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Osteopontin and calbindin gene expression in the eggshell gland as related to eggshell abnormalities
Osteopontin and calbindin gene expression in the eggshell gland as related to eggshell abnormalities H. Arazi,* I. Yoselewitz,* Y. Malka,* Y. Kelner,* O. Genin,† and M. Pines†1 *Extension Service, Ministry of Agriculture & Rural Development, PO Box 28, Bet Dagan 50250, Israel; and †Institute of Animal Sciences, the Volcani Center, Bet Dagan 50250, Israel
Key words: calbindin, osteopontin, eggshell, eggshell gland 2009 Poultry Science 88:647–653 doi:10.3382/ps.2008-00387 A simple increase in shell thickness is not a satisfactory solution because shell thickness affects gas and water exchange, and a thicker shell presents a greater obstacle to the emerging embryo. In addition, thickness is only one of the factors responsible for shell resistance to breakage. The avian egg shell is formed during the passage of the egg through the isthmus and the eggshell gland (ESG), during which the various layers of the shell are assembled sequentially. After fertilization of the ovum in the infundibulum and secretion of albumin in the magnum, the egg enters the isthmus, where various matrix components are secreted and assembled into the shell membranes (Arias et al., 1991). Most of the calcium deposition in the shell occurs while the egg resides in the ESG. It is widely accepted that the organic matrix components play roles in the control of crystallization and eggshell formation (Wu et al., 1994). Biological molecules guide mineralization processes through a series of specific and definable calcium-biomolecule interactions, which lead to the deposition of specific and uniquely ori-
INTRODUCTION The quality of the eggshell is of primary concern to the poultry industry (Hamilton et al., 1979; Roland, 1988; Hunton, 1995). The successful development of chicken embryos 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 (Karlsson and Lilja, 2008). However, the commercial processing and marketing of eggs exposes them to insults, which cause high rates of broken or cracked eggshells, which lead to major economic losses to the egg producer (Dhawale, 2008). Broken eggs cannot be sold as first-quality eggs, and the occurrence of hairline cracks poses the risks of bacterial contamination and of broken and leaking eggs that create problems of internal and external food quality and safety (Bain et al., 2006; Messens et al., 2007). ©2009 Poultry Science Association Inc. Received September 7, 2008. Accepted November 12, 2008. 1 Corresponding author: [email protected]
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only in sections of the pseudostratified epithelium, separated by areas of cells devoid of OPN gene expression, whereas calbindin was expressed at much greater levels throughout the glandular epithelium. Almost no OPN gene expression was observed in the ESG of layers producing corrugated shells, but their pattern of calbindin expression was similar to but somewhat greater than that in ESG that produced normal eggshells. In cases in which eggs had cracks at the sharp or blunt poles, OPN was expressed only at the side opposite to the cracks, whereas calbindin was expressed at both sides equally independent of the cracks. The results suggest that synthesis of the proteins associated with the formation of eggshells with the various abnormalities is controlled by different mechanisms. This may imply that more than 1 strategy will be required to improve eggshell quality.
ABSTRACT Eggshell quality is a major concern to the poultry industry: eggs with poor-quality shells hatch poorly and are rejected in the processing plant. The eggshell gland (ESG) proteins and the matrix proteins, which participate in crystallization, fulfill important functions during formation of the calcified tissues and contribute to the biomechanical properties of the mature product. We selected layers that consistently produced eggshells with specific abnormalities, and continued to do so after molting, and evaluated the expression of 2 genes—osteopontin (OPN) and calbindin—as related to particular eggshell abnormalities. These genes are synthesized by the ESG and appear to participate in the calcification process. When the ESG produces normal eggshells, OPN was expressed uniformly by all of the epithelial cells facing the lumen, and calbindin was expressed by the glandular epithelium. In contrast, in the layers producing pimpled eggs, OPN was expressed
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MATERIALS AND METHODS Birds and Selections for Eggshell Abnormalities A flock of White Leghorns hens were fed a commercial layer diet consisting of 18% CP and 4% calcium, as described previously (Yoselewitz and Balnave, 1989).
During the daily egg collection, the cages containing abnormal and broken shells were recorded. Layers in cages that yielded eggs with abnormal shells for 10 consecutive days were separated and housed in individual cages, the layers responsible for the abnormal eggshells were identified, and the type of abnormality of the shell was recorded, according to Roberts and Brackpool (1995). At the age of 68 wk, the selected layers underwent molting (Bell, 2003) and when they resumed lay, the type of shell abnormality was again recorded. To correlate the specific regions of the ESG corresponding to the blunt and sharp poles, the position of the egg was determined before lay at the time the egg resided within the ESG. Biopsies were taken from the blunt and sharp poles of the same bird and thus can be used as controls, demonstrating that the cells are typical and that the changes observed are not due to any health problems. All animal experiments were carried out according to the guidelines of the Volcani Center Institutional Committee for Care and Use of Laboratory Animals.
