Development of a new, highly sensitive zona pellucida binding assay using a bioluminescence-enhanced detection system

To date, two different zona binding assays have been described in the literature. Both assays, however, require a large quantity of human zonae which vary immensely in quality. Furthermore, an inverted microscope with micromanipulation equipment is necessary, which makes both assays relatively complicated and time-consuming, and requires skilled staff. Therefore, we developed a new, highly sensitive zona binding assay using a bioluminescence-enhanced system which employs a pool of solubilized zona pellucida and is easier for routine use. In the detection system, light emission by the luciferin-luciferase system is measured. Because of the limited availability of human zonae pellucidae, this new assay was first developed in the porcine system. The new bioluminogenic substrate D-luciferin-O-beta-galactopyranoside (Lu-Gal) was synthesized, purified and characterized. Synthesis of Lu-Gal resulted in purity better than 99.998%. Analytical data and spectra were appropriate. In terms of the kinetic data, Lu-Gal is a highly sensitive and specific substrate for beta-galactosidase. Using the given chemical conditions, nonlabelled zonae bound competitively to boar spermatozoa, which resulted in a high sensitivity and specificity. By the addition of 10 nonlabelled zonae, the binding of labelled zonae was almost completely inhibited. Corresponding results were obtained when the bioluminescent system was compared with the hemizona assay. On the other hand, spermatozoa of other species (bull, hamster and man) showed only low binding to the porcine zonae or none at all. Competitive displacement was not observed, indicating the inter-species specificity of the assay.