Gamete fusion: A perspective on egg glycosylphosphatidylinositol-anchored proteins

Fertilization in mammalian organisms is a dramatic and enigmatic event. From the time sperm leave the testicular environment and enter the female reproductive tract there are many biological obstacles that can prevent, or permit, sperm to reach the egg. At the cellular and biochemical levels the process is incompletely understood, therefore, many studies are being conducted to understand the process in its entirety and define the molecular components necessary for fertilization.; This study focuses on egg glycosylphosphatidylinositol-anchored proteins (GPI-anchored proteins) required in sperm-egg fusion, the step prior to embryo formation. GPI-anchored proteins are a group of proteins linked to the extracellular leaflet of the bilayer by a lipid moiety, glycosylphosphatidylinositol. In vitro removal of GPI-anchored proteins from the mouse egg surface with phosphatidylinositol-specific phospholipase C (PI-PLC) results in a block to sperm-egg fusion and a decrease in sperm-egg binding. To test for the in vivo requirement of GPI-anchored proteins, a tissue-specific knockout of the gene PIG-A, which is involved in the first step of glycosylphosphatidylinositol-anchor biosynthesis, was created in mouse. In vivo it was shown that egg GPI-anchored proteins are required for fertility, and in vitro it was shown that the step requiring GPI-anchored proteins was sperm-egg fusion and not sperm-egg binding.; One GPI-anchored protein was identified, CD55, a complement regulatory protein. However, CD55 is not required for fertility, as CD55 knock-out females are fertile. Therefore, mass spectrometric analysis was conducted on a GPI-anchored protein enriched egg supernatant. A preliminary screen of the results revealed three GPI-anchored proteins: alpha-tectorin, stereocilin, and MT4-MMP.; The role of membrane microdomains, GPI-anchored protein- and cholesterol-rich plasma membrane domains, in fertilization was tested. Treatment of the eggs with the cholesterol-sequestering drug methyl beta-cyclodextrin, mbetaCD, a drug that disrupts microdomains, inhibited sperm-egg fusion significantly. In addition, egg microdomains were isolated on sucrose density gradients; CD9, a tetraspanin required for sperm-egg fusion, was found in low-density, lipid raft fractions. The link between GPI-anchored proteins and CD9 was not found in the initial analysis. In conclusion, the evidence suggests that egg microdomains exist and may function in sperm-egg fusion. However, it is not clear.