Preparation of Oviduct Sections and In Situ Hybridization The selected birds were sampled before onset of reproduction and biopsies were taken at 1 h before egg lay. Biopsies of the ESG (2 punch biopsies from each location from 2 birds from each shell-type group were taken) – normal, pimpled, and corrugated shells, and cracks at the sharp and blunt poles – were collected into PBS and fixed overnight in 4% paraformaldehyde in PBS at 4°C. Serial 5-μm sections were prepared after the samples had been dehydrated in graded ethanol solutions, cleared in chloroform, and embedded in Paraplast (McCormick Scientific, St, Louis, MO). For hybridization, the sections were deparaffinized in xylene, rehydrated through a graded series of ethanol solutions, rinsed in distilled water (5 min), and incubated in 2 × Sus scrofa chromosome at 70°C for 30 min. The sections were then rinsed in distilled water and treated with pronase (0.125 mg/mL in 50 mM Tris-HCl, 5 mM EDTA, pH 7.5) for 10 min. After digestion, the slides were rinsed with distilled water, postfixed in 10% formalin in PBS, blocked in 0.2% glycine, rinsed in distilled water, rapidly dehydrated through graded ethanol solutions, and air-dried for several hours. The sections (at least 10 sections/block) were then hybridized with digoxigeninlabeled avian OPN and calbindin probes. For OPN, we used the 820-bp avian OPN probe corresponding to the entire protein-coding sequence (Barak-Shalom et al., 1995; Pines et al., 1996; Lavelin et al., 1998) and for calbindin, we used a 1,300-bp PstI-HincII insert in pGEM vector system (Promega, Madison, WI; Hunziker, 1986). All the preparations for in situ hybridization within each experiment were performed simultaneously with the same probe, and all sections were dipped in emulsion and exposed for the same length of time.
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ented crystalline structures (Weiner and Addadi, 1991). The process of mineralization in the avian eggshell comprises a spatio-temporally defined series of events, which correlate to specific regions along the oviduct (Arias et al., 1993). During the years, various strategies were used to identify eggshell proteins and to associate them with various regions of the shell. They included eggshell extraction (Hincke et al., 1995; Hincke, 1995; Carrino et al.,1997; Gautron et al., 1996) and identification of proteins involved in bone mineralization (Pines et al., 1996; Fernandez et al., 2003). The recent elucidation of the chicken genome provided an opportunity to explore the matrix proteome of the eggshell: more than 500 proteins were found, some unique to the eggshell, some present in other egg compartments, and some in other tissues as well (Mann et al., 2006). To develop strategies to improve eggshell quality, it is important to elucidate the functions of these proteins. In this study, we evaluated the relationship to eggshell abnormalities of the gene expression of 2 proteins – calbindin and osteopontin (OPN) – known to be involved in calcium metabolism of the ESG. Calbindin is a 28,000-kDa calcium-binding protein, which fluctuates in a circadian fashion during the daily egg cycle, in close temporal association with eggshell calcification (Nys et al., 1989; Striem and Bar, 1991; Bar et al., 1992). Calbindin gene expression in the ESG is predominantly Ca2+-dependent and its expression was related to eggshell quality (Nys et al., 1989; Bar et al., 1992). Osteopontin is one of the phosphoproteins found in the eggshell (Fernandez et al., 2003; Mann et al., 2007) and is expressed in the ESG, where massive calcification occurs, also in a circadian fashion during the daily egg cycle but only during the period of shell calcification (Pines et al., 1996; Lavelin et al., 1998, 2000). Upon synthesis, OPN is immediately secreted out of the epithelial cells of the ESG and becomes localized in the core of the unmineralized shell membrane fibers in the bases of the mammillae and in the outermost part of the palisade. It was suggested that OPN could be involved in the mechanism controlling the arrest of eggshell calcification (Fernandez et al., 2003), and the specific occlusion of OPN into calcite during mineralization may influence eggshell structure and thereby modify its fracture resistance (Hincke et al., 2008). In the present study, we evaluated the expression of calbindin and OPN genes in the ESG of layers selected for producing eggs with specific surface abnormalities and with cracks in discrete locations.
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Layers that laid eggs with specific shell malformations were identified among a flock of 2,400 birds. While the birds were 20 to 40 wk of age, the eggshell characteristics were recorded daily. Layers whose eggs consistently had abnormal shells were transferred to individual cages. Of the layers in this age range, 11% were found to lay eggs with various shell irregularities. The distribution of shell types within this consistent group was as follows: pimpled shells (11%), corrugated shells (8%), cracks at the sharp or blunt pole (20 or 2%, respectively), soft shells (35%), without any shell (9%), and normal shells for the rest (15%; Figure 1). Of the eggs laid by the layers selected for their normal eggshells, 93% had normal shells, 1% had soft shells, 3% had corrugated shells, 2% had pimpled shells, and 1% were broken in various locations. To be certain that layers of abnormal eggs produced only eggs with a specific shell abnormality, we selected for further analysis layers that laid eggs with the same shell malformation after molting as they had laid before molting.
