The initial molecular interaction between mouse sperm and the zona pellucida is a complex binding event

Prior to fertilization, mammalian sperm must first bind to the zona pellucida (ZP), a glycoprotein matrix surrounding the egg. Sperm specifically bind to ZP3, an 83-kDa glycoprotein which functions as both an adhesion molecule and as a secretagogue for acrosomal exocytosis (Litscher, E. S., and Wassarman, P. M. (1993) Trends Glycosci. Glycotechnol. 5, 369-388). We used acid solubilized, I-125-labeled ZPs to quantify the initial binding event on mouse spermatozoa. Live sperm could not be used since solubilized ZPs rapidly initiated exocytosis. Instead, acrosome intact mouse sperm were briefly fixed in 1% glutaraldehyde for binding studies using a standard filtration assay. The fixed sperm are suitable for sperm-zona binding assays based on two experiments: 1) incubating either live or fixed sperm in low concentrations of I-125-Zps not sufficient to induce acrosomal exocytosis revealed no differences in binding up to 15 min and 2) solubilized, unlabeled ZPs competed for I-125-ZPs with K-I of approximately 3.78 nM. Sperm-I-125-Zp binding reached equilibrium with a tau(1/2) of similar to 22 min at 37 degrees C. Affinity parameters were calculated using the well substantiated assumption that only ZP3 binds intact mouse sperm. The on-rate constant for association of I-125-Zp binding to the mouse sperm surface was calculated to be 3.2 x 10(6) hr(-1) min(-1). The saturation binding isotherm revealed that there are approximately 30,000 binding sites, ascribed to ZP3, with an EC(50) of 1.29 nM. Further analysis indicated that this binding is complex (Hill coefficient = 1.72), suggesting involvement of multiple receptors on the sperm surface and/or multiple ligand moieties, High and low affinity ZP binding sites on the sperm surface were confirmed by dissociation experiments. I-125-Zp dissociation was clearly biphasic, and kinetic off-rate constants of 0.161 min(-1) and 0.0023 min(-1) were calculated for the low and high affinity sites, respectively. Apparent affinities (K-d values) of 50 nM for the low affinity and 0.72 nM for the high affinity interaction were calculated from the rate constants. These data demonstrate that the initial adhesion event between mouse sperm and the zona pellucida is a high affinity event which is sufficient to tether a sperm to the extracellular matrix prior to the induction of acrosomal exocytosis.