Institute of Animal Sciences, The Volcani Center, Israel
The quality of the eggshell is of primary concern to the poultry industry. A simple increase in shell thickness is not a satisfactory goal, since the thickness accounts for only a small fraction of the shell's resistance to breakage. In order to improve eggshell quality, it is necessary to identify the molecular constituents involved in the mineralization of the eggshell that guide eggshell assembly and mineralization.
Various strategies were used to identify eggshell proteins such as eggshell extraction and protein purification, evaluating the role of proteins involved in other mineralization processes such as bone and the proteomic approach. At present a few hundred proteins are known to be part of the eggshell. Being an extracellular process, eggshell formation is governed by proteins responsible for calcium transport and establishment of the pH gradient needed for crystal formation and proteins that are secreted out, become integrated into the eggshell, regulate the calcification process and become part of the organic shell matrix.
At least three interrelated mechanisms regulate the expression of these genes: the mechanical strain imposed locally by the resident egg; circadian rhythm, probably through systemic hormone secretion; and the calcium flux itself. This occurs in a physiological setting on a daily basis.
The quality of the eggshell is of primary concern to the poultry industry. On the one hand, the successful development of a chicken embryo is dependent upon a robust eggshell for mechanical protection, for protection from infection, for prevention of water loss, and as a primary source of calcium for the embryonic skeleton. On the other hand, the commercial production and marketing of eggs exposes them to insults that cause a high rate of broken or cracked eggshells, which impose major economic losses on the egg producer.
A simple increase in shell thickness is not a satisfactory goal, since shell thickness affects gas and water exchange, and a thicker shell presents a greater obstacle to the emerging embryo. In addition, the thickness accounts for only a small fraction of the shell's resistance to breakage; therefore other characteristics of the shell should be assessed. In order to improve eggshell quality, it is necessary to identify the molecular constituents involved in the mineralization of the eggshell.
Biological molecules guide mineralization processes through a series of specific and definable calcium-biomolecule interactions that lead to the deposition of specific and uniquely oriented crystalline structures (Weiner and Addadi, 1991). Eggshell assembly and mineralization are guided by an array of biomolecules that follow a set of biological principles for the mineralization process. The process of mineralization in the avian eggshell follows a spatiotemporally defined series of events that correlate to specific regions along the oviduct (Arias et al., 1993). Thus, identification of eggshell matrix proteins and their genes, and elucidation of the mechanism and regulation of their synthesis and assembly along the successive segments of the oviduct form a major goal in the process of improving eggshell quality.
The eggshell is formed during passage of the egg through the oviduct, with the various layers of the eggshell assembled sequentially as the egg passes through the successive sectors of the oviduct. After fertilization of the ovum in the infundibulum, and secretion of albumen in the magnum, the egg enters the isthmus 2-3 hours after ovulation. In the isthmus, the granular cells secrete the various components of the shell membranes, such as collagen type X (Arias et al., 1991; 1997). Most of the calcium deposition in the eggshell occurs in the shell gland (ESG) (Creger et al., 1976; Stemberger et al., 1977). Approximately 5-6 g of calcium carbonate is deposited into the chicken eggshell during its formation; most of it during approximately 17 of the 20-hours residence of the egg in the ESG. The rapidity with which this large amount of calcium is deposited makes eggshell mineralization one of the fastest biomineralization processes known.
Involvement of matrix proteins in mineralization
It is widely accepted that the organic matrix components of biologically driven mineralization play a role in the control of crystallization. Extracellular matrix proteins of biomineralized structures influence the strength and shape of the final structure of calcium phosphate (apatite) or calcium carbonate (calcite) by modulating crystal nucleation and growth (Weiner and Addadi, 1991).
Various strategies were used to identify eggshell proteins:
(i) Eggshell extraction and protein purification enabled the eggshell proteins of various avian species to be identified and localized to different regions of the shell. For example, ovocleidin 17 was localized to the palisade and mammillary layers (Hincke et al., 1995), ovalbumin to the mammilary knobs (Hincke, 1995), lysozome and ovotransferrin (Gautron et al., 1997), dermatan sulfate proteoglycan is found in the palisade region (Carrino et al., 1997), keratan sulfate was extracted from the mammilae (Fernandez et al., 1997), ovocalyxin-32 (OCX-32), which is present at high levels in the uterine fluid during the terminal phase of eggshell formation, was localized predominantly to the outer eggshell (Hincke et al., 2003), as were ovocleidin-116 (Mann et al., 2002) and ansocalcin (Lakshminarayanan et al., 2002). The ability of some of these proteins to aggregate in solution or induce nucleation of calcite aggregates has been studied (Lakshminarayanan et al. 2005).