formly by all the epithelial cells of the ESG. Calbindin, on the other hand, was expressed by the glandular epithelium of the ESG, especially by cells below the pseudostratified epithelium. At this stage of the egg cycle, calbindin was expressed at a low level (Figure 2). Eggs with pimpled shells appeared to have small lumps of calcified material all over the shell. Some of these lumps were merely on the surface, whereas others derived from internal membranes. In the ESG of the selected layers that produced pimpled eggs OPN was expressed only in sections of the pseudostratified epithelium that were separated by areas of cells devoid of any OPN gene expression. Calbindin, on the other hand, was expressed throughout the glandular epithelium at much greater levels than were observed in the ESG of layers that produced normal eggshells. Corrugated eggs had a very rough and corrugated surface, probably associated with inability to control and terminate plumping, and with infectious bronchitis as suggested by Roberts and Brackpool (1995). Hardly any OPN gene expression was observed in the ESG of layers that produced shells with corrugated surfaces, and only a few cells expressed the gene. The pattern of calbindin expression was similar to that in layers of normal eggshells although at a somewhat greater level.
OPN and Calbindin Gene Expression in the ESG of Layers of Eggs with Abnormal Shells
OPN and Calbindin Gene Expression in the ESG of Layers of Eggs with Cracks at Discrete Locations
In the ESG of layers that produced eggs with normal shells, the OPN gene was expressed exclusively by the basal pseudostratified epithelium cells with nuclei close to the basement membrane; OPN was expressed uni-
Layers that consistently laid eggs with cracks at the sharp or blunt pole were selected, and ESG biopsies from both poles in each layer were taken for evaluation of OPN and calbindin gene expression. In layers
RESULTS Selection of Layers with Eggshell Abnormalities
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Figure 1. Distribution of eggshell irregularities in layers selected for consistently normal eggshells (A) and those selected for consistently producing eggs with shell abnormalities (B).
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that laid eggs mainly with cracks at the sharp pole, the level and pattern of expression of the OPN and calbindin genes in biopsies taken from the ESG part corresponding to the blunt pole were similar to those in the biopsies taken from layers that produced normal shells (Figure 3). In contrast, biopsies taken from the sharp pole-corresponding part of the ESG that generated eggs with cracks at the sharp pole exhibited almost no OPN gene expression, whereas calbindin expression was similar to that of the ESG of layers with normal eggshells. Similar phenomena were observed when biopsies were taken from layers of eggs mainly with cracks in the blunt pole: biopsies taken the side opposite to the cracks exhibited normal expression of OPN and calbindin genes, whereas those from the location corresponding to the cracks exhibited very low OPN gene expression and normal calbindin expression (Figure 4).
DISCUSSION In this study, we demonstrated for the first time a relationship between irregularities in individual ESG gene expression, on the one hand, and specific shell abnormalities, on the other hand, which suggests that there are differences in the regulation of synthesis of various proteins, during the formation of shells with various irregularities. The enormous number of proteins found in the eggshell suggests a very complex mechanism of regulation of shell formation that would be expected to operate in
different compartments of the oviduct and at a precise time intervals. Most of the proteins found in the shell probably originate in the ESG. In previous studies, eggshell abnormalities were associated with layer lines and lay periods (Kemps et al., 2006), virus infection (Chousalkar and Roberts, 2007), stress (Reynard and Savory, 1999; Lin et al., 2004), photoperiods (Backhouse and Gous, 2005), various nutritional deficiencies, nutritional excesses, and toxic compounds (Dhawale, 2008). The observation that some layers predominantly produced eggs with the same shell irregularities or cracks, and continued to do so even after molting, raised the question of whether this phenomenon is a local and acquired trait of individual layers or is hereditary. A large-scale experiment is needed to address this question. In this study, we evaluated the expression of OPN and calbindin genes synthesized in the ESG of layers that had been selected on the basis of laying mainly eggs with specific shell abnormalities. These 2 genes were chosen for several reasons. • They are both related to calcium metabolism: OPN is intimately involved in the regulation of both physiological and pathological mineralization (Mazzali et al., 2002), and calbindin is known as a facilitator of calcium diffusion (Feher, 1984; Christakos et al., 2007). • Both are associated with eggshell calcification and are expressed in the ESG in a circadian fashion
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Figure 2. Osteopontin (OPN) and calbindin gene expression in the eggshell gland. Eggshell gland biopsies were taken from layers with regular, pimpled, or corrugated eggshells. Osteopontin and calbindin gene expression was evaluated by in situ hybridization. Photographs were taken at low (magnification × 40) and high (magnification × 200) magnifications. In the normal eggshell, OPN was expressed by all of the pseudostratified epithelium cells (arrows). In the pimpled eggs, the arrow heads indicate areas devoid of OPN expression. Calbindin was expressed by the glandular epithelium (dashed arrows).
