Colloidal-gold Immunocytochemical Localization of Osteopontin in Avian Eggshell Gland and Eggshell

Abstract

S U M M A R Y During mineralization of the avian eggshell, there is a sequential and orderly deposition of both matrix and mineral phases. Therefore, the eggshell is an excellent model for studying matrix-mineral relationships and the regulation of mineralization. Osteopontin, as an inhibitor of crystal growth, potently influences the formation of calcium phosphate and calcium carbonate biominerals. The purpose of this study was to characterize matrix-mineral relationships, specifically for osteopontin, in the avian eggshell using high-resolution transmission (TEM) and scanning (SEM) electron microscopy to gain insight into how calcite crystal growth is structured and compartmentalized during eggshell mineralization. Osteopontin was localized at the ultrastructural level by colloidal-gold immunocytochemistry. In EDTAdecalcified eggshell, an extensive matrix network was observed by TEM and SEM throughout all regions and included interconnected fibrous sheets, irregularly shaped aggregates, vesicular structures, protein films, and isolated protein fibers. Osteopontin was associated with protein sheets in the highly mineralized palisades region; some of these features defined boundaries that compartmentalized different eggshell structural units. In fractured and undecalcified eggshell, osteopontin was immunolocalized on the {104} crystallographic faces of calcite-its natural cleavage plane. The specific occlusion of osteopontin into calcite during mineralization may influence eggshell structure to modify its fracture resistance. (J Histochem Cytochem 56:467-476, 2008)

Protein constituents of the eggshell: eggshell-specific matrix proteins

“…(1) ''Egg white'' proteins which are also present in the eggshell-these include ovalbumin, the most abundant egg white protein [23], lysozyme, an antimicrobial protein with hydrolytic activity against peptidoglycans on cell walls of Gram-positive bacteria [24] and ovotransferrin, which sequesters iron necessary for bacterial growth [25]; (2) Ubiquitous proteins that are found in many tissuesexamples are osteopontin, a phosphorylated glycoprotein present in bone and other hard tissues [31][32][33][34][35][36], and clusterin, a widely distributed secretory glycoprotein that is also found in chicken egg white [37]; (3) Eggshell-specific matrix proteins unique to the shell calcification process that are secreted by cells in specific regions of the oviduct where eggshell mineralization is initiated (red isthmus) and continues to completion (uterus). It is these ''eggshell-specific matrix proteins'' which are the primary focus of this review.…”

“…One member, osteopontin, is also an eggshell matrix protein [31][32][33][34][35][36], and regulates calcification in vertebrate biominerals such as bone and teeth [77]. In the eggshell, osteopontin may function during mineralization by inhibiting calcium carbonate precipitation in a phosphorylation-dependent manner [32,78].…”

Ultrastructural matrix–mineral relationships in avian eggshell, and effects of osteopontin on calcite growth in vitro

“…The egg then enters the shell gland pouch (uterus), where mineralization first begins within these initial mammillary base plates (also called knobs, or mammillary tips) located on the outer shell membrane (Wyburn et al, 1973;Fernandez et al, 1997). Granular epithelial cells of the uterus secrete OPN into the uterine fluid which then accumulates in the forming eggshell (Pines et al, 1995;Lavelin et al, 1998Lavelin et al, , 2000Hincke et al, 2008). At this time of early calcification, the CRB and the CRB sac are not yet fully developed (Wyburn et al, 1973).…”

“…OPN is synthesized and secreted by nonciliated granular cells of the surface epithelium lining the lumen of uterus (shell gland) (Pines et al, 1995;Hincke et al, 2008), and its production is stimulated by entry of the egg into the uterus and the associated mechanical strain due to distension of the uterine wall (Lavelin et al, 1998(Lavelin et al, , 2000. This egg-induced expression of OPN leads to release of OPN into the uterine fluid whereupon it subsequently accumulates as part of the eggshell matrix (Pines et al, 1995;Hincke et al, 2008).…”

“…OPN is synthesized and secreted by nonciliated granular cells of the surface epithelium lining the lumen of uterus (shell gland) (Pines et al, 1995;Hincke et al, 2008), and its production is stimulated by entry of the egg into the uterus and the associated mechanical strain due to distension of the uterine wall (Lavelin et al, 1998(Lavelin et al, , 2000. This egg-induced expression of OPN leads to release of OPN into the uterine fluid whereupon it subsequently accumulates as part of the eggshell matrix (Pines et al, 1995;Hincke et al, 2008). While several reports show OPN expression by cells and localization in eggshell matrix (Pines et al, 1995;Fernandez et al, 2003), data is limited with respect to the ultrastructural distribution of OPN, and its relationship with eggshell structure-both in terms of its organization into the shell matrix, and its association with the crystallographic faces of the calcitic mineral.…”