Characterization of Cholinomimetic Agents that Inhibit In Vitro Fertilization in the Mouse Evidence for a Sperm‐Specific Binding Site

Spermatozoa from the cauda epididymidis of the mouse bind 3‐quinuclidinyl benzilate (QNB), a highly specific antagonist of the muscarinic type of acetylcholine receptor; inhibition of in vitro fertilization of mouse eggs parallels this binding. Characterization of 3 H‐QNB binding to mouse spermatozoa by means of Scatchard and Hill plots showed a single class of non‐interacting sites with K D = 5 nM. The stereospecificity of this site was determined by testing the ability of specific probes to displace competitively 3 H‐QNB bound to spermatozoa. Unlabeled QNB, atropine, and scopolamine, which are antagonists of muscarinic receptors, and carbamylcholine and muscarine, which are agonists, competed with 3 H‐QNB for the spermatozoon binding site. Propylbenzilylcholine mustard (PrBCM), a specific, irreversible antagonist of muscarinic sites, did not compete for the 3 H‐QNB binding site. Those compounds that occupied the QNB binding site on mouse spermatozoa also inhibited the fertilization of the zonapellucida‐intact mouse eggs in vitro, whereas PrBCM, which did not occupy the binding site, had no effect on fertilization. There was a close correspondence between the affinity of these compounds for the QNB binding site and their ability to inhibit fertilization. This correspondence held for both agonists and antagonists of muscarinic sites. From these results, we conclude that the 3 H‐QNB binding site of mouse spermatozoa differs from the conventional class of muscarinic sites: while the steric requirements are similar, they are far from identical. This fact suggests that compounds could be synthesized that would be potent inhibitors of fertilization by binding on the sperm surface, but would have no effect on neural tissues. Such compounds would offer a new approach to the design of male fertility control agents.