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New Insight in Eggshell Formation
New Insight in Eggshell Formation I. Lavelin, N. Meiri, and M. Pines1 Institute of Animal Science, Agricultural Research Organization, The Volcani Center, Bet Dagan 50250 Israel protein have been shown to exist in the protein as reactive monoesters that are available for interaction with other ions, among them crystal constituents such as calcium ions. In addition, sulfation of OPN was also found to be associated with mineralization of other tissues. In contrast to the calbindin gene, whose expression is dependent on the calcium flux, the regulation of OPN synthesis is at least in part dependent on the mechanical strain imposed by the resident egg. These results demonstrate the complexity of the regulation of the matrix genes governing eggshell formation.
(Key words: eggshell, osteopontin, mechanical strain, calcification) 2000 Poultry Science 79:1014–1017
INTRODUCTION The quality of the eggshell is of primary concern to the poultry industry. The successful development of chicken embryos is dependent upon a robust eggshell for mechanical protection, protection from infection, 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 and are responsible for major economic losses to the egg producer. Additional losses occur in the highly mechanized washing, grading, and packing procedures. A simple increase in shell thickness is not a satisfactory goal, because shell thickness affects gas and water exchange, and a thicker shell presents a greater obstacle to the emerging embryo. In addition, shell thickness accounts for only a small fraction of the shell resistance; therefore, other characteristics of the shell should be assessed. 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). The process of mineralization in the avian eggshell follows a spatio-temporally defined
Received for publication December 15, 1999. Accepted for publication February 29, 2000. 1 To whom correspondence should be addressed: vlmpines@volcani. agri.gov.il.
series of events that correlate to specific regions along the oviduct (Arias et al., 1993). Eggshell assembly and mineralization are guided by an array of biomolecules that follow a set of biological principles for the mineralization process. Thus, identification of eggshell matrix proteins and their genes along the successive segments of the oviduct is a major goal in the process of improving eggshell quality.
EGGSHELL FORMATION 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 to 3 h after ovulation. In the isthmus, the granular cells secrete 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 eggshell gland (ESG) (Creger et al., 1976; Stemberger et al., 1977). Approximately 5 to 6 g of calcium carbonate is deposited into the chicken eggshell during its formation; most of this occurs during approximately 17 of the 20 h during which the egg resides in the ESG. The rapidity with which this large amount of calcium is deposited makes eggshell
Abbreviation Key: ESG = eggshell gland; OPN = osteopontin; EGF = epidermal growth factor; PTH = parathyroid hormone.
1014
Downloaded from http://ps.oxfordjournals.org/ at University of Southern Queensland on March 14, 2015
ABSTRACT The matrix proteins that participate in crystalization fulfill important functions during the formation of the calcified tissues and contribute to the biomechanical properties of the mature product. We suggest that osteopontin (OPN) is part of an array of macromolecules synthesized and secreted by the cells adjacent to the mineralization front that self-assemble outside the cell and direct crystal formation. The OPN meets the theoretical requirements for involvement in the mineralization process. The phosphorylated residues of acidic phospho-
SYMPOSIUM: SKELETAL BIOLOGY AND RELATED PROBELMS IN POULTRY
mineralization one of the most rapid biomineralization processes known.
INVOLVEMENT OF MATRIX PROTEINS IN MINERALIZATION
1993). The OPN gene is expressed in embryonic skeletal tissue as well as in adult tissues, normal and transformed bone cells, and in some cells of the bone marrow. In addition, OPN is found in certain nonosseous tissues such as kidney, the sensory epithelium of the embryonic ear, and the placental decidua. Chicken OPN has 35% homology in the protein sequence and 65% homology at the nucleotide level with the mammalian OPN (Castagnola, et al., 1991). Expression of the OPN gene, OPN phosphorylation, and secretion are tissue specific and are subject to regulation by many hormones, growth factors, tumor promoters, and oncogenes. For example, expression of OPN in osteoblast-like cells is stimulated by 1,25(OH)2D3, transforming growth factor β, and basic fibroblast growth factor and is inhibited by parathyroid hormone (PTH) and dexamethazone. In contrast, secretion of OPN by rat kidney cells is inhibited by transforming growth factor β and EGF.
