Involvement of carbohydrate moieties of the toad egg vitelline coat in binding with fertilizing sperm

Vitelline coats (VC) were isolated from the eggs of Bufo japonicus, and were added with sperm in reconstituted salt solution, which mimics the physiological role of jelly envelopes, to determine the rates of sperm binding per unit area (0.2 mm2 ) of VC. The rate of sperm binding to VC from uterine eggs was high, but was low to VC from coelomic eggs and eggs activated in 1/20 De Boer's solution (DB) and moderately low to VC from eggs activated in DB. The binding rate increased when VC from coelomic eggs were treated with extracts of the pars recta portion of the oviduct. The sperm that bound to VC were not acrosome-reacted and their binding to VC required both a low salinity, assuring motility of sperm, and sufficiently high levels of Ca2+ and Mg2+ . The rate of sperm binding was reduced by either coexisting solubilized VC materials, periodate-oxidation of VC or the pretreatment of VC with Fab fragments of anti-VC antibodies, which reacted mostly to carbohydrate residues of VC glycoproteins. Sperm-VC binding assays in combination with gel-filtrated VC components revealed that the fractions containing 36-39 kDa components were most effective both in inhibiting the binding and in neutralizing the antibody induced inhibition of binding. These results indicate that carbohydrate moieties in 36-39 kDa glycoproteins of VC, exposed as a result of hydrolysis by the oviducal pars recta protease, are involved in binding with fertilizing sperm.