Capacitated acrosome-intact mouse spermatozoa bind to Sepharose beads coated with functional neoglycoproteins.
Capacitated acrosome-intact mouse spermatozoa bind to the egg's extracellular coat, the zona pellucida (ZP), in a carbohydrate-mediated receptor-ligand manner. The tight irreversible binding of the opposite gametes triggers a signal transduction pathway resulting in the exocytosis of acrosomal contents (i.e., induction of the acrosome reaction [AR]). Previously, we demonstrated that a hexose (mannose) and two amino sugars (N-acetylglucosamine and N-acetylgalactosamine), when covalently conjugated to bovine serum albumin (BSA) (functional neoglycoproteins, ngps), mimicked mZP3 and induced the AR [Biol. Reprod. 60 (1999) 94-101]. To further elucidate the specificity of sperm-ngp interaction and the mZP3 mimicking role of the functional ngps, we have examined binding of the mouse spermatozoa to Sepharose 4B beads coated with the functional and non-functional ngps as well as BSA, ovalbumin (OVA), or asialofetuin (ASF). A significantly greater number of capacitated acrosome-intact spermatozoa bound to the beads coated with functional ngps than the beads coated with non-functional ngps, BSA, OVA, or ASF. The binding was temperature-sensitive and was highest when the sperm-bead assay was carried out at 37 degrees C. Blocking of in vitro capacitation, by including calmodulin antagonists in the incubation medium, prevented sperm from binding to the beads. Furthermore, inclusion of free sugars (mannose, N-acetylglucosamine, or N-acetylgalactosamine) in the binding assay, either individually or as a mixture, inhibited sperm-bead binding in a concentration-dependent manner. Taken together, our data provide evidence strongly suggesting that binding of capacitated spermatozoa to the ngp-coated Sepharose beads is specific. The beads that mimic zona-intact eggs provide an excellent tool for examining pharmacological effects of reagents that alter the sperm function. In addition, the immobilized ngp(s) will be useful as an affinity medium to isolate the sperm surface receptor(s) that recognize and bind to the sugar residues.
Bendahmane
Malika
M
Departments of Obstetrics & Gynecology, Vanderbilt University School of Medicine, Room D-3243 MCN, Nashville, TN 37232-2633, USA.
Tulsiani
Daulat R P
DR
D000177:Acrosome / Q000378:metabolism* / Q000502:physiology / Q000648:ultrastructure; D000818:Animals; D002478:Cells, Cultured; D006023:Glycoproteins / Q000378:metabolism* / Q000502:physiology; D046911:Macromolecular Substances; D008297:Male; D051379:Mice; D008863:Microspheres; D011485:Protein Binding / Q000502:physiology; D015203:Reproducibility of Results; D012680:Sensitivity and Specificity; D012685:Sepharose; D013075:Sperm Capacitation / Q000502:physiology*; D013094:Spermatozoa / Q000166:cytology* / Q000378:metabolism* / Q000502:physiology
D006023:Glycoproteins; D046911:Macromolecular Substances; D012685:Sepharose