OPN AND EGGSHELL FORMATION
Phosphorylated extracellular matrix proteins are thought to play an integral role in the process of the in vivo formation of vertebrate mineralized tissues (Glimcher 1985, 1989). Such proteins were found in almost every mineralized tissue that has been shown to undergo controlled mineralization. Phosphorylated proteins were isolated from the extracellular matrices of dentin (Veis and Perry, 1967) and from mammalian (Denhardt and Guo, 1993) and chicken (Gotoh et al., 1990) bones. Most of these acidic phosphoproteins contain regions in which seven to nine consecutive residues are aspartic acid, which is essential to ensure that calcium bind in such a way that it can interact with the protein and be part of the crystal. One such protein is osteopontin (OPN).
By means of in situ hybridization, OPN gene expression has been detected only in the ESG (Pines et al., 1996), where massive calcification occurs; none was detected in any other part of the oviduct, such as the magnum or isthmus, at any stage of the egg diurnal cycle. The OPN gene was expressed in a circadian fashion during the daily egg cycle only during the period of eggshell calcification. The matching of the oscillation of OPN gene expression with the egg cycle was specific to the ESG and was not found in other OPN-producing tissues such as the kidney. No OPN gene expression was detected in the ESG of a prelaying hen before the onset of reproduction or after forced removal of the egg close to its entry into the ESG. The epithelial cells of the ESG lining the lumen synthesized the OPN (Figure 1). Upon synthesis, OPN is immediately secreted out of the cells and accumulates in the eggshell. Calbindin, a calcium-binding protein, has also been found to be synthesized by the ESG in circadian fashion (Nys et al., 1989; Bar et al., 1992), but, unlike OPN, it was stored in the tubular gland cells of the ESG (Wasserman et al., 1991). The levels of plasma hormones such as PTH and of gonadal and other hormones, as well as the concentration of PTH-related peptide (PTHrP) in the oviduct, oscillate during the diurnal egg cycle and may be involved in the regulation of the matrix protein synthesis.
OSTEOPONTIN: STRUCTURE AND TISSUE DISTRIBUTION
MECHANICAL STRAIN AS INDUCER OF OPN GENE EXPRESSION IN THE ESG
Osteopontin (OPN/2ar/Spp), a phosphorylated glycoprotein, contains 12 phosphoserine residues and a phosphothreonine residue and is devoid of methionine (Prince et al., 1987). The protein binds to hydroxyapatite, facilitates the attachment of cells to tissue-culture plastic, and contains a Gly-Arg-Gly-Asp-Ser (GRGDS) amino acid sequence that serves as a recognition signal for interaction with the αvβ3 cell surface integrin receptor (Ross et al.,
We evaluated the regulation of the OPN gene expression by nonhormonal stimuli, such as calcium flux and mechanical strain, during the daily egg cycle in the oviduct of the laying hen (Lavelin et al., 1998). After the egg enters the ESG, the OPN gene is expressed by the epithelium cells in two waves: first by the basal cells and only then by the apical cells of the epithelium. A reduction in OPN gene expression was observed 1 h prior to lay.
PHOSPHOPROTEINS AND CALCIFICATION
Downloaded from http://ps.oxfordjournals.org/ at University of Southern Queensland on March 14, 2015
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 proteins of the eggshell have already been identified, for example ovocleidin 17 in the palisade and mammillary layers (Hincke et al., 1995), ovalbumin in the mammilary knobs (Hincke, 1995), lysozome and ovotransferrin (Gautron et al., 1997), dermatan sulfate proteoglycan in the palisade region (Carrino et al., 1997), and keratan sulfate on the starting mammilae (Fernandez et al., 1997). Some of the biomolecules, of which there is evidence for a regulatory role in biomineralization, are acidic in nature. In some cases they are located at the interface between the crystal and the extracellular matrix, whereas in others they can actually be found within the crystals. Usually, these acidic proteins are associated with more hydrophobic macromolecules (such as collagen type I in bone) to form extensive three-dimensional structures. Many similar examples have been described for other phyla having mineralized skeletal structures (Weiner and Addadi, 1991).
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formation of the calcified tissues and contribute to the biomechanical properties of the mature product. We suggest that OPN is part of an array of macromolecules synthesized and secreted by the cells adjacent to the mineralization front that self-assemble outside the cell and direct crystal formation. It is expected that the results obtained from similar studies will eventually enable the eggshell mechanical strength to be correlated with the levels of particular molecular components in an eggshell. This information is of the utmost importance for future planning strategies to improve hatchability and reduce eggshell cracking: it could be used by geneticists to select for better eggshell quality and could be incorporated into simulation algorithms for profit maximization in egg-laying operations.
