Useful Mimics for Mammalian Eggs: The Development of Porcine Ovabeads. (2012)

Data from our recently published study have confirmed that initial human sperm-egg binding depends primarily on the recognition of the sialyl-Lewisx sequence presented on glycans associated with human zona pellucida (ZP). There are clear indications that sperm from many species express an egg binding protein (EBP) that has properties associated with classical lectins. Many lectins display Kd values in the nM range for glycoconjugates bearing their ligands, indicating that a lectin-like EBP could mediate robust sperm binding. These findings indicated that a glycoprotein that bears glycans that are similar to those expressed on a ZP matrix could mediate sperm binding in the absence of a ZP protein backbone. Numerous glycomic studies have defined the glycans linked to mammalian ZP glycoproteins, as well as many other unrelated glycoproteins from diverse species. The specific aims of this study were to: (i) identify potential overlaps in mammalian ZP glycosylation with other known glycoproteins; and (ii) determine whether such glycoproteins could mediate the biological activities associated with ZP glycoproteins. Perusal of the glycobiological literature indicated a potential overlap in glycosylation between bovine thyroglobulin (bTg) and the porcine ZP (pZP). pZP glycoproteins express substantial amounts of sulfated and sialylated N- and O-glycans, but neutral tri- and tetraantennary N-glycans terminated with lacNAc (Gal(beta)1-4GlcNAc) sequences have been implicated as the major ligands for porcine sperm-ZP binding. Limited glycomic analyses have been performed on bTg, so this glycoprotein was subjected to modern glycomics and sulfoglycomics analysis. Substantial structural overlaps in glycosylation between bTg and pZP glycoproteins were confirmed. bTg was coupled at saturating concentrations to N-hydroxysuccinimide derivatized agarose beads. Boar sperm were capacitated and incubated under standard pig IVF conditions prior to exposure to bTg coated beads. Sperm bound rapidly and robustly to these beads, just as they did to in vitro matured porcine egg controls. After 30 minutes, the bound sperm underwent acrosomal exocytosis, which was detected by the binding of FITC-labeled peanut agglutinin (Arachis hypogaea) to the exposed inner acrosomal membranes and acrosomal shrouds. Bovine sperm did not bind to these beads under optimal conditions for fertilization, nor did they undergo acrosomal exocytosis. These studies provide compelling evidence that immobilized bTg mediates both boar sperm binding and the induction of acrosomal exocytosis. These results clearly implicate bTg glycans in these biological activities. These beads have been designated as porcine OvaBeads to reflect their capacity to mimic porcine eggs. These beads could be useful for assessing the fertility of boars prior to artificial insemination and IVF cycles, and for further studies on the gamete binding interaction in mammals. (Supported by a Life Sciences Mission Enhancement Reproductive Biology Program (to G.C.); the Food for the 21st Century Program (University of Missouri), and Competitive Grants no. 2007-35203-18274 and 2011-67015-20025 from the USDA NIFA (to P.S.); BBSRC grant BBF0083091 (to A.D.); and a Royal Society International Joint Project Award (to A.D., K.H.K.). MS data were acquired at the NRPGM Core Facilities for Proteomics and Glycomics (Taiwan National Science Council NSC99-3112-B-001-025)).