THE FABRICATION AND COLLAGENOUS SUBSTRUCTURE OF THE EGGSHELL MEMBRANE IN THE ISTHMUS OF THE HEN OVIDUCT

The eggshell of the chicken consists of a bi-layered shell membrane overlaid with a thick, calcified shell matrix. The shell membranes and matrix are deposited onto the egg as it passes through the oviduct. To assess the temporal and spatial aspects of the fabrication of type X collagen within the eggshell extracellular matrix, the immunohistochemical localization of type X collagen was studied in three regions of the hen oviduct (magnum, isthmus and uterus), in the membranes of uncalcified eggshells obtained from the oviduct prior to mineral deposition and in eggshell membrane and calcified eggshell matrix. Additionally, immunohistochemical localization of type I and III collagens was done in order to determine possible co-localization of collagen types or to define tissue compartments. None of the collagen epitopes assayed was found in the shell matrix. Type X collagen epitope was immunohistochemically localized only to the epithelial cell layer lining the isthmus region of the oviduct and in the shell membranes of both uncalcified and calcified eggshells. Antitype III collagen monoclonal antibody delineated the inter-tubular gland connective tissue of the oviduct and was negative in the shell layers under conditions which gave strong connective tissue reactivity. Type I collagen epitope was exposed after pepsin treatment of the tissue and co-localizes with the distribution of type III collagen. Type I collagen co-localized with type X collagen in the shell membranes of uncalcified shells. The type I collagen epitope was reactive in the shell membrane of the uncalcified shells, but could only be detected in calcified shells following pepsin digestion. These observations suggest that the type I collagen epitope is masked during the process of calcification of the shell. Type X collagen is localized to regions adjacent to mineral deposits in the eggshell but is never colocalized with mineral deposits. We hypothesize that extracellular matrices containing type X collagen can function as a mineralization barrier.