FIGURE 1. Immunohistochemistry with avian osteopontin (OPN) antibodies (A) and in situ hybridization with a specific avian OPN probe (B) in the eggshell gland at the time of maximal calcification. Note that OPN is present and the gene is expressed by the epithelial layer facing the lumen.
The calbindin gene, which marks the onset of calcification, was expressed starting 2 h after OPN gene expression. The formation of soft shells was accompanied by a reduction in calbindin but not in OPN gene expression; thus, we concluded that the OPN gene is not regulated by the calcium flux. The application of a mechanical strain comparable to that induced by an egg led to induction of OPN gene expression during a normally quiescent phase in the cyclical expression of this gene. The induction of the gene was time- and strain-dependent and was temporally similar to its induction by the entry of the egg into the ESG. In contrast, the calbindin gene was not affected by mechanical strain. Thus, the ESG of the laying hen provides a system in which to study the effect of a mechanical strain on matrix protein production in vivo, in a relevant physiological setting. The finding suggests that in contrast to expression of the calbindin gene, that of the OPN gene is not regulated by calcium flux but rather by the mechanical strain imposed by the resident egg.
CONCLUSIONS It is most likely that the matrix proteins participating in crystallization fulfill important functions during the
Arias, J. L., M. S. Fernandez, J. E. Dennis, and A. I. Caplan, 1991. Collagens of the chicken eggshell membranes. Connect. Tissue Res. 26:37–45. Arias, J. L., D. J. Fink, S. Q. Xiao, A. H. Heuer, and A. I. Caplan, 1993. Biomineralization and eggshells: Cell-mediated acellular compartments of mineralized extracellular matrix. Int. Rev. Cytol. 145:217–250. Arias, J. L., O. Nakamura, M. S. Fernandez, J. J. Wu, P. Knigge, D. R. Eyre, and A. I. Caplan, 1997. Role of type X collagen on experimental mineralization of eggshell membrane. Connect. Tissue Res. 36:21–33. Bar, A., S. Striem, E. Vax, H. Talpaz, and S. Hurwitz, 1992. Regulation of calbindin messenger RNA and calbindin turnover in intestine and shell gland of the chicken. Am. J. Physiol. 262:R800–R805. Carrino, D. A., J. P. Rodrigues, and A. I. Caplan, 1997. Dermatan sulfate proteoglycans from the mineralized matrix of the avian eggshell. Connect. Tissue Res. 36:175–193. Castagnola, P., P. Bet, R. Quarto, M. Gennari, and R. Cancedda, 1991. cDNA cloning and gene expression of chicken osteopontin. Expression of osteopontin mRNA in chondrocytes is enhanced by trypsin treatment of cells. J. Biol. Chem. 266:9944–9949. Creger, C. R., H. Phillips, and J. J. Scott, 1976. Formation of an eggshell. Poult. Sci. 55:1717–1723. Denhardt, D. T., and X. Guo, 1993. Osteopontin: A protein with diverse functions. FASEB J. 7:1475–1482. Fernandez, M. S., M. Araya., and J. L. Arias, 1997. Eggshell are shaped by a precise spatio-temporal arrangement of sequentially deposited macromolecules. Matrix Biol. 16:13–20. Gautron, J., M. T. Hincke, J. M. Garcia-Ruiz, J. M. DominguezVera, and Y. Nys, 1977. Ovotransferrin and lysozome are constituents in eggshell matrix. Pages 66–73 in: Proceedings VII European Symposium on Quality of Eggs and Eggs Products. J. Kijoski and J. Pikul, ed. Poznan, Poland. Glimcher, M. J., 1985. Pages 607–616 in: Calcium in Biological Systems. R. P. Rubin, G. B. Weiss, and J. W. Putney, ed. Plenum Publishing Co., New York, NY. Glimcher M. J., 1989. Mechanism of calcification: Role of collagen fibrils and collagen-phosphoproteins complexes in vitro and in vivo. Anat. Rec. 224:139–153. Gotoh, Y., L. C. Gerstenfeld, and M. J. Glimcher, 1990. Identification and characterization of the major bone specific phosphoprotein synthesized by cultured embryonic chicken osteoblasts. Eur. J. Biochem. 187:49–58. Hincke, M. T., 1995. Ovalbumin is a component of the chicken eggshell matrix. Connect Tissue Res. 3:227–233. Hincke, M. T., C.P.W. Tsang, M. Courtney, V. Hill, and R. Narbaitz, 1995. Purification and immunohistochemistry of a solu-
