VMO-II Mediates the Binding of the Chalaziferous Layer with the Vitelline Membrane in Quail Eggs

Electron microscopic observations have revealed an electron density gradient in the chalaziferous layer of avian egg. The density is highest in the innermost sub-layer, which is bound with the vitelline membrane, and is thus traditionally referred to as the vitelline membrane outer layer (VMO). After high-concentration salt treatment, two proteins, VMO-I and VMO-II, are liberated from the egg envelopes of quail (Coturnix japonica), which results in the separation of the chalaziferous layer from the vitelline membrane. VMO-I and VMO-II were purified through gel-filtration and subjected to antisera produced in rabbits. The molecular sizes of these two proteins were estimated to be 9 to 15 kDa for VMO-II and 18 kDa for VMO-I. Their amino acid sequences were very similar to those of chickens. Immunofluorescent and immunoelectron micrographs showed that VMO-II was produced by the luminal epithelium and VMO-I by both the luminal and glandular epithelia of the in-fundibulum. Immunogold particles for the labeling of both proteins were distributed throughout the chalaziferous layer, as well as in the chalazae, with a density gradient similar to that mentioned above. In salt-treated envelopes, immunogold labeling was completely absent when stained with anti-VMO-II antiserum and weak with anti-VMO-I antiserum. Immu-nohistochemical studies revealed that purified VMO-II bound with vitelline membranes of salt-treated envelopes, and ligand blotting tests revealed that ZP1 and ZP3 were the molecules bound by VMO-II; the binding was inhibited by various kinds of sugars. VMO-II might play a role in the binding of the chalaziferous layer with the vitelline membrane, a process that leads to the anchoring of the chalaza cord.