Roles of Sulfogalactosylglycerolipid (SGG) in Fertilization: Studies Using a Proteomic Approach.

Abstract

Sulfogalactosylglycerolipid (SGG) is an evolutionarily conserved sulfoglycolipid present at a substantial amount on the surface of mammalian male germ cells. We have shown that SGG is localized to the anterior head plasma membrane (APM) of sperm, the site where sperm interact with the egg zona pellucida (ZP). In addition, SGG participates in the formation of capacitated sperm lipid rafts, as well as in endowment of their ZP binding ability. During the sperm transit through epididymis, SGG also serves as a ligand of arylsulfatase A (ASA), another ZP binding molecule, to tether onto the sperm head surface. All these findings have implicated the roles of SGG in male fertility and suggested that SGG could serve as a biomarker of sperm fertilizing ability and as a potential target for development of a non-hormonal male contraceptive. However, a comprehensive understanding of the molecular mechanisms of SGG in the male reproduction is required. Due to the abundance of SGG on the sperm surface and its adhesion property, we hypothesize that SGG interacts with other proteins synthesized by male germ cells and those originating from epididymal/seminal fluid. These proteins forming cohorts with SGG on the mature sperm surface may be important for fertilization. The objective of our study is to identify these proteins using a proteomic approach. Since the proteins important for sperm-egg binding are known to exist on the sperm APM, we first obtained the total protein profile of APM isolated from pig sperm by nitrogen cavitation. As expected, LTQ-FT mass spectrometry (MS) analysis revealed the presence of many ZP-binding proteins in our APM samples, such as zonadhesin, milk fat globule-EGF factor 8 protein (SED1), and testicular angiotensin-1 converting enzyme; therefore, these APM samples were used as the protein source for the SGG-binding experiments. Proteins were extracted from the APM vesicles using a detergent, octylglucoside. Besides APM proteins, total proteins from spermatogenic cell plasma membranes and epididymal/seminal fluid were also used in this study. SGG binding proteins were prepared by incubating the protein mixture from these various sources with SGG liposomes and the liposome-protein complex was ultracentrifuged. Our trial experiments revealed that ASA and protein(s) with a molecular mass of 27 kDa from the sperm homogenate bound specifically to SGC liposomes. The identity of this 27-kDa protein(s) is being obtained by LC-MS/MS. This information should lead to a better understanding of the significance of SGG in mammalian sperm-egg interaction. Funded by CIHR, NSERC, NIH, and DPST (The Development and Promotion of Science and Technology Talented Project Thailand scholarship). (poster)