Downloaded from http://ps.oxfordjournals.org/ at University of Southern Queensland on March 14, 2015
REFERENCES
SYMPOSIUM: SKELETAL BIOLOGY AND RELATED PROBELMS IN POULTRY ble matrix protein of the chicken eggshell (ovocleidin 17). Calcif. Tissue Int. 56:578–583. Lavelin, I., N. Yarden, S. Ben-Bassat, A. Bar, and M. Pines, 1998. Regulation of osteopontin gene expression during egg shell formation in the laying hen by mechanical strain. Matrix Biol. 17:615–623. Nys, Y., S. Mayel-Afshar, R. Bouillon, H. Van Balen, and D.E.M. Lawson 1989. Increases in calbindin D 28K mRNA in the uterus of the domestic fowl induced by sexual maturity and shell formation. Gen Comp. Endocrinol. 76:322–329. Pines, M., V. Knopov, and A. Bar, 1996. Involvement of osteopontin in egg shell formation in the laying chicken. Matrix Biol. 14:765–771. Prince, C. W., T. Oosawa, W. T. Butler, M. Tomana, A. S. Bhown, M. Bhown, and R. E. Schrohenloher, 1987. Isolation, characterization, and biosynthesis of a phosphorylated glycoprotein from rat bone. J. Biol. Chem. 262:2900–2907.
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Ross, F. P., J. Chappel, J. I. Alvarez, D. Sander, W. T. Butler, M. C. Farach-Carson, K. A. Mintz, P. G. Robey, S. L. Teitelbaum, and D. A. Cheresh, 1993. Interaction between the bone matrix proteins osteopontin and bone sialoprotein and the osteoclast integrin αvβ3 potentiate bone resorption. J. Biol. Chem. 268:9901–9907. Stemberger, B. H., W. J., Mueller, and R. M. Leach, 1977. Microscopic study of the initial stage of eggshell calcification. Poult. Sci. 56:537–543. Veis, A., and A. Perry, 1967. The phosphoprotein of the dentin matrix. Biochemistry 6:2409–2416. Wasserman, R. H., C. A. Smith, C. M. Smith, M. E. Brindak, C. S. Fullmer, L. Krook. J. T. Penniston, and R. H. Kumar, 1991. Immunohistochemical localization of a calcium pump and calbindin-D28K in the oviduct of the laying hen. Histochemistry 96:413–418. Weiner, S., and L. Addadi, 1991. Acidic macromolecules of mineralized tissues: The controllers of crystal formation. Trends Biochem. Sci. 16:252–256. Downloaded from http://ps.oxfordjournals.org/ at University of Southern Queensland on March 14, 2015
(Key words: eggshell, osteopontin, mechanical strain, calcification) 2000 Poultry Science 79:1014–1017
INTRODUCTION The quality of the eggshell is of primary concern to the poultry industry. The successful development of chicken embryos is dependent upon a robust eggshell for mechanical protection, protection from infection, 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 and are responsible for major economic losses to the egg producer. Additional losses occur in the highly mechanized washing, grading, and packing procedures. A simple increase in shell thickness is not a satisfactory goal, because shell thickness affects gas and water exchange, and a thicker shell presents a greater obstacle to the emerging embryo. In addition, shell thickness accounts for only a small fraction of the shell resistance; therefore, other characteristics of the shell should be assessed. 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). The process of mineralization in the avian eggshell follows a spatio-temporally defined
Received for publication December 15, 1999. Accepted for publication February 29, 2000. 1 To whom correspondence should be addressed: vlmpines@volcani. agri.gov.il.
series of events that correlate to specific regions along the oviduct (Arias et al., 1993). Eggshell assembly and mineralization are guided by an array of biomolecules that follow a set of biological principles for the mineralization process. Thus, identification of eggshell matrix proteins and their genes along the successive segments of the oviduct is a major goal in the process of improving eggshell quality.