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during the daily egg formation cycle (Nys et al., 1989; Bar et al., 1992; Pines et al., 1996; Lavelin et al., 1998, 2000). • Association was demonstrated between OPN and calbindin gene expression and reduction of eggshell thickness after xenoestrogen treatment (Kamata et al., 2008). • Xenoestrogen treatment resulted in thinning of the mammillary layer at the same site where OPN was localized (Fernandez et al., 2003; Hincke et al., 2008; Kamata et al., 2008), and • OPN was hypothesized to regulate eggshell formation by inhibiting calcite growth at specific crystallographic faces and compartmental boundaries, thereby creating a biomineralized architecture whose structure provides for the properties and functions of the eggshell (Chien et al., 2008).
In attempting to correlate the expression of these genes with shell cracks and breakage, we compared their expressions in ESG biopsies taken from locations corresponding to the sharp or blunt poles (Figures 3 and 4). Although the levels of calbindin gene expression were similar at both locations irrespective of the location of the cracks, OPN expression was almost completely absent at the location corresponding to the cracks, in contrast to its level at the opposite location. A simple interpretation of these results is that calbindin is not involved in eggshell breakage, at least when it occurs at the eggs poles, whereas reduction in OPN synthesis is directly related to shell breakage. However, if we consider the broader context, these results suggest that normal shell calcification requires a very precise equilibrium between the expressions of OPN, calbindin, and probably various other genes among the matrix
Figure 4. Osteopontin (OPN) and calbindin gene expression in the eggshell gland of layers that produced eggs with cracks in the blunt pole. Osteopontin and calbindin gene expression was evaluated by in situ hybridization. Photographs were taken at low (magnification × 40) and high (magnification × 200) magnifications. In the sharp pole, OPN was expressed by all of the pseudostratified epithelium cells (arrows), whereas no OPN was observed in biopsies taken from the blunt pole. Calbindin was expressed by the glandular epithelium (dashed arrows) in both poles, irrespective of the location of the shell cracks.
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Figure 3. Osteopontin (OPN) and calbindin gene expression in the eggshell gland of layers that produced eggs with cracks in the sharp pole. Osteopontin and calbindin gene expression was evaluated by in situ hybridization. Photographs were taken at low (magnification × 40) and high (magnification × 200) magnifications. In the blunt pole, OPN was expressed by all of the pseudostratified epithelium cells (arrows), whereas no OPN was observed in biopsies taken from the sharp pole. Calbindin was expressed by the glandular epithelium (dashed arrows) in both poles, irrespective of the location of the shell cracks.
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ACKNOWLEDGMENTS The research reported here was supported by the Agricultural Research Organization, the Volcani Center, Bet Dagan, Israel.
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proteins synthesized by the ESG and that any deviation from this equilibrium will cause a specific shell abnormality. Examples of other proteins expressed in a circadian manner in the ESG include: Na+-K+-ATPase, an enzyme important in preserving the volume, pH, and electrical resting potential of cells, and proteoglycans such as glypican-4, which is hypothesized to serve as a coreceptor or modulator, or both, of the activities of various growth factors and to be among the proteoglycans that contribute to the biochemical properties of the mature product (Lavelin et al., 2001, 2002). Both OPN and calbindin are expressed in a circadian fashion during the daily egg cycle but are regulated by different mechanisms: OPN gene expression is regulated by the mechanical forces imposed by the egg entering the ESG, whereas the calbindin gene is regulated by the calcium flux (Lavelin et al., 1998). Thus, any changes in the kinetics of the regulatory processes could cause changes in the ratios between these and other shell matrix proteins at any given time, resulting in various aberrant shell formations that could culminate in surface abnormalities (Figure 2) or cracks in specific locations (Figures 3, 4). The observation that in the ESG of layers with pimpled shells the OPN gene was not expressed uniformly by all cells of the pseudostratified epithelium, in contrast to the situation in layers that produced normal shells (Figure 2), suggests that in addition to central regulatory processes there also may be local regulatory processes. There is no question that a large-scale study and more quantitative methods should be performed. Moreover, the interaction between many of the genes known to be involved in shell calcification should be evaluated. In summary, we demonstrated that OPN and calbindin are differentially expressed in the ESG of layers characterized by their eggshells with surface abnormalities or by cracks at specific sites. These results suggest that there are several different regulation mechanisms controlling the synthesis of the proteins involved in formation of eggshells that exhibit different abnormalities. These observations also raise the question of the efficacy of any single treatment, pharmacological or other, for all shell abnormalities.
