been associated with the absence / reduced or presence a mutant form of PLCζ. In these cases sperm fails to activate eggs after intra-cytoplasmic sperm injection (ICSI). Artificial egg activation is the potential way to trigger Ca2+ oscillations and egg activation. Strontium (Sr2+) is the main artificial agent for this purpose in rodent eggs. The work in this Thesis aims to examine the mechanism of PLCζ or Sr2+ ions to trigger Ca2+ oscillations in mammalian eggs. It was not clear how Sr2+ causes Ca2+ oscillations and why it is only effective in rodents but not human eggs or domestic animals. My studies show that Sr2+ is effective in causing Ca2+ oscillations in mouse eggs over a range of concentrations, but that its actions are influenced by the osmolarity of the medium. Low osmolarity enhances the ability of low concentrations of Sr2+ to cause Ca2+ oscillations. Further investigation revealed that Sr2+ influx is mainly through the reverse mode of the Na+/Ca2+ exchange protein (NCX) which can be controlled by the membrane potential and Na+ gradient across the plasma membrane. Preliminary studies investigated the ability of a modified Sr2+ media that maximizes reverse mode NCX to trigger Ca2+ changes in human eggs. In other studies, various PLCζ-luciferase cRNAs were injected into mouse and human eggs. PLCζ expression in mouse eggs was measured by imaging light due to luciferase iv activity, and Ca2+-oscillations were monitored with Ca2+ sensitive fluorescent dye. Aspects of the structure of PLCζ and the effects and the recent discovery of PLCζ sequence mutations were investigated. Preliminary studies were also carried out to test the ability of recombinant PLCζ protein to cause Ca2+ oscillations in mammalian eggs. It is hoped that these studies might open up new therapies for some male factor infertility couples that acconts 1-5% of failed ICSI.
Identifer | oai:union.ndltd.org:bl.uk/oai:ethos.bl.uk:577925 |
Date | January 2013 |
Creators | Elgmati, Khalil |
Publisher | Cardiff University |
Source Sets | Ethos UK |
Detected Language | English |
Type | Electronic Thesis or Dissertation |
Source | http://orca.cf.ac.uk/50384/ |
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