EGGSHELL FORMATION 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 to 3 h after ovulation. In the isthmus, the granular cells secrete 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 eggshell gland (ESG) (Creger et al., 1976; Stemberger et al., 1977). Approximately 5 to 6 g of calcium carbonate is deposited into the chicken eggshell during its formation; most of this occurs during approximately 17 of the 20 h during which the egg resides in the ESG. The rapidity with which this large amount of calcium is deposited makes eggshell
Abbreviation Key: ESG = eggshell gland; OPN = osteopontin; EGF = epidermal growth factor; PTH = parathyroid hormone.
1014
Downloaded from http://ps.oxfordjournals.org/ at University of Southern Queensland on March 14, 2015
ABSTRACT The matrix proteins that participate in crystalization fulfill important functions during the formation of the calcified tissues and contribute to the biomechanical properties of the mature product. We suggest that osteopontin (OPN) is part of an array of macromolecules synthesized and secreted by the cells adjacent to the mineralization front that self-assemble outside the cell and direct crystal formation. The OPN meets the theoretical requirements for involvement in the mineralization process. The phosphorylated residues of acidic phospho-
SYMPOSIUM: SKELETAL BIOLOGY AND RELATED PROBELMS IN POULTRY
mineralization one of the most rapid biomineralization processes known.
INVOLVEMENT OF MATRIX PROTEINS IN MINERALIZATION
1993). The OPN gene is expressed in embryonic skeletal tissue as well as in adult tissues, normal and transformed bone cells, and in some cells of the bone marrow. In addition, OPN is found in certain nonosseous tissues such as kidney, the sensory epithelium of the embryonic ear, and the placental decidua. Chicken OPN has 35% homology in the protein sequence and 65% homology at the nucleotide level with the mammalian OPN (Castagnola, et al., 1991). Expression of the OPN gene, OPN phosphorylation, and secretion are tissue specific and are subject to regulation by many hormones, growth factors, tumor promoters, and oncogenes. For example, expression of OPN in osteoblast-like cells is stimulated by 1,25(OH)2D3, transforming growth factor β, and basic fibroblast growth factor and is inhibited by parathyroid hormone (PTH) and dexamethazone. In contrast, secretion of OPN by rat kidney cells is inhibited by transforming growth factor β and EGF.
OPN AND EGGSHELL FORMATION
Phosphorylated extracellular matrix proteins are thought to play an integral role in the process of the in vivo formation of vertebrate mineralized tissues (Glimcher 1985, 1989). Such proteins were found in almost every mineralized tissue that has been shown to undergo controlled mineralization. Phosphorylated proteins were isolated from the extracellular matrices of dentin (Veis and Perry, 1967) and from mammalian (Denhardt and Guo, 1993) and chicken (Gotoh et al., 1990) bones. Most of these acidic phosphoproteins contain regions in which seven to nine consecutive residues are aspartic acid, which is essential to ensure that calcium bind in such a way that it can interact with the protein and be part of the crystal. One such protein is osteopontin (OPN).
By means of in situ hybridization, OPN gene expression has been detected only in the ESG (Pines et al., 1996), where massive calcification occurs; none was detected in any other part of the oviduct, such as the magnum or isthmus, at any stage of the egg diurnal cycle. The OPN gene was expressed in a circadian fashion during the daily egg cycle only during the period of eggshell calcification. The matching of the oscillation of OPN gene expression with the egg cycle was specific to the ESG and was not found in other OPN-producing tissues such as the kidney. No OPN gene expression was detected in the ESG of a prelaying hen before the onset of reproduction or after forced removal of the egg close to its entry into the ESG. The epithelial cells of the ESG lining the lumen synthesized the OPN (Figure 1). Upon synthesis, OPN is immediately secreted out of the cells and accumulates in the eggshell. Calbindin, a calcium-binding protein, has also been found to be synthesized by the ESG in circadian fashion (Nys et al., 1989; Bar et al., 1992), but, unlike OPN, it was stored in the tubular gland cells of the ESG (Wasserman et al., 1991). The levels of plasma hormones such as PTH and of gonadal and other hormones, as well as the concentration of PTH-related peptide (PTHrP) in the oviduct, oscillate during the diurnal egg cycle and may be involved in the regulation of the matrix protein synthesis.