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Lavelin, I., N. Meiri, M. Einat, O. Genin, and M. Pines. 2002. Mechanical strain regulation of the chicken glypican-4 gene expression in the avian eggshell gland. Am. J. Physiol. Regul. Integr. Comp. Physiol. 283:R853–R856. Lavelin, I., N. Meiri, O. Genin, R. Alexiev, and M. Pines. 2001. Na+K+-ATPase gene expression in the avian eggshell gland: Distinct regulation in different cell types. Am. J. Physiol. Regul. Integr. Comp. Physiol. 281:R1169–R1176. Lavelin, I., N. Meiri, and M. Pines. 2000. New insight in eggshell formation. Poult. Sci. 79:1014–1017. 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. Lin, H., K. Mertens, B. Kemp, T. Govaerts, B. De Ketelaere, J. Baerdemaeker, E. M. Decuypere, and J. Buyse. 2004. New approach of testing the effect of heat stress on eggshell quality: Mechanical and material properties of eggshell and membrane. Br. Poult. Sci. 45:476–482. Mann, K., B. Macek, and J. V. Olsen. 2006. Proteomic analysis of the acid-soluble organic matrix of the chicken calcified eggshell layer. Proteomics 6:3801–3810. Mann, K., J. V. Olsen, B. Macek, F. Gnad, and M. Mann. 2007. Phosphoproteins of the chicken eggshell calcified layer. Proteomics 7:106–115. Mazzali, M., T. Kipari, V. Ophascharoensuk, J. A. Wesson, R. Johnson, and J. Hughes. 2002. Osteopontin–A molecule for all seasons. Q. J. Med. 95:3–13. Messens, W., K. Griispeerdt, K. De Reu, B. De Ketelaere, K. Mertens, F. Bamelis, B. Kemp, J. De Baerdemaeker, E. Decuypere, and L. Herman. 2007. Eggshell penetration of various types
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Key words: calbindin, osteopontin, eggshell, eggshell gland 2009 Poultry Science 88:647–653 doi:10.3382/ps.2008-00387 A simple increase in shell thickness is not a satisfactory solution because shell thickness affects gas and water exchange, and a thicker shell presents a greater obstacle to the emerging embryo. In addition, thickness is only one of the factors responsible for shell resistance to breakage. The avian egg shell is formed during the passage of the egg through the isthmus and the eggshell gland (ESG), during which the various layers of the shell are assembled sequentially. After fertilization of the ovum in the infundibulum and secretion of albumin in the magnum, the egg enters the isthmus, where various matrix components are secreted and assembled into the shell membranes (Arias et al., 1991). Most of the calcium deposition in the shell occurs while the egg resides in the ESG. It is widely accepted that the organic matrix components play roles in the control of crystallization and eggshell formation (Wu et al., 1994). Biological molecules guide mineralization processes through a series of specific and definable calcium-biomolecule interactions, which lead to the deposition of specific and uniquely ori-
INTRODUCTION The quality of the eggshell is of primary concern to the poultry industry (Hamilton et al., 1979; Roland, 1988; Hunton, 1995). The successful development of chicken embryos 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 (Karlsson and Lilja, 2008). However, the commercial processing and marketing of eggs exposes them to insults, which cause high rates of broken or cracked eggshells, which lead to major economic losses to the egg producer (Dhawale, 2008). Broken eggs cannot be sold as first-quality eggs, and the occurrence of hairline cracks poses the risks of bacterial contamination and of broken and leaking eggs that create problems of internal and external food quality and safety (Bain et al., 2006; Messens et al., 2007). ©2009 Poultry Science Association Inc. Received September 7, 2008. Accepted November 12, 2008. 1 Corresponding author: [email protected]
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only in sections of the pseudostratified epithelium, separated by areas of cells devoid of OPN gene expression, whereas calbindin was expressed at much greater levels throughout the glandular epithelium. Almost no OPN gene expression was observed in the ESG of layers producing corrugated shells, but their pattern of calbindin expression was similar to but somewhat greater than that in ESG that produced normal eggshells. In cases in which eggs had cracks at the sharp or blunt poles, OPN was expressed only at the side opposite to the cracks, whereas calbindin was expressed at both sides equally independent of the cracks. The results suggest that synthesis of the proteins associated with the formation of eggshells with the various abnormalities is controlled by different mechanisms. This may imply that more than 1 strategy will be required to improve eggshell quality.
ABSTRACT Eggshell quality is a major concern to the poultry industry: eggs with poor-quality shells hatch poorly and are rejected in the processing plant. The eggshell gland (ESG) proteins and the matrix proteins, which participate in crystallization, fulfill important functions during formation of the calcified tissues and contribute to the biomechanical properties of the mature product. We selected layers that consistently produced eggshells with specific abnormalities, and continued to do so after molting, and evaluated the expression of 2 genes—osteopontin (OPN) and calbindin—as related to particular eggshell abnormalities. These genes are synthesized by the ESG and appear to participate in the calcification process. When the ESG produces normal eggshells, OPN was expressed uniformly by all of the epithelial cells facing the lumen, and calbindin was expressed by the glandular epithelium. In contrast, in the layers producing pimpled eggs, OPN was expressed
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MATERIALS AND METHODS Birds and Selections for Eggshell Abnormalities A flock of White Leghorns hens were fed a commercial layer diet consisting of 18% CP and 4% calcium, as described previously (Yoselewitz and Balnave, 1989).
