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 pe llucida (ZP), a glycoprotein matrix surrounding the egg. Sperm specifi cally bind to ZP3, an 83-kDa glycoprotein which functions as both an a dhesion molecule and as a secretagogue for acrosomal exocytosis (Litsc her, 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 b e used since solubilized ZPs rapidly initiated exocytosis. Instead, ac rosome 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) i ncubating either live or fixed sperm in low concentrations of I-125-Zp s not sufficient to induce acrosomal exocytosis revealed no difference s in binding up to 15 min and 2) solubilized, unlabeled ZPs competed f or 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 degree s C. Affinity parameters were calculated using the well substantiated assumption that only ZP3 binds intact mouse sperm. The on-rate constan t for association of I-125-Zp binding to the mouse sperm surface was c alculated to be 3.2 x 10(6) hr(-1) min(-1). The saturation binding iso therm revealed that there are approximately 30,000 binding sites, ascr ibed to ZP3, with an EC(50) of 1.29 nM. Further analysis indicated tha t this binding is complex (Hill coefficient = 1.72), suggesting involv ement of multiple receptors on the sperm surface and/or multiple ligan d moieties, High and low affinity ZP binding sites on the sperm surfac e were confirmed by dissociation experiments. I-125-Zp dissociation wa s clearly biphasic, and kinetic off-rate constants of 0.161 min(-1) an d 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 af finity and 0.72 nM for the high affinity interaction were calculated f rom the rate constants. These data demonstrate that the initial adhesi on event between mouse sperm and the zona pellucida is a high affinity event which is sufficient to tether a sperm to the extracellular matr ix prior to the induction of acrosomal exocytosis.