OSTEOPONTIN: STRUCTURE AND TISSUE DISTRIBUTION
MECHANICAL STRAIN AS INDUCER OF OPN GENE EXPRESSION IN THE ESG
Osteopontin (OPN/2ar/Spp), a phosphorylated glycoprotein, contains 12 phosphoserine residues and a phosphothreonine residue and is devoid of methionine (Prince et al., 1987). The protein binds to hydroxyapatite, facilitates the attachment of cells to tissue-culture plastic, and contains a Gly-Arg-Gly-Asp-Ser (GRGDS) amino acid sequence that serves as a recognition signal for interaction with the αvβ3 cell surface integrin receptor (Ross et al.,
We evaluated the regulation of the OPN gene expression by nonhormonal stimuli, such as calcium flux and mechanical strain, during the daily egg cycle in the oviduct of the laying hen (Lavelin et al., 1998). After the egg enters the ESG, the OPN gene is expressed by the epithelium cells in two waves: first by the basal cells and only then by the apical cells of the epithelium. A reduction in OPN gene expression was observed 1 h prior to lay.
PHOSPHOPROTEINS AND CALCIFICATION
Downloaded from http://ps.oxfordjournals.org/ at University of Southern Queensland on March 14, 2015
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 proteins of the eggshell have already been identified, for example ovocleidin 17 in the palisade and mammillary layers (Hincke et al., 1995), ovalbumin in the mammilary knobs (Hincke, 1995), lysozome and ovotransferrin (Gautron et al., 1997), dermatan sulfate proteoglycan in the palisade region (Carrino et al., 1997), and keratan sulfate on the starting mammilae (Fernandez et al., 1997). Some of the biomolecules, of which there is evidence for a regulatory role in biomineralization, are acidic in nature. In some cases they are located at the interface between the crystal and the extracellular matrix, whereas in others they can actually be found within the crystals. Usually, these acidic proteins are associated with more hydrophobic macromolecules (such as collagen type I in bone) to form extensive three-dimensional structures. Many similar examples have been described for other phyla having mineralized skeletal structures (Weiner and Addadi, 1991).
1015
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formation of the calcified tissues and contribute to the biomechanical properties of the mature product. We suggest that OPN is part of an array of macromolecules synthesized and secreted by the cells adjacent to the mineralization front that self-assemble outside the cell and direct crystal formation. It is expected that the results obtained from similar studies will eventually enable the eggshell mechanical strength to be correlated with the levels of particular molecular components in an eggshell. This information is of the utmost importance for future planning strategies to improve hatchability and reduce eggshell cracking: it could be used by geneticists to select for better eggshell quality and could be incorporated into simulation algorithms for profit maximization in egg-laying operations.
FIGURE 1. Immunohistochemistry with avian osteopontin (OPN) antibodies (A) and in situ hybridization with a specific avian OPN probe (B) in the eggshell gland at the time of maximal calcification. Note that OPN is present and the gene is expressed by the epithelial layer facing the lumen.
The calbindin gene, which marks the onset of calcification, was expressed starting 2 h after OPN gene expression. The formation of soft shells was accompanied by a reduction in calbindin but not in OPN gene expression; thus, we concluded that the OPN gene is not regulated by the calcium flux. The application of a mechanical strain comparable to that induced by an egg led to induction of OPN gene expression during a normally quiescent phase in the cyclical expression of this gene. The induction of the gene was time- and strain-dependent and was temporally similar to its induction by the entry of the egg into the ESG. In contrast, the calbindin gene was not affected by mechanical strain. Thus, the ESG of the laying hen provides a system in which to study the effect of a mechanical strain on matrix protein production in vivo, in a relevant physiological setting. The finding suggests that in contrast to expression of the calbindin gene, that of the OPN gene is not regulated by calcium flux but rather by the mechanical strain imposed by the resident egg.