During the daily egg collection, the cages containing abnormal and broken shells were recorded. Layers in cages that yielded eggs with abnormal shells for 10 consecutive days were separated and housed in individual cages, the layers responsible for the abnormal eggshells were identified, and the type of abnormality of the shell was recorded, according to Roberts and Brackpool (1995). At the age of 68 wk, the selected layers underwent molting (Bell, 2003) and when they resumed lay, the type of shell abnormality was again recorded. To correlate the specific regions of the ESG corresponding to the blunt and sharp poles, the position of the egg was determined before lay at the time the egg resided within the ESG. Biopsies were taken from the blunt and sharp poles of the same bird and thus can be used as controls, demonstrating that the cells are typical and that the changes observed are not due to any health problems. All animal experiments were carried out according to the guidelines of the Volcani Center Institutional Committee for Care and Use of Laboratory Animals.
Preparation of Oviduct Sections and In Situ Hybridization The selected birds were sampled before onset of reproduction and biopsies were taken at 1 h before egg lay. Biopsies of the ESG (2 punch biopsies from each location from 2 birds from each shell-type group were taken) – normal, pimpled, and corrugated shells, and cracks at the sharp and blunt poles – were collected into PBS and fixed overnight in 4% paraformaldehyde in PBS at 4°C. Serial 5-μm sections were prepared after the samples had been dehydrated in graded ethanol solutions, cleared in chloroform, and embedded in Paraplast (McCormick Scientific, St, Louis, MO). For hybridization, the sections were deparaffinized in xylene, rehydrated through a graded series of ethanol solutions, rinsed in distilled water (5 min), and incubated in 2 × Sus scrofa chromosome at 70°C for 30 min. The sections were then rinsed in distilled water and treated with pronase (0.125 mg/mL in 50 mM Tris-HCl, 5 mM EDTA, pH 7.5) for 10 min. After digestion, the slides were rinsed with distilled water, postfixed in 10% formalin in PBS, blocked in 0.2% glycine, rinsed in distilled water, rapidly dehydrated through graded ethanol solutions, and air-dried for several hours. The sections (at least 10 sections/block) were then hybridized with digoxigeninlabeled avian OPN and calbindin probes. For OPN, we used the 820-bp avian OPN probe corresponding to the entire protein-coding sequence (Barak-Shalom et al., 1995; Pines et al., 1996; Lavelin et al., 1998) and for calbindin, we used a 1,300-bp PstI-HincII insert in pGEM vector system (Promega, Madison, WI; Hunziker, 1986). All the preparations for in situ hybridization within each experiment were performed simultaneously with the same probe, and all sections were dipped in emulsion and exposed for the same length of time.
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ented crystalline structures (Weiner and Addadi, 1991). The process of mineralization in the avian eggshell comprises a spatio-temporally defined series of events, which correlate to specific regions along the oviduct (Arias et al., 1993). During the years, various strategies were used to identify eggshell proteins and to associate them with various regions of the shell. They included eggshell extraction (Hincke et al., 1995; Hincke, 1995; Carrino et al.,1997; Gautron et al., 1996) and identification of proteins involved in bone mineralization (Pines et al., 1996; Fernandez et al., 2003). The recent elucidation of the chicken genome provided an opportunity to explore the matrix proteome of the eggshell: more than 500 proteins were found, some unique to the eggshell, some present in other egg compartments, and some in other tissues as well (Mann et al., 2006). To develop strategies to improve eggshell quality, it is important to elucidate the functions of these proteins. In this study, we evaluated the relationship to eggshell abnormalities of the gene expression of 2 proteins – calbindin and osteopontin (OPN) – known to be involved in calcium metabolism of the ESG. Calbindin is a 28,000-kDa calcium-binding protein, which fluctuates in a circadian fashion during the daily egg cycle, in close temporal association with eggshell calcification (Nys et al., 1989; Striem and Bar, 1991; Bar et al., 1992). Calbindin gene expression in the ESG is predominantly Ca2+-dependent and its expression was related to eggshell quality (Nys et al., 1989; Bar et al., 1992). Osteopontin is one of the phosphoproteins found in the eggshell (Fernandez et al., 2003; Mann et al., 2007) and is expressed in the ESG, where massive calcification occurs, also in a circadian fashion during the daily egg cycle but only during the period of shell calcification (Pines et al., 1996; Lavelin et al., 1998, 2000). Upon synthesis, OPN is immediately secreted out of the epithelial cells of the ESG and becomes localized in the core of the unmineralized shell membrane fibers in the bases of the mammillae and in the outermost part of the palisade. It was suggested that OPN could be involved in the mechanism controlling the arrest of eggshell calcification (Fernandez et al., 2003), and the specific occlusion of OPN into calcite during mineralization may influence eggshell structure and thereby modify its fracture resistance (Hincke et al., 2008). In the present study, we evaluated the expression of calbindin and OPN genes in the ESG of layers selected for producing eggs with specific surface abnormalities and with cracks in discrete locations.
