Colloidal-gold Immunocytochemical Localization of Osteopontin in Avian Eggshell Gland and Eggshell
Maxwell T. Hincke 1, Yung-Ching Chien 1, Louis C. Gerstenfeld 1 and Marc D. McKee 1*
1 Department of Cellular and Molecular Medicine, University of Ottawa, Ottawa, Ontario, Canada (MTH); Faculty of Dentistry (Y-CC,MDM) and Department of Anatomy and Cell Biology (MDM), McGill University, Montreal, Quebec, Canada; and Department of Orthopaedic Surgery, Boston University, Boston, Massachusetts (LCG)
* To whom correspondence should be addressed. E-mail: marc.mckee{at}mcgill.ca .
Submitted on December 13, 2007
Accepted on 11 January 2008
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Abstract |
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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 in order 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 EDTA-decalcified 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 fibres. Osteopontin was associated with protein sheets in the highly mineralized palisades region; some of these features defined boundaries which 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.
Key Words:
osteopontin, calcite, biomineralization, eggshell, eggshell matrix, chicken
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