Functional Roles of Carbohydrate Moieties of Mammalian Zona Pellucida Proteins in Fertilization
	The plasma membrane of mammalian eggs is surrounded by an extracellular envelope referred to as the zona pellucida. During oogenesis, oocytes become surrounded by the zona pellucida. The zona plays roles in oogenesis, species-specific recognition of sperm, block to polyspermy and protection of the oocyte and embryo until implantation. Studies on the functional roles of the carbohydrate moieties of the zona pellucida have been especially focused on the recognition of sperm (see: Mammalian Fertilization and Sugars). In vitro studies of mouse zona pellucida have established two decades ago that only ZP3 has sperm ligand activity among the three zona components and the ligand activity is ascribed to the O-linked carbohydrate chains of ZP3. Mouse sperm were proposed to bind to the O-linked carbohydrate chains linked to Ser332 and Ser334 of ZP3 (Wassarman model). According to the model that a specific sugar residue of the O-linked chains of ZP3 binds to a lectin-like factor on the plasma membrane of mouse sperm and this binding can account for species-specific recognition between gametes, intense efforts by many research groups have proposed various potential sugar residues as sperm ligands. The model will be proved if knockout mice lacking the potential sperm ligand or lectin-like factor on sperm become infertile. However, to begin with, in 1995 the Gal model was shown to be invalid and the knockout female mice lacking one of glycosyltransferases so far made are fertile. The best studied candidate on mouse sperm plasma membrane that binds to the zona is 1,4-galactosyltransferase I (GalTI). GalTI binds to GlcNAc of ZP3 and this binding induces acrosome reaction of sperm. -hexosaminidase B, which is secreted from cortical granules upon fertilization, removes GlcNAc from the zona, leading to the loss of the sperm ligand activity of the zona. Thus, the GlcNAc model can explain both sperm recognition and block to polyspermy by the zona. Mouse sperm lacking GalTI show reduced binding to ZP3 and reduced induction of acrosome reaction by ZP3, but the mice are fertile. This indicates that not only GlcNAc-GalTI binding but the other molecules are also involved in gamete recognition. Given that in vitro experiments examining mouse gamete recognition suggest the involvement of multiple receptors on sperm or ligands on the zona pellucida, the redundant sperm-binding sites might compensate for the lack of some of the sperm-binding sugar residues (Figure, (1)); therefore the transgenic mice may be fertile.
Figure Two models of sperm-zona recognition and block to polyspermy in mammalian fertilization Mouse sperm persistently bind to two-cell embryos of human ZP2 rescue mice whose endogenouse ZP2 was replaced with human ZP2. Human ZP2 is not processed by a mouse cortical granule enzyme (Figure, (4)). This suggests that the cleavage of sperm ligands on ZP3 by cortical granule glycosidases is not sufficient for the loss of sperm ligand activity of the zona upon fertilization and a change in the supramolecular structure of the zona is also necessary. Sperm may recognize the supramolecular structure rather than the carbohydrate chains of the zona (Supramolecular model, Figure, (3)). The change in supramolecular structure does not occur in the zona of human ZP2 rescue mice and sperm bind persistently to the zona after fertilization. Mouse sperm bind to the zona of human ZP3 rescue mice whose endogenous ZP3 was replaced with human ZP3, but human sperm do not. Since human ZP3 synthesized in mouse oocytes has carbohydrate chains with the same structure as those of endogenous mouse ZP3, this supports the glycan model that the carbohydrate moiety plays an essential role in species-specific recognition of sperm. In the case of pig, the heterocomplex of ZPB and ZPC has sperm-binding activity. The recombinant porcine ZPB and ZPC have carbohydrate chains similar in structure to those of bovine zona and the complex has bovine sperm ligand activity but does not have porcine sperm ligand activity. Thus, more evidence for the glycan model is being reported and the experiments using transgenic mice do not yet completely deny the model. The zona architectures differs among species, so it is possible that the gamete recognition mechanisms also differ among species. Sperm lacking ADAM family members or angiotensin convertase enzyme cannot bind to the zona, showing that these sperm proteins play essential roles in binding of sperm to the zona. We hope that the functional analyses of these proteins will clarify whether or not the carbohydrate moiety of the zona plays an essential role in the recognition of sperm.
	Naoto Yonezawa and Minoru Nakano (Department of Chemistry, Faculty of Science, Chiba University)
	References (1) Hoodbhoy T, Dean J: Insights into the molecular basis of sperm-egg recognition in mammals. Reproduction, 127, 417-422, 2004 (2) Dell A, Chalabi S, Easton RL, Haslam SM, Sutton-Smith M, Patankar MS, Lattanzio F, Panico M, Morris HR, Clark GF: Murine and human zona pellucida 3 derived from mouse eggs express identical O-glycans. Proc. Natl. Acad. Sci. USA, 100, 15631-15636, 2003 (3) Yonezawa N, Kudo K, Terauchi H, Kanai S, Yoda N, Tanokura M, Ito K, Miura K, Katsumata T, Nakano M: Recombinant porcine zona pellucida glycoproteins expressed in Sf9 cells bind to bovine sperm but not to porcine sperm. J. Biol. Chem, 280, 20189-20196, 2005
Feb. 28, 2006