Identification and localization of lysozyme as a component of eggshell membranes and eggshell matrix

The avian eggshell is a composite biomaterial composed of non-calcifying eg gshell membranes and the overlying calcified shell matrix. The calcified sh ell forms in a uterine fluid where the concentration of different protein s pecies varies between the initial, rapid calcification and terminal phases of eggshell deposition. The role of these avian eggshell matrix proteins du ring shell formation is poorly understood. The properties of the individual components must be determined in order to gain insight into their function during eggshell mineralization. In this study, we have identified lysozyme as a component of the uterine fluid by microsequencing, and used western b lotting, immunofluorescence and colloidal-gold immunocytochemistry to docum ent its localization in the eggshell membranes and the shell matrix. Furthe rmore, Northern blotting and RT-PCR indicates that there is a gradient to t he expression of lysozyme message by different regions of the oviduct, with significant albeit low levels expressed in the isthmus and uterus. Lysozym e protein is abundant in the limiting membrane that circumscribes the egg w hite and forms the innermost layer of the shell membranes. It is also prese nt in the shell membranes, and in the matrix of the calcified shell. Calcit e crystals grown in the presence of purified hen lysozyme exhibited altered crystal morphology. Therefore, in addition to its well-known anti-microbia l properties that could add to the protective function of the eggshell duri ng embryonic development, shell matrix lysozyme may also be a structural pr otein which in soluble form influences calcium carbonate deposition during calcification. (C) 2000 Elsevier Science B.V./International Society of Matr ix Biology. All rights reserved.