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Layers that laid eggs with specific shell malformations were identified among a flock of 2,400 birds. While the birds were 20 to 40 wk of age, the eggshell characteristics were recorded daily. Layers whose eggs consistently had abnormal shells were transferred to individual cages. Of the layers in this age range, 11% were found to lay eggs with various shell irregularities. The distribution of shell types within this consistent group was as follows: pimpled shells (11%), corrugated shells (8%), cracks at the sharp or blunt pole (20 or 2%, respectively), soft shells (35%), without any shell (9%), and normal shells for the rest (15%; Figure 1). Of the eggs laid by the layers selected for their normal eggshells, 93% had normal shells, 1% had soft shells, 3% had corrugated shells, 2% had pimpled shells, and 1% were broken in various locations. To be certain that layers of abnormal eggs produced only eggs with a specific shell abnormality, we selected for further analysis layers that laid eggs with the same shell malformation after molting as they had laid before molting.
formly by all the epithelial cells of the ESG. Calbindin, on the other hand, was expressed by the glandular epithelium of the ESG, especially by cells below the pseudostratified epithelium. At this stage of the egg cycle, calbindin was expressed at a low level (Figure 2). Eggs with pimpled shells appeared to have small lumps of calcified material all over the shell. Some of these lumps were merely on the surface, whereas others derived from internal membranes. In the ESG of the selected layers that produced pimpled eggs OPN was expressed only in sections of the pseudostratified epithelium that were separated by areas of cells devoid of any OPN gene expression. Calbindin, on the other hand, was expressed throughout the glandular epithelium at much greater levels than were observed in the ESG of layers that produced normal eggshells. Corrugated eggs had a very rough and corrugated surface, probably associated with inability to control and terminate plumping, and with infectious bronchitis as suggested by Roberts and Brackpool (1995). Hardly any OPN gene expression was observed in the ESG of layers that produced shells with corrugated surfaces, and only a few cells expressed the gene. The pattern of calbindin expression was similar to that in layers of normal eggshells although at a somewhat greater level.
OPN and Calbindin Gene Expression in the ESG of Layers of Eggs with Abnormal Shells
OPN and Calbindin Gene Expression in the ESG of Layers of Eggs with Cracks at Discrete Locations
In the ESG of layers that produced eggs with normal shells, the OPN gene was expressed exclusively by the basal pseudostratified epithelium cells with nuclei close to the basement membrane; OPN was expressed uni-
Layers that consistently laid eggs with cracks at the sharp or blunt pole were selected, and ESG biopsies from both poles in each layer were taken for evaluation of OPN and calbindin gene expression. In layers
RESULTS Selection of Layers with Eggshell Abnormalities
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Figure 1. Distribution of eggshell irregularities in layers selected for consistently normal eggshells (A) and those selected for consistently producing eggs with shell abnormalities (B).
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that laid eggs mainly with cracks at the sharp pole, the level and pattern of expression of the OPN and calbindin genes in biopsies taken from the ESG part corresponding to the blunt pole were similar to those in the biopsies taken from layers that produced normal shells (Figure 3). In contrast, biopsies taken from the sharp pole-corresponding part of the ESG that generated eggs with cracks at the sharp pole exhibited almost no OPN gene expression, whereas calbindin expression was similar to that of the ESG of layers with normal eggshells. Similar phenomena were observed when biopsies were taken from layers of eggs mainly with cracks in the blunt pole: biopsies taken the side opposite to the cracks exhibited normal expression of OPN and calbindin genes, whereas those from the location corresponding to the cracks exhibited very low OPN gene expression and normal calbindin expression (Figure 4).
DISCUSSION In this study, we demonstrated for the first time a relationship between irregularities in individual ESG gene expression, on the one hand, and specific shell abnormalities, on the other hand, which suggests that there are differences in the regulation of synthesis of various proteins, during the formation of shells with various irregularities. The enormous number of proteins found in the eggshell suggests a very complex mechanism of regulation of shell formation that would be expected to operate in
different compartments of the oviduct and at a precise time intervals. Most of the proteins found in the shell probably originate in the ESG. In previous studies, eggshell abnormalities were associated with layer lines and lay periods (Kemps et al., 2006), virus infection (Chousalkar and Roberts, 2007), stress (Reynard and Savory, 1999; Lin et al., 2004), photoperiods (Backhouse and Gous, 2005), various nutritional deficiencies, nutritional excesses, and toxic compounds (Dhawale, 2008). The observation that some layers predominantly produced eggs with the same shell irregularities or cracks, and continued to do so even after molting, raised the question of whether this phenomenon is a local and acquired trait of individual layers or is hereditary. A large-scale experiment is needed to address this question. In this study, we evaluated the expression of OPN and calbindin genes synthesized in the ESG of layers that had been selected on the basis of laying mainly eggs with specific shell abnormalities. These 2 genes were chosen for several reasons. • They are both related to calcium metabolism: OPN is intimately involved in the regulation of both physiological and pathological mineralization (Mazzali et al., 2002), and calbindin is known as a facilitator of calcium diffusion (Feher, 1984; Christakos et al., 2007). • Both are associated with eggshell calcification and are expressed in the ESG in a circadian fashion
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Figure 2. Osteopontin (OPN) and calbindin gene expression in the eggshell gland. Eggshell gland biopsies were taken from layers with regular, pimpled, or corrugated eggshells. Osteopontin and calbindin gene expression was evaluated by in situ hybridization. Photographs were taken at low (magnification × 40) and high (magnification × 200) magnifications. In the normal eggshell, OPN was expressed by all of the pseudostratified epithelium cells (arrows). In the pimpled eggs, the arrow heads indicate areas devoid of OPN expression. Calbindin was expressed by the glandular epithelium (dashed arrows).