CONCLUSIONS It is most likely that the matrix proteins participating in crystallization fulfill important functions during the
Arias, J. L., M. S. Fernandez, J. E. Dennis, and A. I. Caplan, 1991. Collagens of the chicken eggshell membranes. Connect. Tissue Res. 26:37–45. Arias, J. L., D. J. Fink, S. Q. Xiao, A. H. Heuer, and A. I. Caplan, 1993. Biomineralization and eggshells: Cell-mediated acellular compartments of mineralized extracellular matrix. Int. Rev. Cytol. 145:217–250. Arias, J. L., O. Nakamura, M. S. Fernandez, J. J. Wu, P. Knigge, D. R. Eyre, and A. I. Caplan, 1997. Role of type X collagen on experimental mineralization of eggshell membrane. Connect. Tissue Res. 36:21–33. Bar, A., S. Striem, E. Vax, H. Talpaz, and S. Hurwitz, 1992. Regulation of calbindin messenger RNA and calbindin turnover in intestine and shell gland of the chicken. Am. J. Physiol. 262:R800–R805. Carrino, D. A., J. P. Rodrigues, and A. I. Caplan, 1997. Dermatan sulfate proteoglycans from the mineralized matrix of the avian eggshell. Connect. Tissue Res. 36:175–193. Castagnola, P., P. Bet, R. Quarto, M. Gennari, and R. Cancedda, 1991. cDNA cloning and gene expression of chicken osteopontin. Expression of osteopontin mRNA in chondrocytes is enhanced by trypsin treatment of cells. J. Biol. Chem. 266:9944–9949. Creger, C. R., H. Phillips, and J. J. Scott, 1976. Formation of an eggshell. Poult. Sci. 55:1717–1723. Denhardt, D. T., and X. Guo, 1993. Osteopontin: A protein with diverse functions. FASEB J. 7:1475–1482. Fernandez, M. S., M. Araya., and J. L. Arias, 1997. Eggshell are shaped by a precise spatio-temporal arrangement of sequentially deposited macromolecules. Matrix Biol. 16:13–20. Gautron, J., M. T. Hincke, J. M. Garcia-Ruiz, J. M. DominguezVera, and Y. Nys, 1977. Ovotransferrin and lysozome are constituents in eggshell matrix. Pages 66–73 in: Proceedings VII European Symposium on Quality of Eggs and Eggs Products. J. Kijoski and J. Pikul, ed. Poznan, Poland. Glimcher, M. J., 1985. Pages 607–616 in: Calcium in Biological Systems. R. P. Rubin, G. B. Weiss, and J. W. Putney, ed. Plenum Publishing Co., New York, NY. Glimcher M. J., 1989. Mechanism of calcification: Role of collagen fibrils and collagen-phosphoproteins complexes in vitro and in vivo. Anat. Rec. 224:139–153. Gotoh, Y., L. C. Gerstenfeld, and M. J. Glimcher, 1990. Identification and characterization of the major bone specific phosphoprotein synthesized by cultured embryonic chicken osteoblasts. Eur. J. Biochem. 187:49–58. Hincke, M. T., 1995. Ovalbumin is a component of the chicken eggshell matrix. Connect Tissue Res. 3:227–233. Hincke, M. T., C.P.W. Tsang, M. Courtney, V. Hill, and R. Narbaitz, 1995. Purification and immunohistochemistry of a solu-
Downloaded from http://ps.oxfordjournals.org/ at University of Southern Queensland on March 14, 2015
REFERENCES
SYMPOSIUM: SKELETAL BIOLOGY AND RELATED PROBELMS IN POULTRY ble matrix protein of the chicken eggshell (ovocleidin 17). Calcif. Tissue Int. 56:578–583. Lavelin, I., N. Yarden, S. Ben-Bassat, A. Bar, and M. Pines, 1998. Regulation of osteopontin gene expression during egg shell formation in the laying hen by mechanical strain. Matrix Biol. 17:615–623. Nys, Y., S. Mayel-Afshar, R. Bouillon, H. Van Balen, and D.E.M. Lawson 1989. Increases in calbindin D 28K mRNA in the uterus of the domestic fowl induced by sexual maturity and shell formation. Gen Comp. Endocrinol. 76:322–329. Pines, M., V. Knopov, and A. Bar, 1996. Involvement of osteopontin in egg shell formation in the laying chicken. Matrix Biol. 14:765–771. Prince, C. W., T. Oosawa, W. T. Butler, M. Tomana, A. S. Bhown, M. Bhown, and R. E. Schrohenloher, 1987. Isolation, characterization, and biosynthesis of a phosphorylated glycoprotein from rat bone. J. Biol. Chem. 262:2900–2907.
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