(ii) Phosphorylated matrix proteins such as osteopontin (OPN) are believed to play an important role in the process of bone mineralization. The involvement of OPN in bone calcification was deduced from its tissue distribution, its ability to bind calcium, its localization to electron-dense regions of mineralization, and the regulation of its gene expression by calcitrophic hormones such as 1,25(OH)2D3 and parathyroid hormone.
In the hen oviduct, OPN gene expression was detected only in the ESG, where massive calcification occurs, and not in any other part of the oviduct (Pines et al., 1996). The OPN gene was expressed in a circadian fashion during the daily egg cycle, only during the period of eggshell calcification. No OPN gene expression was detected in the ESG of a pre-laying hen before the onset of reproduction, or after forced removal of the egg close to its entrance into the ESG. The epithelial cells of the ESG, which line the lumen, are the source of OPN and, upon synthesis, OPN is immediately secreted out of these cells and localized in the core of the nonmineralized shell membrane fibers in the base of the mammillae and in the outermost part of the palisade). It was suggested that OPN could be part of the mechanism controlling the eggshell calcification arrest (Fernandez et al., 2003).
(iii) The recent elucidation of the chicken genome provided an opportunity to explore the matrix proteome of the eggshell biomineral. More than 500 proteins were identified and were divided among a few functional groups (Mann et al., 2006). Some of the proteins seem to be unique to the eggshell, some are present in other egg compartments and some are to be found in other tissues as well. Interesting questions emerge, such as the role of lipid-binding proteins in a milieu that is almost devoid of lipids, or the presence of proteins with functions such as apoptosis and angiogenesis in surroundings that lack cells or blood vessels.
Regulation of the synthesis of eggshell matrix components
The enormous number of proteins found in the eggshell suggests a very complex mechanism of regulation that would be expected to occur in different compartments of the oviduct and at precise time intervals. The unique circadian fashion of eggshell calcification allowed us to compare ESG gene expression at the time when no egg resides in the ESG and no calcification occurs, with that at the time when the egg resides in the ESG and calcification is at its peak. RNA fingerprinting revealed a set of genes that were differentially induced at the time of calcification (Lavelin et al., 2001, 2002). Some of them, such as Na-K-ATPase, are probably responsible for ion transport and establishing the pH required for calcification; the role of others, such as the proteoglycan glypican-4 is still unknown.
During the past few years, the effect of mechanical force on the regulation of cell functions has been extensively studied. Various stresses or strains, such as hydrostatic or hydrodynamic pressure, tensile or biaxial stretching and fluid shear stress have been studied. The applied forces caused a variety of physiological responses such as increased bone resorption, changes in matrix protein synthesis, cell differentiation, changes in smooth muscle contractility and increased cell migration, all of which involved multiple signal transduction pathways.
It was of great interest to discover that the mechanical strain imposed by the resident egg is coupled to a physiological response and is a major regulator of the expression of genes involved in eggshell calcification. This interpretation was supported by the following observations: the genes are expressed in the ESG only when an egg resides there and imposes a mechanical strain; removal of the mechanical strain caused reduction in the gene expression, and artificial application of a mechanical strain caused their induction to an extent related to the level of the strain (Lavelin et al., 1998, 2001, 2002).
The avian eggshell gland is a tissue specialized in the massive calcium transport needed for eggshell formation. Being an extracellular process, eggshell formation is governed by: (i) proteins responsible for biological processes within the tissue, such as calcium transport and establishment of the pH gradient needed for crystal formation; and (ii) proteins that are secreted out, become integrated into the eggshell, regulate the calcification process and become part of the organic shell matrix. At least three interrelated mechanisms regulate the expression of these genes: the mechanical strain imposed locally by the resident egg; circadian rhythm, probably through systemic hormone secretion; and the calcium flux itself. This occurs in a physiological setting on a daily basis.
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From Proceedings of the "19th Australian Poultry Science Symposium", New South Wales, Australia.