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during the daily egg formation cycle (Nys et al., 1989; Bar et al., 1992; Pines et al., 1996; Lavelin et al., 1998, 2000). • Association was demonstrated between OPN and calbindin gene expression and reduction of eggshell thickness after xenoestrogen treatment (Kamata et al., 2008). • Xenoestrogen treatment resulted in thinning of the mammillary layer at the same site where OPN was localized (Fernandez et al., 2003; Hincke et al., 2008; Kamata et al., 2008), and • OPN was hypothesized to regulate eggshell formation by inhibiting calcite growth at specific crystallographic faces and compartmental boundaries, thereby creating a biomineralized architecture whose structure provides for the properties and functions of the eggshell (Chien et al., 2008).
In attempting to correlate the expression of these genes with shell cracks and breakage, we compared their expressions in ESG biopsies taken from locations corresponding to the sharp or blunt poles (Figures 3 and 4). Although the levels of calbindin gene expression were similar at both locations irrespective of the location of the cracks, OPN expression was almost completely absent at the location corresponding to the cracks, in contrast to its level at the opposite location. A simple interpretation of these results is that calbindin is not involved in eggshell breakage, at least when it occurs at the eggs poles, whereas reduction in OPN synthesis is directly related to shell breakage. However, if we consider the broader context, these results suggest that normal shell calcification requires a very precise equilibrium between the expressions of OPN, calbindin, and probably various other genes among the matrix
Figure 4. Osteopontin (OPN) and calbindin gene expression in the eggshell gland of layers that produced eggs with cracks in the blunt pole. Osteopontin and calbindin gene expression was evaluated by in situ hybridization. Photographs were taken at low (magnification × 40) and high (magnification × 200) magnifications. In the sharp pole, OPN was expressed by all of the pseudostratified epithelium cells (arrows), whereas no OPN was observed in biopsies taken from the blunt pole. Calbindin was expressed by the glandular epithelium (dashed arrows) in both poles, irrespective of the location of the shell cracks.
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Figure 3. Osteopontin (OPN) and calbindin gene expression in the eggshell gland of layers that produced eggs with cracks in the sharp pole. Osteopontin and calbindin gene expression was evaluated by in situ hybridization. Photographs were taken at low (magnification × 40) and high (magnification × 200) magnifications. In the blunt pole, OPN was expressed by all of the pseudostratified epithelium cells (arrows), whereas no OPN was observed in biopsies taken from the sharp pole. Calbindin was expressed by the glandular epithelium (dashed arrows) in both poles, irrespective of the location of the shell cracks.
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ACKNOWLEDGMENTS The research reported here was supported by the Agricultural Research Organization, the Volcani Center, Bet Dagan, Israel.
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proteins synthesized by the ESG and that any deviation from this equilibrium will cause a specific shell abnormality. Examples of other proteins expressed in a circadian manner in the ESG include: Na+-K+-ATPase, an enzyme important in preserving the volume, pH, and electrical resting potential of cells, and proteoglycans such as glypican-4, which is hypothesized to serve as a coreceptor or modulator, or both, of the activities of various growth factors and to be among the proteoglycans that contribute to the biochemical properties of the mature product (Lavelin et al., 2001, 2002). Both OPN and calbindin are expressed in a circadian fashion during the daily egg cycle but are regulated by different mechanisms: OPN gene expression is regulated by the mechanical forces imposed by the egg entering the ESG, whereas the calbindin gene is regulated by the calcium flux (Lavelin et al., 1998). Thus, any changes in the kinetics of the regulatory processes could cause changes in the ratios between these and other shell matrix proteins at any given time, resulting in various aberrant shell formations that could culminate in surface abnormalities (Figure 2) or cracks in specific locations (Figures 3, 4). The observation that in the ESG of layers with pimpled shells the OPN gene was not expressed uniformly by all cells of the pseudostratified epithelium, in contrast to the situation in layers that produced normal shells (Figure 2), suggests that in addition to central regulatory processes there also may be local regulatory processes. There is no question that a large-scale study and more quantitative methods should be performed. Moreover, the interaction between many of the genes known to be involved in shell calcification should be evaluated. In summary, we demonstrated that OPN and calbindin are differentially expressed in the ESG of layers characterized by their eggshells with surface abnormalities or by cracks at specific sites. These results suggest that there are several different regulation mechanisms controlling the synthesis of the proteins involved in formation of eggshells that exhibit different abnormalities. These observations also raise the question of the efficacy of any single treatment, pharmacological or other, for all shell abnormalities.
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