Roles of Sulfogalactosylglycerolipid (SGG) and Its Binding Proteins in Fertilization.

SGG is an evolutionarily conserved sulfoglycolipid present at 10 mole% of total lipids on the surface of mammalian male germ cells. On mature sperm, SGG is localized exclusively on the sperm anterior head plasma membrane (APM), the site where sperm interact with zona pellucida (ZP). We have shown that SGG not only has direct ZP affinity, but also plays an important role in the formation of capacitated sperm lipid rafts, which possess ZP binding ability. Also, SGG and its analog, Sulfogalactosylceramide (SGC), possess affinity for a number of molecules, including heat shock protein 70 (Hsp70) and a ZP-binding protein, arylsulfatase A (ASA), both existing in lipid rafts. Since SGG is present abundantly in sperm APM, we hypothesize that it may recruit other sperm surface proteins important for fertilization to the APM region during capacitation. Synergistic action between SGG and these APM proteins, which may also have SGG affinity, would stabilize the binding of sperm to the ZP. Nitrogen cavitation at 650 psi selectively disrupts the APM. APM vesicles containing lipids and membranous proteins can be pelletted upon ultracentrifugation, whereas peripheral plasma membrane proteins of the APM would remain in the supernatant (SN). Notably, the levels of APM vesicles from capacitated sperm were higher than those from non-capacitated gametes, confirming the significance of APM vesicles in ZP binding. Our proteomic analysis further revealed a number of ZP binding proteins in these vesicles, including SED1, zonadhesin, angiotensin converting enzyme, Sp32, and glutathione S-transferase mu 2 (GST). Some of these proteins may have direct SGG affinity. In fact, our initial attempt to characterize SGG binding proteins using the "SN" fraction indicated that SP-10, Sp32, and GST, all with a similar molecular mass range (25-28 kDa), formed complexes with SGG liposomes, which could then be floated up in a sucrose gradient by ultracentrifugation. Similar experimental approaches are being carried out to identify SGG binding proteins in APM vesicles, with the expectation that some of them would have ZP affinity. Interestingly, chaperone proteins, Hsp70 and Hsp86, were also present in higher amounts in APM vesicles of capacitated sperm. Hsp70 is known to oligomerize upon its binding to SGC. We therefore propose that SGG may induce oligomerization of Hsp70 and perhaps also Hsp86 in the APM entity, especially in capacitated sperm. This would promote the clustering of ZP binding proteins and subsequently their synergistic action in ZP binding. Experiments are underway to validate this hypothesis. Our study should reveal whether SGG can recruit ZP binding proteins to the APM entity via its direct affinity to these proteins and/or indirect influence through their interaction with the chaperone proteins, Hsp70 and Hsp86. Funded by CIHR, NSERC, and DPST (The Development and Promotion of Science and Technology Talented Project Thailand scholarship). (poster)

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